Non-synaptic inhibition between grouped neurons in an olfactory circuit

Abstract

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.

Figure 1. Lateral inhibition of ORNs

a, An olfactory sensillum that houses two ORNs, A and B. Inset: a single-unit recording. “A” has a larger spike amplitude than “B”. b, The two-odor paradigm. c, The ab3 sensillum, whose ORNs express the Or22a and Or85b receptors, which are sensitive to the indicated odorants. d, Top: a sustained stimulus of methyl hexanoate (m-hex, 10⁻⁷ dilution, long blue bar) elicits a response from ab3A (large spikes, ~37 spikes/s). A 500-ms pulse of 2-heptanone (2-hep, 10⁻⁴ dilution, orange bar) activates ab3B (small spikes). The response of ab3A is inhibited by the 2-hep stimulus. Right, averaged responses. Grey traces indicate responses when a pulse of diluent is delivered instead of 2-hep. Shaded areas represent S.E.M.. Inset: blue dots indicate ab3A spikes. Bottom: genetic ablation of ab3B prevented inhibition. e, In flies expressing ChR2* in ab3B (top), a 500-ms pulse of blue light (473 nm, ~10mW/mm²) excited ab3B, which inhibited the response of ab3A to m-hex (~32 spikes/s, 10⁻⁶). The more phasic inhibition is likely due to the kinetics of ChR2-dependent activation. Bottom: flies without ChR2*. f, Top: activation of ab3A by a pulse of m-hex (10⁻⁶) inhibited the response of ab3B to 2-hep (~38 spikes/s, 5×10⁻⁷). Bottom: genetic ablation of ab3A prevented inhibition. Inset: orange dots indicate ab3B spikes. Very large spikes represent the coincidence of A and B spikes. g, ChR2* expressed in ab3A. A pulse of blue light (~25mW/mm²) excited ab3A, inhibiting the response of ab3B to 2-hep (~35 spikes/s, 5×10⁻⁷). n=12 in d–g.

Figure 2. Lateral inhibition in diverse sensilla

Odorants at the tested concentrations activate only one ORN in each sensillum. a–d, Drosophila sensilla. Activation of the target ORN (orange) inhibited the response of the neighboring ORN (blue) to the background odorant. In a, ab1A and ab1B spikes could not be sorted reliably and were grouped. e, In the capitate-peg sensillum of Anopheles, activation of the cpB neuron by 1-octen-3-ol (10⁻⁴) inhibited the response of cpA to CO₂. cpB and cpC spikes were combined. n=11~13. Odor dilutions and A neuron basal activities are in Table S2.

Figure 3. Lateral inhibition is dose-dependent

a, Responses of ab3A and ab3B to a 500-ms pulse of 2-heptanone (orange) superimposed on a background odorant, methyl hexanoate (10⁻⁷ dilution; ~37 spikes/s). At these low concentrations, methyl hexanoate and 2-heptanone selectively activate ab3A and ab3B, respectively. 2-heptanone dilutions are at right. b, Activities of ab3A and ab3B during 2-heptanone pulses. Fit is with the Hill equation. n=12. c, Responses to a pulse of 2-heptanone (10⁻⁴) in presence of varying levels of methyl hexanoate, indicated at right. d, Responses of ab3A during 500-ms exposures to paraffin oil (control) or 2-heptanone with varying concentrations of background methyl hexanoate. n=12. In the absence of sustained stimulation of the A neuron (“no bkg”), strong activation of the B neuron elicited a small increase in the firing of A, which may represent passive depolarization of A resulting from close apposition of the neuronal membranes^,. This effect appears to be overwhelmed by the passive hyperpolarization produced by ephaptic interactions (discussed below) when B is activated during sustained stimulation of A. Differences are significant in all conditions (p<0.002, paired t-test). n=12.

Figure 4. Lateral inhibition does not require synapses

a, ab3 sensilla in flies expressing TNT in ORNs via the Orco promoter . Neurons were exposed to a 500-ms pulse of the ab3B odorant, 2-heptanone (orange, 10⁻⁴), superimposed on the background ab3A odorant, methyl hexanoate (blue, 10⁻⁷). IN: representative interneuron. Right: ab3A activity during a 500-ms exposure to paraffin oil (PO) or 2-heptanone in the presence of methyl hexanoate. Error bars= S.E.M. *, p<0.05, one-way repeated measures ANOVA, multiple comparison versus control group (PO) with Dunnett’s method (n=12). b, T-maze choice between water and 25% ACV or between air and 0.67% CO₂. CO₂ neurons do not express Or genes. (n=9). c, Severed antennae (n=7). d, Cross-correlation analysis of spontaneous spikes from an ab3 sensillum, showing intervals between ab3A spikes and ab3B spikes, binned in 10 ms increments. Each ab3B spike is used as a reference. Another ab3 sensillum gave similar results. e, Recordings made 15 min after introduction of Cd²⁺ (n=12). f, VUAA1 (1 mM) or vehicle (1% DMSO) was delivered via the recording electrode. ab3A responses were recorded for 10 s. *** p<0.001, t-test (n=12).

Figure 5. Lateral inhibition modulates behavior

Activation of ab1A mediates attraction to ACV; activation of ab1C mediates aversion to CO₂. Two of the four ORNs in ab1 are depicted. Preference indices of control (a), Orco-GAL4; UAS-TNT (b), and Orco (c) are shown (mean ± S.E.M.). The ab1A neurons of the TNT-expressing flies respond to ACV but are expected not to transmit information to post-synaptic neurons, whereas ab1A neurons in Orco are expected not to respond to ACV. In each T-maze assay, ~50 flies were allowed two min to choose. In single-odor experiments (black bars) the test arm contained either CO₂ or ACV. *, p<0.05; ***, p <0.001, t-test (n=16). CO₂ was 0.13%; ACV was 100%, pH 7.5. In physiological recordings from Orco flies, ACV did not inhibit the spontaneous firing of the CO₂ neuron.