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. 2019 Dec 13;366(6471):1384-1389.
doi: 10.1126/science.aax8055.

Sensory coding mechanisms revealed by optical tagging of physiologically defined neuronal types

Donghoon Lee  1 , Maiko Kume  1 , Timothy E Holy  2
Affiliations

Sensory coding mechanisms revealed by optical tagging of physiologically defined neuronal types

Donghoon Lee et al. Science. .

Abstract

Neural circuit analysis relies on having molecular markers for specific cell types. However, for a cell type identified only by its circuit function, the process of identifying markers remains laborious. We developed physiological optical tagging sequencing (PhOTseq), a technique for tagging and expression profiling of cells on the basis of their functional properties. PhOTseq was capable of selecting rare cell types and enriching them by nearly 100-fold. We applied PhOTseq to the challenge of mapping receptor-ligand pairings among pheromone-sensing neurons in mice. Together with in vivo ectopic expression of vomeronasal chemoreceptors, PhOTseq identified the complete combinatorial receptor code for a specific set of ligands.

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Figures

Fig. 1.
Fig. 1.. Two-photon photoactivation can tag neurons chosen by activity pattern.
(A) The work flow of physiological optical tagging sequencing (PhOTseq). (B–D) Two-photon calcium imaging of the VNE explanted from a GCaMP5g-p2a-PAmCherry transgenic mouse. (B) Representative images of calcium chemosensory response evoked by either 10 μM A7864 (top left) or 10 μM E1050 (top right). Photoactivation mask (bottom) selects cells responsive to both stimuli. Scale bar: 20 μm. (C) The average GCaMP intensity obtained from masked cells. Black bars: delivery time-course of stimuli. (D) Individual responses (ΔF/F) among masked cells to 10 μM A7864 and 10 μM E1050. (E) Confocal imaging after online two-photon photoactivation. PAmCherry signals (left), PAmCherry signals with two-photon PA mask (middle), and PAmCherry signals with calcium responses to either 10 μM A7864 (cyan) or 10 μM E1050 (green) (right). Red and yellow arrow heads: the examples of PA mask regions associated with single and double PAmCherry-positive neurons, respectively. Scale bar: 20 μm. (F and G) FACS analysis of cells from non-photoactivated tissue (F) or from photoactivated tissue (G). Clusters were marked as P1, P2, P3, P4, and P5. P1 and P2 represent a photoactivated and an experimental control group, respectively. Of the total population, P1 and P2 respectively accounted for 0.013% and 33.9% in (F) or 0.218% and 37.3% in (G).
Fig. 2.
Fig. 2.. PhOTseq identified of VR genes of overlapping cell types.
(A) Example calcium traces (normalized ΔF) from two cells. Amplitude was normalized by the maximum amplitude of all cells recorded. Ligands are listed at the top. (B) Neuronal responses to sulfated steroids. Cells are on columns, stimuli on rows. If not indicated, the ligand concentration is 10 μM. The color bar indicates normalized response. Cluster identities are reported at the top. “*” marks PhOTseq target cell types. “(*)” marks the functional type whose receptor identity was discovered during analysis by ectopic expression in Figure 3. (C) Expression of the 30 most highly expressed VR genes when averaged across all sequenced cells; also shown are three marker genes (Omp, Gnai2, and Gnao1). Cells are on columns, genes on rows. PhOTseq-targeted functional types are shown at the top; cells belonging to these functional clusters were specifically photoactivated and sequenced. The colorbar indicates log-normalized expression level. (D) The proportion of a VSN type in each group shown in (C). Each tick on the horizontal axis represents a different VR gene. Each functional type exhibited only one common VSN type. (E) The expression of five VR genes across different experiment groups. “P” indicates photoactivated cells and “C” indicates non-photoactivated control cells. Asterisk (*) indicates padj < 0.01 (Wilcoxon rank-sum test; padj< 0.01 (Wilcoxon rank-sum test; padj: 2.7×10−11, 2.4×10−10, 1.1×10−20, 1.3×10−40, 2.7×10−30; average fold difference: 6.2, 23.8, 80.7, 75.7, 45.9 for Vmn1r89, Vmn1r86, Vmn1r78, Vmn1r237, Vmn1r58, respectively). (F) Single-cell expression of Vmn1r85 and Vmn1r86 is non-exclusive.
Fig. 3.
Fig. 3.. Ectopic expression enabled functional analysis of vomeronasal receptors.
(A) Expression via AAV injection in the temporal vein of newborn pups. (B) Optical section from light sheet calcium imaging after the ectopic expression of GCaMP-2A-Vmn1r237 in response to different ligands. The apical layer is occupied by dendritic tips (Den) with cell bodies (CB) below. Scale bar: 20 μm (C) Calcium response after ectopic expression of GCaMP-2A-Vmn1r89, -Vmn1r86, -Vmn1r78, -Vmn1r237, -Vmn1r58, or -Vmn1r85. Ligands are identical to those in Figure 2B. (D) Pairwise correlation between the reference clustering and the ectopic responses shown in (C) (left) or autocorrelogram of the reference clustering (right).
Fig. 4.
Fig. 4.. Sequence- and chemoreceptive-similarity are strongly correlated.
(A) An unrooted phylogenetic tree of V1R genes. The five functionally similar (blue) and one distant (red) receptors studied here are marked. The scale bar indicates the number of amino acid substitutions per site. Numbers indicate bootstrap values. (B) Pairwise distances among 204 V1R protein sequences (see Methods). (C) For these deorphanized VRs, the top 20 nearest VRs, based on (B), are rank-ordered. The deorphanized VRs are colored if shown in the table. (D) A 2-dimensional representation of the distance matrix shown in (B). Each dot represents a single VR protein.
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Comment in

  • Cell types exposed by social scent.
    Renninger SL. Renninger SL. Science. 2019 Dec 13;366(6471):1311-1312. doi: 10.1126/science.aaz8969. Science. 2019. PMID: 31831656 No abstract available.

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