InteractiveFly: GeneBrief

Resistant to dieldrin: Biological Overview | References


Gene name - Resistant to dieldrin

Synonyms -

Cytological map position - 67A1-67A1

Function - channel

Keywords - GABA-A receptor, memory acquisition, sleep

Symbol - Rdl

FlyBase ID: FBgn0004244

Genetic map position - 3L:9,143,695..9,170,704 [-]

Classification - Neurotransmitter-gated ion-channel ligand binding domain

Cellular location - transmembrane



NCBI links: Precomputed BLAST | EntrezGene

Recent literature
Tachibana, S., Touhara, K. and Ejima, A. (2015). Modification of male courtship motivation by olfactory habituation via the GABAA receptor in Drosophila melanogaster. PLoS One 10: e0135186. PubMed ID: 26252206
Summary:
A male-specific component, 11-cis-vaccenyl acetate (cVA) works as an anti-aphrodisiac pheromone in Drosophila melanogaster. The presence of cVA on a male suppresses the courtship motivation of other males and contributes to suppression of male-male homosexual courtship, while the absence of cVA on a female stimulates the sexual motivation of nearby males and enhances the male-female interaction. However, little is known how a male distinguishes the presence or absence of cVA on a target fly from either self-produced cVA or secondhand cVA from other males in the vicinity. This study demonstrates that male flies have keen sensitivity to cVA; therefore, the presence of another male in the area reduces courtship toward a female. This reduced level of sexual motivation, however, could be overcome by pretest odor exposure via olfactory habituation to cVA. Real-time imaging of cVA-responsive sensory neurons using the neural activity sensor revealed that prolonged exposure to cVA decreased the levels of cVA responses in the primary olfactory center. Pharmacological and genetic screening revealed that signal transduction via GABAA receptors contributed to this olfactory habituation. It was also found that the habituation experience increased the copulation success of wild-type males in a group. In contrast, transgenic males, in which GABA input in a small subset of local neurons was blocked by RNAi, failed to acquire the sexual advantage conferred by habituation. Thus, this study illustrates a novel phenomenon in which olfactory habituation positively affects sexual capability in a competitive environment.

Mohammad, F., Aryal, S., Ho, J., Stewart, J. C., Norman, N. A., Tan, T. L., Eisaka, A. and Claridge-Chang, A. (2016). Ancient anxiety pathways influence Drosophila defense behaviors. Curr Biol 26: 981-986. PubMed ID: 27020741
Summary:
Anxiety helps us anticipate and assess potential danger in ambiguous situations; however, the anxiety disorders are the most prevalent class of psychiatric illness. Emotional states are shared between humans and other animals, as observed by behavioral manifestations, physiological responses, and gene conservation. Anxiety research makes wide use of three rodent behavioral assays-elevated plus maze, open field, and light/dark box-that present a choice between sheltered and exposed regions. Exposure avoidance in anxiety-related defense behaviors was confirmed to be a correlate of rodent anxiety by treatment with known anxiety-altering agents and is now used to characterize anxiety systems. Modeling anxiety with a small neurogenetic animal would further aid the elucidation of its neuronal and molecular bases. Drosophila neurogenetics research has elucidated the mechanisms of fundamental behaviors and implicated genes that are often orthologous across species. In an enclosed arena, flies stay close to the walls during spontaneous locomotion, a behavior proposed to be related to anxiety. This study tested this hypothesis with manipulations of the GABA receptor, serotonin signaling, and stress. The effects of these interventions were strikingly concordant with rodent anxiety, verifying that these behaviors report on an anxiety-like state. Application of this method was able to identify several new fly anxiety genes. The presence of conserved neurogenetic pathways in the insect brain identifies Drosophila as an attractive genetic model for the study of anxiety and anxiety-related disorders, complementing existing rodent systems.
Raccuglia, D., Yan McCurdy, L., Demir, M., Gorur-Shandilya, S., Kunst, M., Emonet, T. and Nitabach, M. N. (2016). Presynaptic GABA receptors mediate temporal contrast enhancement in Drosophila olfactory sensory neurons and modulate odor-driven behavioral kinetics. eNeuro 3 [Epub ahead of print]. PubMed ID: 27588305
Summary:
Contrast enhancement mediated by lateral inhibition within the nervous system enhances the detection of salient features of visual and auditory stimuli, such as spatial and temporal edges. However, it remains unclear how mechanisms for temporal contrast enhancement in the olfactory system can enhance the detection of odor plume edges during navigation. To address this question, pulses of high odor intensity that induce sustained peripheral responses in olfactory sensory neurons (OSNs) were delivered to Drosophila melanogaster flies. Optical electrophysiology was used to directly measure electrical responses in presynaptic terminals, and it was demonstrated that sustained peripheral responses are temporally sharpened by the combined activity of two types of inhibitory GABA receptors, ionotropic GABAA and metabotropic GABAB inhibitory receptors to generate contrast-enhanced voltage responses in central OSN axon terminals. Furthermore, it was shown how these GABA receptors modulate the time course of innate behavioral responses after odor pulse termination, demonstrating an important role for temporal contrast enhancement in odor-guided navigation.
Seugnet, L., Dissel, S., Thimgan, M., Cao, L. and Shaw, P. J. (2017). Identification of genes that maintain behavioral and structural plasticity during sleep loss. Front Neural Circuits 11: 79. PubMed ID: 29109678
Summary:
Although patients with primary insomnia experience sleep disruption, they are able to maintain normal performance on a variety of cognitive tasks. This observation suggests that insomnia may be a condition where predisposing factors simultaneously increase the risk for insomnia and also mitigate against the deleterious consequences of waking. To gain insight into processes that might regulate sleep and buffer neuronal circuits during sleep loss, three genes, fat facet (faf), highwire (hiw) and the GABA receptor Resistance to dieldrin (Rdl), were manipulated that were differentially modulated in a Drosophila model of insomnia. The results indicate that increasing faf and decreasing hiw or Rdl within wake-promoting large ventral lateral clock neurons (lLNvs) induces sleep loss. As expected, sleep loss induced by decreasing hiw in the lLNvs results in deficits in short-term memory and increases of synaptic growth. However, sleep loss induced by knocking down Rdl in the lLNvs protects flies from sleep-loss induced deficits in short-term memory and increases in synaptic markers. Surprisingly, decreasing hiw and Rdl within the Mushroom Bodies (MBs) protects against the negative effects of sleep deprivation (SD) as indicated by the absence of a subsequent homeostatic response, or deficits in short-term memory. Together these results indicate that specific genes are able to disrupt sleep and protect against the negative consequences of waking in a circuit dependent manner.
Li, Q., Li, Y., Wang, X., Qi, J., Jin, X., Tong, H., Zhou, Z., Zhang, Z. C. and Han, J. (2017). Fbxl4 serves as a clock output molecule that regulates sleep through promotion of rhythmic degradation of the GABAA receptor. Curr Biol [Epub ahead of print]. PubMed ID: 29174887
Summary:
The timing of sleep is tightly governed by the circadian clock, which contains a negative transcriptional feedback loop and synchronizes the physiology and behavior of most animals to daily environmental oscillations. However, how the circadian clock determines the timing of sleep is largely unclear. In vertebrates and invertebrates, the status of sleep and wakefulness is modulated by the electrical activity of pacemaker neurons that are circadian regulated and suppressed by inhibitory GABAergic inputs. This study showed that Drosophila GABAA receptors undergo rhythmic degradation in arousal-promoting large ventral lateral neurons (lLNvs) and their expression level in lLNvs displays a daily oscillation. This study also demonstrated that the E3 ligase Fbxl4 promotes GABAA receptor ubiquitination and degradation and revealed that the transcription of fbxl4 in lLNvs is CLOCK dependent. Finally, it was demonstrated that Fbxl4 regulates the timing of sleep through rhythmically reducing GABA sensitivity to modulate the excitability of lLNvs. This study uncovered a critical molecular linkage between the circadian clock and the electrical activity of pacemaker neurons and demonstrated that CLOCK-dependent Fbxl4 expression rhythmically downregulates GABAA receptor level to increase the activity of pacemaker neurons and promote wakefulness.
Gowda, S. B. M., Paranjpe, P. D., Reddy, O. V., Thiagarajan, D., Palliyil, S., Reichert, H. and VijayRaghavan, K. (2018). GABAergic inhibition of leg motoneurons is required for normal walking behavior in freely moving Drosophila. Proc Natl Acad Sci U S A 115(9): E2115-e2124. PubMed ID: 29440493
Summary:
Walking is a complex rhythmic locomotor behavior generated by sequential and periodical contraction of muscles essential for coordinated control of movements of legs and leg joints. Studies of walking in vertebrates and invertebrates have revealed that premotor neural circuitry generates a basic rhythmic pattern that is sculpted by sensory feedback and ultimately controls the amplitude and phase of the motor output to leg muscles. However, the identity and functional roles of the premotor interneurons that directly control leg motoneuron activity are poorly understood. This study took advantage of the powerful genetic methodology available in Drosophila to investigate the role of premotor inhibition in walking by genetically suppressing inhibitory input to leg motoneurons. For this, an algorithm was developed for automated analysis of leg motion to characterize the walking parameters of wild-type flies from high-speed video recordings. Further, genetic reagents were used for targeted RNAi knockdown of inhibitory neurotransmitter receptors in leg motoneurons together with quantitative analysis of resulting changes in leg movement parameters in freely walking Drosophila. The findings indicate that targeted down-regulation of the GABAA receptor Rdl (Resistance to Dieldrin) in leg motoneurons results in a dramatic reduction of walking speed and step length without the loss of general leg coordination during locomotion. Genetically restricting the knockdown to the adult stage and subsets of motoneurons yields qualitatively identical results. Taken together, these findings identify GABAergic premotor inhibition of motoneurons as an important determinant of correctly coordinated leg movements and speed of walking in freely behaving Drosophila.
Li, X., Ishimoto, H. and Kamikouchi, A. (2018). Auditory experience controls the maturation of song discrimination and sexual response in Drosophila. Elife 7. PubMed ID: 29555017
Summary:
In birds and higher mammals, auditory experience during development is critical to discriminate sound patterns in adulthood. However, the neural and molecular nature of this acquired ability remains elusive. In fruit flies, acoustic perception has been thought to be innate. This study reports, surprisingly, that auditory experience of a species-specific courtship song in developing Drosophila shapes adult song perception and resultant sexual behavior. Preferences in the song-response behaviors of both males and females were tuned by social acoustic exposure during development. This study examined the molecular and cellular determinants of this social acoustic learning and found that GABA signaling acting on the GABAA receptor Rdl in the pC1 neurons, the integration node for courtship stimuli, regulated auditory tuning and sexual behavior. These findings demonstrate that maturation of auditory perception in flies is unexpectedly plastic and is acquired socially, providing a model to investigate how song learning regulates mating preference in insects.

BIOLOGICAL OVERVIEW

In both mammals and insects, neurons involved in learning are strongly modulated by the inhibitory neurotransmitter GABA. The GABAA 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.

GABAA receptors are GABA-gated chloride channels. Accumulating pharmacological and genetic evidence suggests that GABAA 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 GABAA 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 GABAA 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 GABAA 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 GABAA 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 GABAAβ3 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 GABAAα5 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 GABAA 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 GABAA 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 GABAA 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 Rdlf02994, 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 GABAA 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 GABAA receptor and the expression level of gephyrin, a protein involved in GABAA 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 GABAA 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 GABAA 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 GABAA receptor RDL regulates the CS pathway in Drosophila olfactory learning. The conclusion that the GABAA 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 GABAA 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 GABAA 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 GABAA 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 GABAA receptors modulate the CS pathway. Moreover, other studies have shown that the surface expression of GABAA 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 GABAA receptors, again indicating a role for GABAA receptors in the CS pathway. The current results, together with these previous studies, strongly indicate that GABAA receptors regulate the CS pathway for associative learning (Liu, 2009b).

A role for GABAA 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 GABAA 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 GABAA 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 GABAA 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 GABAA 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)

Calcium-stores mediate adaptation in axon terminals of olfactory receptor neurons in Drosophila

In vertebrates and invertebrates, sensory neurons adapt to variable ambient conditions, such as the duration or repetition of a stimulus, a physiological mechanism considered as a simple form of non-associative learning and neuronal plasticity. Although various signaling pathways, as cAMP, cGMP, and the inositol 1,4,5-triphosphate receptor (InsP3R) play a role in adaptation, their precise mechanisms of action at the cellular level remain incompletely understood. In Drosophila odor-induced Ca2+-response in axon terminals of olfactory receptor neurons (ORNs) has been shown to be related to odor duration. In particular, a relatively long odor stimulus (such as 5 s) triggers the induction of a second component involving intracellular Ca2+-stores. A recently developed in-vivo bioluminescence imaging approach was used to quantify the odor-induced Ca2+-activity in the axon terminals of ORNs. Using either a genetic approach to target specific RNAs, or a pharmacological approach, this study showed that the second component, relying on the intracellular Ca2+-stores, is responsible for the adaptation to repetitive stimuli. In the antennal lobes (a region analogous to the vertebrate olfactory bulb) ORNs make synaptic contacts with second-order neurons, the projection neurons (PNs). These synapses are modulated by GABA, through either GABAergic local interneurons (LNs) and/or some GABAergic PNs. Application of GABAergic receptor antagonists, both GABAA or GABAB, abolishes the adaptation, while RNAi targeting the GABABR (a metabotropic receptor) within the ORNs, blocks the Ca2+-store dependent component, and consequently disrupts the adaptation. These results indicate that GABA exerts a feedback control. Finally, at the behavioral level, using an olfactory test, genetically impairing the GABABR or its signaling pathway specifically in the ORNs disrupts olfactory adapted behavior. Taken together, these results indicate that a relatively long lasting form of adaptation occurs within the axon terminals of the ORNs in the antennal lobes, which depends on intracellular Ca2+-stores, attributable to a positive feedback through the GABAergic synapses (Murmu, 2011).

This study provides evidence that the bioluminescent (GFP-aequorin) Ca2+-sensor is sensitive enough to monitor the Ca2+-response following various protocols (duration and repetition-frequency) of odor application. 1 s of odor induces a response which does not significantly decrease if repeated every 5 min, whereas a longer stimulus, such as 5 s, is sufficient to induce a decrease in response following repeated odor stimulations (adaptation). Similarly, using a 5 s odor stimulation and increasing the frequency of repetition to 1-min intervals also induces, in an odor specific manner, a faster adaptation. It was also demonstrated that prolonged odor application (up to 2 min) generates a sustained Ca2+-response within the ORN axon terminals, indicating that the ORNs are capable of responding as long as the odor is presented, of even longer. This work also indicates that the GFP-aequorin probe is not a limiting factor for the detection of the Ca2+-activity. These physiological results (reduction of the Ca2+-activity according to prolonged/sustained odor duration and/or odor repetition) are consistent with previous studies which report that adaptation depends both on the duration of a stimulus and on the frequency of its repetition (Murmu, 2011).

Different physiological approaches, based either on fluorescence brain imaging or electrophysiological techniques have previously reported odor-induced activity in different interconnected neurons in the antennal lobes of different invertebrate model organisms, including honeybees with the goal of deciphering the neural odor code. However, except for one study performed in locusts, which indirectly described a form of adaptation, long-lasting forms of adaptation within ORNs such as that described in this study have not been reported. This is likely due to the experimental design of these previous studies, which either generally took into account the odor-induced signal solely after the response was stabilized (generally after about 5 successive odor applications), or used a shorter odor stimulation duration (< = 1 s), which as demonstrated in this study, is not sufficient to induce detectable and reliable adaptation. Additionally, others have relied on extracellular recordings of the sensillae of the antennae which reflects the activity occurring in the cell-bodies of the ORNs. In this study, monitoring the axon terminals of the ORNs, 5 s odor stimulations, repeated at 5-min intervals, induced a relatively long-lasting adaptation that resembles in term of kinetics, the long-lasting adaptation (LLA) reported in ORNs in salamanders. Indeed and interestingly, the recovery time (15 min for spearmint and octanol and 30 min for citronella) occurs over a similar time scale in salamander ORNs (which are different from the long-lasting olfactory adaptation described in C. elegans. However, in contrast to long-lasting adaptation, which was reported in isolated ORNs, the adaptation described in this study seems to rely on different mechanisms, since it is sensitive to a 'feedback control' provided by GABAergic synaptic transmission within the antennal lobes (Murmu, 2011).

In Drosophila, mutants lacking InsP3R are defective in olfactory adaptive behavior. In vertebrates, different forms of olfactory adaptation have also been reported in the ORNs. First, this study shows in Drosophila that an adaptation mechanism occurs in axon terminal of the ORNs in the antennal lobes. Second, using two independent approaches, pharmacological and genetic, it was shown that odor-induced specific adaptation relies principally on InsP3R and RyR. When these two different receptors are blocked or knocked-down, although some difference (variability) can be observed between different conditions, overall the odor-induced Ca2+-response no longer adapts or is severely affected. More specifically, it seems that the lack of adaptation is due to the non-induction of the 'second delayed and slow rising component' of the Ca2+-response, which is triggered in particular and specific conditions: when the duration of an odor stimulation is relatively long (1 s does not induce it, while 5 s induces an important second component. Alternatively, the second component of the response is also induced and visible particularly on the first and to a lesser extent, on the second odor applications, especially when the odor is successively repeated. This second component gradually vanishes with sequential repetition. That is, adaptation is not directly due to a decrease in the response, but rather indirectly to a defect in presynaptic Ca2+-increase, due to a lack of triggering release of intracellular Ca2+-stores, normally occurring in the first and successive responses following either a sufficiently strong (long stimulus) or repeated stimuli. These results suggest that one of the major intracellular mechanisms of adaptation depends on internal Ca2+-stores. In brief, the intracellular mechanism was blocked that allows the cell to adapt to long lasting or repetitive stimuli. Interestingly, in mammals, in hippocampal CA3 pyramidal neurons, intracellular Ca2+-stores, which are controlled by InsP3R and/or RyR at the presynaptic terminal, have been previously implicated in neurotransmitter release as well as in synaptic plasticity (Murmu, 2011).

In vertebrates, neuronal plasticity related to odor representation occurs at the synapse between the ORNs and the second-order neurons in the olfactory bulb glomeruli, a region analogous to the invertebrate antennal lobes. At this synapse, signal transmission is modulated presynaptically by several mechanisms, a major one being via the metabotropic GABAB receptors. This suppresses presynaptic Ca2+-influx and subsequently transmitter release from the receptor neurons terminal. At least two kinds of presynaptic inhibition (intra- and interglomerular) are mediated by GABAB receptors. Intraglomerular presynaptic inhibition seems to control input sensitivity, while interglomerular presynaptic inhibition seems to increase the contrast of sensory input (although the two studies addressing this question in-vivo show contradictory results). In Drosophila, a similar mechanism seems to occur, as interglomerular presynaptic inhibition, mediated by both ionotropic and metabotropic receptors on the same axon terminal of the ORNs, mediate gain control mechanism, serving to adjust the gain of PN in response to ORN stimulation (Olsen, 2008). Yet another study has suggested that GABAB but not GABAA receptors are involved in presynaptic inhibition (Root, 2008) yielding a contradiction. In this study, by monitoring the Ca2+-release from the axon terminals of ORNs, in experimental conditions that generate a long-lasting form of adaptation, it was shown that GABAergic synaptic transmission plays a role in adaptation. Both ionotropic GABAAR antagonists, bicuculline and picrotoxin, block partially or completely the Ca2+-response, while, CGP54626, a metabotropic GABABR antagonist, also blocks the adaptation, albeit not completely. It should be mentioned that application of picrotoxin per se induces a strong transient Ca2+-release within the axon terminals of the ORNs, even without odor application. This 'transient release effect' likely disturbs the resting state of the neurons, which probably accounts for the important reduction observed in the amplitude of the odor-induced response. Nevertheless, these results suggest that both types of GABA receptors (A and B) are involved in adaptation. Moreover, as proposed by the study of Olsen and Wilson (2008), it cannot be ruled-out that ORNs also express different subtypes of GABAAR (homo- and/or heteromultimers), since the results showed that picrotoxin and particularly bicuculline, two distinct inhibitors of GABAAR, block adaptation. Another possibility is that the effect of the two GABAAR antagonists results from the blockage of GABAAR on other neurons in the antennal lobes, as the LNs or certain PNs (which have not yet been demonstrated). Lastly and unfortunately, this pharmacological approach does not allow distinguishing by which precise neurons this GABAergic-dependent adaptation is mediated. With the goal of clarifying precisely in which neurons GABAergic transmission acts, the metabotropic GABABR (GABABR2-RNAi) or its signaling pathway (UAS-PTX) were blocked directly within ORNs. This yields defects in long-lasting adaptation for several conditions, seemingly in an odor specific manner. Therefore, although GABAergic effects have been described in ORNs of both Drosophila and mammals, to support 'feedback inhibition', this study reports that in different experimental conditions such as a long odor duration (5 s) and/or repetition of the stimulus, it also participates in the adaptation process. Indeed, the results suggest that GABA signaling support a positive (excitatory) feedback control instead of an inhibitory feedback, as formerly reported by other studies. Though these results seem to be contradictory, some explanations can be provided. First, as aforementioned, the experimental conditions are different: this study used a relatively high odor concentration with relatively long odor duration (5 s). In addition, recordings were taken immediately from the first odor application and the successive one, while in the experimental protocol of certain studies, the odor is generally presented several times (priming) before the beginning of recording. Consequently, it seems that these previous studies were performed on already adapted ORNs. This implies that the neuronal network in the antennal lobes was already stimulated, and therefore its dynamics was probably already modified, since as described in this study, an important effect occurs immediately after the first odor application. Moreover, GFP-aequorin allows monitoring, in continuity over a long time period, the intracellular level of calcium with high sensitivity to [Ca2+] (from ~ 10-7 to 10-3). In addition, although it is not possible to precisely assign which glomeruli are activated, this approach allows visualizing simultaneously the odor-induced Ca2+-activity from the entire antennal lobes (the overall depth). Therefore, the outcome of the overall response of the antennal lobes is being monitored, instead of the response from single or a few glomeruli. Finally, in vertebrates it has been reported that in certain experimental conditions, GABA could be excitatory, although this contradiction cannot yet be precisely explained. Furthermore, it seems that a given synapse can display inhibitory effects under one protocol and an excitatory effect with another. Notably, it has been reported that a short stimulation of GABA is inhibitory, while during a long stimulation, the GABA effect can switch from inhibitory to excitatory. Interestingly, this particular 'switching effect' could potentially explain the current 'contradictory' situation reported in this study: in the current experimental conditions, in which a relatively long odor stimulus (5 s) was used, GABA generates an excitatory effect, whereas in previous studies based on short (<1 s) odor stimuli, GABA seems to be inhibitor. This difference in the duration of stimuli could perhaps account for such inverted or 'switching effects' (Murmu, 2011).

To explore the behavioral and functional consequences of disturbing the GABAergic signaling pathway, flies were studied with a GABABR2 (RNAi) ORN-specific genetic knockdown, as well those with a component of its signaling pathway, the G-protein, blocked by the pertussis toxin. Both groups of flies present strong behavioral deficits, as adaptation-disrupted flies are not able to discern between odors and air after 5-min of exposure to odor. Interestingly, control flies reverse their choice preferring odor after a 5-min pre-exposure (adaptation) suggesting that in these experimental conditions, the meaning of the odor changes in the fly's adapted state. These results are consistent with previous studies suggesting that adaptation could serve to extend the operating range of sensory systems over different stimulus intensities. In other terms, adaptation modifies the sensitivity (threshold) to the odor, as previously reported in different organisms, such as C. elegans and vertebrates including humans. This phenomenon is similar to that in other sensory modalities, as in visual system, where light adaptation in photoreceptors sets the gain, allowing vision at both high and low light levels. As previously reported, odors could be repulsive (at high concentrations) or attractive (at low concentrations). In the current experimental conditions in control flies the odors are repulsive. However, after 5-min of preexposure, the flies adapt to this odor concentration, and when tested at the same concentration odors are then likely only weakly perceived and therefore might correspond to an attractive 'weak-odor concentration'. In a former study in similar experimental conditions, it was reported that the flies are attracted by each of these three odors for a weak odor concentration. Interestingly, reverse odor preference has already been reported in C. elegans, resulting from presynaptic changes involving a receptor-like guanylate cyclase (GCY-28) via the diacylglycerol/protein kinase C pathway. Finally, the fact that without pre-exposure all groups of flies preferred the control arm and were repelled by the odorants indicates that the odor acuity of these flies is intact. In other words, odor-adaptation and not odor-acuity is affected in each of these groups of flies. These results strengthen the idea that odor perception and adaptation are indeed two distinct and separable processes (Murmu, 2011).

This study has demonstrated that the adaptation process occurring specifically in the axon terminals of the ORNs depends on intracellular Ca2+-stores, through InsP3 and ryanodine receptors. Moreover, evidence is provided that this Ca2+-release requires synaptic transmission, since it does not occur when the cholinergic receptors are blocked (α-bungarotoxin experiment). It also requires a feedback control through GAB A, since blocking GABAB signaling within ORNs prevents or strongly affects adaptation, suggesting that a local neuronal network mediated by GABAergic neurons is involved (for a more complete overview, see the schematic model of synaptic interactions within the antennal lobes. In complement to the brain imaging data, knocking-down the metabotropic GABABR2, or its signaling pathway specifically in the ORNs, yields olfactory functional behavioral deficits. These results, combined with the results of blocking the InsP3R or RyR suggest that a crucial olfactory integration process that can be ascribed to a form of neuronal plasticity and/or short-term memory occurs directly in the ORNs immediately after the first odor application or during a prolonged odor application. Thus, this effect could resemble the long-lasting form of odor adaptation described previously at the cellular and systems levels in vertebrates, including humans. By extension, it is hypothesized that in humans, the well-known 'odor-specific transient functional anosmia' following a prolonged odor exposure, which results from an adaptation, may also rely on intracellular Ca2+-stores (Murmu, 2011).

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

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

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

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

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

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

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

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

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

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

GABAA receptor-expressing neurons promote consumption in Drosophila melanogaster

Feeding decisions are highly plastic and bidirectionally regulated by neurons that either promote or inhibit feeding. In Drosophila melanogaster, recent studies have identified GABAergic interneurons that act as critical brakes to prevent incessant feeding. These GABAergic neurons may inhibit target neurons that drive consumption. This study tested this hypothesis by examining GABA receptors and neurons that promote consumption. Resistance to dieldrin (RDL), a GABAA type receptor, is required for proper control of ingestion. Knockdown of Rdl in a subset of neurons causes overconsumption of tastants. Acute activation of these neurons is sufficient to drive consumption of appetitive substances and non-appetitive substances and acute silencing of these neurons decreases consumption. Taken together, these studies identify GABAA receptor-expressing neurons that promote Drosophila ingestive behavior and provide insight into feeding regulation (Cheung, 2017).

The dissection of neural circuits that underlie consumption remains an important challenge toward understanding the regulation of feeding behavior. This study identifies neurons that regulate the consumption of non-appetitive and appetitive substances, and depend on the expression of RDL receptor for proper regulation of consumption. These RDL receptor-expressing neurons are able to orchestrate consumption regardless of taste quality, as knockdown of Rdl expression within these neurons not only causes overconsumption of sugar, bitter, and water substances, but tasteless substances as well. Acute activation of these neurons also caused overconsumption of sweet, bitter and water substances, whereas blocking neurotransmission of these neurons results in decreased sucrose consumption in starved flies. These studies reveal a subset of neurons that play a critical role in promoting consumption (Cheung, 2017).

Previous studies have identified two different classes of interneurons that trigger sucrose consumption. FDG neurons are located in the SEZ and respond to sugar stimulation on the proboscis and the cholinergic IN1 neurons respond to sugar stimulation of the internal mouthparts. These two classes of neurons respond selectively to sucrose, suggesting that there is a pathway selective for regulating sucrose consumption. Similarly, ectopic activation of these neurons increased consumption of sucrose but not water or bitter. These studies indicate that consumption of sucrose is regulated independently of consumption of water or bitter and argue for distinct circuits mediating consumption for each class of tastant. The RDL-expressing neurons differ from previously identified consumption neurons because either knockdown of Rdl or optogenetic activation of these neurons elicited consumption not only of appetitive substances, but also of non-appetitive substances. One model suggested by these studies that bears testing is that there may be distinct circuits for sweet, water, and bitter food sources that all converge on the RDL-expressing neurons (Cheung, 2017).

Knockdown of Rdl results in increased consumption of water, sucrose and bitter substances. These RDL neurons may be inhibited by GABAergic neurons such as DSOG1. Previous studies indicate that DSOG1 neurons act as a tonic inhibitor of consumption. Flies with silenced DSOG1 neurons overconsume all taste substances independent of taste quality and nutritional state, very similar to the phenotype observed when activating the RDL neurons in this study. An attractive model is that GABA release from DSOG1 inhibits the RDL neurons, restricting consumption. Indeed, 'studies show that RDL neuronal silencing is able to suppress the DSOG1-silencing phenotype. Although the data are consistent with the model that DSOG1 acts on the RDL neurons, it remains possible that the RDL neurons and DSOG1 influence parallel pathways. Further characterization of the RDL neurons that promote consumption and the DSOG1 neurons that inhibit consumption will enable distinguishing of these models (Cheung, 2017).

This study demonstrates that RDL function in a subset of neurons is critical for the regulation of consumption of all substances, regardless of taste modality. Further studies characterizing these neurons and their interactions with the different neurons that regulate feeding will provide insight into the temporal dynamics and plasticity in feeding decisions (Cheung, 2017).

GABAergic inhibition of leg motoneurons is required for normal walking behavior in freely moving Drosophila

Walking is a complex rhythmic locomotor behavior generated by sequential and periodical contraction of muscles essential for coordinated control of movements of legs and leg joints. Studies of walking in vertebrates and invertebrates have revealed that premotor neural circuitry generates a basic rhythmic pattern that is sculpted by sensory feedback and ultimately controls the amplitude and phase of the motor output to leg muscles. However, the identity and functional roles of the premotor interneurons that directly control leg motoneuron activity are poorly understood. This study took advantage of the powerful genetic methodology available in Drosophila to investigate the role of premotor inhibition in walking by genetically suppressing inhibitory input to leg motoneurons. For this, an algorithm was developed for automated analysis of leg motion to characterize the walking parameters of wild-type flies from high-speed video recordings. Further, genetic reagents were used for targeted RNAi knockdown of inhibitory neurotransmitter receptors in leg motoneurons together with quantitative analysis of resulting changes in leg movement parameters in freely walking Drosophila. The findings indicate that targeted down-regulation of the GABAA receptor Rdl (Resistance to Dieldrin) in leg motoneurons results in a dramatic reduction of walking speed and step length without the loss of general leg coordination during locomotion. Genetically restricting the knockdown to the adult stage and subsets of motoneurons yields qualitatively identical results. Taken together, these findings identify GABAergic premotor inhibition of motoneurons as an important determinant of correctly coordinated leg movements and speed of walking in freely behaving Drosophila (Gowda, 2018).

This report investigated the role of inhibitory premotor input to leg motoneurons in the walking behavior of freely moving Drosophila by suppression of GABAergic inhibitory input to leg motoneurons. The findings indicate that the reduction of inhibitory GABAergic input to leg motoneurons caused by targeted Rdl down-regulation has marked effects on walking behavior. Thus, walking speed was markedly slower compared with controls, and correlated with this, the amplitudes of the leg protraction (swing) and retraction (stance) phases were significantly smaller and the durations of protraction (swing) and retraction (stance) phases were significantly higher than in controls. Moreover, the concurrency state, in which three legs swing together in a tripod gait, was proportionally reduced compared with controls. These prominent effects on walking parameters are similar regardless of whether Rdl down-regulation occurs throughout development or whether it is restricted to the adult stage. Taken together, these findings reveal a prominent, albeit highly specific role of GABAergic premotor inhibitory input to leg motoneurons in the control of normal walking behavior (Gowda, 2018).

The insight into the role of premotor inhibition in walking control reported in this stuyd is the result of two key experimental methodologies. The first is the highly specific genetic access to identified neuronal populations that can now be attained in the Drosophila model system. This is made possible through remarkable targeted expression systems, which together with the availability of libraries of genetically encoded drivers and reporters for molecular manipulation, make it possible to selectively up or down-regulate gene expression in highly specific neuronal populations in intact and freely behaving animals. This study used the Gal4/UAS expression system to achieve targeted genetic access to leg motoneurons and down-regulate GABAergic premotor input to these motoneurons by Rdl RNAi expression. In addition different forms of the Gal80 repressor were used to limit Gal4/UAS targeted expression to adult stages or to specific regions of the central nervous system and, hence, refine the spatiotemporal specificity of the resulting genetic access to motoneurons. Given the wealth of Gal4 drivers and UAS-RNAi reporters currently available, it will be possible to use similar transgenic technology to manipulate other inhibitory and excitatory neurotransmitter receptors in future studies of interneuronal components of the walking circuitry (Gowda, 2018).

The second key method is the development of an advanced automated high-speed video recording and analysis technique that makes it possible to record the protraction (swing) and retraction (stance) phases at high spatiotemporal resolution for each leg in freely walking flies. With this technique, a quantitative assessment of leg motion parameters in freely walking animals can be carried out that can reveal subtle differences in amplitude and phase of movements of individual legs. This quantitative assessment has been critical for uncovering the role of premotor inhibition in walking behavior. Indeed, since the overall leg coordination during walking is unaffected by the reduction of GABAergic input to leg motoneurons, a more conventional qualitative behavioral analysis is unlikely to discern differences in walking between experimental and control animals. Recently, comparable high-resolution recording and analysis methods have been used to quantify leg movement parameters in freely walking flies. These studies have provided important information on walking speed, interleg coordination, and other locomotor parameters and have also documented a role of sensory proprioceptive input to step precision during walking. The fact that these methods for high-resolution recording and analysis are currently available for studying leg movement parameters in freely walking flies should accelerate understanding of walking behavior and the neuronal circuitry involved in its control in Drosophila (Gowda, 2018).

Given the prominent role of inhibitory premotor input to leg motoneurons reported in this study, it will now be important to identify and genetically access the premotor interneurons that provide this inhibitory input. While there is currently little information on the identity of the premotor interneurons that control the activity of leg motoneurons in adult flies, insight into inhibitory premotor interneurons has recently been obtained in larval stages. Thus, in Drosophila larva, a set of inhibitory local interneurons, termed PMSI neurons, have been identified that control the speed of axial locomotion by limiting the burst duration of motoneurons involved in peristaltic locomotion (Kohsaka, 2014). Moreover, a second set of inhibitory premotor interneurons called GVLI neurons have been reported that may be part of a feedback inhibition system involved in terminating each of the waves of motor activity that underlie larval peristalsis (Itakura, 2015). Finally, a pair of segmentally repeated GABAergic interneurons termed GDL neurons have been identified that are necessary for the coordinated propagation of peristaltic motor waves during both forward and backward crawling movements of larvae (Gjorgjieva, 2013). Whether or not these inhibitory premotor interneurons persist into the adult stage and act in the control of walking behavior is not known. Glutamate has previously been suggested to act as an inhibitory neurotransmitter in the Drosophila CNS. To investigate the likelihood that centrally labeled OK371 nonmotoneurons might also play any role in the leg motoneuron inhibition a set of preliminary experiments were carried out involving knockdown of GluCl channels in motoneurons. These experiments reveal enhanced defects in walking behavior with loss of coordination, suggesting there could be a possible role of OK371-labeled glutamatergic interneurons in leg motoneuron inhibition (Gowda, 2018).

In general terms, there are fundamental similarities in the principle mechanisms of locomotion in insects and vertebrates. These mechanistic similarities might also reflect similar motor circuit properties. For example, much like the PMSI neurons in Drosophila, which control the speed of locomotion by limiting motoneuron burst duration, the premotor V1 spinal interneurons in mammals are involved in the regulation of leg motoneuron burst and step cycle duration and thus also likely control the speed of walking movements. Hence, a characterization of the behavioral effects of inhibitory input to leg motoneurons in Drosophila, notably in freely walking flies, is likely to provide useful comparative information for understanding the functional role, and possibly the evolutionary origin, of premotor inhibition in vertebrate locomotory circuitry (Gowda, 2018).

The GABAA 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 GABAA 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 GABAB 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).

Female contact modulates male aggression via a sexually dimorphic GABAergic circuit in Drosophila

Intraspecific male-male aggression, which is important for sexual selection, is regulated by environment, experience and internal states through largely undefined molecular and cellular mechanisms. To understand the basic neural pathway underlying the modulation of this innate behavior, a behavioral assay was established in Drosophila melanogaster, and the relationship between sexual experience and aggression was investigated. In the presence of mating partners, adult male flies exhibited elevated levels of aggression, which was largely suppressed by prior exposure to females via a sexually dimorphic neural mechanism. The suppression involved the ability of male flies to detect females by contact chemosensation through the pheromone-sensing ion channel Ppk29 and was mediated by male-specific GABAergic neurons acting on the GABAA receptor RDL in target cells. Silencing or activating this circuit led to dis-inhibition or elimination of sex-related aggression, respectively. It is proposed that the GABAergic inhibition represents a critical cellular mechanism that enables prior experience to modulate aggression (Yuan, 2013).

Aggression is a complex behavior that is regulated by various internal and external stimuli. To date, however, studies have remained largely focused on the sensory pathways involved in regulating baseline aggression, with less examination of the central components of the underlying neural pathway. Moreover, the close relationship between sex and aggression has been a fascinating topic in both biology and literature, but their intertwined nature and the underlying neurobiological basis have remained elusive. Using a behavioral genetics approach, this study identified a previously unknown neural pathway that underlies the modulation of sex-related male-male aggression in Drosophila by prior contacts with females (Yuan, 2013).

These results suggest that prior female encounter through direct physical contacts activates the pheromone-sensing ppk29 neurons, resulting in inhibition of the central aggression circuit via GABAergic mechanisms involving the RDL GABAA receptor, thereby suppressing the behavioral output for male-male aggression. The three levels of the neural pathway involved in this experience-dependent behavior modification all exhibited sexual dimorphism, consistent with the notion that morphological differences in male and female brains correlate with their distinct behavioral needs. It was possible to modify the aggressive behavior output by manipulating the circuit at each of these three steps, which possibly represented the sequence of the information relay involved in the native behaviors, the sensory input, the information processing and the execution of the behavior. However, it is recognized that the circuit components elucidated by these experiments are clearly only parts of the machinery responsible for aggression modulation. In addition, by identifying RDL as a molecular target for aggression regulation, this study provides an entry point for characterizing the missing link of aggression studies, namely the central neurons that respond to experience-dependent modulation and mediate the execution of aggressive behaviors. Thus, this work provides new insights regarding the intricate interactions between sexual experience and aggression and delineates the underlying mechanisms to inform potential means to suppress excessive aggression (Yuan, 2013).

The female contact-dependent suppression of male aggression may also be viewed as a form of learning-induced plasticity. The learning procedure in this case requires extended physical interactions between the male and the female (over 10 h), which could consist of repeated sessions of male courtship attempts and female rejection. As no obvious defects were observed in aggression suppression in genetic mutants with deficits in courtship conditioning, such as homer, eag, Shaker< and orb2 mutants, this experience-induced suppression of aggression is likely different from conventional courtship conditioning. Another interesting feature of this suppression is that it is long term, yet reversible, lasting up to 2 d after the female encounter. However, it also differs from well-studied long-term memory formation, as no defect was observed in amn mutants, which is required for long-term memory formation. The results implicate the fru+ d5-HT1B+ and GABA+ cluster of neurons in the central brain as the regulators of this suppression, but it remains to be determined whether these neurons are involved in the initiation, acquisition, execution or consolidation phase(s) of this behavior, what takes form as the underlying 'memory trace' and whether plasticity is manifested at the level of the number of neurons activated, neurite arborization, neuronal activity or some other aspect of neuronal signaling (Yuan, 2013).

Notwithstanding the emergence of Drosophila as a successful genetic model for aggression studies and the extensive characterization of its stereotypical motor display of aggression, the strong influence of genetic background over baseline aggression and locomotor activity often complicates Drosophila aggression studies. The current assay avoids such difficulties by consistently eliciting aggression in naive male flies in the presence of females and by inducing a strong suppression of aggression in males with prior female encounter. The small variations among different genetic backgrounds in the behavioral assay make it possible to identify critical cellular and molecular components involved in the regulation of aggression by experience (Yuan, 2013).

One purpose of studying aggression regulation in animal models is to eventually understand the basis of human violence and establish venues to reduce or prevent it. Psychophysiological studies suggest that the failure to maintain an appropriate level of aggression in humans is associated with impaired executive cognitive processes or emotion registration. As an innate behavior built largely on predetermined neural pathways, aggression in Drosophila males can be modulated by prior exposure to females through GABAergic inhibition. This study raises the possibility that an ancient and basic machinery of the central neuronal circuitry, GABAergic inhibition, could be part of a conserved mechanism to modulate the level of aggression in males and ensure proper balance between reproductive competition and individual survival (Yuan, 2013).

Wide awake mediates the circadian timing of sleep onset

How the circadian clock regulates the timing of sleep is poorly understood. This study identifies a Drosophila mutant, wide awake (wake), that exhibits a marked delay in sleep onset at dusk. Loss of Wake in a set of arousal-promoting clock neurons, the large ventrolateral neurons (l-LNvs), impairs sleep onset. Wake levels cycle, peaking near dusk, and the expression of Wake in l-LNvs is Clock dependent. Strikingly, Clock and cycle mutants also exhibit a profound delay in sleep onset, which can be rescued by restoring Wake expression in LNvs. Wake interacts with the GABAA receptor Resistant to Dieldrin (Rdl), upregulating its levels and promoting its localization to the plasma membrane. In wake mutant l-LNvs, GABA sensitivity is decreased and excitability is increased at dusk. It is proposed that Wake acts as a clock output molecule specifically for sleep, inhibiting LNvs at dusk to promote the transition from wake to sleep (Liu, 2014).

The molecular pathways by which the circadian clock modulates the timing of sleep are unknown. This study identified a molecule, Wide Awake, that promotes sleep and is required for circadian timing of sleep onset. The data argue for a direct role for the circadian oscillator in regulating sleep and support a model whereby Wake acts as a molecular intermediary between the circadian clock and sleep. In this model, Wake transmits timing information from the circadian clock to inhibit arousal circuits at dusk, thus facilitating the transition from wake to sleep. wake is transcriptionally upregulated by Clk activity, specifically in LNv clock neurons. Wake levels in l-LNvs rise during the day and peak at the early night, near the wake/sleep transition. This increase in Wake levels upregulates Rdl in l-LNvs, enhancing their sensitivity to GABA signaling and serving to inhibit the l-LNv arousal circuit. In this manner, cycling of Wake promotes cycling of the excitability of l-LNv cells. In wake mutants, l-LNvs lose this circadian electrical cycling; the higher firing rate of these cells at dusk leads to increased release of Pdf, which would act on Pdfr on downstream neurons to inhibit sleep onset. The identity of the GABAergic neurons signaling to the l-LNvs is currently unknown, but if they serve to convey information about sleep pressure from homeostatic circuits, the l-LNvs could serve as a site of integration for homeostatic and circadian sleep regulatory signals (Liu, 2014).

Although Wake is expressed in clock neurons and its levels vary throughout the day, Wake itself is not a core clock molecule, since period length and activity rhythm strength are intact in wake mutants in constant darkness. The effects of Wake on sleep latency are not attributable to alterations in core clock function. In addition, because locomotor rhythm strength is intact in wake mutants, Wake is not a clock output molecule for locomotor rhythms. Rather, Wake is the first clock output molecule shown to specifically regulate sleep timing (Liu, 2014).

Previous studies have demonstrated that Rdl in LNvs regulates sleep in Drosophila. This work further implicates Rdl as a key factor in the circadian modulation of sleep. In mammals, the localization and function of GABAA receptors are regulated by a variety of cytosolic accessory proteins, some of which are associated with the plasma membrane and cytoskeletal elements. The data suggest that Wake acts as an accessory protein for Rdl, upregulating its levels and promoting its targeting to the plasma membrane. Rdl is broadly expressed throughout the adult Drosophila brain, whereas Wake appears more spatially restricted. It is likely that Rdl is regulated by Wake in specific cells (e.g., Wake+ cells), while in other cells that express Rdl but not Wake, other factors are involved. Together, these data suggest a model in which increased GABA sensitivity is required in specific arousal circuits to facilitate rapid and complete switching between sleep/wake states at the appropriate circadian time (Liu, 2014).

Intriguingly, the data, as well as data from the Allen Brain Atlas, suggest that the putative mouse homolog of Wake (ANKFN1) is enriched in the mouse SCN, the master circadian pacemaker in mammals. Specifically, ANKFN1 is expressed in the 'core' region of the SCN, which is analogous to the large LNvs in flies, in that it receives light input and its molecular oscillator does not cycle or cycles weakly in DD. These observations support a potential conservation of Wake function in regulating clock-dependent timing of sleep onset, which will be evaluated by ongoing genetic analysis in mice. The pronounced difficulty of wake flies to fall asleep at lights off is reminiscent of sleep-onset insomnia in humans. Moreover, the most widely used medications for the treatment of insomnia are GABA agonists. Thus, the identification of a molecule that mediates circadian timing of sleep onset by promoting GABA signaling may lead to a deeper understanding of mechanisms underlying insomnia and its potential therapies (Liu, 2014).

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 GABAB 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 GABAB receptor immunoreactivity is seen on these neurons as well as the Cha-Tan neurons. Based on an rdl-Gal4 line, the ionotropic GABAA 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 GABAA receptor desensitization uncouples sleep onset and maintenance in Drosophila

Many lines of evidence indicate that GABA and GABAA 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 GABAA receptor, RdlA302S, 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 RdlA302S mutant flies are resistant to the effects of CBZ on sleep latency and that mutant RDLA302S 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 GABAA receptor signaling dictate sleep latency (Agosto, 2008).

gamma-Aminobutyric acid (GABA) signaling components in Drosophila: immunocytochemical localization of GABAB receptors in relation to the GABAA 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 (GABAAR) for fast inhibitory transmission and on G-protein-coupled receptors (GABABR) for slow and modulatory action. Immunocytochemistry was used to map GABABR sites in the Drosophila CNS, and the distribution was compared with that of the GABAAR 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. GABABR-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 GABABR-IR punctates. However, the mushroom body lobes displayed RDL-IR but not GABABR-IR material, and there were subtle differences in other areas. The vGAT antiserum labeled punctates in the same areas as the GABABR and appeared to display presynaptic sites of GABAergic neurons. Various GAL4 drivers were used to analyze the relation between GABABR 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).

gamma-glutamyl transpeptidase 1 specifically suppresses green-light avoidance via GABA receptors in Drosophila

Drosophila larvae innately show light-avoidance behavior. Compared with robust blue-light avoidance, larvae exhibit relatively weaker green-light responses. In a previous screening for genes involved in larval light-avoidance, compared with control w1118 larvae, larvae with γ-glutamyl transpeptidase 1 (Ggt-1) knockdown or Ggt-1 mutation were found to exhibit higher percentage of green-light avoidance which was mediated by Rh6 photoreceptors. However, their responses to blue light did not change significantly. By adjusting the expression level of Ggt-1 in different tissues, it was found that Ggt-1 in malpighian tubules is both necessary and sufficient for green-light avoidance. These results showed that glutamate levels were lower in Ggt-1 null mutants compared with controls. Feeding Ggt-1 null mutants glutamate can normalize green-light avoidance, indicating that high glutamate concentrations suppressed larval green-light avoidance. However, rather than directly, glutamate affected green-light avoidance indirectly through GABA, the level of which was also lower in Ggt-1 mutants compared with controls. Mutants in glutamate decarboxylase 1, which encodes GABA synthase, and knockdown lines of the GABAA receptor, both exhibit elevated levels of green-light avoidance. Thus, these results elucidate the neurobiological mechanisms mediating green-light avoidance, which was inhibited in wild-type larvae (Liu, 2014).

The ADAR RNA editing enzyme controls neuronal excitability in Drosophila melanogaster

RNA editing by deamination of specific adenosine bases to inosines during pre-mRNA processing generates edited isoforms of proteins. Recoding RNA editing is more widespread in Drosophila than in vertebrates. Editing levels rise strongly at metamorphosis, and Adar5G1 null mutant flies lack editing events in hundreds of CNS transcripts; mutant flies have reduced viability, severely defective locomotion and age-dependent neurodegeneration. On the other hand, overexpressing an adult dADAR isoform with high enzymatic activity ubiquitously during larval and pupal stages is lethal. Advantage was taken of this to screen for genetic modifiers; Adar overexpression lethality is rescued by reduced dosage of the Rdl (Resistant to dieldrin), gene encoding a subunit of inhibitory GABA receptors. Reduced dosage of the Gad1 gene encoding the GABA synthetase also rescues Adar overexpression lethality. Drosophila Adar5G1 mutant phenotypes are ameliorated by feeding GABA modulators. This study demonstrates that neuronal excitability is linked to dADAR expression levels in individual neurons; Adar-overexpressing larval motor neurons show reduced excitability whereas Adar5G1 null mutant or targeted Adar knockdown motor neurons exhibit increased excitability. GABA inhibitory signalling is impaired in human epileptic and autistic conditions, and vertebrate ADARs may have a relevant evolutionarily conserved control over neuronal excitability (Li, 2013).

Actions of agonists, fipronil and Ivermectin on the predominant in vivo splice and edit variant (RDLbd, I/V) of the Drosophila GABA receptor expressed in Xenopus laevis oocytes

Ionotropic GABA receptors are the targets for several classes of insecticides. One of the most widely-studied insect GABA receptors is RDL (resistance to dieldrin), originally isolated from Drosophila melanogaster. RDL undergoes alternative splicing and RNA editing, which influence the potency of GABA. Most work has focussed on minority isoforms. This study reports the first characterisation of the predominant native splice variant and RNA edit, combining functional characterisation with molecular modelling of the agonist-binding region. The relative order of agonist potency is GABA> muscimol> TAC>> beta-alanine. The I/V edit does not alter the potency of GABA compared to RDLbd. Docking calculations suggest that these agonists bind and activate RDLbdI/V through a similar binding mode. TACA and beta-alanine are predicted to bind with lower affinity than GABA, potentially explaining their lower potency, whereas the lower potency of muscimol and isoguvacine cannot be explained structurally from the docking calculations. The A301S (resistance to dieldrin) mutation reduced the potency of antagonists picrotoxin, fipronil and pyrafluprole but the I/V edit had no measurable effect. Ivermectin suppressed responses to GABA of RDLbdI/V, RDLbd and RDLbdI/VA301S. The dieldrin resistant variant also showed reduced sensitivity to Ivermectin. This study of a highly abundant insect GABA receptor isoform will help the design of new insecticides (Lees, 2014).

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 GABAA 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, RDLac and RDLbd (DRC17-1-2), differ in their apparent agonist affinity. This paper reports the cloning of a third splice variant of Rdl, RDLad. Two-electrode voltage clamp electrophysiology was used to investigate agonist pharmacology of this expressed subunit following cRNA injection into Xenopus laevis oocytes. The ECso values for GABA and its analogues isoguvacine, muscimol, isonipecotic acid and 3-amino sulphonic acid on the RDLad homomeric receptor differed from those previously described for RDLac 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 GABAA 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 GABAA 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 RDLac and RDLbd 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 RDLac from RDLad, 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 GABAA and nicotinic receptors (Hosie, 2001).

The known determinants of agonist potency on GABAA receptors align closely to domains A-D of nicotinic receptors. In GABAA 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 GABAA receptors. With this in mind, it is interesting that the differences in the agonist potency and kinetics of recombinant rat GABAA 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).

The role of Rdl in resistance to phenylpyrazoles in Drosophila melanogaster

Extensive use of older generation insecticides may result in pre-existing cross-resistance to new chemical classes acting at the same target site. Phenylpyrazole insecticides block inhibitory neurotransmission in insects via their action on ligand-gated chloride channels (LGCCs). Phenylpyrazoles are broad-spectrum insecticides widely used in agriculture and domestic pest control. So far, all identified cases of target site resistance to phenylpyrazoles are based on mutations in the Rdl (Resistance to dieldrin) LGCC subunit, the major target site for cyclodiene insecticides. This study examined the role that mutations in Rdl have on phenylpyrazole resistance in Drosophila melanogaster, exploring naturally occurring variation, and generating predicted resistance mutations by mutagenesis. Natural variation at the Rdl locus in inbred strains of D. melanogaster included gene duplication, and a line containing two Rdl mutations found in a highly resistant line of Drosophila simulans. These mutations had a moderate impact on survival following exposure to two phenylpyrazoles, fipronil and pyriprole. Homology modelling suggested that the Rdl chloride channel pore contains key residues for binding fipronil and pyriprole. Mutagenesis of these sites and assessment of resistance in vivo in transgenic lines showed that amino acid identity at the Ala301 site influenced resistance levels, with glycine showing greater survival than serine replacement. It was confirmed that point mutations at the Rdl 301 site provide moderate resistance to phenylpyrazoles in D. melanogaster. The beneficial aspects of testing predicted mutations in a whole organism is emphasized to validate a candidate gene approach (Remnant, 2014).

Functions of Rdl orthologs in other species

GABA promotes the competitive selection of dendritic spines by controlling local Ca2+ signaling

Activity-dependent competition of synapses plays a key role in neural organization and is often promoted by GABA; however, its cellular bases are poorly understood. Excitatory synapses of cortical pyramidal neurons are formed on small protrusions known as dendritic spines, which exhibit structural plasticity. This study used two-color uncaging of glutamate and GABA in rat hippocampal CA1 pyramidal neurons and found that spine shrinkage and elimination were markedly promoted by the activation of GABAA receptors shortly before action potentials. GABAergic inhibition suppressed bulk increases in cytosolic Ca2+ concentrations, whereas it preserved the Ca2+ nanodomains generated by NMDA-type receptors, both of which were necessary for spine shrinkage. Unlike spine enlargement, spine shrinkage spread to neighboring spines (<15 μm) and competed with their enlargement, and this process involved the actin-depolymerizing factor ADF/cofilin. Thus, GABAergic inhibition directly suppresses local dendritic Ca2+ transients and strongly promotes the competitive selection of dendritic spines (Hayama, 2013).

Activity-dependent switch of GABAergic inhibition into glutamatergic excitation in astrocyte-neuron networks

Interneurons are critical for proper neural network function and can activate Ca2+ signaling in astrocytes. However, the impact of the interneuron-astrocyte signaling into neuronal network operation remains unknown. Using the simplest hippocampal Astrocyte-Neuron network, i.e., GABAergic interneuron, pyramidal neuron, single CA3-CA1 glutamatergic synapse, and astrocytes, this study found that interneuron-astrocyte signaling dynamically affected excitatory neurotransmission in an activity- and time-dependent manner, and the sign (inhibition vs potentiation) of the GABA-mediated effects were determined. While synaptic inhibition was mediated by GABAA receptors (see Drosophila Rdl), potentiation involved astrocyte GABAB receptors, astrocytic glutamate release, and presynaptic metabotropic glutamate receptors. Using conditional astrocyte-specific GABAB receptor (Gabbr1; see Drosophila metabotropic GABA-B receptor subtype 1) knockout mice, the glial source of the interneuron-induced potentiation was confirmed, and the involvement of astrocytes in hippocampal theta and gamma oscillations in vivo was demonstrated. Therefore, astrocytes decode interneuron activity and transform inhibitory into excitatory signals, contributing to the emergence of novel network properties resulting from the interneuron-astrocyte interplay (Perea, 2016).


REFERENCES

Search PubMed for articles about Drosophila Rdl

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Biological Overview

date revised: 25 April 2018

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