Presynaptic peptidergic modulation of olfactory receptor neurons in Drosophila

The role of classical neurotransmitters in the transfer and processing of olfactory information is well established in many organisms. Neuropeptide action, however, is largely unexplored in any peripheral olfactory system. A subpopulation of local interneurons (LNs) in the Drosophila antannal lobe is peptidergic, expressing Drosophila tachykinins (DTKs). We show here that olfactory receptor neurons (ORNs) express the DTK receptor (DTKR). Using two-photon microscopy, we found that DTK applied to the antennal lobe suppresses presynaptic calcium and synaptic transmission in the ORNs. Furthermore, reduction of DTKR expression in ORNs by targeted RNA interference eliminates presynaptic suppression and alters olfactory behaviors. We detect opposite behavioral phenotypes after reduction and over expression of DTKR in ORNs. Our findings suggest a presynaptic inhibitory feedback to ORNs from peptidergic LNs in the antennal lobe.     

Glutamate is an inhibitory neurotransmitter in the Drosophila olfactory system

Glutamatergic neurons are abundant in the Drosophila central nervous system, but their physiological effects are largely unknown. In this study, we investigated the effects of glutamate in the Drosophila antennal lobe, the first relay in the olfactory system and a model circuit for understanding olfactory processing. In the antennal lobe, one-third of local neurons are glutamatergic. Using in vivo whole-cell patch clamp recordings, we found that many glutamatergic local neurons are broadly tuned to odors. Iontophoresed glutamate hyperpolarizes all major cell types in the antennal lobe, and this effect is blocked by picrotoxin or by transgenic RNAi-mediated knockdown of the GluClα gene, which encodes a glutamate-gated chloride channel. Moreover, antennal lobe neurons are inhibited by selective activation of glutamatergic local neurons using a nonnative genetically encoded cation channel. Finally, transgenic knockdown of GluClα in principal neurons disinhibits the odor responses of these neurons. Thus, glutamate acts as an inhibitory neurotransmitter in the antennal lobe, broadly similar to the role of GABA in this circuit. However, because glutamate release is concentrated between glomeruli, whereas GABA release is concentrated within glomeruli, these neurotransmitters may act on different spatial and temporal scales. Thus, the existence of two parallel inhibitory transmitter systems may increase the range and flexibility of synaptic inhibition.     

Functional feedback from mushroom bodies to antennal lobes in the Drosophila olfactory pathway

Feedback plays important roles in sensory processing. Mushroom bodies are believed to be involved in olfactory learning/memory and multisensory integration in insects. Previous cobalt-labeling studies have suggested the existence of feedback from the mushroom bodies to the antennal lobes in the honey bee. In this study, the existence of functional feedback from Drosophila mushroom bodies to the antennal lobes was investigated through ectopic expression of the ATP receptor P2X₂ in the Kenyon cells of mushroom bodies. Activation of Kenyon cells induced depolarization in projection neurons and local interneurons in the antennal lobes in a nicotinic receptor-dependent manner. Activation of Kenyon cell axons in the βγ-lobes in the mushroom body induced more potent responses in the antennal lobe neurons than activation of Kenyon cell somata. Our results indicate that functional feedback from Kenyon cells to projection neurons and local interneurons is present in Drosophila and is likely mediated by the βγ-lobes. The presence of this functional feedback from the mushroom bodies to the antennal lobes suggests top-down modulation of olfactory information processing in Drosophila.In the fruit fly, the olfactory system is important for identifying food sources, avoiding predators, and recognizing mating partners (1). The primary Drosophila olfactory neurons are the olfactory receptor neurons (ORNs) present in the two olfactory organs of the fruit fly, the antennae and the maxillary palps (2, 3). Chemical stimuli detected by the olfactory receptors in the ORNs are converted into electrical signals, which are transmitted to the secondary neurons, the projection neurons (PNs), in the antennal lobes. The antennal lobes are important olfactory coding centers that consist of PNs as well as inhibitory and excitatory local interneurons (LNs) (4 –6). The PNs receive signals from the primary ORNs and also receive lateral inhibitory/excitatory inputs from the LNs (7 –10). After processing in the antennal lobes, olfactory information is relayed by PNs to the mushroom bodies and the protocerebrum region of the fly brain (11 –13).Feedback is important in sensory processing. For example, in the mammalian thalamocortical system, a large number of thalamus neurons are modulated by cortical feedback mechanisms (14). Such top-down cortical feedback regulation is critical for visual perception (15). Anatomical and functional studies of the mammalian olfactory system indicate that there is also functional feedback from the cortex to the olfactory bulb (16, 17). Several lines of evidence also suggest the existence of feedback in the insect olfactory pathway. Olsen and Wilson (8) showed that spontaneous excitatory postsynaptic activity in PNs is suppressed presynaptically via lateral inhibition by LNs in antennal lobes, suggesting modulation of the primary ORNs by the secondary LNs. In addition, Tanaka et al. (18) demonstrated that odor stimulation can induce spikes and subthreshold membrane potential oscillations in PNs that are phase-locked to odor-elicited local field-potential oscillations in mushroom bodies. These results indicate the existence of feedback within the antennal lobe of Drosophila. Finally, by injecting cobalt into the α-lobe of the mushroom bodies of the honey bee, Rybak and Menzel identified a projection from the α-lobe to the antennal lobe (19). A neuron (the antennal lobe feedback neurons, ALF-1) with similar projection pattern was reported by Kirschner et al. (20). The results of these studies suggest feedback from the mushroom body to the antennal lobe in the honey bee. Based on these observations, we investigated whether functional feedback from mushroom bodies to antennal lobes is present in Drosophila. Drosophila mushroom bodies are predominately composed of Kenyon cells (KCs) (21). Because of the small size of KCs, it is difficult to stimulate these cells using conventional electrophysiological methods. Taking advantage of fly genetics, and the absence of the ionotropic ATP receptor P2X₂ gene in the Drosophila genome (22), Zemelman et al. established a UAS-P2X₂ system for precise activation of specific neurons in the fly brain using exogenous ATP (23). In this study, we have used the UAS-P2X₂ system for activation of KCs in combination with the patch-clamp method for recording of PN and LN activity. Using these techniques, we showed in this study the existence of functional feedback from KCs in the mushroom bodies to PNs and LNs in the antennal lobes.     

Generating sparse and selective third-order responses in the olfactory system of the fly

In the antennal lobe of Drosophila, information about odors is transferred from olfactory receptor neurons (ORNs) to projection neurons (PNs), which then send axons to neurons in the lateral horn of the protocerebrum (LHNs) and to Kenyon cells (KCs) in the mushroom body. The transformation from ORN to PN responses can be described by a normalization model similar to what has been used in modeling visually responsive neurons. We study the implications of this transformation for the generation of LHN and KC responses under the hypothesis that LHN responses are highly selective and therefore suitable for driving innate behaviors, whereas KCs provide a more general sparse representation of odors suitable for forming learned behavioral associations. Our results indicate that the transformation from ORN to PN firing rates in the antennal lobe equalizes the magnitudes of and decorrelates responses to different odors through feedforward nonlinearities and lateral suppression within the circuitry of the antennal lobe, and we study how these two components affect LHN and KC responses.     

The Ataxin-2 protein is required for microRNA function and synapse-specific long-term olfactory habituation

Local control of mRNA translation has been proposed as a mechanism for regulating synapse-specific plasticity associated with long-term memory. We show here that glomerulus-selective plasticity of Drosophila multiglomerular local interneurons observed during long-term olfactory habituation (LTH) requires the Ataxin-2 protein (Atx2) to function in uniglomerular projection neurons (PNs) postsynaptic to local interneurons (LNs). PN-selective knockdown of Atx2 selectively blocks LTH to odorants to which the PN responds and in addition selectively blocks LTH-associated structural and functional plasticity in odorant-responsive glomeruli. Atx2 has been shown previously to bind DEAD box helicases of the Me31B family, proteins associated with Argonaute (Ago) and microRNA (miRNA) function. Robust transdominant interactions of atx2 with me31B and ago1 indicate that Atx2 functions with miRNA-pathway components for LTH and associated synaptic plasticity. Further direct experiments show that Atx2 is required for miRNA-mediated repression of several translational reporters in vivo. Together, these observations (i) show that Atx2 and miRNA components regulate synapse-specific long-term plasticity in vivo; (ii) identify Atx2 as a component of the miRNA pathway; and (iii) provide insight into the biological function of Atx2 that is of potential relevance to spinocerebellar ataxia and neurodegenerative disease.     

Localized olfactory representation in mushroom bodies of Drosophila larvae

Odor discrimination in higher brain centers is essential for behavioral responses to odors. One such center is the mushroom body (MB) of insects, which is required for odor discrimination learning. The calyx of the MB receives olfactory input from projection neurons (PNs) that are targets of olfactory sensory neurons (OSNs) in the antennal lobe (AL). In the calyx, olfactory information is transformed from broadly-tuned representations in PNs to sparse representations in MB neurons (Kenyon cells). However, the extent of stereotypy in olfactory representations in the calyx is unknown. Using the anatomically-simple larval olfactory system of Drosophila in which odor ligands for the entire set of 21 OSNs are known, we asked how odor identity is represented in the MB calyx. We first mapped the projections of all larval OSNs in the glomeruli of the AL, and then followed the connections of individual PNs from the AL to different calyx glomeruli. We thus established a comprehensive olfactory map from OSNs to a higher olfactory association center, at a single-cell level. Stimulation of single OSNs evoked strong neuronal activity in 1 to 3 calyx glomeruli, showing that broadening of the strongest PN responses is limited to a few calyx glomeruli. Stereotypic representation of single OSN input in calyx glomeruli provides a mechanism for MB neurons to detect and discriminate olfactory cues.     

Propagation of olfactory information in Drosophila

Investigating how information propagates between layers in the olfactory system is an important step toward understanding the olfactory code. Each glomerular output projection neuron (PN) receives two sources of input: the olfactory receptor neurons (ORNs) of the same glomerulus and interneurons that innervate many glomeruli. We therefore asked how these inputs interact to produce PN output. We used receptor gene mutations to silence all of the ORNs innervating a specific glomerulus and recorded PN activity with two-photon calcium imaging and electrophysiology. We found evidence for balanced excitatory and inhibitory synaptic inputs but saw little or no response in the absence of direct ORN input. We next asked whether any transformation of activity occurs at successive layers of the antennal lobe. We found a strong link between PN firing and dendritic calcium elevation, the latter of which is tightly correlated with calcium activity in ORN axons, supporting the idea of glomerular propagation of olfactory information. Finally, we showed that odors are represented by a sparse population of PNs. Together, these results are consistent with the idea that direct receptor input provides the main excitatory drive to PNs, whereas interneurons modulate PN output. Balanced excitatory and inhibitory interneuron input may provide a mechanism to adjust PN sensitivity.     

A central neural circuit for experience-independent olfactory and courtship behavior in Drosophila melanogaster

We have studied the function of the major central olfactory pathway in fruit flies. Key elements of this pathway, the projection neurons (PNs), connect the antennal lobes with the lateral protocerebrum both directly and indirectly, the latter via the mushroom bodies (MBs). Transgenic expression of tetanus toxin in the majority of PNs and few MB neurons leads to defects in odor detection and male courtship. Considering behavioral data from flies lacking MBs, our results argue that the direct PN-to-lateral protocerebrum pathway is necessary and sufficient to process these experience-independent behaviors. Moreover, the involvement of an olfactory pathway in male courtship suggests a role of volatile attractive female pheromones in Drosophila.     

Dedicated olfactory neurons mediating attraction behavior to ammonia and amines in Drosophila

Animals across various phyla exhibit odor-evoked innate attraction behavior that is developmentally programmed. The mechanism underlying such behavior remains unclear because the odorants that elicit robust attraction responses and the neuronal circuits that mediate this behavior have not been identified. Here, we describe a functionally segregated population of olfactory sensory neurons (OSNs) and projection neurons (PNs) in Drosophila melanogaster that are highly specific to ammonia and amines, which act as potent attractants. The OSNs express IR92a, a member of the chemosensory ionotropic receptor (IR) family and project to a pair of glomeruli in the antennal lobe, termed VM1. In vivo calcium-imaging experiments showed that the OSNs and PNs innervating VM1 were activated by ammonia and amines but not by nonamine odorants. Flies in which the IR92a⁺ neurons or IR92a gene was inactivated had impaired amine-evoked physiological and behavioral responses. Tracing neuronal pathways to higher brain centers showed that VM1-PN axonal projections within the lateral horn are topographically segregated from those of V-PN and DC4-PN, which mediate innate avoidance behavior to carbon dioxide and acidity, respectively, suggesting that these sensory stimuli of opposing valence are represented in spatially distinct neuroanatomic loci within the lateral horn. These experiments identified the neurons and their cognate receptor for amine detection, and mapped amine attractive olfactory inputs to higher brain centers. This labeled-line mode of amine coding appears to be hardwired to attraction behavior.     

Invariant odor recognition with ON–OFF neural ensembles

Invariant stimulus recognition is a challenging pattern-recognition problem that must be dealt with by all sensory systems. Since neural responses evoked by a stimulus are perturbed in a multitude of ways, how can this computational capability be achieved? We examine this issue in the locust olfactory system. We find that locusts trained in an appetitive-conditioning assay robustly recognize the trained odorant independent of variations in stimulus durations, dynamics, or history, or changes in background and ambient conditions. However, individual- and population-level neural responses vary unpredictably with many of these variations. Our results indicate that linear statistical decoding schemes, which assign positive weights to ON neurons and negative weights to OFF neurons, resolve this apparent confound between neural variability and behavioral stability. Furthermore, simplification of the decoder using only ternary weights ({+1, 0, −1}) (i.e., an “ON-minus-OFF” approach) does not compromise performance, thereby striking a fine balance between simplicity and robustness.

Robustly recognizing a sensory stimulus is a necessity for the survival and propagation of all animals. Since this capability is demonstrated in all sensory systems, it raises the following question: what is the neural basis that underlies this feat of pattern recognition? Most stimuli are encountered in a multitude of ways in natural environments. Often, stimulus features such as intensity, duration, and recurrence could vary. In addition, external perturbances due to changes in environmental conditions (such as changes in humidity or temperature), the presence of other competing cues, or the temporal context (i.e., when it is received in a stimulus sequence) could also change independently of the variation in stimulus-specific features. An additional degree of interference can arise from changes in the sensory circuit due to plastic changes arising either from prior exposures or co-occurrence with other sensory cues. Given the complexity in carrying out the basic task of recognizing a stimulus, we wondered whether there exists a computational framework that can compensate for all these disparate sources of variation and allow robust recognition of a stimulus. In particular, we sought to examine this issue in the well-studied locust olfactory system (1–13).In the locust olfactory system, odorants activate olfactory receptor neurons in the antenna. This signal is transmitted downstream to the antennal lobe (analogous to the vertebrate olfactory bulb) where it drives responses in cholinergic projection neurons (PNs) and GABAergic local neurons (LNs). The interaction between PNs and LNs transforms the sensory input received into complex patterns of spiking activities distributed across ensembles of PNs that become the output of the antennal lobe circuit. Prior work has shown that information about the identity and intensity of an odorant is encoded by spatiotemporal PN activity patterns (5). While individual PN responses were perturbed by manipulating stimulus dynamics (11, 14), stimulus history (15, 16), and presence of background chemicals (7), the ensemble neural patterns still allowed recognition of odorants. Behavioral evidences also support this interpretation and reveal that odorants can be recognized independent of background cues (7) and stimulus history (15).It is worth noting that prior studies examined neural response variabilities that arose due to each of these perturbations in isolation. In natural contexts, such interferences could occur independently or in conjunction with one another. Could robust odor recognition still be achieved? Would an array of schemes that extract information from a variety of response features be necessary for compensating changes associated with each perturbation? Alternately, can the variable neural responses be decoded in a manner that can simultaneously allow invariant odor recognition independent of all these perturbations? If so, what neural response features would be important for achieving this result? We sought to examine these issues in this study.     

Parallel pathways convey olfactory information with opposite polarities in Drosophila

In insects, olfactory information received by peripheral olfactory receptor neurons (ORNs) is conveyed from the antennal lobes (ALs) to higher brain regions by olfactory projection neurons (PNs). Despite the knowledge that multiple types of PNs exist, little is known about how these different neuronal pathways work cooperatively. Here we studied the Drosophila GABAergic mediolateral antennocerebral tract PNs (mlPNs), which link ipsilateral AL and lateral horn (LH), in comparison with the cholinergic medial tract PNs (mPNs). We examined the connectivity of mlPNs in ALs and found that most mlPNs received inputs from both ORNs and mPNs and participated in AL network function by forming gap junctions with other AL neurons. Meanwhile, mlPNs might innervate LH neurons downstream of mPNs, exerting a feedforward inhibition. Using dual-color calcium imaging, which enables a simultaneous monitoring of neural activities in two groups of PNs, we found that mlPNs exhibited robust odor responses overlapping with, but broader than, those of mPNs. Moreover, preferentially down-regulation of GABA in most mlPNs caused abnormal courtship and aggressive behaviors in male flies. These findings demonstrate that in Drosophila, olfactory information in opposite polarities are carried coordinately by two parallel and interacted pathways, which could be essential for appropriate behaviors.In insects, the detection of olfactory cues begins at the peripheral olfactory receptor neurons (ORNs), which transfer the chemical information into neural signals and convey them to the first central relay station—the antennal lobes (ALs) (1–3). After AL local processing, olfactory information is relayed to higher brain regions via different groups of projection neurons (PNs) (4–6). Except some pioneering studies in Hymenopterans (7–11) and Lepidopterans (12), little is known about how these different PNs connect in the olfactory circuit and work physiologically. As the most studied PN type in Drosophila, the cholinergic PNs (mPNs) form the medial antennocerebral tract and convey excitatory signals encoding odor identity and intensity (13–15) that are necessary for the fly to perform appropriate behaviors (16, 17). However, how olfactory information is delivered via pathways mediated by PNs other than mPNs remains to be elucidated. In this study, we focused on the mediolateral antennocerebral tract PNs (mlPNs), which are the second largest PN subset (∼50 mlPNs in each hemisphere) and reported to be largely GABAergic with axons terminating mainly in the lateral horn (LH) (18, 19). Based on the extent of their dendritic arborization, mlPNs can be further categorized into three subtypes: the uniglomerular mlPNs (type 1 mlPNs, mlPN1s); the multiglomerular mlPNs (mlPN2s), which comprise the great majority (>80%) of mlPNs; and the panglomerular mlPNs (mlPN3s) (19). Here we focused on mlPN1s and mlPN2s, which exclusively link ALs with the ipsilateral LH and were labeled by Mz699-Gal4 (hereafter referred to as Mz699-mlPNs, or mlPNs) (20). Using dual whole-cell recordings and optogenetic activation of mlPNs, we examined their connectivity with different groups of AL and LH neurons and identified their main excitatory input neurons and putative output targets. The odor responses of mlPNs were also characterized and compared with those of ORNs and mPNs by dual-color imaging. Finally, we investigated the physiological role of mlPNs at the behavioral level by preferentially silencing the inhibitory output of mlPNs. Our results showed that by conveying olfactory information as inhibitory signals in parallel with the excitatory mPN pathway, the mlPN pathway might play important roles in modulating fly behavior through a novel feedforward inhibition mechansim.     

Brief predator sound exposure elicits behavioral and neuronal long-term sensitization in the olfactory system of an insect

Modulation of sensitivity to sensory cues by experience is essential for animals to adapt to a changing environment. Sensitization and adaptation to signals of the same modality as a function of experience have been shown in many cases, and some of the neurobiological mechanisms underlying these processes have been described. However, the influence of sensory signals on the sensitivity of a different modality is largely unknown. In males of the noctuid moth, Spodoptera littoralis, the sensitivity to the female-produced sex pheromone increases 24 h after a brief preexposure with pheromone at the behavioral and central nervous level. Here we show that this effect is not confined to the same sensory modality: the sensitivity of olfactory neurons can also be modulated by exposure to a different sensory stimulus, i.e., a pulsed stimulus mimicking echolocating sounds from attacking insectivorous bats. We tested responses of preexposed male moths in a walking bioassay and recorded from neurons in the primary olfactory center, the antennal lobe. We show that brief exposure to a bat call, but not to a behaviorally irrelevant tone, increases the behavioral sensitivity of male moths to sex pheromone 24 h later in the same way as exposure to the sex pheromone itself. The observed behavioral modification is accompanied by an increase in the sensitivity of olfactory neurons in the antennal lobe. Our data provide thus evidence for cross-modal experience-dependent plasticity not only on the behavioral level, but also on the central nervous level, in an insect.     

Coordination of central odor representations through transient,non-oscillatory synchronization of glomerular output neurons

At the first stage of processing in the olfactory pathway, the patterns of glomerular activity evoked by different scents are both temporally and spatially dynamic. In the antennal lobe (AL) of some insects, coherent firing of AL projection neurons (PNs) can be phase-locked to network oscillations, and it has been proposed that oscillatory synchronization of PN activity may encode the chemical identity of the olfactory stimulus. It remains unclear, however, how the brain uses this time-constrained mechanism to encode chemical identity when the stimulus itself is unpredictably dynamic. In the olfactory pathway of the moth Manduca sexta,we find that different odorants evoke gamma-band oscillations in the AL and the mushroom body (a higher-order network that receives input from the AL), but oscillations within or between these two processing stages are not temporally coherent. Moreover, the timing of action potential firing in PNs is not phase-locked to oscillations in either the AL or mushroom body, and the correlation between PN synchrony and field oscillations remains low before, during, and after olfactory stimulation. These results demonstrate that olfactory circuits in the moth are specialized to preserve time-varying signals in the insect's olfactory space, and that stimulus dynamics rather than intrinsic oscillations modulate the uniquely coordinated pattern of PN synchronization evoked by each olfactory stimulus. We propose that non-oscillatory synchronization provides an adaptive mechanism by which PN ensembles can encode stimulus identity while concurrently monitoring the unpredictable dynamics in the olfactory signal that typically occur under natural stimulus conditions.     

acj6: a gene affecting olfactory physiology and behavior in Drosophila.

Mutations affecting olfactory behavior provide material for use in molecular studies of olfaction in Drosophila melanogaster. Using the electroantennogram (EAG), a measure of antennal physiology, we have found an adult antennal defect in the olfactory behavioral mutant abnormal chemosensory jump 6 (acj6). The acj6 EAG defect was mapped to a single locus and the same mutation was found to be responsible for both reduction in EAG amplitude and diminished behavioral response, as if reduced antennal responsiveness to odorant is responsible for abnormal chemosensory behavior in the mutant. acj6 larval olfactory behavior is also abnormal; the mutation seems to alter cellular processes necessary for olfaction at both developmental stages. The acj6 mutation exhibits specificity in that visual system function appears normal in larvae and adults. These experiments provide evidence that the acj6 gene encodes a product required for olfactory signal transduction.     

Pheromone responsiveness threshold depends on temporal integration by antennal lobe projection neurons

The olfactory system of male moths has an extreme sensitivity with the capability to detect and recognize conspecific pheromones dispersed and greatly diluted in the air. Just 170 molecules of the silkmoth (Bombyx mori) sex pheromone bombykol are sufficient to induce sexual behavior in the male. However, it is still unclear how the sensitivity of olfactory receptor neurons (ORNs) is relayed through the brain to generate high behavioral responsiveness. Here, we show that ORN activity that is subthreshold in terms of behavior can be amplified to suprathreshold levels by temporal integration in antennal lobe projection neurons (PNs) if occurring within a specific time window. To control ORN inputs with high temporal resolution, channelrhodopsin-2 was genetically introduced into bombykol-responsive ORNs. Temporal integration in PNs was only observed for weak inputs, but not for strong inputs. Pharmacological dissection revealed that GABAergic mechanisms inhibit temporal integration of strong inputs, showing that GABA signaling regulates PN responses in a stimulus-dependent fashion. Our results show that boosting of the PNs’ responses by temporal integration of olfactory information occurs specifically near the behavioral threshold, effectively defining the lower bound for behavioral responsiveness.Olfaction is a key element in many aspects of animal behavior, such as foraging, oviposition, and mate recognition. In many moth species, a special class of odorants called sex pheromones plays a critical role for identification of and orientation to potential mates. Because sex pheromones emitted by females are greatly diluted and dispersed in the air, sophisticated olfactory systems to detect minute amounts of sex pheromones and processing systems to translate subtle peripheral sensory responses into appropriate behavioral responses have evolved in male moths. Theoretical calculations have shown that the detection of 170 molecules of the sex pheromone bombykol [(E,Z)-10,12-hexadecadienol] can trigger sexual behavioral responses in males of the silkmoth Bombyx mori (1). The physical and chemical mechanisms in the antennae, the sensilla, and the olfactory receptor neurons (ORNs) responsible for this remarkably high sensitivity, which can detect even a single molecule, are well understood (2, 3). However, it is unclear how a small number of spikes from a small number of ORNs is processed centrally to allow for high behavioral responsiveness.In moths, pheromone molecules are detected by specialized antennal ORNs that express particular pheromone receptor genes (4–10). The axons of ORNs convey pheromone information to the first olfactory center, the antennal lobe (AL; an analog of the vertebrate olfactory bulb). The AL is composed of a number of glomeruli where ORNs establish connections with two types of neurons: projection neurons (PNs), which relay olfactory information to higher brain regions, and local interneurons (LNs), which are involved in processing olfactory information within the deutocerebrum (11). In particular, male ALs have a specialized pheromone-processing unit called the macroglomerular complex (MGC), which comprises several glomeruli (12). In the silkmoth, the toroid glomerulus in the MGC is known to exclusively process bombykol information (13).Previous reports have shown that the sensitivity of pheromone-responsive ORNs is ∼1,000-fold lower than that of PNs tuned to the same pheromone compound (14, 15), suggesting that the AL network amplifies ORN inputs. One possible source of amplification is the high convergence ratio between ORNs and PNs (16). Considering the small size of most antennae, spatial integration would be likely to occur; however, the fact that weak stimuli activate a few ORNs at best calls for additional mechanisms to explain the high behavioral sensitivity.One candidate explanation is temporal integration, which is a fundamental mechanism for contributing to the amplification of synaptic inputs (17–19). In the olfactory system, the temporal integration properties of AL neurons and their relevance for the initiation of behavior are unknown. Temporal integration in the olfactory system has been challenging to investigate in detail because of the difficulty of accurately controlling olfactory stimuli within a given time period. Under natural conditions, odor molecules are distributed in odor plumes with intermittent local dynamics of odor exposure lasting >100 ms, followed by odor-free air lasting several hundreds of milliseconds (20–24). However, another study showed that “bursts” of odor within a plume structure can be as little 10–20 ms (25, 26). Therefore, the stimulation of pheromone-responsive ORNs must be controlled in the millisecond range.To examine the role of temporal integration in the generation of low thresholds for pheromone-induced behavioral responses, we generated GAL4/UAS transgenic silkmoths expressing channelrhodopsin-2 (ChR2), a light-gated ion channel from green algae, in bombykol-responsive ORNs. The transgenic silkmoths showed light-activated pheromone orientation behavior, and we succeeded in controlling the activity of bombykol-responsive ORNs with single-spike resolution. Using paired-pulse photostimulation at various interstimulus intervals (ISIs), we tested whether temporal integration in the AL network could be the basis of the high behavioral sensitivity.     

Auditory circuit in the Drosophila brain

Most animals exhibit innate auditory behaviors driven by genetically hardwired neural circuits. In Drosophila, acoustic information is relayed by Johnston organ neurons from the antenna to the antennal mechanosensory and motor center (AMMC) in the brain. Here, by using structural connectivity analysis, we identified five distinct types of auditory projection neurons (PNs) interconnecting the AMMC, inferior ventrolateral protocerebrum (IVLP), and ventrolateral protocerebrum (VLP) regions of the central brain. These auditory PNs are also functionally distinct; AMMC-B1a, AMMC-B1b, and AMMC-A2 neurons differ in their responses to sound (i.e., they are narrowly tuned or broadly tuned); one type of audioresponsive IVLP commissural PN connecting the two hemispheres is GABAergic; and one type of IVLP-VLP PN acts as a generalist responding to all tested audio frequencies. Our findings delineate an auditory processing pathway involving AMMC→IVLP→VLP in the Drosophila brain.     

Effects of ethanol on excitatory and inhibitory synaptic transmission in rat cortical neurons

BACKGROUND: The gamma-aminobutyric acid-A (GABA(A)) receptor and glutamate receptors are among the most important target sites for the behavioral effects of ethanol. However, data in the literature concerning the ethanol modulation of the GABA(A) and glutamate receptors have been controversial. The activity of the neuronal nicotinic acetylcholine (ACh) receptors (nAChRs) has recently been reported to be potently augmented by ethanol. The activation of nAChRs is also known to cause the release of various neurotransmitters including GABA and glutamate. Thus, ethanol potentiation of nAChRs is expected to stimulate the GABAergic and glutamatergic systems. METHODS: Whole-cell patch clamp experiments were performed using rat cortical neurons in primary culture to record spontaneous miniature inhibitory postsynaptic currents (mIPSCs) and spontaneous miniature excitatory postsynaptic currents (mEPSCs). RESULTS: Two types of neurons were distinguished: bipolar neurons possessed alpha4beta2 nAChRs generating a steady current in response to 30 nM ACh, and multipolar neurons that did not generate a current by ACh application. Acetylcholine greatly increased the frequency of mEPSCs and mIPSCs in bipolar neurons but not in multipolar neurons. The amplitude of neither type of neuron was affected by ACh. Ethanol at 10 to 100 mM suppressed the amplitude of mEPSCs while augmenting the amplitude of mIPSCs in both bipolar and multipolar neurons, indicating the direct action on the respective receptors. In bipolar neurons, ACh plus 100 mM ethanol greatly increased the frequency of mIPSCs beyond the levels achieved by ACh alone, while no such increases were observed in multipolar neurons. CONCLUSIONS: It is concluded that ethanol stimulation of nAChRs modulates the activity of both glutamate and GABA receptors in rat cortical bipolar neurons.     

Signaling between periglomerular cells reveals a bimodal role for GABA in modulating glomerular microcircuitry in the olfactory bulb

In the mouse olfactory bulb glomerulus, the GABAergic periglomerular (PG) cells provide a major inhibitory drive within the microcircuit. Here we examine GABAergic synapses between these interneurons. At these synapses, GABA is depolarizing and exerts a bimodal control on excitability. In quiescent cells, activation of GABA_A receptors can induce the cells to fire, thereby providing a means for amplification of GABA release in the glomerular microcircuit via GABA-induced GABA release. In contrast, GABA is inhibitory in neurons that are induced to fire tonically. PG–PG interactions are modulated by nicotinic acetylcholine receptors (nAChRs), and our data suggest that changes in intracellular calcium concentrations triggered by nAChR activation can be amplified by GABA release. Our results suggest that bidirectional control of inhibition in PG neurons can allow for modulatory inputs, like the cholinergic inputs from the basal forebrain, to determine threshold set points for filtering out weak olfactory inputs in the glomerular layer of the olfactory bulb via the activation of nAChRs.The balance of excitation and inhibition is critical for the normal functioning of brain networks. Timed inhibition of principal neurons modulates circuit output and contributes to network synchrony and oscillation. GABAergic interneurons play a key role in regulating these network properties (1, 2). Recent findings (e.g., ref. 3), however, have compelled us to move away from a simple view of transmission in the brain, in which glutamate and GABA represent the major excitatory and inhibitory transmitter systems, to a more nuanced interpretation of their roles.GABAergic neurotransmission has both inhibitory and excitatory effects in the CNS. Whereas the inhibitory actions of GABA on principal neurons in different brain regions have been examined extensively, studies of excitatory GABA have focused mostly on the developmental aspects of neuronal growth and synapse formation (4, 5). Recent evidence suggests that GABA can be excitatory in mature neurons as well (with the term “mature” here referring to neurons that are integral parts of established brain networks) (3, 6).Dynamic GABAergic signaling between inhibitory interneurons is less well understood. The common assumption is that GABAergic signaling between these interneurons would lead to disinhibition of principal neurons in a circuit. Excitatory GABA signaling between these interneurons, on the other hand, could serve as a means for amplification of principal cell inhibition. A combination of the two could effectively buffer interneuron firing rates and possibly normalize circuit output in a given area (7).The modularity in brain circuits allows for application of principles gleaned from the study of one defined circuit to other circuits as well. In the olfactory bulb (OB) glomerulus, the GABAergic periglomerular (PG) cells provide a large fraction of the inhibitory drive for information transfer between the olfactory nerve (ON) and mitral cells (MCs), the principal neurons. In this system, the existence of PG–PG synapses has been demonstrated (8), and GABA has been suggested to be depolarizing, yet inhibitory, on these neurons (9). Whether these synapses participate in glomerular signaling either during odor input or during neuromodulation of glomerular output is not yet known.In this paper, we report that GABAergic connections between PG cells have a bimodal effect on excitation depending on the previous activity state of the neurons. Excitation of PG cells by GABA can lead to amplification of glomerular inhibition via GABA-induced GABA release (GIGR). GABA release from PG cells modulates glomerular output on the activation of nicotinic acetylcholine receptors (nAChRs), wherein weak signals from the ON are filtered out while stronger ones are transmitted (10). Our results suggest that bimodal signaling by GABA could be important in determining set points for inhibition thresholds in the glomerular microcircuit.     

Effect of juvenile hormone on the central nervous processing of sex pheromone in an insect

Behavioral sex pheromone responsiveness in the male moth Agrotis ipsilon was previously shown to be controlled by juvenile hormone (JH). However, this morphogenetic hormone did not change the sensitivity of antennae to sex pheromones. To analyze the possible involvement of JH in the central integration of the female-produced sex pheromone, we investigated the pheromone response of olfactory antennal lobe (AL) interneurons in male A. ipsilon as a function of age and JH status by using intracellular recordings. When the antennae were stimulated with the sex pheromone blend, the sensitivity of olfactory AL neurons increased with age, as does the JH-dependent behavioral and physiological development of A. ipsilon males. Furthermore, males surgically deprived of JH showed a significant decrease in the sensitivity of the AL neurons. JH injection in operated or in young males restored or induced, respectively, a high sensitivity of the AL neurons. JH seems likely to be involved in the plasticity of the adult insect brain by modulating the central nervous processing of olfactory information, thus allowing mate recognition and reproduction at the optimal time.