Postsynaptic odorant concentration dependent inhibition controls temporal properties of spike responses of projection neurons in the moth antennal lobe

Abstract

Although odorant concentration-response characteristics of olfactory neurons have been widely investigated in a variety of animal species, the effect of odorant concentration on neural processing at circuit level is still poorly understood. Using calcium imaging in the silkmoth (Bombyx mori) pheromone processing circuit of the antennal lobe (AL), we studied the effect of odorant concentration on second-order projection neuron (PN) responses. While PN calcium responses of dendrites showed monotonic increases with odorant concentration, calcium responses of somata showed decreased responses at higher odorant concentrations due to postsynaptic inhibition. Simultaneous calcium imaging and electrophysiology revealed that calcium responses of PN somata but not dendrites reflect spiking activity. Inhibition shortened spike response duration rather than decreasing peak instantaneous spike frequency (ISF). Local interneurons (LNs) that were specifically activated at high odorant concentrations at which PN responses were suppressed are the putative source of inhibition. Our results imply the existence of an intraglomerular mechanism that preserves time resolution in olfactory processing over a wide odorant concentration range.

Figure 1. Different concentration-response characteristics in dendrites and somata of antennal lobe (AL) projection neurons (PNs).

A, Schematic diagram of loading a calcium indicator into PNs with a micropipette by local electroporation (left). The toroid glomerulus processing bombykol is delineated by a dashed line. Fluorescence images of labeled PNs (middle) and the response to 1000 ng bombykol in false colors (right) are shown. Dendritic and somatic regions of interest (ROIs) are indicated by boxes. D: dorsal, M: medial. Scale bar: 50 µm. B, Representative time courses of PN responses to bombykol stimuli in the dendrites (left) and a soma (right). Black bars under time courses indicate the stimulus. C, Concentration-response characteristics of PN dendrites (magenta) and somata (green). Calcium responses were integrated over 3 s from stimulus onset. (P<0.05 for significant differences indicated by different letters associated with the data groups shown as means±SEM, n = 6 for dendrites and n = 17 for somata, one-way repeated measures ANOVA followed by Tukey-Kramer test).

Figure 2. Relation between calcium and spike responses in PNs.

A, Fluorescence images of PNs labeled with calcium indicator (top) and the response to 1000 ng bombykol in false colors (bottom). Spike responses were recorded from the labeled soma indicated by the arrowhead. The microelectrode for loose-patch recording is delineated by dashed lines and the ROI in the dendritic region is marked. D: dorsal, M: medial. Scale bars: 50 µm. B, Representative PN spike responses to bombykol stimuli. The responses were recorded from the soma shown in (A). Black bar under the spike responses indicates stimulus. C, Concentration-response characteristics of PN spike responses. The response amplitudes were quantified using peak instantaneous spike frequency (peak ISF, left) and spike response duration (right). (P<0.05 for significant differences indicated by different letters associated with the data groups shown as means±SEM, n = 6, one-way repeated measures ANOVA followed by Tukey-Kramer test). D, Representative time courses of convoluted PN spike responses (left) and simultaneously acquired calcium responses in the soma (middle) and the dendrites (right). Same sample as shown in (A) and (B). E, Time courses of correlation coefficients between PN spike convolutions and calcium responses in the dendrites (magenta), and between the spike convolutions and calcium responses of the somata (green) (*: P<0.05 between both correlation coefficients at each time point, n = 6, Wilcoxon signed-rank test, data shown as means±SEM). For the first 200 ms following stimulus onset, correlation coefficients are not available because spike responses had a longer latency for all stimulus concentrations.

Figure 3. Concentration-response characteristics of PNs under picrotoxin (PTX) treatment.

A, Representative time courses of PN responses to bombykol stimuli in the dendrites (left) and a soma (right) under PTX treatment. Black bars under time courses indicate stimulus. B, Concentration-response characteristics in PN dendrites (left) and somata (right) before (black) and under PTX treatment (magenta). Calcium responses were integrated over 3 s following stimulus onset (P<0.05 for significant differences indicated by different letters associated with the data groups shown as means±SEM, n = 6 at dendrites and n = 17 at somata, one-way repeated measures ANOVA followed by Tukey-Kramer test).

Figure 4. Calcium responses of local interneurons (LNs) to bombykol stimuli.

A, Schematic diagram of loading the calcium indicator into LNs with a micropipette by local electroporation. The toroid glomerulus is delineated by a dashed line. B, Two-dimensional projection of confocal sections (left, extending over 180 µm from the AL surface) and a single confocal optical section (middle) of labeled LNs. Scale bars: 100 µm. The arrowhead in the projection image indicates the injection site. LN branches innervating the MGC are resolved in the optical section. Stack projection images of the medial cell cluster (MC, right, top, extending over 60 µm in depth) and the lateral cell cluster (LC, right, bottom, extending over 60 µm in depth) are enlarged. Brightness and contrast of the images were adjusted at the same level for both cell clusters. The outlines of both cell clusters are delineated by dashed lines. Scale bars: 10 µm. C, Representative calcium response of LNs to 5000 ng bombykol in false colors. D, Representative time courses of LN calcium responses, same sample as in (C). Black bar under time courses indicates stimulus. E, Concentration-response characteristics of LNs in MGC (magenta) and in the OGR (green). Calcium responses were integrated over 3 s following stimulus onset (*: P<0.05, between both regions at given concentration, n = 7, Wilcoxon signed-rank test, means±SEM). D: dorsal, M: medial, MGC: macroglomerular complex, OGR: ordinary glomerular region. The outline of the AL is delineated by dashed lines.