InteractiveFly: GeneBrief

Resistant to dieldrin: Biological Overview | References

In both mammals and insects, neurons involved in learning are strongly modulated by the inhibitory neurotransmitter GABA. The GABA_A receptor, Resistance to dieldrin (Rdl), is highly expressed in the Drosophila mushroom bodies (MBs), a group of neurons playing essential roles in insect olfactory learning. Flies with increased or decreased expression of Rdl in the MBs were generated. Olfactory associative learning tests showed that Rdl overexpression impaired memory acquisition but not memory stability. This learning defect is due to disrupting the physiological state of the adult MB neurons rather than causing developmental abnormalities. Remarkably, Rdl knockdown enhanced memory acquisition but not memory stability. Functional cellular imaging experiments showed that Rdl overexpression abolished the normal calcium responses of the MBs to odors while Rdl knockdown increased these responses. Together, these data suggest that RDL negatively modulates olfactory associative learning, possibly by gating the input of olfactory information into the MBs (Liu, 2007).

Neurons comprising the neural circuits that mediate learning are modulated by the inhibitory neurotransmitter γ-amino butyric acid (GABA). For instance, the hippocampus, which is involved in the formation of multiple types of memories in mammalian organisms, is densely innervated by GABAergic interneurons. The insect mushroom bodies (MBs), which similarly are involved in the formation of multiple types of memories, are also subject to GABAergic modulation (Perez-Orive, 2002; Yasuyama, 2002). These and other similar observations make it clear that a deep understanding of the molecular and systems neuroscience properties that underlie memory formation will not emerge until a detailed knowledge of when and where GABAergic modulation occurs and how this modulation alters the function of the cells and networks that mediate memory formation.

GABA_A receptors are GABA-gated chloride channels. Accumulating pharmacological and genetic evidence suggests that GABA_A receptors participate in the cellular and circuit mechanisms underlying learning and memory, but the current information is inconsistent and lacks depth. Several prior studies have used intraperitoneal or intracerebroventricular injection of GABA_A receptor agonists or antagonists and monitored effects on behavior. However, the widespread effects caused by this approach make it impossible to assign behavioral changes to any specific population of neurons. Better spatial resolution for the pharmacological effects has been achieved by injecting drugs into specific brain regions either before or after training or just prior to testing, and in several cases, receptor agonists have inhibited behavioral performance, and antagonists have facilitated it. However, these studies fail to provide information regarding the specific cell type affected within the targeted region and the identity of the targeted GABA receptor. Furthermore, they provide no information about how the pharmacological agents affect the information processing relevant to learning mechanisms by the neurons. Moreover, the simplistic idea that GABA_A receptor agonists and antagonists/inverse agonists may decrease and increase behavioral performance, respectively, remains controversial because of reports to the contrary (Liu, 2007 and references therein).

Genetic dissections of GABA_A receptor function using viable knockouts have provided more specific information regarding the receptor type involved, but they lack information about how information processing is altered, the neurons involved in the behavior being tested, and whether the behavioral results are due to a physiological disruption of GABA_A function or a developmental insult secondary to the developmental loss of the receptor. Moreover, the controversies regarding the direction of behavioral change (improve versus impair) with decreased receptor function remain. For instance, DeLorey (1998) reported that GABA_Aβ3 knockout mice showed impaired performance several days after training in a step-through passive avoidance task and contextual fear conditioning, but Collinson (2002) and Crestani (2002) reported GABA_Aα5 mutant mice to have enhanced performance in a match-to-place version of the water maze test and in trace fear conditioning, respectively. Although genetic dissections point to the inadequacy of pharmacological manipulations by emphasizing receptor-specific functions, the use of whole-animal knockouts fails to offer reliable conclusions about where and how GABA_A receptors influence the complex neural circuitry underlying learning and memory (Liu, 2007).

This study probed the role of GABAergic modulation using Drosophila olfactory learning as a model because of the ability to bidirectionally alter the expression of specific GABA_A receptors in identified populations of neurons of the adult and to probe how these modulations alter the information processing capabilities of the neurons. In Drosophila, at least three genes are thought to encode GABA_A receptors: resistance to dieldrin (Rdl), GABA and glycine-like receptor of Drosophila (Grd), and ligand-gated chloride channel homologue 3 (Lcch3). Rdl is by far the best characterized of the three molecularly and through functional expression experiments (Hosie, 1997; Buckingham, 2005). RDL also has an important role in insecticide resistance (ffrench-Constant, 2004). This receptor is highly expressed in the Drosophila antennal lobes (ALs) and the MBs (Harrison, 1996), both of which are essential structures required for the acquisition, storage, and retrieval of olfactory memory (Liu, 2007).

One attractive idea for the role of GABAergic inhibition of neurons involved in learning is that the inhibition serves to sparsen sensory representations to make learning easier and recall faster (Perez-Orive, 2002; Olshausen, 2004). The projection neurons in the insect AL receive information about odors from olfactory receptor neurons in the antennae and transmit this information to higher-order structures including the MBs and the lateral horn (LH). The AL projection neurons exhibit robust firing when the animal senses an odor, but the robustness of the response in the postsynaptic MB neurons is sparsened because of postulated feedforward GABAergic inhibition received from the LH (Perez-Orive, 2002). Unfortunately, there remains no direct experimental evidence in favor of or against this hypothesis (Liu, 2007).

In this study, flies were generated with elevated or decreased expression of Rdl in the MBs and the learning performance of these flies was assayed along with the calcium responses in the MBs produced by odor and electric shock stimulation. The results indicate that the level of memory acquisition is inversely related to the level of RDL expression in the MB neurons, indicating that RDL in the MBs inhibits olfactory learning. This inhibition of learning is due to the expression level of Rdl at the time of learning, rather than to developmental alterations that may occur in the neural circuit from perturbing Rdl expression during development. Furthermore, the calcium response of MB neurons to odor stimulation is also inversely related to the level of Rdl expression in these neurons, indicating that the expression level of Rdl gates the receipt of information about the conditioned stimulus during olfactory learning (Liu, 2007).

The reported expression pattern (Harrison, 1996) of Rdl in the adult Drosophila brain was verified. A polyclonal antibody was developed that recognizes RDL protein by immunoblotting. The abundance of this protein was reduced in flies heterozygous for two different null and homozygous lethal alleles of Rdl, Rdl¹ and Rdl^f02994, confirming the ability of this antibody to detect the RDL protein. The expression pattern of Rdl in the central brain by was characterized by immunohistochemistry. The RDL protein was detected throughout the ALs, the MBs, and the central complex. In the MBs, RDL was detected both in the dendrites (calyces) and the axons (α, α′, β, β′, γ lobes and peduncles), but no RDL signal was observed in the cell bodies of MB neurons (Liu, 2007).

Both overexpression and knockdown strategies with tissue and time-specific control were used to probe the role of the GABA_A receptor RDL in olfactory learning. The abundant endogenous expression of Rdl in the olfactory nervous system strongly suggests a critical role in odor perception, discrimination, and learning. The results conclusively show a physiological role for Rdl in olfactory learning. Overexpression of Rdl in the MBs impaired learning, while knockdown of Rdl in the same neurons enhanced learning. The data also show that RDL is involved in memory acquisition but not memory stability. These behavioral data along with functional imaging results indicate that the GABAergic system inhibits olfactory learning, probably by gating the level of olfactory information into the MBs (Liu, 2007).

The Rdl gene exhibits extensive alternative splicing. Exons 3 and 6 of the Rdl gene have two alternative splice forms each, so that the Rdl gene encodes four different isoforms, all of which are found in RNA isolated from early embryos (ffrench-Constant, 1993a). When expressed in Xenopus oocytes, the proteins produced from alternative splicing show differential responses to agonists (Hosie, 2001), suggesting different physiological properties for the isoforms in vivo. Since the detailed temporal and spatial expression pattern of each isoform in the adult fly brain has not been reported, the antigen and all RNAi constructs against sequences common to all known isoforms were designated. Therefore those isoform(s) that are expressed in the adult MBs and those that are responsible for inhibiting olfactory learning cannot be identify (Liu, 2007).

Prior studies have shown that GABAergic inhibition shapes odor-evoked spatiotemporal activity patterns in the Drosophila ALs (Wilson, 2005). GABA receptor function in the honeybee AL has also been shown to be required for fine, but not coarse, odor discrimination, by using picrotoxin to inhibit AL GABA receptors (Stopfer, 1997). These observations raise the issue of why the c772-Gal4, MB {Gal80}; UAS-Rdli flies exhibit normal olfactory learning. Although it is possible that c772-Gal4 drives expression in AL interneurons other than those involved in olfactory discrimination, which would explain the observation, the more likely explanation is that the odors used in this study are quite disparate, allowing for the normal learning of these odors. This predicts that a phenotype may emerge in tests of these flies for fine odor discrimination (Liu, 2007).

One possible role for the GABAergic inhibition of the MB neurons is to sparsen the odor representations (Perez-Orive, 2002). Sparsening of sensory representations has been proposed as a simplification that the nervous system makes to allow easier and faster encoding and retrieval of memories (Olshausen and Field, 2004). In its simplest form, the sparsening hypothesis for GABAergic inhibition of the MBs predicts that lessening the inhibition by reducing Rdl expression should make the representations more complex and more difficult to learn, whereas enhanced acquisition with reduced Rdl expression was observed. Rather than facilitating and enhancing memory formation by the sparsening of representations, these results are more consistent with the alternative idea that the GABAergic system inhibits learning (Liu, 2007).

What is the purpose of a neural system that inhibits learning? One possibility is that this inhibitory system may provide a necessary balance for the acquisition of different forms of memory. Extinction is an active form of learning occurring when the repeated presentation of a CS alone causes a gradual decrease in the conditioned response in a previously conditioned animal. The surface expression of the GABA_A receptor and the expression level of gephyrin, a protein involved in GABA_A receptor clustering, have been reported to decrease in the basolateral amygdala of the rat after fear conditioning, yet these GABAergic markers significantly increase after extinction training (Chhatwal, 2005), suggesting that the GABAergic system has opposing roles for conditioning and extinction. Preliminary data also show that Rdl knockdown reduced extinction, supporting the hypothesis that the GABAergic system inhibits conditioning while enhancing extinction (Liu, 2007).

Second, this inhibitory system could serve as a noise filter for information transmission from the ALs to the MBs. The projection neurons of the ALs convey olfactory information to at least two third-order olfactory areas: the MBs and the lateral horns. The MBs are required for olfactory learning, and the lateral horns are thought to be involved in establishing odor identity. Excitatory local neurons were discovered in the ALs. These neurons may be involved in signal amplification by providing cross excitation to projection neurons that are innervated by olfactory receptor neurons that are not responsive to the test odor. The net result is enhanced and more generalized output from the ALs. While this signal amplification could potentially be beneficial for odor detection and discrimination in the lateral horns, it could also introduce extra noise and be detrimental to the MBs for learning about odors in a specific way relative to their importance. By reducing the activity of the MB neurons, the GABAergic system could potentially reduce the time window for coincidence detection in the MBs, thus inhibiting generalized learning and facilitating selective learning. Thus, the GABAergic system may be a noise filter needed by the MBs for optimal learning (Liu, 2007).

Finally, the inhibitory system on the MBs may allow learning to occur through the mechanism of inhibiting the inhibition. There are two major questions of focus for future investigations relative to this idea. One key question is whether learning alters the abundance or function of RDL receptors in the MB neurons. This change could serve to lessen the inhibitory constraints on MB neurons postconditioning, thus potentiating the effect of the trained odor. The surface expression of GABA_A receptors has been reported to decrease in the basolateral amygdala after fear conditioning in rodents (Chhatwal, 2005). A related question is whether there are learning-induced changes that occur in the presynaptic GABAergic extrinsic neurons that innervate the MBs. Although these neurons and potential learning-induced changes are yet to be identified in Drosophila, it has been reported that classical olfactory discrimination conditioning of the mouse alters the release of neurotransmitters in the olfactory bulb, including the release of GABA (Brennan, 1998). Presynaptic changes in the release of GABA due to conditioning might also have similar effects by potentiating CS responses after learning. Glutamate released by repetitive activation of the Schaffer collateral triggers a heterosynaptic and persistent depression of GABA release onto CA1 pyramidal neurons (Chevaleyre, 2003). The appropriate paired stimulation of MB neurons by CS and US could in a related way produce a retrograde signal to depress GABA release and thus potentiate learning mediated by the MB neurons (Liu, 2007).

The GABA_A receptor RDL suppresses the conditioned stimulus pathway for olfactory learning

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).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Plasticity of local GABAergic interneurons drives olfactory habituation

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

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

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

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

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

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

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

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

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

The GABA_A receptor RDL acts in peptidergic PDF neurons to promote sleep in Drosophila

Sleep is regulated by a circadian clock that times sleep and wake to specific times of day and a homeostat that drives sleep as a function of prior wakefulness. Flies display the core behavioral features of sleep, including relative immobility, elevated arousal thresholds, and homeostatic regulation. Sleep-wake modulation was assessed by a core set of circadian pacemaker neurons that express the neuropeptide PDF. It was found that disruption of PDF function increases sleep during the late night in light:dark and the first subjective day of constant darkness. Flies deploy genetic and neurotransmitter pathways to regulate sleep that are similar to those of their mammalian counterparts, including GABA. RNA interference-mediated knockdown of the GABA_A receptor gene, Resistant to dieldrin (Rdl), in PDF neurons reduces sleep, consistent with a role for GABA in inhibiting PDF neuron function. Patch-clamp electrophysiology reveals GABA-activated picrotoxin-sensitive chloride currents on PDF+ neurons. In addition, RDL is detectable most strongly on the large subset of PDF+ pacemaker neurons. These results suggest that GABAergic inhibition of arousal-promoting PDF neurons is an important mode of sleep-wake regulation in vivo (Chung, 2009).

It is proposed that GABA release inhibits large LNv output and PDF release to reduce wake, suggesting an important role for GABA inhibition. In this model, the circadian clock times PDF neuron activation and PDF release during the late night and following day to promote waking behavior. Of note, a similar arousal-promoting function for circadian pacemaker neurons has been described in mammals. This is also approximately the time when the large LNv have been shown to be more depolarized and have higher levels of spontaneous activity. RDL receptors on LNv soma and on fibers in the accessory medulla suggest that GABA may regulate LNv excitability. It is interesting that GABA is also an important neurotransmitter in mammalian circadian pacemaker neurons, capable of reducing their spontaneous activity. In addition, RDL receptors on PDF varicosities in the optic lobe may function presynaptically to regulate PDF release. GABA may also act through metabotropic GABA_B receptors, which have been described in the sLNv, but their function in circadian or sleep behavior is unknown. GABAergic signaling may affect the function of the transcription factor ATF2, which is important for PDF neuron function in sleep. Changes in PDF neuron function may in turn act by antagonizing sleep-promoting circuits that exist within the mushroom bodies as well as the pars intercerebralis (PI). Of note, the PI appears to express the PDF receptor. Identifying the anatomic targets of PDF as well as the neural sources of GABAergic inputs will be important for further defining sleep-wake circuits in Drosophila (Chung, 2009).

Glutamate, GABA and Acetylcholine Signaling Components in the Lamina of the Drosophila Visual System

Synaptic connections of neurons in the Drosophila lamina, the most peripheral synaptic region of the visual system, have been comprehensively described. Although the lamina has been used extensively as a model for the development and plasticity of synaptic connections, the neurotransmitters in these circuits are still poorly known. Thus, to unravel possible neurotransmitter circuits in the lamina of Drosophila, Gal4 driven green fluorescent protein in specific lamina neurons was combined with antisera to gamma-aminobutyric acid (GABA), glutamic acid decarboxylase, a GABA_B type of receptor, L-glutamate, a vesicular glutamate transporter (vGluT), ionotropic and metabotropic glutamate receptors, choline acetyltransferase and a vesicular acetylcholine transporter. It is suggested that acetylcholine may be used as a neurotransmitter in both L4 monopolar neurons and a previously unreported type of wide-field tangential neuron (Cha-Tan). GABA is the likely transmitter of centrifugal neurons C2 and C3 and GABA_B receptor immunoreactivity is seen on these neurons as well as the Cha-Tan neurons. Based on an rdl-Gal4 line, the ionotropic GABA_A receptor subunit RDL may be expressed by L4 neurons and a type of tangential neuron (rdl-Tan). Strong vGluT immunoreactivity was detected in a-processes of amacrine neurons and possibly in the large monopolar neurons L1 and L2. These neurons also express glutamate-like immunoreactivity. However, antisera to ionotropic and metabotropic glutamate receptors did not produce distinct immunosignals in the lamina. In summary, this paper describes novel features of two distinct types of tangential neurons in the Drosophila lamina and assigns putative neurotransmitters and some receptors to a few identified neuron types (Kolodziejczyk, 2008; full text of article).

Modulation of GABA_A receptor desensitization uncouples sleep onset and maintenance in Drosophila

Many lines of evidence indicate that GABA and GABA_A receptors make important contributions to human sleep regulation. Pharmacological manipulation of these receptors has differential effects on sleep onset and sleep maintenance insomnia. Sleep is regulated by GABA in Drosophila; a mutant GABA_A receptor, Rdl^A302S, specifically decreases sleep latency, the length of time that it takes to accomplish the transition from full wakefulness to sleep. The drug carbamazepine (CBZ) has the opposite effect on sleep; it increases sleep latency as well as decreasing sleep. Behavioral and physiological experiments indicated that Rdl^A302S mutant flies are resistant to the effects of CBZ on sleep latency and that mutant RDL^A302S channels are resistant to the effects of CBZ on desensitization, respectively. These results suggest that this biophysical property of the channel, specifically channel desensitization, underlies the regulation of sleep latency in flies. These experiments uncouple the regulation of sleep latency from that of sleep duration and suggest that the kinetics of GABA_A receptor signaling dictate sleep latency (Agosto, 2008).

gamma-Aminobutyric acid (GABA) signaling components in Drosophila: immunocytochemical localization of GABA_B receptors in relation to the GABA_A receptor subunit RDL and a vesicular GABA transporter

γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in insects and is widely distributed in the central nervous system (CNS). GABA acts on ion channel receptors (GABA_AR) for fast inhibitory transmission and on G-protein-coupled receptors (GABA_BR) for slow and modulatory action. Immunocytochemistry was used to map GABA_BR sites in the Drosophila CNS, and the distribution was compared with that of the GABA_AR subunit RDL. To identify GABAergic synapses, an antiserum was raised to the vesicular GABA transporter (vGAT). For general GABA distribution, an antiserum to glutamic acid decarboxylase (GAD1) and a gad1-GAL4 was used to drive green fluorescent protein. GABA_BR-immunoreactive (IR) punctates were seen in specific patterns in all major neuropils of the brain. Most abundant labeling was seen in the mushroom body calyces, ellipsoid body, optic lobe neuropils, and antennal lobes. The RDL distribution is very similar to that of GABA_BR-IR punctates. However, the mushroom body lobes displayed RDL-IR but not GABA_BR-IR material, and there were subtle differences in other areas. The vGAT antiserum labeled punctates in the same areas as the GABA_BR and appeared to display presynaptic sites of GABAergic neurons. Various GAL4 drivers were used to analyze the relation between GABA_BR distribution and identified neurons in adults and larvae. These findings suggest that slow GABA transmission is very widespread in the Drosophila CNS and that fast RDL-mediated transmission generally occurs at the same sites (Enell, 2007).

GABA receptors containing Rdl subunits mediate fast inhibitory synaptic transmission in Drosophila neurons

GABAergic inhibition in Drosophila, as in other insects and mammals, is important for regulation of activity in the CNS. However, the functional properties of synaptic GABA receptors in Drosophila have not been described. This study reports that spontaneous GABAergic postsynaptic currents (sPSCs) in cultured embryonic Drosophila neurons are mediated by picrotoxin-sensitive chloride-conducting receptors. A rapid increase in spontaneous firing in response to bath application of picrotoxin demonstrates that these GABA receptors mediate inhibition in the neuronal networks formed in culture. Many of the spontaneous GABAergic synaptic currents are sodium action potential independent [miniature IPSCs (mIPSCs)] but are regulated by external calcium levels. The large variation in mIPSC frequency, amplitude, and kinetics properties between neurons suggests heterogeneity in GABA receptor number, location, and/or subtype. A decrease in the mean mIPSC decay time constant between 2 and 5 d, in the absence of a correlated change in rise time, demonstrates that the functional properties of the synaptic GABA receptors are regulated during maturation in vitro. Finally, neurons from the GABA receptor subunit mutant Rdl exhibit reduced sensitivity to picrotoxin blockade of the mIPSCs and resistance to picrotoxin-induced increases in spontaneous firing frequency. This demonstrates that Rdl containing GABA receptors play a direct role in mediating synaptic inhibition in Drosophila neural circuits formed in culture (Lee, 2003).

The abundant expression of acetylcholine and GABA, throughout the Drosophila CNS, suggests that these classic neurotransmitters play a major role in mediating fast synaptic transmission. Recent electrophysiological studies have provided insights into the functional aspects of excitatory cholinergic transmission in Drosophila neurons. However, virtually nothing was known about the properties of inhibitory GABAergic synaptic transmission. The current data demonstrate that the receptors mediating GABAergic currents in Drosophila neurons are PTX sensitive, heterogeneous with respect to their biophysical properties, and regulated during maturation in culture. The GABAergic currents are inhibitory, and Rdl encoded subunits contribute to the population of receptors meditating fast inhibitory synaptic transmission in neuronal networks formed in culture (Lee, 2003).

A reversal potential near the chloride equilibrium potential and blockade by low (1 µM) concentrations of PTX, a potent antagonist of insect GABA receptors (Sattelle, 1990), suggested that the spontaneous IPSCs recorded in the embryonic neurons are mediated by GABA-gated chloride channels. This conclusion is also supported by the finding that, although puffing of GABA or glutamate evokes chloride currents in the cultured neurons, as predicted from a study of larval motor neurons (Rohrbough, 2002), only the GABA-evoked currents are effectively blocked by 1 µM PTX. This PTX concentration does not significantly reduce the glutamate-evoked currents. These data strongly support the hypothesis that the receptors mediating spontaneous synaptic currents are GABA-gated, as opposed to glutamate-gated, chloride channels (Lee, 2003).

GABA acts primarily as an inhibitory neurotransmitter in the adult CNS of both vertebrates and invertebrates (Mody, 1994; Hosie, 1997). However, there is abundant evidence that GABA can be excitatory during early development (Ben-Ari, 1997). Recently, it has been reported that a blockade of GABA_A receptors in the neonatal rodent brain can induce increases in neuronal excitation. This indicates that GABAergic transmission can also serve an inhibitory role in hippocampal and cortical circuits during early development in mammals (Lamsa, 2000; Palva, 2000; Wells, 2000). Using a similar strategy, blocking GABA receptors by bath application of PTX, an increase was observed in spontaneous neuronal firing in the Drosophila cultures. This demonstrates that embryonic neurons form spontaneously active circuits in culture and GABAergic transmission can mediate inhibition in these networks, even at this early developmental stage in Drosophila. Future studies will be necessary to determine whether GABA can be depolarizing and/or elicit action potentials, as observed in some neurons from the early postnatal rodent brain, in subpopulations of Drosophila neurons (Lee, 2003).

At many chemical synapses, some portion of the vesicular release of neurotransmitter is action potential (AP) independent. In cultured embryonic Drosophila neurons, much of the spontaneous release of GABA at synapses appears to occur in the absence of sodium spikes in presynaptic neurons. A blockade of the mIPSCs by removal of external calcium or the addition of cobalt demonstrates that these events are dependent on flux of calcium through voltage-gated channels in the presynaptic neurons. Spontaneously occurring calcium-dependent APs, could result in transient changes in levels of calcium in the presynaptic terminals that would in turn regulate vesicular release of GABA. However, this seems unlikely in the Drosophila cultures because, although many of the neurons fire spontaneous sodium APs in normal saline, regenerative spikes have not been observed in the presence of TTX. Alternatively, small fluctuations in the resting membrane potential could regulate the opening of voltage-gated calcium channels in the presynaptic terminals. The resulting changes in intracellular calcium levels would in turn influence the frequency of spontaneous fusion events. This seems plausible, given the relatively depolarized resting potential of the embryonic neurons and calcium channels that activate at voltages as low as -60 mV. In a similar manner, the unexpected appearance of mIPSCs in bursts could arise from regular oscillations in the membrane potential of a population of presynaptic GABAergic neurons in which peaks, associated with high intracellular calcium levels, may trigger the release of multiple quanta. Calcium imaging studies, in combination with electrophysiological recording, should be useful in elucidating the mechanisms underlying the AP-independent burst activity observed in the embryonic Drosophila neurons (Lee, 2003).

In mammals, there is a high degree of heterogeneity in GABA receptor properties in neurons from different regions of the CNS. Factors influencing functional heterogeneity include receptor subunit composition and desensitization rates (Vicini, 1999). In addition, the functional properties of receptors mediating synaptic currents in individual neurons can change during development. For example, there is a maturational progression from mIPSCs with slow to more rapid decay kinetics correlated with changes in receptor subunit composition and populations of cerebellar, hippocampal, cortical, and thalamic neurons. The heterogeneity in the decay kinetics of currents recorded from embryonic Drosophila neurons during the first week in culture is consistent with the expression of multiple receptor subtypes. This is not surprising, given that the cultures are prepared from whole embryos and therefore contain neurons from all parts of the nervous system. The shift in mean decay time constant, 1.5- to 2-fold during the first week in culture, suggests that functional properties of receptors mediating the GABAergic mIPSCs in Drosophila neurons are also subject to regulation during maturation. In rodent cortical neurons, a decrease in mPSC decay time constant, both in vivo and in dissociated cell culture, demonstrated that signals necessary for initiating the changes in GABA receptor function can be retained in dissociated cell culture. Although no parallel study has been conducted on GABAergic mIPSCs in Drosophila in vivo, preliminary data suggest that the GABAergic mIPSC decay rate in CNS neurons cultured from late-stage pupae are faster than those seen even in the older embryo cultures. This is consistent with the changes occurring over time in culture representing maturation that normally occurs in the animal. The evolutionary conservation of this change supports the hypothesis that alterations in GABA receptor kinetics play an important role in shaping early neural circuitry (Lee, 2003).

Cloning and expression studies have been important in defining the role of two Drosophila GABA receptor subunit genes, Rdl and LCCH3, in the formation of functional GABA-gated ion channels (Hosie, 1997). Pharmacological analysis of wild-type and Rdl mutant neurons now provide the first insights into the subunit composition of synaptic GABA receptors in Drosophila. The PTX-sensitive mIPSCs in wild-type neurons are not blocked by bicuculline methylchloride (BMC). This pharmacological profile is similar to homomultimeric GABA-gated chloride channels encoded by the Rdl GABA receptor subunit gene when expressed in Xenopus oocytes (ffrench-Constant, 1993b; full text of article ) and Sf2 cells (Zhang, 1995). A significant reduction of the sensitivity of the mIPSCs to blockade by PTX in Rdl mutant versus wild-type neurons confirmed that Rdl-encoded subunits contribute to the population of functionally active synaptic GABA receptors (Lee, 2003).

The Rdl mutant neurons in culture exhibit a 5- to 10-fold reduction in PTX sensitivity based on the comparison with the wild-type dose-response curve. In contrast, the GABA-evoked currents mediated by mutant Rdl channels expressed in oocytes are ~100-fold less sensitive to PTX blockade than the wild-type Rdl channels (ffrench-Constant, 1993b; full text of article). This suggests that synaptic GABA receptors containing Rdl in the neurons are heteromultimers rather than homomultimers. It does not seem likely that the receptors are Rdl- and LCCH3-encoded heteromultimers because expression studies indicate that these form PTX-insensitive, BMC-sensitive receptors (Zhang, 1995). In addition, antibody staining has shown that Rdl protein is localized in the synaptic neuropil in embryos and larval CNS, whereas LCCH3 is found primarily in the cell bodies, making it unlikely that they interact in vivo (Aronstein, 1996). Therefore, the synaptic receptors may include additional subunits, perhaps encoded by GRD (Harvey, 1994) or other as yet uncharacterized GABA receptor genes (Lee, 2003).

The resistance to PTX-induced increases in neuronal firing rates in Rdl mutant cultures demonstrates that Rdl subunit-containing GABA receptors actively mediate synaptic inhibition in Drosophila neural circuits. Therefore, it is possible that synaptically localized Rdl-containing receptors are involved in higher-order functions such as GABA receptor-mediated synchronization of neural activity known to be important in olfactory information-processing locusts (MacLeod, 1996). Manipulation of Rdl expression in Drosophila should make it possible to test this hypothesis (Lee, 2003).

Alternative splicing of a Drosophila GABA receptor subunit gene identifies determinants of agonist potency

Alternative splicing of the Drosophila Rdl gene yields four ionotropic GABA receptor subunits. The two Rdl splice variants cloned to date, RDL_ac and RDL_bd (DRC17-1-2), differ in their apparent agonist affinity. This paper reports the cloning of a third splice variant of Rdl, RDL_ad. Two-electrode voltage clamp electrophysiology was used to investigate agonist pharmacology of this expressed subunit following cRNA injection into Xenopus laevis oocytes. The EC_so values for GABA and its analogues isoguvacine, muscimol, isonipecotic acid and 3-amino sulphonic acid on the RDL_ad homomeric receptor differed from those previously described for RDL_ac and DRC17-1-2 receptors. In addition to providing a possible physiological role for the alternative splicing of Rdl, these data delineate a hitherto functionally unassigned region of the N-terminal domain of GABA receptor subunits, which affects agonist potency and aligns closely with known determinants of potency in nicotinic acetylcholine receptors. Thus, using expression in Xenopus oocytes, differences have been demonstrated in agonist potency for the neurotransmitter GABA (and four analogues) between splice variant products of the Drosophila melanogaster Rdl gene encoding homomer-forming GABA receptor subunits (Hosie, 2001).

There is mounting evidence that subunits encoded by insect Rdl genes underly the characteristic pharmacology of the bicuculline-insensitive GABA receptors that predominate in insect nervous systems. cRNAs exhibiting a high identity (>80%) with those encoding Drosophila RDL subunits, have been cloned from a variety of insect species; these subunits are widely distributed in insect CNS. Although the intron-exon structures of Rdl genes from insect species other than Drosophila melanogaster and Aedes aegyptae remain to be determined, there is evidence to suggest that some of these may also be alternatively spliced. To date, multiple RDL isoforms have been isolated in Drosophila and in other insects such as Blatella germanica (the German cockroach, where four homologues have been identified (Kaku, 1994); all differ in the region corresponding to that encoded by exon 6 of the Drosophila Rdl gene. Indeed, in some cases, the putative D. melanogaster and B. germanica subunits differ at homologous residues and the identities of these variant residues are preserved in different species (Hosie, 2001).

It is, therefore, possible that Rdl genes are alternatively spliced in a number of insect species. The present study demonstrates that the 10 amino acid differences, which result from the alternative splicing of exon 6 of D. melanogaster Rdl, confer a three-fold change in the potencies of GABA and its analogues. Thus, by generating small changes in agonist sensitivity, the alternative splicing of Rdl genes may serve to increase functional diversity in insect GABA receptors. Such a mechanism would distinguish insects from vertebrates, where the principal mechanism for increasing GABA receptor diversity appears to be the co-assembly of different subunit classes and isoforms, which are encoded by separate genes. While the difference in agonist potency conferred by splice variants of D. melanogaster Rdl is not great, it is similar to that observed for certain recombinant GABA_A receptors containing different α subunit isoforms. Just how significant this is depends very much on the synaptic concentration of GABA; high micromolar concentrations would saturate Rdl variants. The physiological role of Rdl splice variants may, therefore, be to yield GABA receptors with differing single-channel kinetics. In line with this, the kinetics of recombinant GABA_A receptor channels depends on their subunit composition, and is affected by the presence of different α isoforms. While the M2 domain of α subunits is highly conserved, the most variant region in the N-terminal domain of α isoforms corresponds to the region of RDL subunits encoded by exon 6. With this in mind, it may be worth performing a detailed study of the kinetics of different RDL receptors. Such a detailed comparison of kinetics has yet to be undertaken; however, no differences in the single-channel conductances of RDL_ac and RDL_bd have been observed (Hosie, 2001).

The alternative splicing of D. melanogaster Rdl provides further evidence for a conserved structure-function relationship for the cys-loop family of neurotransmitter receptors, which includes nAChRs and strychnine-sensitive glycine receptors. In the best studied members of this family, nAChRs of vertebrate muscle, the agonist binding sites are considered to lie at the interfaces of two subunit pairs and are composed of a number of discrete regions of the N-terminal domain of each subunit. Six such regions have been identified, loops A-F although further determinants lying outside these domains have also been identified. Loops A, B and C lie on the nicotinic α subunit, while the remainder lie on the adjacent face of another subunit, normally a non-α subunit. The subunits encoded by the Rdl gene, like the α7-9 nAChR subunits of vertebrates, are capable of forming functional homomers. The residues, which differentiate RDL_ac from RDL_ad, lie between loops B and C and thus encompass a region equivalent to loop E of nAChRs. In nAChRs, the residues of loop E appear to interact directly with agonists and their substitution confers marked changes in agonist potency. Although the majority of the substitutions in the RDL subunit variants are not conservative they affect relatively small changes in agonist potency, suggesting that rather than interacting directly with the agonist molecule they may influence the shape of the binding site and/or its coupling to the channel gate. However, the changes in RDL are naturally occurring, with a possible physiological role, and could therefore be expected to produce subtle differences in receptor function rather than the dramatic changes associated with site-directed mutagenesis of vertebrate GABA_A and nicotinic receptors (Hosie, 2001).

The known determinants of agonist potency on GABA_A receptors align closely to domains A-D of nicotinic receptors. In GABA_A receptors, residues homologous to domains B and C have been identified on the β subunit while a residue in the homologue of domain D has been identified in the α subunit. Similarly, determinants of agonist potency are located in bicuculline-insensitive vertebrate GABA receptor ρ subunits at positions homologous to domains A, B and C. However, an equivalent of domain E has yet to be recognised in vertebrate ionotropic GABA receptors. The results of the present study demonstrate that naturally occurring substitutions of amino acids in this region affect the potency of GABA on RDL homomers. Whether this region of the N-terminal domain also affects the potency of agonists on vertebrate GABA receptors remains to be determined. However, with the exception of the signal peptide and the extreme N-terminus of the mature subunit, this is the least conserved region in the N-terminal domain of invertebrate α subunit isoforms, and it is the α subunits which would be expected to contribute domain E to the agonist binding site of GABA_A receptors. With this in mind, it is interesting that the differences in the agonist potency and kinetics of recombinant rat GABA_A receptors containing α1 or α3 subunits could be accounted for primarily by differences in the transition rates underlying agonist association and dissociation (Hosie, 2001).

The present study demonstrates that a naturally occurring substitution of residues in the extracellular region of Drosophila GABA receptors underlies moderate changes in the potency of the natural agonist, GABA, and may therefore serve a physiological role. The region containing these changes aligns with determinants of agonist potency on nAChRs. It is therefore possible that this region may also affect either the agonist responses of vertebrate GABA receptors, or the kinetics of channel opening (Hosie, 2001).

Search PubMed for articles about Drosophila Rdl

Agosto, J., et al. (2008). Modulation of GABAA receptor desensitization uncouples sleep onset and maintenance in Drosophila. Nat. Neurosci. 11(3): 354-9. PubMed citation: 18223647

Aronstein, K., Auld, V., ffrench-Constant, R. (1996). Distribution of two GABA receptor-like subunits in the Drosophila CNS. Invert Neurosci 2: 115-120. PubMed citation: 9372158

Ben-Ari, Y., Khazipov, R., Leinekugel, X., Caillard, O. and Gaiarsa, J.-L. (1997). GABA_A, NMDA and AMPA receptors: a developmentally regulated 'menage a trois.' Trends Neurosci 20: 523-529. PubMed citation: 9364667

Brennan, P. A., et al. (1998). Changes in neurotransmitter release in the main olfactory bulb following an olfactory conditioning procedure in mice. J. Neuroscience 87: 583-590. PubMed citation: 9758225

Buckingham, S. D., et al. (2005). Insect GABA receptors: splicing, editing, and targeting by antiparasitics and insecticides. Mol. Pharm. 68: 942-951. PubMed citation: 16027231

Chevaleyre, V. and Castillo, P. E. (2003). Heterosynaptic LTD of hippocampal GABAergic synapses: a novel role of endocannabinoids in regulating excitability. Neuron 38: 461-472. PubMed citation: 12741992

Chhatwal, J. P., et al. (2005). Regulation of gephyrin and GABA_A receptor binding within the amygdala after fear acquisition and extinction. J. Neurosci. 25: 502-506. PubMed citation: 15647495

Chung, B. Y., et al. (2009). The GABA_A receptor RDL acts in peptidergic PDF neurons to promote sleep in Drosophila. Curr. Biol. 19: 386-390. PubMed Citation: 19230663

Collinson, N., et al. (2002). Enhanced learning and memory and altered GABAergic synaptic transmission in mice lacking the a5 subunit of the GABA_A receptor. J. Neurosci. 22: 5572-5580. PubMed citation: 12097508

Crestani, F., et al. (2002). Trace fear conditioning involves hippocampal a5 GABA_A receptors. Proc. Natl. Acad. Sci. 99: 8980-8985. PubMed citation: 12084936

Das, S., et al. (2011). Plasticity of local GABAergic interneurons drives olfactory habituation. Proc. Natl. Acad. Sci. 108(36): E646-54. PubMed Citation: 21795607

DeLorey, T. M., et al. (1998). Mice lacking the beta3 subunit of the GABA_A receptor have the epilepsy phenotype and many of the behavioral characteristics of Angelman syndrome. J. Neurosci. 18: 8505-8514. PubMed citation: 9763493

Enell, L., Hamasaka, Y., Kolodziejczyk, A. and Nässel, D. R. (2007). gamma-Aminobutyric acid (GABA) signaling components in Drosophila: immunocytochemical localization of GABA_B receptors in relation to the GABA_A receptor subunit RDL and a vesicular GABA transporter. J. Comp. Neurol. 505(1): 18-31. PubMed citation: 17729251

ffrench-Constant, R. H. and Rocheleau, T. A. (1993a). Drosophila gamma-aminobutyric acid receptor gene Rdl shows extensive alternative splicing. J. Neurochem. 60: 2323-2326. PubMed citation: 7684073

ffrench-Constant, R. H., et al. (1993b). A point mutation in Drosophila GABA receptor confers insecticide resistance. Nature 363: 449-451. PubMed citation: 8389005; Online text

ffrench-Constant, R. H., Daborn, P. J. and Le Goff, G. (2004). The genetics and genomics of insecticide resistance. Trends Genet. 20(3): 163-70. PubMed Citation: 15036810

Gerdjikov, T. V., et al. (2008). Hippocampal alpha 5 subunit-containing GABA A receptors are involved in the development of the latent inhibition effect. Neurobiol. Learn. Mem. 89: 87-94. PubMed Citation: 17638582

Harris, J. A. and Westbrook, R. F. (1998). Evidence that GABA transmission mediates context-specific extinction of learned fear. Psychopharmacology 140: 105-115. PubMed Citation: 9862409

Harrison, J. B., et al. (1996). Immunocytochemical mapping of a C-terminus anti-peptide antibody to the GABA receptor subunit, RDL in the nervous system in Drosophila melanogaster. Cell Tissue Res. 284: 269-278. PubMed citation: 8625394

Harvey, R. J., et al. (1994). Sequence of a Drosophila ligand-gated ion-channel polypeptide with an unusual amino-terminal extracellular domain. J. Neurochem. 62: 2480-2483. PubMed citation: 8189252

Hosie, A. M., Aronstein, K., Sattelle, D. B. and ffrench-Constant, R. H. (1997). Molecular biology of insect neuronal GABA receptors. Trends Neurosci 20: 578-583. PubMed citation: 9416671

Hosie, A. M., Buckingham, S. D., Presnail, J. K. and Sattelle, D. B. (2001). Alternative splicing of a Drosophila GABA receptor subunit gene identifies determinants of agonist potency. Neuroscience 102(3): 709-14. PubMed citation: 11226707

Kaku, K. and Matsumura, F. (1994). Identification of the site of mutation within the M2 region of the GABA receptor of the cyclodiene-resistant German cockroach. Comp. Biochem. Physiol. Pharmacol. Toxicol. Endocrinol. 108: 367-376

Kim, Y. C., Lee, H. G. and Han, K. A. (2007). D1 dopamine receptor dDA1 is required in the mushroom body neurons for aversive and appetitive learning in Drosophila. J. Neurosci. 27: 7640-7647. PubMed Citation: 17634358

Kolodziejczyk, A., Sun, X., Meinertzhagen, I. A. and Nässel, D. R. (2008). Glutamate, GABA and acetylcholine signaling components in the lamina of the Drosophila visual system. PLoS ONE 3(5): e2110. PubMed citation: 18464935

Lamsa, K., Palva, J. M., Ruusuvuori, E., Kaila, K. and Taira, T. (2000). Synaptic GABA(A) activation inhibits AMPA-kainate receptor-mediated bursting in the newborn (P0-P2) rat hippocampus. J Neurophysiol 83: 359-366. PubMed citation: 10634879

Larkin, A., et al. (2010). Central synaptic mechanisms underlie short-term olfactory habituation in Drosophila larvae. Learn Mem. 17(12): 645-53. PubMed Citation: 21106688

Lee, D., Su, H. and O'Dowd, D. K. (2003). GABA receptors containing Rdl subunits mediate fast inhibitory synaptic transmission in Drosophila neurons. J. Neurosci. 23(11): 4625-34. PubMed citation: 12805302

Liu, X., Krause, W. C. and Davis, R. L. (2007). GABA_A receptor RDL inhibits Drosophila olfactory associative learning. Neuron 56(6): 1090-102. PubMed citation: 18093529

Liu, X. and Davis, R. L. (2009a). The GABAergic anterior paired lateral neuron suppresses and is suppressed by olfactory learning. Nat. Neurosci. 12: 53-59. PubMed Citation: 19043409

Liu, X., Buchanan, M. E., Han, K. A. and Davis, R. L. (2009b). The GABA_A receptor RDL suppresses the conditioned stimulus pathway for olfactory learning. J. Neurosci. 29(5): 1573-9. PubMed Citation: 19193904

MacLeod, K. and Laurent, G. (1996). Distinct mechanisms for synchronization and temporal patterning of odor-encoding neural assemblies. Science 274: 976-979. PubMed citation: 8875938

Mody, I., De Koninck, Y. D., Otis, T. S. and Soltesz, I. (1994)/ Bridging the cleft at GABA synapses in the brain. Trends Neurosci 17: 517-525. PubMed citation: 7532336

Olshausen, B. A. and Field, D. J.(2004). Sparse coding of sensory inputs. Curr. Opin. Neurobiol. 14: 481-487. PubMed citation: 15321069

Palva, J. M., et al. (2000). Fast network oscillations in the newborn rat hippocampus in vitro. J Neurosci 20: 1170-1178. PubMed citation: 10648721

Perez-Orive, J., et al. (2002). Oscillations and sparsening of odor representations in the mushroom body. Science 297: 359-365. PubMed citation: 12130775

Riemensperger, T., Voller, T., Stock, P., Buchner, E. and Fiala, A. (2005). Punishment prediction by dopaminergic neurons in Drosophila. Curr. Biol. 15: 1953-1960. PubMed Citation: 16271874

Rohrbough, J. and Broadie, K. (2002). Electrophysiological analysis of synaptic transmission in central neurons of Drosophila larvae. J. Neurophysiol. 88: 847-860. PubMed citation: 12163536

Sachse, S., Rueckert, E., Keller, A., Okada, R., Tanaka, N. K., Ito, K. and Vosshall, L. B. (2007). Activity-dependent plasticity in an olfactory circuit. Neuron 56: 838-850. PubMed Citation: 18054860

Sattelle, D. B. (1990). GABA receptors of insects. Adv. Insect. Physiol. 22: 1-113

Stopfer, M., Bhagavan, S., Smith, B. H. and Laurent, G. (1997). Impaired odour discrimination on desynchronization of odour-encoding neural assemblies. Nature 390: 70-74. PubMed citation: 9363891

Vicini, S. (1999). New perspectives in the functional role of GABA_A channel heterogeneity. Mol. Neurobiol. 19: 97-110. PubMed citation: 10371465

Wilson, R. I. and Laurent, G. (2005). Role of GABAergic inhibition in shaping odor-evoked spatiotemporal patterns in the Drosophila antennal lobe. J. Neurosci. 25: 9069-9079. PubMed citation: 16207866

Yasuyama, K., Meinertzhagen, I. A. and Schurmann, F. W. (2002). Synaptic organization of the mushroom body calyx in Drosophila melanogaster. J. Comp. Neurol. 445: 211-226. PubMed citation: 11920702

Zhang, H.-G., et al. (1995). Subunit composition determines picrotoxin and bicuculline sensitivity of Drosophila -aminobutyric acid receptors. Mol Pharmacol 48: 835-840. PubMed citation: 7476913

date revised: 20 March 2012

Home page: The Interactive Fly © 2008 Thomas Brody, Ph.D.