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 links for GABA-B-R1: Precomputed BLAST | EntrezGene

NCBI links for GABA-B-R2: Precomputed BLAST | EntrezGene

NCBI links for GABA-B-R3: Precomputed BLAST | EntrezGene
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.

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).

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).

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).

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).

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: 25 November 2014

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