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 suboesophageal 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 suboesophageal 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 (fig. S4B). 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).