Quantitative differences in neuronal subpopulations between mouse and human dorsal root ganglia demonstrated with RNAscope in situ hybridization

Abstract

Next-generation transcriptomics in combination with imaging-based approaches have emerged as powerful tools for the characterization of dorsal root ganglion (DRG) neuronal subpopulations. The mouse DRG has been well characterized by many independently conducted studies with convergent findings, but few studies have directly compared expression of population markers between mouse and human. This is important because of our increasing reliance on the mouse as a preclinical model for translational studies. Although calcitonin gene-related peptide (CGRP) and P2X purinergic ion channel type 3 receptor (P2X3R) have been used to define peptidergic and nonpeptidergic nociceptor subpopulations, respectively, in mouse DRG, these populations may be different in other species. To directly test this, as well as a host of other markers, we used multiplex RNAscope in situ hybridization to elucidate the distribution of a multitude of unique and classic neuronal mRNAs in peptidergic (CGRP-expressing) and nonpeptidergic (P2X3R-expressing) nociceptor subpopulations in mouse and human DRG. We found a large overlapping CGRP and P2X3R neuronal subpopulation in human, lumbar DRG that was not present in mouse. We also found differential expression in a variety of mRNAs for transient receptor potential channels, cholinergic receptors, potassium channels, sodium channels, and other markers/targets. These data offer insights into the spatial and functional organization of neuronal cell subpopulations in the rodent and human DRG and support the idea that sensory system organizational principles are likely different between both species.

Figure 1.. Distribution of peptidergic (CALCA; CGRP) and non-peptidergic (P2RX3; P2X3R) populations in mouse and human DRG.

A) Representative 20x images of mouse and human DRG labeled with RNAscope in situ hybridization for CALCA (green) and P2RX3 (blue) mRNA. Mouse and human DRG were costained for NF200 protein (purple) and DAPI (purple), respectively. B) Pie chart representation of CALCA and P2RX3 subpopulations in mouse and C) human DRG. D) The total number of CALCA-expressing neurons (all CALCA, and CALCA/P2RX3 neurons) was significantly increased in human DRG compared to mouse, but the population of P2RX3-expressing neurons (all P2RX3 and CALCA/P2RX3 neurons) was unchanged. The percentage of CALCA/P2RX3 co-expressing neurons was significantly increased in human DRG compared to mouse. E) Histogram with Gaussian distribution displaying the diameters of CALCA, P2RX3, and CALCA/P2RX3 co-expressing neurons in mouse and F) human DRG. G) Sensory neuron subpopulations were further divided in mouse DRG based on expression with Neurofilament-200 (NF200). H) Histogram with Gaussian distribution displaying the size distribution of Calca, P2rx3, and NF200 sensory neuron subpopulations in mouse DRG. Two way ANOVA with Bonferroni ****p<0.0001. Scale bar = 50 μm.

Figure 2.. Distribution of SCN10A (Nav1.8) mRNA in human DRG.

A) Representative 20x images of human DRG labeled with RNAscope in situ hybridization for CALCA (green) and P2RX3 (blue), SCN10A (red), and DAPI (purple). B) Pie chart representation of all SCN10A-positive sensory neuron subpopulations in human DRG. SCN10A was expressed exclusively in neurons that co-expressed CALCA and P2RX3. C) SCN10A was expressed in 71% of all sensory neurons in human DRG. D) Histogram with Gaussian distribution displaying the size profile of all SCN10A-positive neurons in human DRG. Scale bar = 50 μm.

Figure 3.. Distribution of SCN9A mRNA and Nav1.7 protein in human DRG.

A) Representative 20x images of human DRG labeled with RNAscope in situ hybridization for CALCA (green) and P2RX3 (blue), SCN9A (red), and DAPI (purple). B) Pie chart representation of all Scn9a-positive sensory neuron subpopulations in human DRG. C) SCN9A was expressed in 96% of sensory neurons in human DRG. D) Histogram with Gaussian distribution displaying the size profile of all SCN9A -positive neurons in human DRG. E) Representative 20x images of Nav1.7 (red) protein staining using a knockout-validated monoclonal antibody. The negative control was imaged at the same settings (no primary with secondary antibody). The negative control shows background lipofuscin (large globular structures) and myelin autofluorescence. F) Nav1.7 protein was expressed in 90.5% of sensory neurons in human DRG. G) Histogram with Gaussian distribution displaying the size profile of all Nav1.7 protein-positive neurons in human DRG. 20x scale bar = 50 μm.

Figure 4.. Distribution of KCNS1 (Kv9.1) mRNA in mouse and human DRG.

A) Representative 20x images of mouse and human DRG labeled with RNAscope in situ hybridization for CALCA (green), P2RX3 (blue), and KCNS1 (red) mRNA. Mouse and human DRG were costained for NF200 protein (purple) and DAPI (purple), respectively. B) Representative 100x overlay image showing CALCA (green), P2RX3 (blue), KCNS1 (red) and DAPI (purple) signal in human DRG. White arrow points toward a KCNS1-positive neuron. C) Pie chart representation of all KCNS1-positive sensory neuron subpopulations in mouse and D) human DRG. E) KCNS1 was expressed in 55.5% of sensory neurons in mouse DRG and 42.4% in human DRG. F) Histogram with Gaussian distribution displaying the size profile of all KCNS1-positive neurons in mouse and G) human DRG. Unpaired t-test, p>0.5. 20x scale bar = 50 μm. 100x scale bar = 10 μm.

Figure 5.. Distribution of HCN1 mRNA in mouse and human DRG.

A) Representative 20x images of mouse and human DRG labeled with RNAscope in situ hybridization for CALCA (green), P2RX3 (blue), and HCN1 (red) mRNA. Mouse and human DRG were costained for NF200 protein (purple) and DAPI (purple), respectively. B) Representative 100x overlay image showing CALCA (green), P2RX3 (blue), HCN1 (red) and DAPI (purple) signal in human DRG. White arrows point toward HCN1-positive neurons. C) Pie chart representation of all HCN1-positive sensory neuron subpopulations in mouse and D) human DRG. E) HCN1 was expressed in 70.9% of sensory neurons in mouse DRG and 94.4% in human DRG. F) Histogram with Gaussian distribution displaying the size profile of all HCN1-positive neurons in mouse and G) human DRG. 20x scale bar = 50 μm. 100x scale bar = 10 μm.

Figure 6.. Distribution of CHRNA9 mRNA in mouse and human DRG.

A) Representative 20x images of mouse and human DRG labeled with RNAscope in situ hybridization for CALCA (green), P2RX3 (blue), and CHRNA9 (red) mRNA. Mouse and human DRG were costained for NF200 protein (purple) and DAPI (purple), respectively. B) Representative 100x overlay image showing CALCA (green), P2RX3 (blue), CHRNA9 (red) and DAPI (purple) signal in human DRG. White arrows point toward CHRNA9-positive neurons. C) Pie chart representation of all CHRNA9-positive sensory neuron subpopulations in mouse and human DRG. D) CHRNA9 mRNA was not detected in mouse DRG but was expressed in 25.6% of sensory neurons in human DRG. E) Histogram with Gaussian distribution displaying the size profile of all CHRNA 9-positive neurons in human DRG. 20x scale bar = 50 μm. 100x scale bar = 10 μm.

Figure 7.. Distribution of TRPV1 mRNA in mouse and human DRG.

A) Representative 20x images of mouse and human DRG labeled with RNAscope in situ hybridization for CALCA (green), P2RX3 (blue), and TRPV1 (red) mRNA. Mouse and human DRG were costained for NF200 protein (purple) and DAPI (purple), respectively. B) Representative 100x overlay image showing CALCA (green), P2RX3 (blue), TRPV1 (red) and DAPI (purple) signal in human DRG. White arrows point toward Trpv1-positive neurons. C) Pie chart representation of all TRPV1-positive sensory neuron subpopulations in mouse and D) human DRG. E) TRPV1 was expressed in significantly more neurons in human DRG (74.7%) than in mouse DRG (32.4%). F) Histogram with Gaussian distribution displaying the size profile of all TRPV1-positive neurons in mouse and G) human DRG. Unpaired t-test, **p<0.01. 20x scale bar = 50 μm. 100x scale bar = 10 μm.

Figure 8.. Distribution of TRPA1 mRNA in mouse and human DRG.

A) Representative 20x images of mouse and human DRG labeled with RNAscope in situ hybridization for CALCA (green), P2RX3 (blue), and TRPA1 (red) mRNA. Human DRG was costained for DAPI (purple). B) Representative 40x overlay image showing CALCA (green), P2RX3 (blue), Trpa1 (red) and DAPI (purple) signal in human DRG. White arrows point toward TRPA1-positive neurons. C) Pie chart representation of all TRPA1-positive sensory neuron subpopulations in mouse and D) human DRG. E) TRPA1 was expressed in significantly more neurons in mouse DRG (55.2%) than in human DRG (16.3%). F) Histogram with Gaussian distribution displaying the size profile of all TRPA1-positive neurons in mouse and G) human DRG. Unpaired t-test, ***p<0.001. 20x scale bar = 50 μm. 40x scale bar = 20 μm.