Sensory Adaptation to Chemical Cues by Vomeronasal Sensory Neurons

Evaluating sensory adaptation to monomolecular AOS ligands with population VSN Ca²⁺ imaging. A, VNO imaging setup using OCPI (light sheet) microscopy, which enables volumetric imaging of thousands of VSNs in the intact vomeronasal epithelium. B, Experimental design for accelerating protocol of stimulus acquisition. Two monomolecular ligands, DCA and CA, were applied in blocks of six trials (three each for DCA and CA) with the noted recovery periods within each block. C, D, Example images taken from a single frame of a 51-frame z-stack showing the responses of VSNs to repeated stimulation with 10 µM DCA in “Block 1” (180 s recovery, C) and “Block 5” (60 s recovery, D). Arrowheads mark the position of a DCA-responsive cell. Scale bar: 100 µm. Symbols refer to derivative images in E–G and traces shown in H. E–G, Normalized change in fluorescence (ΔF/F) of VSNs on trial 1 of 3 (left) and trial 3 of 3 (right) in Block 1 (180 s recovery, E), Block 5 (60 s recovery, F), and Block 7 (15 s recovery, G). Arrowheads mark the same cell as in C, D. H, Across-trial responses of a DCA-selective cell (top, same as the cell shown in C–G), a DCA- and CA-responsive cell (middle), and a CA-selective cell (bottom). Symbols refer to the responses noted in C–G.

VSN responses to monomolecular bile acid ligands in the accelerating intertrial interval paradigm. A, Clustering of VSN responses to 10 µM bile acids reveals three populations: those that respond to DCA-only, CA and DCA (CA + DCA), and CA-only. Each column represents ΔF/F for a single VSN across six trials of each stimulus (all trials from blocks 1 and 2). Includes responses from 486 VSNs across four tissues (four animals). Two experiments presented DCA first and two presented CA first. B, Normalized response patterns of clustered VSNs across all blocks/trials. Shown are VSNs from two experiments in which DCA was presented first. Each row is a VSN and each column a single trial. Blocks are noted by vertical ticks. C, Adaptation across and within each stimulus block, arranged by cluster. Box plots indicate the median (dark line) and 25–75% interquartile range of the per-VSN normalized response magnitude of the first trial (t1) of each block. Solid black lines/symbols indicate the degree of intrablock adaptation ([t3-t1]/[t1+offset]). D, Reduction in cluster discriminability (d’) resulting from inter- and intratrial adaptation.

VSN responses to monomolecular bile acid ligands in the decelerating intertrial interval paradigm. A, Stimulus delivery design. B, Example ΔF/F images showing the responses of VSNs to repeated stimulation with 10 µM DCA and 10 µM CA in “Block 1” (15 s recovery, left) and “Block 7” (180 s recovery, right). White and red arrowheads mark VSNs in the CA+DCA and CA-only clusters, respectively. C, Normalized ΔF/F responses of clustered VSNs across blocks/trials. Shown are VSNs from three experiments in which DCA was presented first. Note that cross-adaptation is largely restricted to the CA+DCA cluster. D, Interblock and intrablock adaptation, arranged by cluster. E, In stark contrast to the accelerating protocol (gray line), discriminability between the DCA-only and CA-only clusters in the decelerating protocol (black line) remained high except in trials separated by 15 s.

Electrophysiological recordings of evoked firing activity from individual VSNs. A, Schematic representation of a VNO coronal slice and a patch pipette in preparation for electrophysiological recordings from a VSN. B, Representative recordings in the on-cell loose-patch configuration from a single VSN showing the firing activity in response to high K⁺ solution (top), diluted urine (1:50; middle), or diluted artificial urine (1:50; bottom). Time of stimulus presentation is indicated by the top bars. C, Raster plots of firing activity from the same VSN in B. The recovery time between individual stimuli was at least 2 min. D, Heat map showing the average spike frequency of the traces in C in bins of 1 s. The firing frequency increased in response to urine, or to high K⁺ solution as a positive control, but not in response to artificial urine.

Spike frequency adaptation to repeated mouse urine stimulation of VSNs. A, Representative loose-patch recordings from two individual VSNs in response to double-pulse stimulations with diluted urine (1:50). A 5 s urine pulse was delivered, followed by a second identical pulse with ISIs ranging from 5 to 60 s. The recovery time between each double-pulse experiment was at least 2 min. In black, a urine-responsive VSN did not fire in response to a second pulse of urine after 5 s, showed a reduced firing frequency to the second urine pulse at 10 s ISI, and a recovery at 60 s ISI. In pink, another VSN showing a smaller reduction in firing frequency at 5- and 10-s ISIs compared to the previous VSN. B, Heat map of normalized mean firing frequency from double-pulse urine stimulations at different ISIs for 14 VSNs. The mean frequency during the second urine pulse was normalized to the mean frequency evoked by the first pulse. VSNs shown in A are indicated by black and green circles. C, Box and scatter plots of the normalized frequency at different ISIs. Each dot represents an individual VSN at a given ISI. In the box plot horizontal lines represent the median, lower and upper box boundaries represent the first and third quartile, respectively, and upper and lower whiskers represent the 5th and 95th percentile. Statistics: Tukey–Nemenyi test after Friedman test: *p < 0.01.