A mouse DRG genetic toolkit reveals morphological and physiological diversity of somatosensory neuron subtypes

Abstract

Dorsal root ganglia (DRG) somatosensory neurons detect mechanical, thermal, and chemical stimuli acting on the body. Achieving a holistic view of how different DRG neuron subtypes relay neural signals from the periphery to the CNS has been challenging with existing tools. Here, we develop and curate a mouse genetic toolkit that allows for interrogating the properties and functions of distinct cutaneous targeting DRG neuron subtypes. These tools have enabled a broad morphological analysis, which revealed distinct cutaneous axon arborization areas and branching patterns of the transcriptionally distinct DRG neuron subtypes. Moreover, in vivo physiological analysis revealed that each subtype has a distinct threshold and range of responses to mechanical and/or thermal stimuli. These findings support a model in which morphologically and physiologically distinct cutaneous DRG sensory neuron subtypes tile mechanical and thermal stimulus space to collectively encode a wide range of natural stimuli.

Keywords: dorsal root ganglion; genetic tools; mechanosensation; population code; somatosensation; sparse labeling; thermosensation; transcriptional heterogeneity.

Figure 1.. The transcriptional landscape of DRG sensory neurons informs the creation of tools for genetic access to neuronal subtypes.

(A) UMAP visualizations of DRG scRNA-seq data alongside putative sensory neuron subtype identities. (B) Violin plots displaying expression profiles of the marker genes used to generate mouse recombinase lines. TPT: tags per ten thousand. (C) Targeting constructs and validation for the genetic tools using in situ hybridization. Smr2^T2a-Cre mice were crossed to Rosa26^LSL-ReaChR mice; Trpm8^T2a-FlpO mice were crossed to Avil^Cre; Ai195 (TIGRE^{LSL-jGCaMP7s-FSF}) mice; the remainder were labeled using neonatal I.P. injections of an AAV carrying a Cre-dependent GFP reporter. Scale bar = 50 μm. (D) Summary of the specificity and efficiency of the genetic tools using the labeling strategies in (C). Specificity refers to the percentage of labeled cells expressing the corresponding marker among labeled cells; efficiency refers to the percentage of labeled cells with the marker among cells expressing the marker.

Figure 2.. Morphological diversity of genetically labeled DRG subtypes revealed by sparse labeling.

(A) Reconstructed examples of hairy skin whole mount AP staining of a single Aβ SAI-LTMR labeled using TrkC^CreER; an Aβ RA-LTMR labeled using Ret^CreER; an Aδ-LTMR labeled using TrkB^CreER and a C-LTMR labeled using TH^2A-CreER. All the driver lines in (A) are crossed to Brn3a^cKOAP. (B) Reconstructed examples of an Aβ field-LTMR labeled using TrkC^CreER, Brn3a^cKOAP and an CGRP-η neuron labeled using Bmpr1b^Cre (AAV-CAG-FLEX-PLAP injection into hairy skin). (C) Reconstructed examples of free-nerve endings of individual TRPM8⁺, CYSLTR2⁺, SSTR2⁺, MRGPRD⁺, MRGPRB4⁺ and MRGPRA3⁺ neurons. See Methods for sparse labeling approaches. (D) Reconstructed examples of free nerve endings of an SMR2⁺ neuron. (E) Summary of anatomical receptive field sizes of genetically labeled DRG neuron subtypes. The dashed line separates the hair follicle-associated endings and the “free-nerve” endings. The scale of the Y axis is log2. N = 17, 35, 63, 90, 125, 54, 7, 40, 95, 53, 18, 84, 76 (from left to right). Images of each subtype were obtained from more than 3 animals, 4–5 weeks old. (F) Summary of number of hair follicles innervated by individual neurons of different subtypes. N = 7, 37, 45, 89, 86, 50, left to right. (G) Summary of branching density of free nerve ending neurons. N = 7, 22, 12, 15, 6, 5, from left to right. All scale bars 500 μm (A-D). The data for Aβ SAI-LTMRs, Aβ RA-LTMRs, and Aβ field-LTMRs are replotted or reconstructed from Bai et al..

Figure 3.. Characterization of the spinal cord terminals of genetically labeled cutaneous DRG subtypes.

(A) Schematics of central projections of DRG neurons. The red frame refers to the region of interest shown in (B). (B) Representative images of central terminals in lumbar spinal cord of genetically labeled cutaneous DRG subtypes, co-stained with CGRP and IB4. The right column shows quantification of the depth of spinal cord terminals relative to CGRP and IB4 signal. Trpm8^T2a-FlpO were crossed to Avil^CreER; Ai65 mice; TrkB^CreER and Ret^CreER were crossed to Advillin^FlpO; Ai65 mice; the remainder were labeled using neonatal I.P. injections of an AAV carrying a Cre-dependent GFP reporter. All images are of the same scale. Scale bar is 50 μm. Depth quantifications are from spinal sections from over three animals per subtype. (C) Comparison of axon terminal depth for the cutaneous DRG subtypes, relative to CGRP and IB4 labeled axons.

Figure 4.. Indentation force space is tiled by the different mechanical thresholds of DRG neuron subtypes.

(A) Left: Schematic of in vivo DRG calcium imaging and the indentation stimulus. Right: Representative field of view from Th^2A-CreER; Ai148 (baseline fluorescence, scale bar 50μm). To express GCaMP in distinct DRG subtypes, Bmpr1b^T2a-Cre mice were crossed to Ai95; MrgprA3^Cre mice were crossed to Ai96; Trpm8^T2a-FlpO mice were crossed to Avil^Cre; Ai195; other recombinase mouse lines were crossed to Ai148. (B) Representative calcium signals and threshold distributions for each DRG neuron subtype responding to 0.5-second step indentations. Left in each box: Traces from a total of three trials for the same example neuron (scale bars: 20% ΔF/F, Y axis; 10s, X axis. Right in each box: Number of traces with indicated threshold. (C) Summary of threshold distributions for each DRG neuron subtype across the range of indentation forces. The percentage of trials with a corresponding threshold are coded by brightness levels. Percentages for TRPM8⁺ and SSTR2⁺ neurons, which didn’t respond to indentation, were set to zero. (D) Summary of average response amplitudes for each DRG subtype across the range of indentation forces. The response amplitudes (ΔF/F) were normalized to responses at 75mN for each neuron and then averaged for each subtype. The normalized amplitudes for TRPM8⁺ and SSTR2⁺ neurons were set to zero, as in (C).

Figure 5.. Responses to diverse mechanical and thermal stimuli by distinct DRG neuron subtypes.

(A) Heatmaps of calcium signals under a range of mechanical and thermal stimuli. VF-6g, VF-10g and VF-26g refer to 6-gram, 10-gram and 26-gram von Frey filaments. VF-8g-Array refers to an array (5*5) of custom made 8-gram von Frey hair covering the same area as the Peltier device (See Methods). Baseline temperature: 32°C. Each row of the heatmap represents responses of an individual neuron. The vertical scale bars on the right refer to 10 neurons. The horizontal scale bar represents 20 seconds. (B-D) Summaries of response amplitudes (ΔF/F) for air puff (B), stroke (C), and pinch (D) stimuli, normalized to each neuron’s maximum responses to all stimuli. Neuron counts from left to right are 33, 91, 38, 98, 131, 71, 81, 81, 89, 58, and 128. Neurons of each subtype were imaged from 3–5 animals, age 4–6 weeks.

Figure 6.. Temperature is reported as absolute or relative by distinct sensory neuron subtypes.

(A) Summary of thermal thresholds for DRG neuron subtypes, with percentage at each threshold indicated by the brightness level. Aβ RA-LTMRs and Aδ-LTMRs were set to zero due to lack of thermal responses. (B) Summary of response amplitudes (ΔF/F) for DRG neuron subtypes, normalized to each neuron’s maximum response to all stimuli, and averaged within each subtype. (C) Diverse response profiles to cooling. C-LTMRs exhibited transient responses to temperature decrease. TRPM8⁺ neurons showed three types of responses: cooling-activated (top), cooling-activated and warmth-inhibited (middle), and cooling-and-heat activated (bottom). (D) Diverse response profiles to warmth or heat. MRGPRB4⁺, CYSLTR2⁺ and MRGPRA3⁺ neurons exhibited two types of responses: neurons responding to relative increase (top) and only to absolute temperature (bottom). SSTR2⁺ and MRGPRD⁺ neurons responded to absolute warmth/heat. (E) SMR2⁺ neurons exhibit responses to heat and/or cold. Scale bars in C-E: 20% ΔF/F (Y-axis), 10s (X-axis). Individual traces are shown in gray, and the average trace in blue. Dashed vertical lines mark temperature change. From C-E, neuron counts (plotted/total imaged) are provided in the right.

Figure 7.. Polymodality of DRG neuron subtypes.

(A-C) Distributions of tuning preferences for each DRG subtype. Each neuron is represented by a dot, positioned based on its maximum response magnitude to mechanical (M), heat (H), or cold (C) stimuli. Dot color shows relative preference for these modalities, with white dots near the center indicating tuning to all three. Subtypes exhibiting strong preference to mechanical stimuli are in (A), those with polymodal responses in (B), and those primarily responding to thermal stimuli in (C).