InteractiveFly: GeneBrief

: Biological Overview | References

Three invertebrate GABA_B receptor subtypes (D-GABA_BR1, R2 and R3) have been isolated from Drosophila. While D-GABA_BR1 and R2 show high sequence identity to mammalian GABA_BR1 and R2, respectively, the receptor D-GABA_BR3 seems to be an insect-specific subtype with no known mammalian counterpart so far. All three Drosophila GABA_BR subtypes are expressed in the embryonic central nervous system. In situ hybridization of Drosophila embryos shows that two of the D-GABA_BRs (GABA_BR1 and R2) are expressed in similar regions, suggesting a coexpression of the two receptors, while the third GABA_BR (GABA_BR3) displays a unique expression pattern. In agreement with these results D-GABA_BR1 and R2 could only be functionally characterized when the two subtypes are coexpressed either in Xenopus laevis oocytes or mammalian cell lines, while GABA_BR3 was inactive in any combination (Mezler, 2001). These observations or consonant with the idea that GABA_B 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 GABA_B receptor (GABA_BR) 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 GABA_BR expression. ORNs that sense the aversive odorant CO₂ do not express GABA_BRs nor exhibit any presynaptic inhibition. In contrast, pheromone-sensing ORNs express a high level of GABA_BRs 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 GABA_BRs 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 GABA_A receptor, which is sensitive to the antagonist picrotoxin, and the slow metabotropic GABA_B 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 GABA_BRs 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 GABA_BR 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 GABA_BRs 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 GABA_BR expression and behavioral experiments indicate that GABA_BR 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 GABA_BRs reveals a scalable presynaptic inhibition to suppress olfactory response at high odor concentrations. Pharmacological and molecular experiments suggest that GABA_BRs 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 GABA_BR expression. The importance of presynaptic GABA_BRs in olfactory localization was investigated, and it was found that reduction of GABA_BR 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 GABA_BR2 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 GABA_BR 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 GABA_BR 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 GABA_BR, 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 GABA_BR 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 GABA_BR in olfactory localization. GABA_BR-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 GABA_BRs 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 GABA_BR 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 GABA_AR and GABA_BR are expressed in ORNs to mediate presynaptic inhibition and that GABA_AR 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 GABA_BRs but not GABA_ARs are involved in presynaptic inhibition. To resolve these discrepancies further molecular experiments will be important to determine conclusively whether ORNs express GABA_AR 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 GABA_BR 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 GABA_A and GABA_B 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 GABA_B receptor antagonist blocked the late phase of this inhibition, but had only a modest effect on the early phase. Adding a GABA_A antagonist to the GABA_B antagonist blocked the residual early portion of the inhibition. The GABA_A antagonist alone had no effect. Taken together, these results suggest that both GABA_A and GABA_B receptors are present on the same ORN axon terminals, and either GABA_A or GABA_B receptors alone are sufficient to mediate substantial inhibition of EPSCs just after GABA release. The late phase of inhibition evidently involves only GABA_B 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 GABA_A and GABA_B components of EPSC inhibition are associated with an increase in the paired-pulse ratio. This implies that the independent actions of both GABA_A and GABA_B receptors are at least partially presynaptic. As a further test of this model, GABA_B 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 GABA_B antagonist and completely blocked by the GABA_A antagonist. As a negative control, this phenotype was confirmed to require both the ORN-specific promoter and the toxin transgene. This demonstrates that GABA_B receptors inhibit ORN-PN synapses at a purely presynaptic locus (Olsen, 2008).

If activation of either GABA_A or GABA_B 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 GABA_A or a GABA_B 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 GABA_A and GABA_B receptors. This arrangement is unusual but not unique; for example, there are several instances of GABA_A and GABA_B 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 GABA_A receptors were required for the a brief early phase of inhibition after odor onset, while GABA_B 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).

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 GABA_B 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 (Dachs, 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 (Dachs, 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 (Dachs, 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 GABA_B 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 GABA_B 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 GABA_B receptor (Dachs, 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 (Dachs, 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 GABA_B-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 GABA_B receptors by the ORNs innervating different glomeruli, and using previously unpublished data from that study, a greater expression of GABA_B 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 (Dachs, 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 (Dachs, 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 (Dachs, 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 (Dachs, 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 GABA_A 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 GABA_B-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 (Dachs, 2009).

Glutamate, GABA and acetylcholine signaling components in the lamina of the Drosophila visual system

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

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 GABA_A- and a GABA_B-type antagonist, respectively. Whereas GABA_A receptors shape PN odor responses during the early phase of odor responses, GABA_B receptors mediate odor-evoked inhibition on longer time scales. The patterns of odor-evoked GABA_B-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 GABA_A receptors (Harrison, 1996). However, GABA_A 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 GABA_A receptors. In contrast, GABAergic inhibition of PNs is mediated by both GABA_A and GABA_B 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 GABA_B receptor antagonist and that blocking GABA_B 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 GABA_B 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 GABA_B 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 GABA_B 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 GABA_B 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 GABA_B 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-LN_vs, in Drosophila that utilizes GABA as a slow inhibitory neurotransmitter. Intracellular calcium levels were monitored in dissociated larval s-LN_vs with the calcium-sensitive dye Fura-2. GABA decreased intracellular calcium in the s-LN_vs 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 GABA_B receptor (GABA_B-R) antagonist, CGP54626, but not by an ionotropic receptor antagonist, picrotoxin. The GABA_B-R agonist, 3-APMPA, produced a response similar to GABA. An antiserum against one of the Drosophila GABA_B-Rs (GABA_B-R2) labeled the dendritic regions of the s-LN_vs in both adults and larvae, as well as the dissociated s-LN_vs. It was found that some GABAergic processes terminate at the dendrites of the LN_vs, as revealed by GABA immunostaining and a GABA-specific GAL4 line (GAD1-gal4). These results suggest that the s-LN_vs receive slow inhibitory GABAergic inputs that decrease intracellular calcium of these clock neurons and block their calcium cycling. This response is mediated by postsynaptic GABA_B 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).

Abbott, L. F. and Luo, S. X. (2007). A step toward optimal coding in olfaction. Nature Neurosci. 10: 1342-1343. PubMed Citation: 17965649

Bettler, B., Kaupmann, K., Mosbacher, J. and Gassmann, M. (2004). Molecular structure and physiological functions of GABAB receptors. Physiol. Rev. 84: 835-867. PubMed Citation: 15269338

Bhandawat, V., et al. (2007). Sensory processing in the Drosophila antennal lobe increases reliability and separability of ensemble odor representations. Nat. Neurosci. 10: 1474-1482. PubMed Citation: 17922008

Chen, G., van den Pol, A. N. (1998). Presynaptic GABAB autoreceptor modulation of P/Q-type calcium channels and GABA release in rat suprachiasmatic nucleus neurons. J. Neurosci. 18: 1913-1922. PubMed Citation: 9465016

Dacks, A. M., Christensen, T. A. and Hildebrand, J. G. (2008). Modulation of olfactory processing in the antennal lobe of Manduca sexta by serotonin. J Neurophysiol. 99(5): 2077-2085. PubMed Citation: 18322001

Dacks, A. M., Green, D. S., Root, C. M., Nighorn, A. J., and Wang, J. W. (2009). Serotonin modulates olfactory processing in the antennal lobe of Drosophila. J. Neurogenet. 23(4): 366-77. PubMed Citation: 19863268

Galvez, T., et al. (2001). Allosteric interactions between GB1 and GB2 subunits are required for optimal GABA(B) receptor function. EMBO J. 20: 2152-2159. PubMed Citation: 11331581

Gribkoff, V. K., Pieschl, R. L. and Dudek, F. E. (2003). GABA receptor- mediated inhibition of neuronal activity in rat SCN in vitro: pharmacology and influence of circadian phase. J. Neurophysiol. 90: 1438-1448. PubMed Citation: 12750413

Hamasaka, Y., Wegener, C. and Nässel, D. R. (2005). GABA modulates Drosophila circadian clock neurons via GABA_B receptors and decreases in calcium. J. Neurobiol. 65(3): 225-40. PubMed Citation: 16118795

Kaupmann, K., et al. (1998). Human gamma-aminobutyric acid type B receptors are differentially expressed and regulate inwardly rectifying K+ channels. Proc. Natl. Acad. Sci. 95: 14991-14996. PubMed Citation: 9844003

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

Mazzoni, E. O., Desplan, C. and Blau, J. (2005). Circadian pacemaker neurons transmit and modulate visual information to control a rapid behavioral response. Neuron 45: 293-300. PubMed Citation: 15664180

Mezler, M., Muller, T., Raming, K. (2001). Cloning and functional expression of GABAB receptors from Drosophila. Europ. J. Neurosci. 13: 477-486. PubMed Citation: 11168554

Ng, M., et al. (2002). Transmission of olfactory information between three populations of neurons in the antennal lobe of the fly. Neuron 36: 463-474. PubMed Citation: 12408848

Olsen, S. R. and Wilson, R. I. (2008). Lateral presynaptic inhibition mediates gain control in an olfactory circuit. Nature 452: 956-960. PubMed Citation: 18344978

Petri, B., Homberg, U., Loesel, R. and Stengl, M. (2002). Evidence for a role of GABA and Mas-allatotropin in photic entrainment of the circadian clock of the cockroach Leucophaea maderae. J. Exp. Biol. 205: 1459-1469. PubMed Citation: 11976357

Petzold, G. C., Hagiwara, A. and Murthy, V. N. (2009). Serotonergic modulation of odor input to the mammalian olfactory bulb. Nat Neurosci. 12(6): 784-91. PubMed Citation: 19430472

Root, C. M., et al. (2008). A presynaptic gain control mechanism fine-tunes olfactory behavior. Neuron 59(2): 311-21. PubMed Citation: 18667158

Shang, Y., et al. (2007). Excitatory local circuits and their implications for olfactory processing in the fly antennal lobe. Cell 128(3): 601-612. PubMed Citation: 17289577

Stocker, R. F., Heimbeck, G., Gendre, N. and de Belle, J. S. (1997). Neuroblast ablation in Drosophila P[GAL4] lines reveals origins of olfactory interneurons. J. Neurobiol. 32: 443-456. PubMed Citation: 9110257

Wilson, R. I. and Laurent, G. (2005). Role of GABAergic inhibition in shaping odor-evoked spatiotemporal patterns in the Drosophila antennal lobe. J. Neurosci. 25(40): 9069-79. 16207866

date revised: 10 January 2011

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