InteractiveFly: GeneBrief

GABA-B-R1, GABA-B-R2 and GABA-B-3: Biological Overview | References

Gene names - metabotropic GABA-B receptor subtype 1, metabotropic GABA-B receptor subtype 2 and metabotropic GABA-B receptor subtype 3

Synonyms -

Cytological map positions - 35B8-35B8, 93F9-93F9 and 21E2-21E2

Function - GABA receptor

Keywords - presynaptic inhibition, neuromodulation, olfaction, antennal lobe, brain, optic lobe

Symbols - GABA-B-R1, GABA-B-R2 and GABA-B-3

FlyBase ID: FBgn0260446, FBgn0027575 and FBgn0031275

Genetic map positions - 2L:15,017,154..15,034,704 [+], 3R:17,596,055..17,601,562 [-] and 2L:752,800..762,142 [+]

Classification - metabotropic transmembrane receptors for gamma-aminobutyric acid (GABA)

Cellular location - surface transmembrane

NCBI link for GABA-B-R1: EntrezGene
NCBI link for GABA-B-R2: EntrezGene
NCBI links for GABA-B-R3: EntrezGene

GABA-B-R1 orthologs: Biolitmine
GABA-B-R2 orthologs: Biolitmine
GABA-B-R3 orthologs: Biolitmine
Recent literature
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
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.
Li, F., Sami, A., Noristani, H. N., Slattery, K., Qiu, J., Groves, T., Wang, S., Veerasammy, K., Chen, Y. X., Morales, J., Haynes, P., Sehgal, A., He, Y., Li, S. and Song, Y. (2020). Glial Metabolic Rewiring Promotes Axon Regeneration and Functional Recovery in the Central Nervous System. Cell Metab. PubMed ID: 32941799
Axons in the mature central nervous system (CNS) fail to regenerate after axotomy, partly due to the inhibitory environment constituted by reactive glial cells producing astrocytic scars, chondroitin sulfate proteoglycans, and myelin debris. This study investigated this inhibitory milieu, showing that it is reversible and depends on glial metabolic status. Glia can be reprogrammed to promote morphological and functional regeneration after CNS injury in Drosophila via increased glycolysis. This enhancement is mediated by the glia derived metabolites: L-lactate and L-2-hydroxyglutarate (L-2HG). Genetically/pharmacologically increasing or reducing their bioactivity promoted or impeded CNS axon regeneration. L-lactate and L-2HG from glia acted on neuronal metabotropic GABA(B) receptors to boost cAMP signaling. Local application of L-lactate to injured spinal cord promoted corticospinal tract axon regeneration, leading to behavioral recovery in adult mice. These findings revealed a metabolic switch to circumvent the inhibition of glia while amplifying their beneficial effects for treating CNS injuries.

Three invertebrate GABAB receptor subtypes (D-GABABR1, R2 and R3) have been isolated from Drosophila. While D-GABABR1 and R2 show high sequence identity to mammalian GABABR1 and R2, respectively, the receptor D-GABABR3 seems to be an insect-specific subtype with no known mammalian counterpart so far. All three Drosophila GABABR subtypes are expressed in the embryonic central nervous system. In situ hybridization of Drosophila embryos shows that two of the D-GABABRs (GABABR1 and R2) are expressed in similar regions, suggesting a coexpression of the two receptors, while the third GABABR (GABABR3) displays a unique expression pattern. In agreement with these results D-GABABR1 and R2 could only be functionally characterized when the two subtypes are coexpressed either in Xenopus laevis oocytes or mammalian cell lines, while GABABR3 was inactive in any combination (Mezler, 2001). These observations or consonant with the idea that GABAB receptors function as heterodimers (see review by Bettler, 2004).

Early sensory processing can play a critical role in sensing environmental cues. This study investigated the physiological and behavioral function of gain control at the first synapse of olfactory processing in Drosophila. Olfactory receptor neurons (ORNs) express the GABAB receptor (GABABR) and its expression expands the dynamic range of ORN synaptic transmission that is preserved in projection neuron responses. Strikingly, it was found that different ORN channels have unique baseline levels of GABABR expression. ORNs that sense the aversive odorant CO2 do not express GABABRs nor exhibit any presynaptic inhibition. In contrast, pheromone-sensing ORNs express a high level of GABABRs and exhibit strong presynaptic inhibition. Furthermore, a behavioral significance of presynaptic inhibition was revealed by a courtship behavior in which pheromone-dependent mate localization is impaired in flies that lack GABABRs in specific ORNs. Together, these findings indicate that different olfactory receptor channels may employ heterogeneous presynaptic gain control as a mechanism to allow an animal's innate behavioral responses to match its ecological needs (Root, 2008).

The stereotypic organization of the Drosophila olfactory system and the identification of the family of odorant receptor genes make the fly an attractive system to study olfactory mechanisms. An adult fly expresses about 50 odorant receptor genes and each ORN typically expresses just one or a few receptor genes. ORNs detect odors in the periphery and send axons to glomeruli in the antennal lobe. Each glomerulus receives axons from about 20 ORNs expressing the same receptor genes and dendrites of a few uniglomerular projection neurons (PNs), which propagate olfactory information to higher brain centers. This numerically simple olfactory system coupled with genetic markers to label most of the input channels provides an opportunity to dissect synaptic function and information processing (Root, 2008).

The Drosophila antennal lobe is populated with GABAergic local interneurons (LNs) that release GABA in many if not all glomeruli (Ng, 2002). GABA exerts its modulatory role via two distinct receptor systems, the fast ionotropic GABAA receptor, which is sensitive to the antagonist picrotoxin, and the slow metabotropic GABAB receptor, which is sensitive to the antagonist CGP54626. Pharmacological blockade of the GABA receptors demonstrate that GABA-mediated hyperpolarization suppresses PN response to odor stimulation in a non-uniform fashion (Ng, 2002; Wilson, 2005; Shang, 2007). Electron microscopy studies of the insect antennal lobe show that GABAergic LNs synapse with PNs, which support the well established olfactory mechanism of lateral inhibition. GABAergic LNs also synapse onto ORNs and imaging studies in mouse suggest that activation of GABABRs in ORN terminals suppress neurotransmitter release in ORNs (Root, 2008).

It was hypothesized that setting the appropriate olfactory gain for environmental cues is important for adjusting an organism's sensitivity to its environment. A recent study shows that GABABR mediated presynaptic inhibition provides a mechanism to modulate olfactory gain (Olsen, 2008). Electrical recordings show that interglomerular presynaptic inhibition suppresses the olfactory gain of PNs to potentially increase the dynamic range of the olfactory response. Likewise, gain modulation may not be uniform among different glomeruli, which could reflect a tradeoff between sensitivity and dynamic range in different olfactory channels. For example, high sensitivity may be crucial for some environmental cues, such as those that require an immediate behavioral response, whereas a larger dynamic range may be more advantageous for other odors where precise spatial and temporal information may be critical for optimal performance (Root, 2008).

This study investigated the physiological and behavioral function of gain control in early olfactory processing. Interneuron-derived GABA was shown to activate GABABRs on ORN terminals, reducing the gain of ORN-to-PN synaptic transmission. Different types of ORNs exhibit different levels of presynaptic inhibition and this heterogeneity in presynaptic inhibition is preserved in antennal lobe output projection neurons. Interestingly, pheromone-sensing ORNs exhibit high levels of GABABR expression and behavioral experiments indicate that GABABR expression in a population of pheromone ORNs is important for mate localization, suggesting that presynaptic gain control is important for the olfactory channel-specific fine-tuning of behavior (Root, 2008).

Two-photon imaging was used to monitor activity in selective neural populations in the antennal lobe. Specific blockade of GABABRs reveals a scalable presynaptic inhibition to suppress olfactory response at high odor concentrations. Pharmacological and molecular experiments suggest that GABABRs are expressed in primary olfactory receptor neurons. Furthermore, the level of presynaptic inhibition is different in individual glomerular modules, which is tightly linked to the level of GABABR expression. The importance of presynaptic GABABRs in olfactory localization was investigated, and it was found that reduction of GABABR expression in the presynaptic terminal of ORNs impairs the ability of male flies in locating potential mates (Root, 2008).

Heterogeneity was found in the levels of presynaptic inhibition among different glomeruli. Varying GABABR2 expression level in ORNs with molecular manipulations is sufficient to produce predictable alterations in presynaptic inhibition in specific glomeruli. Together these experiments argue that presynaptic GABABR expression level is a determinant of glomerulus-specific olfactory gains in the antennal lobe. A recent report revealed that there is a non-linear transformation between ORNs and PNs that is heterogeneous between glomeruli (Bhandawat, 2007). In other words, PNs innervating a given glomerulus have a unique response range for its ORN input. Given that ORNs are the main drivers of PN response (Olsen, 2007; Root, 2007), it is plausible that the heterogeneity in presynaptic inhibition contributes to the heterogeneity in ORN to PN transformations observed by Bhandawat and colleagues. Additionally, heterogeneity in GABA release by LNs (Ng, 2002) could also contribute to heterogeneity in presynaptic inhibition. It is interesting to note that when presynaptic inhibition is abolished, heterogeneity remains in the input-output curves of PN response to the four different odors in these experiments, suggesting that other mechanisms such as probability of vesicle release contribute to the heterogeneity as well (Root, 2008).

Theoretical analysis of antennal lobe coding has recently suggested that the non-linear synaptic amplification in PNs observed by Bhandawat and colleagues provides an efficient coding mechanism for the olfactory system (Abbott, 2007). According to this model, the optimal distribution of firing rates across a range of odorants should be flat without clusters. Firing rates of a given ORN responses cluster in an uneven distribution. Conversely, PNs exhibit a more equalized firing rate distribution than ORNs (Bhandawat, 2007). According to the optimum coding theory, the high amplification of ORN to PN transformation generates a more even distribution of PN firing rates that should facilitate odor discrimination. However, this model of olfactory coding poses a potential problem. The high gain in this synaptic amplification reduces the dynamic range of PNs, causing a loss of information about concentration variation that could be important for an animal to localize odor objects. Presynaptic inhibition may provide a mechanism to expand the dynamic range of the olfactory system. For some glomerular modules that mediate innate behaviors such as avoidance of the stress odorant CO2, there is a potential trade off for odor sensitivity and dynamic range. The lack of GABABR in the CO2 sensing ORNs could be important to maintain high sensitivity (Root, 2008).

Pheromones play an important role in Drosophila mating behaviors and the current results indicate that pheromone sensing ORNs have high levels of GABABR, which is correlated with a high level of presynaptic inhibition in these ORNs. Mate localization is impaired in the absence of presynaptic inhibition in one pheromone sensing glomerulus. It is interesting to note that in addition to the pheromone sensing ORNs, the palpal ORNs also exhibit high GABABR expression level. Although the behavioral role of the palpal ORNs has not been determined, it is possible that they are also important for odor object localization. There are two potential mechanisms for the role of GABABR in olfactory localization. GABABR-mediated activity-dependent suppression of presynaptic transmission on a short time scale provides a mechanism for dynamic range expansion. On a longer time scale, activity-dependent suppression provides a mechanism for adaptation, hence a high pass filter to allow the detection of phasic information. Further experiments will be necessary to determine which property is important for olfactory localization (Root, 2008).

Intraglomerular and interglomerular presynaptic inhibition mediated by GABABRs have been described in the mammalian olfactory system. Intraglomerular presynaptic inhibition was suggested as a mechanism to control input sensitivity while maintaining the spatial maps of glomerular activity. Interglomerular presynaptic inhibition was proposed as a mechanism to increase the contrast of sensory input. A recent report revealed a similar gain control mechanism by interglomerular presynaptic inhibition in the Drosophila olfactory system (Olsen, 2008) where GABABR expression in ORNs was shown to scale the gain of PN responses. Interestingly, most if not all of the presynaptic inhibition was suggested to be lateral. In contrast, this study study does not seek to distinguish between intra- and interglomerular presynaptic inhibition, however evidence was found that the VA1lm glomerulus receives significant intraglomerular presynaptic inhibition. Thus, despite significant differences between the insect and mammalian olfactory systems, the inhibitory circuit in the first olfactory processing center appears remarkably similar (Root, 2008).

Based on whole cell recordings of PNs in response to ORN stimulation, Olsen (2008) suggests that both GABAAR and GABABR are expressed in ORNs to mediate presynaptic inhibition and that GABAAR signaling is a large component of lateral presynaptic inhibition. In contrast, this study, which employed direct optical measurements of presynaptic calcium and synaptic vesicle release, suggests that GABABRs but not GABAARs are involved in presynaptic inhibition. To resolve these discrepancies further molecular experiments will be important to determine conclusively whether ORNs express GABAAR and whether the receptor contributes to gain control. Furthermore, the antennal lobe is a heterosynaptic system comprised of at least three populations of neurons that include ORNs, LNs and PNs. Therefore, how these different populations of neurons respond to GABA signaling and what contribution they make to olfactory processing in the antennal lobe is a critical question for future investigation (Root, 2008).

This study has demonstrated differential presynaptic gain control in individual olfactory input channels and its contribution to the fine-tuning of physiological and behavioral responses. Synaptic modulation by the intensity of receptor signaling is reminiscent of the mammalian nervous system where expression levels of AMPA glutamate receptors play an important role in regulating synaptic efficacy. Furthermore, presynaptic regulation of GABABR signaling provides a mechanism to modulate the neural activity of individual input channels without much interference with overall detection sensitivity because this mechanism of presynaptic inhibition would only alter responses to high intensity stimuli. In parallel, it is tempting to speculate that global modulation of interneuron excitability should alter the amount of GABA release across channels, thus providing a multi-channel dial of olfactory gain control that may reflect the internal state of the animal (Root, 2008).

Lateral presynaptic inhibition mediates gain control in an olfactory circuit

Olfactory signals are transduced by a large family of odorant receptor proteins, each of which corresponds to a unique glomerulus in the first olfactory relay of the brain. Cross-talk between glomeruli has been proposed to be important in olfactory processing, but it is not clear how these interactions shape the odor responses of second-order neurons. In the Drosophila antennal lobe (a region analogous to the vertebrate olfactory bulb), most inter-glomerular input to identified second-order olfactory neurons was selectively removed. This was found to broaden the odor tuning of these neurons, implying that inter-glomerular inhibition dominates over inter-glomerular excitation. The strength of this inhibitory signal scales with total feedforward input to the entire antennal lobe, and has similar tuning in different glomeruli. A substantial portion of this inter-glomerular inhibition acts at a presynaptic locus, and the results imply this is mediated by both GABAA and GABAB receptors on the same nerve terminal (Olsen, 2008).

A sensory stimulus generally triggers activity in multiple neural processing channels, each of which carries information about some feature of that stimulus. The concept of a processing channel has a particularly clear anatomical basis in the first relay of the olfactory system, which is typically divided into glomerular compartments. Each glomerulus receives input from many first-order olfactory receptor neurons (ORNs), all of which express the same odorant receptor. Each second-order neuron receives direct ORN input from a single glomerulus, and thus all the first- and second-order neurons corresponding to a glomerulus constitute a discrete processing channel. An odorant typically triggers activity in multiple glomeruli, and local interneurons that interconnect glomeruli provide a substrate for cross-talk between channels (Olsen, 2008).

The Drosophila antennal lobe is a favored model for investigating olfactory processing because it contains only ~50 glomeruli, each of which corresponds to an identified type of ORN and an identified type of postsynaptic projection neuron (PN). Several recent studies of the Drosophila antennal lobe have produced divergent views of the relative importance of inter-glomerular connections. One model proposes that PN odor responses are almost completely determined by feedforward excitation. This model ascribes little importance to cross-talk between glomerular processing channels. An alternative model proposes that inter-glomerular connections make an important contribution to shaping PN odor responses. However, this has not been demonstrated by showing a change in PN odor responses when lateral inputs to that PN are removed. (The word 'lateral' is used as a synonym for 'inter-glomerular') (Olsen, 2008).

In principle, several features of olfactory processing in the Drosophila antennal lobe could reflect either intra- or inter-glomerular events. For example, most PNs are more broadly tuned to odors than their presynaptic ORNs. This could reflect a purely intra-glomerular nonlinear process, such as short-term synaptic depression at ORN-PN connections. Alternatively, it could be due to the fact that lateral excitatory connections exist between glomeruli. It is also unclear whether inhibitory epochs in PN odor responses reflect inter- or intra-glomerular events. Many GABAergic interneurons form connections between glomeruli, but several recent studies have failed to observe any inter-glomerular inhibition. These studies removed all the direct ORN inputs to an identified PN, and asked whether lateral input could be evoked in that PN by olfactory stimulation of other ORN types. In all cases, lateral inputs to PNs were excitatory. This raises the possibility that inter-glomerular inhibition might not exist, and inhibitory PN odor responses might merely reflect intra-glomerular feedback. This is an important issue because intra- and inter-glomerular inhibition have different consequences for how olfactory representations are transformed in this circuit (Olsen, 2008).

This study addressed three questions: Do inter-glomerular interactions make a substantial contribution to PN odor responses? Do these interactions include lateral inhibition? If so, how does this occur, and why has it been difficult to observe? (Olsen, 2008).

What happens to PN odor responses when most lateral input to that PN is removed. Advantage was taken of the fact that the fly has two olfactory organs. About 90% of ORNs are contained in the antennae, with 10% in the maxillary palp. Palp ORNs express odorant receptors not expressed in the antennae, and project to palp glomeruli that are distinct from glomeruli targeted by antennal ORNs. Antennal and palp glomeruli are interconnected by local interneurons. Acute removal of the antennae eliminates 90% of the input to the antennal lobe, and thus most excitatory drive to local interneuron. If lateral connections are mainly excitatory, then removing the antennae should decrease the odor responses of palp PNs (Olsen, 2008).

This experiment was performed in two different palp glomeruli (VM7 and VC1). Surprisingly, removing the antennae increased most of the odor responses of these PNs. No odor responses were decreased. This implies that most of the odors normally evoke lateral inhibitory input to these glomeruli, and this outweighs the effect of lateral excitatory input (Olsen, 2008).

The input-output function of each glomerulus was examined by plotting the strength of each PN odor response versus the strength of the cognate ORN response to the same odor. These input-output functions were nonlinear, which is typical for most glomeruli. When most lateral input to PNs were removed, the nonlinearity persisted, and PNs became even more broadly tuned. This argues that broad PN tuning results mainly from purely intra-glomerular mechanisms. These could include short-term synaptic depression at ORN-PN connections, and/or an intrinsic ceiling on PN firing rates. Lateral excitation should tend to broaden PN tuning even more, but lateral inhibition evidently counteracts this (Olsen, 2008).

One clue to the significance of lateral inputs is that in the intact antennal lobe circuit, PN responses cannot be predicted purely on the basis of feedforward excitatory inputs. Two odors can elicit similar responses in an ORN, but divergent responses in a postsynaptic PN. For example, pentyl acetate and 4-methyl phenol evoke similar activity in VM7 ORNs, but not in VM7 PNs, implying that these odors recruit different lateral inputs to this glomerulus. After antennae were removed, these odors evoked similar responses in VM7 PNs. Overall, removing the antenna increased the correlation between the ranked odor preferences of ORNs and their cognate PNs (Olsen, 2008).

Several recent studies have failed to observe lateral inhibition in the Drosophila antennal lobe. These studies silenced all direct ORN inputs to a PN, and focused on lateral input to that PN evoked by stimulating ORNs presynaptic to other glomeruli. It was reasoned that some lateral inhibition might target ORN axon terminals; if so, this would only be observed when the direct ORN inputs to a PN are active. GABAergic inhibition at ORN axon terminals has been described previously in the olfactory bulb, and synapses from GABAergic interneurons onto ORN axon terminals have been found in an insect antennal lobe (Olsen, 2008).

To test the idea that some lateral inhibition is presynaptic, it was asked how lateral input to glomerulus VM7 depends on the activity of VM7 ORNs. Each VM7 ORN fires spontaneously at ~10 spikes/s, and consequently these PNs are bombarded by spontaneous spike-driven EPSPs. When VM7 ORNs were silenced by removing the maxillary palps, large spontaneous EPSPs disappeared in these PNs. In this experimental configuration, stimulating the antennae with odorants depolarized VM7 PNs, which is consistent with previous reports that lateral input is excitatory when direct ORN inputs are silent. Next, it was asked whether preserving spontaneous activity in VM7 ORNs would allow observation of lateral inhibition. To prevent odor-evoked activity in VM7 ORNs, the maxillary palps were covered with a plastic shield. It was inferred that the shield did not prevent spontaneous activity in VM7 ORNs because normal spontaneous EPSPs were observed in VM7 PNs. The shield was clearly an effective barrier to odors because it blocked the normal strong excitatory response to ethyl butyrate in VM7 PNs. Rather, when the palps were shielded, stimulating the antennae with odorants suppressed spontaneous EPSPs in VM7 PNs. This was accompanied by hyperpolarization of the membrane potential, which would reflect (at least in part) the removal of ongoing depolarization produced by EPSP bombardment (Olsen, 2008).

For all 20 odors tested, lateral input depolarized VM7 PNs when their cognate ORNs were absent, and hyperpolarized these PNs when their ORNs were spontaneously active. This argues that a substantial component of lateral inhibition acts at a presynaptic locus. This does not mean that all lateral inhibition is presynaptic; indeed there is evidence for an additional postsynaptic component (Olsen, 2008).

When palp ORNs were shielded, different odors evoked different amounts of inhibition in glomerulus VM7. It is hypothesized that this odor tuning could explain why antennal removal disinhibits some VM7 PN odor responses more than others. To test this, the amount of inhibition evoked by each odor was tested in the shielded-palps experiment, and compared to the change in PN spiking responses to that odor after antennal removal. These two measures were well-correlated, which argues that these two experimental paradigms measure the same underlying phenomenon (Olsen, 2008).

The odor tuning of lateral input must reflect the connectivity of the local interneurons that mediate this inhibition. Many individual GABAergic interneurons innervate all glomeruli (Wilson, 2005; Shang, 2007; Stocker, 1997), suggesting that lateral inhibition to each glomerulus might reflect pooled input from all ORNs. If so, then the size of lateral input to a glomerulus should correlate with the total ORN activity evoked by that odor. Total ORN activity is estimated by summing the spiking responses of each antennal ORN type; this measure was found to predict the strength of lateral inhibition evoked by each odor in the shielded-palps experiment (Olsen, 2008).

If lateral inhibition to all glomeruli scales with total ORN activity, then lateral inhibitory input to each glomerulus would show the same odor tuning. It has alread been shown that the odor tuning of lateral input to VM7 is a good predictor of which odor responses were most disinhibited in VM7 PNs after antennal removal. As expected, it also partially predicted which odor responses were most disinhibited in a different palp glomerulus, VC1. However, this correlation was weaker than the correlation with disinhibition in VM7. This leaves open the possibility that there may be some differences in the odor tuning of lateral inhibitory input to different glomeruli (Olsen, 2008).

The results suggest that much of the lateral inhibition in this circuit acts by suppressing ORN-PN synaptic transmission. To test this, ORN-PN synaptic strength was monitored in one glomerulus while recruiting lateral input to that glomerulus. Recordings were made from an identified PN while electrically stimulating the ipsilateral antennal nerve to evoke excitatory postsynaptic currents (EPSCs). Next, an odor was used to stimulate ORNs in the remaining intact antenna (and the maxillary palps). Because most glomeruli receive bilateral ORN input, olfactory stimulation of the contralateral antenna drives activity in ipsilateral glomeruli. Finally, odors were prevented from recruiting direct ORN input to the recorded PN by mutating the odorant receptor gene normally expressed by its ORNs (Olsen, 2008).

As predicted, olfactory stimulation of the contralateral antenna inhibited EPSCs evoked by ipsilateral nerve stimulation. It was possible to mimic this inhibition by iontophoresing GABA into the antennal lobe neuropil. A GABAB receptor antagonist blocked the late phase of this inhibition, but had only a modest effect on the early phase. Adding a GABAA antagonist to the GABAB antagonist blocked the residual early portion of the inhibition. The GABAA antagonist alone had no effect. Taken together, these results suggest that both GABAA and GABAB receptors are present on the same ORN axon terminals, and either GABAA or GABAB receptors alone are sufficient to mediate substantial inhibition of EPSCs just after GABA release. The late phase of inhibition evidently involves only GABAB receptors (Olsen, 2008).

Presynaptic inhibition is generally associated with a change in the way a synapse responds to paired electrical pulses. This study found that both the GABAA and GABAB components of EPSC inhibition are associated with an increase in the paired-pulse ratio. This implies that the independent actions of both GABAA and GABAB receptors are at least partially presynaptic. As a further test of this model, GABAB signaling was genetically abolished selectively in presynaptic ORNs. An ORN-specific promoter was ised to drive expression of pertussis toxin, a selective inhibitor of some types of G-proteins. In these flies, GABA still inhibited ORN-PN EPSCs, but now this inhibition had a briefer duration than in wild-type flies. Unlike in wild-type flies, this inhibition was completely resistant to the GABAB antagonist and completely blocked by the GABAA antagonist. As a negative control, this phenotype was confirmed to require both the ORN-specific promoter and the toxin transgene. This demonstrates that GABAB receptors inhibit ORN-PN synapses at a purely presynaptic locus (Olsen, 2008).

If activation of either GABAA or GABAB receptors is sufficient to mediate strong lateral inhibition, then blockade of both receptors should be required to mimic the removal of lateral input. To test this, again recordings were made from palp PNs in glomerulus VM7. Normally, the odor pentyl acetate weakly excites VM7 ORNs and inhibits VM7 PNs. When most lateral input is removed (by removing the antennae), this odor strongly excites these PNs. This disinhibition could not be inhibited by applying either a GABAA or a GABAB receptor antagonist alone. However, the two antagonists together produced strong disinhibition that resembled the effect of removing the antennae (Olsen, 2008).

Many previous studies have shown that odors can inhibit spiking in olfactory bulb mitral cells and antennal lobe projection neurons, but in principle this inhibition could be purely intra-glomerular. Experiments in vitro have revealed several types of inter-glomerular circuits, but some of these circuits are evidently not recruited by olfactory stimuli. This study has directly demonstrated an important role for inhibitory interactions between olfactory glomeruli in vivo (Olsen, 2008).

The results argue that a substantial component of inter-glomerular inhibition occurs at a presynaptic locus. Previous studies in other species have shown that GABA can inhibit release from ORN axon terminals. The results imply that in Drosophila this is mediated by both GABAA and GABAB receptors. This arrangement is unusual but not unique; for example, there are several instances of GABAA and GABAB inhibition at the same presynaptic site in other neural circuits. Ionotropic and metabotropic receptors act with different kinetics, and so this arrangement might ensure that inhibition spans a broad time window. Although both receptor types were co-active during most of the odor response, it was noticed that GABAA receptors were required for the a brief early phase of inhibition after odor onset, while GABAB receptors were required for the long, late phase (Olsen, 2008).

This study has shown that lateral inhibitory input to a glomerulus roughly scales with total ORN activity. This would imply that the odor tuning of GABAergic input to each glomerulus is approximately similar. However, the effects of lateral input may nevertheless be somewhat glomerulus-specific. Even if the odor tuning of GABA release were identical in all glomeruli, the efficacy of presynaptic inhibition will vary with presynaptic membrane potential. As a result, the same inhibitory signal should be more effective in some glomeruli than in others. Also, in some glomeruli, lateral inhibition might be outweighed by lateral excitation. This would explain why other studies have found that some PNs can be excited by odors that do not excite their cognate ORNs (Olsen, 2008).

In sum, it is proposed that this form of gain control represents a flexible balance between sensitivity and efficiency. When total ORN input is weak, lateral inhibition is minimal, and ORN-PN synapses are strong. When an odorant recruits vigorous ORN input to many glomeruli, GABAergic interneurons inhibit ORN neurotransmitter release. This should prevent a stimulus from saturating the dynamic range of many PN types simultaneously. Because this mechanism suppresses responses that are strong and redundant, it may tend to decrease cross-correlations between the output of different glomeruli, and thus promote a more efficient neural code for odors (Olsen, 2008).

Drosophila pacemaker neurons require G protein signaling and GABAergic inputs to generate twenty-four hour behavioral rhythms

Intercellular signaling is important for accurate circadian rhythms. In Drosophila, the small ventral lateral neurons (s-LN(v)s) are the dominant pacemaker neurons and set the pace of most other clock neurons in constant darkness. This study shows that two distinct G protein signaling pathways are required in LN(v)s for 24 hr rhythms. Reducing signaling in LN(v)s via the G alpha subunit Gs, which signals via cAMP, or via the G alpha subunit Go, which signals via Phospholipase 21c, lengthens the period of behavioral rhythms. In contrast, constitutive Gs or Go signaling makes most flies arrhythmic. Using dissociated LN(v)s in culture, Go and the metabotropic GABA(B)-R3 receptor were shown to be required for the inhibitory effects of GABA on LN(v)s, and reduced GABA(B)-R3 expression in vivo lengthens period. Although no clock neurons produce GABA, hyperexciting GABAergic neurons disrupts behavioral rhythms and s-LN(v) molecular clocks. Therefore, s-LN(v)s require GABAergic inputs for 24 hr rhythms (Dahdal, 2010).

Insulin signaling, lifespan and stress resistance are modulated by metabotropic GABA receptors on insulin producing cells in the brain of Drosophila

Insulin-like peptides (ILPs) regulate growth, reproduction, metabolic homeostasis, life span and stress resistance in worms, flies and mammals. A set of insulin producing cells (IPCs) in the Drosophila brain that express three ILPs (DILP2, 3 and 5) have been the main focus of interest in hormonal DILP signaling. Little is, however, known about factors that regulate DILP production and release by these IPCs. This study shows that the IPCs express the metabotropic GABA(B) receptor (GBR), but not the ionotropic GABA(A) receptor subunit RDL. Diminishing the GBR expression on these cells by targeted RNA interference abbreviates life span, decreases metabolic stress resistance and alters carbohydrate and lipid metabolism at stress, but not growth in Drosophila. A direct effect of diminishing GBR on IPCs is an increase in DILP immunofluorescence in these cells, an effect that is accentuated at starvation. Knockdown of irk3, possibly part of a G protein-activated inwardly rectifying K(+) channel that may link to GBRs, phenocopies GBR knockdown in starvation experiments. These experiments suggest that the GBR is involved in inhibitory control of DILP production and release in adult flies at metabolic stress and that this receptor mediates a GABA signal from brain interneurons that may convey nutritional signals. This is the first demonstration of a neurotransmitter that inhibits insulin signaling in its regulation of metabolism, stress and life span in an invertebrate brain (Enell, 2009).

Gamma-aminobutyric acid (GABA)-mediated neural connections in the Drosophila antennal lobe

Inhibitory synaptic connections mediated by gamma-aminobutyric acid (GABA) play important roles in the neural computation of the brain. To obtain a detailed overview of the neural connections mediated by GABA signals, this study analyzed the distribution of the cells that produce and receive GABA in the Drosophila adult brain. Relatively small numbers of the cells, which form clusters in several areas of the brain, express the GABA synthesis enzyme Gad1. In contrast, many cells scattered across the brain express ionotropic GABA(A) receptor subunits (Lcch3 and Rdl) and metabotropic GABA(B) receptor subtypes (GABA-B-R1, -2, and -3). To analyze the expression of these genes in distinct identified cell types, focus was placed on the antennal lobe, where GABAergic neurons play important roles in odor coding. By combining fluorescent in situ hybridization and immunolabeling against GFP expressed with cell-type-specific GAL4 driver strains, the percentage of the cells that produce or receive GABA was quantified for each cell type. GABA was synthesized in the middle antennocerebral tract (mACT) projection neurons and two types of local neurons. Among them, mACT neurons had few presynaptic sites in the antennal lobe, making the local neurons essentially the sole provider of GABA signals there. However, not only these local neurons but also all types of projection neurons expressed both ionotropic and metabotropic GABA receptors. Thus, even though inhibitory signals are released from only a few, specific types of local neurons, the signals are read by most of the neurons in the antennal lobe neural circuitry (Okada, 2009).

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).

Odor mixtures of opposing valence unveil inter-glomerular crosstalk in the Drosophila antennal lobe

Evaluating odor blends in sensory processing is a crucial step for signal recognition and execution of behavioral decisions. Using behavioral assays and 2-photon imaging, this study has characterized the neural and behavioral correlates of mixture perception in the olfactory system of Drosophila. Mixtures of odors with opposing valences elicit strong inhibition in certain attractant-responsive input channels. This inhibition correlates with reduced behavioral attraction. Defined subsets of GABAergic interneurons provide the neuronal substrate of this computation at pre- and postsynaptic loci via GABAB- and GABAA receptors, respectively. Intriguingly, manipulation of single input channels by silencing and optogenetic activation unveils a glomerulus-specific crosstalk between the attractant- and repellent-responsive circuits. This inhibitory interaction biases the behavioral output. Such a form of selective lateral inhibition represents a crucial neuronal mechanism in the processing of conflicting sensory information (Mohamed, 2019).

This study analyzed the integration of binary odor mixtures of opposing hedonic valences and demonstrate how glomerular-specific inhibition and crosstalk results in an appropriate behavioral output. Glomeruli that strongly respond to the attractive odor are inhibited by the repellent odor in the mixture, which is mediated by defined subsets of GABAergic local interneurons (LNs; see Circuit model for glomerulus-specific crosstalk in the fly AL). Heterogeneity in responses to mixtures has been shown in previous studies where excitation of some glomeruli by one of the mixture components can inhibit the glomeruli activated by the other component. Similar to invertebrates, evidence for non-linearity of mixture interactions has been reported in individual mitral/tufted cells (PNs analogs) in the olfactory bulb of vertebrates. As an alternative scenario it is also conceivable that instead of inhibiting the attractant-coding pathway to shift the behavior towards aversion, the response of the repellent-responsive glomeruli could be boosted via lateral excitation. Lateral excitation has been described to drive synergistic interaction between the binary mixture of cis-vaccenyl acetate and vinegar. Although odors representing sex and food are mutually reinforcing, a binary mixture of odors with opposing valences means a conflicting input. It is therefore postulated that, in contrast to reinforcing input, conflicting sensory input is processed via lateral inhibition in the fly AL. An assumption that would be intriguing to be tested in the future (Mohamed, 2019).

No inhibition of the attractant-responsive glomeruli when stimulating with MIX(+) (a binary mixture of ethyl acetate and benzaldehyde). This lack of inhibition is probably due to the strong ORN input leading to high presynaptic firing rates in the attractant-responsive glomeruli. Consequently, lateral inhibition deriving from the aversive circuit has only a low impact and does not decrease the excitation of the attractant-responsive glomeruli (Mohamed, 2019).

Obviously not all glomeruli that are activated by an attractive odor are inhibited by a repellent in a mixture and might not contribute to the attractiveness of an odor. This observation makes sense in the light of accumulating evidence suggesting that the innate behavioral output is correlated either to the summed weights of specific activated glomeruli or to the activity of single processing channels. The latter argument is supported by the finding that only very few, special glomeruli seem to be valence-specific and induce clear attraction or aversion behavior upon artificial activation (Mohamed, 2019).

It is important to mention that the subset of repellent-responsive glomeruli does also respond to non-aversive and even partly attractive odors, such as E2-hexenal and ethyl benzoate. However, an attractive odorant may indeed activate some aversive input channels beside their main activation of the attractive circuitry (or the other way around). What actually matters is the behavioral output that is consequently elicited when a specific glomerulus becomes activated. For example, ORNs that respond to CO2 are also activated by ethyl benzoate and E2-hexenal. However, the CO2 circuit has been clearly demonstrated to mediate behavioral aversion. Following this argument, artificial activation of glomeruli DL1 and/or DL5 leads to aversive behavior, while silencing DM1 and DM4 abolished attraction to the attractant. These experiments provide evidence that activation of the repellent- and attractant-responsive glomeruli causes a valence-specific behavior, and can therefore be defined as attractive or aversive input channels, respectively (Mohamed, 2019).

Interestingly, one exception was observed in the data set: although the repellent odor geosmin reduced the attraction to balsamic vinegar in the mixture, no mixture inhibition was observed. The detection of geosmin is one of the rare cases, where an odor is detected by only one receptor type and consequently activates only one glomerulus. Similar specialized pathways have been described for the detection of sex pheromones and CO2. Glomeruli processing these ecologically labeled lines differ from broadly tuned glomeruli with regard to their neuronal composition. Hence, it is conceivable that the narrowly tuned geosmin-responsive glomerulus does not exhibit strong interglomerular interactions and has therefore a different impact on the attractant-responsive glomeruli. Mixture interactions between geosmin and attractive odors might be implemented in higher processing centers which contain circuit elements mediating interactions between odors (Mohamed, 2019).

Lateral inhibition, which is believed to enhance contrast and to facilitate discrimination of similar stimuli, is an important motif throughout the nervous system. In mice, dense center-surround inhibition refines mitral cell representation of a glomerular map56, while other evidence showed that lateral inhibition can be rather selective and biased between different mitral cells. In accordance with the olfactory bulb, the AL exhibits broad, selective or even both forms of lateral inhibition, whereby certain glomeruli can show different sensitivities towards an inhibitory input. Lateral inhibition in the Drosophila AL is largely mediated through GABA. Most of the GABAergic inhibition in the Drosophila AL has been shown to take place predominantly on the presynaptic site mediated through GABAA and GABAB receptors. In addition, PNs also receive GABAergic inhibition via GABAA and/or GABAB receptors from LNs. Notably, this study found that two out of four attractant-responsive glomeruli are inhibited on the pre- and postsynaptic levels (via GABAB- or GABAA-receptors), while the other two glomeruli are inhibited only presynaptically through GABAB-type receptors. Previous results have so far shown that GABAA-type receptors contribute weakly to lateral inhibition and shape the early phase of odor responses. However, the data demonstrate that GABAA-type receptors largely mediate mixture-induced inhibition during the full period of the odor presentation which is reminiscent to tonic inhibition in the mammalian system (Mohamed, 2019).

This study shows that mixture-induced lateral inhibition of the attractant-responsive glomeruli was abolished when GABA synthesis was silenced in mostly patchy LNs. Hence the data suggest, in consistency with previous studies, that LNs with more selective innervations mediate glomerulus-specific interactions and rather contribute to mixture processing, while pan-glomerular LNs (e.g., GH298-Gal4 and H24-Gal4), that globally release GABA, might be involved in gain control (Mohamed, 2019).

Interestingly, the repellent-responsive glomeruli DL1 and DL5 did not show any mixture interaction, but mediate the lateral inhibition of the attractant-responsive glomeruli. Two possible scenarios would provide the neuronal substrate for this mechanism dependent on either the donor (i.e. LNs) or the receiver (i.e. glomerulus) side. First, since glomeruli vary dramatically in their GABA sensitivity and consequently their sensitivity to LN activation14, lateral inhibition is heterogeneous across different glomeruli. Second, lateral inhibition is biased among different glomeruli due to a glomerulus-specific synaptic distribution of pre- and postsynapses of GABAergic LNs, i.e. the GABA release is not uniform. This assumption is supported by data revealing that GABAergic LNs possess a higher density of postsynapses in DL1 and DL5 than in the attractant-responsive glomeruli. In line with the current findings, EM based data from the larvae AL describe GABAergic, oligoglomerular 'choosy' LNs with a clear polarity contributing to postsynaptic inhibition for most glomeruli, while they receive inputs from only a small glomerular subset. Hence, there is strong evidence that some glomeruli can drive lateral inhibition in other glomeruli. Both scenarios could either occur separately or reinforce each other. Moreover, it might be ecological relevant not to inhibit the input of the aversive pathways since these are associated with life-threatening situations that should be coded reliably and rather override an attractive input (Mohamed, 2019).

In contrast to expectations, sole photoactivation of DL1 or DL5 or stimulation with the repellent alone did not induce inhibition in the attractant-responsive glomeruli. This might be due to the low spontaneous activity of ORNs innervating the attractant-responsive glomeruli, which correlates with spontaneous fluctuations in the membrane potential of the postsynaptic PNs. Consequently, inhibitory responses (i.e. hyperpolarizations) are difficult to capture with calcium imaging (Mohamed, 2019).

In other sensory systems, lateral inhibitory connections of neuronal subsets involved in sensory processing have been elucidated in great detail, such as in the retina of mice or the rat visual cortex. Also for the Drosophila AL, previous studies suggested that glomerular subgroups are connected via inhibitory LNs. However, these studies could neither pinpoint the precise connections nor their significance for behavioral perception. The data provide evidence for a specific inhibitory crosstalk between identified glomeruli and substantiate the existence of selective lateral inhibition in the fly AL. The postulated network circuits offer insights into the principle of sensory integration. It will be intriguing to see whether neuron-specific crosstalk represents a general phenomenon to integrate multiple and rather conflicting input channels in other sensory modalities (Mohamed, 2019).

A GABAergic feedback shapes dopaminergic input on the Drosophila mushroom body to promote appetitive long-term memory

Memory consolidation is a crucial step for long-term memory (LTM) storage. However, a clear picture of how memory consolidation is regulated at the neuronal circuit level is still lacking. This study took advantage of the Drosophila olfactory memory center, the mushroom body (MB), to address this question in the context of appetitive LTM. The MB lobes, which are made by the fascicled axons of the MB intrinsic neurons, are organized into discrete anatomical modules, each covered by the terminals of a defined type of dopaminergic neuron (DAN) and the dendrites of a corresponding type of MB output neuron (MBON). An essential role has been revealed of one DAN, the MP1 neuron, in the formation of appetitive LTM. The MP1 neuron is anatomically matched to the GABAergic MBON MVP2, which has been attributed feedforward inhibitory functions recently. This study used behavior experiments and in vivo imaging to challenge the existence of MP1-MVP2 synapses and investigate their role in appetitive LTM consolidation. MP1 and MVP2 neurons form an anatomically and functionally recurrent circuit, which features a feedback inhibition that regulates consolidation of appetitive memory. This circuit involves two opposite type 1 and type 2 dopamine receptors (the type 1 DAMB and the type 2 dD2R) in MVP2 neurons and the metabotropic GABAB-R1 receptor in MP1 neurons. It is proposed that this dual-receptor feedback supports a bidirectional self-regulation of MP1 input to the MB. This mechanism displays striking similarities with the mammalian reward system, in which modulation of the dopaminergic signal is primarily assigned to inhibitory neurons (Pavlowsky, 2018).

Formation of a memory engram is a multi-step process, from encoding the relevant information to the final storage of memory traces. Describing the neuronal architecture and functions that underlie each step of this process is crucial to understanding memory ability. In Drosophila, a very fine knowledge is available of the anatomy of the mushroom body (MB), the major olfactory integrative brain center, as well as its input and output neurons. The mapping to these circuits of various functional modalities occurring at the different stages of memory encoding, storage, and recall is also quite advanced (Pavlowsky, 2018).

Drosophila MBs are paired structures including ~2,000 intrinsic neurons per brain hemisphere. These neurons receive dendritic input from the antennal lobes through projection neurons in the calyx area on the posterior part of the brain. Their axons form a fascicle, called a peduncle, that traverses the brain to the anterior part, where axons branch to form horizontal and vertical lobes according to three major branching patterns (α/β, α'/β' and γ). MB lobes are tiled by spatially segregated presynaptic projections from dopamine neurons (DANs), on the one hand, and dendrites of MB output neurons (MBONs), on the other hand. DANs and MBONs are matched to form defined anatomical compartments that are increasingly considered as independent functional units. On several of these compartments, it was shown that DAN activity can induce heterosynaptic plasticity at the MB/MBON synapse, which could be a cellular substrate of memory encoding (Pavlowsky, 2018).

In addition to this canonical anatomical motif of the DAN/MB intrinsic neurons/MBON triad, electron microscopy connectome reconstruction in the larval brain has evidenced recently that DANs have direct synaptic connections to their matched MBONs. In the adult, direct DAN-to-MBON synapses have also been observed in several compartments of the MB vertical lobes (Pavlowsky, 2018).

MB activity is regulated by a broad spectrum of neuromodulatory input, among which tonic dopamine signaling plays an important role in the regulation of memory persistence or expression. In particular, it has been shown that sustained rhythmic activity of the MP1 DAN, also named PPL1-γ1pedc and which innervates the γ1 module and the α/β peduncle, is crucial after conditioning to enable the consolidation of both aversive and appetitive long-term memory (LTM), the most stable memory forms that rely on de novo protein synthesis. The MP1 neuron is anatomically matched with the MVP2 neuron, a GABA-ergic MBON that shows a complex arborization. The MVP2 neuron, also named MBON-γ1pedc > α/β, possesses two dendritic domains on the γ1 and peduncle compartments. On the ipsilateral side, MVP2 has presynaptic projections on MB vertical and medial lobes and also targets brain areas outside MB where other MBONs project. In particular, MVP2 neurons mediates a feedforward inhibition of specific MBONs involved in aversive and appetitive memory retrieval (Perisse, 2016). Interestingly, MVP2 neurons also send a presynaptic projection onto the contralateral peduncle (Aso, 2014; Perisse, 2016), a place of MP1 presynaptic coverage. Hence, the anatomy of the MP1-MVP2 neurons is compatible with the existence of feedback circuitry. This study tested experimentally the existence of such a functional feedback in the context of appetitive LTM formation (Pavlowsky, 2018).

Appetitive memory results from the paired delivery of an odorant and a sugar to starved flies. Only one pairing is sufficient to form both short-term memory (STM) and LTM, but it was shown that these two memory phases stem from distinct properties of the reinforcing sugar: although the sweetness of the sugar is sufficient so that flies form appetitive STM, the formation of LTM requires that the conditioning is made with a caloric sugar. The nutritional value of the reinforcing sugar translates in the fly brain as a post-ingestive sustained rhythmic signaling from MP1 neurons that is necessary to consolidate LTM. At the cellular level, STM and LTM stem from parallel and independent memory traces located in distinct subsets of MB neurons; respectively, γ neurons and α/β neurons. Several MB output circuits have been involved in the retrieval of appetitive STM (MBON-γ2α'1), LTM (MBON-α3, MBON-α1), or both (M4/M6, also named MBON-γ5β'2a/MBON-β'2mp), providing as many candidate synaptic sites of memory encoding (Pavlowsky, 2018).

This work confirmed that post-training MP1 activity is required for LTM formation, but it was shown in addition that this activity must be temporally restricted. MP1 activity is self-regulated through an inhibitory feedback by MVP2 neurons. Immediately after conditioning, the oscillatory activity of MP1 is enhanced and MVP2 is inhibited. After about 30 min, MVP2 is activated, terminating the period of MP1 increased signaling, which, this study shows is a requirement for proper LTM formation. It is proposed that the bidirectional action of this feedback loop is based at the molecular level on the sequential involvement of two antagonist dopamine receptors, the type 1 DAMB and the type 2 dD2R on one side and the metabotropic GABAB-R1 receptor on the other side (Pavlowsky, 2018).

This work describes a functional inhibitory feedback from an MBON, the GABA-ergic MVP2 neuron, to the dopaminergic neuron of the same MB module, the MP1 neuron. Anatomical data from synaptic staining and electron microscopy, as well as the requirement of a specific GABA receptor in MP1 neurons for appetitive LTM, lead to the hypothesis of a direct connection between MVP2 and MP1 neurons, although alternative scenarios featuring plurisynaptic circuits involving additional GABAergic neurons cannot be ruled out at this stage. Using time-resolved manipulation of neuronal activity, it was shown that this feedback circuit is involved in the first hour after appetitive conditioning for LTM formation. It was already known, and confirmed in this study, that the activity of MP1 neurons, in the form of regular calcium oscillations, is necessary in the first 30-45 min after conditioning to build LTM. Strikingly, in the present work, it was shown that, after this initial time period, the activity of MP1 neurons is not merely dispensable but rather deleterious for LTM formation, since activating MP1 neurons from 0.5 hr to 1 hr after conditioning caused an LTM defect (Pavlowsky, 2018).

Conversely, it was found that, in that time interval where MP1 neuron activity is deleterious, MVP2 neurons need to be active for normal LTM performance. Imaging experiments showed that blocking MVP2 neurons increased the persistence of MP1 neuron oscillations, up to more than 1 hr post-conditioning. The same effect was observed when the GABAB-R1 receptor was knocked down in MP1 neurons. Interestingly, blocking MVP2 neurons or GABA-ergic signaling in MP1 neurons mostly affected the frequency and the regularity of MP1 calcium signals, without markedly increasing their amplitude. Hence MVP2 neurons seem to be involved in terminating the period of sustained oscillatory signaling from MP1 neurons rather than merely decreasing MP1 activity. However, in the first 0.5 hr after conditioning, MVP2 neuron activity is not simply dispensable but also deleterious for LTM. Since MVP2 neurons have an inhibitory effect on MP1 activity, it is likely that MVP2 neurons have to be inhibited to let MP1 oscillations occur. Strikingly, this study established that MVP2 neurons are modulated by dopamine signaling through two receptors: DAMB, a type 1 activating receptor; and dD2R, a type 2 inhibitory receptor. Although these two receptors have opposite downstream effects, both are required in MVP2 for normal LTM performance. Overall, the results evidence that the MP1-MVP2 feedback circuit is functionally designed to allow the onset of LTM-gating oscillations only on a precise time windows of about 0.5 hr after conditioning (Pavlowsky, 2018).

It is proposed that MP1 activity is self-regulated through a dual receptor mechanism that controls MVP2 feedback. Initially, the ongoing activity of MP1 neurons inhibits MVP2 neurons through the dD2 receptor, which allows for sustained MP1 activity. In a second step, DAMB is activated in MVP2 neurons to enable the inhibitory feedback that shuts off MP1 oscillations. This model unifies molecular data and the results obtained from time-resolved thermogenetic manipulation of neuronal activity; unfortunately, such temporality of receptor involvement cannot be tested with RNAi-based knockdown (Pavlowsky, 2018).

DAMB and dD2R are two G-protein-coupled dopamine receptors. Although dD2R is a clear homolog of mammalian D2 receptor, and is negatively coupled to cAMP synthesis, the molecular mechanisms downstream of DAMB appear to be more diverse. It was shown that DAMB activation can stimulate cAMP synthesis, similarly to the function of a type 1 receptor, likely through Gβγ-coupled signaling. Surprisingly, it was recently shown that DAMB-mediated dopamine signaling could transiently inhibit the spiking of sleep-promoting neurons through the same G-protein pathway. Additionally, it was shown that DAMB can also activate downstream calcium signaling from intracellular calcium stores. In the current model, MVP2 neurons need, at one point, to be activated to dampen MP1 oscillations, so activating functions of DAMB seem to be more relevant in the present environment. Interestingly, physiological measurements in a heterologous system showed that cAMP activation occurs within tens of minutes, while calcium activation occurs on much shorter timescales. The delayed requirement of MVP2 activity (starting ~30 min after conditioning) seems to be more consistent with an activation of the cAMP pathway. It would be helpful in the future to decipher the molecular mechanism downstream of DAMB involved in this feedback loop. The sequential activation of two distinct dopamine receptors could be due to different affinities for dopamine. Indeed, pharmacological studies show that D2R-like receptors have a higher affinity toward dopamine compared to the D1-like receptors in mammals. However, in the specific case of Drosophila D2R and DAMB, similar dopamine affinities for both receptors were reported (0.5 μM for D2R [52 and 0.1-1 μM for DAMB, although these are all obtained from in vitro preparations of cultured cells. There could be also be subtler differences of activation kinetics based both on the quantity and on the mode of dopamine release by MP1 neurons (Pavlowsky, 2018).

MP1 neurons and MVP2 neurons have been shown to play crucial roles in both aversive and appetitive memories. During aversive conditioning, MP1 neurons mediate the unconditioned stimulus, which is thought to involve dDA1 activation in MB neurons. In a recent report, it was shown that suppressing the activity of MVP2 neurons during an odor presentation leads to the formation of an aversive memory toward this odor. In light of this result, these authors proposed that the role of steady-state MVP2 activity is to prevent the formation of irrelevant memory from insignificant stimuli. Given the role of MP1 in the signaling of negative stimuli during aversive learning, this finding and its interpretation are fully consistent with the existence of an inhibitory feedback from MVP2 neurons to MP1 neurons, as reported in the present work. MP1 neurons are also central in the formation of LTM after conditioning. Tonic signaling through slow oscillations of MP1 neurons gates the formation of aversive LTM after spaced training. The same kind of sustained post-training signaling builds LTM after appetitive conditioning. Both in aversive and appetitive paradigms, this LTM-gating function involves DAMB signaling in MB neurons. After aversive spaced training, it was shown that DAMB activation triggers an upregulation of MB energy metabolism, which starts the consolidation of LTM. Finally, MP1 neurons also regulate the retrieval of appetitive STM. MP1 inhibition in starved flies, through suppressive dNPF signaling, allows integration of the appetitive motivational state with the expression of MB-encoded memory trace during retrieval to allow for the expression of appetitive STM. This involves enhanced feedforward inhibition from MVP2 neurons to the M4/M6 MBONs that mediate appetitive memory retrieval. The fact that MP1 inhibition goes along with enhanced MVP2 activity is consistent with the fact that baseline MP1 activity can drive an inhibition of MVP2 through dD2R, as is reported in this study. This may explain why a knockdown of dD2R in MVP2 neurons, by indiscriminately disturbing this MP1-MVP2 inhibitory link, would impair the odor-specific message carried by M4/M6 neurons for memory retrieval and cause an STM defect. All these findings illustrate how the sophistication of MP1 neuron involvement in memory is tightly linked to the diversity of receptors and neuronal targets that it can activate. A finer understanding of these processes calls for higher resolution physiological measurements to understand how the various dopamine receptors are sensitive to different modalities or kinetics of dopamine release (Pavlowsky, 2018).

Recently, it was shown that acquisition and consolidation of appetitive LTM also rely on a positive-feedback circuit involving the α1 MB compartment, dopaminergic PAM-α1, and glutamatergic MBON-α1 neurons (Ichinose, 2015). Thus, consolidation of appetitive memory involves two different recurrent circuits that share common features, such as the MBON's dual functions in consolidation and retrieval of memory. MP1 neurons are activated after a conditioning with a nutritious sugar, which is necessary for LTM formation. PAM-α1 neurons are activated during conditioning and probably mediate the coincidence detection between sugar intake and odor perception within MB neurons. The recurrent activity of the α1 compartment loop is also necessary for proper LTM formation, presumably to stabilize a nascent memory trace. Interestingly, the electron microscopy reconstruction of the adult MB vertical lobes recently showed that MVP2 neurons form direct synapses with MBONs in the α2 and α3 modules and, probably, in the α1 compartment as well. Therefore, the two feedback circuits may not be independent, and MVP2 neurons may also mediate a feedforward input from the MP1/MVP2 loop to the PAM-α1/MBON-α1 loop. The dD2R-mediated inhibition of MVP2 neurons by MP1 activity immediately after conditioning could, therefore, help in maintaining the recurrent activity in the α1 compartment (Pavlowsky, 2018).

In conclusion, this study shown here that a negative-feedback loop functions to control appetitive LTM formation, likely involving two antagonist dopaminergic receptors. This negative-feedback loop is strikingly similar to one recently described in the mammalian mesolimbic system in which feedback from inhibitory neurons prevents the over-activation of dopaminergic neurons (Edwards, 2017). These two circuits have at least three common features: they rely on the metabotropic receptors DA1 and GABABR1; they comprise dopaminergic and inhibitory neurons, which are monosynaptically connected in mammals, and possibly also in Drosophila; and they are involved in the memory acquisition of motivationally relevant stimuli. These shared properties of negative-feedback loops highlight how similar strategies exist at both the network and molecular levels to regulate certain related behaviors across species (Pavlowsky, 2018).

Mechanism underlying starvation-dependent modulation of olfactory behavior in Drosophila larva

Starvation enhances olfactory sensitivity that encourage animals to search for food. The molecular mechanisms that enable sensory neurons to remain flexible and adapt to a particular internal state remain poorly understood. The roles of GABA and insulin signaling in starvation-dependent modulation of olfactory sensory neuron (OSN) function was studied in the Drosophila larva. The GABAB-receptor and insulin-receptor play important roles during OSN modulation. Using an OSN-specific gene expression analysis, this study explored downstream targets of insulin signaling in OSNs. The results suggest that insulin and GABA signaling pathways interact within OSNs and modulate OSN function by impacting olfactory information processing. It was further shown that manipulating these signaling pathways specifically in the OSNs impact larval feeding behavior and its body weight. These results challenge the prevailing model of OSN modulation and highlight opportunities to better understand OSN modulation mechanisms and their relationship to animal physiology (Slankster, 2020).

Starvation increases olfactory sensitivity that enhances an animal's search for food. This has been shown in insects, worms, and mammals including humans. However, the mechanisms by which an animal's starved state modulates sensory neuron function remain poorly understood. Understanding of these mechanisms significantly improved in the last decade or so from studies that showed how neuromodulators enable changes in the gain of peripheral sensory inputs. The prevailing mechanistic model for olfactory sensory neuron (OSN) modulation by the animal's starved state is that during the animal's starved-state, lower insulin signaling frees production of the short neuropeptide F receptor (sNPFR1), which increases sNPF signaling. Higher sNPF signaling increases presynaptic facilitation of OSNs, which leads to enhanced responses to odors. Interestingly, insulin and neuropeptide Y (the mammalian ortholog of sNPF) signaling also feature in the vertebrate olfactory bulb (Slankster, 2020).

However, the above model is incomplete and several questions remain. For instance, the model does not account for the role of GABA signaling, which plays important roles during both starvation and olfactory behavior in flies and mammals. The model also does not account for interactions between GABA and insulin signaling pathways that are known to impact neuromodulation in both fly and mammalian systems: For instance, GABAB-Receptor (GABABR) mediates a GABA signal from fly brain interneurons, which may be involved in the inhibitory control of Drosophila insulin like peptide (DILP) production; In mammalian CNS neurons, insulin increases the expression of GABAAR on the postsynaptic and dendritic membranes; GABA administration to humans resulted in a significant increase in circulating Insulin levels under both fasting and fed conditions. Finally, the model does not account for the ultimate targets of insulin/GABA/sNPF signaling that alter OSN sensitivity to odors and its function (Slankster, 2020).

The above questions are significant because the mechanisms driving neural circuit modulations are fundamental to understanding of how neural circuits support animal cognition and behavior. If these mechanisms are better understood, it would be possible to learn how flexibility and the ability to adapt to a particular internal state are built into the sensory circuit. Understanding the mechanisms by which the starved state of an animal modulates its olfactory sensitivity and thereby controls its food-search behavior is important for both olfactory and appetite research. Finally, this connection cannot be ignored in light of the obesity epidemic and the demonstration that obese adults have reduced olfactory sensitivity (Slankster, 2020).

This study builds upon the prevailing model and argue that GABA and insulin signaling pathways interact within OSNs to mediate starvation-dependent modulation of its function and that defects in these signaling pathways impact larval food-search and feeding behaviors, which in turn impact weight gain. The Drosophila larval system is used in this study to demonstrate evidence in support of this argument. Using larval behavior assays, this study shows that GABABR and insulin receptor (InR) are required for starvation dependent increases in larval olfactory behavior. Using a novel OSN-specific gene expression analysis, this study shows that insulin and GABA signaling pathways interact within OSNs and modulate OSN function by impacting odor reception, olfactory information processing, and neurotransmission. Finally, this study shows that manipulating these signaling pathways specifically in the OSNs impact larval feeding behavior and its body weight (Slankster, 2020).

Insulin and GABA signaling pathways interact within OSNs and likely modulate OSN function by impacting odor reception (Orco), olfactory information processing (Rut), and/or neurotransmission (Syt1). Defects in GABA/insulin signaling pathways impact the animal's feeding behavior and body weight. These findings suggest a hitherto unsuspected role for GABA signaling in starvation-dependent modulation of OSN function, a role that is likely downstream of insulin signaling. They also raise questions about how individual OSNs may be differentially modulated by the animal's starved state. Finally, these findings imply a potential relationship between nutrient sensing and animal physiology (Slankster, 2020).

GABA and insulin signaling play important roles during both starvation and olfactory behavior. While GABA signaling in different regions of the animal brain is known to mediate starvation-dependent behavior, its role in specific olfactory neurons during starvation is unclear. Similarly, insulin has long been considered as an important mediator of state dependent modulation of feeding behavior. However, its precise role in olfactory neurons during starvation is controversial. According to the prevailing model, insulin signaling decreases upon starvation. However, a previous study showed that there is a three-fold increase in DILP-6 (Drosophila Insulin like Peptide) mRNA expression in larval tissue including fat bodies upon starvation (Slaidina, 2009), which is inconsistent with the model described in this paper. While the significance of DILP-6 increase in larval tissue during starvation is as yet unclear, consistent with the prevailing model, this study shows that InR and DILP-6 expression in larval head samples decrease upon starvation (Slankster, 2020).

This study also shows that higher insulin signaling increases expression levels of GABABRs in OSNs. This result is in line with several other studies in flies and mammals that have suggested possible interactions between GABA signaling and insulin signaling in different regions of the brain. The most relevant example supporting the current observation is noted in mice where insulin increases the expression of GABAARs on the postsynaptic and dendritic membranes of CNS neurons (Wan, 1997). Other examples show how GABA signaling might influence insulin signaling. For instance, in flies, GABA signaling from interneurons has been shown to affect insulin signaling by regulating DILP production (Enell, 2010); In humans, GABA administration significantly increases circulating insulin levels under both fasting and fed conditions; In diabetic rodent models, combined oral administration of GABA and an anti-diabetic drug (Sitagliptin) promoted beta cell regeneration and reduced blood glucose levels. Overall, this study adds to this growing body of literature and strongly suggests that GABA and insulin signaling pathways interact within larval OSNs to mediate OSN modulation (Slankster, 2020).

It is noted that starvation enhanced larval attraction toward only a subset of the odors tested. A related question in the field is whether starvation enhances an animal's ability to detect food-odors or all odors. Studies are inconclusive so far. Some studies have shown that starvation enhances an animal's ability to detect both food-related odors and nonfood-related odors. While similar results have also been shown in humans, the findings regarding the relevance of odor to feeding are rather mixed. This study along with previous studies raise the possibility that starvation differentially modulates individual OSNs. Indeed, individual OSNs exhibit functional diversity that may lend them to differential modulation by the animal's internal state. This diversity may stem from heterogeneous GABABR levels on the terminals of individual OSNs that determine differential presynaptic gain control. It is reasonable to speculate that heterogeneous GABABR and/or InR levels in individual OSNs could contribute to differential modulation of OSNs by the animal's starved state, which in turn impacts behavior toward only a subset of odors (Slankster, 2020).

An inability to regulate sensitivity to food odors at appropriate times leads to irregular feeding habits, which in turn leads to weight gain. Obesity researchers will readily acknowledge that while several obvious risk factors for obesity (e.g., genetics, nutrition, metabolism, environment etc.) have been heavily researched, the relationship between nutrient sensing/sensory behavior and obesity remains grossly understudied. The present study sets the stage to further explore this relationship. Interestingly, several of the signaling molecules described in this study that play a role in OSN modulation have also been implicated in hyperphagia and obesity phenotypes. For instance, overexpression of sNPF in Drosophila and NPY injection in the hypothalamus of rats leads to increased food-intake and bigger and heavier phenotypes. Genetically obese rats have low levels of insulin in the brain including in the olfactory bulb and imbalanced insulin signaling via insulin receptors is associated with obesity phenotypes. Adenylyl cyclase (rut) deficient mice were found to be obese and both Adenylyl cyclase and Synaptotagmin have been targeted for anti-obesity drug development. These studies provide added significance to the observation that manipulating mechanisms mediating starvation-dependent modulation of OSNs impact feeding behavior and weight gain in larvae (Slankster, 2020).

Indeed, food odors can be powerful appetitive cues. A previous study showed that larvae engage in appetitive cue-driven feeding behavior and that this behavior required NPF signaling within dopaminergic neurons in higher-order olfactory processing centers (Wang, 2013). The current studies show that manipulating GABABR signaling in first-order OSNs impact appetitive cue-driven feeding behavior in larvae. While it remains to be seen whether parallel regulations during different stages of olfactory information processing impact feeding behavior, further studies are needed to reveal the mechanistic relationship between GABABR/InR signaling in OSNs, feeding behavior, and changes in body-weight (Slankster, 2020).

Based on the evidence so far, a motivating model is proposed for future investigations (see Model for OSN modulation). In this model, InR expressed on the terminals of larval OSNs act as sensors for the internal state of the animal. Its concerted activity with GABABR impacts OSN function either at the level of odor reception by affecting the expression of Orco or at the level of olfactory signal transduction by affecting the expression of Rut or at the level of neurotransmission by affecting the expression of Syt1 and sNPFR1. It is acknowledged that more exhaustive gene expression analyses are required to identify other molecular players downstream of InR and GABABR. It would also be valuable to investigate the relationship between InR expression levels on the terminals of individual OSNs and the sensitivity of individual OSNs to modulation by the animal's starved state (Slankster, 2020).

A valid concern in this study is that an innate attraction of larvae toward an odorant does not necessarily equate to food-search behavior. However, it is argued that attractiveness toward an odor source is a reliable measure of food-search behavior because an animal's ability to efficiently smell and move toward an odor source necessarily predicates most forms of such behavior. Another possibility to be considered is that changes in OSN sensitivity, food-search and/or feeding behaviors are independently regulated. For instance, it has been noted that starvation-induced hyperactivity in adult Drosophila was independently regulated from food consumption behavior in the flies. Blocking octopamine signaling in a small group of octopaminergic neurons located in the subesophageal zone (SEZ) of the fly brain neurons eliminated starvation induced hyperactivity but not the increase in food consumption. While such a possibility cannot be ruled out, the evidence presented in this study support the argument that starvation induced-changes in OSN function is related to the observed changes in food search and feeding behaviors. It is acknowledged that other studies have opted to keep larvae on sucrose with the intention of starving them of amino acids and other nutrients. So, the non-starved conditions in the present study actually represents partial starvation of macronutrients other than sugar. This was done to control the nutrient intake in the non-starved state with the intention of measuring the impact of individual macronutrients on OSN modulation in future studies. Finally, while this study tested the hypothesis that increases in body-weight of mutant genotypes are due to altered food consumption, alternate hypotheses that body-weight increases may be due to altered metabolism or increased fat accumulation haven not been tested(Slankster, 2020).

Overall, this study conducted in a simple, tractable, and highly conserved model system builds upon the prevailing model of starved-state dependent modulation of OSN function. It highlights and offers unique opportunities that are now possible to address the inadequate understanding of OSN modulation mechanisms at the resolution of single neurons, which in turn would lead to a better understand how flexibility and the ability to adapt to a particular internal state are built into the sensory circuit (Slankster, 2020).

Presynaptic inhibition of dopamine neurons controls optimistic bias

Regulation of reward signaling in the brain is critical for appropriate judgement of the environment and self. In Drosophila, the protocerebral anterior medial (PAM) cluster dopamine neurons mediate reward signals. This study shows that localized inhibitory input to the presynaptic terminals of the PAM neurons titrates olfactory reward memory and controls memory specificity. The inhibitory regulation was mediated by metabotropic gamma-aminobutyric acid (GABA) receptors clustered in presynaptic microdomain of the PAM boutons. Cell type-specific silencing the GABA receptors enhanced memory by augmenting internal reward signals. Strikingly, the disruption of GABA signaling reduced memory specificity to the rewarded odor by changing local odor representations in the presynaptic terminals of the PAM neurons. The inhibitory microcircuit of the dopamine neurons is thus crucial for both reward values and memory specificity. Maladaptive presynaptic regulation causes optimistic cognitive bias (Yamagata, 2021).

Regulation of reward signaling in the brain is critical for maximizing positive outcomes and for avoiding futile costs of the behaviors at the same time. Across animal phyla, dopamine neurons are primarily involved in reward processing. In the fruit fly Drosophila melanogaster, a subset of dopamine neurons in the protocerebral anterior medial (PAM) cluster mediates the reinforcement property of sugar reward. In olfactory learning, dopamine input to the mushroom body (MB) causes changes in preference of a simultaneously presented odor by modulating the output of odor-representing MB intrinsic neurons, Kenyon cells (KCs). Such associative presentations of odor and electric shocks were reported to change the activity of MB-projecting dopamine neurons. Recent studies suggest that axon terminals of the dopamine neurons locally integrate olfactory inputs to function as multiple independent units, though such subcellular reward processing has yet to be examined (Yamagata, 2021).

The results of this study indicate that presynaptic modulation of the PAM neurons is a critical component for determining the magnitude of dopaminergic reward signals. Notably, abolition of the local GABAergic input to the PAM terminals not only enhanced the internal reward intensity but compromised memory specificity. These behavioral alterations can be explained by a dual physiological role of GABA-B-R3, that is, the gain control and the spatial segmentation of dopaminergic reward signals in the PAM terminals. As the behavioral traits caused by the downregulation of GABA-B-R3 are characteristic in optimism, presynaptic control of reward signals may underlie such a cognitive bias. It would be fruitful to examine if a similar subcellular modulation of punishment-mediating neurons conversely leads to the pessimistic bias (Yamagata, 2021).

Serotonin modulates olfactory processing in the antennal lobe of Drosophila

Sensory systems must be able to extract features of environmental cues within the context of the different physiological states of the organism and often temper their activity in a state-dependent manner via the process of neuromodulation. The effects of the neuromodulator serotonin on a well-characterized sensory circuit, the antennal lobe of Drosophila melanogaster, was examined using two-photon microscopy and the genetically expressed calcium indicator, G-CaMP. Serotonin enhances sensitivity of the antennal lobe output projection neurons in an odor-specific manner. For odorants that sparsely activate the antennal lobe, serotonin enhances projection neuron responses and causes an offset of the projection neuron tuning curve, most likely by increasing projection neuron sensitivity. However, for an odorant that evokes a broad activation pattern, serotonin enhances projection neuron responses in some, but not all, glomeruli. Further, serotonin enhances the responses of inhibitory local interneurons, resulting in a reduction of neurotransmitter release from the olfactory sensory neurons via GABAB receptor-dependent presynaptic inhibition, which may be a mechanism underlying the odorant-specific modulation of projection neuron responses. These data suggest that the complexity of serotonin modulation in the antennal lobe accommodates coding stability in a glomerular pattern and flexible projection neuron sensitivity under different physiological conditions (Dacks, 2009).

It was asked if the responses of DM2 and VM2 PNs are modulated by 5HT in the presence of strong lateral activity. Electrical stimulation of the antennal nerve generates uniform activation of ORNs and 5HT application resulted in equal proportional enhancement of PN responses in the VM2 and DM2 glomeruli. Thus, the responses of VM2 PNs can be modulated by 5HT even in the presence of strong lateral influences, suggesting that the odorant-specific 5HT-modulation is likely due to heterogeneous lateral interactions that are revealed with the application of specific odorants (such as IAA). The equal proportional enhancement of the responses of VM2 and DM2 PNs to electrical stimulation, in combination with the enhancement of VM2 PN responses to 3-heptanol, eliminate the possibility that the differential enhancement of responses of VM2 and DM2 PNs to IAA is due to differences in the expression levels of 5HT receptors (Dacks, 2009).

To address the possibility that the differential enhancement of PN responses by 5HT is due to a differential increase in the responses of ORNs (thus increasing the amount of input to the PNs), the effects of serotonin on ORN neurotransmitter release was examined in flies bearing the Or83b-Gal4 and UAS-spH transgenes. These flies express the activity reporter, synaptopHluorin, in all ORNs that express the relatively ubiquitous Or83b protein. Surprisingly, 5HT significantly decreased the strength of ORN responses to electrical stimulation of the antennal nerve. This could either have been due to a direct decrease in the excitability of ORNs or an enhancement of the presynaptic inhibition impinging upon ORNs, which in Drosophila is mediated by GABAB receptor expression on ORNs (Olson, 2008; Root, 2008). To test the possibility that 5HT enhances presynaptic inhibition, the GABAB receptor antagonist, CGP54626 (as described in Root, 2008), was appled to block any presynaptic inhibition experienced by the ORNs. and then 5HT was applied. When 5HT was applied to preparations that had been pretreated with CGP54626, there was no longer any effect of 5HT on ORN response, suggesting that the attenuation of ORN responses by 5HT was due to an enhancement of the influence of GABAergic LNs (Dacks, 2009).

To further confirm that 5HT modulates the activity of GABAergic LNs, the effects were examined of 5HT on the responses of flies bearing the UAS-GCaMP and GAD1-Gal4 transgenes, which label most of the GABAergic LNs within the AL. Serotonin application enhanced the responses of these LNs. Further, there was no statistically significant difference in the magnitude of response enhancement for LNs in the VM2 glomerulus, compared to the DM2 glomerulus. This indicated that the differential enhancement of PN responses to IAA was not likely due to a greater enhancement of those LNs innervating the VM2 glomerulus. To determine if there was a difference in the level of expression of GABAB receptors expressed by ORNs innervating the VM2 and DM2 glomeruli, the reporter expression levels were measure in flies bearing the GABABR2-Gal4 and UAS-CD8GFP transgenes, which had been analyzed in Root (2008). There was a statistically significant difference in the levels of GABAB receptors expressed by ORNs innervating the VM2 glomerulus, compared to those innervating the DM2 glomerulus. These results suggest that while 5HT enhances the responses of PNs and GABAergic LNs, the resultant increase in presynaptic inhibition impinging upon the ORNs may have balanced the enhancement of PN responses in those glomeruli in which the ORNs express high levels of the GABAB receptor (Dacks, 2009).

This study has investigated modulation of different neuronal populations in the Drosophila AL by 5HT, with two-photon imaging of neural activity in response to odor and electrical stimulation. Serotonin appears to have two primary effects: an enhancement of PN and LN responses and a concomitant increase in lateral interactions mediated by LNs. The sex pheromone, cVA, and the odorants, ethyl hexanoate and 3-heptanol, selectively activate the DA1, DM2, and VM2 glomeruli, respectively, and the response strength of PNs in these glomeruli is enhanced by 5HT. Studies of the AL in moths have established that the excitability of AL neurons is enhanced by 5HT (see Dacks, 2008), which causes a reduction of two K+ conductances. The resultant increase in membrane resistance should, therefore, decrease the amount of input current required to elicit a response, thus increasing the sensitivity of AL neurons. The results are consistent with these effects of 5HT observed in the ALs of moth. Serotonin causes Drosophila PNs to respond to intensities of antennal nerve stimulation that were previously subthreshold. It is possible that 5HT causes a similar reduction in K+ conductances in Drosophila AL neurons; however, further biophysical studies are necessary to determine if this is the case (Dacks, 2009).

When the effects were examined of 5HT on the responses of PNs to odors that activated large portions of the AL, evidence was found that 5HT modulated lateral interactions within the AL. For the odor, IAA, 5HT enhanced the responses of PNs in many (including the DM2), but not all, glomeruli (in particular, the VM2). The responses of the VM2 PNs to 3-heptanol and antennal nerve stimulation were enhanced by 5HT, indicating that the differential effects of 5HT on IAA responses were due to modulation of lateral interactions within the AL and not due to the VM2 PNs being immune to the effects of 5HT. Serotonin attenuated the responses of ORNs in a GABAB-receptor-dependent manner and enhanced the responses of GABAergic LNs, indicating that 5HT enhanced the presynaptic inhibition impinging upon ORNs. Root (2008) reported that there is heterogeneous expression of GABAB receptors by the ORNs innervating different glomeruli, and using previously unpublished data from that study, a greater expression of GABAB receptors by ORNs innervating the VM2 glomerulus was observed, compared to the DM2 glomerulus, which likely explains the differences in the effects of 5HT on the responses of PNs in these glomeruli for IAA. This does not, however, preclude the possibility that 5HT modulates lateral inhibition or excitation impinging directly upon PNs. There is a rich diversity of LNs interconnecting the glomeruli of the AL. In Drosophila, different glomeruli receive variable innervation from LNs, and the lateral interactions between glomeruli are nonuniform. Heterogeneous inhibitory interactions within the AL have been demonstrated in other insect species, and so, modulation of LN activity by 5HT likely has nonuniform effects across glomeruli (Dacks, 2009).

Comparing what is known already from moths with what was found in Drosophila, several underlying principles begin to emerge. This study shows that, as in moths, 5HT enhances the responses of both PNs and LNs in the AL of Drosophila. Further, this did not simply result in a simple increase in activity across the entirety of the AL, but rather that the modulation of the lateral network resulted in an enhancement of presynaptic inhibition, likely resulting, at least in part, in an odor-dependant enhancement of responses, as in the case for the odor IAA. It should be noted that the VM2 glomerulus produced weaker responses to IAA, compared to the other glomeruli that were enhanced. This suggests that 5HT may serve to enhance strongly activated glomeruli within the pattern of AL activation and enhance suppression of more weakly activated glomeruli. Serotonin may enhance only certain features of the AL representation that are key for the behavioral sensitivity of flies or moths to a given odor, and the effects of 5HT on the local network, at least in part, result in a dampening mechanism to prevent a nonspecific enhancement of all activity (potentially via an enhancement of presynaptic inhibition (Dacks, 2009).

Both flies and moths possess a single morphologically similar neuron (the contralaterally projecting, serotonin-immunoreactive, deutocerebral, or CSD neuron) that innervates the antennal lobes, and electron microscopy studies in the moth revealed serotonergic synaptic release sites within the AL. The levels of 5HT in the AL of moths vary throughout the day, reaching their peak when moths are most active and reaching their trough when moths are least active. Recordings in moths have demonstrated that this neuron responds to mechanosensory stimulation of the antennae by wind, suggesting that this neuron releases 5HT in the AL when the moths are actively tracking odor sources. Most, if not all, behaviors exhibited by moths are, at least in part, mediated by olfactory stimuli, and so, it appears that the CSD neuron affects olfactory processing only when the moths (and, potentially, flies) are most likely to be using the olfactory system. This could, therefore, subserve a function to modulate the acuity and sensitivity of the antennal lobe, based on the arousal state of the individual animal (Dacks, 2009).

A sensory system must temper its function based on the current physiological state. Neuromodulation accommodates state-dependent adjustment of neural function, whereby synaptic strength is altered by affecting presynaptic release of primary neurotransmitters or the effective synaptic current in postsynaptic cells. By adjusting the excitability of the cellular elements within a network, neuromodulators can alter network properties. In moths, the levels of 5HT in the AL cycle throughout the day, peaking when moths are most active, and 5HT injection increases olfactory behavioral sensitivity, suggesting that 5HT may be a mechanism by which arousal modifies the performance of the AL to best suit the physiological state. Elevating 5HT levels promotes sleep in Drosophila via the 5HT1A receptors. However, the 5HT1A receptor is not required for the enhancement by 5HT of PN responses to cVA in the AL of Drosophila. Circadian entrainment in Drosophila requires the 5HT1B and 5HT2 receptors. Four 5HT receptor genes, 5HT1A, 5HT1B, 5HT2, and 5HT7, have been identified in the Drosophila genome. Therefore, it is possible that different 5HT receptors participate in different neural circuits to regulate complex behavioral traits in response to the release of 5HT (Dacks, 2009).

Findings from this study suggest that in addition to similarity in the anatomical organization of the olfactory system, neuromodulation is similar in different animal species as well. In the AL of Manduca, 5HT enhances the responses of both PNs and LNs and causes odorant-dependent modulation of olfactory responses. In the rat, 5HT causes a depolarization of the membrane potential of some mitral cells in the olfactory bulb and a hyperpolarization of other mitral cells that is blocked by GABAA receptor antagonists. Further, a recent study in mice found, similar to Drosophila, that the responses of ORNs were attenuated by 5HT, an effect mediated by GABAB-receptor-dependent presynaptic inhibition (Petzhold, 2009). This suggests that across diverse phyla, 5HT enhances the responses of output neurons from the primary olfactory neuropil, yet concurrently modulates the activity of the local circuitry. The activation of specific sets of local interneurons by a given odor results in patterns of lateral excitation and inhibition, which are likely enhanced by 5HT. The overall effect of this form of modulation is an enhancement of certain response features over others, although future experiments with genetic manipulations of 5HT receptors in specific populations of AL neurons should shed light on the behavioral significance of the apparent selective modulation of specific olfactory response features by 5HT (Dacks, 2009).

Presynaptic gain control drives sweet and bitter taste integration in Drosophila

The sense of taste is critical in determining the nutritional suitability of foods. Sweet and bitter are primary taste modalities in mammals, and their behavioral relevance is similar in flies. Sweet taste drives the appetitive response to energy sources, whereas bitter taste drives avoidance of potential toxins and also suppresses the sweet response. Despite their importance to survival, little is known about the neural circuit mechanisms underlying integration of sweet and bitter taste. This study describes a presynaptic gain control mechanism in Drosophila that differentially affects sweet and bitter taste channels and mediates integration of these opposing stimuli. Gain control is known to play an important role in fly olfaction, where GABAB receptor (GABABR) mediates intra- and interglomerular presynaptic inhibition of sensory neuron output. In the taste system, gustatory receptor neurons (GRNs) responding to sweet compounds were found to express GABABR, whereas those that respond to bitter do not. GABABR mediates presynaptic inhibition of calcium responses in sweet GRNs, and both sweet and bitter stimuli evoke GABAergic neuron activity in the vicinity of GRN axon terminals. Pharmacological blockade and genetic reduction of GABABR both lead to increased sugar responses and decreased suppression of the sweet response by bitter compounds. A model is proposed in which GABA acts via GABABR to expand the dynamic range of sweet GRNs through presynaptic gain control and suppress the output of sweet GRNs in the presence of opposing bitter stimuli (Chu, 2014).

GABAB receptors play an essential role in maintaining sleep during the second half of the night in Drosophila melanogaster

GABAergic signalling is important for normal sleep in humans and flies. This study has advance the current understanding of GABAergic modulation of daily sleep patterns by focusing on the role of slow metabotropic GABAB receptors in Drosophila. It was asked whether GABAB-R2 receptors are regulatory elements in sleep regulation in addition to the already identified fast ionotropic Rdl GABAA receptors. By immunocytochemical and reporter-based techniques it was shown that the pigment dispersing factor (PDF)-positive ventrolateral clock neurons (LNv) express GABAB-R2 receptors. Downregulation of GABAB-R2 receptors in the large PDF neurons (l-LNv) by RNAi reduced sleep maintenance in the second half of the night, whereas sleep latency at the beginning of the night that was previously shown to depend on ionotropic Rdl GABAA receptors remained unaltered. The results confirm the role of the l-LNv neurons as an important part of the sleep circuit in D. melanogaster and also identify the GABAB-R2 receptors as the thus far missing component in GABA-signalling that is essential for sleep maintenance. Despite the significant effects on sleep, no changes were observed changes in circadian behaviour in flies with downregulated GABAB-R2 receptors, indicating that the regulation of sleep maintenance via l-LNv neurons is independent of their function in the circadian clock circuit (Gmeiner, 2013).

The fruit fly has become a well-accepted model for sleep research. As in mammals, it has been shown that the sleep-like state of Drosophila is associated with reduced sensory responsiveness and reduced brain activity, and is subject to both circadian and homeostatic regulation. Similarly to in humans, monaminergic neurons (specifically dopaminergic and octopaminergic neurons) enhance arousal in fruit flies, whereas GABAergic neurons promote sleep (Agosto, 2008). As in humans, GABA advances sleep onset (reduces sleep latency) and prolongs total sleep (increases sleep maintenance). Brain regions possibly implicated in the regulation of sleep in D. melanogaster are the pars intercerebralis, the mushroom bodies and a subgroup of the pigment dispersing factor (PDF)-positive neurons called the l-LNv neurons. The l-LNv belong to the circadian clock neurons, indicating that in flies, as in mammals, the sleep circuit is intimately linked to the circadian clock and that the mechanisms employed to govern sleep in the brain are evolutionarily ancient (Gmeiner, 2013).

The l-LNv are conspicuous clock neurons with wide arborisations in the optic lobe, fibres in the accessory medulla -- the insect clock centre -- and connections between the brain hemispheres. Thus, the l-LNv neurons are anatomically well suited to modulate the activity of many neurons. In addition, their arborisations overlap with those of monaminergic neurons. Several studies show that they indeed receive dopaminergic, octopaminergic and GABAergic input and that they control the flies' arousal and sleep. Furthermore, the l-LNv are directly light sensitive and promote arousal and activity in response to light, especially in the morning (Gmeiner, 2013).

A part of the sleep-promoting effect of GABA on the l-LNv has been shown to be mediated via the fast ionotropic GABAA receptor Rdl (Resistance to dieldrin) (Agosto, 2008). Rdl Cl- channels are expressed in the l-LNv (Agosto, 2008) and, similar to mammalian GABAA receptors, they mediate fast inhibitory neurotransmission. As expected, GABA application reduced the action potential firing rate in the l-LNv, whereas application of picrotoxin, a GABAA receptor antagonist, increased it (McCarthy, 2011). Furthermore, an Rdl receptor mutant with prolonged channel opening and consequently increased channel current significantly decreased sleep latency of the flies after lights-off, whereas the downregulation of the Rdl receptor via RNAi increased it (Agosto, 2008; Gmeiner, 2013 and references therein).

Nevertheless, the manipulation of the Rdl receptor had no effect on sleep maintenance. Because the latter is significantly reduced after silencing the GABAergic neurons (Parisky, 2008), other GABA receptors must be responsible for maintaining sleep. Suitable candidates are slow metabotropic GABAB receptors that are often co-localised with ionotropic GABAA receptors (Enell, 2007). In Drosophila, like in mammals, the metabotropic GABAB receptors are G-protein-coupled seven-transmembrane proteins composed of two subunits, GABAB-R1 and GABAB-R2 (Kaupmann, 1998; Mezler, 2001). The GABAB-R1 is the ligand binding unit and GABAB-R2 is required for translocation to the cell membrane and for stronger coupling to the G-protein (Kaupmann, 1998; Galvez, 2001). This study shows that the l-LNv do express metabotropic GABAB-R2 receptors and that these receptors are relevant for sleep maintenance but not for sleep latency. Thus, metabotropic and ionotropic GABA receptors are cooperating in sleep regulation (Gmeiner, 2013).

This study shows that metabotropic GABAB-R2 receptors are expressed on the PDF-positive clock neurons (LNv neurons), and that their downregulation in the l-LNv by RNAi results in: (1) a higher activity level throughout the day and night and (2) reduced sleep maintenance in the second half of the night. Neither sleep onset nor circadian rhythm parameters were affected by the downregulation. It is concluded that GABA signalling via metabotropic receptors on the l-LNv is essential for sustaining sleep throughout the night and for keeping activity at moderate levels throughout the 24-h day (preventing flies from hyperactivity). A major caveat of RNAi is off-target effects, particularly when Gal4 drivers are expressed in large numbers of non-target neurons. Though GABAB-R2 was downregulated in only eight neurons per brain hemisphere and the behavioural effects of the knockdown experiments were carefully correlated with observation and measures of GABAB-R2 immunostaining in the s-LNv and l-LNv, it is still possible that some effects were due to off-target knockdown of other membrane proteins. Nevertheless, given the fact that no such effects have been reported in the previous paper that used the same GABAB-R2 RNAi line (Root, 2008), it is thought unlikely that the behavioural effects described in this study were due to off-target knockdown of other genes (Gmeiner, 2013).

The results are in line with a former study describing the location of GABAB receptors in D. melanogaster (Hamasaka, 2005). The ionotropic GABAA receptor Rdl has also been identified on the l-LNv neurons and has been shown to regulate sleep, but its downregulation delayed only sleep onset and did not perturb sleep maintenance (Parisky, 2008). In contrast, silencing GABAergic signalling influenced sleep onset and sleep maintenance, indicating that GABA works through the fast Rdl receptor, and also implying a longer-lasting signalling pathway. GABAB receptors are perfect candidates in mediating slow but longer-lasting effects of GABA. Often, GABAA and GABAB receptors cooperate in mediating such fast and slow effects. For example, in the olfactory system, GABAA receptors mediate the primary modulatory responses to odours whereas GABAB receptors are responsible for long-lasting effects (Wilson, 2005; Gmeiner, 2013 and references therein).

In D. melanogaster, GABAB receptors consist of the two subunits GABAB-R1 and GABAB-R2, and only the two units together can efficiently activate the metabotropic GABA signalling cascade (Galvez, 2001; Mezler, 2001). In the current experiments, only GABAB-R2 was downregulated, but this manipulation should also have decreased the amount of functional GABAB-R1/GABAB-R2 heterodimers and, therefore, reduced GABAB signalling in general. Taking into account that sleep maintenance in the second half of the night was already significantly impaired by an ~46% reduction in detectable GABAB-R2 immunostaining intensity in the l-LNv clock neurons, it can be assumed that GABAB receptors account for an even larger portion of the sleep maintenance than detected in these experiments. Thus GABAB receptors play a crucial role in mediating GABAergic signals to the l-LNv neurons, which are needed to sustain sleep throughout the night. This is mainly due to the maintenance of extended sleep bout durations in the second half of the night. When signalling by the GABAB receptor is reduced, sleep bouts during this interval are significantly shortened, leading to less total sleep (Gmeiner, 2013).

Most importantly, this study confirmed the l-LNv as important components in regulation of sleep and arousal (Agosto, 2008; Parisky, 2008; Kula-Eversole, 2010; Shang, 2011). In contrast, the s-LNv seem to be not involved in sleep-arousal regulation but are rather important for maintaining circadian rhythmicity under DD (reviewed by Helfrich-Förster, 2007). One caveat in clearly distinguishing the function of s-LNv and l-LNv is the fact that both cell clusters express the neuropeptide PDF and, as a consequence, Pdf-GAL4 drives expression in both subsets of clock neurons. Though no significant GABAB-R2 knock-down in the s-LNv is seen, it cannot be completely excluded that GABAB-R2 was slightly downregulated in these clock neurons and that this knock-down contributes to the observed alterations in sleep. To restrict the knock-down to the s-LNv the R6-GAL4 line was used that is expressed in the s-LNv but not in the l-LNv. Neither a reduction in GABAB-R2 staining intensity in the s-LNv nor any effects on sleep in the second half of the night was seen. The lack of any visible GABAB-R2 downregulation in the s-LNv with R6-GAL4 is in agreement with observations of Shafer and Taghert (Shafer, 2009), who could completely downregulate PDF in the s-LNv using Pdf-GAL4 but not using R6-GAL4. Thus, R6-GAL4 is a weaker driver than Pdf-GAL4 and is obviously not able to influence GABAB-R2 in the s-LNv. Nevertheless, in the current experiments the R6-Gal4-driven GABAB-R2 RNAi led to flies that had slightly higher diurnal activity levels and less diurnal rest than the control flies. This suggests that GABAB-R2 was downregulated somewhere else. When checking the R6-GAL4 expression more carefully it was found that R6-GAL4 was not restricted to the brain, but was also present in many cells of the thoracic and especially the abdominal ganglia. Given the broad expression of GABAB-R2, a putative knock-down in the ventral nervous system is likely to affect locomotor activity (Gmeiner, 2013).

The results on the l-LNv certainly do not exclude a role of GABA in the circadian clock controlling activity rhythms under DD conditions (here represented by the s-LNv). In mammals, GABA is the most abundant neurotransmitter in the circadian clock centre in the brain -- the suprachiasmatic nucleus. GABA interacts with GABAA and GABAB receptors, producing primarily but not exclusively inhibitory responses through membrane hyperpolarisation. GABA signalling is important for maintaining behavioural circadian rhythmicity, it affects the amplitude of molecular oscillations and might contribute to synchronisation of clock cells within the suprachiasmatic nucleus. The same seems to be true for fruit flies. The s-LNv neurons of adults alter cAMP levels upon GABA application on isolated brains in vitro (Lelito, 2012). Hyperexcitation of GABAergic neurons disrupts the molecular rhythms in the s-LNv and renders the flies arrhythmic (Dahdal, 2010). Thus, GABA signalling affects the circadian clock in the s-LNv. Flies with downregulated GABAB-R2 receptors were found to have slightly longer free-running periods than the control flies, but this turned out to be only significant in comparison with Control 2 and not to Control 1. Dahdal (2010) found similar small effects on period after downregulating GABAB-R2 receptors, but a significant period lengthening after downregulating GABAB-R3 receptors. This indicates that GABA signals via GABAB-R3 receptors to the s-LNv and was confirmed in vitro in the larval Drosophila brain by Ca2+ imaging (Dahdal, 2010). Nevertheless, the study of Dahdal does not rule out that GABA signals via GABAB-R3 plus GABAB-R2 receptors on the adult s-LNv. This study found a rather strong expression of GABAB-R2 receptors in these clock neurons, and were not able to downregulate it significantly by RNAi, although dicer2 was used as amplification. Dahdal did not use dicer2, and they also did not measure the effectiveness of the downregulation of GABAB-R2 by RNAi immunocytochemically directly in the s-LNv. Thus, the exact GABAB receptors that mediate GABA responses in the adult s-LNv need still to be determined (Gmeiner, 2013).

In summary, it is concluded that the l-LNv subgroup of the PDF-positive clock neurons is a principal target of sleep-promoting and activity-repressing GABAergic neurons and sits at the heart of the sleep circuit in D. melanogaster. Thus, the sleep circuitry of flies is clearly more circumscribed and simpler than that of mammals. Mammals have many targets of sleep-promoting GABAergic neurons, and the circadian clock seems to have a mainly modulatory and less direct influence on sleep (Mistlberger, 2005). The fly sleep circuitry may therefore have condensed the mammalian arousal and sleep stimulating systems (e.g., monaminergic, cholinergic, peptidergic and GABAergic systems) into a simpler and more compact region, which seems to largely coincide with the eight PDF-positive l-LNv cells of the circadian circuit (Gmeiner, 2013).

Sleep-promoting effects of threonine link amino acid metabolism in Drosophila neuron to GABAergic control of sleep drive

Emerging evidence indicates the role of amino acid metabolism in sleep regulation. This study demonstrates sleep-promoting effects of dietary threonine (SPET) in Drosophila. Dietary threonine markedly increased daily sleep amount and decreased the latency to sleep onset in a dose-dependent manner. High levels of synaptic GABA or pharmacological activation of metabotropic GABA receptors (GABAB-R) suppressed SPET. By contrast, synaptic blockade of GABAergic neurons or transgenic depletion of GABAB-R in the ellipsoid body R2 neurons enhanced sleep drive non-additively with SPET. Dietary threonine reduced GABA levels, weakened metabotropic GABA responses in R2 neurons, and ameliorated memory deficits in plasticity mutants. Moreover, genetic elevation of neuronal threonine levels was sufficient for facilitating sleep onset. Taken together, these data define threonine as a physiologically relevant, sleep-promoting molecule that may intimately link neuronal metabolism of amino acids to GABAergic control of sleep drive via the neuronal substrate of sleep homeostasis (Ki, 2019).

The circadian clock and sleep homeostasis are two key regulators that shape daily sleep behaviors in animals. In stark contrast to the homeostatic nature of sleep, the internal machinery of sleep is vulnerable to external (e.g., environmental change) or internal conditions (e.g., genetic mutation) that lead to adaptive changes in sleep behaviors. Sleep behavior is conserved among mammals, insects, and even lower eukaryotes. Since the identification of the voltage-gated potassium channel Shaker as a sleep-regulatory gene in Drosophila, fruit flies have been one of the most advantageous genetic models to dissect molecular and neural components that are important for sleep homeostasis and plasticity (Ki, 2019).

To date, a number of sleep-regulatory genes and neurotransmitters have been identified in animal models as well as in humans. For instance, the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is known to have a sleep-promoting role that is conserved in invertebrates and vertebrates. Hypomorphic mutations in mitochondrial GABA-transaminase (GABA-T) elevate GABA levels and lengthen baseline sleep in flies (Chen, 2015). The long sleep phenotype in GABA-T mutants accompanies higher sleep consolidation and shorter latency to sleep onset, consistent with the observations that pharmacological enhancement of GABAergic transmission facilitates sleep in flies and mammals, including humans. In addition, resistance to dieldrin (Rdl), a Drosophila homolog of the ionotropic GABA receptor, suppresses wake-promoting circadian pacemaker neurons in adult flies to exert sleep-promoting effects. Similarly, 4,5,6,7-tetrahydroisoxazolo[5,4 c]pyridin-3-ol (THIP), an agonist of the ionotropic GABA receptor, promotes sleep in insects and mammals (Ki, 2019).

Many sleep medications modulate GABAergic transmission. A prominent side effect of anti-epileptic drugs relevant to GABA is causing drowsiness. Conversely, glycine supplements improve sleep quality in a way distinct from traditional hypnotic drugs, minimizing deleterious cognitive problems or addiction. In fact, glycine or D-serine acts as a co-agonist of N-methyl-D-aspartate receptors (NMDARs) and promotes sleep through the sub-type of ionotropic glutamate receptors. Emerging evidence further supports the roles of amino acid transporters and metabolic enzymes in sleep regulation. In particular, it has been demonstrated that starvation induces the expression of metabolic enzymes for serine biosynthesis in Drosophila brains, and elevates free serine levels to suppress sleep via cholinergic signaling (Sonn, 2018). These observations prompted a hypothesis that other amino acids may also display neuro-modulatory effects on sleep behaviors (Ki, 2019).

The molecular and neural machinery of sleep regulation intimately interacts with external (e.g., light, temperature) and internal sleep cues (e.g., sleep pressure, metabolic state) to adjust the sleep architecture in animals. Using a Drosophila genetic model, this study has investigated whether dietary amino acids could affect sleep behaviors, through this investigation SPET was discovered. Previous studies have demonstrated that the wake-promoting circadian pacemaker neurons are crucial for timing sleep onset after lights-off in LD cycles. In addition, WAKE-dependent silencing of clock neurons and its collaborative function with RDL have been suggested as a key mechanism in the circadian control of sleep onset. However, the current evidence indicates that SPET facilitates sleep onset in a manner independent of circadian clocks. It was further elucidated that SPET operates likely via the down-regulation of metabotropic GABA transmission in R2 EB neurons, a neural locus for generating homeostatic sleep drive (Ki, 2019).

Both food availability and nutritional quality substantially affect sleep behaviors in Drosophila. Sucrose contents in food and their gustatory perception dominate over dietary protein to affect daily sleep. Starvation promotes arousal in a manner dependent on the circadian clock genes Clock and cycle as well as neuropeptide F (NPF), which is a fly ortholog of mammalian neuropeptide Y. On the other hand, protein is one of the nutrients that contribute to the postprandial sleep drive in Drosophila and this observation is possibly relevant to SPET. While Leucokinin (Lk) and Lk receptor (Lkr) play important roles in dietary protein-induced postprandial sleepand in starvation-induced arousal, comparable SPET was observed between hypomorphic mutants of Lk or Lkr and their heterozygous controls. Therefore, SPET and its neural basis reveal a sleep-regulatory mechanism distinct from those involved in sleep plasticity relevant to food intake (Ki, 2019).

What will be the molecular basis of SPET? Given the general implication of GABA in sleep promotion, a simple model will be that a molecular sensor expressed in a subset of GABAergic neurons (i.e., LN) directly responds to an increase in threonine levels, activates GABA transmission, and thereby induces sleep. Several lines of evidence, however, favored the other model that dietary threonine actually down-regulates metabotropic GABA transmission in R2 EB neurons, de-represses the neural locus for generating homeostatic sleep drive, and thereby enhances sleep drive. The latter model does not necessarily conflict with sleep-promoting effects of genetic or pharmacological conditions that generally elevate GABA levels or enhance GABAergic transmission since those effects will be the net outcome of activated GABA transmission via various sub-types of GABA receptors expressed in either wake- or sleep-promoting neurons and their (Ki, 2019).

The structural homology among threonine, GABA, and their metabolic derivatives (e.g., alpha-ketobutyrate and gamma-hydroxybutyrate) led to the hypothesis that these relevant chemicals may act as competitive substrates in enzymatic reactions for their overlapping metabolism. Consequently, dietary threonine may limit the total flux of GABA-glutamate-glutamine cycle possibly through substrate competition, decreases the size of available GABA pool, and thereby down-scales GABA transmission for SPET. This accounts for why genetic or pharmacological elevation of GABA levels rather suppresses SPET. Threonine, GABA, and their derivatives may also act as competitive ligands for metabotropic GABA receptors, explaining weak GABA responses in R2 EB neurons of threonine-fed flies. Biochemical and neural evidence supportive of this hypothesis is quite abundant. It has been previously shown that alpha-ketobutyrate, GABA, and the ketone body beta-hydroxybutyrate act as competitive substrates in common enzymatic reactions. Moreover, functional interactions of beta-hydroxybutyrate or gamma-hydroxybutyrate with GABAergic signaling have been well documented. Finally, threonine and GABA derivatives have anti-convulsive effects, which further support their common structural and functional relevance to GABAergic signaling (Ki, 2019).

The removal of the amino group is the initial step for amino acid metabolism, and various transaminases mediate its transfer between amino acids and alpha-keto acids. On the other hand, a group of amino acids (i.e., glutamate, glycine, serine, and threonine) has their own deaminases that can selectively remove the amino group. The presence of these specific deaminases is indicative of active mechanisms that individually fine-tune the baseline levels of these amino acids in metabolism, and possibly in the context of other physiological processes as well. This idea is further supported by the conserved roles of glutamate, glycine, and serine as neurotransmitters or neuromodulators important for brain function, including sleep regulation. In fact, serine, glycine, and threonine constitute a common metabolic pathway, and threonine may contribute indirectly to glycine- or serine-dependent activation of sleep-promoting NMDAR. Nonetheless, this study found that sleep-modulatory effects of dietary glycine were distinct from SPET and thus, it is speculated that threonine may act as an independent neuromodulator, similar to other amino acids with their dedicated deaminases (Ki, 2019).

While several lines of the data support that threonine is likely to be an endogenous sleep driver in fed conditions, it wa recently demonstrated that starvation induces serine biosynthesis in the brain and neuronal serine subsequently suppresses sleep via cholinergic signaling (Sonn, 2018). These two pieces of relevant works establish a compelling model that the metabolic pathway of serine-glycine-threonine functions as a key sleep-regulatory module in response to metabolic sleep cues (e.g., food ingredients and dietary stress). It is further hypothesized that the adaptive control of sleep behaviors by select amino acids and their conserved metabolic pathway suggests an ancestral nature of their sleep regulation. Future studies should address if the serine-glycine-threonine metabolic pathway constitutes the sleep homeostat that can sense and respond to different types of sleep needs. In addition, it will be interesting to determine if this metabolic regulation of sleep is conserved among other animals, including humans (Ki, 2019).

Activity-dependent regulation of astrocyte GAT levels during synaptogenesis

Astrocytic uptake of GABA through GABA transporters (GATs) is an important mechanism regulating excitatory/inhibitory balance in the nervous system; however, mechanisms by which astrocytes regulate GAT levels are undefined. This study found that at mid-pupal stages the Drosophila melanogaster CNS neuropil was devoid of astrocyte membranes and synapses. Astrocyte membranes subsequently infiltrated the neuropil coordinately with synaptogenesis, and astrocyte ablation reduced synapse numbers by half, indicating that Drosophila astrocytes are pro-synaptogenic. Shortly after synapses formed in earnest, GAT was upregulated in astrocytes. Ablation or silencing of GABAergic neurons or disruption of metabotropic GABA receptor 1 and 2 (GABABR1/2) signaling in astrocytes led to a decrease in astrocytic GAT. Notably, developmental depletion of astrocytic GABABR1/2 signaling suppressed mechanosensory-induced seizure activity in mutants with hyperexcitable neurons. These data reveal that astrocytes actively modulate GAT expression via metabotropic GABA receptor signaling and highlight the importance of precise regulation of astrocytic GAT in modulation of seizure activity (Muthukumar, 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).

Role of GABAergic inhibition in shaping odor-evoked spatiotemporal patterns in the Drosophila antennal lobe

Drosophila olfactory receptor neurons project to the antennal lobe, the insect analog of the mammalian olfactory bulb. GABAergic synaptic inhibition is thought to play a critical role in olfactory processing in the antennal lobe and olfactory bulb. However, the properties of GABAergic neurons and the cellular effects of GABA have not been described in Drosophila, an important model organism for olfaction research. Whole-cell patch-clamp recording, pharmacology, immunohistochemistry, and genetic markers have been used to investigate how GABAergic inhibition affects olfactory processing in the Drosophila antennal lobe. This study shows that many axonless local neurons (LNs) in the adult antennal lobe are GABAergic. GABA hyperpolarizes antennal lobe projection neurons (PNs) via two distinct conductances, blocked by a GABAA- and a GABAB-type antagonist, respectively. Whereas GABAA receptors shape PN odor responses during the early phase of odor responses, GABAB receptors mediate odor-evoked inhibition on longer time scales. The patterns of odor-evoked GABAB-mediated inhibition differ across glomeruli and across odors. LNs display broad but diverse morphologies and odor preferences, suggesting a cellular basis for odor- and glomerulus-dependent patterns of inhibition. Together, these results are consistent with a model in which odors elicit stimulus-specific spatial patterns of GABA release, and as a result, GABAergic inhibition increases the degree of difference between the neural representations of different odors (Wilson, 2005).

Smell begins when odor molecules interact with olfactory receptor neurons (ORNs). ORNs then project to the brain following anatomical rules common to species as evolutionarily distant as flies and rodents. Briefly, the odor sensitivity of a particular ORN is specified by the expression of a single olfactory receptor gene. All the ORNs that express a particular receptor send their axons to the same glomeruli in the brain. There, ORNs make synapses with second-order neurons [mitral cells (in vertebrates) or projection neurons (in insects)] (Wilson, 2005).

What happens when signals reach these second-order olfactory neurons is determined by complex local circuitry. One obstacle to understanding this circuitry is the sheer number of input channels in the mammalian olfactory system. The rat olfactory bulb contains ~1000 glomeruli; in contrast, the Drosophila antennal lobe contains just ~40 glomeruli. This, along with the genetic advantages of Drosophila, makes the fruit fly a useful model for investigating olfactory processing (Wilson, 2005).

A given odor excites many Drosophila antennal lobe projection neurons (PNs) but inhibits others. These odor-evoked inhibitory epochs can last from ~100 ms to several seconds. Similar odor-evoked inhibition has also been observed in other insects and in olfactory bulb mitral cells. Some odor responses of mitral cells and PNs are purely inhibitory. Other responses are multiphasic, in which an inhibitory epoch follows or precedes an excitatory epoch. These temporal patterns are cell and odor dependent and have been proposed to encode information about the stimulus. However, the mechanism of these 'slow' patterns is not fully understood (Wilson, 2005).

One possibility is that inhibitory epochs represent periods when principal neurons are synaptically inhibited by GABAergic local neurons (LNs). GABA-immunoreactive LNs are present in the adult antennal lobe of several species and in the larval Drosophila antennal lobe (Python, 2002). Antennal lobe LNs can synaptically inhibit PNs, and the antennal lobe is strongly immunoreactive for GABAA receptors (Harrison, 1996). However, GABAA antagonists do not block odor-evoked slow inhibition or slow temporal patterns in PNs. Therefore, these inhibitory epochs have been hypothesized to reflect a metabotropic conductance or the action of a different inhibitory neurotransmitter. Alternatively, inhibition of PNs could be caused by inhibition of ORNs (Wilson, 2005).

This study investigated the mechanisms of odor-evoked inhibition in PNs. It was confirmed that many Drosophila antennal lobe LNs are GABAergic. GABA receptors contribute to odor-evoked inhibition of PNs on both fast and slow time scales, and GABA-mediated slow inhibition increases the diversity of odor-evoked responses among PNs. This is consistent with models that invoke GABAergic inhibition to increase the discriminability of olfactory representations (Wilson, 2005).

As in the olfactory bulb, each glomerulus in the Drosophila antennal lobe contains four main classes of neurons: (1) the axon terminals of ORNs, (2) the dendrites of PNs that convey information from ORNs to higher brain centers, (3) neurites from LNs that interconnect glomeruli, and (4) the centrifugal axonal projections of neurons that relay information to the antennal lobe from higher brain centers. Recent studies have illuminated the development, morphology, and physiology of Drosophila ORNs and PNs. Drosophila LNs, in contrast, have not received much attention. LNs have been noted in Golgi-impregnated antennal lobes, but remarkably little is known about the number, morphology, and connectivity of these cells or about their impact on other antennal lobe neurons. If adult LNs are also GABAergic, and if GABA is inhibitory (as it is in other insects), then LNs could participate in sculpting the inhibitory epochs prominent in many PN odor responses. In the larval Drosophila antennal lobe, many LNs are immunopositive for GABA. In the adult, it has been shown that many somata around the antennal lobes express the GABA biosynthetic enzyme glutamic acid decarboxylase (Wilson, 2005).

This study confirms that many adult Drosophila antennal LNs are GABAergic. Using confocal immunofluorescence microscopy with an anti-GABA antibody, many GABA-positive somata were observed in the vicinity of the antennal lobe neuropil. To identify LNs, flies were used in which a large subpopulation of these cells were genetically labeled. In these flies (GAL4 enhancer trap line GH298), reporter gene activity labels a cluster of somata lateral to the antennal lobe neuropils. The neurites of these neurons collectively fill the antennal lobes, reminiscent of the morphology of LNs identified in Golgi impregnations. When whole-cell patch-clamp recordings were made from the somata of GFP-positive cells in GH298-GAL4, UAS-CD8GFP flies, intrinsic properties characteristic of LNs were observed, namely high input resistances and action potentials with amplitude >40 mV. It was also confirmed with single-cell biocytin fills that these GFP-positive neurons were indeed LNs. When GH298-GAL4,UAS-CD8GFP brains stained for GABA were visualized using dual-channel confocal microscopy, it was found that most GFP-positive somata were also GABA positive. About one-fifth of the GFP-positive somata did not stain for GABA. These neurons may contain a different neurotransmitter, or the staining may not have been sensitive enough to detect low levels of GABA. The possibility cannot be excluded that these GABA-negative neurons are not LNs (Wilson, 2005).

It was then confirmed that GABA hyperpolarizes antennal lobe neurons. In LNs, these results imply that inhibition is mediated entirely by GABAA receptors. In contrast, GABAergic inhibition of PNs is mediated by both GABAA and GABAB receptors. Thus, synaptic inhibition onto PNs and LNs is functionally specialized (Wilson, 2005).

How might GABAergic inhibition contribute to olfactory processing in the Drosophila antennal lobe? Recent studies using optical measurements of neural activity have concluded that ORN and PN odor responses are very similar and that the antennal lobe is merely a relay station that faithfully transmits ORN signals to PNs without alteration. These conclusions imply that synaptic inhibition in the antennal lobe may exist merely to control global excitability and may not play an important role in representing information about the stimulus. However, the optical reporters used in these studies lack temporal resolution, have limited dynamic range, and may not be sensitive to inhibitory events. Whole-cell patch-clamp recordings from Drosophila PNs show prominent inhibitory epochs in many odor responses, generating odor-dependent spatiotemporal response patterns. Such complex temporal patterns are not present in the responses of ORNs, implying that they arise in the antennal lobe and thus represent a transformation of the olfactory code between the first and second layers of olfactory processing. These temporal patterns are reminiscent of those seen in olfactory bulb mitral cells and in other insects (Wilson, 2005).

A common notion in olfaction is that such spatiotemporal patterns represent lateral interactions, the net effect of which is to amplify contrast. This idea has taken two main forms. The first proposes a contrast-enhancement mechanism akin to that seen in the retina. According to this model, specific mutual inhibitory interactions exist between principal neurons in nearby glomeruli with similarly tuned ORN inputs. When a principal neuron is activated strongly by an odor, it will trigger lateral inhibition of its neighbors to suppress weak responses to that odor, sharpening the difference between their tuning curves. A different hypothesis is that lateral interactions exist in a more distributed manner. Odors are represented as stimulus-specific sequences of neuronal ensembles. The stimulus is represented both by the identity of the active neurons and the time when they are active. According to this model, the net effect of interglomerular interactions is not to prune away weak responses. Rather, inhibitory interactions may coexist with excitatory interactions (or relief-of-inhibition mechanisms), such that new principal neuron responses appear as others disappear. Because each stimulus is represented by an evolving neural ensemble, the available coding space is expanded. Again, the outcome of this process is thought to be a progressive decorrelation, such that overlap is reduced between stimulus representations (Wilson, 2005).

Both these models predict that eliminating odor-evoked inhibitory epochs in second-order olfactory neurons will increase the similarity between the spatiotemporal activity patterns produced in these neurons by different odors. This study reports that odor-evoked inhibitory epochs in Drosophila PNs are mostly suppressed by a GABAB receptor antagonist and that blocking GABAB receptors decreases the coefficient of variation among PN peristimulus-time histograms. These results are consistent with models in which lateral interactions between principal and local neurons increase the degree of difference between the neural representations of different odors (Wilson, 2005).

It is important to point out that the effect of the GABAB antagonist on PN odor responses may be mediated partly by presynaptic effects on ORN axon terminals or by indirect effects via other excitatory inputs to PNs. Determining the locus of this effect will require additional experiments using cell type-specific genetic manipulations. However, because GABAB receptors mediate much of the direct effect of GABA on PNs, it seems likely that the effect of CGP54626, a compound that blocks the late inhibitory epoch in a PN odor response, on odor-evoked PN activity is attributable at least in part to postsynaptic GABAB receptors (Wilson, 2005).

Finally, it should be noted that two conceptually distinct kinds of temporal patterns can in principle coexist among second-order olfactory neurons. Slow temporal patterns are punctuated by inhibitory epochs on the timescale of tens to thousands of milliseconds. In this study, it was shown that these slow patterns in the Drosophila antennal lobe are sensitive to a GABAB antagonist. Distinct from this is fast inhibition, which synchronizes the firing of principal neurons on time scales of several milliseconds and is sensitive to picrotoxin. Fast, odor-evoked synchronous oscillations occur in the olfactory systems of many organisms and are required for fine olfactory discrimination in the honeybee. There is little evidence for such oscillatory synchronization among Drosophila PNs. These observations deserve additional investigation but suggest that different organisms may emphasize different strategies for olfactory processing (Wilson, 2005).

Theoretical models of olfactory processing that invoke synaptic inhibition to increase the contrast between different stimulus representations presume nonuniform connectivity between inhibitory and principal neurons. In insects, a GABAergic LN can arborize across the entire antennal lobe, and so it is not obvious that single LNs will make connections preferentially with particular glomeruli. In this study, it was found that the neurites of single LNs form spatially heterogeneous patterns in the antennal lobe. This finding alone does not prove that individual LNs make connections preferentially in the glomeruli in which their dendrites are most dense; for example, average synaptic strength could be higher in glomeruli with fewer neurites. However, individual LNs also displayed specific odor preferences. This supports the idea that the odor tuning of individual LNs might be correlated with which glomeruli were preferentially innervated by that LN. According to this model, LN odor tuning would be biased toward the tuning of the excitatory neurons innervating those glomeruli. Drosophila LNs receive excitatory input from PNs. In other insect species, LNs are also known to receive direct input from ORNs (Wilson, 2005).

Consistent with these conclusions, a functional imaging study of the Drosophila antennal lobe has found that each odor stimulus evokes GABA release in some glomeruli more than others. Furthermore, these spatial patterns of GABA release are odor dependent (Ng, 2002). That study measured synaptic release from all GABAergic neurons simultaneously. This investigation has now been extended to single LNs, the morphological and functional diversity of which suggests a cellular mechanism for how the pattern of GABA release can be nonuniform and odor dependent. Ultimately, a test of this idea should come from correlating the morphology of single LNs with their odor preferences. Recent studies have reported the odor tuning of a large subset of Drosophila olfactory receptors and the mapping of each receptor to a specific ORN type. Once it is know which ORN type corresponds to each glomerulus, it should be possible to design experiments of this type more systematically (Wilson, 2005).

GABA modulates Drosophila circadian clock neurons via GABAB receptors and decreases in calcium

Circadian clocks play vital roles in the control of daily rhythms in physiology and behavior of animals. In Drosophila, analysis of the molecular and behavioral rhythm has shown that the master clock neurons are entrained by sensory inputs and are synchronized with other clock neurons. However, little is known about the neuronal circuits of the Drosophila circadian system and the neurotransmitters that act on the clock neurons. This study provides evidence for a new neuronal input pathway to the master clock neurons, s-LNvs, in Drosophila that utilizes GABA as a slow inhibitory neurotransmitter. Intracellular calcium levels were monitored in dissociated larval s-LNvs with the calcium-sensitive dye Fura-2. GABA decreased intracellular calcium in the s-LNvs and blocked spontaneous oscillations in calcium levels. The duration of this response was dose-dependent between 1 nM and 100 microM. The response to GABA was blocked by a metabotropic GABAB receptor (GABAB-R) antagonist, CGP54626, but not by an ionotropic receptor antagonist, picrotoxin. The GABAB-R agonist, 3-APMPA, produced a response similar to GABA. An antiserum against one of the Drosophila GABAB-Rs (GABAB-R2) labeled the dendritic regions of the s-LNvs in both adults and larvae, as well as the dissociated s-LNvs. It was found that some GABAergic processes terminate at the dendrites of the LNvs, as revealed by GABA immunostaining and a GABA-specific GAL4 line (GAD1-gal4). These results suggest that the s-LNvs receive slow inhibitory GABAergic inputs that decrease intracellular calcium of these clock neurons and block their calcium cycling. This response is mediated by postsynaptic GABAB receptors (Hamasaka, 2005).

In Drosophila, the circadian clock located in the brain is essential for timing of different daily activities. Circadian signals driving these rhythms are generated by cooperation of several sets of neuronal clocks in the brain. Of these clock neurons, the LNvs, known to release the neuropeptide PDF as their main output factor, are the ones necessary for maintaining the circadian activity and eclosion rhythms under constant conditions. This study has focused attention on identifying neuronal inputs to these LNvs. Using specific GAL4 lines combined with immunocytochemistry, it was found that GABAergic neurons send their axons to the dendrites of the LNvs in the larval brain. Dissociated larval LNvs kept in culture respond to GABA and a specific agonist of GABABRs with a decrease in intracellular calcium. This response was blocked by a GABAB, but not a GABAA, receptor antagonist (Hamasaka, 2005).

Furthermore, the dendrites of the LNvs and the dissociated larval LNvs express GABABR2 immunoreactivity. Immunolabeling and GAD1-GAL4 expression showed that the LNvs are not GABAergic, which in turn suggests that the GABABRs on the LNv branches in the LOC are postsynaptic. This is in accordance with the common notion that the dorsal terminals of the LNvs are the output area and release site of PDF, whereas the branches in the larval optic center LOC are dendrites with input sites. Congruent with this is the localization of the GFP-tagged vesicle marker synaptobrevin, which is concentrated in the dorsal terminals, but not in the dendrites, when driven in the larval LNvs with the pdf-GAL4. Collectively, these data suggest that the LNvs receive GABAergic inputs, possibly mediating slow inhibitory postsynaptic potentials. Like in the larvae, GABAergic neurons in the adult brain invade the dendritic region of the LNvs, and this region was also immunostained with the antiserum against GABABR2. The four LNvs in the larva survive metamorphosis and are designated s-LNvs in the adult brain where they remain the main pacemakers of the clock. The effect of GABA on the adult LNvs was not tested, it is likely that they also respond to GABA (Hamasaka, 2005).

It was found that the GABA-induced decrease in intracellular calcium in the dissociated larval LNvs is blocked by the GABABR antagonist, CGP54626, but not by the chloride channel blocker PiTX, known to be a selective GABAAR antagonist. Further support for presence of GABABRs was that the GABABR agonist 3-APMPA mimics the response to GABA in the dissociated LNvs. This could mean that the GABA action on these neurons is mediated solely by the slower GABABR type of receptors. Both 3-APMPA and CGP54626 have been shown to be effective on Drosophila GABABRs in earlier studies, although some other agonists and antagonists commonly used in mammalian studies were not. Several investigations have in fact shown that the insect GABABRs display a somewhat different pharmacology than their mammalian counterparts. It is satisfactory to note that the EC50 values of the GABA and 3-APMPA responses determined in dissociated Drosophila neurons are in accordance with those obtained when expressing the Drosophila GABABR1/R2 complex in Xenopus oocytes (Metzler, 2001). In Drosophila three different GABABRs are known (D-GABABR1-3; Metzler, 2001), whereas only two types, GABABR1-2, have been characterized in mammals. To be functional the GABABRs in Drosophila and mammals have to form heterodimers of GABABR1 and GABABR2 (Galvez, 2001; Mezler, 2001). The Drosophila GABABR3 alone or in combination with either of the other two receptors is not functional in the expression systems tested so far (Mezler, 2001). Because GABABR1 and 2 require heterodimerization to function, it is likely that they are always coexpressed in neurons. Thus, although only antiserum to GABABR2 was used to demonstrate the presence of receptors on LNvs, it is likely that GABABR1 is also expressed in these neurons and that the GABABR2-IR punctae represent functional GABABR sites (Hamasaka, 2005).

The GABABRs commonly couple to the Gαo/Gαi type of G-proteins (see Kaupmann, 1998; Bettler, 2004). Typically, activated postsynaptic GABABRs increase the K+ conductance through GIRK and other K+ channels, which generally leads to a hyperpolarization (see Bettler, 2004). GABABRs can also inhibit voltage-dependent Ca2+ channels both at presynaptic (see Bettler, 2004) and postsynaptic sites. In different heterologous expression systems, activation of the Drosophila GABABR1/R2 leads to a decrease in cyclic AMP and an increase of the K+ conductance by stimulating GIRKs (Mezler, 2001). In identified insect neurons, activation of the native GABABRs caused increases of potassium conductance resulting in a stable and PiTX-insensitive slow hyperpolarization. Rests using the broad spectrum K+ channel blockers Cs+, TEA, and 4-AP did not affect the calcium response to GABA. Incubation in Ba2+ appeared to partially block the GABABR response, suggesting that GABA activates Ba2+-sensitive K+ channels in the LNvs. Ba2+, however, can bind to Fura-2 with a slightly larger Kd than Ca2+, but with a similar effect on the fluorescence ratio. Thus, it is possible that Ba2+ masked the decrease in intracellular calcium level induced by GABA and therefore the data do not provide conclusive evidence for the involvement of GIRKs or other K+ channels in the GABA response of the LNvs. The lack of clear effects of the different K+ channel blockers may on the other hand point to the alternative possibility that activation of the GABABRs of the LNvs leads to an inhibition of voltage-dependent Ca2+ channels, as described in mammals (Hamasaka, 2005).

A decrease of intracellular Ca2+ upon application of a GABAB agonist, as seen in the LNvs, has also reported for cricket mushroom body Kenyon cells in short-term culture, but occurred only when these cells were kept in high (20 mM) instead of low (2 mM) Ca2+ concentration in the saline (Hamasaka, 2005).

The LNvs, however, responded to a GABAB agonist at an extracellular Ca2+ concentration of 1.8 mM. This suggests that the LNvs, at least under the culture conditions that were used, are depolarized above the basal membrane potential. In fact, the basal-free Ca2+ concentration was found to be nearly twice as high in LNvs than in other peptidergic neurons. Furthermore, most of the cultured LNvs displayed spontaneous fluctuations in the free intracellular Ca2+ concentration that were not observed in other peptidergic neurons. These fluctuations were blocked by GABA and a GABABR agonist. In summary so far, the results suggest that the membrane activity of the LNvs can be modulated by GABAergic input via GABABRs (Hamasaka, 2005).

In adult flies, three types of neuronal inputs to the LNvs have been reported: the photoreceptor axons of the eyelets and the axons of two sets of clock neurons, DN1 and DN3. In larvae only the axons of the BO photoreceptors and DN1 clock neurons have been identified as putative inputs to LNvs. The GABAergic neuron processes contacting the LNv dendrites appear distinct from any of these clock neurons or photoreceptors, suggesting that the GABAergic neurons constitute a novel input pathway to the LNvs. It was also excluded that any of the LNvs express GABA and thus utilize the GABABRs as autoreceptors. However, the full morphology of the GABA- and GAD1-expressing neurons could not be elucidated and it is not yet possible to suggest a circuit/pathway for the GABAergic inputs (Hamasaka, 2005).

This study has shown that GABA can change the level of intracellular calcium in the LNvs. Although the circuits supplying the GABAergic input to the LNvs remain to be elucidated, the importance of the LNvs as circadian pacemakers makes it reasonable to assume that the GABAergic inputs play a role in the modulation of circadian activity. Interestingly, it has been demonstrated that the membrane potential of clock neurons is critical for the generation of circadian rhythms in Drosophila (Hamasaka, 2005).

Electrical silencing of the LNvs, using a modified form of open-rectifier K+ channel and an inwardly rectifying K+ channel, eliminates the circadian oscillation of a molecular clock component, TIM, in both larvae and adults. As a consequence the adult flies exhibit arrhythmic locomotor behavior in constant darkness. Previously, it was shown that activation of nicotinic acetylcholine receptors induces increases in intracellular calcium levels in dissociated larval LNvs. This, in conjunction with the cholinergic nature of larval photoreceptors of the BO, suggests that light information to the LNvs is transmitted by acetylcholine. Thus, the inhibitory modulation of the LNvs by GABA might either serve to alter responses to the depolarizating light inputs from the larval photoreceptors or to mediate other slow inhibitory actions, including synchronization of activity of LNvs and other neurons (Hamasaka, 2005).

A role for GABA in circadian clocks in the brain has been shown both in insects and vertebrates. In the cockroach, Leucophaea maderae, microinjection of GABA shifts the locomotor activity rhythm in a fashion similar to that obtained with light pulses (Petri, 2002). GABAergic neurons of the optic lobes invade the accessory medulla where the L. maderae pacemakers are located, and it was suggested that GABA mediates light inputs to the circadian clock (Petri, 2002). The substantial GABAergic projections to the LNv dendrites in the adult brain of Drosophila seem to partly originate from the optic lobes. However, the imaginal optic lobes have not yet developed in the third instar larvae. Thus a direct photoreceptor input via GABAergic neurons, as in the cockroach, seems unlikely in the Drosophila larvae, but cannot be excluded for adults. Instead the GABAergic processes invading the LOC appear to be derived from cell bodies laterally in the midbrain and to be part of neurons projecting into the central brain or even contralaterally to the other hemisphere. No GABA immunoreactivity or GAD1 expression was detected in any of the LNvs or other clock neurons. Thus the GABAergic neurons do not seem to be part of direct local circuits involved in synchronization of the LNvs in one hemisphere. On the other hand, the GABAergic neurons could play a role in synchronizing activity in LNvs with other (clock) neurons or the LNvs in the contralateral hemisphere. The GABAergic neurons could also relay signals from interneurons that carry sensory information, such as light, odors, or temperature. It should be noted that GABAergic axon branches were observed in the vicinity of the dorsal terminals of the LNvs in the adult CNS. In fact, some terminals of the LNvs appeared GABABR2-IR in the adult CNS. This might suggest that GABABRs control release of PDF presynaptically in the adult CNS. In the mammalian SCN, GABABRs exert substantial effects on neuronal activity (Gribkoff, 2003), phase-shift the circadian clock, and inhibit light-induced c-Fos expression and field potentials. However, all these effects of GABABRs seem to be due to a presynaptic block of transmitter release rather than a postsynaptic hyperpolarization (Chen, 1998; Hamasaka, 2005 and references therein).

In conclusion, this study has identified novel input neurons contacting the dendrites of the main circadian pacemaker neurons, the LNvs, in Drosophila. These input neurons are GABAergic and may play a modulatory role by inducing slow inhibitory postsynaptic potentials in the LNvs via GABABRs. Because the LNvs are the master circadian pacemakers it is reasonable to assume that the GABAergic inputs play a role in regulation of circadian activity. A recent article demonstrated that the larval circadian pacemakers (LNvs) also play a role in transmitting rapid photosensory signals critical for photophobic behavior (Mazzoni, 2005). These findings shed new light on the larval LNvs and urge consideration of GABAergic inputs also in terms of modulation of rapid behavioral responses. The functional role of the GABAergic input remains to be elucidated, for instance by studies using GABABR knockout flies. Furthermore, it will be important to delineate the circuits supplying the GABAergic input to the LNvs (Hamasaka, 2005).

GABA suppresses neurogenesis in the adult hippocampus through GABAB receptors

Adult neurogenesis is tightly regulated through the interaction of neural stem/progenitor cells (NSCs) with their niche. Neurotransmitters, including GABA activation of GABAA receptor ion channels, are important niche signals. This study shows that adult mouse hippocampal NSCs and their progeny express metabotropic GABAB receptors. Pharmacological inhibition of GABAB receptors stimulated NSC proliferation and genetic deletion of GABAB1 receptor subunits increased NSC proliferation and differentiation of neuroblasts in vivo. Cell-specific conditional deletion of GABAB receptors supports a cell-autonomous role in newly generated cells. These data indicate that signaling through GABAB receptors is an inhibitor of adult neurogenesis (Giachino, 2013).

Olfactory control of blood progenitor maintenance

Drosophila hematopoietic progenitor maintenance involves both near neighbor and systemic interactions. This study shows that olfactory receptor neurons (ORNs) function upstream of a small set of neurosecretory cells that express GABA. Upon olfactory stimulation, GABA from these neurosecretory cells is secreted into the circulating hemolymph and binds to metabotropic GABAB receptors expressed on blood progenitors within the hematopoietic organ, the lymph gland. The resulting GABA signal causes high cytosolic Ca2+, which is necessary and sufficient for progenitor maintenance. Thus, the activation of an odorant receptor is essential for blood progenitor maintenance, and consequently, larvae raised on minimal odor environments fail to sustain a pool of hematopoietic progenitors. This study links sensory perception and the effects of its deprivation on the integrity of the hematopoietic and innate immune systems in Drosophila (Shim, 2013).

Niche-dependent mechanisms of hematopoietic progenitor development and maintenance have been extensively described in both vertebrate and invertebrate literature. Mechanisms independent of the niche that operate at a more systemic level but affect progenitor development have recently started to emerge (Shim, 2013).

This study describes a signal that originates from the brain and regulates blood progenitor maintenance. This pathway is independent of the nutritional signal that involves Drosophila insulin and TOR. Olfaction-dependent sensory stimulation relays systemic cues from the central nervous system to the undifferentiated blood progenitors by regulating physiological levels of GABA secreted into the blood stream. GABA is expressed in a small number of neurosecretory cells of the brain, and the release of GABA from this class of neurosecretory cells is critically dependent on olfactory stimulation. Olfactory dysfunction decreases GABA expression in neurosecretory cells and also reduces systemic GABA levels in the circulating blood. Blood progenitors express the metabotropic GABAB receptor, which enables them to respond to GABA, raising the concentration of their cytosolic calcium essential for inhibition of premature differentiation and maintenance of the progenitors. This control is lost when either the olfactory neurons or their network partners in the olfactory glomeruli are disrupted. A consequence of the above mechanism is that wild-type Drosophila larvae reared on odor-limited media have dramatically reduced systemic GABA levels, and consequently, their blood progenitors precociously differentiate. Upon blocking olfaction, GABA levels in the entire central brain region are reduced, but it is the two GABA-expressing neurosecretory cells in each lobe of the central brain that are important in controlling GABA secreted into the aorta that controls hematopoiesis (Shim, 2013).

Within the lymph gland, the GABAB receptor is expressed in the blood progenitors and is downregulated as the cells differentiate. Binding of GABA to GABABR maintains high cytosolic Ca2+ in the progenitors, a prerequisite for their remaining undifferentiated. The differentiated blood cells have very low or undetectable levels of Ca2+ and are also unresponsive to its alterations. Downstream of elevated Ca2+, the functions of Calmodulin and CaMKII are essential for progenitor maintenance. Events further downstream currently remain unclear. In principle, Ca2+ could directly or indirectly interact with either ROS or Wg-related pathways shown to be important for progenitor maintenance (Shim, 2013).

Accumulating evidence has shown that the mammalian nervous system also regulates innate immune responses through hormonal and neuronal routes. Sympathetic and parasympathetic nervous systems directly innervate into immune organs, whereas neuroendocrine factors control inflammation at a systemic level. Furthermore, immune cells express receptors for various neuronal factors, supporting the idea that there are contributions of the nervous system to immunity. Brain dysfunction, including certain neurodegenerative diseases, generate heightened immune reaction, as the central nervous system is generally thought to inhibit immune responses. The mammalian hematopoietic niche is innervated, and cells within the niche express a Ca2+-sensing receptor on their surface that they utilize to home toward the periendosteal compartment. However, a direct involvement of secreted GABA or olfaction in the maintenance of hematopoietic progenitors has not been demonstrated in any other system (Shim, 2013).

GABA is conserved from bacteria to plants and animals. In plants, GABA functions as a metabolite, a signaling molecule, and in stress response. In vertebrates, GABA function has been primarily studied in neurotransmission, but it also functions as a metabol and in developmental signaling in both embryonic tissue and in adult regeneration. This study could readily detect GABA secreted into the Drosophila hemolymph. This is not unprecedented, as GABA can be measured in the bloodstream of many mammals, including humans. Interestingly, GABABR is expressed in primary human HSCs, and its expression is higher in immature stem cells than in more mature progenitors. GABA function in human HSCs remains unclear, and it is not known whether its function is controlled through a sensory signal as has been described in this study in Drosophila (Shim, 2013).

In addition to the universally used developmental pathways such as Hh, Dpp, and Wg, Drosophila blood precursors utilize several unusual pathways for their development. For example, physiologically generated ROS functions as a signaling molecule that allows the blood progenitors to differentiate, whereas increased ROS, resulting from infection, is a stress signal that causes rapid expansion of this differentiation process. It was shown that insulin maintains the progenitor population during normal development, and starvation is a stress condition that causes a drop in insulin levels and allows premature differentiation. Similarly, Hif-alpha, stabilized under normoxic conditions by physiologically generated NO, binds Notch and maintains a class of blood cells, whereas hypoxic conditions sensed as a stress stabilize additional amounts of Hif-alpha and increase the number of these blood cells. To summarize, in all the above instances, examples are seen of signals that are used by the myeloid precursors for their normal development in a programmed manner, and the same signals cause rapid expansion of these blood cells upon conditions of stress. In the fly, the conditions that favor blood differentiation, including reduced olfaction, are normally initiated during pupariation when the need for increased numbers of macrophages is critical. As a bonus, these same pathways can cause increased differentiation earlier in larval life when activation of these pathways is perceived as a stress response. This response is reflected in mutational studies (Shim, 2013).

Overall, this study describes the mechanism for coordinating inputs from olfactory stimulation to maintain blood progenitors via regulation of systemic GABA levels. As olfaction is an important sensory input for the larva, inability to sense odor could be interpreted as an important stress response. Anosmic larvae cannot survive in a competitive environment due to lack of food-searching behavior. Furthermore, a recent study has shown that OR56a senses a microbial odorant to avoid unsuitable breeding and feeding sites. Thus, proper olfaction promotes survival, both by allowing improved competition within a brood and through avoidance of infectious organisms. Increased hematopoietic differentiation in the absence of odor input could also be beneficial to the larva in mounting an immune response, although this remains to be proven in future studies. In humans, loss of olfaction has been associated with abnormalities in many parts of the brain, and impaired olfaction leads to amplified inflammation in mammals. The current data in Drosophila highlight that sensory stress response can directly influence developmental and cell fate decisions of blood progenitors. Whether this is also relevant to higher organisms with more complicated blood lineages remains to be explored (Shim, 2013).

Gamma-aminobutyric acid B receptor 1 mediates behavior-impairing actions of alcohol in Drosophila: adult RNA interference and pharmacological evidence

In addition to their physiological function, metabotropic receptors for neurotransmitter gamma-aminobutyric acid (GABA), the GABA(B) receptors, may play a role in the behavioral actions of addictive compounds. Recently, GABA(B) receptors were cloned in fruit flies (Drosophila melanogaster), indicating that the advantages of this experimental model could be applied to GABA(B) receptor research. RNA interference (RNAi) is an endogenous process triggered by double-stranded RNA and is being used as a tool for functional gene silencing and functional genomics. This study shows how cell-nonautonomous RNAi can be induced in adult fruit flies to silence a subtype of GABA(B) receptors, GABA(B)R1, and how RNAi combined with pharmacobehavioral techniques (including intraabdominal injections of active compounds and a computer-assisted quantification of behavior) can be used to functionally characterize these receptors. Injection of double-stranded RNA complementary to GABA(B)R1 into adult Drosophila selectively destroys GABA(B)R1 mRNA and attenuates the behavioral actions of the GABA(B) agonist, 3-aminopropyl-(methyl)phosphinic acid. Moreover, both GABA(B)R1 RNAi and the GABA(B) antagonist CGP 54626 reduced the behavior-impairing effects of ethanol, suggesting a putative role for the Drosophila GABA(B) receptors in alcohol's mechanism of action. This Drosophila model can be used for further in vivo functional characterization of GABA(B) receptor subunits and their involvement in the molecular and systemic actions of addictive substances (Dzitoyeva, 2003).

No ligand binding in the GB2 subunit of the GABA(B) receptor is required for activation and allosteric interaction between the subunits

The GABA(B) receptor plays important roles in the tuning of many synapses. Although pharmacological differences have been observed between various GABA(B)-mediated effects, a single GABA(B) receptor composed of two subunits (GB1 and GB2) has been identified. Although GB1 binds GABA, GB2 plays a critical role in G-protein activation. Moreover, GB2 is required for the high agonist affinity of GB1. Like any other family 3 G-protein-coupled receptors, GB1 and GB2 are composed of a Venus Flytrap module (VFTM) that usually contains the agonist-binding site and a heptahelical domain. So far, there has been no direct demonstration that GB2 binds GABA or another endogenous ligand. This study has further refined the GABA-binding site of GB1 and characterized the putative-binding site in the VFTM of GB2. None of the residues important for GABA binding in GB1 appeared to be conserved in GB2. Moreover, mutation of 10 different residues, alone or in combination, within the possible binding pocket of GB2 affects neither GABA activation of the receptor nor the ability of GB2 to increase agonist affinity on GB1. These data indicate that ligand binding in the GB2 VFTM is not required for activation. Finally, although in either GB1 or the related metabotropic glutamate receptors most residues of the binding pocket are conserved from Caenorhabditis elegans to human, no such conservation is observed in GB2. This suggests that the GB2 VFTM does not constitute a binding site for a natural ligand (Kniazeff, 2002).

Cloning and functional expression of GABA(B) receptors from Drosophila

The neurotransmitter GABA (gamma-aminobutyric acid) functions as the major inhibitory neurotransmitter in the central nervous system of vertebrates and invertebrates. In vertebrates GABA signals both through ionotropic receptors [GABA(A), GABA(C)], which induce fast synaptic inhibitory responses, and through metabotropic receptors [GABA(B)], which play a fundamental role in the reduction of presynaptic transmitter release and postsynaptic inhibitory potentials. Whilst GABA(A) and GABA(C) receptors have been cloned from vertebrates as well as invertebrates, GABA(B) receptors have only been identified in vertebrate species to date, although indirect evidence suggests their existence in arthropods, too. This paper reports the cloning of three putative invertebrate GABA(B) receptor subtypes [D-GABA(B)R1, R2 and R3] isolated from Drosophila melanogaster. Whilst D-GABA(B)R1 and R2 show high sequence identity to mammalian GABA(B)R1 and R2, respectively, the receptor D-GABA(B)R3 seems to be an insect-specific subtype with no known mammalian counterpart so far. All three D-GABA(B)R subtypes are expressed in the embryonic central nervous system. In situ hybridization of Drosophila melanogaster embryos shows that two of the D-GABA(B)Rs [D-GABA(B)R1 and R2] are expressed in similar regions, suggesting a coexpression of the two receptors, whilst the third D-GABA(B)R [D-GABA(B)R3] displays a unique expression pattern. In agreement with these results it has only been possible to functionally characterize D-GABA(B)R1 and R2 when the two subtypes are coexpressed either in Xenopus laevis oocytes or mammalian cell lines, whilst D-GABA(B)R3 was inactive in any combination. The pharmacology of the coexpressed D-GABA(B)R1/2 receptor was different from the mammalian GABA(B)Rs: e.g. baclofen, an agonist of mammalian GABA(B)Rs, showed no effect (Mezler, 2001).

Functions of Metabotropic GABA-B receptor orthologs in other species

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).


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Biological Overview

date revised: 26 August 2020

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