Non-synaptic inhibition between grouped neurons in an olfactory circuit.

Diverse sensory organs, including mammalian taste buds and insect chemosensory sensilla, show a marked 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 neighbouring ORN. Mechanistically, such lateral inhibition does not depend on synapses and is probably mediated by ephaptic coupling. Moreover, lateral inhibition in the periphery can modulate olfactory behaviour. 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 CO(2)-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

In vivo regulation of AMPA receptors by their TARP auxiliary subunits

Ion channels are often modulated by auxiliary subunits. Transmembrane AMPA receptor regulatory proteins (TARPs) are auxiliary subunits for AMPA-type glutamate receptors. These receptors are responsible for much of the fast excitatory synaptic transmission in the brain, and their mobility is thought to contribute to memory storage mechanisms. Although TARPs are known to modify AMPA receptor trafficking and gating in vitro, their contribution to in vivo AMPA receptor function is less clear.By generating mice lacking multiple TARP family members, we found that TARPs are functionally redundant. Single TARP knockout mice are viable, while those lacking multiple isoforms are often lethal. Consistent with molecular redundancy, AMPA receptor transmission in cerebellar Golgi cells is unaffected in single TARP knockout mice, but nearly eliminated in double knockouts. Unexpectedly, the remaining AMPA receptors have a different subunit composition, suggesting that TARPs may preferentially traffic GluR2 containing receptors.Our studies also highlight a role for TARPs in inhibitory neurons because AMPA receptor function in cerebellar Purkinje cells and Golgi cells is reduced in TARP knockout mice. Additionally, the loss of TARPs reduces the decay time of interneuron EPSCs. This indicates that AMPA receptor biophysical properties make a significant contribution to the time course of synaptic events, even at fast-decaying interneuron synapses.Our studies investigating TARP function in vivo also led to the discovery that TARPs profoundly change AMPA receptor pharmacology. Whereas CNQX is a competitive antagonist on AMPA receptors alone, it is a partial agonist on receptors containing any member of the TARP family and elicits depolarizing currents in neurons throughout the brain. By obtaining the crystal structure of CNQX bound to the AMPA receptor ligand-binding domain, we determined that CNQX induces a small amount of domain closure. Based on our findings, we propose an expanded model of AMPA receptor gating such that TARPs increase the likelihood that domain closure leads to channel opening, i.e. they increase the coupling efficiency. Together our studies demonstrate that TARPs are an integral component of AMPA receptors in the central nervous system

Molecular Profiling of the Drosophila Antenna Reveals Conserved Genes Underlying Olfaction in Insects

Repellent odors are widely used to prevent insect-borne diseases, making it imperative to identify the conserved molecular underpinnings of their olfactory systems. Currently, little is known about the molecules supporting odor signaling beyond the odor receptors themselves. Most known molecules function in one of two classes of olfactory sensilla, single-walled or double-walled, which have differing morphology and odor response profiles. Here, we took two approaches to discover novel genes that contribute to insect olfaction in the periphery. We transcriptionally profiled Drosophila melanogaster amos mutants that lack trichoid and basiconic sensilla, the single-walled sensilla in this species. This revealed 187 genes whose expression is enriched in these sensilla, including pickpocket ion channels and neuromodulator GPCRs that could mediate signaling pathways unique to single-walled sensilla. For our second approach, we computationally identified 141 antennal-enriched (AE) genes that are more than ten times as abundant in D. melanogaster antennae as in other tissues or whole-body extracts, and are thus likely to play a role in olfaction. We identified unambiguous orthologs of AE genes in the genomes of four distantly related insect species, and most identified orthologs were expressed in the antenna of these species. Further analysis revealed that nearly half of the 141 AE genes are localized specifically to either single or double-walled sensilla. Functional annotation suggests the AE genes include signaling molecules and enzymes that could be involved in odorant degradation. Together, these two resources provide a foundation for future studies investigating conserved mechanisms of odor signaling

Non-synaptic inhibition between grouped neurons in an olfactory circuit.

Diverse sensory organs, including mammalian taste buds and insect chemosensory sensilla, show a marked 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 neighbouring ORN. Mechanistically, such lateral inhibition does not depend on synapses and is probably mediated by ephaptic coupling. Moreover, lateral inhibition in the periphery can modulate olfactory behaviour. 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 CO(2)-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

An RNA-Seq Screen of the <i>Drosophila</i> Antenna Identifies a Transporter Necessary for Ammonia Detection

<div><p>Many insect vectors of disease detect their hosts through olfactory cues, and thus it is of great interest to understand better how odors are encoded. However, little is known about the molecular underpinnings that support the unique function of coeloconic sensilla, an ancient and conserved class of sensilla that detect amines and acids, including components of human odor that are cues for many insect vectors. Here, we generate antennal transcriptome databases both for wild type <i>Drosophila</i> and for a mutant that lacks coeloconic sensilla. We use these resources to identify genes whose expression is highly enriched in coeloconic sensilla, including many genes not previously implicated in olfaction. Among them, we identify an ammonium transporter gene that is essential for ammonia responses in a class of coeloconic olfactory receptor neurons (ORNs), but is not required for responses to other odorants. Surprisingly, the transporter is not expressed in ORNs, but rather in neighboring auxiliary cells. Thus, our data reveal an unexpected non-cell autonomous role for a component that is essential to the olfactory response to ammonia. The defective response observed in a <i>Drosophila</i> mutant of this gene is rescued by its <i>Anopheles</i> ortholog, and orthologs are found in virtually all insect species examined, suggesting that its role is conserved. Taken together, our results provide a quantitative analysis of gene expression in the primary olfactory organ of <i>Drosophila</i>, identify molecular components of an ancient class of olfactory sensilla, and reveal that auxiliary cells, and not simply ORNs, play an essential role in the coding of an odor that is a critical host cue for many insect vectors of human disease.</p></div

The loss of ammonia response localizes to the <i>Amt</i> gene.

<p>(A) Traces on the left show that the responses to a 500 ms pulse of 0.1% ammonia are similar in flies heterozygous for the <i>Amt¹</i> transposon and the <i>Df(3R)BSC471</i> deficiency that removes ∼30 kb including <i>Amt</i> and nine other genes. In contrast, <i>Amt¹</i>/<i>Df(3R)BSC471</i> flies have greatly reduced responses to ammonia (n = 9 each, p<0.0001). Averaged data are shown in the graph on the right. (B) The lack of response to 0.1% ammonia in ac1 sensilla from <i>Amt¹</i> flies is rescued by the addition of a genomic fragment containing <i>Amt</i> and the neighboring gene <i>Hsc70-4</i> (n = 8 each, p<0.0001). (C) The response to ammonia in <i>Amt¹</i> mutant flies is also rescued by transgenic expression of <i>UAS-Amt</i> under the control of an <i>Amt-Gal4</i> promoter (n = 9 each, p<0.0001).</p

Chemosensory gene expression in the wild-type antennal third segment.

<p>(A) Third antennal segment (arrowhead) on a <i>Drosophila</i> head. (B) Scanning electron micrograph of the antennal surface with a coeloconic sensillum (C), trichoid sensillum (T) and small basiconic sensillum (SB) labeled. (C) Diagram of a generic sensillum containing an olfactory receptor neuron (ORN) whose dendrite is surrounded by sensillar lymph. The sensillum and antennal surface is covered with a cuticle, and pores in the sensillum cuticle permit airborne odors to enter the sensillum lymph. The sensillum is separated from other sensilla by the epithelium. Auxiliary cells, including tormogen (To), trichogen (Tr) and thecogen (Th) cells, surround the ORN. (D) Members of the <i>Or</i> and (E) <i>IR</i> olfactory receptor gene families detected in the Canton-S (CS) third antennal segment with at least 1 read per million mapped reads (RPM) in each of three samples. Genes are listed by decreasing reads per million mapped reads per kilobase of gene length (RPKM). Two <i>IR</i> genes, <i>IR75b</i> and <i>IR75c,</i> were annotated as a single gene, <i>CG14586,</i> at the time of the gene mapping (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004810#pgen.1004810.s010" target="_blank">Dataset S1</a>), and are therefore represented by a single bar in the graph. (F) <i>Gr</i> genes. (G) <i>Obp</i> genes. (H) <i>Trp</i> family genes. (A) is from <a href="http://cedar.bio.indiana.edu/~ggrumbli/highrespackage" target="_blank">http://cedar.bio.indiana.edu/~ggrumbli/highrespackage</a>, (B) is from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004810#pgen.1004810-Shanbhag1" target="_blank">[3]</a>, and (C) is adapted from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004810#pgen.1004810-Gu1" target="_blank">[116]</a>.</p

The <i>Amt¹</i> defect is restricted to the ammonia response of ac1 sensilla.

<p>(A) Odor responses of ac1 sensilla in <i>Amt¹</i>. Ammonia, 2-oxovaleric acid, and pyrrolidine each activate one of the three ORNs in ac1; only the ammonia response was impaired (n = 10–11, p<0.0001). (B–D) Response profiles of other sensillar types appeared normal (n = 8–9 each).</p

Expression of <i>Amt.</i>

<p>(A) RT-PCR analysis of <i>Amt</i> expression in CS. <i>Synaptogmin</i> was used as a positive control. (B) Whole-mount confocal image of a third antennal segment of an <i>Amt-GAL4; UAS-mCD8::GFP</i> fly. GFP expression is seen in large, amorphous auxiliary cells, but not in neurons. White arrowheads indicate the sacculus here and in panels E and F. Scale bar  = 30 µm. (C) Confocal image of an <i>in situ</i> hybridization to an antennal section from an <i>Amt-GAL4; UAS-mCD8::GFP</i> fly using antisense probes for <i>Amt</i> (red) and <i>GFP</i> (green). The two probes co-localize. (D, E, G, H), confocal images of antennal sections labeled with an antisense probe for <i>Amt</i> (red) and an antibody against GFP (green) driven by (D) <i>IR76b-Gal4</i>, (E) <i>IR8a-Gal4</i>, (G) <i>IR92a-Gal4</i>, and (H) <i>IR76a-Gal4</i>. <i>IR76b-Gal4</i> and <i>IR8a-Gal4</i> are co-receptors that label at least one ORN in each surface coeloconic sensillum type (ac1–4). <i>IR8a-Gal4</i> also labels coeloconic ORNs in the third chamber of the sacculus. <i>Amt</i> is detected in larger neighboring auxiliary cells in a subset of the coeloconic sensilla. (F) Confocal image of an <i>in situ</i> hybridization to an antennal section from a CS fly using antisense probes for <i>Amt</i> (red) and <i>Obp84a</i> (green). <i>Amt</i> is expressed in different auxiliary cells from those that express <i>Obp84a</i>, which is also expressed in coeloconic sensilla. (G) Expression of <i>Amt</i> on the antennal surface is found surrounding the IR92a ammonia receptor-expressing neurons, which are in ac1. (H) <i>Amt</i> is not detected in ac4 sensilla, which contain ORNs that express <i>IR76a</i>. (I, J) Higher magnification images of (F) and (G) respectively.</p

Functional categorization of 250 genes depleted in <i>ato.</i>

<p>(A) The ten genes that show the most signification depletion in <i>ato</i>, based on p-value. Each is color-coded according to the categories in panel (B). (B) Categorization of the 250 genes that show at least four-fold reduction in <i>ato</i> with a FDR <0.01. Putative functions were determined by examination of automated gene annotation and BLAST searches to identify similar proteins. (C) Gene Ontology (GO) term analysis using the program AmiGO identified four level two terms that were significantly enriched among the 250 <i>ato</i>-depleted genes compared to all <i>D. melanogaster</i> genes listed in FlyBase. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004810#pgen.1004810.s007" target="_blank">Figure S7</a> for the complete analysis of all level two GO terms. (D) Significance of enrichment of lower level terms, which form a subset of higher level terms (<i>e.g.</i> “detection of stimulus” is a subset of “response to stimulus”). Lower level terms were selected to illustrate the types of functions most enriched in the dataset. The number of <i>ato</i>-depleted genes annotated with each GO term is indicated. We note that the ion channel genes depleted in <i>ato</i> include two members of the <i>ppk</i> family, <i>ppk25</i> and <i>ppk10</i>, two members of the <i>Trp</i> family, <i>nanchung</i> and <i>inactive</i>, and four potassium channel genes: <i>shaw-like (shawl), TWIK-related acid-sensitive K⁺ channel (Task7), Inwardly rectifying potassium channel 1 (Irk1)</i>, and <i>CG1756</i>. Depleted genes described by “transmembrane signaling receptor activity” included six GPCRs: <i>5-HT2, 5-HT7, CG43795, frizzled 3 (fz3), Pigment-dispersing factor receptor (Pdfr),</i> and <i>CG18208</i>.</p