dunce

DEVELOPMENTAL BIOLOGY

See the embryonic expression pattern of dnc at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.

Larval

Mushroom bodies (MBs) are the centers for olfactory associative learning and elementary cognitive functions in the arthropod brain. In order to understand the cellular and genetic processes that control the early development of MBs, high-resolution neuroanatomical studies of the embryonic and post-embryonic development of the Drosophila MBs have been performed. In the mid to late embryonic stages, the pioneer MB tracts extend along Fasciclin II (Fas II)-expressing cells to form the primordia for the peduncle and the medial lobe. As development proceeds, the axonal projections of the larval MBs are organized in layers surrounding a characteristic core, which harbors bundles of actin filaments. Mosaic analyses reveal sequential generation of the MB layers, in which newly produced Kenyon cells project into the core to shift to more distal layers as they undergo further differentiation. Whereas the initial extension of the embryonic MB tracts is intact, loss-of-function mutations of fas II causes abnormal formation of the larval lobes. Mosaic studies demonstrate that Fas II is intrinsically required for the formation of the coherent organization of the internal MB fascicles. Furthermore, ectopic expression of Fas II in the developing MBs results in severe lobe defects, in which internal layers also are disrupted. These results uncover unexpected internal complexity of the larval MBs and demonstrate unique aspects of neural generation and axonal sorting processes during the development of the complex brain centers in the fruit fly brain (Kurusu, 2002).

Studies of MB development with mosaic clones have shown that MB neurons in the adult brain are classified into three groups that project dorsally to the alpha and alpha' lobes and medially to the ß, ß' and gamma lobes. Based on this classification, all the Kenyon cells born before the mid-third larval instar belong to the gamma group. Only in the late third instar, the second group of neurons projecting into the alpha' and ß' lobes is produced. In this study, using various MB markers, it has been demonstrated that the larval Kenyon cells can be further subdivided into distoproximal concentric groups surrounding each of the neuroblasts. Furthermore, the axonal projections of the Kenyon cells are also organized into concentric layers in the peduncle and lobes. Axons of newly born Kenyon cells project into the core that is constituted of densely packed thin fibers rich in actin filaments (Kurusu, 2002).

Distoproximal expression patterns of nuclear regulatory genes in the larval MB cell clusters have been described. In particular, whereas ey is expressed in all the MB cells, including the neuroblasts and ganglion mother cells (GMCs), dac is expressed in differentiated Kenyon cells but not in the centrally located proliferating cells. GAL4 MB markers, such as 201Y and c739, are expressed in an outer group of the differentiated Kenyon cells that is located several cell diameters away from the proliferating neuroblasts (Kurusu, 2002).

While the four MB neuroblasts continue dividing up to the late pupal stage supplying increasing numbers of Kenyon cells, the newly formed larval MB axons follow the medial and the dorsal lobe projections that were pioneered at the embryonic stage with a concomitant increase in the sizes of the lobes. By contrast, a set of genes is turned on in the Kenyon cells after the hatching of the first instar larvae in slightly different patterns in both the cell bodies and their projections. As development proceeds, these differential gene expression patterns became more evident in the second instar larval stage. While the Dac protein is expressed in most of the Kenyon cells, dnc-lacZ is expressed in a small subset of cells peripherally positioned in each of the Kenyon cell clusters originated by the four MB neuroblasts. Expression of 201Y is detected in another subset of cells located more centrally in each of the Kenyon cell clusters, whereas c739 is widely expressed in most of the Kenyon cells (Kurusu, 2002).

Remarkably, these differential expression patterns observed in the Kenyon cells were topologically reflected in their axonal projections in the peduncle and lobes: dnc-lacZ is detected in the outermost surface layer of the peduncle and lobes; 201Y is detected in both the surface and middle layers; and c739 is detected in most axons, a pattern similar to that of FAS II (Kurusu, 2002).

As development proceeds further, further subdivisions emerge in the third instar larval stage with increasing numbers of Kenyon cells and their axons. Moreover the expression patterns of the 201Y and c739 markers change in both cell bodies and their projections; 201Y is then detected in many of the Kenyon cells and their projections, obscuring the 201Y peripheral pattern in the previous larval instar; c739 is then detected in a group of cells located near each of the neuroblasts. The axons of the c739-expressing cells project into an inner layers of the peduncle and lobes. By contrast, dnc-lacZ is maintained in the peripheral subdivisions both in the Kenyon cells and their projections. Double staining with anti-Fas II antibody confirms discrete internal organization of the peduncle and lobes, which are concentrically subdivided into at least three layers surrounding a core that is not labeled with the MB markers, including Fas II (Kurusu, 2002).

Interestingly, the reporter molecule for dnc-lacZ exhibits a characteristic patchy appearance in the calyx, peduncle and lobes, suggesting uneven distribution of the dnc-lacZ fibers. Indeed, higher magnification of the calyces double labeled with anti-ß-gal and anti-synaptotagmin antibodies reveals extensive arborization of the dnc-lacZ expressing neurons around the synaptic terminals, which are likely to represent the afferent terminals of axonal collaterals of the antennocerebral neurons (Kurusu, 2002).

Based on these expression profiles of nuclear regulatory genes and GAL4 markers in the cell bodies, it is suggested that the Kenyon cells that are labeled with both Dac and 201Y project their axons into the concentric layers that also are labeled with Fas II. However, the proximally located Kenyon cells that are labeled with DAC but not 201Y may correspond to the newly differentiated MB neurons that project thin fibers into the core of the peduncle and lobes. Recently described (using a DsRed variant) has been a similar concentric generation of Kenyon cell fibers in the surrounding layers of the peduncle and lobes, in which younger axons extend into the inner layer to shift older fibers into the outer layers. Clonal studies on the larval projection patterns support this temporal order of layer generation and further show that axons of the newly produced Kenyon cells first project into the core as actin-rich thin fibers to shift to the surrounding layers as they undergo further differentiation (Kurusu, 2002).

Adult

Antibodies to the dnc PDE show that the most intensely stained regions in the adult brain are the mushroom body neuropil--areas previously implicated in learning and memory. In situ hybridization demonstrated that DNC RNA is enriched in the mushroom body perikarya. The mushroom bodies of third instar larval brains are also stained intensely by the antibody, suggesting that the dnc PDE plays an important role in these neurons throughout their development (Nighorn, 1991).

Effects of Mutation or Deletion

Several chromosomal deletions and inversions that remove increasingly larger portions of the dnc gene from its 5' end and progressively more of the five known transcription start sites (tss) were used to assess the functions of the various transcriptional units. Surprisingly, the dnc PDE activity, female fertility, mushroom body expression, learning, and memory are unaffected by the removal of tss1 and tss2. tss3 is required for elevated mushroom body expression but not for female fertility nor initial learning. tss4 contributes to learning and the female fertility function, whereas tss5 contributes to female fertility. The results indicate that the structural complexity of the gene is of biological significance, with individual transcriptional units serving different biological functions (Qui, 1993).

The numbers of terminal varicosities and branches are increased in dnc mutants, suggesting a role for dunce in axonogenesis. Such increase is suppressed in dunce-rutabaga 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, which display greatly enhanced nerve activity as a result of reduction in different K+ currents. The expanded projections in dnc are further enhanced in double mutants of dnc with either eag or Sh, an effect that could again be suppressed by rut. The results provide evidence for altered plasticity of synaptic morphology in memory mutants dnc and rut and suggests a role of cAMP cascade in mediating activity-dependent synaptic plasticity (Zhong, 1992).

Increase in synaptic growth in eag, Shaker and dunce mutants is accompanied by approximately 50% reduction in synaptic levels of Fasciclin2. This decrease in Fas2 is both necessary and sufficient for presynaptic sprouting; Fas2 mutants that decrease Fas2 levels by 50% lead to sprouting similar to eag, Shaker and dunce mutants, while Fas2 transgenetic animals that maintain synaptic Fas 2 levels suppress sprouting in eag, Shaker and dunce mutants. However, Fas2 mutants that cause a 50% increase in bouton number do not alter synaptic strength; rather, evoked release from single boutons has a reduced quantal content, suggesting that the wild-type amount of release machinery is distributed throughout more boutons. Thus these results show a requirement for the presynaptic downreguation of Fas2 in activity and cAMP-induced synaptic sprouting. It is speculated that activity or cAMP may trigger the down-regulation of synaptic Fas2 by actively removing it from the presynaptic terminal (Schuster, 1996).

Since Fas2 mutants lead to an increase in number of boutons without affecting synaptic strength, and increased cAMP in dnc mutants increases both synaptic structure and quantal content, there must be other elements downstream of cAMP, but not downstream from Fas2, that are involved in increasing quantal content. CREB, the cyclic AMP response element-binding protein, mediates the transcriptional requirement of cAMP-dependent long-term synaptic change. Thus CREB is a candidate for the cAMP target responsible for increasing quantal content. CREB acts in parallel with FAS2 to cause an increase in synaptic strength. Expression of an endogenous CREB repressor, dCREB2-b (an isoform of CREB), in dunce mutants blocks functional but not structural plasticity. Expression of the activator isoform, dCREB2-a, increases synaptic strength, by increasing presynaptic transmitter release at single boutons, but only in Fas2 mutants that increase bouton number. Strong overexpression of dCREB2-a results in a significant increase in quantal content, independent of genetic background and with little effect on bouton number. Thus CREB-mediated increase in synaptic strength is due to increased presynaptic transmitter release and expression of dCREB2-a in a Fas2 mutant background genetically reconstitutes cAMP-dependent plasticity. Thus cAMP initiates parallel changes in CREB and Fas2 to achieve long term synaptic enhancement (Davis, G. W. 1996).

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).

One target of cAMP modulation of synaptic function is ion channels. In Drosophila, cAMP has been shown to modulate K+ conductances, and a hyperpolarized resting potential has bee reported in mutant dunce muscle. In dunce mutants, synaptic plasticity in motor end plates can be restored by K+ channel blockers, suggesting that the effects of cAMP are mediated by K+ channel conductances. In support of this notion, abnormal spontaneous spikes and altered firing patterns in correlation with altered K+ conductances are observed in embryonic neuroblasts ('giant neurons') from dnc and rut mutants (Zhao, 1997). Despite this, the fundamental question remains open: whether mushroom body (mrb) neurons from Drosophila mutants deficient in the cAMP cascade exhibit altered K+ conductances. Outward current modulation by cAMP was investigated in wild type (wt) and dunce (dnc) Drosophila larval neurons. dnc is deficient in a cAMP phosphodiesterase and has altered memory. Outward current modulation by cAMP was investigated by acute or chronic exposure to cAMP analogs. The analysis included a scrutiny of outward current modulation by cAMP in neurons from the mrb. In Drosophila, the mrb are the centers of olfactory acquisition and retention. Based on outward current patterns, neurons are classified into four types. Downmodulation of outward currents induced by acute application of cAMP analogs is reversible and is found only in type I and type IV neurons. In the general wt neuron population, approximately half of the neurons exhibit cAMP-modulated, 4-aminopyridine (4-AP)-sensitive currents. Because mrb neurons are expected to contribute only 1% to 5% of total neurons, it is concluded that downregulation by cAMP of K+ conductances occurs in many more neurons than are expected to be contributed by the mrb. However, a significantly larger fraction of mrb neurons in wt (70%) is endowed with cAMP-modulated, 4-AP-sensitive currents. Only 30% of the dnc neurons display outward currents modulated by cAMP. The deficit of cAMP-modulated outward currents is most severe in neurons derived from the mrb of dnc individuals. Only 4% of the mrb neurons of dnc are cAMP-modulated. The dnc defect can be induced by chronic exposure of wt neurons to cAMP analogs. These results document for the first time a well defined electrophysiological neuron phenotype in correlation with the dnc defect. Moreover, this study demonstrates that in dnc mutants such a deficiency affects most severely neurons in brain centers of acquisition and retention. A suitable candidate to account for the maintained, 4-AP-sensitive outward currents downmodulated by cAMP is the K+ channel encoded for by Shaw. Downmodulation of K+ currents by cyclic nucleotide may operate indirectly through protein kinase A (Delgado, 1998).

Neural circadian pacemakers can be reset by light, and the resetting mechanism may involve cyclic nucleotide second messengers. Pacemaker resetting and free-running activity rhythms have been examined in flies mutant for dunce and for DC0 [DCO is the catalytic subunit of cAMP-dependent protein kinase (PKA). dnc mutants exhibit augmented light-induced phase delays and shortened circadian periods, which indicate altered pacemaker function. Light-induced phase advances are normal in dnc, suggesting a selective effect on one component of the pacemaker resetting response. Circadian rhythms in cAMP content in head tissues demonstrate that dnc mutations increase the amplitude of daily cAMP peaks. These results show that cAMP levels are not chronically elevated in the dnc mutant. A role for cAMP signaling in circadian processes is also suggested by an analysis of DC0 mutants, which have severe kinase deficits and display arrhythmic locomotor activity (Levine, 1994).

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 (Engel, 1996).

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 (Engel, 1996).

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).

dunce mutations are semi-dominant for initial learning and genetic variants carrying the enzymatically hypomorphic dnc2 mutation produce learning scores lower than those of the amorphic dncM11. There are no discernable effects of the different dunce mutations on memory formation 30 to 180 min after training. These results are consistent with a model of memory formation, in which dunce is hypothesized to disrupt acquisition and/or short-term memory (Tully, 1993).

Synaptic transmission was examined in Drosophila memory mutants. In both dunce and rutabaga 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. The results suggest that the cAMP cascade plays a role in synaptic facilitation and potentiation and indicate that synaptic plasticity is altered in Drosophila memory mutants (Zhong, 1991).

There is a muscle potassium-selective channel that is directly and reversibly activated by cAMP in a dose-dependent fashion. Activation is specific and it cannot be mimicked by a series of agents that include AMP, cGMP, ATP, inositol trisphosphate, and Ca2+. Channel current records obtained from larval muscle in different dunce mutants possessing abnormally high levels of cAMP show that, in the mutants, the channel displays an increased probability of opening (Delgado, 1991).

Various K+ currents in Drosophila muscles are affected by altered cAMP cascades in dunce and rutabaga mutants. 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. Results from voltage-clamp studies indicated that both IA and IK are increased in dnc alleles. Normal muscle fibers treated with dibutyryl-cAMP showed a similar increase of IA, but no significant effect on IK. In contrast to the dnc alleles, the rut mutations appeared to enhance ICS greatly while leaving the amplitude of other currents largely unchanged. In addition, the dibutyryl-cAMP-induced increase in IA is not observed in rut fibers. Caffeine and W7, which are known to interfere with several second messenger pathways, also modulate K+ currents in larval muscle fibers. The currents in dnc and rut fibers show strikingly altered responses to caffeine and W7 (Zhong, 1993)

The opposing biochemical defects of rutabaga and dunce allow rut mutants to partially suppress the female sterility caused by elevated cyclic AMP levels in dunce flies. 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 (Feany, 1990).

Two partial suppressors of the female sterility phenotype have been selected in an X chromosome containing a dunce null mutation. Both suppressors are associated with reduced AC2 activity. Complementation analyses suggest that both are alleles of the learning mutant rutabaga. 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, although some do develop to adults. These adult progeny exhibit morphological defects confined mostly to the second and third thoracic segments or to the first five abdominal segments. These observations demonstrate that the dunce gene is required in adult females for egg laying and that the dunce gene provides an essential maternal function required for normal development of the zygote. Clonal analysis, employing the dominant female-sterile mutation ovoD1, demonstrates that the former requirement for PDE activity resides in somatic cells and that the latter requirement resides in germ line cells. Female germ line cells homozygous for a dunce null mutation produce oocytes that fail to develop. Thus, homozygous dunce null-mutant zygotes develop to adults solely because of the enzyme or mRNA present in the oocytes of heterozygous mothers. 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).

Conditional expression of dunce transgenes in dnc adults shortly before training significantly improves learning over nontransgenic controls. Remarkably, behavioral rescue is also observed after induction of a transgene carrying a rat counterpart of dunce. Induction of the transgenes in adult dnc females confers partial rescue of the female sterility phenotype. These data are consistent with a major physiological requirement for the gene's activity in the learning process and show that a rat counterpart can substitute functionally for the Drosophila gene (Dauwalder, 1995).

The genetic complementation patterns of both behavioral and lethal alleles at the stoned locus have been characterized. Mutations at two loci, dunce and shibire, act synergistically with certain stoned temperature sensitive mutations to cause lethality, but fail to interact with stnC (Petrovich, 1993).

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).

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 Drosophila mutants amnesiac, dunce, and rutabaga were isolated after associative conditioning tests, during which animals were trained to associate the presence of an odor with that of electric shocks (ES). In the absence of conditioning, the odor avoidance (OA) of these mutants is normal, indicating that their poor associative conditioning performance is attributable to specific learning or memory deficits. However, the OA of the mutants is greatly decreased after their exposure to ES. This effect can last for hours. These results strongly suggest that part of the defect displayed by these mutants in associative conditioning tests does not correspond to a learning or memory deficit but might arise from abnormal sensitivity to stressful stimuli. OA after ES of two previously characterized dnc mutants was examined. Df(1)N79f specifically decreases Dnc expression in the mushroom bodies, leading to a normal level of learning but decreased memory. Df(1)N79f mutants displays a normal OA after ES. Df(1)N64j15 affects the entire brain expression of Dnc, leading to decreased learning and memory. Df(1)N64j15 animals show a strong decrease of their OA after ES. Thus, the lack of Dnc 'general' expression is most likely responsible for the OA defect, which would be responsible for the apparent learning defect after conditioning. In contrast, the Dnc phosphodiesterase accumulated in the mushroom bodies is thought to be involved specifically in memory formation (Preat, 1998).

It is well known that cAMP signaling plays a role in regulating functional plasticity at central glutamatergic synapses. However, in the Drosophila CNS, where acetylcholine is thought to be a primary excitatory neurotransmitter, cellular changes in neuronal communication mediated by cAMP remain unexplored. In this study the effects of elevated cAMP levels on fast excitatory cholinergic synaptic transmission were examined in cultured embryonic Drosophila neurons. Chronic elevation in neuronal cAMP [in dunce neurons or wild-type neurons grown in dibutyryl-cAMP (db-cAMP)] results in an increase in the frequency of cholinergic miniature EPSCs (mEPSCs). The absence of alterations in mEPSC amplitude or kinetics suggests that the locus of action is presynaptic. Furthermore, a brief exposure to db-cAMP induces two distinct changes in transmission at established cholinergic synapses in wild-type neurons: a short-term increase in the frequency of spontaneous action potential-dependent synaptic currents and a long-lasting, protein synthesis-dependent increase in the mEPSC frequency. A more persistent increase in cholinergic mEPSC frequency induced by repetitive, spaced db-cAMP exposure in wild-type neurons is absent in neurons from the memory mutant dunce. These data demonstrate that interneuronal excitatory cholinergic synapses in Drosophila, like central excitatory glutamatergic synapses in other species, are sites of cAMP-dependent plasticity. In addition, the alterations in dunce neurons suggest that cAMP-dependent plasticity at cholinergic synapses could mediate changes in neuronal communication that contribute to memory formation (Lee, 2000).

Thus neuronal cAMP levels can regulate functional plasticity, independent of differentiation, at cholinergic synapses between cultured Drosophila neurons. Although numerous studies have demonstrated that the cAMP signaling cascade plays a role in modulating plasticity at central glutamatergic (hippocampus) synapses and presumed glutamatergic (Aplysia) synapses, this is the first direct evidence that cAMP regulates plasticity at central cholinergic synapses by mediating fast excitatory transmission. Since acetylcholine appears to be a primary excitatory neurotransmitter in the fly brain, these data are consistent with the hypothesis that cAMP-dependent plasticity at cholinergic synpases mediates changes in neuronal communication in the Drosophila CNS. Although the majority of fast excitatory synaptic transmission in the mammalian brain is mediated by glutamate, recent reports indicate the presence of fast synaptic signaling via nAChRs in both the rodent hippocampus and visual cortex. In light of these findings in Drosophila, it seems likely that cAMP may also be important in modulating fast cholinergic synaptic transmission in the mammalian CNS (Lee, 2000).

The data demonstrating a significant increase in mEPSC frequency in three different dunce alleles, isolated in two independent screens, strongly support the hypothesis that mutations in dunce, a gene encoding a cAMP phosphodiesterase, result in alterations in cholinergic synaptic transmission. The phosphodiesterase activity is higher in the dnc¹ versus dnc² mutant. However, the cAMP levels in these two dunce alleles are similar and significantly higher than wild-type. The similarity in the mEPSC frequency in dnc¹ and dnc² and the observation that chronic exposure to db-cAMP induces a concentration-dependent increase in mEPSC frequency in wild-type neurons, are consistent with the suggestion that elevated levels of cAMP in the mutant regulate mEPSC frequency. The smaller increase in mEPSC frequency in neurons within the dnc^M11 cultures (as compared to dnc¹ and dnc² cultures) is not unexpected. The dnc^M11 cultures are genetically heterogenous with only 25% of the neurons homozygous for the mutant allele. In contrast, in the dnc¹ and dnc² cultures, all of the neurons are genetically homozygous for the mutations in the dnc locus. Assessment of mEPSC frequency after experimental manipulations resulting in a reduction in cAMP levels in dnc mutant neurons will be important in further examining the role of cAMP in regulation of synaptic transmission in the mutant neurons (Lee, 2000).

In this study, brief exposures of cultured wild-type neurons to db-cAMP demonstrate that the cAMP-signaling cascade is involved in both short-term and long-lasting modulation of activity at cholinergic synapses in Drosophila. The short-term change is characterized by a rapid onset increase in cholinergic sEPSC frequency in differentiated wild-type neurons. Because there are no immediate changes in action potential (AP) independent synaptic transmission, and the increase in sEPSC frequency is blocked in the presence of a protein kinase inhibitor (staurosporine), it suggests that the alterations involve posttranslational modifications of existing proteins that do not affect the synaptic machinery involved in mediating constitutive release. The increase in AP-dependent release is transient in that the majority of the elevation in synaptic current frequency observed 24 hr after cAMP exposure could be accounted for by AP-independent synaptic currents. In Aplysia, it has been shown that cAMP, through protein kinase A (PKA), mediates a phosphorylation-induced reduction in conductance of existing potassium channels resulting in a rapid, transient increase in neuronal excitability contributing to short-term facilitation. Phosphorylation induces a rapid and reversible decrease in outward potassium currents in Drosophila neurons. This suggests that an increase in AP duration and/or excitability, similar to that seen in Aplysia sensory neurons, may contribute to the rapid cAMP-induced increase in sEPSC frequency at cholinergic synapses in Drosophila neurons (Lee, 2000).

The long-lasting change induced by brief db-cAMP exposure in cultured Drosophila neurons is characterized by a delayed onset increase in mEPSC frequency that peaks 24 hr after exposure and is blocked by cycloheximide. Time course, requirement for de novo protein synthesis, and the indication that the changes are presynaptic (absence of changes in the biophysical properties of each mEPSC), are all consistent with cAMP-inducing synaptic growth resulting in an increase in the number of functionally identical cholinergic release sites. A similar mechanism has been proposed to underlie cAMP-induced long-term facilitation in Aplysia where studies in cell culture have revealed that enhancement of synaptic strength between identified sensory and motor neurons, observed at 24 hr after the stimulus, requires protein and RNA synthesis and the growth of new synaptic connections between the neurons. However, further analysis will be necessary to determine if cAMP regulates evoked transmitter release at cholinergic synapses in Drosophila and if so whether the mechanism involves regulation of the number of synaptic sites or affects other processes such as efficacy of transmission at each bouton (Lee, 2000).

In Aplysia cell culture, while a single pulse (1x) of serotonin can induce cAMP-dependent short-term facilitation, repetitively spaced (5x) application of serotonin is necessary to induce cAMP-dependent long-term facilitation. Consolidation of changes induced by spaced stimuli involve cAMP response element-binding protein (CREB)-mediated gene transcription. A role for cAMP-CREB-mediated transcription has also been demonstrated in long-term potentiation at glutamatergic synapses in the rodent hippocampus. Furthermore, studies in Drosophila indicate that cAMP-initiated changes in CREB activity play a role in long-term synaptic enhancement at peripheral glutamatergic synapses. The observation that repetitive spaced treatments with db-cAMP induces a more persistent change than a single db-cAMP treatment, suggests that plasticity at cholinergic synapses induced by spaced exposure to db-cAMP in Drosophila involves activation of CREB-mediated gene transcription. It will be possible to test this hypothesis by examining cAMP-dependent modulation of cholinergic transmission in neurons from transgenic flies carrying CREB activators and CREB inhibitors (Lee, 2000).

The data clearly demonstrate that cAMP plays an important role in short-term modulation of transmission, as well as initiating events that contribute to long-lasting synaptic changes requiring new protein synthesis, at interneuronal cholinergic synapses in Drosophila. Several lines of evidence support the hypothesis that the cAMP-dependent regulation of cholinergic plasticity reported in the cultured neurons is likely to represent a mechanism involved in modulation of functional transmission important for behavior in the adult fly. (1) It was found that repetitive spaced exposure to db-cAMP induces a more persistent increase in mEPSC frequency than an equivalent length single exposure in wild-type Drosophila neurons. These results represent a remarkable parallel to those of behavioral studies in Drosophila, demonstrating that repetitive spaced trails, where an olfactory stimulus is paired with a foot shock, induces more persistent memory than an equivalent number of training trials presented in the absence of a rest interval between trials in wild-type flies. (2) The data from dnc mutant neurons reveals that chronic disruption of cAMP signaling in neurons, previously shown to result in associative learning deficits in the fly, alters the basal levels of AP-independent transmission at cholinergic synapses. Even more significant is the finding that a persistent increase in mEPSC frequency could not be induced by spaced exposure to db-cAMP in the dnc mutant neurons. The inability of dnc neurons to respond to transient, activity-induced changes in cAMP levels that are likely to occur during olfactory training episodes in the adult fly, could contribute to the reduced performance index when compare with wild-type. Finally, although there are no studies directly demonstrating cholinergic synaptic transmission in the adult Drosophila CNS, cholinergic transmission takes place at synapses in the mushroom body of the adult honeybee, an anatomical structure critical for learning and memory in Drosophila as well as the honeybee. Taken together these findings support the hypothesis that cholinergic synapses in Drosophila, similar to central glutamatergic synapses in other species, are sites of cAMP-dependent synaptic plasticity important for learning and memory. Insights gained from functional studies in this Drosophila culture system, well suited to molecular genetic and biochemical manipulations, will be useful in delineating the molecular mechanisms underlying modulation of central synaptic transmission, a process thought to contribute to learning and memory in all animals (Lee, 2000).

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).

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).

Dunce role in oogenesis

Because of the potential role of the G protein-coupled receptor kinase 2 gene in G protein signaling it was hypothesized that Gprk2 is involved in a signaling pathway that utilizes cAMP as a second messenger. To examine this hypothesis, tests were performed for genetic and biochemical interactions between dunce and Gprk2 mutants; dunce is able to suppress the sterility defect of a gprk2 mutant. Similarly, gprk2 mutation is able to rescue the viability and sterility defects of dunce mutants. Results of the biochemical analysis of cAMP levels in the dunce;gprk2 mutant combinations further strengthen the genetic findings. These results suggest that Gprk2 is involved in a receptor-mediated signaling pathway that utilizes cAMP as a second messenger (Lannutti, 2001).

The genetic interaction between dunce and Gprk2 was examined through the effects of these two genes on oogenesis. A mutant allele of Gprk2, called gprk2⁶⁹³⁶, has decreased fertility as a result of reduced levels of egg laying and hatching, and developing egg chambers display defects in the formation of anterior structures. Similarly, many alleles of dunce are sterile, with an ovary phenotype that resembles gprk2⁶⁹³⁶. Introduction of a single copy of a hypomorphic or null allele of dunce into the gprk2⁶⁹³⁶ background suppresses all of these defects to a significant degree. Suppression is also observed when a single copy of gprk2⁶⁹³⁶ is introduced into a dunce background. Like rutabaga mutants (rutabaga encodes a calcium/calmodulin-dependent adenylate cyclase), gprk2⁶⁹³⁶ has reduced levels of cAMP. Ovaries from gprk2⁶⁹³⁶ females contain about one third the normal amount of cAMP. In addition, in every mutant combination where fertility is increased, cAMP levels are closer to wild type levels. These results suggest that Gprk2 is functioning in a cAMP-signaling pathway and that the underlying basis of the interaction between Gprk2 and dunce is a normalization of cAMP levels (Lannutti, 2001).

Because of the possibility that dunce may be acting in a common pathway with Gprk2, the dunce ovarian phenotype was reexamined. Females homozygous for hypomorphic (dnc²) and null (dnc^M14 ) alleles have been shown to lay few or no eggs. This has been attributed to defects in the chorion and vitelline membrane. In addition to these defects, nurse cell dumping is incomplete in egg chambers from dnc²homozygous females. This incomplete cytoplasmic transfer may cause or contribute to the formation of misshapen dorsal appendages. Incomplete cytoplasmic dumping is also apparent in dnc^M14 homozygotes and dnc²/dnc^M14 transheterozygotes. In both of these genotypes, egg chambers have severely truncated dorsal appendages or the dorsal appendages fail to form altogether. The percentage of egg chambers that fail to complete cytoplasmic dumping does not increase significantly in the stronger allelic combinations, probably because many egg chambers fail to develop to that stage (Lannutti, 2001).

In addition to the dumping and dorsal appendage defects, ovaries from dunce mutant mothers contain degenerating egg chambers. In dnc² homozygotes, 70% of stages 10B and 11 egg chambers are degenerating, as determined by the presence of condensed, DAPI-bright nuclei. In dnc²/dnc^M14 and dnc^M14 homozygotes, egg chamber degeneration is detected in earlier stages (beginning at stage 6) and occurs more frequently. All three aspects of the dunce ovary phenotype, the incomplete cytoplasmic dumping, the malformed dorsal appendages, and the degeneration of egg chambers, resemble those of the gprk2⁶⁹³⁶ mutant (Lannutti, 2001).

There is a marked increase in fertility in allelic combinations of dunce and gprk2. This increase suggests that there should be a corresponding improvement in the ovary morphology of combinations of both mutants. To test this, a quantitative analysis of the ovaries from the different mutant combinations was performed. The feature of the dunce phenotype that is most readily quantified is egg chamber degeneration; this defect can be assayed by the presence of DAPI-bright nuclei. In dnc² homozygotes, DAPI-bright, nurse cell nuclei were observed in 39% of stages 9 and 10 egg chambers. This defect is more severe in dnc²/dnc^M14 transheterozygotes and dnc^M14/dnc^M14 homozygotes. In these genotypes 52% (dnc²/dnc^M14) and 58% (dnc^M14/dnc^M14) of stages 9 and 10 egg chambers display a degenerating phenotype. These numbers are an underestimate of the level of degeneration in dnc²/dnc^M14 and dnc^M14/dnc^M14 allelic combinations because egg chambers often degenerate earlier in oogenesis. As a result, ovaries from dnc²/dnc^M14 and dnc^M14/dnc^M14 females produce few egg chambers past stage 11. One copy of gprk2⁶⁹³⁶, which suppresses the sterility of the two dunce alleles, also suppresses degeneration in the dnc²/dnc², dnc²/dnc^M14, and dnc^M14/dnc^M14 mutants. Although degeneration is dramatically reduced in all three genotypes, rescue is not complete. These results support the suppression of sterility that is observed in dunce;gprk2⁶⁹³⁶ allelic combinations (Lannutti, 2001).

Mutations in the dunce gene cause an increase in embryonic and adult cAMP levels (two- to fivefold, depending on the allele) and most dunce alleles are sterile. In contrast, mutations in rutabaga cause a slight decrease in cAMP levels but are completely fertile. Double mutants of rutabaga and dunce have intermediate levels of cAMP and fertility is partially restored. Similarly, one copy of gprk2⁶⁹³⁶ partially restores fertility in dunce mutants. By analogy to the dunce-rutabaga interaction, the simplest explanation for the mutual suppression of gprk2⁶⁹³⁶ and dunce is that cAMP levels in the ovaries of gprk2⁶⁹³⁶ homozygotes are lower than those in wild type flies. In agreement with this suggestion, it was found that cAMP levels in the ovaries of gprk2⁶⁹³⁶ homozygotes are almost threefold lower than those in wild type females. Furthermore, the suppression of the gprk2⁶⁹³⁶ phenotype by dunce (and vice versa) is reflected in the cAMP levels of the dunce, gprk2⁶⁹³⁶ allelic combinations. Introducing a single copy of the dnc² or dnc^M14 mutant into gprk2⁶⁹³⁶ homozygotes resulted in a wild-type cAMP content, threefold higher than that in gprk2⁶⁹³⁶ homozygotes. The gprk2⁶⁹³⁶ mutant also suppresses the elevated levels of cAMP seen in mutant dunce alleles. In short, in every mutant combination in which an increase in fertility is observed, the cAMP content in the ovaries changes in the expected direction. Taken together, these results strongly support the hypothesis that Gprk2 is involved in a signaling pathway that utilizes cAMP as a second messenger (Lannutti, 2001).

It is interesting that gprk2⁶⁹³⁶ and dunce mutants have similar ovary phenotypes, even though they have opposite effects on cAMP levels in the ovaries. It appears that cAMP levels must be maintained at an optimum level; both an increase and a decrease cause defects in dorsal appendage formation, cytoplasmic dumping, and probably other maternal and zygotic functions. This effect has also been documented in assays of learning in the fly. Both rutabaga and dunce mutants cause defects in learning and memory, although they cause opposite changes in cAMP levels. Apparently, the level of cAMP must be tightly regulated for proper functioning of cAMP-dependent pathways (Lannutti, 2001 and references therein).

Another parallel with the dunce, rutabaga studies is the observation that the degree of suppression does not always correlate with the level of cAMP in the ovaries. In earlier studies it has been shown that a dunce, rutabaga double mutant (homozygous for both alleles) is still defective in learning, although cAMP levels are rescued. Similarly, rescue of fertility is not complete, even in combinations that produced cAMP levels very close to those of wild type. For example, the ovaries from dnc²/+;gprk2⁶⁹³⁶ /gprk2⁶⁹³⁶ females have cAMP levels that are not statistically different from wild type levels. However, the fertility of these females is still statistically less than that in wild type. There are several possible explanations for this effect. (1) It has been suggested that the kinetics of cAMP metabolism are as important as the level of cAMP per se. Similarly, the subcellular distribution of cAMP may play an equally important role in fertility. (2) The Gprk2 and dunce genes may have functions that are not dependent on one another. For example, because there are many heptahelical receptors in Drosophila and only two known GRKs, it is likely that Gprk2 protein phosphorylates many different receptor types. If some of these receptors act through second messengers other than cAMP, then they would not be rescued by a decrease in the dosage of dunce. Similarly, Dunce is the major source of phosphodiesterase activity in Drosophila. Therefore, a decrease in phosphodiesterase activity could disrupt signaling from Gprk2-independent receptors. (3) Gprk2 and Dunce could carry out functions that are not directly related to production or metabolism of second messengers. For example, mammalian GRKs have been shown to interact with cytoskeletal proteins, suggesting that they have a potential scaffolding or docking function (Lannutti, 2001 and references therein).

In germline clones of dnc^M14, eggs are laid but fail to hatch, suggesting that dunce activity is required in the somatic cells for egg laying and in the germline for hatching. This appears to contradict the data suggesting a germline requirement for dunce in egg laying. However, there are several possible explanations for this apparent discrepancy. (1) The analysis of egg laying from females with dnc^M14germline clones could not be quantitative because the ovarioles do not all contain clones. Therefore, while the data demonstrate that somatic expression of dunce is necessary for egg laying, it does not exclude a role in the germline as well. (2) By the same argument, a role for Gprk2 activity in the somatic cells has not been ruled out. Although Gprk2 expression in the follicle cells cannot be detected, it is possible that a low level of somatic expression plays a role in egg laying. (3) Although the Gprk2 gene plays a role in regulating the level of cAMP in the ovaries, it has not been directly shown that Gprk2 and dunce are acting in the same molecular pathway. Perhaps dunce and Gprk2 act in different signaling pathways that are active in somatic and germline cells, respectively, and the interaction of the two pathways is necessary for normal levels of egg laying (Lannutti, 2001).

In summary, analysis of the interaction between gprk2⁶⁹³⁶ and dunce mutants demonstrates that the regulation of cAMP synthesis and metabolism is critical for development. The gprk2⁶⁹³⁶ mutant has not only reduced levels of cAMP but also decreased egg laying and hatching. These defects were all suppressed by weak and null alleles of dunce. Similarly, the increased levels of cAMP and sterility of dunce are suppressed by the gprk2⁶⁹³⁶ mutant. Further analysis of the developmental functions of gprk2⁶⁹³⁶ in vivo will continue to complement the biochemical characterization of this protein (Lannutti, 2001).

A screen was performed for female sterile mutations on the X chromosome of Drosophila and new loci were identified that are required for developmental events in oogenesis: new alleles of previously described genes were identified as well. The screen has identified genes that are involved in cell cycle control, intracellular transport, cell migration, maintenance of cell membranes, epithelial monolayer integrity and cell survival or apoptosis. New roles are described for the genes dunce, brainiac and fs(1)Yb, and new alleles of Sex lethal, ovarian tumor, sans filles, fs(1)K10, singed, and defective chorion-1 have been identified (Swan, 2001).

Analysis of a spatial orientation memory in Drosophila

Flexible goal-driven orientation requires that the position of a target be stored, especially in case the target moves out of sight. The capability to retain, recall and integrate such positional information into guiding behaviour has been summarized under the term spatial working memory. This kind of memory contains specific details of the presence that are not necessarily part of a long-term memory. Neurophysiological studies in primates indicate that sustained activity of neurons encodes the sensory information even though the object is no longer present. Furthermore they suggest that dopamine transmits the respective input to the prefrontal cortex, and simultaneous suppression by GABA spatially restricts this neuronal activity. Fruit flies possess a similar spatial memory during locomotion. Using a new detour setup, flies are shown to be able to remember the position of an object for several seconds after it has been removed from their environment. In this setup, flies are temporarily lured away from the direction towards their hidden target, yet they are thereafter able to aim for their former target. Furthermore, it was found that the GABAergic (stainable with antibodies against GABA) ring neurons (Hanesch, 1998) of the ellipsoid body in the central brain are necessary and their plasticity is sufficient for a functional spatial orientation memory in flies. The protein kinase S6KII (ignorant; Putz, 2004) is required in a distinct subset of ring neurons to display this memory. Conditional expression of S6KII in these neurons only in adults can restore the loss of the orientation memory of the ignorant mutant. The S6KII signalling pathway therefore seems to be acutely required in the ring neurons for spatial orientation memory in flies (Neuser, 2008).

Previous studies have shown that walking flies heading for an object maintain their direction even when the target disappears. This persistence of orientation can last for several seconds, indicating that flies store the position of, or the path towards, the hidden object for further targeting. It is therefore proposed that flies form a spatial memory for objects that is similar to the working memory in vertebrates. To investigate this putative memory in Drosophila a detour paradigm for walking flies was established. Single flies were put into a cylindrical virtual-reality arena, in which two dark vertical stripes were presented at opposite sides. Normally, flies patrol between the two visual objects for a considerable length of time. In the new paradigm, the stripes disappeared when the fly crossed the invisible midline of the circular walking platform, and a new target appeared laterally at a 90° angle to the fly. In most cases wild-type flies turned towards this new target if it was presented for more than 500 ms. After the fly had oriented itself towards the new object (deviation of the fly's longitudinal body axis from the ideal course to the stripe below +/-15°), this target also disappeared within 1 s and no objects were visible to the fly. It was then determined whether the fly turned back to continue its approach to its initial, but still invisible, target. The walking traces reveal a direct course towards the former location of the first target. The flies therefore retained positional information on the former object, although it was no longer present in the environment (Neuser, 2008).

Wild-type (Canton-S) flies recall the old target and integrate it into a guided behaviour with a median frequency of 80% as measured in ten consecutive trials for each fly. Longer presentation of the distracter stripe did not significantly change the percentage of positive choices. These data strongly suggest that flies stored the relative position of the first target in a spatial orientation memory for at least 4 s. To exclude the possibility that flies used chemical traces of former runs for their orientation the absolute positions of the stripes were randomly changed after each trial. As a result of this randomization, flies had to update their memory continuously. Moreover, no training effect could be observed, because the frequency of positive turns did not change during the ten consecutive trials. Similar performances were observed when two opposing distracters were presented to the fly. This orientation memory for vanished objects is considered to be to be idiothetic. Because no visible landmarks were presented to the fly after the distracter disappeared, the fly could not use a stored reference picture of the environment for its guidance. It is therefore suggested that the fly uses online stored information of its own angle towards the former target, a strategy known as path integration. Path integration has been shown to be used by other insects, such as ants and bees, to navigate through a familiar landscape (Neuser, 2008).

In an attempt to localize this type of memory to discrete parts of the insect brain, several mutant lines with structural central-complex defects of Drosophila were analysed. The central complex is composed of four different neuropils and has been implicated in supervising motor output during locomotion. First tests showed that the persistence of orientation towards a removed target is reduced or lost whenever the ellipsoid body of the central complex was defective. Therefore the ellipsoid body open mutant (ebo^KS263) was tested in the detour paradigm; these flies did not show a preference for the first target after the detour, suggesting that an intact ellipsoid body is required for establishing a spatial orientation memory. In contrast, the use of hydroxyurea to ablate the mushroom bodies, which are important in olfactory memory, did not disturb the orientation memory (Neuser, 2008).

One prominent type of neuronal cells of the ellipsoid body is the group of GABAergic ring neurons. The fibres of these neurons run in a prominent tract, the RF tract (ring-neuron and tangential fan-shaped-body neuron tract), and form bushy thin endings in the ipsilateral lateral triangle and bleb-like endings in the ellipsoid body. Four different kinds of ring neuron (R1-R4) can be distinguished by their arborization pattern around the ellipsoid body canal. R1-R3 neurons project outwards from the ellipsoid body canal, whereas the arborization of R1 is restricted to the inner zone, that of R2 to the outer zone, and that of R3 to both zones. R4 neurons project from the periphery inwards and arborize in the outermost zone. It was next proposed that the ring neurons might be necessary for the orientation memory. The GAL4/UAS system was used to silence distinct subsets of ring neurons through the expression of tetanus toxin (TNT) by using the GAL4 driver lines c232, c481 and c105. For temporal control, TNT was induced conditionally by using the temperature-sensitive GAL4 repressor GAL80^ts under the control of the ubiquitous Tubulin promoter (Tub-GAL80^ts). Experimental and control flies were raised at 18°C, tested within the detour paradigm, and retested after the induction of TNT. Pairwise comparison revealed that the preference for the original target was lost whenever the toxin was expressed in ring neurons of the ellipsoid body. This finding confirms the hypothesis that the ellipsoid-body ring neurons are necessary components of the orientation memory (Neuser, 2008).

To investigate which molecular pathways are involved in this kind of memory, focus was placed on the cyclic-AMP signalling pathway. Variable levels of cAMP have been shown to have a crucial function in memory formation during associative learning in Drosophila. cAMP levels are modulated by the opposing actions of adenylyl cyclases and cAMP phosphodiesterases. Mutants for the adenylyl cyclase gene rutabaga (rut¹ and rut²⁰⁸⁰) were unable to target visual objects and could not be tested in the paradigm. Therefore mutants of the dunce gene (dnc), which encodes a cAMP phosphodiesterase, were tested in the detour paradigm. The dnc¹ mutant is a hypomorph and displays about half of the enzyme activity in the wild type. dnc¹ mutant flies show deficits in several paradigms of associative classical learning and operant conditioning. In contrast, dnc¹ mutants showed no defects in the detour paradigm, indicating that a tight modulation of cAMP levels might not be critically required for spatial orientation memory (Neuser, 2008).

Another molecule involved in memory formation in Drosophila is a member of the ribosomal serine kinase family. ignorant (ign) encodes the S6 kinase II (S6KII), which interacts with mitogen-activated protein (MAP) kinase signalling in Drosophila. S6KII does not seem to be involved in cAMP signalling pathways. The null allele ign^58/1 has been shown to be defective in classical aversive conditioning and operant learning (Putz, 2004). Therefore ign^58/1 flies were tested in the detour paradigm. Although the mutants readily targeted visible objects, they showed no directional preference for the position of the original target after it disappeared, suggesting that they had lost their memory. In contrast, walking speed, walking activity and orientation towards visual objects were similar to those of the wild type. Next whether ign is required in the ring neurons targeted by c232-GAL4 was tested with the use of a UAS-ign RNA-mediated interference (RNAi) effector line. RNAi silencing in these ring neurons decreased the performance by half. This decrease in memory constitutes only a partial phenocopy of the null mutant, because the performance was not significantly different from that of the wild type or ign^58/1. Nevertheless, this result is interpreted to suggest that ign is required in the ring neurons for spatial orientation memory (Neuser, 2008).

To address the question of whether restoring S6KII levels is sufficient for regaining memory, neuron-specific rescue experiments were performed in the ign^58/1 mutant background. S6KII was expressed pan-neuronally with Appl-GAL4 and elav-GAL4, and also specifically in the R3 and R4 ring neurons with c232-GAL4. In all three cases a complete rescue was observed. Next, whether ign function in the R3 and R4 ring neurons is acutely required for orientation memory was examined. Therefore, again use was made of the GAL80^ts transgene to rescue the ign phenotype only in the adult. Conditional expression of S6KII only in the R3 and R4 ring neurons resulted in a perfect rescue of the ign mutant. This result -- that acute S6KII expression in the R3 and R4 ring neurons accomplished a complete rescue -- reveals that this very narrow subset of cells is sufficient for regaining a functional orientation memory. It has been reported that Drosophila S6KII negatively regulates extracellular signal-regulated kinases (ERKs) by acting as a cytoplasmic anchor of the MAP kinase. Further studies will determine whether the MAP kinase signalling pathway is required for this kind of memory task (Neuser, 2008).

It is concluded that the relevant ring neurons use the inhibitory neurotransmitter GABA. Their circuitry and interconnections within the ellipsoid body are not yet known. Expression of the dDA1 dopamine receptor in the ellipsoid body has recently been shown. It is therefore possible that the same neurotransmitter systems as those used for visual-spatial memory in the monkey prefrontal cortex are used to establish orientation memory in the central complex of flies (Neuser, 2008).

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.