rutabaga

DEVELOPMENTAL BIOLOGY

Adult

RNA in situ hybridization and immunohistochemistry demonstrate that the expression of the rutabaga gene is markedly elevated in the mushroom bodies of normal flies (Han, 1992).

Most attempts to localize physical correlates of memory in the central nervous system (CNS) rely on ablation techniques. This approach has the limitation of defining just one of an unknown number of structures necessary for memory formation. The Drosophila rutabaga type I Ca(2+)/CaM-dependent adenylyl cyclase (AC) gene has been used to determine in which CNS region AC expression is sufficient for memory formation. Using pan-neural and restricted CNS expression with the GAL4 binary transcription activation system, the memory defect of the rutabaga mutant has been rescued in a fast robust spatial learning paradigm. The ventral ganglion, antennal lobes, and median bundle are likely the CNS structures sufficient for rutabaga AC- dependent spatial learning (Zars, 2000a).

Effects of Mutation or Deletion

dunce mutants, which have elevated cAMP concentrations, show an increase in the numbers of terminal varicosities and branches. Such increase was suppressed in dnc/rut double mutants by rut mutations, which reduce cAMP synthesis. More profuse projections of larval motor axons have also been reported in double-mutant combinations of ether a go-go (eag) and Shaker (Sh) alleles. These display greatly enhanced nerve activity as a result of reduction in different K+ currents. Combinations of dnc and rut with eag and Sh mutations have been examined to explore the possible relation between activity- and cAMP-induced morphological changes. The expanded projections in dnc are further enhanced in double mutants of dnc with either eag or Sh, an effect that could be suppressed by rut (Zhong, 1992).

Genetic studies using Drosophila have advanced an understanding of the molecular mechanisms upon which different forms of learning are based, including habituation, but the relevant neural components of the learning pathways have not been as fully explored. A well defined neural circuit that underlies an escape response can be habituated, providing excellent opportunities for studying the physiological parameters of learning in a functional circuit in the fly. Compared with other forms of conditioning, relatively little is known of the physiological mechanisms responsible for habituation.

The giant nerve fiber pathway mediates a jump-and-flight escape response to visual stimuli. In the tethered fly, the jump may also be triggered electrically at multiple sites. The jump-and-flight response exhibits various parameters of habituation, including frequency-dependent decline in responsiveness, spontaneous recovery, and dishabituation by a novel stimulus (attributable to plasticity in the brain).

Mutations of rutabaga that diminish cAMP synthesis reduce the rate of habituation, whereas dunce mutations that increase cAMP levels lead to a detectable but moderate increase in habituation rates. Surprisingly, habituation is extremely rapid in dunce/rutabaga double mutants. This corresponds to the extreme defects shown with other learning tasks in double mutants, and demonstrates that defects of the rutabaga and dunce products interact synergistically in ways that could not have been predicted on the basis of simple counterbalancing biochemical effects.

Although habituation is localized to afferent neurons that innervate the giant fiber, cAMP mutations also affect performance in thoracic portions of the pathway on a millisecond time scale not otherwise accounted for by behavioral plasticity. More significantly, spontaneous recovery and dishabituation are not as clearly affected as is habituation in mutants; this indicates that these processes may not overlap entirely in terms of cAMP-regulating mechanisms. The analysis of the habituation of the giant fiber response in available learning and memory mutants could be a crucial step toward realizing the promise of memory mutations to elucidate mechanisms in neural circuits that underlie behavioral plasticity (Engel, 1996).

In both dunce and rutabaga mutant larvae, voltage-clamp analysis of neuromuscular transmission reveals impaired synaptic facilitation and post-tetanic potentiation as well as abnormal responses to direct application of dibutyryl cAMP. In addition, the calcium dependence of transmitter release is shifted in dunce (Zhong, 1991).

Four distinct K+ currents have been identified in Drosophila larval muscle fibers, i.e. the voltage-activated transient IA and delayed IK and the Ca(2+)-activated fast ICF and slow ICS. Both IA and IK are increased in dnc alleles. Normal muscle fibers treated with cyclic AMP show a similar increase of IA, but no significant effect on IK. In contrast to the dnc alleles, the rut mutations appear to enhance ICS greatly while leaving the amplitude of other currents largely unchanged. In addition, the cAMP-induced increase in IA is not observed in rut mutant fibers. The fact that not all dnc and rut mutant defects can be mimicked or reversed by acute application of cAMP suggests that long-term modulation of K+ currents by cAMP may involve mechanisms distinct from the short-term effect of cAMP (Zhong, 1993)

Selection of mutations that suppress dunce sterility has led to the isolation of two rutabaga alleles. The alleles (rut2 and rut3) decrease basal adenylate cyclase activity but, unlike the original rutabaga mutation, leave the calcium/calmodulin-stimulated activity intact. Behaviorally, the two alleles also differ from rut1. One of the mutations partially rescues the dunce learning defect, and flies bearing both alleles learn. Calcium responsiveness may thus be the crucial component of adenylate cyclase activity required for associative learning (Feany, 1990).

Females homozygous for dunce null mutations that abolish PDE activity do not deposit eggs. The suppressors exhibit differential effects on egg deposition and production of progeny; double-mutant females deposit many eggs that fail to hatch, but some develop to adults. These adult progeny exhibit morphological defects that are confined mostly to the second and third thoracic segments or to the first five abdominal segments. Mutant alleles of rutabaga act in the germ line cells to partially suppress the developmental defects caused by dunce mutations. Thus the rutabaga gene, as well as the dunce gene, functions in both somatic and germ line cells (Bellen, 1987).

Mutants of the Drosophila dunce and rutabaga genes, which encode a cAMP-specific phosphodiesterase and a calcium/calmodulin-responsive adenylyl cyclase, respectively, are deficient in short-term memory. Altered synaptic plasticity has been demonstrated at neuromuscular junctions in these mutants, but little is known about how their central neurons are affected. This problem was examined by using the "giant" neuron culture, which offers a unique opportunity to analyze mutational effects on neuronal activity and the underlying ionic currents in Drosophila. On the basis of instantaneous frequency and first latency of spikes evoked by current steps, four categories of firing patterns (tonic, adaptive, delayed, and interrupted) were identified in wild-type neurons, revealing interesting parallels to those commonly observed in vertebrate CNS neurons. The distinct firing patterns are correlated with expression of different ratios of 4-aminopyridine- and tetraethylammonium-sensitive K+ currents. Subsets of dnc and rut neurons display abnormal spontaneous spikes and altered firing patterns. Altered frequency coding in mutant neurons was demonstrated further by using stimulation protocols involving conditioning with previous activity. Abnormal spike activity and reduced K+ current remain in double-mutant neurons, suggesting that the opposite effects on cAMP metabolism by dnc and rut do not counterbalance the mutual functional defects. The aberrant spontaneous activity and altered frequency coding in different stimulus paradigms may present problems in the stability and reliability of neural circuits for information processing during certain behavioral tasks, raising the possibility of modulation in neuronal excitability as a cellular mechanism underlying learning and memory (Zhao, 1997).

In response to suprathreshold step current injections, wild-type neurons of different categories follow a defined temporal pattern in firing frequency, and each operates within a restricted frequency range. In contrast, erratic firing patterns in subsets of dnc and rut mutant neurons deviate from a clear scheme of frequency coding for each cell category. Some details of the abnormalities are noteworthy. (1) The periodic bursting activity of single mutant neurons reaches an instantaneous spike frequency as high as 120 Hz, whereas the maximum instantaneous frequency seldom approaches 30 Hz in wild-type controls. Such bursting activities apparently occur more frequently in tonic and delayed neurons than in adaptive neurons. (2) Unlike wild-type neurons that return to quiescence at the termination of stimulation, some mutant neurons frequently generate prolonged firing activities outlasting current steps for seconds. These long-lasting potentials seem to be more frequent in neurons of rut than those of dnc. (3) Extreme cases of abnormal patterns of regenerative potentials were found in subpopulations of mutant neurons that do not fall into the four categories in response to step current injections. Additional subtleties of mutational effects on neuronal excitability have been revealed with stimulation paradigms involving preconditioning, such as a progressive increment of stimulation strength in the ramp or long-duration depolarization in the twin-pulse protocol. In general, mutant neurons displayed in these two paradigms show considerably greater variability than wild-type controls. Moreover, the overall trend found in each category of wild-type controls with a twin-pulse paradigm becomes blurred in dnc and rut mutant neurons. So far, these paradigms have examined only short-term plasticity in neuronal excitability. The long-term effects of conditioning by prolonged previous activity on firing patterns in Drosophila neurons must await further investigation (Zhao, 1997 and references).

Synchronous activities and oscillations at characteristic firing frequencies in neuronal populations are thought to be important for the proper functioning of isolated neuronal networks of the rat hippocampus and neocortex. Recently, theoretical analysis and computational modeling have proposed that multiple short-term memory events could be represented by oscillatory activities in a network, with each memory event stored at a different high-frequency subcycle imbedded in a low-frequency oscillation. Progress made in insects reveals that the frequency of field potential oscillations in the mushroom bodies of the locust is odor-dependent, with the processing of different features of olfactory information distributed among neural subassemblies. The observed aberrant spontaneous activity, disrupted frequency coding, and abnormal modulation by previous conditioning in dnc and rut neurons of Drosophila might present problems in the stability of neural circuits and the reliability of information processing, causing poor performance in certain learning tasks in mutants. These results thus lend strong support for the notion that in addition to the well established synaptic mechanisms, modulation of neuronal excitability represents a potentially important cellular mechanism for learning and memory processes (Zhao, 1997 and references).

Upon exposure to ethanol, adult Drosophila display behaviors that are similar to acute ethanol intoxication in rodents and humans. Within minutes of exposure to ethanol vapor, flies first become hyperactive and disoriented and then uncoordinated and sedated. After approximately 20 min of exposure they become immobile, but nevertheless recover 5-10 min after ethanol is withdrawn. cheapdate, a mutant with enhanced sensitivity to ethanol, has been identified as a contributory factor, using an inebriometer to measure ethanol-induced loss of postural control. An inebriometer is a device that allows a quantitative assessment of ethanol-induced loss of postural control. The inebriometer is an approximately 4 ft long glass column containing multiple oblique mesh baffles through which ethanol vapor is circulated. To begin a "run," about 100 flies are introduced into the top of the inebriometer. With time, flies lose their ability to stand on the baffles and gradually tumble downward. As they fall out of the bottom of the inebriometer, a fraction collector is used to gather them at 3 min intervals, at which point they are counted. The elution profile of wild-type control flies follows a normal distribution; the mean elution time (MET), approximately 20 min at a standard ethanol vapor concentration, is inversely proportional to their sensitivity to ethanol. A genetic screen was carried out to isolate P element-induced mutants with altered sensitivity to ethanol intoxication using the inebriometer as the behavioral assay. One X-linked mutation isolated in this screen was named cheapdate (chpd) to reflect the increased ethanol sensitivity displayed by hemizygous mutant male flies. chpd males elute from the inebriometer with a MET of 15 min compared with 20 min for the wild-type controls. This increased ethanol sensitivity of chpd males was observed at all ethanol vapor concentrations tested. Genetic and molecular analyses reveals that cheapdate is an allele of the memory mutant amnesiac. amnesiac has been postulated to encode a neuropeptide that activates the cAMP pathway. Consistent with this, it is found that the enhanced ethanol sensitivity of cheapdate can be reversed by treatment with agents that increase cAMP levels or PKA activity. Conversely, genetic or pharmacological reduction in PKA activity results in increased sensitivity to ethanol (Moore, 1998).

Flies carrying mutations in three molecules involved in cAMP signaling were tested for response to ethanol: (1) rutabaga (rut), encoding the Ca²⁺-calmodulin-sensitive AC; (2) dunce (dnc), encoding the major cAMP-phosphodiesterase (PDE), and (3) DCO, encoding the major catalytic subunit of cAMP-dependent protein kinase (PKA-C1). Males hemizygous for rut mutations display an ethanol-sensitive phenotype similar to that of amn mutants. Flies heterozygous for the loss-of-function DCO alleles, which show reduced cAMP-stimulated PKA activity, also display increased ethanol sensitivity (homozygotes cannot be tested because they die as embryos). Ethanol sensitivity of males hemizygous for dnc mutations, however, are nearly normal. These data show that flies unable to increase cAMP levels normally (such as rut and possibly amn) or to respond properly to increased cAMP levels (such as DCO/+) are more sensitive to ethanol-induced loss of postural control. The converse, however, is not observed; dnc flies, whose cAMP levels are several times higher than wild type, display nearly normal ethanol sensitivity, a phenotype that is also observed in males doubly mutant for dnc and amn. Unexpectedly, whereas both rut and amn are ethanol sensitive, males doubly mutant for rut and amn are not significantly different from control (Moore, 1998).

In mammalian cells and tissues, ethanol potentiates receptor-mediated cAMP signal transduction; the mechanisms underlying this effect, however, remain poorly understood. While a direct link between cAMP signaling and ethanol-induced behaviors has not been established in mammals, the responses to acute ethanol are thought to be mediated by alterations in the function of various ligand-gated ion channels. Certain subtypes of GABA_A and NMDA receptors are potentiated and inhibited by ethanol, respectively, and both these channels can be phosphorylated by PKA in cells, tissues, or heterologous expression systems. It is tempting to speculate that PKA phosphorylation of neurotransmitter receptors is altered by ethanol and that this contributes to the behavior of the inebriated animal (Moore, 1998 and references).

A hybrid system was used to explore the relationship between Neurofibromin 1 and the PKA pathway. PACAP38 is mammalian protein that belongs to the vasoactive intestinal polypeptide-secretin-glucagon peptide family. Mutations in Drosophila genes rutabaga, Ras1 and Raf1 eliminate the response of flies to PACAP38. PACAP38 functions as a ligand for G protein-coupled receptors in vertebrates and in flies is known to stimulate cAMP synthesis inducing a 100-fold enhancement in K+ currents by coactivating both Rutabaga-adenylyl cyclase-cAMP and Ras-Raf kinase pathways (Zhong, 1995a). Mutations in rutabaga, Ras1 and Raf1 eliminate the response to PACAP38. Activation of both cAMP and Ras-Raf pathways together, but not alone, mimics the PACAP38 response (Zhong, 1995a). The involvement of Ras in the PACAP38 response has led to an investigation into the effect of Nf1 mutations. The purpose of this study was to further test whether Nf1 acts in the Ras or PKA pathways (Guo, 1997).

The PACAP38 enhancement of potassium currents is eliminated in Nf1 mutants. Perfusion of PACAP38 to the neuromuscular junction induces an inward current followed by a 100-fold enhancement of K+ currents in wild-type larvae. In Nf1 mutants, the inward current remains mostly intact, but the enhancement of K+ currents is abolished. Because the inward current is not affected in Nf1 mutants, it appears that PACAP38 receptors are normally activated by the peptide in these mutants. To control for the involvement of potential developmental effects of Nf1 mutants, wild type heat shock inducable Nf1 was induced in Nf1 mutants and the response to PACAP38 was studied. The PACAP mediated enhancement requires heat shock induction of Nf1. Because PACAP38 is a vertebrate peptide, the response induced by endogenous PACAP38-like neuropeptide (Zhong, 1995b) was tested. High-frequency stimulation (40Hz) applied to motor axons through a suction pipette increases K+ currents, presumably by causing the release of PACAP38-like peptides (Zhong, 1995b). The evoked PACAP38-like response is also eliminated in NF1 mutants. Induced expression of constitutively active Ras or active Raf neither blocks nor mimicks the PACAP38 response, suggesting that failure to negatively regulate Ras-Raf signaling does not explain the defective PACAP38 response in Nf1 mutants (Guo, 1997).

What is the role of the cyclic AMP pathway in PACAP38 responses? Application of cAMP analogs to Nf1 mutants restores the normal response to PACAP38. The cAMP analogs are effective if applied any time before or within 2 min after application of PACAP38. Application of cAMP analogs also restores the response to PACAP38 in rutabaga mutants, but not in Ras mutants. Thus, Nf1 appears to regulate the rutabaga-encoded adenylyl cyclase rather than the Ras-Raf pathway. Moreover, the Nf1 defect is rescued by the exposure of cells to pharmacological treatment that increased concentrations of cAMP, such as forskolin, which stimulates G-protein coupled adenylyl cyclase activity. Exploration of the mechanism by which NF1 influences G protein-mediated activation of adenylyl cyclase may lead to new insights into the mechanisms of G protein-mediated signal transduction and the pathogenesis, and possibly the treatment, of human type 1 neurofibromatosis (Guo, 1997).

The tumor-suppressor gene Neurofibromatosis 1 (Nf1) encodes a Ras-specific GTPase activating protein (Ras-GAP). In addition to being involved in tumor formation, NF1 has been reported to cause learning defects in humans and Nf1 knockout mice. However, it remains to be determined whether the observed learning defect is secondary to abnormal development. The Drosophila NF1 protein is highly conserved, showing 60% identity of its 2,803 amino acids with human NF1. Previous studies have suggested that Drosophila NF1 acts not only as a Ras-GAP but also as a possible regulator of the cAMP pathway that involves the rutabaga (rut)-encoded adenylyl cyclase. Because rut was isolated as a learning and short-term memory mutant, the hypothesis has been pursued that NF1 may affect learning through its control of the Rut-adenylyl cyclase/cAMP pathway. NF1 has been shown to affect learning and short-term memory independent of its developmental effects. G-protein-activated adenylyl cyclase activity consists of NF1-independent and NF1-dependent components, and the mechanism of the NF1-dependent activation of the Rut-adenylyl cyclase pathway is essential for mediating Drosophila learning and memory (Guo, 2000).

To test whether the NF1-dependent learning defect involves the cAMP pathway, learning scores of NF1 ^P2 and rut ¹ single-mutant, and rut ¹; NF1 ^P2 double-mutant flies were compared. The learning scores of all three mutant genotypes are very similar. The learning score of another double mutant, dunce (dnc) rut ¹, is reduced when compared with either single mutants, which indicates that the two mutations exert additive effects on learning even though both gene products are involved in the cAMP cascade (Rut-adenylyl cyclase (AC) for synthesizing cAMP, and Dnc-phosphodiesterase for degrading cAMP. Therefore, the absence of any further reduction of learning in the double mutant rut ¹;NF1 ^P2 suggests that both gene products function closely in the cAMP pathway (Guo, 2000).

This idea is supported by studies of NF1 mutant flies carrying a transgene encoding a mutant catalytic subunit of cAMP-dependent protein kinase (PKA*), which is constitutively active. Sustained expression of this PKA subunit rescues the small body size phenotype of NF1 mutants. Heat-shock induction of the constitutively active PKA should, in principle, bypass the requirement for the Rut-AC and all other molecules upstream of normal PKA activation. The hsp70-PKA* transgene completely rescues the learning defect of NF1^P1 when the flies are raised at room temperature. NF1^P2 mutants are partially rescued by the transgene at room temperature, but show complete rescue with heat shock (37°C, 30 min), or with a shift to 25°C overnight before being tested. In addition, NF1 mutations also cause a short-term memory defect (3- and 8-h retention) that is also fully rescued by heat-shock induction of PKA*. To determine whether expression of hsp70-PKA* induces a nonspecific enhancement of learning, leaky or induced expression of hsp-PKA* in the wild-type background does not increase the learning score even if flies are undertrained. For undertraining, flies were subjected to 3 repeats of electric shock in a single training trial instead of 12 trials. It is concluded that the PKA* effect is not nonspecific and that the learning defect observed in NF1 mutants can be rescued by induction of PKA activity. Therefore, the biochemical deficiency in the NF1 mutants must reside upstream of PKA induction in the cAMP pathway (Guo, 2000).

These behavioral analyses corroborate electrophysiological data that indicate that NF1 might exert its effect through regulation of the activation of Rut-AC. Biochemical assays provide direct evidence to support the idea. Previous experiments have shown that Rut-AC expressed in a cell line can be stimulated not only by Ca²⁺/calmodulin, but also by reagents that stimulate G-proteins, including GTPgammaS and AIF^-₄. AC activity has been examined in membrane fractions of adult brain tissues. The basal level of AC activity is very similar in the control (K33) and NF1 mutant membranes, but the GTPgammaS-stimulated AC activity is markedly reduced in NF1^P1 and NF1^P2 mutant membranes. However, significant GTPgammaS-stimulated activity occurs above the basal level in the mutants. Overexpression of NF1 in control flies does not increase AC activity, whereas the reduction in stimulated AC activity seen in NF1 mutants is mostly rescued by acutely induced expression of the NF1 transgene, indicating that NF1 is indeed able to regulate cAMP synthesis. Thus, GTPgammaS-stimulated AC activity consists of two components: one that is NF1 dependent and one that is NF1 independent. To determine whether the NF1-dependent AC activity is due to Rutabaga, rut ¹ and rut ¹;NF1 ^P2 mutant flies were examined. The basal and GTPgammaS-stimulated levels of AC activity are very similar in the single mutant, rut ¹ , and in the double mutant, rut ¹;NF1 ^P2. Thus, the NF1 mutation has no impact on AC activity in the absence of Rut-AC. In other words, the NF1-dependent cAMP activity is mediated through Rut-AC (Guo, 2000).

The Ca²⁺ dependence of the NF1 effect was examined to determine how Rut-AC is involved. Membrane fractions extracted from abdominal tissues were used because the Ca²⁺-dependent Rut-AC activity is easier to detect. The data are consistent with a report that the Ca ²⁺-dependent peak of AC activity is missing in rut mutants. Again, the NF1 mutation has no effects on AC activity across Ca ²⁺ concentrations without Rut-AC. Moreover, the Ca²⁺-dependent peak of Rut-AC activity is dependent on G-protein stimulation but not on the presence of NF1 (Guo, 2000).

Together, these results reveal a new mechanism that describes how G-proteins activate the cAMP pathway for normal learning and memory. The G-protein-activated AC activity is both NF1 dependent and NF1 independent. The NF1-dependent component involves Rut-AC. One possibility for how NF1 regulates AC activity is that NF1 acts as a GAP not only for the small G-protein Ras but also for heterotrimeric G-proteins. Thus, NF1 is required for a functional interaction between AC and heterotrimeric G-proteins, similar to the involvement of IRA, a Ras-GAP that is distantly related to NF1, in the Ras-activated cAMP pathway in yeast (Guo, 2000).

Another possibility is that NF1 regulates AC activity independent of its role as a Ras-GAP. Therefore, NF1 may be important for coordinating activities of multiple signal-transduction pathways. Nevertheless, the expression of the tumor-suppressor gene NF1 and its regulation of the Rut-AC signal-transduction pathway are critical to the biochemical processes underlying olfactory learning in Drosophila. Similar mechanisms are to be expected in vertebrates (Guo, 2000).

Relatively little is known about the regulation of ion channels, particularly that of Ca2+ channels, in Drosophila. Physiological and pharmacological differences between invertebrate and mammalian L-type Ca2+ channels raise questions on the extent of conservation of Ca2+ channel modulatory pathways. An examination was made of the role of the cyclic adenosine monophosphate (cAMP) cascade in modulating the dihydropyridine (DHP)-sensitive Ca2+ channels in the larval muscles of Drosophila, using mutations and drugs that disrupt specific steps in this pathway. The L-type (DHP-sensitive) Ca2+ channel current is increased in dunce mutants, which have high cAMP concentration owing to cAMP-specific phosphodiesterase (PDE) disruption. The current is decreased in the rutabaga mutants, where adenylyl cyclase (AC) activity is altered, thereby decreasing the cAMP concentration. The dunce effect is mimicked by 8-Br-cAMP, a cAMP analog, and IBMX, a PDE inhibitor. The rutabaga effect is rescued by forskolin, an AC activator. H-89, an inhibitor of protein kinase-A (PKA), reduces the current and inhibits the effect of 8-Br-cAMP. The data suggest modulation of L-type Ca2+ channels of Drosophila via a cAMP-PKA mediated pathway. While there are differences in L-type channels, as well as in components of cAMP cascade, between Drosophila and vertebrates, main features of the modulatory pathway have been conserved. The data also raise questions on the likely role of DHP-sensitive Ca2+ channel modulation in synaptic plasticity, and learning and memory, processes disrupted by the dnc and the rut mutations (Bhattacharya, 1999).

The effects of chronically lowered cyclic adenosine monophosphate (cAMP) on the morphology and physiology of the Drosophila larval neuromuscular junction has been investigated using two fly lines in which cAMP is significantly lower than normal in the nervous system: (1) transgenic flies in which Dunce is overexpressed in the nervous system, and (2) flies mutant for the rutabaga gene (rut1) that have reduced adenylyl cyclase activity. In comparison with controls, larvae with reduced cAMP exhibit a smaller number of synaptic varicosities. This effect is more pronounced in transgenic larvae, in which the reduction of neural cAMP is more pronounced. Synaptic transmission is also reduced in both cases, as evidenced by smaller excitatory junctional potentials (EJPs). Synaptic currents recorded from individual synaptic varicosities of the neuromuscular junction indicate almost normal transmitter release properties in transgenic larvae and a modest impairment in rut1 larvae. Thus, reduction in EJP amplitude in transgenic larvae is primarily due to reduced innervation, while in rut1 larvae it is attributable to the combined effects of reduced innervation and a mild impairment of transmitter release. It is concluded that the major effect of chronically lowered cAMP is reduction of innervation rather than impairment of transmitter release properties (Cheung, 1999).

At Drosophila neuromuscular junctions, there are two synaptic vesicle pools, namely the exo/endo cycling pool (ECP) and the reserve pool (RP). An extracellularly applied fluorescent dye, FM1-43, is incorporated into synaptic vesicles in nerve terminals during endocytosis and subsequently released by exocytosis. Using this dye, the two synaptic vesicle pools, ECP and RP have been identified in the larval NMJ. Vesicles in ECP can be loaded with FM1-43 by high K+ stimulation and are located at the periphery of individual boutons. Loaded dye is completely released by a second challenge of high K+ saline. Both pools are loaded with FM1-43 by enhancing endocytosis with cyclosporin A or by incubating at room temperature after complete depletion of vesicles in shibire at a nonpermissive temperature. The dye in ECP can be unloaded by a second challenge of high K+ saline, while vesicles in RP still maintain the dye. The RP appears to be more broadly distributed toward the center of individual boutons. In this report, this distribution is referred to as being in the center of the bouton because of its appearance in fluorescence microscopy without intending any implication as to its composition or exact boundary. In the animals whose RP is disconnected from the cycling pathway by treatment with cytochalasin D, high-frequency stimulation causes an accelerated decline of synaptic potentials, while low-frequency stimulation does not, suggesting that RP is required for sustaining high rate release of transmitter (Kuromi, 2000 and references therein).

During high-frequency nerve stimulation, vesicles in RP are recruited for release, and endocytosed vesicles are incorporated into both pools, whereas with low-frequency stimulation, vesicles are incorporated into and released from ECP. Release of vesicles from RP can be detected electrophysiologically after emptying vesicles in the ECP of transmitter by a H+ pump inhibitor. Recruitment from RP is depressed by inhibitors of steps in the cAMP/PKA cascade and enhanced by their activators. In rutabaga (rut) mutants, which have low cAMP levels, mobilization of vesicles from RP during tetanic stimulation is depressed, while it is enhanced in dunce (dnc) mutants, which have high cAMP levels (Kuromi, 2000).

The present electrophysiological studies have shown that, in wild type larvae, recruitment of synaptic vesicles from RP induced by 10 Hz stimulation for 10 s continues for some period, even after tetanic stimulation, but such recruitment is not observed in rut and does not continue after tetanic stimulation in dnc. Reduced recruitment of vesicles from RP in rut mutants may be caused by a failure in Ca2+/calmodulin-dependent cAMP production. However, even in rut, repeated tetanic stimulation does recruit vesicles from RP, and enhanced synaptic potentials continue after tetanic stimulation. A similar phenomenon is observed in rut after treatment with db-cAMP. Thus, it is likely even in rut that cAMP production can occur through another pathway during tetanic stimulation. FM1-43 loading experiments show that during high-frequency stimulation, some recycling vesicles are incorporated into RP in wild type, whereas recycling vesicles are minimally stored in RP in dnc mutants. Although RP may be refilled slowly with vesicles recycled or transported by axonal flow, the high level of cAMP produced by tetanic stimulation causes a marked translocation of vesicles from RP to ECP, resulting in no net accumulation of recycling vesicles into RP in dnc. Together, these findings suggest that recruitment of vesicles from RP to ECP is one of the mechanisms that control synaptic efficacy and plasticity (Kuromi, 2000).

Larval molting in Drosophila, as in other insects, is initiated by the coordinated release of the steroid hormone ecdysone, in response to neural signals, at precise stages during development. Using genetic and molecular methods, the roles played by two major signaling pathways in the regulation of larval molting have been examined in Drosophila. Previous studies have shown that mutants for the Inositol 1,4,5-trisphosphate receptor gene (Itpr) are larval lethals. In addition, they exhibit delays in molting that can be rescued by exogenous feeding of 20-hydroxyecdysone. Mutants for adenylate cyclase (rut) synergize, during larval molting, with Itpr mutant alleles, indicating that both cAMP and InsP3 signaling pathways function in this process. The two pathways act in parallel to affect molting, as judged by phenotypes obtained through expression of dominant negative and dominant active forms of protein kinase A (PKA) in tissues that normally express the InsP3 receptor. Furthermore, these studies predict the existence of feedback inhibition through protein kinase A on the InsP3 receptor by increased levels of 20-hydroxyecdysone (Venkatesh, 2001).

Interestingly, steroid secretion by the adrenal fasciculata-reticularis cells of mammalian adrenal glands in response to adrenocorticotrophic hormone occurs through the cAMP pathway, while InsP₃-mediated Ca²⁺ release is required for the steroidogenic action of Angiotensin II on adrenal glomerulosa cells. An increase in cytosolic Ca²⁺ levels is thought to affect multiple steps in mammalian steroid biosynthesis, including one crucial step that requires the transfer of endogenous cholesterol from the outer to the inner mitochondrial membrane. The data presented in this study support a similar model in which 20-hydroxyecdysone levels are regulated through activation of both InsP₃ and cAMP signals. The presence of multiple genes encoding adenylate cyclases allows rut mutant alleles to proceed through molting normally. Presumably, however, activity of the alternate adenylate cyclase(s) is dependent on InsP₃ receptor function since removal of the Itpr gene in rut mutant backgrounds leads to phenotypes that are synergistic. Activation of the two second messenger pathways probably occurs in the ring gland via PTTH and other as yet unidentified neural factors. Alternate explanations whereby InsP₃ and/or cAMP signaling are required for PTTH release from neurons or during conversion of 20-hydroxyecdysone precursors to 20-hydroxyecdysone cannot be ruled out at this stage. In either event the two pathways act in parallel to maintain 20-hydroxyecdysone levels perhaps via nonoverlapping downstream targets (Venkatesh, 2001).

The dunce and rutabaga mutations of Drosophila affect a cAMP-dependent phosphodiesterase and a Ca²⁺/CaM-regulated adenylyl cyclase, respectively. These mutations cause deficiencies in several learning paradigms and alter synaptic transmission, growth cone motility, and action potential generation. The cellular phenotypes either are Ca²⁺ dependent (neurotransmission and motility) or mediate a Ca²⁺ rise (action potential generation). However, interrelations among these defects have not been addressed. Conditions have been established for fura-2 imaging of Ca²⁺ dynamics in the 'giant' neuron culture system of Drosophila. Using high K+ depolarization of isolated neurons, a larger, faster, and more dynamic response was observed from the growth cone than the cell body. This Ca²⁺ increase depends on an influx through Ca²⁺ channels and is suppressed by the Na+ channel blocker TTX. Altered cAMP metabolism by the dnc and rut mutations reduces response amplitude in the growth cone while prolonging the response within the soma. The enhanced spatial resolution of these larger cells allow an analysis of Ca²⁺ regulation within distinct domains of mutant growth cones. Modulation by a previous conditioning stimulus was altered in terms of response amplitude and waveform complexity. Furthermore, rut disrupts the distinction in Ca²⁺ responses observed between the periphery and central domain of growth cones with motile filopodia (Berke, 2002).

This study describes the first use of fura-2 imaging for intracellular Ca²⁺ in a dissociated culture system of Drosophila. Unlike in vivo preparations such as the neuromuscular junction, optical imaging in culture can relate subcellular Ca²⁺ dynamics throughout different regions of single neurons. The spatial resolution offered by optical imaging complements electrophysiological studies of Drosophila giant neurons in culture and may indicate the functional significance of membrane excitability differences in subneuronal regions. The two approaches in combination will greatly enhance the neurogenetic study of Ca²⁺-dependent processes involved in neuronal development and physiology (Berke, 2002).

The initial characterization of regional differences in high K⁺-induced Ca²⁺ regulation in the soma and growth cone indicates that both Ca²⁺ and Na⁺ channels are involved. Drosophila central neurons in culture contain two types of Ca²⁺ currents (L and T type) distinguished by their activation voltage, decay kinetics, voltage dependence, and underlying single-channel activities. In other species, electrical recordings from the growth cone indicate that similar L- and T-type Ca²⁺ channels are expressed throughout the neuron, but that channel density may be higher and the channels more clustered in the growth cone. Such variation may relate to findings that the growth cone and soma differ in sensitivity and response kinetics during depolarization. In future investigations, it will be interesting to use identified mutations in Na⁺ and Ca²⁺ channel genes to dissect their involvement in Ca²⁺ regulation throughout the neuron (Berke, 2002).

Local Ca²⁺ levels regulate filopodial formation and play an important role in directing growth cone turning in cultured neurons from other species. The data from Drosophila show that the magnitude of the high K⁺ response is larger in the periphery of motile, as opposed to nonmotile, growth cones. Distinct regions within the growth cone exhibit differences in the localization of cytoskeletal elements and cytoplasmic organelles. It may be questioned whether the cytokinesis inhibition technique used to generate giant neurons affects the distribution of Ca²⁺ channels in the soma and growth cone because of actin cytoskeletal disruption. However, previous electrophysiological studies did not detect differences in action potential and ion current properties with and without the removal of cytochalasin B. This result is consistent with a preliminary study indicating that differences in response characteristics between the soma and growth cone are still evident in embryonic cultures made in the absence of CCB. In future studies, untreated larval neurons in culture can be used to examine Ca²⁺ signaling in the same dnc and rut growth cones that display retarded motility (Berke, 2002).

The analysis of these well studied mutants by fura-2 imaging has revealed previously unknown mutant phenotypes and suggests the usefulness of Ca²⁺ imaging for a wide range of other mutations. dnc and rut decrease sensitivity to high K⁺ stimulation for a cytosolic Ca²⁺ increase while affecting both the activity dependence and spatial distribution of [Ca²⁺] within motile and nonmotile growth cones. Chronic changes in cAMP metabolism imposed by the dnc and rut mutations decrease sensitivity most strongly in the growth cone while prolonging the Ca²⁺ increase only in the soma (Berke, 2002).

It is known that dnc and rut alter the modulation of K⁺ currents gated by the Sh and eag channel subunits. Moreover, enhanced spike activity has been detected with patch-clamp recordings from the soma of dnc and rut giant neurons. Such altered excitability may explain the prolonged soma response observed and should be further examined during patch-clamp experiments with high K⁺ depolarization. Electrophysiological studies of other currents in dnc and rut neurons have been lacking, but a Ba²⁺ current flowing through wild-type Ca²⁺ channels increases during the application of cAMP analogs. Further support for Ca²⁺ channel defects stems from findings that dnc and rut increase and decrease an L-type (dihydropyridine-sensitive) Ca²⁺ current in larval muscle, phenotypes that are mimicked by short-term pharmacological manipulations on wild-type muscles. However, some dnc and rut physiological phenotypes at the larval neuromuscular junction cannot be mimicked by acute pharmacological treatments and are attributed to long-term, developmental effects of the mutations. Different aspects of the Ca²⁺ signaling phenotypes may be caused by either acute or chronic effects. Therefore, the combination of genetic and pharmacological analyses will offer a more comprehensive picture of how the cAMP pathway regulates neuronal Ca²⁺ (Berke, 2002).

In Drosophila, rest shares features with mammalian sleep, including prolonged immobility, decreased sensory responsiveness and a homeostatic rebound after deprivation. To understand the molecular regulation of sleep-like rest, the involvement of a candidate gene, cAMP response-element binding protein (CREB), was investigated. The duration of rest is inversely related to cAMP signaling and CREB activity. Acutely blocking CREB activity in transgenic flies does not affect the clock, but increases rest rebound. CREB mutants also have a prolonged and increased homeostatic rebound. In wild types, in vivo CREB activity increases after rest deprivation and remains elevated for a 72-hour recovery period. These data indicate that cAMP signaling has a non-circadian role in waking and rest homeostasis in Drosophila (Hendricks, 2001).

The daily rest of flies carrying mutations and/or transgenes that alter cAMP signaling was examined at several points in the pathway. dunce flies have a mutation in the phosphodiesterase enzyme and therefore have increased cAMP. The null mutant (dnc^ML) rests significantly less than the background yw strain. Similarly, increasing PKA activity in flies with a heat-shock-inducible transgene of the catalytic subunit of PKA significantly decreases daily rest durations compared to pre-heat-shock rest levels. Decreased adenylyl cyclase enzyme activity and thus decreased cAMP characterize rutabaga (rut) mutants, which rest more than the Canton S background strain. Similarly, S162 flies that carry a mutation that abolishes dCREB2 activity rest more than their comparison group (siblings without the mutation). The mutation is a stop codon just upstream of the basic leucine-zipper motif of the dCREB2 gene (Hendricks, 2001).

Habituation is a fundamental form of behavioral plasticity that permits organisms to ignore inconsequential stimuli. This study describes the habituation of a locomotor response to ethanol and other odorants in Drosophila, measured by an automated high-throughput locomotor tracking system. Flies exhibit an immediate and transient startle response upon exposure to a novel odor. Surgical removal of the antennae, the fly's major olfactory organs, abolishes this startle response. With repeated discrete exposures to ethanol vapor, the startle response habituates. Habituation is reversible by a mechanical stimulus and is not due to the accumulation of ethanol in the organism, nor to non-specific mechanisms. Ablation or inactivation of the mushroom bodies, central brain structures involved in olfactory and courtship conditioning, results in decreased olfactory habituation. In addition, olfactory habituation to ethanol generalizes to odorants that activate separate olfactory receptors. Finally, habituation is impaired in rutabaga, an adenylyl cyclase mutant isolated based on a defect in olfactory associative learning. These data demonstrate that olfactory habituation operates, at least in part, through central mechanisms. This novel model of olfactory habituation in freely moving Drosophila provides a scalable method for studying the molecular and neural bases of this simple and ubiquitous form of learning (Cho, 2004).

Neuronal activity and adenylate cyclase in environment-dependent plasticity of axonal outgrowth in Drosophila

The development of the nervous system is influenced by environmental factors. Among all environmental factors, temperature belongs to a unique category. Besides activating some specific sensory pathways, it exerts nonspecific, pervasive effects directly on the entire nervous system, especially in exothermic species. This study uses mutants to genetically discover how temperature affects nerve terminal arborization at larval neuromuscular junctions of Drosophila. It is known that hyperexcitability in K+ channel mutants leads to enhanced ramification of larval nerve terminals. Elevated cAMP levels in dunce mutants with reduced phosphodiesterase activity also cause enhanced arborization. These genetic alterations are thought to perturb mechanisms relevant to activity-dependent neural plasticity, in which neuronal activity activates the cAMP pathway, and consequently affect nerve terminal arborization by regulating expression of adhesion molecules. This study demonstrates the robust influence of rearing temperature on motor nerve terminal arborization. Analysis of ion channel and cAMP pathway mutants indicates that this temperature-dependent plasticity is mediated via neuronal activity changes linked to mechanisms controlled by the rutabaga-encoded adenylyl cyclase (Zhong, 2004).

From these observations, four conclusions can be drawn. (1) Developmental temperature is a robust environmental factor that influences neuronal outgrowth in larval neuromuscular junctions of Drosophila. (2) Critical temperature-dependent neuronal growth is mediated by neural activity, although temperature may exert nonspecific pervasive effects on cellular or molecular activities. (3) Nerve terminal arborization increases with activity level but becomes suppressed beyond an optimal activity level. (4) Rut-regulated cAMP pathways play an essential role in mediating activity-dependent nerve terminal arborization. The result suggests that presynaptic Rut activity is critical (Zhong, 2004).

It is conceivable that neuronal activity may be generally increased at higher rearing temperatures in flies. For instance, transient K⁺ currents inactivate faster at increased temperatures, which should allow higher-frequency firing of action potentials. It is also noted that a wings-down phenotype presumably resulting from extreme hyperexcitability is observed only in eag Sh double mutants but not in the corresponding single mutants at room temperature. However, this phenotype could be found among a fraction of eag or Sh single mutants reared at 30°C. Thus, it is logical to speculate that increased neural activity at higher rearing temperatures leads to modification of nerve terminal arborization. The present study provides several lines of evidence in support of this idea, as summarized below. It appears that temperature increase and hyperexcitability mutations exert similar influences on nerve terminal arborization. The effect of a small increase in temperature (from RT to 25°C) is equivalent to that of single eag or Sh mutations, whereas a large increase (from RT to 30°C) affects arborization similar to that of the double mutants. More conclusive evidence comes from the observation that temperature-induced enhancement of arborization can be suppressed by the no action potential (nap) mutation, in which neuronal activity is lowered because of a reduced number of Na⁺ channels. Moreover, both activity- and temperature-dependent arborization are linked to the cAMP pathway. Both Sh (at 25°C)- and temperature (at 30°C)-induced enhancement in arborization are suppressed by the rut mutation (Zhong, 2004).

The cAMP pathway has been suggested to be a necessary component in visual experience-dependent cortical plasticity of ocular dominance and has been shown to be a critical signal transduction pathway in mediating synaptic reorganization during long-term memory formation in Aplysia. Previous studies have indicated that elevated cAMP levels in dnc mutants lead to enhancement of arborization at the larval neuromuscular junction, and this enhanced ramification in dnc mutants can be suppressed by the rut mutation, as shown in dnc rut double mutants. This establishes that cAMP is able to influence arborization, but its role in mediating this activity-dependent arborization has not been resolved previously. In this study, it is clearly demonstrated in rut and rut Sh double mutants that arborization is not enhanced (even at high temperatures or in hyperexcitability mutants) if rut-encoded adenylyl cyclase activity is removed. In contrast, dnc-encoded cAMP-specific phosphodiesterase is not a component directly mediating activity-dependent plasticity. Arborization in dnc mutants still varies with temperature in a striking manner, whereas hyperexcitability and temperature are unable to alter arborization in rut mutants (Zhong, 2004).

It is interesting to note that motor nerve terminal arborization is reduced in dnc, Sh, and eag Sh mutants reared at 30°C. This observation has prompted the proposal that there is an optimal level of activity, hence of cAMP, for promoting axon outgrowth and arborization. In other words, there is a bell-shaped relationship curve between neuronal activity and ramification of arbors: motor nerve terminal arborization is enhanced with an increase in activity and will become suppressed with additional increases in activity. In fact, a similar relationship has been suggested between intracellular calcium concentrations and growth cone formation and neurite outgrowth in cultured neurons. In summary, the results presented demonstrate that developmental temperature is a robust environmental factor that influences neuronal outgrowth, and that temperature-dependent neuronal growth is mediated by neural activity. The effect of rut and dnc at different developmental temperatures and their interaction with channel mutations demonstrate an essential role of the Rut-regulated cAMP pathway in developmental neural plasticity in response to environmental changes (Zhong, 2004).

Pharmacogenetic rescue in time and space of the rutabaga memory impairment by using Gene-Switch

The GAL4-based Gene-Switch system has been engineered to regulate transgene expression in Drosophila in both time and space. A Gene-Switch transgene was constructed in which Gene-Switch expression is restricted spatially by a defined mushroom body enhancer. This system allows Gene-Switch to be active only in the mushroom bodies and only on administration of the pharmacological Gene-Switch ligand RU486. This line was used to drive the expression of a rutabaga cDNA in otherwise rutabaga mutant flies. Induction of the rutabaga cDNA in the mushroom bodies only during adulthood, or during adulthood along with the larval and pupal developmental stages, corrects the olfactory memory impairment found in rutabaga mutants. Induction of the cDNA only during the larval and pupal stages was inconsequential to performance in olfactory memory tasks. These data indicate that normal rutabaga function must be expressed in adulthood for normal memory and conclusively delimit the time and space expression requirements for correcting the rutabaga memory impairment. Such combined pharmacogenetic regulation of transgene expression now allows this time and space dissection to be made for other behavioral mutants (Mao, 2004; full text of article).

Rutabaga is the most thoroughly characterized of all Drosophila memory mutants. Previous studies have shown that the gene encodes for a calcium:calmodulin-dependent adenylyl cyclase and that this cyclase is preferentially expressed in the axons of mushroom body neurons. These observations, combined with the knowledge that the products of other learning genes like dunce show preferential expression in the mushroom bodies, prompted two major and important questions. First, are mushroom bodies the site of action of the adenylyl cyclase in terms of promoting memory formation? This possibility was strongly predicted to be the case, given the preferential expression in these neurons and the fact that other gene products important for learning exhibit a similar expression pattern. This prediction was confirmed through standard GAL4-mediated rescue experiments. The second question, whose answer, surprisingly, was unknown, was whether the adenylyl cyclase is important for the development of brain structures such as mushroom bodies that are required for memory, or whether the requirement of the cyclase is restricted to adult stages. The possibility that developmental defects underlie the impairment in memory was made stronger by studies that demonstrated that the mutants do have an altered nervous system and the discovery that mutation of the rut homolog in the mouse produces an alteration in barrel fields of the somatosensory cortex and in the refinement of axonal projections from retinal ganglion cells (Mao, 2004).

This study has provided through the Gene-Switch pharmacogenetic approach described here, an answer to this question. Expression of rut only in the adult mushroom bodies is sufficient for the rescue of the memory impairment of rut mutants, and expression of rut in the mushroom bodies during development is inconsequential to adult odor memory formation. This result was achieved in part through the construction of an RU486-inducible, mushroom body Gene-Switch line P{MB-Switch}12-1. This line should be very valuable for similar pharmacogenetic studies in which the experimenter requires control over the timing of gene expression in the mushroom bodies. Moreover, nearly identical results were obtained with a second system for controlling transgene expression in time and space. This system, named TARGET, utilizes a temperature-sensitive repressor of GAL4 (GAL80^ts) to modulate GAL4 activity with temperature shifts (Mao, 2004).

Even though the requirement for adenylyl cyclase activity for odor learning has been delimited to the adult brain, its specific role in learning processes is still uncertain. The most attractive possibility is that the adenylyl cyclase is coupled to neurotransmitter receptors that register the unconditioned stimulus during learning, or provide a neuromodulatory role for the actual association of the conditioned stimulus and the unconditioned stimulus. An alternative idea is that the adenylyl cyclase is required for the maturation of some other physiological process or cellular components and, in its absence, the neurons remain incompetent to support memory formation (Mao, 2004).

The Gene-Switch system, like TARGET, requires several hours to days for complete induction. This time course is to be expected for any transcriptionally based system, which requires an applied inducer to alter the activity of a transcription factor to initiate the subsequent processes of transcription, RNA processing, translation, posttranslational modification, and cellular targeting. The system should be extremely effective for answering questions regarding broad temporal expression requirements such as the one posed here. Questions that require more acute induction and deinduction, such as whether any given gene product is required for the formation of memories, their stability, or their retrieval, will likely require the development of gene expression systems that operate at the posttranslational level, to turn on or turn off the activity of any given protein (Mao, 2004).

Waking experience affects sleep need in Drosophila: Experience-dependent changes in sleep need require dopaminergic modulation, cAMP signaling, and a particular subset of long-term memory genes

Sleep is a vital, evolutionarily conserved phenomenon, whose function is unclear. Although mounting evidence supports a role for sleep in the consolidation of memories, until now, a molecular connection between sleep, plasticity, and memory formation has been difficult to demonstrate. Drosophila as a model to investigate this relation; the intensity and/or complexity of prior social experience stably modifies sleep need and architecture. Furthermore, this experience-dependent plasticity in sleep need is subserved by the dopaminergic and adenosine 3',5'-monophosphate signaling pathways and a particular subset of 17 long-term memory genes (Ganguly-Fitzgerald, 2006).

Sleep is critical for survival, as observed in the human, mouse, and fruit fly, and yet, its function remains unclear. Although studies suggest that sleep may play a role in the processing of information acquired while awake, a direct molecular link between waking experience, plasticity, and sleep has not been demonstrated. Advantage was taken of Drosophila genetics and the behavioral and physiological similarities between fruit fly and mammalian sleep to investigate the molecular connection between experience, sleep, and memory (Ganguly-Fitzgerald, 2006).

Drosophila is uniquely suited for exploring the relation between sleep and plasticity for at least two reasons. (1) Fruit flies sleep. This is evidenced by consolidated periods of quiescence associated with reduced responsiveness to external stimuli and homeostatic regulation -- the increased need for sleep that follows sleep deprivation. (2) Drosophila has been successfully used to elucidate conserved mechanisms of plasticity. For example, exposure to enriched environments, including the social environment, affects the number of synapses and the size of regions involved in information processing in vertebrates and Drosophila. In the fruit fly, these structural changes occur in response to experiential information received within a week of emergence from pupal cases. Although brain plasticity is not limited to this period, the first week of emergence does coincide with the development of complex behaviors in Drosophila, including sleep. Hence, daytime sleep, which accounts for about 40% of total sleep in adults, is highest immediately after eclosion and stabilizes to adult levels 4 days after emergence (Ganguly-Fitzgerald, 2006).

To assess the impact of waking experience during this period of brain and behavioral development, individuals from the wild-type C-S strain were exposed to either social enrichment or impoverishment immediately at eclosion and were tested individually for sleep 5 days later. Socially enriched individuals (E), exposed to a group of 30 or more males and females (1:1 sex ratio) before being tested, slept significantly more than their socially impoverished (I) siblings, who were housed individually. This difference in sleep [DeltaSleep (E)] was restricted to daytime sleep. Socially enriched individuals consolidated their daytime sleep into longer bouts of ~60 min compared with their isolated siblings, who slept in 15-min bouts. In contrast, nighttime sleep was unaffected by prior social experience, corresponding with observations that daytime sleep is more sensitive to sex, age, genotype, and environment, when compared with nighttime sleep. This effect of social experience on sleep persisted over a period of days. Moreover, it was a stable phenotype: When socially enriched, longer-sleeping individuals and socially impoverished, shorter-sleeping siblings were sleep-deprived for 24 hours, they defended their respective predeprivation baseline sleep quotas by returning to these levels after a normal homeostatic response (Ganguly-Fitzgerald, 2006).

Experience-dependent modifications in sleep have long been observed in humans, rats, mice, and cats. But what is the nature of the experiential information that modifies sleep need in genetically identical Drosophila? Differences in sleep need in socially enriched and socially impoverished individuals were not a function of the space to which they were exposed -- flies reared in 2-cc tubes slept the same as those reared in 40-cc vials. Neither did it arise out of differences in reproductive state or sexual activity between the two groups: Socially impoverished mated and virgin individuals slept the same, as did socially enriched individuals from mixed-sex or single-sex groups. Further, differences in sleep were not a reflection of differences in overall activity (measured as infrared beam breaks) between the two groups. Although social context can reset biological rhythms, mutations in clock (Clk^jerk), timeless (tim⁰¹), and cycle (cyc⁰¹) disrupt circadian rhythms but had no effect on experience-dependent responses in sleep need (Ganguly-Fitzgerald, 2006).

Because social interaction requires sensory input, fly strains that were selectively impaired in vision, olfaction, and hearing were evaluated . Blind norpA homozygotes failed to display a response in sleep to waking experience: Sleep need in norpA mutants did not increase after exposure to social enrichment. In contrast, norpA/+ heterozygotes with restored visual acuity slept more when previously socially enriched. Attenuating visual signals by rearing wild-type (C-S) flies in darkness also abolished the effect of waking experience on sleep. Compromising the sense of smell while retaining visual acuity also blocked experience-dependent changes in sleep need: Socially enriched smellblind¹ mutants slept the same as their impoverished siblings. As confirmation, neurons carrying olfactory input to the brain were specifically silenced [Or83b-Gal4/UAS-TNT, and it was observed that sleep in these flies was also not affected by prior waking experience. Auditory cues, however, did not affect the relation between experience and sleep. Finally, sleep need in individual Drosophila increased with the size of the social group to which they were previously exposed. Socially isolated flies slept the least, whereas those exposed to social groups of 4, 10, 20, 60, and 100 (1:1 sex ratio) showed proportionately increased daytime sleep need. When rendered blind, however, flies did not display this relation between sleep need and the intensity of prior social interactions (Ganguly-Fitzgerald, 2006).

If sensory stimulation received during a critical period of juvenile development directs the maturation of the adult sleep homeostat, then subsequent environmental exposure should not affect adult sleep time and consolidation. Alternatively, if experience-dependent modifications in sleep are a reflection of ongoing plastic processes, this phenomenon would persist in the adult. It was observed that sleep in flies was modified by their most recent social experience regardless of juvenile experience. Shorter sleeping socially impoverished adults became longer sleepers when exposed to social enrichment before being assayed. Conversely, longer sleeping socially enriched flies became shorter sleepers after exposure to a period of social isolation. Moreover, repeated switching of exposure between the two social environments consistently modified sleep, reflecting an individual's most recent experience (Ganguly-Fitzgerald, 2006).

An estimation of neurotransmitter levels in whole brains revealed that short-sleeping, socially impoverished individuals contained one-third as much dopamine as their longer-sleeping, socially stimulated isogenic siblings. Silencing or ablating the dopaminergic circuit in the brain [TH-Gal4/UAS-TNT and TH-Gal4/UAS-Rpr specifically abolished response to social impoverishment in individuals that were reared in social enrichment. Similar results were obtained when endogenous dopamine levels were aberrantly increased, by disrupting the monoamine catabolic enzyme, arylalkylamine N-acetyltransferase, in Dat^lo mutants. Hence, abnormal up- or down-regulation of the dopaminergic system prevented behavioral plasticity in longer sleeping, socially enriched individuals when switched to social impoverishment (Ganguly-Fitzgerald, 2006).

The observation that dopaminergic transmission affects experience-dependent plasticity in sleep need is particularly compelling, given its role as a modulator of memory. Mutations in 49 genes implicated in various stages of learning and memory were screened to assess their impact on experience-dependent changes in sleep need. Of these, only mutations in short- and long-term memory genes affected experience-dependent plasticity in sleep need. Mutations in dunce (dnc¹) and rutabaga (rut²⁰⁸⁰) have opposite effects on intracellular levels of adenosine 3',5'-monophosphate (cAMP), but are both correlated with short-term memory loss. In dnc¹ mutants, waking experience had no impact on subsequent sleep need. This effect was partially rescued in dnc¹/+ heterozygotes, but complete rescue was only achieved when a fully functional dunce transgene was introduced into the null mutant background. rut²⁰⁸⁰, however, selectively abolished the ability of socially enriched adults to demonstrate decreases in sleep after exposure to social impoverishment, which was reminiscent of aberrant dopaminergic modulation. Similarly, of the long-term memory genes screened, 17 (~40%) specifically disrupted the change in sleep need in socially enriched adults after exposure to social impoverishment. For example, overexpression of the Drosophila CREB gene repressor, dCREB-b, resulted in socially enriched flies that continued to be longer sleepers even after exposure to social impoverishment. As a control, overexpression of the dCREB-a activator yielded wild-type phenotypic read out. It is noteworthy that not all long-term memory mutants had a disrupted relation between experience and sleep. Instead, the particular subset of genes identified, only half of which are expressed in the mushroom bodies, may specifically contribute to pathways that underlie sleep-dependent consolidation of memories (Ganguly-Fitzgerald, 2006).

Finally, to assess the correlation between sleep and memory, male flies trained for a courtship conditioning task that generated long-term memories were measured for sleep after training. Males whose courtship attempts are thwarted by nonreceptive, recently mated females or by males expressing aphrodisiac pheromones form long-term associative memories as evidenced by subsequently reduced courtship of a receptive virgin female. Trained males that formed long-term memories slept significantly more than their untrained siblings and wake controls (ones that were sleep-deprived while the experimental flies were being trained). Exposure to a virgin female did not alter sleep need. As before, this increase in sleep was associated with longer daytime sleep bouts in trained individuals compared with controls. Further, sleep deprivation for 4 hours immediately after training abolished training-induced changes in sleep-bout duration, as well as courtship memory. Although these results are intriguing, invertebrate memory is particularly sensitive to extinction by mechanical perturbations. However, gentle handling that ensured wakefulness, but not mechanical stimulation, immediately following training, also abolished subsequent courtship memory. Furthermore, sleep deprivation per se did not affect the formation of long-term memory: Trained flies that were allowed to sleep unperturbed for 24 hours and then subjected to 4 hours of sleep deprivation retained courtship memory (Ganguly-Fitzgerald, 2006).

In summary, this study has demonstrate a rapid and dynamic relation between prior social experience and sleep need in Drosophila. In particular, experience-dependent changes in sleep need require dopaminergic modulation, cAMP signaling, and a particular subset of long-term memory genes, supporting the hypothesis that sleep and neuronal activity may be inexorably intertwined. These observations are compelling given two recent studies have demonstrating a central role of the mushroom bodies in sleep regulation and emphasize the importance of establishing Drosophila as a model system to investigate the molecular pathways underlying sleep and plasticity (Ganguly-Fitzgerald, 2006).

Roles for Drosophila mushroom body neurons in olfactory learning and memory

Olfactory learning assays in Drosophila have revealed that distinct brain structures known as mushroom bodies (MBs) are critical for the associative learning and memory of olfactory stimuli. However, the precise roles of the different neurons comprising the MBs are still under debate. The confusion surrounding the roles of the different neurons may be due, in part, to the use of different odors as conditioned stimuli in previous studies. This study investigated the requirements for the different MB neurons, specifically the α/ß versus the γ neurons, and whether olfactory learning is supported by different subsets of MB neurons irrespective of the odors used as conditioned stimuli. The rutabaga (rut)-encoded adenylyl cyclase was expressed in either the γ or α/ß neurons and the effects were examined on restoring olfactory associative learning and memory of rut mutant flies. A temperature-sensitive shibire (shi) transgene was expressed in these neuron sets and the effects of disrupting synaptic vesicle recycling on Drosophila olfactory learning was examined. These results indicate that although odor-pair-specific learning was not detected using GAL4 drivers that primarily express in γ neurons, expression of the transgenes in a subset of α/ß neurons resulted in both odor-pair-specific rescue of the rut defect as well as odor-pair-specific disruption of learning using shi^ts1 (Akalal, 2006).

Drosophila olfactory learning is typically assayed using olfactory classical conditioning. Using this assay, several memory mutants and the genes involved in olfactory memory formation have been identified. This assay has also been used to investigate the roles of the different MB lobes. In each case, GAL4 drivers that express in distinct lobes of the MBs were used. When G-protein signaling was disrupted using pan-MB GAL4 drivers (238y, c747, and c309) to express a constitutively activated stimulatory heterotrimeric GTP-binding protein α-subunit (Gα_s*), associative olfactory learning was reported to be completely abolished. Using 201y-GAL4, a line that expresses extensively in the γ lobes but only in the narrow core elements of the α/ß lobes, learning was only reduced by ~50%. In a study that investigated the effects of expressing shi^ts1 using c739-GAL4, an α/ß driver, and 201y-GAL4, a significant impairment of performance was observed when neurotransmission was transiently inactivated through the α/ß lobes with only a slight, but nonsignificant, decrease in memory performance observed using 201y-GAL4, suggesting a greater role for MB α/ß lobes in olfactory memory. Other experiments involving rescue of the rut mutant defect by expressing a wild-type rut cDNA showed that memory was restored to wild-type levels using broad-MB GAL4 drivers (247, c772, and 30y) and the γ lobe driver H24-GAL4. However, memory was only partially rescued using 201y-GAL4, and no rescue was observed using the GAL4 drivers 189y and 17d, which both express primarily in the MB α/ß lobes. This, therefore, suggested a greater role of MB γ lobes in olfactory learning. The apparent contradictions among each of these studies could be due to the fact that in each of these experiments different combinations of odors were used for the olfactory learning assay: MCH-OCT, MCH-BEN, and OCT-BEN. To resolve the apparent discrepancies and inconsistencies among the different studies, this study used three commonly used odor combinations (MCH-OCT, MCH-BEN, and OCT-BEN) and two different assays, shi^ts1 inactivation of neurotransmission as well as rescue of the rut memory defect, to examine the roles of the different neurons comprising the MB lobes (Akalal, 2006).

Expressing transgenes using the two γ lobe GAL4 drivers NP1131 and H24 did not produce any odor-pair-specific learning effects. Using these γ drivers to express a rut cDNA in a rut mutant background results in a partial rescue of the learning defect for each of the odor combinations tested. The γ driver of choice has typically been H24-GAL4, but the expression pattern of this driver is not limited to the γ neurons. In fact, it expresses very robustly in the ALs, and one concern is that this might affect performance scores since the learning assay is based on olfactory cues. The use of NP1131-GAL4 that expresses primarily in the γ neurons and a small subset of α'/ß' neurons is important to validate the results for H24-GAL4. A prior study using H24-GAL4 to rescue the rut learning defect resulted in performance scores that were statistically indistinguishable from wild-type flies using MCH and BEN. However, among the different lines that were observed to rescue the rut defect, H24-GAL4 yielded the lowest scores, and another γ driver, 201y-GAL4, used in the same study failed to rescue the defect. Yet another study that used H24-GAL4 to restore a rut cDNA in a rut mutant background failed to produce rescue of the defect using MCH and OCT as odorants. The current data suggest that driving a rut cDNA in γ neurons results in rut levels that partially rescue the defect. Although no odor-pair-specific learning was detected using the two γ drivers tested, this does not preclude the possibility that when γ drivers that express in other subsets of γ neurons are discovered, this would remain the case. It is likewise important to note that the three odorants used in in the current study do not represent the whole repertoire of odors that a fly responds and learns to. Thus, it remains possible that the γ neurons labeled by NP1131-GAL4 and H24-GAL4 may still be involved in the odor-pair-specific associative learning of other smells (Akalal, 2006).

Rut rescue using GAL4 drivers that express in the α/ß neurons reveals some odor-pair-specific effects. Performance scores for flies carrying c739-GAL4 together with the UAS-rut transgene in a rut mutant background show no rescue of the rut defect for all odor combinations tested. In contrast, driving a rut cDNA using 17d-GAL4 shows partial rescue of the rut defect when MCH-BEN and OCT-BEN are used for the assay but not for MCH-OCT. To address the observed contrast in the rescue using the different odor combinations for these two lines, the expression patterns for the two α/ß drivers was compared. The expression level for c739-GAL4 is greater than 17d-GAL4, and it appears that only a subset of neurons, a thin core, is labeled for 17d-GAL4. Using antibodies raised against glutamate, a further subdivision of the lobes has been described as a slender core of glutamatergic neurons, the α and ß core neurons (αc and ßc), that lie posteriorly and are partly enclosed by the α and ß neurons. Whether the core neurons that are marked by 17d-GAL4 correspond to the glutamatergic subdivision of the α/ß lobes remains to be determined. One explanation for the observed odor-pair-specific effects is that 17d-GAL4 and c739-GAL4 express in nonoverlapping regions of the MB α/ß neurons. Behavioral experiments were performed on flies that were 2–5 d old, and although the neuron counts make clear that the expression of c739-GAL4 is broader than 17d-GAL4, it was not possible to confirm whether there is nonoverlapping expression at the α/ß core. Preliminary data suggest, however, that during this time period, the spatial expression of 17d-GAL4 is in the core region, while c739-GAL4 expression is more peripheral with a slight overlap of expression as a ring around this core. Additional experiments are needed to extend these observations (Akalal, 2006).

What is the significance of organizing the Drosophila MBs into different lobes, and is there differential representation of odors in the different lobes? These findings indicate that to answer this question one must take into account the choice of odor pairs used in the olfactory assay. The differential effects seen when using different odor combinations for 17d-GAL4 suggest that even though this driver expresses in a subset of α/ß neurons, some of these neurons must be important in MCH-BEN and OCT-BEN learning since driving rut using c739-GAL4, a line that expresses in a greater number of α/ß neurons, is insufficient to see a partial rescue of the rut learning defect. Clearly, this partial rescue is more than a mass effect of rut-expressing MB neurons. This raises an important point: It may not be accurate to describe GAL4 driver patterns based solely on lobe-specificity, since different GAL4 drivers may highlight subsets of the neurons comprising individual lobes. The significance of achieving complete rescue when the double GAL4 c739;H24 was used to express UAS-rut in a rut mutant background is still unclear. Although it is tempting to speculate that this complete rescue occurs by completing a spatial circuit that involves odorant representation in both α/ß and γ neurons, more experiments need to be performed to investigate whether it may just represent the massed effect of expression in more MB neurons (Akalal, 2006).

Examination of the odor-evoked activity in Drosophila MB neurons by expressing a green fluorescent protein-based Ca²⁺ indicator, G-CaMP, have revealed remarkable spatial stereotypy. In fact, several studies have shown that stereotypical anatomical and functional organization can be found at the different levels of the insect olfactory pathway. Each olfactory receptor neuron (ORN) likely expresses a single olfactory receptor (OR) gene, and ORNs that express the same OR genes converge on a common glomerulus in the AL, resulting in a stereotyped projection pattern. A near complete map of ORN connectivity constructed through a systematic survey of Drosophila OR expression has validated the principles of 'one neuron-one receptor' and 'one glomerulus-one receptor.' Different odors activate different combinations of ORs, and individual receptors can mediate both excitatory and inhibitory responses to different odors in the same cell. In addition, a topographic organization of the AL has been described wherein ORNs in distinct sensilla types project into distinct regions of the AL. At the level of the MB neuron cell bodies and the calyx, different odors evoked distribution patterns of fluorescence that were odor-specific and conserved across flies, resulting in stereotyped responses for BEN-, MCH-, and OCT-evoked fluorescence activity at both the wide-field and single-neuronal level. The current results demonstrate an odor-pair-specific effect with 17d-GAL4 using two odor combinations that have BEN as one of the odors. Several studies have indicated that Drosophila processes BEN differently from other odors. In fact, although surgical removal of the antennae and palps of wild-type flies results in the abolishment of MCH and OCT avoidance, BEN avoidance is only partially affected in both T-maze and arena paradigms, suggesting that BEN is sensed through other nonolfactory pathways. To eliminate naive odor bias, experiments are usually performed in a counterbalanced design, with half of the flies used in the calculation of the performance index being trained to the first odor and the other half to the second odor. An examination of the half P.I. scores for these experiments does not reveal obvious asymmetries between BEN and the two counter-odors. Moreover, the fact that the odor-pair-specific effects are seen in both rescue as well as disruption of memory experiments suggests that it is the expression of transgenes in the subset of neurons defined by 17d-GAL4 that confers the odor-pair-specific behavioral phenotypes observed in this study (Akalal, 2006).

The existence of such stereotypical anatomical and functional organization at the various levels of the Drosophila olfactory pathway may explain the odor-pair-specific rut rescue and shi^ts1-mediated disruption of learning that was observed in this study. The spatial pattern of odor-evoked fluorescence activity for BEN has been reported to occur mostly in the center of the calyx, OCT-evoked activities distribute more laterally and medially, while MB neurons that displayed fluorescence transients in response to MCH occur primarily in the top and middle portions of the soma layer. To investigate whether different lobes of the MBs receive olfactory information from different subsets of AL glomeruli, the spatial correlation between MB neurons and projection neurons (PNs) have been examined. The MB dendrites of γ neurons were found to preferentially occupy the center of the calyx, and although the dendrites of the α'/ß' and α/ß core and surface neurons were more widespread across the calyx, their distribution was slightly nuanced. Based on these observations, it is speculated that olfactory learning using different odors is, in part, a function of the relationships between the expression pattern of the GAL4 driver and the degree and pattern of overlap and nonoverlap in the populations of MB neurons that respond to the odor combinations chosen. This is a more complex picture than just having the different odors mapping to distinct lobes of the MBs, and, in fact, a shift toward describing GAL4 drivers based on actual patterns of expression may be more useful than describing them solely on MB lobe-specificity. Ultimately, determining the precise manner by which odors are encoded in the Drosophila brain and how this links to specific behavioral outputs will require careful analyses of the expression patterns of the GAL4 drivers and the representation of the odors in the different MB neurons (Akalal, 2006).

Distinct memory traces for two visual features in the Drosophila brain

The fruit fly can discriminate and remember visual landmarks. It analyses selected parts of its visual environment according to a small number of pattern parameters such as size, colour or contour orientation, and stores particular parameter values. Like humans, flies recognize patterns independently of the retinal position during acquisition of the pattern (translation invariance). The central-most part of the fly brain, the fan-shaped body, contains parts of a network mediating visual pattern recognition. Short-term memory traces have been identified of two pattern parameters -- elevation in the panorama and contour orientation. These can be localized to two groups of neurons extending branches as parallel, horizontal strata in the fan-shaped body. The central location of this memory store is well suited to mediate translational invariance (Liu, 2006).

A fly tethered to a torque meter, with its head (and hence its eyes) fixed in space, can control its orientation with respect to the artificial scenery in a flight simulator. In this set up, the fly is conditioned to avoid certain flight directions relative to virtual landmarks and recognizes these visual patterns for up to at least 48 h. Visual pattern recognition in Drosophila has been studied in some detail. Flies store values of at least five pattern parameters: size, colour, elevation in the panorama, vertical compactness, and contour orientation. Moreover, they memorize spatial relations between parameter values. The neuronal substrate underlying visual pattern recognition is little understood in any organism (Liu, 2006).

In Drosophila, memory traces can be localized to groups of neurons in the brain. Using the enhancer GAL4/UAS expression system, short-term memory traces of aversive and appetitive olfactory conditioning have been assigned to output synapses of subsets of intrinsic neurons of the mushroom bodies (MBs). The Rutabaga protein -- a type 1 adenylyl cyclase that is regulated by Ca²⁺/Calmodulin and G protein, and is considered a putative convergence site of the unconditioned and conditioned stimulus in olfactory associative learning, selectively restores olfactory learning if expressed in these cells in an otherwise rutabaga (rut)-mutant animal. Moreover, expressing a mutated constitutively activating Galpha_s protein (Galpha_s*) in the MBs interferes with olfactory learning. Blocking the output from these neurons during memory retrieval has the same effect, while blocking it during acquisition has no effect. Interestingly, memory traces for other learning tasks seem to reside in other parts of the brain: for remembering its location in a dark space, the fly seems to rely on a rut-dependent memory trace (Zars, 2000) in neurons of the median bundle and/or the ventral ganglion (Liu, 2006).

The present study localizes short-term memory traces for visual pattern recognition to the fan-shaped body (FB), the largest component of the central complex (CX; also called the central body in other species). The CX is a hallmark of the arthropod brain. It has been characterized functionally as a pre-motor centre with prominent, but not exclusive, visual input. In the locust, large-field neurons sensitive to the e-vector orientation of polarized light have been described in the CX. Because of its repetitive structure and the precisely ordered overlay of fiber projections from the two hemispheres in the FB, neighbourhood relations of visual space might still be partially preserved at this level (retinotopy). Using the genetic approach, this study shows that a small group of characteristic stratified neurons in the FB house a memory trace for the pattern parameter 'elevation', and a different set of neurons forming a parallel stratum contain a memory trace for 'contour orientation' (Liu, 2006).

Of ten mutants with structural abnormalities in the CX, all were impaired in visual pattern recognition. They were able to fly straight and to avoid heat, yet they failed to remember the patterns. Did they really lack the memory or had they lost their ability to discriminate between patterns? Fortunately, individual flies often display spontaneous preferences for one of the patterns. In three lines, these preferences were consistent enough to reveal intact pattern discrimination, suggesting that aberrant circuitry of the central complex can affect visual learning independent of visual pattern discrimination (Liu, 2006).

Since the developmental and structural defects in these mutants are not well characterized, the GAL4/UAS system was used to acutely interfere with CX function. A GAL4 driver line (c205-GAL4) was used with expression in parts of the CX and, the gene for tetanus toxin light chain (CntE) was used as the effector. CntE blocks neurons by cleaving neuronal Synaptobrevin, a protein controlling transmitter release. For temporal control, the temperature-sensitive GAL4-specific silencer GAL80 was added under the control of a tubulin promoter (tub-GAL80^ts). Flies (UAS-CntE/+; tub-GAL80^ts/c205-GAL4) were raised at 19 °C, and were transferred for 14 h to the restrictive temperature (30 °C) just before the behavioural experiment to induce GAL4-driven toxin expression. Flies kept at the low temperature showed normal memory scores, while after inactivation of GAL80^ts no pattern memory was observed. Again, flight control and heat avoidance were normal, and Fourier analysis confirmed that flies at the high temperature had retained their ability to tell the patterns apart. As with the structural mutants, interrupting the circuitry of the CX by tetanus toxin expression seemed to specifically interfere with visual pattern memory. In addition, the use of tub-GAL80^ts excluded the possibility that toxin expression in unknown tissues during development might cause the memory impairment in the adult. These results do not, as yet, address the question of memory localization (Liu, 2006).

Visual pattern memory in the flight simulator requires an intact rut gene. Mutant rut flies (rut²⁰⁸⁰) showed normal visual flight control, heat avoidance and pattern discrimination. To confirm that the defect was indeed due to the mutation in the rut gene rather than an unidentified second-site mutation, rut was rescued by the expression of the wild-type rut cDNA (UAS-rut⁺) using the pan-neuronally expressing driver line elav-GAL4. Indeed, flies of the genotype rut²⁰⁸⁰/Y;elav-GAL4/UAS-rut⁺ have normal memory (Liu, 2006).

Visual pattern memory in the flight simulator has been shown to depend upon at least two kinds of behavioural plasticity: (1) an associative classical (pavlovian) memory trace is formed linking a particular set of values of pattern parameters to heat; (2) the fly's control of the panorama operantly facilitates the formation of this memory trace (Brembs, 2000). Either of the two processes might depend upon the Rut cyclase (Liu, 2006).

To address this issue, rut mutant flies were tested in a purely classical variant of the learning paradigm. During training, panorama motion was uncoupled from the fly's yaw torque and the panorama was slowly rotated around the fly. Heat was made contingent with the appearance of the 'punished' pattern in the frontal quadrant of the fly's visual field. All other parameters were kept as described. For testing memory, panorama motion was coupled again to yaw torque and the fly's pattern preference was recorded as usual. Even in the absence of operant facilitation, visual pattern memory required the intact rut gene. Therefore, the rut-dependent memory trace investigated in this study represents the association of a property of a visual pattern with the reinforcer (Liu, 2006).

As a first step in localizing the memory trace, it was asked in which neurons of the rut mutant expression of the wild-type rut gene would be sufficient to restore learning. To this end, a total of 27 driver lines expressing GAL4 in different neuropil regions of the brain was used to drive the UAS-rut⁺ effector gene in the rut mutant background. The parameter 'elevation' was measured. With seven of the driver lines, pattern memory was restored (104y, 121y, 154y, 210y, c5, c205 and c271) (Liu, 2006).

Comparison of the expression patterns of the 27 lines allowed the putative site of the memory trace to be narrowed down to a small group of neurons in the brain. The seven rescuing lines all showed transgene expression in a stratum in the upper part of the FB. In three of them staining is rather selective. It comprises, in addition to the FB, only a layer in the medulla, several cell clusters in the subesophageal ganglion and a few other scattered neurons (Liu, 2006).

Evidently, rut⁺ expression in the MBs is neither necessary (104y, c5, c205, 154y) nor sufficient for rescue. This result is in line with the earlier observation that elimination of more than 90% of the MBs by hydroxyurea treatment of first-instar larvae has no deleterious effect on visual pattern memory. The MBs were ablated in one group of rescue flies (rut²⁰⁸⁰/Y;UAS-rut⁺/ +;c271/+). They showed full visual pattern memory (Liu, 2006).

Although GAL4 expression in the optic lobes is prominent in all seven rescuing lines, it occurs in distinctly different layers that do not overlap. For instance, in 104y expression is restricted to layer 2, whereas in 210y it is found only in the serpentine layer (layer 7). A similar situation is found for the s ganglion, although there the staining patterns are more difficult to evaluate. Finally, expression in the ellipsoid body is again not necessary (104y, c5, c205, 154y) or sufficient (c232, 78y, 7y, and so on) for rescue. Thus, the expression patterns favour the conclusion that the neurons of the upper stratum of the FB might be the site of the memory trace for the parameter 'elevation' in visual pattern memory (Liu, 2006).

Neurons in this stratum, labelled in all seven rescuing lines, have a very characteristic shape. Their cell bodies are located just lateral to the calyces. Their neurites run slightly upward in an antero-medial direction, forming an upward-directed tufted arborization just behind the alpha/alpha'-lobe of the MB. From there, the fiber turns sharply down and backward towards the midline just in front of the FB. Finally, it turns horizontally backward, spreading as a sharp stratum through all of the FB across the midline. These neurons have been described in Golgi preparations. They belong to a larger group of tangential FB neurons called F neurons. Besides the stratum in FB, most of them have an arborization in a particular part of the unstructured neuropil. The layer stained in 104y, and the other six rescuing lines, is tentatively classify as layer 5 (from bottom upward), and hence provisionally the neurons are called F5, although, without further markers, it is difficult to reliably number the layers. In summary, expression of Rut cyclase in F5 neurons rescues the rut-dependent memory defect for pattern elevation, whereas no rescue effect is observed in any of 20 strains without expression of Rut cyclase in F5 neurons (though Rut cyclase was expressed in other regions of the brain). Hence, a rut-dependent memory trace for pattern elevation may reside in F5 neurons (Liu, 2006).

This finding does not exclude the possibility that memory is redundant, and that other rut-dependent memory traces for pattern elevation might be found elsewhere. Therefore, it was asked whether plasticity in the F5 neurons is necessary for visual pattern memory. The Rut cyclase is regulated by G protein signaling, and olfactory learning/memory can be blocked by a constitutively active form of the Galpha_s protein subunit (Galpha_s*). The Galpha_s* mutant protein was expressed in the FB using the driver line c205, and the flies were tested for their memory of 'elevation'. Memory was fully suppressed. Since in olfactory learning, overexpression of the wild-type protein does not interfere with learning, these results support the hypothesis that continuous upregulation of Rut cyclase in the F5-neurons interferes with visual short-term memory, implying that F5 neurons are the only site of a rut-dependent memory trace for pattern elevation (Liu, 2006).

The patterns used in the experiments so far exclusively addressed the parameter 'elevation' (upright and inverted Ts or horizontal bars at different elevations). It was of interest to discover whether the mutant defect in rut and the Rut rescue in the F5 neurons affects only this parameter, or whether it applies to other pattern parameters as well. Therefore, the study looked at to two further parameters: 'size' and 'contour orientation'. Three driver lines -- c205, NP6510 and NP2320 -- were chosen showing different expression patterns in the FB. In the line NP6510, as in c205, a group of F neurons is marked. They are putatively classified as F1, since their horizontal stratum lies near the lower margin of the FB. Their cell bodies form a cluster in the dorso-frontal cellular cortex above the antennal lobes. Like the F5 neurons, they have large arborizations in the dorsal unstructured neuropil. The line NP2320 expresses the driver in columnar neurons running perpendicular to the strata of F neurons, with their cell bodies scattered singly or in small groups between the calyces. Since they seem to have no arborizations outside the FB, they are tentatively classified as pontine neurons (Liu, 2006).

Initially, it was shown that pattern memory requires the rut gene for each of the three parameters. Next, the Rut rescue flies were studied (for example, rut²⁰⁸⁰/Y;c205/UAS-rut⁺). In the line c205, memory was restored only for 'elevation', not for 'size' or 'contour orientation'. Correspondingly, the memory impairment by expression of dominant-negative Galpha_s* in this driver line should be specific for 'elevation', as is indeed the case. With the driver line NP6510, memory was not restored for either 'elevation' or for 'size,' but memory was restored for 'contour orientation'. The third driver line, NP2320, labelling columnar neurons of the FB, did not restore the memory for any of the three pattern parameters. Among the 27 GAL4 lines, a second was found with a very similar expression pattern as NP6510 (NP6561). The P-element insertions in the two lines are only 124 nucleotides apart from each other. Like NP6510, NP6561 restores the memory for 'contour orientation' but not for 'size' or 'elevation'. These results strongly suggest that memory traces for distinct visual pattern parameters are located in different parts of the FB, and that, in addition to the memory trace in F5 neurons, a memory trace for the parameter 'contour orientation' is located in F1 neurons (Liu, 2006).

A pertinent question in rescue experiments is whether the rescue is due to the provision of an acute function in the adult or to the avoidance of a developmental defect. Therefore, the tub-GAL80^ts transposon was added to the system. The driver lines c205 and NP6510 were chosen. Groups of adult males (for example, rut²⁰⁸⁰/Y;+/tub-GAL80^ts;NP6510/UAS-rut⁺), raised at 19°C, were kept as adults for 14 h at 19°C or 30°C. Afterwards, pattern memory for the corresponding pattern parameter was tested. In both cases, flies that had been kept at 30°C showed normal memory, indicating that Rut cyclase induced just a few hours before the experiment had restored an immediate neuronal function rather than preventing a developmental defect. This conclusion was further supported by the finding that Galpha_s* expression in the adult (using tub-GAL80^ts) was sufficient to disrupt memory (Liu, 2006).

Several conclusions can be drawn from the above results. Memory traces in Drosophila are associated with specific neuronal structures: odor memories with the MBs, visual memories with the CX, and place memory (tentatively) with the median bundle. Memory traces are not stored in a common all-purpose memory centre. Even within the visual domain, memories for distinct pattern parameters are localized within distinct structures: a rut-dependent short-term memory trace for the pattern parameter 'elevation' to F5 neurons, and a corresponding memory trace for 'contour orientation' to F1 neurons. Moreover, if the constitutively activating Galpha_s* protein indeed interferes with the regulation of Rut cyclase, it follows that the brain contains no other redundant rut-dependent memory traces for these pattern parameters. The Rut-mediated plasticity is necessary and sufficient, at least in F5 neurons. As in the earlier examples, the memory traces are confined to relatively small numbers of neurons. At least in flies, and probably in insects in general, memory traces appear to be part of the circuitry serving the respective behaviour (Liu, 2006).

This study provides a first glimpse of the circuitry within a neural system for visual pattern recognition. Though the picture is far from complete, it invites (and may guide) speculation. The FB is a fiber matrix of layers, sectors and shells. The F1- and F5-neurons form two sharp parallel horizontal strata in this matrix. If the width of the FB represents the azimuth of visual space as has been proposed, the horizontal strata of the F neurons would be well suited to mediate translation invariance. In any case, it is satisfying to find a translation invariant memory trace in the CX where visual information from both brain hemispheres converges. These first components of the circuitry may encourage modelling efforts for pattern recognition in small visual systems (Liu, 2006).

G(o) signaling is required for Drosophila associative learning; Tests of genetic interaction with rutabaga

Heterotrimeric G(o) is one of the most abundant proteins in the brain, yet relatively little is known of its neural functions in vivo. This study demonstrates that G(o) signaling is required for the formation of associative memory. In Drosophila, pertussis toxin (PTX) is a selective inhibitor of G(o) signaling. The postdevelopmental expression of PTX within mushroom body neurons robustly and reversibly inhibits associative learning. The effect of G(o) inhibition is distributed in both γ- and α/β-lobe mushroom body neurons. However, the expression of PTX in neurons adjacent to the mushroom bodies does not affect memory. PTX expression also does not interact genetically with a rutabaga adenylyl cyclase loss-of-function mutation. Thus, G(o) defines a new signaling pathway required in mushroom body neurons for the formation of associative memory (Ferris, 2006).

An associative memory is one that links external stimuli to particular events, such that the stimuli come to predict the events. In the negatively reinforced olfactory associative learning assay of Drosophila, flies are presented with an odor (conditional stimulus paired, CS⁺) paired with an electric shock (unconditioned stimulus, US). The flies are then presented with a second odor (conditioned stimulus unpaired, CS^-). The associative memory is measured as the conditioned avoidance of the CS⁺ in a T-maze. The disruption of the cyclic AMP (cAMP) signaling pathway within Drosophila leads to reduced learning scores. The effect of cAMP disruption has been mapped back to the mushroom body neurons through the targeted expression of a constitutively active G(s)α and by rescuing the rutabaga type I adenylyl cyclase (rut) phenotype with targeted expression of a rut cDNA. It is thought that the cAMP pathway controls the association between the CS⁺ and the US within the mushroom body neurons (Ferris, 2006).

The G(o) heterotrimeric protein is thought to be the most abundant membrane protein in the vertebrate brain and is activated both by numerous G protein-coupled receptors (GPCRs) and by amyloid precursor protein. Although G(o) can participate in diverse signaling pathways, only a few specific in vivo functions have been ascribed to this molecule. In Drosophila, G(o)α47A is the only gene encoding the alpha subunit of G(o), and it is expressed throughout the adult brain. The G(o) protein is much more abundant in the heads of rutabaga (rut) and dunce learning mutants than in the heads of wild-type flies, suggesting a possible role for G(o) in memory formation (Ferris, 2006).

The S1 subunit of PTX from Bordetella pertussis catalyzes the transfer of an ADP-ribose onto the Gα subunit of the vertebrate G(i/o/t) heterotrimeric G proteins, preventing these proteins from binding to activated GPCRs. In Drosophila, PTX is a selective enzymatic inhibitor of G(o) signaling: Drosophila does not have a transducin homolog, and the G(i)α65A protein does not contain the PTX recognition site, whereas G(o)α does; PTX will ADP-ribosylate a single protein in Drosophila, as seen in western blots and after isoelectric focusing; and PTX comigrates with G(o)α and is immunoprecipitated by independent G(o)α-specific antibodies (Ferris, 2006).

To determine if G(o) is a mediator of associative memory, a PtxA transgene was expressed within the mushroom body neurons. The P{UASPTX}16 transgenic line was selected because the basal expression of PTX is low in this line and because PTX can be induced by Gal4, albeit in small amounts. G(o)α47A loss-of-function mutant embryos die during embryogenesis owing to defects in nervous system and mesoderm development. In keeping with this result, it was found that the induction of PTX within the developing mesoderm or nervous system also results in embryonic lethality, indicating that this toxin is functional when expressed early in development (Ferris, 2006).

The role of G(o) in associative memory was examined by inducing PTX expression within the adult mushroom bodies with the P{MBSwitch}12 Gene-Switch driver. The resulting induction abolished the immediate associative memory 3 min after training, which is frequently taken as a measure of learning. Although the PTX-uninduced P{MBSwitch}12/P{UASPTX}16 flies also showed reduced learning, their scores were not significantly lower than the PTX-uninduced P{UASPTX}16/+ control group. The induction of PTX within the mushroom body did not alter naïve sensitivities to either odorants or electric shock, indicating that PTX expressed in the mushroom bodies does not affect the perception of the stimuli (Ferris, 2006).

The severity of the PTX learning phenotype might result from the death of the mushroom body neurons. This hypothesis was tested by examining the integrity of the mushroom bodies after the induction of PTX and by establishing whether the associative learning phenotype was reversible. Because Gene-Switch has slow off-rate kinetics, the Gal80^ts system was used with the P247 Gal4 driver. P247 drives expression in ~700 α/β- and γ-lobe mushroom body neurons. Two independent Gal80^ts transgenes were used to ensure more complete inhibition of Gal4 at 18°C. After inducing PTX for 12 h at 32°C, 3-min memory was almost entirely abolished. Using antibodies to downstream of receptor kinase (DRK), which preferentially mark the mushroom body, it was found that PTX induction did not alter either the gross structure of the mushroom bodies or the expression of DRK. Similar results were found using antibodies to cAMP-dependent protein kinase 1 (DCO). It was also found that the effect of PTX was reversible: although a 2-h induction of PTX within mushroom body neurons produced significant inhibition of 3-min memory, this effect was completely reversed after 6 d. Therefore, the effect of PTX on learning is not due to the death of the mushroom body neurons (Ferris, 2006).

Next, whether the effect of PTX on learning was specific to the mushroom bodies was examined. PTX was induced in the R3 and R4d neurons of the ellipsoid body and separately in the dorsally paired medial (DPM) neurons, which innervate the mushroom bodies. The induction of PTX with the Gal80^ts system in either set of neurons did not affect performance in the learning assay, suggesting that PTX is cell autonomous. Moreover, PTX induction in the DPM neurons had no effect on 60-min memory. The inhibition of neurotransmission in DPM neurons by the shibire^ts transgene completely blocks 60-min memory but has no effect on 3-min memory. Thus, PTX and shibire^ts have different effects in the DPM neurons, indicating that PTX is not a general inhibitor of neurotransmission (Ferris, 2006).

Experiments were conducted to see whether the requirement for G(o) signaling in olfactory associative learning is dispersed throughout the different neurons of the mushroom body lobes, or if the requirement is limited to a subset of these neurons. Several genes have been identified that are preferentially expressed in the different mushroom body lobes, indicating that these lobes have distinct molecular repertoires; however, direct tests for lobe function have yet to provide unequivocal and differentiated roles for the constituent neurons in associative learning. The c772 Gal4 line drives expression in ~800 neurons of the α/β and γ lobes. It was found that a 12-h induction with c772 was sufficient to ablate the associative memory, whereas a 2-h induction was not. There were no differences in the naïve avoidance of odor or shock between the c772/Gal80^ts20; PTX/Gal80^ts2 PTX-induced and PTX-uninduced experimental groups. There were, however, some differences in naïve odor avoidance between the c772/Gal80^ts20; PTX/Gal80^ts2 PTX-induced group and the control genotypes, suggesting that PTX induction in non-mushroom-body neurons by c772 may affect odor perception or discrimination and that the Gal80^ts inhibition may not be complete in these neurons. The differences in odor avoidance may also participate in the severe c772/PTX phenotype, although it is unlikely to have a major effect on learning as naïve avoidance scores were not significantly different in the within-genotype control group. It is likely that differences in expression levels between c772 and P247 account for the different time courses in the inhibition of learning by PTX between these two lines. The 12-h induction of PTX in the γ-lobe neurons marked by 1471 caused a substantial, but not complete, loss of 3-min memory, as did the expression of PTX in the α/β-lobe neurons marked by c739. Thus, G(o) signaling is required for 3-min memory in both the γ and α/β neurons of the mushroom body as defined by the 1471 and c739 drivers, respectively. In contrast, PTX driven by the α/β-lobe driver 17d did not have an observable effect on 3-min memory. The mushroom body neurons defined by 17d are most likely the core neurons of the α/β lobe, which may be functionally distinct from the other neurons of the α/β lobe, since they are insensitive to the effects of PTX in associative memory and have no effect on the rescue of the rut learning phenotype. The fact that associative memory formation was affected by PTX induction in the α/β- and γ-lobe neurons, but not in the putative α/β core neurons, defines a new requirement for G(o) signaling in these lobes for learning and memory and should further help dissect the memory process in these neurons (Ferris, 2006).

Next it was considered whether the G(o) pathway interacts genetically with the rut adenylyl cyclase in associative memory formation. The persistent activation of vertebrate G(o) may initially lead to the short-term inhibition of type I adenylyl cyclase, followed by the increased responsiveness of this enzyme to G(s)α stimulation, known as heterologous sensitization or supersensitization. Thus, PTX may be interfering with the down regulation of rut activity by G(o), resulting in neurons with too much adenylyl cyclase activity. Alternatively, PTX may inhibit the heterologous sensitization of rut by G(o), leaving the neurons with too little cAMP after G(s) activation. The former hypothesis predicts that a reduction in rut activity may partially suppress the PTX phenotype, whereas the latter suggests that the reduction in rut may act synergistically with PTX. These predictions were tested by looking for a genetic interaction between a mild induction of PTX in the subset of mushroom body neurons defined by P247 and a single copy of rut²⁰⁸⁰. This rut mutation demonstrates a semidominant haploinsufficiency, indicating that learning is extremely sensitive to the activity levels of this enzyme. It was found that the performance of the rut²⁰⁸⁰/+; Gal80^ts20/+; PTX/P247, Gal80^ts2 PTX-induced flies was reduced, but not significantly, as compared to that of the Gal80^ts20/+; PTX/P247, Gal80^ts2 flies. This result suggests an additive interaction between PTX and the rut²⁰⁸⁰ heterozygote, but there was evidently neither suppression nor a synergistic relationship between PTX and one copy of rut²⁰⁸⁰. The independence of G(o) function during learning from rut was further assessed in rut homozygotes. The performance of the rut²⁰⁸⁰; Gal80^ts20/+; PTX/P247, Gal80^ts2 PTX-induced flies was significantly worse than that of either the PTX-induced flies or the rut homozygous flies. Thus, G(o) signaling has functions in olfactory learning and memory within the mushroom body neurons defined by P247 that are independent of rut (Ferris, 2006).

This study has shown, through the postdevelopmental induction of PTX expression within mushroom bodies, that activation of G(o) is required during the physiological events, which lead to associative memory formation. The severity of the learning phenotype in PTX-induced flies coupled with the lack of genetic interaction with rut²⁰⁸⁰ strongly suggests that the function of G(o) in associative learning and memory is largely independent of the cAMP pathway. Additional members of this new associative learning pathway are currently unknown. One possibility is that, similar to the role of the G(o) in the vertebrate dorsal root ganglia, the Drosophila G(o) may participate in learning through the inhibition of voltage-gated Ca²⁺ channels (VGCCs). These Ca²⁺ channels are thought to be activated by the odor-induced depolarization of the mushroom body neurons, leading to the release of synaptic vesicles and the CS pathway activation of rut. It is plausible that the negative regulation of the VGCCs may be necessary to restrict the number of activated synapses during learning. Nevertheless, it is now clear that the in vivo functions of G(o) include the formation of associative memories in Drosophila (Ferris, 2006).

The GABA_A receptor RDL suppresses the conditioned stimulus pathway for olfactory learning: Genetic interactions with NF1

Assigning a gene's function to specific pathways used for classical conditioning, such as conditioned stimulus (CS) and unconditioned stimulus (US) pathway, is important for understanding the fundamental molecular and cellular mechanisms underlying memory formation. Prior studies have shown that the GABA receptor RDL inhibits aversive olfactory learning via its role in the Drosophila mushroom bodies (MBs). This study describes the results of further behavioral tests to further define the pathway involvement of RDL. The expression level of Rdl in the MBs influenced both appetitive and aversive olfactory learning, suggesting that it functions by suppressing a common pathway used for both forms of olfactory learning. Rdl knock down failed to enhance learning in animals carrying mutations in genes of the cAMP signaling pathway, such as rutabaga and NF1, suggesting that RDL works up stream of these functions in CS/US integration. Finally, knocking down Rdl or over expressing the dopamine receptor dDA1 in the MBs enhanced olfactory learning, but no significant additional enhancement was detected with both manipulations. The combined data suggest that RDL suppresses olfactory learning via CS pathway involvement (Liu, 2009b).

The level of Rdl expression in the MBs affects the calcium response observed in these neurons when animals are presented with odor but not shock stimulus. This provided the basis for hypothesizing that RDL might specifically regulate the CS pathway for olfactory learning. Data presented in this study shows that the level of Rdl expression the MBs influences both aversive and appetitive olfactory learning, which share a common CS pathway. Thus, these observations are consistent with the CS pathway-specific hypothesis. Rdl knock down failed to produce enhanced learning when combined with mutations of either the rut or NF1 gene, both of which may be involved in the process of integration of CS and US information. This observation argues against the possibility that RDL acts downstream of CS/US integration, providing further support for RDL's role in the CS pathway (Liu, 2009b).

Prior experiments have shown that blocking neurotransmitter release from dopaminergic neurons impairs aversive olfactory learning but not appetitive olfactory learning, while blocking the synthesis of octopamine impairs appetitive olfactory learning but not aversive olfactory learning. This is consistent with the simple model that the neuromodulators are involved in US pathways for learning, with octopamine delivering only appetitive US (sugar) and dopamine delivering only aversive US (electric shock). This model also suggests that increasing the expression level of dDA1 will increase aversive US input, and thereby enhance aversive learning, as long as other factors such as dopamine release are not limiting. This possibility was tested, and evidence is provided for increased performance with increased expression of dDA1 in the MBs. Since knocking down Rdl increases the CS signal, it follows that combining over-expression of dDA1 with knock down of Rdl might enhance learning synergistically, and produce an even greater enhancement of learning. However, no synergism between these two was detected: although dDA1 over-expression alone and Rdl knock down alone both enhance olfactory learning, the combined treatments failed to produce a significantly higher performance score than either treatment alone. Two possible hypotheses can account for these results. The learning enhancement of either treatment produces performance close to ceiling levels, where no further enhancement can be detected. Alternatively, the dDA1 receptor, and thus the dopamine system, plays some role in the CS pathway that overlaps with RDL, such that the two learning enhancing effects do not sum. The authors prefer the later possibility for two reasons. (1) Functional imaging of the dopaminergic neurons projecting to the MBs using calcium reporters has revealed that these neurons respond not only to shock stimuli presented to the fly, but also to odor stimuli (Riemensperger, 2005). This indicates that the response properties of these neurons are not specific to the US pathway, which is predicted by the 'US pathway only' hypothesis. Rather, dopaminergic neurons respond to the CS and are therefore intertwined in some way with the CS pathway. (2) Flies mutant for the dDA1 gene exhibit impairment in both aversive and appetitive olfactory learning, both of which can be rescued by expressing dDA1 in the MBs (Kim, 2007). This observation suggests that dDA1 may play a role in the CS pathway like RDL. An overriding conclusion is that the model envisioning aversive and appetitive specific US pathway roles for dopamine and octopamine, respectively, is overly simplistic (Liu, 2009b).

The results suggest that the GABA_A receptor RDL regulates the CS pathway in Drosophila olfactory learning. The conclusion that the GABA_A receptor modulates the CS pathway for learning is not limited to either insects or learning supported by olfactory cues. During taste aversion learning in mice, pre-exposure to the CS of the tastant alone causes latent inhibition where the mice show reduced learning to the CS after pairing the CS with the US. This phenomenon is distinctly absent in male mice carrying a point mutation in the α5 subunit of the GABA_A receptor, which is highly expressed in the hippocampus (Gerdjikov, 2008). Since CS information is the only stimulus presented during the pre-exposure period, these results support the role of GABA_A receptors in regulating the CS pathway. Extinction is another type of learning where repeated exposure to the CS alone after CS/US conditioning reduces the CR. Systemic administration of a GABA_A receptor antagonist blocks the development and expression of extinction in rats during contextual fear learning (Harris, 1998). Since extinction trials are composed of the CS exposure by itself, these results also indicate that GABA_A receptors modulate the CS pathway. Moreover, other studies have shown that the surface expression of GABA_A receptors increases in the basolateral amygdala after extinction trials following fear conditioning (Chhatwal, 2005). These results indicate that CS exposure alone during extinction is sufficient to modulate the cellular trafficking of GABA_A receptors, again indicating a role for GABA_A receptors in the CS pathway. The current results, together with these previous studies, strongly indicate that GABA_A receptors regulate the CS pathway for associative learning (Liu, 2009b).

A role for GABA_A receptors in suppressing learning by regulating the CS pathway has at least two broad implications. (1) It suggests that the receptors provide a gate to the association center (MBs). Other molecules may also provide similar gates, but learning must overcome this negative influence for memory formation to occur. This gate is probably nonspecific relative to odor type, that is, the GABA_A receptor gate suppresses learning to most or all odors. It follows that learning must mobilize cellular mechanisms for overriding the gate. These could be at the level of the presynaptic GABAergic neurons, such that the presynaptic neurons release less neurotransmitter after learning, or they could be at the level of the postsynaptic receptor, with receptor expression, sensitivity, or conductance altered by learning. Evidence has been provided for a reduced presynaptic release following learning (Liu, 2009b), but postsynaptic mechanisms may occur as well (Chhatwal, 2005). (2) Events or processes that alter the salience of the CS and its ability to enter into associations might function via altering the presynaptic GABAergic release or the postsynaptic GABA_A receptors. For instance, spaced conditioning is generally more effective in producing long-lasting memories compared with massed conditioning. It is possible that the rest period between spaced conditioning trials allows for receptor desensitization, producing a more effective subsequent training trial. Memory acquisition becomes more difficult with age. It could be that aging alters the fluidity of the GABA_A receptor gate, making acquisition more difficult (Liu, 2009b).

Differential roles of the fan-shaped body and the ellipsoid body in Drosophila visual pattern memory

The central complex is a prominent structure in the Drosophila brain. Visual learning experiments in the flight simulator, with flies with genetically altered brains, revealed that two groups of horizontal neurons in one of its substructures, the fan-shaped body, were required for Drosophila visual pattern memory. However, little is known about the role of other components of the central complex for visual pattern memory. This study shows that a small set of neurons in the ellipsoid body, which is another substructure of the central complex and connected to the fan-shaped body, is also required for visual pattern memory. Localized expression of rutabaga adenylyl cyclase in either the fan-shaped body or the ellipsoid body is sufficient to rescue the memory defect of the rut²⁰⁸⁰ mutant. RNA interference of rutabaga was performed in either structure, and it was found that they both were required for visual pattern memory. Additionally, the above rescued flies were tested under several visual pattern parameters, such as size, contour orientation, and vertical compactness, and differential roles were revealed of the fan-shaped body and the ellipsoid body for visual pattern memory. This study defines a complex neural circuit in the central complex for Drosophila visual pattern memory (Pan, 2009).

This study reports that a subset of the ellipsoid body neurons are necessary for Rut-dependent visual pattern memory, in addition to the previously described horizontal neurons in the fan-shaped body (Liu, 2006). These substructures of the central complex play different roles in visual pattern memory. Moreover, the experiments revealed that the choice of mutant allele was crucial when using the rutabaga rescue strategy (Pan, 2009).

To localize the physical correlates of memory in the Drosophila central nervous system, one usually utilizes two distinct ways: (1) functional knockdown of 'memory genes' or neural transmission in specific brain regions, and (2) functional rescue by targeted expression of a 'memory gene' in the respective mutant. The former defines a structure necessary for memory formation, while the latter identifies a structure that is sufficient. rutabaga is such a gene that is involved in many forms of learning and memory in Drosophila. Functional rescue of the rut²⁰⁸⁰ mutant in olfactory aversive learning, olfactory reward learning, spatial learning, and visual pattern memory in tethered flight revealed distinct brain structures for memory formation: mushroom bodies for aversive olfactory learning, projection neurons or mushroom bodies for olfactory reward learning, median bundle for spatial learning, and fan-shaped body for visual pattern memory. It should be noted that all these rescue experiments were done in the rut²⁰⁸⁰ mutant. The mutation is caused by a P-element insertion 155 bp upstream of the rut gene, which leads to a reduced rut level. The results of rescue experiments demonstrate that using a hypomorphic mutant allele is not optimal for determining the sufficiency of a brain region for a given task. For example, although restoring Rut function in the fan-shaped body rescued visual pattern memory successfully, this cannot exclude the involvement of other regions owing to the residual Rut activity in the rut²⁰⁸⁰ flies. Therefore, it is crucial to perform rescue experiments in a null mutant. rut¹ appears to be a suitable candidate, as the point mutation in the gene leads to a complete loss of Rut activity in both cultured cells and head homogenate extracts. Overexpression of rut⁺ in either the fifth layer (F5 neurons) alone of the fan shaped body or overexpresssion in a subset of large field neurons in the ellipsoid body that are called 'R neurons' (R2/R4m neurons) neurons alone in the rut¹ mutant failed to restore visual pattern memory, implying that neither the fan-shaped body nor ellipsoid body neurons were sufficient. However, a combination of these two regions did succeed in rescuing the rut¹ memory defect. Taken together with the RNAi results that indicated necessary roles of both the F5 and R2/R4m neurons, it could be concluded that these fan-shaped body and ellipsoid body neurons seemed to be the sufficient brain regions where Rut functions, in the rut¹ mutant, to form visual pattern memory (Pan, 2009).

Currently the exact role of rut in the fan-shaped body and ellipsoid body is not known; however, it can be inferred from previous studies on the larval neuromuscular junction that rut may mediate synaptic plasticity in these neurons. It is assumed that rut-dependent synaptic plasticity may be lost in the rut¹ or RNAi silencing flies, but only compromised in the rut²⁰⁸⁰ flies. The results could be interpreted as that the loss of rut-dependent synaptic plasticity in either the fan-shaped body or ellipsoid body impaired visual pattern memory, but flies with a compromised fan-shaped body and a restored ellipsoid body, or a compromised ellipsoid body and a restored fan-shaped body, could form stable, wild-type memories. It seems that an operating range for the underlying neural circuit exists. Complete loss of rut-dependent synaptic plasticity in either the fan-shaped body or ellipsoid body moves the circuit out of the operating range, while restoring either of the compromised fan-shaped body or ellipsoid body can bring the circuit back into the operating range (Pan, 2009).

It has been found in many insects including Drosophila that the central complex is involved in visual signal processing and motor control. However, the exact roles of the central complex substructures are not well understood. What was known until now is that the F1 neurons are necessary for visual pattern memory for 'contour orientation' and F5 neurons for 'elevation,' which raises the possibility that visual signals are processed in the fan-shaped body and distinct F neurons are responsible for different visual pattern parameters. Recently, the R2/R4m neurons in the ellipsoid body were proved to be involved in ethanol sensitivity and tolerance, and later in olfactory long-term memory consolidation. In this study, the R2/R4m neurons were found to be required for visual pattern memory for all tested parameters and thus may be parameter independent. However, the exact role of the R2/R4m neurons for visual pattern memory could not be determined yet (Pan, 2009).

These studies indicated that Rut function in the central complex was crucial for Drosophila visual pattern memory; however, there might be some other Rut-independent neurons that also contribute to the neural circuit. Future work should focus on loss-of-function studies by blocking neural signaling in targeted subsystems. Furthermore, a temporal dissection of memory acquisition and retrieval would help arriving at an understanding of how the different neuropils are involved. As the F and R neurons are all large field neurons that connect to other brain regions or other parts of the central complex, it is also crucial to identify the upstream and downstream neurons. Although the picture is far from complete, it seems that the central complex might be the major center for visual pattern memory. Studying such adaptive behaviors from a single gene to multiple types of neurons within a circuit is a challenging but indispensable step to unravel the neural basis of complex behaviors (Pan, 2009).

Use-dependent plasticity in clock neurons regulates sleep need in Drosophila

Sleep is important for memory consolidation and is responsive to waking experience. Clock circuitry is uniquely positioned to coordinate interactions between processes underlying memory and sleep need. Flies increase sleep both after exposure to an enriched social environment and after protocols that induce long-term memory. This study found that flies mutant for rutabaga, period, and blistered were deficient for experience-dependent increases in sleep. Rescue of each of these genes within the ventral lateral neurons (LNVs) restores increased sleep after social enrichment. Social experiences that induce increased sleep were associated with an increase in the number of synaptic terminals in the LNV projections into the medulla. The number of synaptic terminals was reduced during sleep and this decline was prevented by sleep deprivation (Donlea, 2009).

Although sleep is a process that is necessary for survival, the functions of sleep are unknown. Sleep is regulated by circadian influences and is important for consolidation of long-term memory (LTM). Additionally, LTM is modulated by circadian mechanisms. Because the relationship between sleep, memory, and circadian rhythms seem to be phylogenetically conserved, Drosophila can be used to explain mechanisms that coordinate these processes. Drosophila show an increase in daytime sleep after exposure to socially enriched environments. Similarly, an increase in sleep after courtship conditioning is necessary for LTM (Donlea, 2009).

Increased sleep after social enrichment is dependent upon genes that are required for learning and memory, including genes that alter cyclic adenosine monophosphate signaling. Although newly eclosed flies that are mutant for the adenylyl cyclase rutabaga (rut²⁰⁸⁰) show increased sleep after social enrichment, 3 to 4 day-old adult rut mutants do not respond to changes in the social environment. Elevating wild-type rut in adult flies with an RU486-inducible driver rescued experience-dependent increases in sleep in adult rut mutants; vehicle-treated siblings showed no increase in sleep. To identify circuits that mediate experience-dependent increases in sleep, a series of GAL4 lines was used to drive wild-type rut expression in brain circuits. Expression of UAS-rut using pdf-GAL4 restored the increase in daytime sleep and daytime sleep-bout duration, although to a lesser extent than GSelav. The expression pattern of pdf-GAL4 is limited to the ventral lateral neurons (LN_Vs), a group of clock neurons that express pigment-dispersing factor (pdf). Although pdf is the only known output from the LN_Vs, flies mutant for pdf show a wild-type increase in sleep (Donlea, 2009).

Given this role of clock cells, the clock gene period (per), which is expressed in the LN_Vs and is required for LTM, was examined. Rescue of wild-type per using a 7.2-kb fragment of the per genomic sequence (per⁰¹; per+7.2-2) restored expression of PER at CT0 within the LN_Vs as well as the dorsal lateral neurons, LN_Ds; mutant flies carrying a null mutation, per⁰¹, expressed no PER. Although per⁰¹ mutants showed no increase in sleep after social enrichment, per⁰¹;per+7.2-2 flies displayed normal experience-dependent increases in sleep. per⁰¹ mutants have no LTM when tested 48 hours after training and only show a transient increase in sleep. per⁰¹;per+7.2-2 flies displayed LTM and increases in sleep. Although per levels are low in mutants for Clock and cycle, both acquire LTM and increase sleep after social enrichment. Thus, only a very small amount of per may be required to support increased sleep and LTM (Donlea, 2009).

To further investigate the role of synaptic plasticity in clock cells, the Drosophila homolog for serum response factor (SRF), blistered (bs), was used. In mice, SRF is essential for activity-induced gene expression and plays an important role in synaptic long-term potentiation (Ramanan, 2005) and in contextual habituation (Etkin, 2006). bs retains a 93% identity with SRF within the DNA-binding MCM1-ARG80-Agamous-Deficiens-SRF (MADS) domain. Social enrichment elevated the transcription of bs in wild-type Canton-S (Cs) flies. Mutants carrying a P element inserted into the bs gene (P{GAL4}bs¹³⁴⁸) do not increase sleep after social enrichment. This deficit was also found in flies carrying either of two other mutant alleles for bs (bs² and bs³) and was present in flies that are homozygous for mutant bs alleles and flies that have been outcrossed to either Cs or to flies carrying the In(2LR)Px⁴ deficiency. The P-element insertion in bs¹³⁴⁸ preserves the MADS domain; similar N-terminal truncated mutant SRF acts as dominant negative. BS is expressed throughout the brain, including pdf-expressing LN_Vs. When UAS-egfp was driven by P{GAL4}bs¹³⁴⁸, expression was restricted to a small number of neurons, including the LN_Vs. Expression of bs using P{GAL4}bs¹³⁴⁸ to drive either of two wild-type bs (UAS-bs) constructs rescued experience-dependent increases in sleep. Moreover, inducing bs expression within the LN_Vsusing pdf-GAL4 increased sleep after social enrichment (Donlea, 2009).

To establish whether expression of bs is required for LTM, flies carrying the P{GAL4}bs¹³⁴⁸ mutant allele were tested using courtship conditioning. Although P{GAL4}bs¹³⁴⁸/+ flies acquire short-term memory, LTM was impaired. Rescue of wild-type bs using P{GAL4}bs¹³⁴⁸ restored LTM. Next, the GAL4 repressor cry-GAL80 was used to block UAS-bs expression within the LNs. Although UAS-bs/+;cry-gal80/+ control flies showed significant courtship suppression, P{GAL4}bs¹³⁴⁸/UAS-bs;cry-GAL80/+ flies had no LTM, which suggests a role for the LNs, although a role for the dorsal neurons (DNs) cannot be excluded. Although SRF deletion in mouse forebrain results in neurons with abnormal morphology (Knoll, 2006), the morphology of LN_Vs in mutant P{GAL4}bs¹³⁴⁸/+ flies did not differ from that of LN_Vs in P{GAL4}bs¹³⁴⁸/UAS-bs rescue flies. All three mutants for bs had intact circadian rhythms and showed anticipatory activity before light-dark transitions; only bs³ flies show an altered period under constant darkness. These findings suggest that there are no developmental abnormalities in the LN_Vs in bs mutants (Donlea, 2009).

Hypomorphic alleles for bs prevent proper wing development through interactions with Epidermal growth factor receptor (Egfr) signaling. Because Egfr alters sleep in Drosophila (Foltenyi, 2007), interactions between bs and Egfr may regulate responses to social experience. After social enrichment, transcription of Egfr was significantly elevated in Cs flies. The Egfr genomic sequence contains several CC(A/T)6GG CArG elements that can be bound by bs to promote transcription, and transcription of Egfr was significantly reduced in bs mutants. Thus, P{GAL4}bs¹³⁴⁸ was used to drive expression of a constitutively active Egfr construct (UAS-Egfr*). Although P{GAL4}bs¹³⁴⁸/+ mutants showed no change in sleep after social enrichment, activation of Egfr in P{GAL4}bs¹³⁴⁸/+;UAS-Egfr*/+ flies increased sleep. Conversely, the expression of a dominant-negative construct for Egfr (UAS-Egfr^DN) using pdf-GAL4 prevented increases in sleep after social enrichment (Donlea, 2009).

A recent theory proposes that a function of sleep is to downscale synaptic connections. Moreover, structural plasticity can be induced by environmental manipulation in Drosophila. To quantify the effect of social enrichment on the number of post-synaptic terminals in LN_V projections, pdf-GAL4 was used to drive expression of a green fluorescent protein (GFP)-tagged construct of the postsynaptic protein discs-large (UAS-dlgWT-gfp). After 5 days of social enrichment, LN_V projections into the medulla of pdf-GAL4/+;;UAS-dlgWT-gfp/+ flies contained significantly more GFP-positive terminals. Although it has not been demonstrated that the labeled synaptic terminals are functional, these tools have been used to quantify synapses. The expression of the UAS-dlgWT-GFP marker did not alter synaptic function in a wild-type background and did not prevent the increase in sleep when expressed using pdf-GAL4 after social enrichment. To determine the effect of waking on synapse number, socially isolated pdf-GAL4/+;;UAS-dlgWT-gfp/+ flies and their enriched siblings either were allowed to sleep ad libitum or were sleep deprived for 48 hours after social enrichment. Although the number of dlg-GFP positive terminals remained elevated in sleep-deprived socially enriched flies, terminal number was significantly reduced in siblings that were allowed to sleep. Similarly, the number of presynaptic terminals in LN_V projections into the medulla using a GFP-tagged construct of the presynaptic protein synaptobrevin (UAS-VAMP-GFP) in pdf-GAL4/+;UAS-VAMP-GFP/+ flies was increased. After 48 hours of recovery, socially enriched pdf-GAL4/+;UAS-VAMP-GFP/+ flies had a reduced number of VAMP-GFP-positive presynaptic terminals relative to their sleep-deprived siblings. A recent study has reported a clock-dependent remodeling in the axonal terminals of the PDF circuit that is highest during the day. Recent data indicates that hyperexcitation of a subset of the LN_Vs suppresses sleep in Drosophila. Together with the current results, these data suggest that the PDF circuit is well suited to test the hypothesis that sleep acts to downscale synaptic connections that are potentiated during waking experience (Donlea, 2009).

Dynamics of learning-related cAMP signaling and stimulus integration in the Drosophila olfactory pathway

Functional imaging with genetically encoded calcium and cAMP reporters was used to examine the signal integration underlying learning in Drosophila. Dopamine and octopamine modulated intracellular cAMP in spatially distinct patterns in mushroom body neurons. Pairing of neuronal depolarization with subsequent dopamine application revealed a synergistic increase in cAMP in the mushroom body lobes, which was dependent on the rutabaga adenylyl cyclase. This synergy was restricted to the axons of mushroom body neurons, and occurred only following forward pairing with time intervals similar to those required for behavioral conditioning. In contrast, forward pairing of neuronal depolarization and octopamine produced a subadditive effect on cAMP. Finally, elevating intracellular cAMP facilitated calcium transients in mushroom body neurons, suggesting that cAMP elevation is sufficient to induce presynaptic plasticity. These data suggest that rutabaga functions as a coincidence detector in an intact neuronal circuit, with dopamine and octopamine bidirectionally influencing the generation of cAMP (Tomchik, 2009).

This study presents data suggesting that rut mediates a synergistic increase in cAMP when dopamine and neuronal depolarization (via acetylcholine stimulation) are paired, an effect that exhibits temporal pairing requirements similar to those of behavioral conditioning. This provides strong evidence that the rut-encoded AC is a molecular coincidence detector, a long-hypothesized yet never adequately tested idea. The Drosophila rut AC is similar to the mammalian type I AC (AC1) in that it is sensitive to stimulation by both G_αs and Ca²⁺/CaM. When expressed in HEK cells, mammalian AC1 is stimulated by G_s-coupled receptors only if this stimulation is paired with calcium elevation, in which case a synergistic elevation of cAMP is observed. Similarly, the only rut-dependent effect observed was the synergistic increase in cAMP following pairing of dopamine and neuronal depolarization (Tomchik, 2009).

cAMP responses were observed to either acetylcholine or dopamine applied in isolation in both wild-type and rut¹ flies. This suggests that there are additional ACs in the mushroom bodies that respond to unpaired dopamine and acetylcholine but do not generate synergistic increases in cAMP upon coincident depolarization. Several identified and putative ACs could underlie these responses in rut¹ mutants (DAC39E, DAC78C, DAC76E, CG32158, CG32301, and CG32305). It is unlikely that the responses to acetylcholine-induced depolarization in rut¹ mutants are due to calcium-induced AC activation; previous studies did not detect any calcium sensitive cyclase activity in rut¹ mutants. There are several possible explanations for why such increases in cAMP are observed in rut¹ mutants. Remaining ACs could be directly activated by depolarization or via muscarinic acetylcholine receptors that positively couple cAMP. Alternatively, the DPM neurons could be downstream of the mushroom body neurons and provide feedback on the mushroom bodies. The DPM neurons are believed to release a peptide that stimulates cAMP. This alternative model emphasizes the need to evaluate responses within the context of a neural circuit, as allowed by the preparation used for these studies (Tomchik, 2009).

Synergistic increases in cAMP were observed when acetylcholine was paired with dopamine, but the opposite effect occurred when acetylcholine was paired with octopamine. Various Drosophila tyramine/octopamine receptors can simulate or inhibit the production of cAMP, which may explain the inhibition of cAMP generation when octopamine is paired with acetylcholine. This inhibitory effect does not require rut, and is therefore likely mediated by other cyclases. There are several implications of this finding in terms of the role of octopamine in conditioning. Previous data have suggested that dopamine and octopamine may relay the aversive and appetitive US, respectively. If this model is correct, then the data suggest that an appetitive stimulus could suppress the responses of mushroom body neurons to a subsequent CS. However, dopamine plays a role in both appetitive and aversive learning . This opens the alternate possibility that dopamine could relay both appetitive and aversive US, with octopamine playing a different role in mushroom body physiology (Tomchik, 2009).

Data from the preparation that was used suggest that elevating cAMP is sufficient to induce plasticity in the mushroom bodies, facilitating the calcium responses of mushroom body neurons to stimulation by acetylcholine. Therefore, cAMP appears to be both necessary and sufficient for neuronal plasticity in the mushroom body. There are several mechanisms by which cAMP could facilitate responses of mushroom body neurons. cAMP could elevate membrane potential by activating cyclic nucleotide-gated channels, which are expressed in the Drosophila antennal lobes and mushroom body neurons (among other areas). cAMP has been shown to have direct effects on a K⁺-selective ion channel. In addition, cAMP could affect neuronal excitability via activation of protein kinase A (PKA). Likely targets of PKA phosphorylation include Na⁺ and K⁺ channels, modulation of which can influence neuronal excitability. In cricket mushroom body neurons, Na⁺-activated K⁺ channels are modulated by dopamine and octopamine, as well as cAMP/PKA and cGMP/PKG pathways. Notably, presynaptic facilitation in Aplysia sensory neurons relies on modulation of potassium channels by cAMP/PKA (Tomchik, 2009).

Plasticity in the Drosophila mushroom body shares some features with the plasticity that underlies the siphon withdrawal reflex in Aplysia. In addition to having a large presynaptic component, with cAMP increases being both necessary and sufficient, this type of plasticity involves increased influx of calcium into the presynaptic terminal. In both Drosophila and Aplysia, the plasticity appears to be heterosynaptic, requiring input from neurons releasing serotonin in Aplysia and dopamine (and/or octopamine) in Drosophila. In Aplysia, facilitation of sensory neuron-motor neuron synapses exhibits temporal requirements similar to those of behavioral conditioning. Likewise, this study found that the synergistic generation of cAMP has temporal requirements similar to those of differential behavioral conditioning in flies. Thus, the synergistic increases in cAMP could underlie the plasticity that drives some of the behavioral modification following learning in Drosophila. However, there must be at least one other pathway for plasticity in Drosophila, because performance is reduced, but not eliminated, in rut mutants (Tomchik, 2009).

Different sets of mushroom body neurons appear to have different temporal roles in learning and memory, with synaptic transmission from α/β neurons being required during memory retrieval and synaptic transmission from α'/β' neurons and DPM neurons being required during learning and early memory consolidation. That begs the question: which neurons are responsible for registering CS/US coincidence and initially triggering memory formation? One possibility is that CS/US coincidence is initially registered in the α'/β' neurons and then the memory is sequentially transferred to the α/β neurons during consolidation. Alternatively, CS/US coincidence could be registered in parallel across both sets of neurons. The data suggest that initial learning could be triggered in parallel across both the α/β and α'/β' neurons. Synergistic increases are observed in cAMP in both the α and α' lobes, and increases in cAMP facilitated the responses of axons in both areas as well. This makes sense given that rut is expressed at high levels in both the α/β and α'/β' lobes, suggesting that the molecular machinery for coincidence detection is present in both. It seems likely that the initial coincidence will occur in parallel across different subsets of mushroom body α/β and α'/β' neurons, depending on how the specific odor is encoded in the mushroom body (Tomchik, 2009).

Central synaptic mechanisms underlie short-term olfactory habituation in Drosophila larvae

Naive Drosophila larvae show vigorous chemotaxis toward many odorants including ethyl acetate (EA). Chemotaxis toward EA is substantially reduced after a 5-min pre-exposure to the odorant and recovers with a half-time of ~20 min. An analogous behavioral decrement can be induced without odorant-receptor activation through channelrhodopsin-based, direct photoexcitation of odorant sensory neurons (OSNs). The neural mechanism of short-term habituation (STH) requires the (1) Rutabaga adenylate cyclase; (2) transmitter release from predominantly GABAergic local interneurons (LNs); (3) GABA-A receptor function in projection neurons (PNs) that receive excitatory inputs from OSNs; and (4) NMDA-receptor function in PNs. These features of STH cannot be explained by simple sensory adaptation and, instead, point to plasticity of olfactory synapses in the antennal lobe as the underlying mechanism. These observations suggest a model in which NMDAR-dependent depression of the OSN-PN synapse and/or NMDAR-dependent facilitation of inhibitory transmission from LNs to PNs contributes substantially to short-term habituation (Larkin, 2010).

Experience-induced plasticity of synapses is believed to be a fundamental mechanism of learning and memory. However, central synaptic changes that underlie memory have not been clearly defined, even for relatively simple nonassociative learning processes such as habituation (Larkin, 2010).

During habituation, unreinforced exposure to a repeated or prolonged stimulus results in a reversible decrease in response to that stimulus. Habituation probably serves as an important building block for more complex cognitive function. By allowing unchanging or irrelevant stimuli to be ignored, it allows cognitive resources to be focused on more salient stimuli (Larkin, 2010 and references therein).

The neural basis of short-term habituation (STH) is best studied in the marine snail, Aplysia californica. Here STH (lasting ~30 min) of the defensive gill-withdrawal reflex in response to tactile stimulation of the siphon is thought to arise from presynaptic depression of transmitter release at sensorimotor synapses. However, even here, presynaptic plasticity may not be cell-autonomous, potentially requiring, for instance, activity of yet-to-be-identified interneurons (Larkin, 2010).

Several forems of habituation have been described in Drosophila and are often shown to require the function of genes that regulate cAMP-dependent forms of associative memory. For instance, habituation of proboscis extension reflex as well as odor-evoked startle reflex in adult Drosophila requires rutabaga (rut)-encoded Ca2+/calmodulin-sensitive adenylyl cyclase. In addition, habituation of the ethanol-induced startle response requires the shaggy/GSK-3 signaling pathway. Despite such pioneering observations, the mechanisms of these various forms of habituation, even whether the primary neuronal changes are purely sensory or involve plasticity of central synapses (involving centrally located interneurons that may integrate various different kinds of modulatory, inhibitory, and excitatory inputs), remain poorly understood (Larkin, 2010).

Recent advances in understanding the circuitry that underlies Drosophila olfactory behavior, as well as the development of new tools to perturb identified neurons in vivo, has opened the opportunity for understanding mechanisms of olfactory habituation at the level of the underlying neural circuitry (Larkin, 2010).

In the larval olfactory system, 21 olfactory sensory neurons (OSNs), each expressing a single odorant receptor (together with the broadly expressed Or83b co-receptor), synapse, respectively, onto 21 cognate projection neurons (PNs) within 21 glomeruli in the larval antennal lobe (AL). Local, predominantly GABAergic interneurons (LNs) synapse widely within the antennal lobe, interlinking different glomeruli. Various neuromodulatory synapses also form on the larval antennal lobe and mushroom body. Thus, odorant-stimulated signals in sensory neurons are processed in the antennal lobe, modulated by motivational or emotional states, and relayed through projection neurons to higher brain centers (Larkin, 2010).

Previous work has shown that in Drosophila larvae, olfactory chemotaxis decreases after odorant pre-exposure. This study shows that this behavioral habituation, alternatively referred to as 'adaptation' by some previous investigators, arises from mechanisms of synaptic plasticity. This study demonstrates that odorant receptor activation is not necessary for olfactory habituation; however, local interneuron activity and projection neuron signaling is necessary. These observations suggest a model in which habituation occurs by a pathway in which NMDA receptors in projection neurons signal depression of OSN-PN synapses and/or facilitation of LN-PN synapses (Larkin, 2010).

Previous studies have not clearly discriminated between peripheral and central mechanisms. Indeed, the term 'adaptation,' better applied to sensory neuron changes such as receptor desensitization, has often been used interchangeably with the term 'habituation', which is usually restricted to behavioral changes arising from central synaptic mechanisms (Larkin, 2010). .

The form of larval olfactory STH characterized in this study displays at least some of the defining behavioral characteristics of habituation. First, there is a behavioral decrement in response to repeated or sustained application of a particular stimulus. Second, STH shows spontaneous recovery with time in the absence of the habituating stimulus. And third, STH is susceptible to dishabituation when habituated larvae are presented with of a strong or noxious stimulus. The property of dishabituation is particularly significant, as an important way of distinguishing between habituation and either fatigue or sensory adaptation. Dishabituation shows that the habituated animal retains the capability to respond and suggests that the attenuated behavioral response arises from some form of active suppression. Thus, the behavioral data suggest (1) that the term 'habituation' may be better used in place of 'adaptation,' while referring to the behavioral phenomenon that was studied; and (2) that STH probably arises from central synaptic mechanisms, rather than sensory neuron adaptation (Larkin, 2010).

Three main lines of data support the conclusion that STH arises from a central synaptic mechanism that resides in the antennal lobe, rather than from adaptation of olfactory receptor signaling in the OSN. First, behavioral decrements similar to STH can be induced by direct depolarization of OSNs, indicating that STH may potentially be induced by processes stimulated by activation action-potential firing in OSNs, independently of olfactory receptor activation. Second, and more striking, STH requires synaptic-vesicle exocytosis from local interneurons during the process of odorant exposure, when STH is being established. This requirement is incompatible with an exclusively sensory mechanism. Third, STH requires the function of NMDA receptors on postsynaptic projection neurons. This last observation also provides a particularly strong argument for a synaptic mechanism, indicating a need for plasticity of OSN and/or LN synapses made onto dendrites of projection neurons in the antennal lobe. Given that OSNs are excitatory and LNs are primarily inhibitory, it appears most likely that NMDAR functions in PNs to depress excitatory OSN-PN synapses and/or to potentiate inhibition by strengthening the LN-PN synapse. It is suggestd that the LN-PN mechanism may be involved because (1) LN transmission seems necessary for both induction and expression of habituation; and (2) the process of dishabituation could be attractively explained as arising from the inhibition of local inhibitory synapses through descending neuromodulation. A requirement for facilitation of the LN-PN synapse would be consistent with previous studies (Sachse, 2007) showing that adult-long-term olfactory habituation is associated with an increase in odor-evoked calcium fluxes in GABAergic processes within the Drosophila antennal lobe (Larkin, 2010).

Based both on experimental and theoretical arguments, a simple model is suggested for short-term olfactory habituation. Since this is a model, no claim is being made to to having ruled out additional major contributing mechanisms, It is suggested that during initial odorant pre-exposure, dendritic NMDA receptors on projection neurons detect and respond to membrane depolarization occurs coincident with transmitter release from LNs. Calcium entry through dendritic NMDA receptors may trigger a local retrograde signal required for facilitation of transmitter release from the LNs. Although existing data do not rule out functions for rutabaga in higher larval brain centers, it is suggested that either the generation of a retrograde signal in PN dendrites or the presynaptic response of LNs to this signal could be dependent on the rut adenylate cyclase. In habituated animals, facilitation of GABA release would reduce odor-evoked projection neuron outputs to higher brain centers, thereby reducing olfactory behavior. As NMDAR signaling would only occur at active glomeruli, this mechanism can account not only for the observed odor selectivity of habituation, but also the instances of cross-habituation (Larkin, 2010).

Such a model also naturally suggests a hypothesis for the mechanism of dishabituation: namely, that dishabituating stimuli cause release of neuromodulators that act to reduce GABA release from local inhibitory synapses (Larkin, 2010).

Given the remarkable similarities in the anatomical organization of insect and mammalian olfactory systems, a significant conservation of olfactory mechanisms would be expected. In rodents, at least two forms of habituation have been described, lasting 2-3 and 30-60 min, respectively: the latter equivalent in timescale to larval STH described in this study. Consistent with a similar underlying mechanism, the more persistent form of olfactory habituation can be blocked by an N-methyl-D-aspartate (NMDA) receptor antagonist in the olfactory bulb, a structure homologous to the insect antennal lobe. Thus, larval STH described in this study has some similarities to a previously characterized form of mammalian olfactory habituation. Analysis of the underlying mechanisms is therefore likely to provide directly transferable insights in mammalian olfaction. The data make the prediction that the activity of mammalian olfactory interneurons, either periglomerular or granule cells, is critical for the establishment and display of at least one timescale of olfactory habituation (Larkin, 2010).

In addition to providing some insight into mechanisms of olfactory habituation in mammals, it possible that circuit mechanisms of larval olfactory habituation are relevant to other forms of behavioral habituation. In at least three previous instances, increased inhibition has been associated with attenuated behavior. For example, habituation of an escape reflex mediated by the lateral giant fibers in the crayfish has been associated with enhanced GABAergic transmission onto giant fibers. Similarly, LTP of inhibitory synapses controlling excitability of the Mauthner cell has been associated with reduced escape behavior in goldfish. Furthermore, ethanol, a potentiator of GABA synapses, has been shown to enhance habituation of a motor pathway in the frog spinal cord. Could these different instances of habituation all involve circuit mechanisms similar to those used in Drosophila larval olfactory behavior (Larkin, 2010)?

In all brain regions, principal/projection neurons are subject to inhibitory feedback modulation and a pathway that has been appreciated as potentially essential for neuronal homeostasis. Potentiation of inhibitory feedback triggered by the pattern of principle cell activation would be predicted to preferentially dampen this particular output pattern. Thus, the circuit mechanism suggest in this study is theoretically generalizable to other and more complex forms of habituation. Further experiments will be required to determine the validity of this very testable hypothesis (Larkin, 2010).

The importance of habituation has been underlined by the fact that deficits in sensory gating and pre-pulse inhibition (PPI), processes with similarities to habituation, have been linked with various neurological problems, including autism and schizophrenia. Indeed, a circuit model for understanding schizophrenia has specifically proposed that altered negative feedback in the hippocampus may underlie both positive and negative symptoms of schizophrenia (Larkin, 2010).

In addition, defects in habituation or habituation-like processes have been described in Fragile X syndrome and migraines. It has also been shown to have important effects relating to learning disabilities, age-related changes in learning, and substance abuse. If mechanisms of olfactory habituation prove to be general, then studies of olfactory plasticity may prove relevant for other forms of cognition as well as for human neurological disease (Larkin, 2010).

A distinct set of Drosophila brain neurons required for neurofibromatosis type 1-dependent learning and memory

Nonspecific cognitive impairments are one of the many manifestations of neurofibromatosis type 1 (NF1). A learning phenotype is also present in Drosophila melanogaster that lack a functional neurofibromin gene (nf1). Multiple studies have indicated that Nf1-dependent learning in Drosophila involves the cAMP pathway, including the demonstration of a genetic interaction between Nf1 and the rutabaga-encoded adenylyl cyclase (Rut-AC). Olfactory classical conditioning experiments have previously demonstrated a requirement for Rut-AC activity and downstream cAMP pathway signaling in neurons of the mushroom bodies. However, Nf1 expression in adult mushroom body neurons has not been observed. This study addresses this discrepancy by demonstrating (1) that Rut-AC is required for the acquisition and stability of olfactory memories, whereas Nf1 is only required for acquisition, (2) that expression of nf1 RNA can be detected in the cell bodies of mushroom body neurons, and (3) that expression of an nf1 transgene only in the alpha/beta subset of mushroom body neurons is sufficient to restore both protein synthesis-independent and protein synthesis-dependent memory. These observations indicate that memory-related functions of Rut-AC are both Nf1-dependent and -independent, that Nf1 mediates the formation of two distinct memory components within a single neuron population, and that understanding of Nf1 function in memory processes may be dissected from its role in other brain functions by specifically studying the alpha/beta mushroom body neurons (Buchanan, 2010).

Neurofibromatosis Type 1 (NF1) is an autosomal, dominant genetic disorder that afflicts approximately one in every 3500 individuals. Like other clinical manifestations of NF1, expression and penetrance of cognitive phenotypes varies and may include deficiencies of visual-spatial processing, executive function, and attention. Homologs of human Nf1 in mouse and Drosophila melanogaster share significant identity at the protein level, and animal models in both species were developed shortly after the human Nf1 gene was cloned. Both models demonstrate cognitive phenotypes, and insights gained through animal studies have shed light on the genetic and biochemical basis of these defects (Buchanan, 2010).

Drosophila has been utilized extensively for expanding basic understanding of memory, making it ideal for investigating NF1 cognitive deficits. After olfactory classical conditioning, Drosophila form protein synthesis-independent early memories (PSI-EM), comprised of short-term memory (STM) tested at 3 minutes after training, middle-term memory (MTM) often tested at 3 hours after training, and protein synthesis-dependent long-term memory (PSD-LTM), tested at 24 hours after conditioning. The nf1 mutant flies demonstrate deficiencies in PSI-EM and PSD-LTM. A current model postulates that Nf1 contributes to PSI-EM through stimulation of the rutabaga-encoded adenylyl cyclase (Rut-AC). Stimulation of Gα_s-dependent AC activity requires only the Nf1 C-terminal domain. The PSI-EM phenotypes of nf1, rut-AC, and nf1/rut-AC mutants are similar, both genes are required at the time of learning, and either ubiquitous expression of a constitutively active protein kinase A (hsPKA*) transgene or neuronal expression of a Nf1 C-terminal domain transgene rescues the nf1 phenotype. Furthermore, the current model also postulates that Nf1 contributes to PSD-LTM through regulation of Ras via its GAP-related domain (GRD). Stimulation of Ras-dependent AC activity is absent in nf1 mutants, but transgenic expression of the Nf1-GRD restores this activity and improves the PSD-LTM phenotype of nf1 mutant (Buchanan, 2010).

It is surprising that endogenous Nf1 expression has not been observed in adult mushroom body (MB) neurons. MB neurons are essential for olfactory memory formation, and Rut-AC is preferentially expressed in these neurons. Rescue experiments demonstrated that transgenic expression of rut-AC in α/β and γ MB neurons restores normal memory in homozygous mutants. If Nf1 indeed stimulates Rut-AC activity during learning, it is probably expressed, and required, in MB neurons. This study explores whether Nf1 and Rut-AC are involved in the same operational phase of learning, whether they are expressed in the same neurons, whether both are required in the same neurons for rescue of PSI-EM, and whether the Ras-mediated function of Nf1 is required in overlapping neurons. A role is reported for Rut-AC in memory stability that is Nf1-independent, nf1 expression in MB neurons was observed, and a requirement for nf1 expression in α/β MB neurons was observed for both PSI-EM and PSD-LTM (Buchanan, 2010).

Regardless of species being studied, neurofibromin is involved in many different brain activities including, but not limited to, cognitive processes, circadian rhythms, cortical development, and glial development. Even within the cognitive realm, Nf1 function depends on the context of specific training conditions. Protein synthesis-independent short- and middle-term memories appear to require an activation of Rut-AC by Nf1, whereas protein synthesis-dependent long-term memory requires an additional modulation of Ras activity. By rescuing the performance of homozygous mutants, this study has demonstrated that expression of Nf1 in adult α/β mushroom body neurons is sufficient to support all forms of Nf1-dependent memory. A requirement was revealed for Nf1 during acquisition. Together, thee observations expand current understanding of Nf1 and Rut-AC functions and challenge current models of mushroom body neuron activity in olfactory memory formation (Buchanan, 2010).

Rut-AC is required in both α/β and γ mushroom body neurons for complete rescue of rut STM deficits (Akalal, 2006), yet this study has shown that Nf1 is required only in the α/β mushroom body neurons. It is unclear why Rut-AC activation would only require Nf1 in one subset of neurons. One possibility is that the Nf1 stimulation of Rut-AC in α/β neurons during acquisition may indirectly facilitate, through unknown signals, Rut-AC activity in the γ neurons. Prior results have been interpreted to suggest that there are communication loops that exist between certain types of mushroom body neurons, and with extrinsic mushroom body neurons for normal learning and consolidation. A similar process could allow Rut-AC activation in γ neurons to be indirectly dependent upon Nf1 in α/β mushroom body neurons. Alternatively, it could be that the Rut-AC is dependent upon Nf1 in the α/β mushroom body neurons for its role in learning but Nf1-independent in γ mushroom body neurons (Buchanan, 2010).

For rescue of protein synthesis-dependent long-term memory (PSD-LTM), both Rut-AC and Nf1 expression are required only in α/β neurons, suggesting that their interaction is necessary to support this form of memory as well. A recent study concluded that the Nf1-GAP-related domain, which has been shown to mediate an adenylyl cyclase activity (Hannan, 2006), is necessary and sufficient for Nf1-dependent LTM (Ho, 2007). In contrast to Rut-AC, this adenylyl cyclase activity is stimulated by Ras and is Gα_s-independent. It is important to note, however, that the Nf1-GRD domain only partially rescued the LTM phenotype of nf1 mutants. Full rescue of LTM required a full-length nf1 transgene. Together, these data and and the current study suggest that Nf1 simultaneously mediates the activation of both AC signaling pathways in α/β neurons to facilitate new protein synthesis and the formation of long-lasting memory (Buchanan, 2010).

Early work on the role of Rut-AC in olfactory associative memory suggested that this adenylyl cyclase plays a role in behavioral acquisition. Using an olfactory avoidance assay, it was suggested that rutabaga mutants could obtain normal performance with more intense training. A delay was observed in the acquisition of olfactory memory in rutabaga mutants, which require 3 times the amount of training as controls to overcome. A similar delay in acquisition was discovered for nf1 mutants, consistent with the hypothesis that Nf1 is required for G-protein activation of Rut-AC during learning. The results also demonstrate that Rut-AC is essential for the stability of olfactory memory. However, this function is independent of an interaction with Nf1. It is believed that the association of Nf1 and Rut-AC may be transient, only required for the initial activation of Rut-AC in its role as a molecular coincidence detector in α/β neurons (Tomchik, 2009). If this model were true, memory stability would therefore require continued stimulation of Rut-AC molecules via an independent and perhaps spatially distinct mechanism that does not require Nf1 (Buchanan, 2010).

Recent efforts have attempted to assign temporal and operational phases of olfactory memory processing to distinct regions within the adult olfactory system. Upon pairing of odor and electric shock, new projection neuron synapses are recruited to the odor representatio. Pairing dopamine application with neuronal depolarization in adult brain preparations results in a Rut-AC-dependent synergistic increase of cAMP in both α and α’ lobes (Tomchik, 2009), and memory acquisition requires synaptic transmission from α’/β’ neurons. Although it was demonstrated that both Nf1 and Rut-AC are required for memory acquisition, neither of these need be expressed in α'/β' neurons. It is therefore proposed that memory acquisition cannot be thought of as a specific event involving a distinct neuronal subset. Rather, a model is envisioned in which the pairing of odor and electric shock induces a change on the neuronal systems level. Each individual neuron subset may register this change in a different way, but every change is in some way necessary for memory acquisition as a whole. Additional work will be required to determine whether memory consolidation, retrieval, or processing of longer-term memories also require plasticity throughout the entire olfactory system (Buchanan, 2010).

It is clear from the data herein that Nf1 function is required in the adult brain, in α/β neurons defined by the c739-gal4 driver, for PSI-EM formation and for PSD-LTM formation. By identifying a minimal region in which Nf1 expression is required, it is now possible to isolate its role in memory formation from others that may occur in the brain. This mapping promises a more accurate analysis of Nf1-dependent memory and insights into both memory processing as a whole and into the cognitive deficits associated with Neurofibromatosis Type 1 (Buchanan, 2010).

Plasticity of local GABAergic interneurons drives olfactory habituation

Despite its ubiquity and significance, behavioral habituation is poorly understood in terms of the underlying neural circuit mechanisms. This study presents evidence that habituation arises from potentiation of inhibitory transmission within a circuit motif commonly repeated in the nervous system. In Drosophila, prior odorant exposure results in a selective reduction of response to this odorant. Both short-term (STH) and long-term (LTH) forms of olfactory habituation require function of the rutabaga-encoded adenylate cyclase in multiglomerular local interneurons (LNs) that mediate GABAergic inhibition in the antennal lobe; LTH additionally requires function of the cAMP response element-binding protein (CREB2) transcription factor in LNs. The odorant selectivity of STH and LTH is mirrored by requirement for NMDA receptors and GABA_A receptors in odorant-selective, glomerulus-specific projection neurons (PNs). The need for the vesicular glutamate transporter in LNs indicates that a subset of these GABAergic neurons also releases glutamate. LTH is associated with a reduction of odorant-evoked calcium fluxes in PNs as well as growth of the respective odorant-responsive glomeruli. These cellular changes use similar mechanisms to those required for behavioral habituation. Taken together with the observation that enhancement of GABAergic transmission is sufficient to attenuate olfactory behavior, these data indicate that habituation arises from glomerulus-selective potentiation of inhibitory synapses in the antennal lobe. It is suggested that similar circuit mechanisms may operate in other species and sensory systems (Das, 2011)

A key observation is that rut function is uniquely required in adult-stage GABAergic local interneurons for STH and LTH. This observation contrasts with the rut requirement in mushroom-body neurons for olfactory aversive memory. The demonstration of fundamentally different neural mechanisms used in olfactory habituation and olfactory-associative memory elegantly refutes a proposal of the Rescorla-Wagner model that habituation (and extinction) may be no more than associations made with an unconditioned stimulus of zero intensity (Das, 2011)

The requirement for rut in inhibitory LNs also indicates that intrinsic properties of multiglomerular LNs change during habituation. However, logic, as well as anatomical and functional imaging data, indicate that glomerulus-selective plasticity must be necessary if LN changes produce odorant-selective habituation. A potentially simple mechanism for glomerulus-specific potentiation of LN terminals is suggested by the specific requirement for postsynaptic NMDAR in odorant-responsive glomeruli (Das, 2011)

The observation that LTH and STH show similar dependence on rut, NMDAR, VGLUT, GABAA receptors, and transmitter release from LN1 cells indicates a substantially shared circuit mechanism for the two timescales of habituation. The data point to a model in which transient facilitation of GABAergic synapses underlies STH; long-lasting potentiation of these synapses through CREB and synaptic growth-dependent processes underlies LTH. This finding differs in three ways from synaptic facilitation that underlies Aplysia sensitization. First, it refers to inhibitory synapses, with potentiation that may involve a specific heterosynaptic mechanism similar to that used for inhibitory Long Term Potentiation (iLTP) in the rodent ventral tegmentum. Second, by presenting evidence for necessary glutamate corelease from GABAergic neurons, it proposes the involvement of a relatively recently discovered synaptic mechanism for plasticity. Third, it posits an in vivo mechanism to enable glomerulus- specific plasticity of LN terminals (Das, 2011)

It is pleasing that, in all instances tested, physiological and structural plasticity induced by 4-d odorant exposure requires the same mechanisms required for behavioral LTH. When taken together, these different lines of experimental evidence come close to establishing a causal connection between behavioral habituation and accompanying synaptic plasticity in the antennal lobe (Das, 2011)

It is important to acknowledge that, although the current experiments show that plasticity of LN-PN synapses contributes substantially to the process of behavioral habituation, it remains possible that plasticity of other synapses, such as of recently identified excitatory inputs made onto inhibitory LNs, also accompany and contribute to olfactory habituation (Das, 2011)

The conserved organization of olfactory systems suggests that mechanisms of olfactory STH and LTH could be conserved across species. Although this prediction remains poorly tested, early observations indicate that a form of pheromonal habituation in rodents, termed the Bruce effect, may arise from enhanced inhibitory feedback onto mitral cells in the vomeronasal organ (Das, 2011)

Less obviously, two features of the circuit mechanism that we describe suggest that it is scalable and generalizable. First, selective strengthening of inhibitory transmission onto active glomeruli can be used to selectively dampen either uniglomerular (CO2) or multiglomerular (EB) responses; thus, the mechanism is scalable. Second, the antennal lobe/olfactory bulb uses a circuit motif commonly repeated throughout the brain, in which an excitatory principal cell activates not only a downstream neuron but also local inhibitory interneurons, which among other things, limit principal cell excitation (Das, 2011)

It is possible that, in nonolfactory regions of the brains, a sustained pattern of principal neuron activity induced by a prolonged, unreinforced stimulus could similarly result in the specific potentiation of local inhibition onto these principal neurons. Subsequently, the pattern of principal cell activity induced by a second exposure to a now familiar stimulus would be selectively gated such that it would create only weak activation of downstream neurons. In this manner, the circuit model that is proposed for olfactory habituation could be theoretically generalized. More studies are expected to test the biological validity of this observation (Das, 2011)

Distinct molecular underpinnings of Drosophila olfactory trace conditioning

Trace conditioning is valued as a simple experimental model to assess how the brain associates events that are discrete in time. This study adapted an olfactory trace conditioning procedure in Drosophila by training fruit flies to avoid an odor that is followed by foot shock many seconds later. The molecular underpinnings of the learning are distinct from the well-characterized simultaneous conditioning, where odor and punishment temporally overlap. First, Rutabaga adenylyl cyclase (Rut-AC), a putative molecular coincidence detector vital for simultaneous conditioning, is dispensable in trace conditioning. Second, dominant-negative Rac expression, thought to sustain early labile memory, significantly enhances learning of trace conditioning, but leaves simultaneous conditioning unaffected. It was further shown that targeting Rac inhibition to the mushroom body (MB) but not the antennal lobe (AL) suffices to achieve the enhancement effect. Moreover, the absence of trace conditioning learning in D1 dopamine receptor mutants is rescued by restoration of expression specifically in the adult MB. These results suggest the MB as a crucial neuroanatomical locus for trace conditioning, which may harbor a Rac activity-sensitive olfactory 'sensory buffer' that later converges with the punishment signal carried by dopamine signaling. The distinct molecular signature of trace conditioning revealed in this study should contribute to the understanding of how the brain overcomes a temporal gap in potentially related events (Shuai, 2011).

In trace conditioning, the conditional stimulus (CS) and the unconditional stimulus (US) are separated in time by a stimulus- free interval. This so-called 'trace interval' can last for a fraction of a second in eyeblink conditioning but many seconds in fear conditioning, which poses a challenging question: how does the brain overcome this temporal gap to form the association between the CS and US? Intriguingly, trace conditioning in mammals engages neural substrates fundamentally different from delay conditioning, where the CS precedes but also temporally overlaps with the US. Early evidence comes from lesion studies with experimental animals showing that acquisition of trace conditioning requires intact hippocampal formation and medial prefrontal cortex, whereas delay conditioning can occur even with the entire forebrain removed. Later studies involving human subjects further validate the involvement of different brain circuits in these two conditioning variants and even suggest, more surprisingly, that conscious awareness might be a prerequisite for trace but not delay conditioning. It is then hypothesized that the participation of hippocampus and neocortex, as well as the associated higher cognitive function, is necessary in trace conditioning to maintain a representation of the CS or CS/US contingency so as to bridge the temporal gap. However, little is known about what form this representation takes and how it eventually converges with the US (Shuai, 2011 and references therein).

This study characterized trace conditioning in the fruit fly and used mutant analyses to show that it is distinct from the well-characterized simultaneous conditioning at the molecular level. These data complement the mammalian circuit-level studies and, more importantly, open up a molecular understanding of the internal trace that the brain uses to bridge the temporal gap (Shuai, 2011).

Odor footshock pairing elicits robust learning in fruit flies. The current study adapted this assay to study trace conditioning simply by modifying the timing relationship between the CS+ odor and the US punishment. To mimic the widely used simultaneous conditioning paradigm, CS^- presentation is kept at 45 s after the punishment. Single-trial training is sufficient to elicit considerable learning performance; the learning index for OCT and 4-methycyclohexanol (MCH) is ~35 for trace conditioning at a trace interval of 30 s. Although a portion of the score (~10) might be attributed to attraction to the CS^- via backward conditioning, the behavioral results clearly indicate a marked ability of fruit flies to associate events that are temporally discrete (Shuai, 2011).

One remarkable finding of the current study is that flies devoid of Rut-AC perform normally in trace conditioning. This result is interesting in view of the belief that dually regulated adenylyl cyclase plays a central role in invertebrate associative learning. The function of Rut-AC is best described as a molecular coincidence detector that is synergistically activated by the CS-evoked calcium entry and the US-evoked G protein-coupled receptor activation. It has been hypothesized that the stimulus-free gap in trace conditioning can be bridged by the temporal integration property of Rut-AC. However, the current results disagree with this hypothesis. The normal or even higher performance of rut-deficient mutants suggests that CS-US association in trace conditioning may recruit separate molecular machineries or occur in a distinct group of neurons. Also pertinent to this study is that cAMP levels in the prefrontal cortex negatively influence working memory performance. Therefore, whereas cAMP signaling is essential for some learning tasks, it is dispensable or even detrimental for others (Shuai, 2011).

Another intriguing finding is that induced expression of dominant-negative Rac enhances the learning of trace but not simultaneous conditioning. Notably, no learning enhancement was observed in a number of simultaneous conditioning variants with altered training parameters, including lowered odor concentration and conditioned intensity discrimination in the current work, as well as reduced shock pulses and lowered shock voltage in a previous report. Thus, the differential effects are not explained by a ceiling effect or other ancillary factors. Trace conditioning testing was performed almost immediately (within 3 min) after the training, rendering a better retention of the acquired associative memory also unlikely. Trace conditioning becomes less efficient as trace interval increases, indicating that an inner trace of the odor gradually degrades with time. It is therefore speculated that inhibition of Rac activity might preserve this transient 'sensory buffer' so as to facilitate trace conditioning. In the learning of simultaneous conditioning, the co-occurrence of odor and shock makes it possible to process the CS and US information automatically, e.g., via simple convergence on coincidence detection molecules like Rut-AC; hence the requirement of an olfactory sensory buffer is superfluous, which explains the lack of enhancement from Rac inhibition. The above speculation is particularly attractive considering a recently established role of Rac in the forgetting of a cold-shock sensitive early associative memory. It appears that the perdurance of two short-lived memory forms, one registered after a passive olfactory experience and lasting tens of seconds and the other registered after an associative reinforcement and lasting several hours, are both sensitive to Rac signaling manipulation (Shuai, 2011).

Drac1(N17) takes effect in the MB, the center for olfactory learning and sensory integration in insects. The localization of the Drac1 (N17) effect, combined with the full rescue of the dDA1 mutant phenotype in the MB, implies a possible trace conditioning model in which the MB bridges the temporal gap by holding a short-term sensory buffer of the odor, which later converges with the reinforcement signal carried by dopamine signaling. In accordance with this model, two recent studies in fruit fly and honey bee found no correlation between trace conditioning behavior and the postodor calcium response patterns in olfactory sensory neurons and projection neurons of the AL. Both studies pointed out the likelihood that the sensory buffer relevant to trace conditioning is in neurons downstream of the AL, most likely in the MB. Nonetheless, the AL may still retain odor information in biochemical signals other than calcium or in shortterm synaptic plasticityThe rapidly evolving molecular imaging techniques in fruit flies may help to delineate the nature of the putative sensory buffer and how it interacts later with a biologically significant stimulus (Shuai, 2011).

Another remaining puzzle is that both simultaneous and trace conditioning, although recruiting different molecular mechanisms, rely on the MB as a mutual crucial site. This seems at variance with the view from mammalian studies, where trace conditioning recruits neural circuits distinct from delay conditioning. Species or paradigm differences might explain the discrepancy, but it awaits to be fully addressed by future studies exploring whether brain regions outside the MB are additionally engaged in trace conditioning in fruit flies and, more importantly, whether various MB subdivisions contribute differentially to these two conditioning variants (Shuai, 2011).

G(o) activation is required for both appetitive and aversive memory acquisition in Drosophila

Heterotrimeric G(o) is an abundant brain protein required for negatively reinforced short-term associative olfactory memory in Drosophila. G(o) is the only known substrate of the S1 subunit of pertussis toxin (PTX) in fly, and acute expression of PTX within the mushroom body neurons (MB) induces a reversible deficit in associative olfactory memory. This study demonstrates that the induction of PTX within the α/β and γ lobe MB neurons leads to impaired memory acquisition without affecting memory stability. The induction of PTX within these MB neurons also leads to a significant defect in an optimized positively reinforced short-term memory paradigm; however, this PTX-induced learning deficit is noticeably less severe than found with the negatively reinforced paradigm. Both negatively and positively reinforced memory phenotypes are rescued by the constitutive expression of G(o)α transgenes bearing the Cys(351)Ile mutation. Since this mutation renders the G(o) molecule insensitive to PTX, the results isolate the effect of PTX on both forms of olfactory associative learning to the inhibition of the G(o) activation (Madalan, 2011).

The acute expression of PTX within the α/β and γ lobe neurons defined by the P247 driver is sufficient to inhibit both aversive and appetitive short-term olfactory memories. It was further shown through transgenic rescue experiments that the PTX inhibition of both aversive and appetitive short-term memories requires the G(o)α _Cys351 ADP-ribosylation site. PTX will only ribosylate heterotrimers (not individual α subunits), and the consequence of this ribosylation is inhibition of the heterotrimer activation. The inhibition of G(o) signaling by PTX is, therefore, extremely specific; since the ADP-ribosylated G(o) heterotrimers cannot be activated, they do not generate ectopic Gβ/γ subunits, nor do they sequester free Gβ/γ subunits away from other Gα subunits. Hence, the PTX loss-of-learning phenotype and the rescue of this deficit with the expression of G(o)α_Cys351Iso demonstrates that G(o) activation is required within the mushroom body neurons for the formation of short-term olfactory associative memories (Madalan, 2011).

Since anatomically distinct regions of the mushroom bodies have distinct roles in associative memory acquisition, stabilization, and recall, the identification of neurons that require G(o) activation provides important insight into the function of this signaling pathway during memory formation. Previously, it was found that PTX would partially affect negatively reinforced learning when expression was limited to either the α/β or γ mushroom body neurons, but when expressed in both subpopulations of neurons, as defined by the P247 and c772 Gal4 lines, it would almost completely eliminate memory formation. In contrast, the inhibition of G(o) activation within the α/β core neurons or within the DPM neurons had no effect on aversive memories. This studt further delineated the G(o) requirements during memory formation by excluding α'/β' lobe neurons. The DPM and α'/β' neurons are likely involved in a recurrent circuit that during both appetitive and aversive memory acquisition supports the consolidation of memories within the α/β lobe neurons of the mushroom bodies. The activation of G(o) is, therefore, required for aversive memory formation outside of the acquisition and stabilization events that occur within this α'/β'-DPM neuron circuit (Madalan, 2011).

These data highlight potential functions for this G(o) activation in memory formation. The stability of negatively reinforced olfactory memories appeared unaffected by the inhibition of G(o) activation, which suggests that G(o) activation is required during memory formation but not subsequently. In this last experiment, however, possible effects on memory stability may be hidden by the low performance found in flies expressing PTX within their α/β and γ lobe neurons and in the control flies trained with a single CS-US pairing. G(o) activation is also unlikely to be required for the initial encoding of CS strength or identity within the α/β and γ lobe neurons but may be involved in processing the electric shock or in subsequent memory formation processes. This latter conjecture is based partially on the fact that overtraining in the negatively reinforced learning paradigm will not compensate for the learning defect as would be expected if PTX reduced the salience of the odor stimuli. Moreover, the inhibition of G(o) activation differentially affected performance in the appetitive and aversive paradigms, which is again inconsistent with a change in odor strength or identity. The lower performance asymptote found in the acquisition curve after G(o) inhibition could, however, reflect a defect in processing the electric shock, in memory formation, or in reinforcement events acting downstream from this step, as well as in multiple processes. Although transducing the signal for the electric shock within the α/β and γ lobe neurons could account for the severely reduced learning found with PTX expression in both neuron types, it would fail to account for the phenotype found in appetitive memory. G(o) may also be required for a process shared by both appetitive and aversive memory systems that provides more general information about properties of the reinforcement, such as its predictive value, and, as such, could influence memory strength (Madalan, 2011).

The receptor(s) responsible for activating G(o) during memory formation and the downstream effectors are currently unknown. G(o) signaling pathways in vertebrates are better studied than in Drosophila and can offer insight into possible pathways for activation and effectors during Drosophila associative memory formation. G(o) is an extremely abundant membrane protein in the vertebrate brain, comprising between 1%ñ2% of total membrane protein, suggesting a common role in neural signal transduction. In vertebrate cells, G(o) is typically activated by GPCRs but can also be activated by non-GPCR receptors such as Amyloid Precursor Protein and GAP-43. The GPCRs that activate G(o) are typically of the G(i/o) coupled family and will generally inhibit neural activity; these include opioid receptors, different neuropeptide receptors, subtypes of mGluR receptors, and GABA_B receptors. However, the specificity of GPCR coupling to specific classes of G proteins is not absolute and may strongly depend on the cellular context of the GPCRs. The promiscuous coupling of GPCRs may also be regulated through post-transcriptional modification. For example, the palmitoylation of the Endothelin_B receptor results in a shift in coupling from G(i) to G(q), and the phosphorylation of the β2-adrengenic receptor by PKA leads to a G(s) to G(i) shift in coupling. Hence, it is not possible to a priori eliminate any GPCR from consideration based on receptor type and preferred coupling. Given this stipulation, one possible candidate for G(o) activating GPCRs within the α/β and γ lobe neurons is the dDA1 receptor. The dDA1 dopamine receptor is prominently expressed in the α/β and γ lobe neurons of the mushroom bodies where G(o) activation is required. Moreover, similar to the effects of PTX, dDA1 mutants display severe impairments in aversive memory and less severe impairments in appetitive memory. However, dDA1, which can activate cAMP synthesis, has not been shown capable of coupling to G(o) (Madalan, 2011).

Few G(o) effectors have been demonstrated in vertebrates, and in most cases, it is the βγ subunits that are responsible for actuating signaling. Presynaptic voltage-gated Ca²⁺ channels represent a major effector for G(o). G-protein βγ-subunits inhibit N-type (Ca_v2.2) and P/Q-type (Ca_v2.1) presynaptic Ca²⁺ channels involved in neurotransmitter release, causing a positive shift in their voltage dependence of activation. The inhibition is lifted by high-frequency action potentials. N-type channels also undergo a voltage-independent inhibition that is mediated by the direct binding of G(o)α to the α_1B subunit, resulting in an inhibition of Ca²⁺ current even after strong depolarization. These effects of activated G(o) on the presynaptic voltage-gated Ca²⁺ channels were described in cultured sensory neurons from embryonic chick dorsal root ganglion and set in motion by noradrenaline (NA), γ-aminobutyric acid (GABA), serotonin (5-HT), enkephalin, and somatostatin GPCRs. This voltage-gated Ca²⁺ channels effector pathway for G(o) is also present in central neurons, e.g., in Purkinje cerebellar neurons, elicited through GABA_B receptors, and in sympathetic neurons, e.g., effected through presynaptic β2-adrenergic autoreceptors, adenosine A1 receptors, and E2 (PGE2). The Drosophila cacophony voltage-gated Ca²⁺ channel α subunit may be a target for G(o) subunits during memory formation (Madalan, 2011 and references therein).

Pheromone binding to the VR2 receptors in rodent vomeronasal organs activates G(o), liberating Gβ/γ, which then activates phospholipase Cβ to mediate pheromone signal transduction. In Drosophila, plcβ21 is coexpressed with G(o)α in essentially the entire nervous system. Recently, Dahdal et al. found that Plcβ21 is an effector for G(o) in the Drosophila LN_vs neurons. Hence, the loss of Plcβ21 activation within the α/β or γ lobe neurons may account for the loss of short-term memory found after PTX expression (Madalan, 2011).

Lastly, the activation of adenylyl cyclase is an important G-protein signaling pathway involved in associative memory formation. In vertebrates, G(o) does not appear to signal through adenylyl cyclase. In Drosophila, G(o) signaling within the mushroom body neurons during memory formation is, at least partially, if not wholly, independent of the rut adenylyl cyclase. This conclusion was based on several considerations including the significantly stronger phenotype of PTX inhibition as compared to rut mutants. Additionally, PTX expression within the α/β lobe neurons will inhibit short-term memory, whereas rut activity within these neurons is not necessary for negatively reinforced short-term memory. Moreover, when PTX was lightly induced in rut²⁰⁸⁰ homozygotes or heterozygotes, PTX displayed additivity in the short-term memory phenotype. When these PTX- rut²⁰⁸⁰ 'double-mutant' experiments were performed, rut²⁰⁸⁰ was reported to be an amorph or, at least, a severe-hypomorph; more recent data indicates that rut mRNA levels are at ~25% wild-type levels in the rut²⁰⁸⁰ allele. Hence, it remains possible that the additivity found between PTX expression and the rut²⁰⁸⁰ may be due to residual activity in the rut²⁰⁸⁰ allele. Nevertheless, the learning phenotype for rut²⁰⁸⁰ is as strong as the reported amorphic rut¹ allele, and it even displays haploinsufficiency, arguing that a high level of the enzyme is required to support memory formation. This, together with the strength of the phenotypic differences and the differences in anatomical requirements for G(o) activation and rut, argues against a direct interaction between the cAMP pathway and G(o) activation during negatively reinforced memory formation. In positively reinforced memory, rut is required in the α'/β' neurons and the projection neurons of the antennal lobe. Thus, the role for G(o) activation in positively reinforced memory also maps outside the rut domains and, therefore, is also rut-independent (Madalan, 2011).

In summary, the activation of G(o) is an essential signaling event for associative memory formation. The G(o) pathway is required for the formation of both appetitive and aversive memories within the α/β and γ lobe neurons. Further elucidation of this pathway within these neurons will likely provide fundamental information on the molecular events underlying memory formation (Madalan, 2011).

Altered gene regulation and synaptic morphology in Drosophila learning and memory mutants

Genetic studies in Drosophila have revealed two separable long-term memory pathways defined as anesthesia-resistant memory (ARM) and long-lasting long-term memory (LLTM). ARM is disrupted in

radish (rsh) mutants, whereas LLTM requires CREB-dependent protein synthesis. Although the downstream effectors of ARM and LLTM are distinct, pathways leading to these forms of memory may share the cAMP cascade critical for associative learning. Dunce, which encodes a cAMP-specific phosphodiesterase, and rutabaga, which encodes an adenylyl cyclase, both disrupt short-term memory. Amnesiac encodes a pituitary adenylyl cyclase-activating peptide homolog and is required for middle-term memory. This study demonstrates that the Radish protein localizes to the cytoplasm and nucleus and is a PKA phosphorylation target in vitro. To characterize how these plasticity pathways may manifest at the synaptic level, synaptic connectivity was assayed and an expression analysis was performed to detect altered transcriptional networks in rutabaga, dunce, amnesiac, and radish mutants. All four mutants disrupt specific aspects of synaptic connectivity at larval neuromuscular junctions (NMJs). Genome-wide DNA microarray analysis revealed approximately 375 transcripts that are altered in these mutants, suggesting defects in multiple neuronal signaling pathways. In particular, the transcriptional target Lapsyn, which encodes a leucine-rich repeat cell adhesion protein, localizes to synapses and regulates synaptic growth. This analysis provides insights into the Radish-dependent ARM pathway and novel transcriptional targets that may contribute to memory processing in Drosophila (Guan, 2011).

Drosophila has proven to be a powerful model for identifying gene products involved in learning and memory based on olfactory, visual, and courtship behavioral assays. How proteins identified in these studies regulate neuronal function or physiology to specifically alter behavioral plasticity is an ongoing area of investigation. Using the well-characterized 3rd instar larval NMJ as a model glutamatergic synapse, the effects on synaptic connectivity were compared of several learning mutants that alter cAMP signaling (dnc¹, rut¹, amn¹) with the poorly characterized ARM mutant rsh¹. Each mutant altered synaptic connectivity at NMJs in a specific manner, suggesting that changes in neuronal connectivity in the CNS might contribute to the behavioral defects found in these strains. The observations in dnc¹ and rut¹ are similar to previous studies of synaptic morphology in these mutants. Gene expression was assayed in the mutants using microarray analysis, which revealed many neuronal transcripts that were transcriptionally altered. A long-term goal is to link transcriptional changes in specific loci to the behavioral and morphological defects found in learning and memory mutants (Guan, 2011).

Experimental approaches to define the biochemical transition from short-term plasticity to long-term memory storage have suggested a key role for cAMP signaling. At the molecular level, one of the best-characterized pathways for STM has been described for gill withdrawal reflex facilitation in Aplysia. In this system, conditioned stimuli act through a serotonergic G protein-coupled receptor pathway to activate adenylyl cyclase in the presynaptic sensory neuron, resulting in the synthesis of cAMP. cAMP activates PKA, which phosphorylates a presynaptic potassium channe, leading to prolonged calcium influx and enhanced neurotransmitter release from the sensory neuron. Insights into the LLTM pathway in Aplysia have implicated CREB function. Robust training or stimulation with serotonin induces translocation of the catalytic subunit of PKA into the nucleus, where it activates the transcription factor CREB-1 and inhibits the transcriptional suppressor CREB-2. CREB-1 acts on additional transcription factors to produce specific mRNAs that are transported to dendrites and captured by activated synapses. Local synthesis of new proteins and subsequent growth of synaptic connections is predicted to underlie long-term memory in the system. It is likely that similar molecular pathways exist in other species. Transgenic Drosophila with inducible inhibition of PKA show memory impairment. PKA is also activated during hippocampal LTP induction in mammals, and transgenic mice that express an inhibitor of PKA have defective LTP and hippocampal-dependent memory, suggesting a general role for cAMP/PKA in the transition from learning to memory storage (Guan, 2011).

In addition to CREB-dependent LLTM, which requires transcription and translation for its formation, the Radish-dependent ARM pathway represents a distinct long-term memory storage mechanism. These various memory pathways partially overlap in time. Three hours after training ~50% of memory is stored as STM, with the rest present as ARM, which is formed immediately after training in flies and can last for days depending on training intensity. ARM is not blocked by agents that disrupt electrical activity in the brain, suggesting that a biochemical pathway for ARM is likely initiated by learning stimuli, but does not require continued neuronal excitation for its expression. ARM is also not as sensitive to translation inhibition, as a 50% reduction of protein synthesis by cycloheximide does not affect ARM, but blocks LLTM (Guan, 2011).

Similar to the role of CREB in LLTM, Radish appears to be a key regulator of the ARM phase of memory. In contrast to the molecular pathways underlying STM (cAMP/PKA cascade) and LLTM (PKA/CREB), the signaling mechanisms mediating ARM are unknown. Unfortunately, the amino acid sequence of the radish locus gives little insight into its function, as it lacks known structural motifs or domains. Radish contains a serine/arginine-rich sequence with very limited homology to splicing factors, hinting that it may be involved in RNA processing. The Radish protein also contains PKA phosphorylation sites and multiple NLS sites within its sequence. Consistent with these sequence features, This study found that Radish is phosphorylated by PKA in vitro, linking ARM to the cAMP/PKA pathway. By generating a GFP-tagged Radish transgenic animal, it was possible to characterize Radish localization. Radish was prominently localized to cell bodies of neurons in the CNS, but was enriched in the nucleus in other cell types such as salivary gland and muscle cells. Given the overlap between several of the NLS and PKA sites in Radish, it will be interesting to explore whether the phosphorylation state of Radish regulates its subcellular distribution. An attractive hypothesis is that activated PKA phosphorylates Radish at synapses, resulting in transport to the nucleus with accompanying effects on transcription or RNA processing that would modify long-term synaptic function. Given that ARM can last for days, a change in nuclear function is an attractive biological underpinning, even though ARM has been suggested to be a translation-independent form of memory. Given that general protein synthesis was reduced by only 50% in the previous studies, it is quite possible that ARM and LLTM have different thresholds for translational inhibition (Guan, 2011).

In terms of synaptic modifications in rsh¹ mutants, this study found that larval NMJ synapses were altered compared with controls. Specifically, rsh¹ mutants had shorter axonal projections onto target muscles and displayed more synaptic boutons within the innervated region. These alterations gave rise to a more compact innervation pattern than observed in controls. Overgrowth of synapses at larval NMJs was also observed in dnc¹ mutants, whereas reduced innervation length was found in rut¹ mutants. As such, rsh¹ mutant NMJs display a unique phenotype compared with mutants that increase or decrease cAMP levels. The molecular mechanisms by which Radish regulates synaptic growth are unclear. Radish could directly interface with growth regulators at the synapse in a PKA-dependent fashion. Indeed, an interaction between Radish and Rac1 was found in a high-throughput yeast two-hybrid screen for interacting Drosophila proteins. Rac1 is a Rho family GTPase that regulates neuronal and synaptic morphology via reorganization of the cytoskeleton. Rac1 function has also been linked to PAK1 and the Fragile-X Mental Retardation protein (FMRP), which alter synaptic and behavioral plasticity in mammals. Recently, Rac activity has been linked to memory decay in Drosophila (Shuai. 2010), indicating that a Radish-Rac link might control memory processing via alterations in cytoskeletal modulation of synaptic function or stability. Although it is possible that Radish regulates synaptic properties through a Rac1 interaction, no robust Rac1-Radish interaction was observed in either yeast-two hybrid or GST pull-down experiments. No Radish-GFP enrichment was observed at larval synapses where the synaptic growth defect was quantified, although the protein was present in larval axons. As such, it may be that NMJ defects in rsh¹ arise through downstream effects secondary to the loss of Radish function in a neuronal compartment besides the synapse (Guan, 2011).

To further explore this possibility and examine links between rsh and the STM pathway, genome-wide microarray studies were performed on several learning and memory mutants. Although there were some shared transcriptional changes between rsh¹ and the other mutants (dnc¹, rut¹, amn¹), most of the changes in rsh¹ were unique. Although linking these changes to a direct effect on the underlying biology will require more work, several interesting loci were identified that could contribute to synaptic plasticity defects. The Drosophila NFAT homolog, a transcription factor that binds to the activity-regulated AP-1 (Fos/Jun) dimer, was robustly up-regulated by sevenfold in rsh¹ mutants. The RNA-binding protein smooth (sm) was also up-regulated in rsh¹ mutants. Mutations in sm have been shown to alter axonal pathfinding. Other genes that were transcriptionally altered in rsh¹ mutants and that would be predicted to influence synaptic connectivity were the Sh potassium channel, the adapter protein Disabled, and the Lapsyn cell adhesion protein. The potential role of Lapsyn was intriguing, as LRR-containing proteins have been implicated in the regulation of neurite outgrowth and synapse formation. In particular, netrin-G ligand and synaptic-like adhesion molecule (SALM) are known LRR proteins that regulate neuronal connectivity and synapse formation. In Drosophila, LRR repeat proteins have been implicated in motor neuron target selection. Given the roles of other LRR-containing proteins in the regulation of neuronal connectivity, this study explored whether Lapsyn might also function in this pathway. Lapsyn was up-regulated by neuronal activity in addition to being up-regulated in rsh¹, making it an interesting transcriptional target to assay for a role in synaptic modification (Guan, 2011).

Lapsyn mRNA expression was broadly up-regulated in the brain by neuronal activity, suggesting a potential widespread effect on neuronal function. Lapsyn-GFP transgenic protein targeted to the presynaptic terminal, partially overlapping with the periactive zone, a region of the nerve terminal enriched in proteins that regulate synaptic vesicle endocytosis and synaptic connectivity. Animals lacking Lapsyn died at the end of embryogenesis, although the early stages of nervous system formation appeared normal. It was possible to partially rescue Lapsyn mutants with neuronal expression of a Lapsyn transgene, indicating an essential function for the protein in the nervous system. Rescue to adulthood required expression outside the nervous system, suggesting Lapysn is likely to have functions in other tissue types as well. Manipulations of Lapsyn expression in the nervous system resulted in distinct defects in synaptic connectivity at the NMJ. Heterozygotes expressing only a single copy of the Lapsyn gene displayed supernumerary satellite bouton formation, a phenotype commonly associated with mutants that disrupt synaptic endocytosis or that alter the transmission or trafficking of synaptic growth factors through the endosomal system. This increase in satellite boutons in Lapsyn heterozygotes suggests that the protein plays a role in the regulation of synaptic growth signaling. Overexpression of Lapsyn, as induced by activity or observed in rsh¹ mutants, also elicited a change in synaptic growth, resulting in an increase in overall bouton number at larval NMJs. Thus, regulation of Lapsyn levels modulate synaptic growth mechanisms at NMJs. Lapsyn mutant heterozygotes also display defects in larval associative learning, although this phenotype could not be rescued with pan-neuronal overexpression. The lack of a specific rescue makes it unclear whether the learning defects are linked to a non-Lapsyn function, or if a more specific spatial and temporal expression of Lapsyn is required for functional rescue (Guan, 2011).

How Lapsyn participates in synaptic signaling is currently unclear. The closest mammalian homologs of Lapsyn are the NGL family of synaptic adhesion molecules. Three isoforms are found in mammals, NGL-1, NGL-2, and NGL-3, which interact with netrin-G1, netrin-G2, and the receptor tyrosine phosphatase LAR, respectively. NGL-1 promotes axonal outgrowth, whereas NGL-2 is capable of triggering synapse formation. The interaction of NGL-3 with LAR is intriguing, as the Drosophila LAR homolog has been shown to bind the heparan sulfate proteoglycans Syndecan and Dallylike to regulate synaptic growth at the NMJ. The homology between Lapsyn and the mammalian NLG family is restricted to the extracellular LRR domain, with no homology observed in the intracellular C terminus. The three mammalian NLGs also lack homology to each other at the C terminus, except for the presence of a PDZ-binding domain at the end of the intracellular domain. It will be important to identify binding partners for Lapsyn at the synapse to define how it may regulate synaptic adhesion or signaling between the pre- and postsynaptic compartments to regulate synaptic growth. Likewise, additional studies into the Radish-dependent ARM phase of memory may reveal how rsh-dependent changes in Lapsyn levels contribute to the synaptic and behavioral defects of this memory mutant (Guan, 2011).

Cyclic adenosine monophosphate metabolism in synaptic growth, strength, and precision: neural and behavioral phenotype-specific counterbalancing effects between dnc phosphodiesterase and rut adenylyl cyclase mutations

Two classic learning mutants in Drosophila, rutabaga (rut) and dunce (dnc), are defective in cyclic adenosine monophosphate (cAMP) synthesis and degradation, respectively, exhibiting a variety of neuronal and behavioral defects. This study asked how the opposing effects of these mutations on cAMP levels modify subsets of phenotypes, and whether any specific phenotypes could be ameliorated by biochemical counter balancing effects in dnc rut double mutants. This study at larval neuromuscular junctions (NMJs) demonstrates that dnc mutations caused severe defects in nerve terminal morphology, characterized by unusually large synaptic boutons and aberrant innervation patterns. Interestingly, a counterbalancing effect led to rescue of the aberrant innervation patterns but the enlarged boutons in dnc rut double mutant remained as extreme as those in dnc. In contrast to dnc, rut mutations strongly affect synaptic transmission. Focal loose-patch recording data accumulated over 4 years suggest that synaptic currents in rut boutons were characterized by unusually large temporal dispersion and a seasonal variation in the amount of transmitter release, with diminished synaptic currents in summer months. Experiments with different rearing temperatures revealed that high temperature (29-30°C) decreased synaptic transmission in rut, but did not alter dnc and wild-type (WT). Importantly, the large temporal dispersion and abnormal temperature dependence of synaptic transmission, characteristic of rut, still persisted in dnc rut double mutants. To interpret these results in a proper perspective, previously documented differential effects of dnc and rut mutations and their genetic interactions in double mutants on a variety of physiological and behavioral phenotypes were reviewed. The cases of rescue in double mutants are associated with gradual developmental and maintenance processes whereas many behavioral and physiological manifestations on faster time scales could not be rescued. Factors that could contribute to the effectiveness of counterbalancing interactions between dnc and rut mutations for phenotypic rescue are discussed (Ueda, 2012).

It has been demonstrated that cAMP levels are decreased in rut. The results clearly contrast the differential effects of disruptions in synthesis and degradation of cAMP on synaptic function and nerve terminal morphology. Mutations in dnc, including dnc¹, dnc^M11, and dnc^M14, can lead to severe defects in nerve terminal branching and bouton morphology. Aside from this study, previous reports have documented in identified larval muscles that total bouton numbers and motor terminal branching pattern are severely affected by dnc, but these defects were not detected in rut. A similar situation has been reported in the adult CNS: axon terminal growth in the mushroom body is enhanced in dnc but is not affected in rut. In contrast, rut and dnc mutations both have clear effects on synaptic transmission but in distinct manners. Increased cAMP levels in dnc could enhance transmitter release (as indicated by increased ejp sizes with a minimal disturbance in the temporal precision of the release process. In comparison, rut mutations more severely disrupt temporal control of release, regardless of the rearing temperature. In addition, the rearing temperature affects the amplitude of synaptic transmission in rut, with strongly depressed transmission at high temperature. This likely reflects a decrease in vesicle release because the miniature ejp size was unaltered at different temperatures (data not shown) (Ueda, 2012).

A number of mutant alleles of the rut gene have been described in the literature of developmental studies, but the alleles frequently used in neurogenetic experiments are limited to rut¹, rut², rut³, rut¹⁰⁸⁴, and rut²⁰⁸⁰. Furthermore, only three mutant alleles have been biochemically characterized in Drosophila: rut¹, rut², and rut³. It should be mentioned that these rut mutations can cause significant decrease in total cAMP synthesis despite the fact that there are at least four adenylyl cyclase (AC) homologous genes that have been identified molecularly and biochemically in Drosophila. This raises the possibility that rut may represent a major AC gene but all AC genes may play differential roles in regulating cAMP levels, depending on their subcellular localization and conditions to activate their actions. As demonstrated in this study as well as in earlier reports, a general pattern of relative severity among several rut mutant alleles is observed across different phenotypes, as represented in the following sequence: rut¹ = rut¹⁰⁸⁴ = rut² = rut³ = WT (Ueda, 2012).

Compared to the AC genes, there appears to be fewer PDE homologous genes and only two genes are known for their cAMP degradation action besides dnc. However, dnc gene products are represented by more than 10 splicing variants as opposed to 2 rut splicing variants. There are a large number of dnc mutant alleles reported in literature but only a small number of them are frequently used in neurogenetic studies, i.e., dnc¹, dnc², dnc^M11, and dnc^M14. Interestingly, a consistent pattern of phenotypic severity can be observed across different phenotypes among these four alleles: dnc^M11 = dnc^M14 = dnc¹ = dnc² (Ueda, 2012).

A comparison of their effects on a variety of phenotypes includes PDE enzyme activity disruption, defective growth cone motility of cultured neurons, enhanced growth of larval NMJ, enhanced K ⁺ and Ca^{2 +} currents in larval muscles, decrease in the larval motor neuron firing frequency upon depolarization, increase in whole-cell ejps or ejcs, and decrease in activity-dependent facilitation of synaptic transmission at larval NMJ, decrease in the habituation rate of olfactory jump response and odor-electric shock association in adult flies, and female sterility. In a different approach, overexpression of a UAS-dnc ⁺ transgene in motor neurons results in reduced NMJ growth and decreased ejp size even in larvae reared at room temperature. These phenotypes demonstrated the effects of increased cAMP degradation in contrast to those caused by dnc mutations (Ueda, 2012).

When considering their mechanisms of action, several reported phenotypic effects of dnc alleles may be complicated by the implications of contributions from the genetic background. Notably, the dnc^M11 mutant line has been reported to affect protein kinase C (PKC) activity in addition to PDE. In addition, the severity of dnc¹ may in fact be more extreme than reported, since dnc¹ has been shown to be female sterile once a second-site mutation near the dnc locus is removed from the original fertile line. It is possible that many dnc¹ lines used in neurogenetic investigations contain this mutation in the background (Ueda, 2012).

A number of experimental paradigms have been used to characterize behavioral and physiological phenotypes of dnc and rut mutants with defined quantitative parameters. For a majority of phenotypes examined, dnc and rut mutations do not lead to opposite effects on these quantitative indices, even though they alter the cAMP levels in opposite directions. Only for certain phenotypes, the dnc and rut mutations affect the parameters in opposite directions. For example, in larval neuromuscular synaptic boutons, mobilization of synaptic vesicles from the reserve pool to exo/endo cycling pool is suppressed in rut and enhanced in dnc. Similarly, the number of docked vesicles at synapses is decreased in rut and increased in dnc. Ca^{2 +} current measured in larval muscles is decreased in rut and increased in dnc. Hyperexcitability-induced overgrowth of larval NMJ can be suppressed by rut but enhanced by dnc. Similarly, dnc and rut mutations exert opposite effects on Kenyon cell axon counts in the mushroom body of developing adult flies. Finally, habituation rate of the giant fiber escape circuit is decreased by rut and increased by dnc (Ueda, 2012).

In contrast, for some other phenotypes, rut alleles have no apparent effects while dnc mutants display clear alterations. For instance, the larval NMJ terminal projection pattern and adult mushroom body axonal terminal growth were altered in dnc but not in rut. Moreover, identified K ⁺ currents in larval muscles are increased in dnc but unaltered in rut. In these cases, increased cAMP levels can produce abnormalities but underlying mechanisms may be tolerant to depleted cAMP levels (Ueda, 2012).

For another group of phenotypes, dnc and rut mutations can affect separate parameters and sometimes produce superficially similar effects by altering a parameter in the same direction. Such cases include decreased growth cone motility, irregular action potential firing pattern, and modified intracellular Ca^{2 +} dynamics in cultured neurons. In larval neuromuscular junctions, both dnc and rut decrease synchronicity of synaptic transmitter release and presynaptic facilitation of neuromuscular transmission. During post-eclosion development of adult flies, both dnc and rut mutations enhance the axon terminal growth of mechanosensory cells and decrease the structural and functional adaptation of the olfactory system to odor exposure. Neither dnc nor rut mutants respond to environmental or social deprivation in modifying Kenyon cell axon counts of young adults. Mutations of either dnc or rut decreases habituation rate of the proboscis extension reflex and olfactory avoidance and jump response, and the performance indices of both classical and operant conditioning. Studies on alcohol response have demonstrated increased sensitivity in rut alleles but no apparent change in dnc alleles. Although it is reassuring to observe opposite effects of dnc and rut mutations on some of the quantitative parameters, it should be noted that it is not straightforward in associating most of the indicators with the defective mechanisms directly regulated by cAMP signaling. Dysfunction in AC and PDE may exert opposite effects on some cell biological mechanisms or neural circuit components but can still lead to apparently similar deficiencies of a cellular function or behavioral task (Ueda, 2012).

Some insights may be gained through examining the genetic interactions between dnc and rut in double mutants about how rut AC and dnc PDE are involved in particular aspects of physiological or behavioral plasticity. At the present time, only a limited number of reports document the resultant phenotypes in dnc rut double mutants. Significantly, the majority of the single-mutant phenotypes of dnc or rut mutations do not become less severe in dnc rut double mutants, even though the overall cAMP levels are largely restored. The phenotypes that are not rescued in double mutants include increased bouton size in dnc and impaired synchronicity of transmitter release in larval NMJs, irregular firing of cultured neurons, and habituation and olfactory associative learning of adult flies (Ueda, 2012).

However, a few cases of successful rescue in double mutants have been described. Decreased growth cone motility in dnc and rut neurons in culture can be restored by combining two mutations and the overgrowth and altered projection patterns of dnc larval motor terminals is suppressed in dnc rut. Interestingly, none of the above cases of successful rescue involve opposite effects of dnc and rut single-mutant phenotypes. Notably, both cases of restoration involve a particular allele, rut¹. The allele rut¹ is different from other alleles with characterized AC enzyme activity (rut² and rut³) in that the Ca^{2 +} /CaM-dependent activation of AC is eliminated in rut¹ flies, but retained in rut² and rut³. Unlike rut¹, the allele rut² is not able to rescue the dnc mutational effects of enhanced larval NMJ growth and irregular firing in cultured neurons. In the present study of NMJ focal recording, it was clears that rut² did not affect the precision in release timing (ejc peak time) and ejc amplitudes, although rut¹ decreased the temporal precision of release (increased variability in ejc peak time) and the ejc amplitude significantly. It will be helpful if further experiments are performed on additional allele combinations of dnc and rut to delineate the role of Ca^{2 +} -dependent regulation of AC in specific phenotypes of interest (Ueda, 2012).

In addition to peculiarities of enzymatic properties in mutant alleles, e.g., rut¹ AC devoid of Ca^{2 +} /calmodulin (CaM) sensitivity, other factors influencing interactions between dnc and rut must be considered. As summarized above, counterbalancing rescue of dnc and rut phenotypes in double mutants is likely to be exceptions rather than a general rule. Therefore, it would be desirable to identify the conditions and factors that could facilitate their counterbalancing interactions, which may provide insights into the orchestration of dnc PDE and rut AC underlying the phenotype of interest (Ueda, 2012).

First, it is important to consider the temporal and spatial characteristics of expression and operation of these enzymes. In the temporal domain, their effects on a variety of phenotypes are mediated through integration among different biochemical pathways and cellular processes, some of which may function with rapid kinetics, whereas others may represents slow accumulation of products through a number of steps. Some of the resultant phenotypes may require continuous adjustment in response to internal or environmental conditions while others may appear relatively permanent and irreversible, possibly associated with developmental events (Ueda, 2012).

The spatial factors to be considered include the cellular expression and subcellular localization of the enzymes. To the best of our knowledge, there is little information about whether dnc PDE and rut AC are colocalized in molecular assemblies or aggregates within certain functional domains in specific neuronal cell types. Close proximity of AC and PDE localization facilitates local regulation of cAMP levels within a short time. Certain cellular processes with slower kinetic steps also facilitate integration of dnc and rut interactions, extending their balancing acts to a broader spatial range (Ueda, 2012).

For the few examples of successful counterbalancing rescue, growth cone motility seems to be a continuous adjustment by cAMP on a time scale of tens of seconds to minutes. This relatively slow kinetics makes it possible to readily manipulate the cAMP signaling pathway, e.g., bath application of db-cAMP increases rut growth cones motility, mimicking dnc counter balancing effects in double-mutant growth cones. Some developmental or maintenance processes, such as axonal path finding, branch formation, target interaction, and synaptogenesis, are also slow adjustment processes (in the order of hours to days). In these cases, restoration of cAMP levels through long-range interactions of AC and PDE may be sufficient to rescue the single-mutant phenotype. For example, dnc defects in larval motor terminal growth are suppressed by rut¹ (Ueda, 2012).

In contrast, defects in some physiological properties (K ⁺ currents, neuronal firing, and transmitter release timing) and behavioral conditioning (habituation and classical conditioning) cannot be rescued by combining dnc and rut, which sometimes leads to even more extreme deficiencies, e.g., the extremely rapid habituation in dnc rut. One possibility is the requirement of dynamic cAMP regulation within a short time period (millisecond to second range) during which a counterbalancing act is difficult to achieve. Another possible explanation is the requirement of unimpaired cellular machinery laid down during development (e.g., proper channel and receptor localization) and functional connectivity among synaptic partners (inhibitory and excitatory elements in the circuit) underlying behavioral phenotypes under consideration. Deviation from coordinated actions of such subcellular machinery or circuit components will make it difficult to obtain compensatory rescue (Ueda, 2012).

It should be noted that well-defined abnormalities in central fiber projection have been reported in dnc and rut single mutants that reflect the alterations in peripheral motor terminals in larval NMJs. Furthermore, dnc PDE and rut AC are preferentially expressed in mushroom bodies, which are important in odor-associated learning. Therefore, it is reasonable to speculate that defects in higher functions, including classical associative learning and habituation, may involve anatomical defects in the CNS, such as altered dendritic arbors and synaptic connections detectable in certain defined circuits, in addition to potential changes in synaptic physiology (Ueda, 2012).

Cell-specific expression and subcellular localization of AC and PDE isoforms may affect dnc and rut single-mutant, as well as double-mutant phenotypes. These include splicing variants of the dnc and rut gene as well as the products of their homologous genes. Such complexity needs to be considered in the interpretation of dnc and rut interactions in order to appreciate contributions of individual splicing variants and to delineate influence from their homologous genes (Ueda, 2012).

Finally, cross-talk between the cAMP and other signaling pathways can also modify dnc and rut phenotypes. For example, variety of signalling pathways are known to converge onto the CREB transcription factor. It is also established that not only the cAMP cascade but also other signaling pathways, including PKG and CaMKII, can modify larval NMJ physiology and morphology as well as adult habituation, courtship conditioning and classical conditioning. It will be of particular interest to establish the consequences of such genetic interactions across signaling pathways. Double mutant analysis in conjunction with transgenic and genomic approaches remains a powerful and profitable direction for revealing the genetic network underlying neural and behavioral plasticity (Ueda, 2012).

Gamma neurons mediate dopaminergic input during aversive olfactory memory formation in Drosophila

Mushroom body (MB)-dependent olfactory learning in Drosophila provides a powerful model to investigate memory mechanisms. MBs integrate olfactory conditioned stimulus (CS) inputs with neuromodulatory reinforcement (unconditioned stimuli, US), which for aversive learning is thought to rely on dopaminergic (DA) signaling to DopR, a D1-like dopamine receptor expressed in MBs. A wealth of evidence suggests the conclusion that parallel and independent signaling occurs downstream of DopR within two MB neuron cell types, with each supporting half of memory performance. For instance, expression of the Rutabaga (Rut) adenylyl cyclase in γ neurons is sufficient to restore normal learning to rut mutants, whereas expression of Neurofibromatosis 1 (NF1) in α/β neurons is sufficient to rescue NF1 mutants. DopR mutations are the only case where memory performance is fully eliminated, consistent with the hypothesis that DopR receives the US inputs for both γ and α/β lobe traces. This study demonstrates, however, that DopR expression in γ neurons is sufficient to fully support short- and long-term memory. It is argued that DA-mediated CS-US association is formed in γ neurons followed by communication between γ and α/β neurons to drive consolidation (Qin, 2012).

Because DopR is thought to mediate the US information, identification of the spatial requirements of this receptor pinpoints the initial site of CS-US coincidence detection. To date, most genetic and circuit manipulations suggest that olfactory memory performance at a given retention interval can be dissected into distinct and independently disruptable mechanisms acting in parallel in distinct neuronal cell types. For example, the STM defects of rut and NF1 can be rescued with expression in γ for rut and β/γ neurons for NF1. Experimental dissections of the circuits required for LTM have suggested a major role for β/γ neurons as well as for ellipsoid body (eb) and DAL neurons. Such findings have been interpreted as supporting the idea of independent signaling for parallel memory traces as well as sequential action in different cell types to support a single memory mechanism. The current findings demonstrate that DopR expression in MBs is sufficient to support both rut-dependent and rut-independent forms of CS-US association leading to STM, as well as to consolidated ARM and LTM. This conclusion also generalizes to three different combinations among five different odors, providing strong evidence that the functional distinctions between KC classes are not artifacts caused by differences in the population of neurons involved in coding each odor percept. With each of these odor combinations and memory phases, there also was no case where expression in α/β or α'/β' populations was sufficient or necessary to provide substantial rescue of dumb2 (a piggyBac insertion in the first intron of the DopR locus) mutants (Qin, 2012).

Together, this set of findings pinpoints the DopR-mediated inputs for STM, MTM, ARM, and LTM to the γ neuron population of MB KCs. This conclusion is consistent with findings from previous attempts to map the subset of DA neurons that convey the US to MBs using either inhibition or activation of neural transmission to block or mimic the US signal. In these studies, the largest magnitude effects were seen with stimulation of MB-MP1, a neuron in the PPL1 cluster of DA neurons (although it should be noted that smaller magnitude effects also were seen for several other DA cell types), which is sufficient to substitute for the US. Although inhibition of MB-MP1 neurons has not been demonstrated to block learning, these DA neurons likely participate in mediating at least a portion of the US stimulus for aversive conditioning. MB-MP1 neurons project to the base of the peduncle, occupied by the axons of α/β neurons and the heel of the MB, which is comprised largely of γ neurons. As an independent validation of the hypothesis that these MB-MP1 neurons provide direct input to γ neurons, the GFP reconstituted across the synapse (GRASP) method was used to visualize putative synaptic connections in the heel between these two cell types (Qin, 2012).

The fact that γ lobe expression of DopR is sufficient to restore not only STM but also both ARM and LTM is noteworthy. Previous attempts to map the neural circuits for olfactory memory have revealed roles for α/β lobes in particular for consolidated memory. Because massed and spaced training experiments consist of repetitive training rather than the single training trial used for STM and MTM, differences in circuit requirements could in principle derive from training paradigm-dependent differences in the CS-US association circuit, as appears to be true for appetitive reinforcement. But this appears not to be the case for DopR function in aversive reinforcement, because full rescue of these consolidated forms of memory were obtained with γ lobe expression of DopR (Qin, 2012).

How can this conclusion be reconciled with the requirement for downstream signaling molecules within α/β lobe neuron, as well as in downstream eb neurons and dorsal-anterior-lateral (DAL) neurons? Three possible explanations are seen, that are not mutually exclusive. First, it is possible that US information is deconstructed into more than one pathway, mediated by different receptors. These could include additional DA receptors, or other neurotransmitter systems such as serotonin. It is worth noting that DA inputs to MBs also have been implicated in hunger/satiety modulation of appetitive memory retrieval, and DopR signaling also has been implicated in several forms of arousal that in principle could represent a component of the reinforcement signal that could be separate from a more specific perceptual representation of the shock experience. The findings nevertheless lead to the conclusion that any additional US information depends critically on DopR-mediated DA signaling in the γ lobe population of neurons. A second possibility worth considering stems from the finding that output from α/β lobe, eb, and DAL neurons are each required for retrieval depending on the retention interval measured. Thus a model cannot be formally ruled out in which all of the functional impacts of various manipulations of α/β lobe derive from defects in retrieval. This would be difficult to fathom for cases such as NF1 rescue of STM and Rut function for LTM, but in principle this interpretation is possible. The third possibility is that consolidation of the γ lobe CS-US association involves signaling within α/β lobe neurons, as well as in downstream eb neurons and DAL neurons. Such a model predicts communication between the γ lobe and the rest of MBs during training and/or afterward (Qin, 2012).

Suppression of inhibitory GABAergic transmission by cAMP signaling pathway: alterations in learning and memory mutants

The cAMP signaling pathway mediates synaptic plasticity and is essential for memory formation in both vertebrates and invertebrates. In the fruit fly Drosophila melanogaster, mutations in the cAMP pathway lead to impaired olfactory learning. These mutant genes are preferentially expressed in the mushroom body (MB), an anatomical structure essential for learning. While cAMP-mediated synaptic plasticity is known to be involved in facilitation at the excitatory synapses, little is known about its function in GABAergic synaptic plasticity and learning. Using whole-cell patch-clamp techniques on Drosophila primary neuronal cultures, this study demonstrates that focal application of an adenylate cyclase activator forskolin (FSK) suppressed inhibitory GABAergic postsynaptic currents (IPSCs). A dual regulatory role of FSK on GABAergic transmission was observed, where it increases overall excitability at GABAergic synapses, while simultaneously acting on postsynaptic GABA receptors to suppress GABAergic IPSCs. Further, it was shown that cAMP decreased GABAergic IPSCs in a PKA-dependent manner through a postsynaptic mechanism. PKA acts through the modulation of ionotropic GABA receptor sensitivity to the neurotransmitter GABA. This regulation of GABAergic IPSCs is altered in the cAMP pathway and short-term memory mutants dunce and rutabaga, with both showing altered GABA receptor sensitivity. Interestingly, this effect is also conserved in the MB neurons of both these mutants. Thus, this study suggests that alterations in cAMP-mediated GABAergic plasticity, particularly in the MB neurons of cAMP mutants, account for their defects in olfactory learning (Ganguly, 2013).

Ca2+/CaM dependent adenylate cyclase (AC) produces cAMP and is also known to function as a co-incidence detector during learning in both Drosophila and Aplysia. In addition, AC-dependent cAMP activation changes the strength of Drosophila excitatory synapses which may be the cellular mechanism underlying learning and memory. Although inhibitory synaptic transmission is equally important for proper neuronal communication, the effects of cAMP at the inhibitory GABAergic synapses have remained unexplored. This study shows that forskolin (FSK), an activator of cAMP, suppresses the frequency of inhibitory GABAergic IPSCs in Drosophila primary neuronal cultures. A concentration dependent effect of FSK on GABAergic IPSCs was observed in the same physiological range as described in recent imaging studies in intact fly brains. Further cAMP was shown to decrease GABAergic IPSCs in a PKA-dependent manner through a postsynaptic mechanism (Ganguly, 2013).

Sparsening of odor representation through GABAergic inhibition in the mushroom body (MB) neurons is thought to be a possible mechanism for information storage in locusts (Perez-Orive, 2002). GABAergic local neurons are known to be involved in olfactory information processing in Drosophila (Wilson, 2005; Olsen, 2008) indicating that GABAergic transmission plays a crucial role in shaping odor response. The MB shows extensive GABAergic innervation in both locusts (Perez-Orive, 2002) and Drosophila (Yasuyama, 2002). This, along with the observation that cAMP pathway genes like dunce and rutabaga are preferentially expressed in the MB (Davis, 2011), indicates that cAMP mediated GABAergic plasticity may be important for learning in Drosophila. Consistent with this hypothesis, this study observed altered cAMP mediated GABAergic IPSCs in the cAMP mutants dnc1 and rut1. The effect of cAMP on suppression of GABAergic currents was less pronounced in the mutants. This suggests that the altered inhibition contributes to their observed learning defects. In fact, recent studies have shown that GABAA RDL receptors expressed in the MB and GABAergic neurons projecting to the MB are essential for olfactory learning (Liu, 2007; Liu, 2009). It is thus possible that altered cAMP mediated GABAergic plasticity at the MB neurons may account for some forms of the learning defects in Drosophila (Ganguly, 2013).

GABAergic IPSCs are known to act through picrotoxin-sensitive postsynaptic GABA receptors in both Drosophila embryonic and pupal neuronal cultures. This study observed that the suppression of GABAergic IPSCs by FSK is completely abolished in the presence of a membrane impermeable PKA inhibitor restricted to the postsynaptic neuron. This indicates that PKA may modulate GABAergic IPSCs by regulating GABA receptor sensitivity by phosphorylation, similar to what has been suggested in the mammalian hippocampus (Ganguly, 2013).

There are three known ionotrophic GABA receptor gene homologs in Drosophila - RDL, LCCH3 and GRD. Amongst them, the GABA RDL subunit is widely expressed in several regions of the Drosophila brain and its expression in the MB is inversely correlated to olfactory learning. Therefore, RDL-containing GABA receptors may play an important role in cAMP-dependent synaptic plasticity and thus be involved in learning and memory. The data suggests that the majority of synaptic GABA receptors contain the RDL subunit while a small fraction of synaptic GABA receptors lack RDL, providing evidence of heterogeneous synaptic GABA receptors in Drosophila for the first time. However, it is still not known what particular subunit of GABA receptors is involved in regulation of cAMP-dependent GABAergic plasticity. Based on the observation that RDL containing GABA receptors mediate the majority of GABAergic IPSCs in Drosophila primary neuronal cultures, the action of FSK on GABAergic IPSCs is probably through the GABA RDL subunit. While the detailed molecular mechanism remains to be explored, it is proposed that PKA-mediated phosphorylation of RDL subunits and subsequent GABA receptor internalization may occur in the postsynaptic region. In this scenario, the only functional synaptic GABA receptors will be those lacking the RDL subunit at the postsynaptic regions. This will account for a decrease in mIPSC frequency with response to FSK, while leaving mIPSC amplitude almost unchanged (Ganguly, 2013).

Although GABA receptor subunits would be the target of cAMP-PKA signaling the possibility that other molecules can be phosphorylated and then indirectly regulate GABA receptor subunits can still not be rule out. Future work using heterologous expression systems for GABA subunits will help to determine whether GABA receptors are directly phosphorylated (Ganguly, 2013).

Recordings from embryonic and pupal MB neurons of both dunce and rutabaga mutants show a defect in ionotropic GABA receptor response in the presence of FSK. Interestingly, this response to FSK is similar in both the mutants despite their contrasting levels of cellular cAMP. Recent imaging studies in the rutabaga mutant have shown that AC is required for co-incidence detection in the MB neurons. FSK application also fails to increase PKA to wild-type levels in the MB neurons of rutabaga. Thus in the current experiments, the changes in receptor response in rutabaga can be explained by a lack of increase in cAMP/PKA levels due to defects in FSK-mediated AC activation (Ganguly, 2013).

The dunce mutants with high levels of cAMP also show defects in short-term memory due to alterations in the spatiotemporal restriction of dunce phosphodiesterase to the Drosophila MB. Further, the dunce MB neurons show an increase in PKA levels on FSK application similar to the wild-type strains. These findings suggest that FSK-mediated inhibition of GABA receptor should be greater in dunce neurons. However, in the current results the dunce and rutabaga mutants, despite having opposing effects on cellular cAMP levels, showed very similar FSK mediated effects on GABAergic IPSCs. Several other studies have also shown that dunce and rutabaga have similar defects in growth cone motility, excitatory synaptic plasticity and more importantly, short-term memory. Even though the effect of FSK on GABAergic IPSCs in dunce and rutabaga mutants is similar, it is very likely that the molecular mechanisms underlying these responses differ in the two mutants. It has been shown that elevated cAMP signaling reduces phosphorylation in rat kidney cells through activation of protein phosphatase 2A. In addition, increased PKA activity in mouse hippocampus hyper-phosphorylates several downstream molecular targets including a tyrosine phosphatase (STEP), correlates with decreased phosphodiesterase protein (PDE4) levels and results in memory defects. Therefore, it is tempting to speculate that high levels of cAMP due to the dunce mutation leads to the activation of phosphatase(s) and thus reduces the effects of FSK as seen in the current study. Taken together, all these findings strongly suggest that the disruption of cellular cAMP homeostasis can alter inhibitory GABAergic synaptic plasticity and hence cause defects in olfactory learning, although the underlying mechanisms leading to this effect can be different (e.g. reduced PKA activity in rut1 versus increased phosphatase activity in dnc1) (Ganguly, 2013).

Strengthening in the efficacy of excitatory transmission underlies enhanced synaptic plasticity such as hippocampal long-term potentiation (LTP) and facilitation (LTF) in Aplysia. It is thus possible that the suppression of inhibitory transmission by a common second messenger like cAMP, which can enhance excitatory synaptic transmission, may lead to synaptic strengthening. Previous work has shown that the cAMP activator FSK increases excitability at the cholinergic synapses in Drosophila primary neuronal cultures. However the effect of FSK on other synapses like the GABAergic synapses has not been explored. This study shows that FSK elevates overall cellular excitability at GABAergic synapses as demonstrated by the increase in spontaneous AP frequency. Moreover, when PKA in the postsynaptic neuron is completely blocked by an inhibitor, an increase is seen in the frequency of GABAergic IPSCs. Together with previous studies on cholinergic synapses (Yuan, 2007), the current results indicate that FSK/cAMP act as common molecules regulating globally presynaptic excitability at both the cholinergic as well as GABAergic synapses. It is also noted that FSK inhibits the response of postsynaptic GABA receptors in a specific manner leading to a decrease in GABAergic synaptic strength. These studies demonstrate a novel dual regulatory role of cAMP by showing that it increases overall presynaptic function on one hand; and, acts specifically on postsynaptic GABA receptors to decrease GABAergic plasticity on the other. This action of cAMP could result in global increases in excitability and learning (Ganguly, 2013).

AKAPs act in a two-step mechanism of memory acquisition

Defining the molecular and neuronal basis of associative memories is based upon behavioral preparations that yield high performance due to selection of salient stimuli, strong reinforcement, and repeated conditioning trials. One of those preparations is the Drosophila aversive olfactory conditioning procedure where animals initiate multiple memory components after experience of a single cycle training procedure. This study explored the analysis of acquisition dynamics as a means to define memory components and revealed strong correlations between particular chronologies of shock impact and number experienced during the associative training situation and subsequent performance of conditioned avoidance. Analyzing acquisition dynamics in Drosophila memory mutants revealed that rutabaga (rut)-dependent cAMP signals couple in a divergent fashion for support of different memory components. In case of anesthesia-sensitive memory (ASM) this study identified a characteristic two-step mechanism that links rut-AC1 to A-kinase anchoring proteins (AKAP)-sequestered protein kinase A at the level of Kenyon cells, a recognized center of olfactory learning within the fly brain. It is proposed that integration of rut-derived cAMP signals at level of AKAPs might serve as counting register that accounts for the two-step mechanism of ASM acquisition (Scheunemann, 2013).

Conditioned odor avoidance is subject to a general dichotomy since multiple memory components are engaged in control of behavior. This is usually analyzed at two time points, i.e., 3 min and 3 h after training. At 3 min, basal and dynamic STM are separable by genetic means as revealed by opposing phenotypes of rut¹ and dnc¹ mutants. Moreover, those components are also separable due to characteristic differences in their acquisition dynamics as revealed by different effects of shock number. A similar dichotomy applied to 3 h memory when ASM and ARM were separable by means of amnestic treatment. It was striking that basal STM and consolidated ARM were instantaneously acquired, resulting in a front line of protection by eliciting conditioned avoidance after a singular experience of a CS/US pairing. Interestingly, consolidated ARM (as defined by means of resisting amnestic treatment) was installed quickly after training. However, it remains to be addressed at the genetic and molecular level, whether 3 h ARM linearly results from the functionally similar basal SMT component (Scheunemann, 2013).

In contrast, the two components of dynamic STM and labile ASM acquire in a dynamic fashion but are clearly dissociated from each other by characteristic chronologies of CS/US pairings required for their acquisition. However, either component contributes to behavioral performance in addition to the appropriate instantaneous component, and hence, increases avoidance probability during the test situation. Considering a potential benefit from avoiding aversive situations this overall dichotomy of behavioral control seems plausible and is also reflected at the genetic level since rut-dependent cAMP signals are limited to support of dynamic but not instantaneous memory components. Rut-dependent STM and ASM, however, are dissociated by means of shock impact and discontinuous formation of ASM is limited to situations where animals repeatedly experience high-impact CS/US pairings within a predefined time window. Experience that does not meet this criterion, however, is not discounted but adds to continuously acquired dynamic STM. By functional means these two components are thus clearly separated but commonly dependent on rut-derived cAMP signals within the KC layer, forming ties between genetically and functionally defined memory components (Scheunemann, 2013).

Genetic dissection of memory has a long-standing history in Drosophila and provided a powerful means to define molecular, cellular, and neuronal networks involved in regulation of conditioned odor avoidance. Among others, the cAMP-signaling cascade has been identified as one central tenet of aversive odor memory foremost by means of two single-gene mutants affecting either a Ca²⁺-sensitive type 1 adenylyl cyclase (AC1) and/or a cAMP-specific type 4 phosphodiesterase (PDE4) affected in the Drosophila learning mutants rutabaga (rut-AC1) and dunce (dnc-PDE4). Although originally isolated due to poor performance in the aversive odor learning paradigm, a general dichotomy has been recently revealed that separates memory components by their dependency on either rut-AC1 or dnc-PDE4 function, and the view was established that two different types of cAMP signals are engaged during the single-cycle training procedure (Scheunemann, 2012). A similar dichotomy is observed at level of acquisition dynamics and suggests that rut-dependent cAMP signals are limited to formation of dynamically acquired memory components, i.e., dynamic STM and ASM. Interestingly, rut-dependent cAMP is also required for long-term memory (LTM), which acquires after spaced and repeated training sessions. Downstream the signaling cascade, however, appropriate cAMP signals are differently channeled to either support LTM in a CREB-dependent manner, ASM via tomosyn-dependent plasticity, or basal STM via synapsin-dependent regulation of synaptic efficacy. It appears that the chronology of CS/US pairings is an important determinant of which downstream effect is triggered and hence molecular mechanisms must be installed that are sensitive to the temporal order of training (Scheunemann, 2013).

At the level of molecular scaffolds, literature suggests that AKAPs serve the integration of cAMP with other signaling processes and are crucially involved in the control of a plethora of cellular functions in any organ. For example, AKAP79 coordinates cAMP and Ca²⁺ signaling in neurons to control ion channel activities. The recognized design principle of AKAPs to serve as molecular switch is well in line with the recognized two-step register mechanism involved in ASM formation. An increasing body of evidence shows that AKAPs are involved in memory processing across phyla and accordantly those studies revealed a contribution for support of matured, but not immediate memories. Communality among all those AKAP-dependent memories is the need for repeated and temporally organized training sessions, i.e., only spaced training sessions are effective to induce protein synthesis-dependent LTM in flies and mammals. Similarly, ASM requires the precise timing of two training sessions and mechanistically acts via an 'activated' state generated by the initial CS/US pairing that persists within the brain for ~5 min. Such temporal integration might well take place at level of AKAPs within the KC layer to operate rut-AC1-dependent cAMP signals finally onto phosphorylation of tomosyn. Identification of the particular AKAPs involved in two-step ASM formation will require further analysis of appropriate mutants. To date, only four Drosophila AKAPs are characterized, i.e., rugose, a 550 kDa protein that impacts on STM performance probably via molecular domains other than its AKAP function; yu/spoonbill that supports LTM; and Nervy and AKAP²⁰⁰ have not been tested for their impact on aversive odor memory (Scheunemann, 2013).

Together, the benchmarking of Drosophila aversive odor memory performance by means of acquisition dynamics that were demonstrated in this study will provide a valuable tool since dynamic aspects of acquisition are obviously informative and add to the steady-state condition determined by the single-cycle training procedure (Scheunemann, 2013).

Novel role for ecdysone in Drosophila conditioned behavior: Linking GPCR-mediated non-canonical steroid action to cAMP signaling in the adult brain

DopEcR, a G-protein coupled receptor for ecdysteroids, is involved in activity- and experience-dependent plasticity of the adult central nervous system. Remarkably, a courtship memory defect in rutabaga (Ca²⁺/calmodulin-responsive adenylate cyclase) mutants is rescued by DopEcR overexpression or acute 20E feeding, whereas a memory defect in dunce (cAMP-specific phosphodiestrase) mutants is counteracted when a loss-of-function DopEcR mutation is introduced. A memory defect caused by suppressing dopamine synthesis is also restored through enhanced DopEcR-mediated ecdysone signaling, and rescue and phenocopy experiments revealed that the mushroom body (MB) - a brain region central to learning and memory in Drosophila - is critical for the DopEcR-dependent processing of courtship memory. Consistent with this finding, acute 20E feeding induced a rapid, DopEcR-dependent increase in cAMP levels in the MB. The multidisciplinary approach demonstrates that DopEcR mediates the non-canonical actions of 20E and rapidly modulates adult conditioned behavior through cAMP signaling, which is universally important for neural plasticity. This study provides novel insights into non-genomic actions of steroids, and opens a new avenue for genetic investigation into an underappreciated mechanism critical to behavioral control by steroids (Ishimoto, 2013).

Steroid hormones are essential modulators of a broad range of biological processes in a diversity of organisms across phyla. In the adult nervous system, the functions of steroids such as estrogens and glucocorticoids are of particular interest because they have significant effects on the resilience and adaptability of the brain, playing essential roles in endocrine regulation of behavior. Reflecting their importance in neural functions, steroid hormones are implicated in the etiology and pathophysiology of various neurological and psychiatric disorders, and are thus often targeted in therapies. The biological actions of steroids are mediated mainly by nuclear hormone receptors - a unique class of transcription factors that activate or repress target genes in a steroid-dependent manner. Substantial evidence suggests, however, that steroid hormones can also exert biological effects quickly and independently of transcriptional regulation, by modulating intracellular signaling pathways. Such 'non-genomic' effects might be induced by direct allosteric regulation of ion channels, including receptors for GABA and NMDA. Alternatively, in certain contexts, non-genomic steroid signaling could be mediated by classical nuclear hormone receptors acting as effector molecules in the cytosol (Ishimoto, 2013).

G-protein coupled receptors (GPCRs) that directly interact with steroids have the potential to play an important role in non-genomic steroid signaling. So far, however, only few GPCRs have been identified as bona fide steroid receptors in vertebrates. The G-protein coupled estrogen receptor 1 (GPER, formally known as GPR30) is the best studied GPCR that is responsive to steroids. Pharmacological and gene knockout approaches suggest that this protein has widespread roles in the reproductive, nervous, endocrine, immune and cardiovascular systems (Prossnitz, 2011). Although other G-protein coupled receptors were predicted to be responsive to steroids (e.g., the Gq-coupled membrane estrogen receptor and estrogen receptor-X), their molecular identity is not known (Qiu, 2006; ToranAllerand, 2002). Overall, the physiological roles of the GPCR-mediated actions of steroids and the underlying molecular mechanisms remain poorly understood, and sometimes controversial, in spite of their importance. In particular, it is unknown how this non-canonical steroid mechanism influences neural functions and complex behaviors (Ishimoto, 2013).

Drosophila genetics has been extensively used to study the roles and mechanisms of action of steroid hormones in vivo. The major steroid hormone in Drosophila is the molting hormone 20-hydroxy-ecdysone (20E), which orchestrates a wide array of developmental events, including embryogenesis, larval molting and metamorphosis. Recent studies revealed that 20E also plays important roles in adult flies, regulating: the innate immune response, stress resistance, longevity, the formation of long-term courtship memory and the active/resting state. In general, the functions of 20E during development and adulthood are thought to be executed by ecdysone receptors (EcRs), members of the evolutionarily conserved nuclear hormone receptor family (Ishimoto, 2013).

In addition to canonical ecdysone signaling via EcRs, Srivastava (2005) identified a novel GPCR called DopEcR, and showed that it propagates non-genomic ecdysone signaling in vitro. DopEcR shares a high level of amino-acid sequence similarity with vertebrate β-adrenergic receptors. In situ hybridization and microarray data revealed that DopEcR transcripts are preferentially expressed in the nervous system. In heterologous cell culture systems, DopEcR is localized to the plasma membrane and responds to dopamine as well as ecdysteroids (ecdysone and 20E), modulating multiple, intracellular signaling cascades (Srivastava, 2005). Furthermore, Inagaki (2012) recently detected DopEcR expression in the sugar-sensing gustatory neurons of adult flies, and showed that DopEcR-mediated dopaminergic signaling enhances the proboscis extension reflex during starvation. Nonetheless, little is known about whether DopEcR functions as a steroid receptor in vivo, and about how it drives responses in the central nervous system (CNS) to modulate complex behaviors. This study reports that DopEcR mediates non-genomic ecdysone signaling in the adult brain, and that it is critical for memory processing. It was also shown that, during memory processing, DopEcR transmits information via novel steroid signals that interact with the cAMP pathway, a signaling cascade that is universally important for neuronal and behavioral plasticity. This genetic study thus uncovers underappreciated GPCR-mediated functions and mechanisms of action that employ non-canonical steroid signaling to regulate the adult nervous system and, thereby, behavior (Ishimoto, 2013).

This study used genetic, pharmacological, and behavioral approaches in Drosophila to demonstrate that the steroid hormone 20E rapidly regulates behavioral plasticity via a non-genomic mechanism that is mediated by the GPCR-family protein DopEcR. This non-canonical steroid signaling pathway was found to have strong functional interactions with the classical 'memory genes' rut and dnc, which encode the central components of the cAMP pathway. The identification of 20E as an important modulator of cAMP signaling in the adult Drosophila brain reveals an unprecedented opportunity - that of taking advantage of fly genetics to dissect the molecular and cellular mechanisms responsible for the non-genomic steroid signaling that underlies neuronal and behavioral plasticity (Ishimoto, 2013).

Electrophysiological analyses revealed that the adult giant-fiber (GF) pathway of DopEcR mutant flies is more resistant to habituation than that of control flies. Direct excitation of GF or its downstream elements would lead to a short-latency response of the dorsal longitudinal flight muscle (DLM), which could follow high-frequency stimuli up to several hundred Hz. In contrast, the afferent input to the GF leads to a long-latency response that is labile and fails to follow repetitive stimulation well below 100 Hz and displays habituation even at 2-5 Hz. Although there is the possibility that DopEcR-positive thoracic neurons may modulate thoracic motor outputs and contribute to certain parameters of the habituation process not characterized in this study, the more effective modulation would occur in the more labile element afferent to the GF circuit rather than the robust GF-PSI-DLMn downstream pathway (PSI referring to peripherally synapsing interneuron), which is responsible for the reliability of the escape reflex. Thus, the mutant phenotype in habituation indicates that DopEcR positively controls activity-dependent suppression of neuronal circuits afferent to the GF neurons in the brain (Ishimoto, 2013).

Moreover, the finding that DopEcR and rut mutants have a similar GF habituation phenotype raises the possibility that DopEcR positively regulates cAMP levels in the relevant neurons following repetitive brain stimulation. Besides GF habituation, Drosophila displays olfactory habituation, which is mediated by the neural circuit in the antennal lobe. Interestingly, Das (2011) found that olfactory habituation is induced by enhancement of inhibitory GABAergic transmission, and that rut function is required for this neuronal modulation. Similar modulation of GABAergic transmission may also be responsible for habituation of the GF pathway. It will be interesting to examine whether and how DopEcR contributes to the regulation of rut and enhanced GABAergic transmission in GF habituation (Ishimoto, 2013).

Several studies already suggested that 20E has rapid, EcR-independent effects in Drosophila and other invertebrate species. For example, 20E was shown to reduce the amplitude of excitatory junction potentials at the dissected Drosophila larval neuromuscular junction (NMJ), and to do so within minutes of direct application (Ruffner, 1999). Whereas treatment with 20E did not change the size and shape of the synaptic currents generated by spontaneous release, it led to a reduction in the number of synaptic vesicles released by the motor nerve terminals following electrical stimulation. A similar effect of 20E was observed in crayfish, and it was suggested that the suppression of synaptic transmission by 20E may account for the quiescent behavior of molting insects and crustaceans. These observations suggested that 20E suppresses synaptic efficacy under certain conditions by modulating presynaptic physiology through a non-genomic mechanism. It is possible that such actions of 20E are mediated by DopEcR. To detail the mechanisms underlying DopEcR-dependent neural plasticity, it will be worthwhile to determine if and how DopEcR contributes to 20E-induced, rapid synaptic suppression at the physiologically accessible larval NMJ, and to determine the extent to which non-genomic mechanisms of steroid actions are shared between the larval NMJ and the adult brain (Ishimoto, 2013).

One surprising finding made in this study is that ecdysone signaling can modify the phenotypes associated with mutations in the classic 'memory genes', namely rut and dnc, through the actions of DopEcR. rut and dnc encode central components of the cAMP pathway, which is required for memory processing in vertebrates as well as invertebrates. The demonstration that genetically and/or pharmacologically enhancing DopEcR-mediated ecdysone signaling restores the courtship memory phenotype of loss-of-function rut mutants suggests that 20E-mediated DopEcR activation triggers an outcome similar to rut activation, i.e., increased cAMP levels. This assumption is supported by the finding that loss-of-function dnc mutants restore courtship memory when DopEcR activity is suppressed. A similar restoration of the dnc memory phenotype also occurs in a dnc and rut double mutant, again supporting the idea that DopEcR positively regulates cAMP production (Ishimoto, 2013).

The results of rescue and phenocopy experiments indicate that the MB is critical for the DopEcR-dependent processing of courtship memory. Although the endogenous expression pattern of DopEcR is not known, DopEcR is thus likely to modulate cAMP levels in the MB in response to 20E during courtship conditioning. A new Gal4 line has been generated in which a portion of the first coding exon of DopEcR is replaced with a DNA element that contains the Gal4 cDNA whose translation initiation codon is positioned exactly at the DopEcR translation start site. When this line was used to drive UAS-GFP, the reporter gene was widely expressed in the adult brain with prominent signals in the MB. This preliminary result strongly indicates the endogenous expression of DopEcR in the MB. It has also been directly shown that cAMP levels in the MB increase rapidly in flies fed 20E, and that this increase does not occur when DopEcR expression is down-regulated specifically in the MB. Taken together, these findings suggest that DopEcR expressed in the MB responds to 20E and acts upstream of cAMP signaling in a cell-autonomous manner (Ishimoto, 2013).

Surprisingly, enhancement of DopEcR-mediated ecdysone signaling restored courtship memory in flies harboring a strong hypomorphic allele of rut (rut¹⁰⁸⁴). A similar result was obtained even in mutants harboring a presumptive rut null allele rut¹. These results suggest that, upon stimulation by 20E, DopEcR may be able to signal via another adenylyl cyclase that can compensate for the lack of Rut. This interesting possibility requires further investigation (Ishimoto, 2013).

This study has focused on the roles and mechanisms of action of DopEcR-mediated, non-genomic ecdysone signaling. Since it has been found that 20E levels rise in the head during courtship conditioning (Ishimoto, 2009), the data presented in this study suggest that DopEcR is activated by 20E during conditioning, triggers a rise in cAMP levels and induces physiological changes that subsequently suppress courtship behavior. This interpretation assumes that 20E directly activates DopEcR to increase cAMP levels. Previous cell-culture studies suggested that DopEcR also responds to dopamine to modulate intracellular signaling (Srivastava, 2005). Furthermore, Inagaki (2012) has demonstrated that flies respond to starvation by sensitizing gustatory receptor neurons to sugar via dopamine/DopEcR signaling. It is therefore necessary to consider whether dopamine is directly involved in the processing of courtship memory through DopEcR. There is a possibility that 20E initially stimulates the production and/or release of dopamine, and that it in turn activates DopEcR and elevates cAMP levels to induce courtship memory. This possibility is thought unlikely because even when courtship memory is disrupted by pharmacological suppression of dopamine synthesis, 20E feeding can compensate for decreased dopamine and allow restoration of memory. Although dopamine plays a significant role in courtship memory, the results suggest that DopEcR does not act as the major dopamine receptor in this particular learning paradigm. The possibility is thus favored that dopamine contributes to courtship memory in parallel with, or upstream of, DopEcR-mediated ecdysone signaling. Consistent with this view, Keleman (2012) reported that the formation of courtship memory depends on the MB γ neurons, which express DopR1 dopamine receptors, receiving dopaminergic inputs. Notably, the current results indicate that the processing of courtship memory requires DopEcR expression in the αβ, but not γ, neurons of the MB, which makes it unlikely that DopEcR is directly influenced by the dopaminergic neurons innervating γ neurons (Ishimoto, 2013).

Ecdysone signaling through nuclear EcRs is necessary for forming long-term courtship memory that lasts at least 5 days, but appears not to have a significant effect on short-term courtship memory (Ishimoto, 2009). In contrast, we found that DopEcR-mediated ecdysone signaling is critical for habituation and 30-minute courtship memory. These findings suggest that DopEcR and EcRs control distinct physiological responses to courtship conditioning, and that the former regulates short-term memory, while the latter regulates long-term memory. Although non-genomic actions of steroid hormones have been implicated in vertebrate learning and memory, such actions have been attributed mainly to the classical nuclear hormone receptors that function outside of the nucleus and exert roles distinct from those of steroid-activated transcription factors. Although recent evidence has shown that membrane-bound receptors independent of the classical estrogen receptors are involved in estradiol-induced consolidation of hippocampal memory, the molecular identities of these proteins have not been established. The current findings in this study provide a novel framework for dissecting GPCR-mediated steroid signaling at the molecular and cellular levels. Furthermore, future analysis of the functional interplay between genomic and non-genomic steroid signaling pathways is expected to reveal novel mechanisms through which steroid hormones regulate plasticity of the nervous system and other biological phenomena (Ishimoto, 2013).

The neuro-ecology of Drosophila pupation behavior

Many species of Drosophila form conspecific pupa aggregations across the breeding sites. These aggregations could result from species-specific larval odor recognition. To test this hypothesis larval odors of D. melanogaster and D. pavani, two species that coexist in the nature, were tested. When stimulated by those odors, wild type and vestigial (vg) third-instar larvae of D. melanogaster pupated on conspecific larval odors, but individuals deficient in the expression of the odor co-receptor Orco randomly pupated across the substrate, indicating that in this species, olfaction plays a role in pupation site selection. Larvae are unable to learn but can smell, the Synapsin (Syn^97CS) and rut strains of D. melanogaster, did not respond to conspecific odors or D. pavani larval cues, and they randomly pupated across the substrate, suggesting that larval odor-based learning could influence the pupation site selection. Thus, Orco, Syn97CS and rut loci participated in the pupation site selection. When stimulated by conspecific and D. melanogaster larval cues, D. pavani larvae also pupated on conspecific odors. The larvae of D. gaucha, a sibling species of D. pavani, did not respond to D. melanogaster larval cues, pupating randomly across the substrate. In nature, D. gaucha is isolated from D. melanogaster. Interspecific hybrids, which result from crossing pavani female with gaucha males clumped their pupae similarly to D. pavani, but the behavior of gaucha female x pavani male hybrids was similar to D. gaucha parent. The two sibling species show substantial evolutionary divergence in organization and functioning of larval nervous system. D. melanogaster and D. pavani larvae extracted information about odor identities and the spatial location of congener and alien larvae to select pupation sites. It is hypothesized that larval recognition contributes to the cohabitation of species with similar ecologies, thus aiding the organization and persistence of Drosophila species guilds in the wild (Del Pino, 2014).

Metabolic learning and memory formation by the brain influence systemic metabolic homeostasis

Metabolic homeostasis is regulated by the brain, but whether this regulation involves learning and memory of metabolic information remains unexplored. This study use a calorie-based, taste-independent learning/memory paradigm to show that Drosophila form metabolic memories that help in balancing food choice with caloric intake; however, this metabolic learning or memory is lost under chronic high-calorie feeding. Loss of individual learning/memory-regulating genes causes a metabolic learning defect, leading to elevated trehalose and lipid levels. Importantly, this function of metabolic learning requires not only the mushroom body but also the hypothalamus-like pars intercerebralis, while NF-κB activation in the pars intercerebralis mimics chronic overnutrition in that it causes metabolic learning impairment and disorders. Finally, this study evaluated this concept of metabolic learning/memory in mice, suggesting that the hypothalamus is involved in a form of nutritional learning and memory, which is critical for determining resistance or susceptibility to obesity. In conclusion, these data indicate that the brain, and potentially the hypothalamus, direct metabolic learning and the formation of memories, which contribute to the control of systemic metabolic homeostasis (Zhang, 2015).

This work consists of a series of studies in Drosophila: these animals were found to temporarily develop metabolic learning to balance food choice with caloric intake. In Drosophila research, sugar has often been used for studying the appetitive reward value of food taste. Of interest, recent research has suggested that fruit flies can distinguish caloric values from the taste property of food. Using tasteless sorbitol as a carbohydrate source to generate an environmental condition that contained NC versus HC food, this study revealed that Drosophila can develop a form of metabolic learning and memory independently of taste, by which flies are guided to have a preference for normal caloric environment rather than high-caloric environment. However, this form of metabolic memory does not seem robust, as it is vulnerably diminished under genetic or environmental influences. It is postulated that this vulnerability to overnutrition is particularly prominent for mammals (such as C57BL/6J mice), and overnutritional reward-induced excess in caloric intake can quickly become dominant. This effect can be consistently induced in Drosophila when learning/memory-regulating genes are inhibited in the brain or the hypothalamus-like PI region. It was observed that each of these genetic disruptions led to impaired metabolic learning, resulting in increased caloric intake and, on a chronic basis, the development of lipid excess and diabetes-like phenotype. Indeed, it has been documented that chronic high-sugar feeding is sufficient to cause insulin resistance, obesity and diabetes in Drosophila. It is yet unclear whether this metabolic learning can induce an appetitive memory of normal caloric environment or an aversive memory of high-caloric environment. Regardless, the findings in this work have provoked a stimulating question, that is, whether this form of metabolic learning and memory is present in the mammals and, if so, whether this mechanism can be consolidated to improve the control of metabolic physiology and prevent against diseases. These mouse studies may provide an initial support to this concept and strategy, but clearly, in-depth future research is much needed (Zhang, 2015).

In light of the underlying neural basis for this metabolic learning, this study indicates that multiple brain regions are required, including the hypothalamus-like PI region in addition to the MB (equivalent to the hippocampus in mammals), which is classically needed for learning and memory formation. Anatomically, the PI region is located in the unpaired anteromedial domain of the protocerebral cortex, which is near the calyces of the MB and the dorsal part of the central complex (another brain region for regulating learning and memory). Functionally, the PI region has been demonstrated to coordinate with the MB in regulating various physiological activities in Drosophila. Thus, it is very possible that some PI neurons present nutritional information to the MB and thus induce metabolic learning and memory formation. However, the underlying detailed mechanism is still unknown, especially if this process involves a role of dilps, which represent the prototypical neuropeptides produced by the PI neurons. Considering that the PI region in flies is equivalent to the mammalian hypothalamus, this study was extended to mouse models by comparatively analysing A/J versus C57BL/6J mice—which are known to have different diet preference as well as different susceptibilities to obesity development. While A/J mice showed a learning process of distinguishing NC versus HC food, C57BL/6J mice failed to do so. It is particularly notable that this difference of learning and memory between these two strains is associated with differential expression profiles of learning/memory genes in the hypothalamus rather than the hippocampus. This finding, in conjunction with the Drosophila study, highlights a potential that the hypothalamus has a unique role in mediating metabolic learning and memory formation. Although the mouse experiments cannot exclude the impacts from the taste/smell properties of the studied food, the results demonstrated that there is a form of nutritional memory, which seems dissociable from the memory of overnutritional reward. These initial observations in mice lend an agreement with findings in Drosophila, suggesting that the brain and potentially the hypothalamus can link nutritional environment to a form of metabolic learning and memory homeostasis (Zhang, 2015).

From a disease perspective, metabolic learning in Drosophila is impaired under chronic overnutrition, and the mouse study was in line with this understanding. This response to overnutrition is useful when famine is outstanding; however, it is a dilemma when metabolic disease is of concern, much like the scenario pertaining to leptin resistance under chronic overnutrition, whereas an increase in leptin sensitivity is demanded to reduce obesity. Recently, it was established that NF-κB-dependent hypothalamic inflammation links chronic overnutrition to the central dysregulation of metabolic balance. This study showed that activation of the NF-κB pathway in the PI region weakened the function of metabolic learning and, conversely, NF-κB inhibition in this region provided a protective effect against chronic overnutrition-impaired metabolic learning. These findings are in alignment with the literature, for example, pan-neuronal NF-κB inhibition was shown to improve activity-dependent synaptic signalling and cognitive function including learning and memory formation, and persistent NF-κB activation inhibits neuronal survival and the function of learning and memory formation. Hence, overnutrition-induced neural NF-κB activation has a negative impact on metabolic learning and memory formation in regulating metabolic homeostasis homeostasis (Zhang, 2015).

To summarize the findings in this work, a series of behavioural studies was performed revealing that Drosophila have a form of metabolic learning and memory, through which the flies are directed to balance food choice with caloric intake in relevant environments. Several learning/memory-regulating genes including rut, dnc and tequila are involved in this function, and brain regions including the PI in addition to the MB are required to induce this mechanism. On the other hand, metabolic learning is impaired under chronic overnutrition through NF-κB activation, leading to excess exposure to calorie-enriched environment, which causes metabolic disorders. Overall, metabolic learning and memory formation by the brain and potentially the hypothalamus play a role in controlling metabolic homeostasis homeostasis (Zhang, 2015).