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1.  Olfactory Processing and Behavior Downstream from Highly Selective Receptor Neurons 
Nature neuroscience  2007;10(5):623-630.
In either the vertebrate nose or the insect antenna, most olfactory receptor neurons (ORNs) respond to multiple odors. However, some ORNs respond to just a single odor, or at most to a few highly related odors. It has been hypothesized that narrowly-tuned ORNs project to narrowly-tuned neurons in the brain, and that these dedicated circuits mediate innate behavioral responses to a particular ligand. Here we have investigated neural activity and behavior downstream from two narrowly-tuned ORN types in Drosophila. We found that genetically ablating either of these ORN types impairs innate behavioral attraction to their cognate ligand. Neurons in the antennal lobe postsynaptic to one of these ORN types are, like their presynaptic ORNs, narrowly tuned to a pheromone. However, neurons postsynaptic to the second ORN type are broadly tuned. These results demonstrate that some narrowly-tuned ORNs project to dedicated central circuits, ensuring a tight connection between stimulus and behavior, whereas others project to central neurons which participate in the ensemble representations of many odors.
doi:10.1038/nn1881
PMCID: PMC2838507  PMID: 17417635
2.  Selective gating of glutamatergic inputs to excitatory neurons of amygdala by presynaptic GABAb receptor 
Neuron  2009;61(6):917-929.
GABAb receptor (GABAbR)-mediated suppression of glutamate release is critical for limiting glutamatergic transmission across the central nervous system. Here we show that, upon tetanic stimulation of afferents to lateral amygdala, presynaptic GABAbR-mediated inhibition only occurs in glutamatergic inputs to principle neurons (PNs), but not to interneurons (INs), despite the presence of GABAbR in terminals to both types of neurons. The selectivity is caused by differential local GABA accumulation; it requires GABA reuptake, and parallels distinct spatial distributions of presynaptic GABAbR in terminals to PNs and INs. Moreover, GABAbR-mediated suppression of theta-burst induced long-term potentiation (LTP) occurs only in the inputs to PNs, but not to INs. Thus, target cell-specific control of glutamate release by presynaptic GABAbR orchestrates the inhibitory dominance inside amygdala and may contribute to prevention of non-adaptive defensive behaviors.
doi:10.1016/j.neuron.2009.01.029
PMCID: PMC2673456  PMID: 19324000
3.  Presynaptic Facilitation by Neuropeptide Signaling Mediates Odor-Driven Food Search 
Cell  2011;145(1):133-144.
Internal physiological states influence behavioral decisions. We have investigated the underlying cellular and molecular mechanisms at the first olfactory synapse for starvation modulation of food search behavior in Drosophila. We found that a local signal by short neuropeptide F (sNPF) and a global metabolic cue by insulin are integrated at specific odorant receptor neurons (ORNs) to modulate olfactory sensitivity. Results from two-photon calcium imaging show that starvation increases presynaptic activity via intraglomerular sNPF signaling. Expression of sNPF and its receptor (sNPFR1) in Or42b neurons is necessary for starvation-induced food search behavior. Presynaptic facilitation in Or42b neurons is sufficient to mimic starvation-like behavior in fed flies. Furthermore, starvation elevates the transcription level of sNPFR1 but not that of sNPF, and insulin signaling suppresses sNPFR1 expression. Thus, starvation increases expression of sNPFR1 to change the odor map, resulting in more robust food search behavior.
doi:10.1016/j.cell.2011.02.008
PMCID: PMC3073827  PMID: 21458672
Drosophila; olfaction; sNPF; gain control; feeding behavior; two-photon imaging; Insulin; PI3K; wortmannin; LY294002
4.  Differential Regulation of GABAB Receptor Trafficking by Different Modes of N-methyl-d-aspartate (NMDA) Receptor Signaling* 
The Journal of Biological Chemistry  2014;289(10):6681-6694.
Background: Heterodimerization of GABAB1 and GABAB2 subunits is required for functional GABABRs.
Results: GABABR subunits are differentially regulated by activation of synaptic or extrasynaptic NMDARs.
Conclusion: GABABR trafficking and function is regulated by NMDARs.
Significance: GABABRs are potential targets for treating diseases such as stroke and cerebral ischemia.
Inhibitory GABAB receptors (GABABRs) can down-regulate most excitatory synapses in the CNS by reducing postsynaptic excitability. Functional GABABRs are heterodimers of GABAB1 and GABAB2 subunits and here we show that the trafficking and surface expression of GABABRs is differentially regulated by synaptic or pathophysiological activation of NMDA receptors (NMDARs). Activation of synaptic NMDARs using a chemLTP protocol increases GABABR recycling and surface expression. In contrast, excitotoxic global activation of synaptic and extrasynaptic NMDARs by bath application of NMDA causes the loss of surface GABABRs. Intriguingly, exposing neurons to extreme metabolic stress using oxygen/glucose deprivation (OGD) increases GABAB1 but decreases GABAB2 surface expression. The increase in surface GABAB1 involves enhanced recycling and is blocked by the NMDAR antagonist AP5. The decrease in surface GABAB2 is also blocked by AP5 and by inhibiting degradation pathways. These results indicate that NMDAR activity is critical in GABABR trafficking and function and that the individual subunits can be separately controlled to regulate neuronal responsiveness and survival.
doi:10.1074/jbc.M113.487348
PMCID: PMC3945329  PMID: 24425870
G Protein-coupled Receptors (GPCR); GABA Receptors; Glutamate Receptor Ionotropic (AMPA, NMDA); Neurodegeneration; Neurotransmitter Receptors; Receptor Endocytosis; Receptor Recycling; GABAB Receptor; Chem-LTP; Oxygen-glucose Deprivation (OGD)
5.  Methamphetamine-evoked depression of GABAB receptor signaling in GABA neurons of the VTA 
Neuron  2012;73(5):978-989.
Psychostimulants induce neuroadaptations in excitatory and fast inhibitory transmission in the ventral tegmental area (VTA). Mechanisms underlying drug-evoked synaptic plasticity of slow inhibitory transmission mediated by GABAB receptors and G protein-gated inwardly rectifying potassium (GIRK/Kir3) channels, however, are poorly understood. Here, we show that one day after methamphetamine (METH) or cocaine exposure, both synaptically-evoked and baclofen-activated GABABR-GIRK currents were significantly depressed in VTA GABA neurons, and remained depressed for 7 days. Presynaptic inhibition mediated by GABABRs on GABA terminals was also weakened. Quantitative immunoelectron microscopy revealed internalization of GABAB1R and GIRK2, which occurred coincident with dephosphorylation of Ser783 in GABAB2R, a site implicated in regulating GABABR surface expression. Inhibition of protein phosphatases recovered GABABR-GIRK currents in VTA GABA neurons of METH-injected mice. This psychostimulant-evoked impairment in GABABR signaling removes an intrinsic brake on GABA neuron spiking, which may augment GABA transmission in the mesocorticolimbic system.
doi:10.1016/j.neuron.2011.12.031
PMCID: PMC3560416  PMID: 22405207
6.  GISP: a novel brain-specific protein that promotes surface expression and function of GABAB receptors 
Journal of neurochemistry  2007;100(4):1003-1017.
Synaptic transmission depends on the regulated surface expression of neurotransmitter receptors, but many of the cellular processes required to achieve this remain poorly understood. To better define specific mechanisms for the GABAB receptor (GABABR) trafficking, we screened for proteins that bind to the carboxy-terminus of the GABAB1 subunit. We report the identification and characterization of a novel 130-kDa protein, GPCR interacting scaffolding protein (GISP), that interacts directly with the GABAB1 subunit via a coiled-coil domain. GISP co-fractionates with GABABR and with the postsynaptic density and co-immunoprecipitates with GABAB1 and GABAB2 from rat brain. In cultured hippocampal neurons, GISP displays a punctate dendritic distribution and has an overlapping localization with GABABRs. When co-expressed with GABABRs in human embryonic kidney cells, GISP promotes GABABR surface expression and enhances both baclofen-evoked extracellular signal-regulated kinase (ERK) phosphorylation and G-protein inwardly rectifying potassium channel (GIRK) currents. These results suggest that GISP is involved in the forward trafficking and stabilization of functional GABABRs.
doi:10.1111/j.1471-4159.2006.04271.x
PMCID: PMC3315443  PMID: 17241134
A-kinase anchoring protein; cultured neurons; GABAB receptor; GPCR interacting scaffolding protein; hippocampus; receptor trafficking
7.  Excitatory Local Circuits and Their Implications for Olfactory Processing in the Fly Antennal Lobe 
Cell  2007;128(3):601-612.
Summary
Conflicting views exist of how circuits of the antennal lobe, the insect equivalent of the olfactory bulb, translate input from olfactory receptor neurons (ORNs) into projection neuron (PN) output. Synaptic connections between ORNs and PNs are one-to-one, yet PNs are more broadly tuned to odors than ORNs. The basis for this difference in receptive range remains unknown. Analyzing a Drosophila mutant lacking ORN input to one glomerulus, we show that some of the apparent complexity in the antennal lobe’s output arises from lateral, interglomerular excitation of PNs. We describe a previously unidentified population of cholinergic local neurons (LNs) with multiglomerular processes. These excitatory LNs respond broadly to odors but exhibit little glomerular specificity in their synaptic output, suggesting that PNs are driven by a combination of glomerulus-specific ORN afferents and diffuse LN excitation. Lateral excitation may boost PN signals and enhance their transmission to third-order neurons in a mechanism akin to stochastic resonance.
doi:10.1016/j.cell.2006.12.034
PMCID: PMC2866183  PMID: 17289577
8.  Depression of glutamate and GABA release by presynaptic GABAB receptors in the entorhinal cortex in normal and chronically epileptic rats 
Neuro-Signals  2007;15(4):202-215.
Presynaptic GABAB-receptors (GABABR) control glutamate and GABA release at many synapses in the nervous system. In the present study we used whole-cell patch clamp recordings of spontaneous excitatory and inhibitory synaptic currents in the presence of TTX to monitor glutamate and GABA release from synapses in layer II and V of the rat entorhinal cortex (EC) in vitro. In both layers the release of both transmitters was reduced by application of GABABR agonists. Quantitatively, the depression of GABA release in layer II and layer V, and of glutamate release in layer V was similar, but glutamate release in layer II was depressed to a greater extent. The data suggest that the same GABABR may be present on both GABA and glutamate terminals in the EC, but that the heteroreceptor may show a greater level of expression in layer II. Studies with GABABR antagonists suggested that neither the auto- nor the heteroreceptor was consistently tonically activated by ambient GABA in the presence of TTX. Studies in EC slices from rats made chronically epileptic using a pilocarpine model of temporal lobe epilepsy revealed a reduced effectiveness of both auto- and heteroreceptor function in both layers. This could suggest that enhanced glutamate and GABA release in the EC may be associated with the development of the epileptic condition.
doi:10.1159/000098515
PMCID: PMC2504722  PMID: 17215590
GABAB receptors; entorhinal cortex; glutamate release; GABA release; epilepsy
9.  Sensory Processing in the Drosophila Antennal Lobe Increases the Reliability and Separability of Ensemble Odor Representations 
Nature neuroscience  2007;10(11):1474-1482.
Here we describe several fundamental principles of olfactory processing in the Drosophila antennal lobe (the analog of the vertebrate olfactory bulb), based on a systematic analysis of input and output spike trains of seven identified glomeruli. Repeated presentations of the same odor elicit more reproducible responses in second-order projection neurons (PNs) than in their presynaptic olfactory receptor neurons (ORNs). PN responses rise and accommodate rapidly, emphasizing odor onset. Furthermore, weak ORN inputs are amplified in the PN layer but strong inputs are not. This nonlinear transformation broadens PN tuning, and produces more uniform distances between odor representations in PN coding space. Additionally, a portion of a PN’s odor response profile is not systematically related to its direct ORN inputs, likely reflecting lateral connections between glomeruli. Finally, we show that a linear discriminator classifies odors more accurately using PN spike trains as compared to an equivalent number of ORN spike trains.
doi:10.1038/nn1976
PMCID: PMC2838615  PMID: 17922008
10.  Distinct functions of acj6 splice forms in odor receptor gene choice 
Individual olfactory receptor neurons (ORNs) selectively express one or a small number of odor receptors from among a large receptor repertoire. The expression of an odor receptor dictates the odor response spectrum of the ORN. The process of receptor gene choice relies in part on a combinatorial code of transcription factors. In Drosophila, the POU domain transcription factor Acj6 is one element of the transcription factor code. In acj6 null mutants, many ORNs do not express an appropriate odor receptor gene and thus are not correctly specified. We find that acj6 is alternatively spliced to yield many structurally distinct transcripts in the olfactory organs. We generate flies that express single splice forms of acj6 in an acj6− background. We find that different splice forms are functionally distinct; they differ in their abilities to specify ORN identities. Some individual splice forms can fully rescue the specification of some ORNs. Individual splice forms can function both positively and negatively in receptor gene regulation. ORNs differ in their requirements for splice forms; some are not fully rescued by any single splice form tested, suggesting that some ORNs may require the combinatorial action of multiple splice forms. Late expression of some acj6 splice forms is sufficient to rescue some ORN classes, consistent with a direct role for Acj6 isoforms in receptor gene expression. The results indicate that alternative splicing may add another level of richness to the regulatory code that underlies the process of odor receptor gene choice.
doi:10.1523/JNEUROSCI.6292-09.2010
PMCID: PMC2858447  PMID: 20371823
olfaction; Drosophila; Acj6; POU-domain; odor receptor; splicing
11.  GABAB receptor GTP-binding is decreased in the prefrontal cortex but not the hippocampus of aged rats 
Neurobiology of Aging  2011;33(6):1124.e1-1124.e12.
GABAB receptors (GABABRs) have been linked to a wide range of physiological and cognitive processes and are of interest for treating a number of neurodegenerative and psychiatric disorders. As many of these diseases are associated with advanced age, it is important to understand how the normal aging process impacts GABABR expression and signaling. Thus, we investigated GABABR expression and function in the prefrontal cortex (PFC) and hippocampus of young and aged rats characterized in a spatial learning task. Baclofen-stimulated GTP-binding and GABABR1 and GABABR2 proteins were reduced in the PFC of aged rats but these reductions were not associated with spatial learning abilities. In contrast, hippocampal GTP-binding was comparable between young and aged rats but reduced hippocampal GABABR1 expression was observed in aged rats with spatial learning impairment. These data demonstrate marked regional differences in GABABR complexes in the adult and aged brain and could have implications for both understanding the role of GABAergic processes in normal brain function and the development of putative interventions that target this system.
doi:10.1016/j.neurobiolaging.2011.11.011
PMCID: PMC3310948  PMID: 22169202
Aging; Baclofen; Spatial learning; GABABR; PFC; Memory
12.  Non-synaptic inhibition between grouped neurons in an olfactory circuit 
Nature  2012;492(7427):66-71.
Summary
Diverse sensory organs, including mammalian taste buds and insect chemosensory sensilla, show a striking compartmentalization of receptor cells. However, the functional impact of this organization remains unclear. Here we show that compartmentalized Drosophila olfactory receptor neurons (ORNs) communicate with each other directly. The sustained response of one ORN is inhibited by the transient activation of a neighboring ORN. Mechanistically, such lateral inhibition does not depend on synapses and is likely mediated by ephaptic coupling. Moreover, lateral inhibition in the periphery can modulate olfactory behavior. Together, the results show that integration of olfactory information can occur via lateral interactions between ORNs. Inhibition of a sustained response by a transient response may provide a means of encoding salience. Finally, a CO2-sensitive ORN in the malaria mosquito Anopheles can also be inhibited by excitation of an adjacent ORN, suggesting a broad occurrence of lateral inhibition in insects and possible applications in insect control.
doi:10.1038/nature11712
PMCID: PMC3518700  PMID: 23172146
13.  Functional Roles for Synaptic-Depression within a Model of the Fly Antennal Lobe 
PLoS Computational Biology  2012;8(8):e1002622.
Several experiments indicate that there exists substantial synaptic-depression at the synapses between olfactory receptor neurons (ORNs) and neurons within the drosophila antenna lobe (AL). This synaptic-depression may be partly caused by vesicle-depletion, and partly caused by presynaptic-inhibition due to the activity of inhibitory local neurons within the AL. While it has been proposed that this synaptic-depression contributes to the nonlinear relationship between ORN and projection neuron (PN) firing-rates, the precise functional role of synaptic-depression at the ORN synapses is not yet fully understood. In this paper we propose two hypotheses linking the information-coding properties of the fly AL with the network mechanisms responsible for ORNAL synaptic-depression. Our first hypothesis is related to variance coding of ORN firing-rate information — once stimulation to the ORNs is sufficiently high to saturate glomerular responses, further stimulation of the ORNs increases the regularity of PN spiking activity while maintaining PN firing-rates. The second hypothesis proposes a tradeoff between spike-time reliability and coding-capacity governed by the relative contribution of vesicle-depletion and presynaptic-inhibition to ORNAL synaptic-depression. Synaptic-depression caused primarily by vesicle-depletion will give rise to a very reliable system, whereas an equivalent amount of synaptic-depression caused primarily by presynaptic-inhibition will give rise to a less reliable system that is more sensitive to small shifts in odor stimulation. These two hypotheses are substantiated by several small analyzable toy models of the fly AL, as well as a more physiologically realistic large-scale computational model of the fly AL involving glomerular channels.
Author Summary
Understanding the intricacies of sensory processing is a major scientific challenge. In this paper we examine the early stages of the olfactory system of the fruit-fly. Many experiments have revealed a great deal regarding the architecture of this system, including the types of neurons within it, as well as the connections those neurons make amongst one another. In this paper we examine the potential dynamics produced by this neuronal network. Specifically, we construct a computational model of this early olfactory system and study the effects of synaptic-depression within this system. We find that the dynamics and coding properties of this system depend strongly on the strength, and sources of, synaptic-depression. This work has ramifications for understanding the coding properties of other insect olfactory systems, and perhaps even other sensory modalities in other animals.
doi:10.1371/journal.pcbi.1002622
PMCID: PMC3426607  PMID: 22927802
14.  Presynaptic GABAB Receptor Regulates Activity-Dependent Maturation and Patterning of Inhibitory Synapses through Dynamic Allocation of Synaptic Vesicles 
Accumulating evidence indicate that GABA regulates activity-dependent development of inhibitory synapses in the vertebrate brain, but the underlying mechanisms remain unclear. Here we combined live imaging of cortical GABAergic axons with single cell genetic manipulation to dissect the role of presynaptic GABAB receptors (GABABRs) in inhibitory synapse formation in mouse. Developing GABAergic axons form a significant number of transient boutons but only a subset was stabilized. Synaptic vesicles in these nascent boutons are often highly mobile in the course of tens of minutes. Activation of presynaptic GABABRs stabilized mobile vesicles in nascent boutons through the local enhancement of actin polymerization. Inactivation of GABABRs in developing basket interneurons resulted in aberrant pattern of bouton size distribution, reduced bouton density and reduced axon branching, as well as reduced frequency of miniature inhibitory currents in postsynaptic pyramidal neurons. These results suggest that GABABRs along developing inhibitory axons act as a local sensor of GABA release and promote presynaptic maturation through increased recruitment of mobile vesicle pools. Such release-dependent validation and maturation of nascent terminals is well suited to sculpt the pattern of synapse formation and distribution along axon branches.
doi:10.3389/fncel.2012.00057
PMCID: PMC3512030  PMID: 23227002
GABAB receptor; synaptic vesicle dynamics; live cell imaging; actin polymerization; FRET; inhibitory synapses; activity-dependent development
15.  A spiking neural network model of self-organized pattern recognition in the early mammalian olfactory system 
Olfactory sensory information passes through several processing stages before an odor percept emerges. The question how the olfactory system learns to create odor representations linking those different levels and how it learns to connect and discriminate between them is largely unresolved. We present a large-scale network model with single and multi-compartmental Hodgkin–Huxley type model neurons representing olfactory receptor neurons (ORNs) in the epithelium, periglomerular cells, mitral/tufted cells and granule cells in the olfactory bulb (OB), and three types of cortical cells in the piriform cortex (PC). Odor patterns are calculated based on affinities between ORNs and odor stimuli derived from physico-chemical descriptors of behaviorally relevant real-world odorants. The properties of ORNs were tuned to show saturated response curves with increasing concentration as seen in experiments. On the level of the OB we explored the possibility of using a fuzzy concentration interval code, which was implemented through dendro-dendritic inhibition leading to winner-take-all like dynamics between mitral/tufted cells belonging to the same glomerulus. The connectivity from mitral/tufted cells to PC neurons was self-organized from a mutual information measure and by using a competitive Hebbian–Bayesian learning algorithm based on the response patterns of mitral/tufted cells to different odors yielding a distributed feed-forward projection to the PC. The PC was implemented as a modular attractor network with a recurrent connectivity that was likewise organized through Hebbian–Bayesian learning. We demonstrate the functionality of the model in a one-sniff-learning and recognition task on a set of 50 odorants. Furthermore, we study its robustness against noise on the receptor level and its ability to perform concentration invariant odor recognition. Moreover, we investigate the pattern completion capabilities of the system and rivalry dynamics for odor mixtures.
doi:10.3389/fncir.2014.00005
PMCID: PMC3916767  PMID: 24570657
pattern recognition; olfactory bulb; piriform cortex; large-scale neuromorphic systems; spiking neural network; BCPNN; concentration invariance; pattern rivalry
16.  Functional Differences between Global Pre- and Postsynaptic Inhibition in the Drosophila Olfactory Circuit 
The Drosophila antennal lobe is subdivided into multiple glomeruli, each of which represents a unique olfactory information processing channel. In each glomerulus, feedforward input from olfactory receptor neurons (ORNs) is transformed into activity of projection neurons (PNs), which represent the output. Recent investigations have indicated that lateral presynaptic inhibitory input from other glomeruli controls the gain of this transformation. Here, we address why this gain control acts “pre”-synaptically rather than “post”-synaptically. Postsynaptic inhibition could work similarly to presynaptic inhibition with regard to regulating the firing rates of PNs depending on the stimulus intensity. We investigate the differences between pre- and postsynaptic gain control in terms of odor discriminability by simulating a network model of the Drosophila antennal lobe with experimental data. We first demonstrate that only presynaptic inhibition can reproduce the type of gain control observed in experiments. We next show that presynaptic inhibition decorrelates PN responses whereas postsynaptic inhibition does not. Due to this effect, presynaptic gain control enhances the accuracy of odor discrimination by a linear decoder while its postsynaptic counterpart only diminishes it. Our results provide the reason gain control operates “pre”-synaptically but not “post”-synaptically in the Drosophila antennal lobe.
doi:10.3389/fncom.2012.00014
PMCID: PMC3309306  PMID: 22470334
Drosophila; antennal lobe; odor discriminability; presynaptic inhibition; postsynaptic inhibition; gain control; decorrelation; concentration invariance
17.  Odor Coding in the Maxillary Palp of the Malaria Vector Mosquito Anopheles gambiae 
Current biology : CB  2007;17(18):1533-1544.
Summary
Background
Many species of mosquitoes, including the major malaria vector Anopheles gambiae, utilize carbon dioxide (CO2) and 1-octen-3-ol as olfactory cues in host-seeking behaviors that underlie their vectorial capacity. However, the molecular and cellular basis of such olfactory responses remains largely unknown.
Results
Here, we use molecular and physiological approaches coupled with systematic functional analyses to define the complete olfactory sensory map of the An. gambiae maxillary palp, an olfactory appendage that mediates the detection of these compounds. In doing so, we identify three olfactory receptor neurons (ORNs) that are organized in stereotyped triads within the maxillary-palp capitate-peg-sensillum population. One ORN is CO2-responsive and characterized by the coexpression of three receptors that confer CO2 responses, whereas the other ORNs express characteristic odorant receptors (AgORs) that are responsible for their in vivo olfactory responses.
Conclusions
Our results describe a complete and highly concordant map of both the molecular and cellular olfactory components on the maxillary palp of the adult female An. gambiae mosquito. These results also facilitate the understanding of how An. gambiae mosquitoes sense olfactory cues that might be exploited to compromise their ability to transmit malaria.
doi:10.1016/j.cub.2007.07.062
PMCID: PMC3113458  PMID: 17764944
18.  Inhibition of Olfactory Receptor Neuron Input to Olfactory Bulb Glomeruli Mediated by Suppression of Presynaptic Calcium Influx 
Journal of neurophysiology  2005;94(4):2700-2712.
We investigated the cellular mechanism underlying presynaptic regulation of olfactory receptor neuron (ORN) input to the mouse olfactory bulb using optical-imaging techniques that selectively report activity in the ORN pre-synaptic terminal. First, we loaded ORNs with calcium-sensitive dye and imaged stimulus-evoked calcium influx in a slice preparation. Single olfactory nerve shocks evoked rapid fluorescence increases that were largely blocked by the N-type calcium channel blocker ω-conotoxin GVIA. Paired shocks revealed a long-lasting suppression of calcium influx with ~40% suppression at 400-ms interstimulus intervals and a recovery time constant of ~450 ms. Blocking activation of postsynaptic olfactory bulb neurons with APV/CNQX reduced this suppression. The GABAB receptor agonist baclofen inhibited calcium influx, whereas GABAB antagonists reduced paired-pulse suppression without affecting the response to the conditioning pulse. We also imaged transmitter release directly using a mouse line that expresses synaptopHluorin selectively in ORNs. We found that the relationship between calcium influx and transmitter release was superlinear and that paired-pulse suppression of transmitter release was reduced, but not eliminated, by APV/CNQX and GABAB antagonists. These results demonstrate that primary olfactory input to the CNS can be presynaptically regulated by GABAergic interneurons and show that one major intracellular pathway for this regulation is via the suppression of calcium influx through N-type calcium channels in the pre-synaptic terminal. This mechanism is unique among primary sensory afferents.
doi:10.1152/jn.00286.2005
PMCID: PMC1282456  PMID: 15917320
19.  Development of GPCR Modulation of GABAergic Transmission in Chicken Nucleus Laminaris Neurons 
PLoS ONE  2012;7(4):e35831.
Neurons in the nucleus laminaris (NL) of birds act as coincidence detectors and encode interaural time difference to localize the sound source in the azimuth plane. GABAergic transmission in a number of CNS nuclei including the NL is subject to a dual modulation by presynaptic GABAB receptors (GABABRs) and metabotropic glutamate receptors (mGluRs). Here, using in vitro whole-cell patch clamp recordings from acute brain slices of the chick, we characterized the following important but unknown properties pertaining to such a dual modulation: (1) emergence of functional GABA synapses in NL neurons; (2) the temporal onset of neuromodulation mediated by GABABRs and mGluRs; and (3) the physiological conditions under which GABABRs and mGluRs are activated by endogenous transmitters. We found that (1) GABAAR-mediated synaptic responses were observed in about half of the neurons at embryonic day 11 (E11); (2) GABABR-mediated modulation of the GABAergic transmission was detectable at E11, whereas the modulation by mGluRs did not emerge until E15; and (3) endogenous activity of GABABRs was induced by both low- (5 or 10 Hz) and high-frequency (200 Hz) stimulation of the GABAergic pathway, whereas endogenous activity of mGluRs was induced by high- (200 Hz) but not low-frequency (5 or 10 Hz) stimulation of the glutamatergic pathway. Furthermore, the endogenous activity of mGluRs was mediated by group II but not group III members. Therefore, autoreceptor-mediated modulation of GABAergic transmission emerges at the same time when the GABA synapses become functional. Heteroreceptor-mediated modulation appears at a later time and is receptor type dependent in vitro.
doi:10.1371/journal.pone.0035831
PMCID: PMC3335798  PMID: 22545142
20.  The Drosophila melanogaster Phospholipid Flippase dATP8B Is Required for Odorant Receptor Function 
PLoS Genetics  2014;10(3):e1004209.
The olfactory systems of insects are fundamental to all aspects of their behaviour, and insect olfactory receptor neurons (ORNs) exhibit exquisite specificity and sensitivity to a wide range of environmental cues. In Drosophila melanogaster, ORN responses are determined by three different receptor families, the odorant (Or), ionotropic-like (IR) and gustatory (Gr) receptors. However, the precise mechanisms of signalling by these different receptor families are not fully understood. Here we report the unexpected finding that the type 4 P-type ATPase phospholipid transporter dATP8B, the homologue of a protein associated with intrahepatic cholestasis and hearing loss in humans, is crucial for Drosophila olfactory responses. Mutations in dATP8B severely attenuate sensitivity of odorant detection specifically in Or-expressing ORNs, but do not affect responses mediated by IR or Gr receptors. Accordingly, we find dATP8B to be expressed in ORNs and localised to the dendritic membrane of the olfactory neurons where signal transduction occurs. Localisation of Or proteins to the dendrites is unaffected in dATP8B mutants, as is dendrite morphology, suggesting instead that dATP8B is critical for Or signalling. As dATP8B is a member of the phospholipid flippase family of ATPases, which function to determine asymmetry in phospholipid composition between the outer and inner leaflets of plasma membranes, our findings suggest a requirement for phospholipid asymmetry in the signalling of a specific family of chemoreceptor proteins.
Author Summary
The olfactory systems of insects are fundamental to critical behaviours such as finding mates, food and host plants. Insects can detect a wide range of environmental cues using three different families of olfactory receptor proteins. Why insects have three different families of receptor genes, and how they function together, is not fully understood. Here we identified a new gene, dATP8B, which is critically and specifically required for the function of only one of these receptor families in Drosophila. dATP8B is a member of the P4-type ATPases, or phospholipid flippases; these enzymes function in establishing a difference or asymmetry in lipid composition between the outer and inner leaflets of plasma membranes. This is thought to be important for many cellular membrane processes; however, specific functions of individual flippase proteins are not well described. We find that dATP8B is required for the function of the odorant receptor family, but not the ionotropic-like and gustatory receptor families. This further highlights the functional differences between these receptor families and suggests a role for phospholipids in the signalling of a specific family of receptor proteins.
doi:10.1371/journal.pgen.1004209
PMCID: PMC3961175  PMID: 24651716
21.  Avoiding DEET through insect gustatory receptors 
Neuron  2010;67(4):555-561.
Summary
DEET is the most widely used insect repellent worldwide. In Drosophila olfactory receptor neurons (ORNs), DEET is detected through a mechanism that employs the olfactory receptor, OR83b. However, it is controversial as to whether ORNs respond directly to DEET or whether DEET blocks the response to attractive odors. Here, we showed that DEET suppressed feeding behavior in Drosophila and this effect was mediated by gustatory receptor neurons (GRNs). DEET was potent in suppressing feeding as <0.1% DEET elicited aversive behavior. Inhibition of feeding by DEET required multiple gustatory receptors (GRs), which were expressed in inhibitory GRNs. DEET stimulated action potentials in GRNs that respond to aversive compounds, and this response was lost in Gr32a, Gr33a and Gr66a mutants. Since 0.02% DEET elicited action potentials, we conclude that DEET directly activates of GRNs. We suggest that the effectiveness of DEET in pest control owes to its dual action in inducing avoidance simultaneously via GRNs and ORNs.
doi:10.1016/j.neuron.2010.07.006
PMCID: PMC2929391  PMID: 20797533
22.  Differential GABAB-Receptor-Mediated Effects in Perisomatic- and Dendrite-Targeting Parvalbumin Interneurons 
The Journal of Neuroscience  2013;33(18):7961-7974.
Inhibitory parvalbumin-containing interneurons (PVIs) control neuronal discharge and support the generation of theta- and gamma-frequency oscillations in cortical networks. Fast GABAergic input onto PVIs is crucial for their synchronization and oscillatory entrainment, but the role of metabotropic GABAB receptors (GABABRs) in mediating slow presynaptic and postsynaptic inhibition remains unknown. In this study, we have combined high-resolution immunoelectron microscopy, whole-cell patch-clamp recording, and computational modeling to investigate the subcellular distribution and effects of GABABRs and their postsynaptic effector Kir3 channels in rat hippocampal PVIs. Pre-embedding immunogold labeling revealed that the receptors and channels localize at high levels to the extrasynaptic membrane of parvalbumin-immunoreactive dendrites. Immunoreactivity for GABABRs was also present at lower levels on PVI axon terminals. Whole-cell recordings further showed that synaptically released GABA in response to extracellular stimulation evokes large GABABR-mediated slow IPSCs in perisomatic-targeting (PT) PVIs, but only small or no currents in dendrite-targeting (DT) PVIs. In contrast, paired recordings demonstrated that GABABR activation results in presynaptic inhibition at the output synapses of both PT and DT PVIs, but more strongly in the latter. Finally, computational analysis indicated that GABAB IPSCs can phasically modulate the discharge of PT interneurons at theta frequencies. In summary, our results show that GABABRs differentially mediate slow presynaptic and postsynaptic inhibition in PVIs and can contribute to the dynamic modulation of their activity during oscillations. Furthermore, these data provide evidence for a compartment-specific molecular divergence of hippocampal PVI subtypes, suggesting that activation of GABABRs may shift the balance between perisomatic and dendritic inhibition.
doi:10.1523/JNEUROSCI.1186-12.2013
PMCID: PMC3814621  PMID: 23637187
23.  Optogenetically Induced Olfactory Stimulation in Drosophila Larvae Reveals the Neuronal Basis of Odor-Aversion behavior 
Olfactory stimulation induces an odor-guided crawling behavior of Drosophila melanogaster larvae characterized by either an attractive or a repellent reaction. In order to understand the underlying processes leading to these orientations we stimulated single olfactory receptor neurons (ORNs) through photo-activation within an intact neuronal network. Using the Gal4-UAS system two light inducible proteins, the light-sensitive cation channel channelrhodopsin-2 (ChR-2) or the light-sensitive adenylyl cyclase (Pacα) were expressed in all or in individual ORNs of the larval olfactory system. Blue light stimulation caused an activation of these neurons, ultimately producing the illusion of an odor stimulus. Larvae were tested in a phototaxis assay for their orientation toward or away from the light source. Here we show that activation of Pacα expressing ORNs bearing the receptors Or33b or Or45a in blind norpA mutant larvae induces a repellent behavior away from the light. Conversely, photo-activation of the majority of ORNs induces attraction towards the light. Interestingly, in wild type larvae two ligands of Or33b and Or45a, octyl acetate and propionic ethylester, respectively, have been found to cause an escape reaction. Therefore, we combined light and odor stimulation to analyze the function of Or33b and Or45a expressing ORNs. We show that the larval olfactory system contains a designated neuronal pathway for repellent odorants and that activation of a specific class of ORNs already determines olfactory avoidance behavior.
doi:10.3389/fnbeh.2010.00027
PMCID: PMC2889724  PMID: 20577637
Drosophila; olfaction; photo-activation; optogenetics; olfactory behavior; electrophysiology; channelrhodopsin-2; photo-activated adenylyl cyclase
24.  Excitatory Interactions Between Olfactory Processing Channels in the Drosophila Antennal Lobe 
Neuron  2007;54(1):89-103.
Summary
Each odorant receptor gene defines a unique type of olfactory receptor neuron (ORN) and a corresponding type of second-order neuron. Because each odor can activate multiple ORN types, information must ultimately be integrated across these processing channels to form a unified percept. Here we show that, in Drosophila, integration begins at the level of second-order projection neurons (PNs). We genetically silence all the ORNs that normally express a particular odorant receptor, and find that PNs postsynaptic to the silent glomerulus receive substantial lateral excitatory input from other glomeruli. Genetically confining odor-evoked ORN input to just one glomerulus reveals that most PNs postsynaptic to other glomeruli receive indirect excitatory input from the single ORN type that is active. Lateral connections between identified glomeruli vary in strength, and this pattern of connections is stereotyped across flies. Thus, a dense network of lateral connections distributes odor-evoked excitation between channels in the first brain region of the olfactory processing stream.
doi:10.1016/j.neuron.2007.03.010
PMCID: PMC2048819  PMID: 17408580
25.  A new Drosophila POU gene, pdm3, acts in odor receptor expression and axon targeting of olfactory neurons 
Olfaction depends on the differential activation of olfactory receptor neurons (ORNs) and on the proper transmission of their activities to the brain. ORNs select individual receptors to express, and they send axons to particular targets in the brain. Little is known about the molecular mechanisms underlying either process. We have identified a new Drosophila POU gene, pdm3, that is expressed in ORNs. Genetic analysis shows that pdm3 is required for odor response in one class of ORNs. We find that pdm3 acts in odor receptor expression in this class, and that the odor response can be rescued by the receptor. Another POU gene, acj6, is required for receptor expression in the same class, and we find a genetic interaction between the two POU genes. The results support a role for a POU gene code in receptor gene choice. pdm3 is also expressed in other ORN classes in which it is not required for receptor expression. For two of these classes pdm3 is required for normal axon targeting. Thus this mutational analysis, the first for a POU class VI gene, demonstrates a role for pdm3 in both of the processes that define the functional organization of ORNs in the olfactory system.
doi:10.1523/JNEUROSCI.2063-08.2008
PMCID: PMC2572001  PMID: 18614681
POU gene; odor receptor; axon targeting; Drosophila; maxillary palp; antenna

Results 1-25 (584016)