Characterization of sensory neuronal subtypes innervating mouse tongue

Abstract

The tongue is uniquely exposed to water-soluble environmental chemicals that may lead to injury or tumorigenesis. However, comparatively little research has focused on the molecular and functional organization of trigeminal ganglia (TG) afferent neurons innervating the tongue. The current study identified and characterized lingual sensory neurons based on a neuronal subtype classification previously characterized in the dorsal root ganglion (DRG) neurons. We employed immunohistochemistry on transgenic reporter mouse lines as well as single-cell PCR of known markers of neuronal subtypes to characterize neuronal subtypes innervating the tongue. Markers expressed in retrogradely labeled TG neurons were evaluated for the proportion of neurons expressing each marker, intensity of expression, and overlapping genes. We found that tongue-innervating sensory neurons primarily expressed CGRP, TRPV1, TrkC, 5HT3A and Parvalbumin. These markers correspond to peptidergic and a subgroup of non-peptidergic C-nociceptors, peptidergic A nociceptors, proprioceptors and myelinated low-threshold mechanoreceptors (LTMRs). Interestingly, as reported previously, we also found several differences between TG and DRG neurons indicating the need for single-cell sequencing of neuronal types based on tissue type within all TG as well as DRG neurons.

Fig 1. Expression of subtype markers in lingual sensory neurons.

1% WGA-488 or WGA-568 was injected in tongues of mice bilaterally and 3 days later, TG tissues were harvested for immunostaining. Transgenic reporter mouse lines were used to determine the types of sensory neuron innervating mouse tongue. a. Number of Neurons expressing the marker and co-localizing with WGA were counted from 2–3 animals per group. Data are presented as mean +/- SEM of percentage of neurons expressing each marker. b. Each TG tissue sections from each retrogradely labeled transgenic mouse was stained with antibodies specific for either TRPV1, CGRP or Calbindin. Data are presented as WGA-labeled neurons expressing both markers. c. Representative immunostaining of TrkC-TDT, WGA and TRPV1, CGRP or Calbindin. d. Representative immunostaining of Parv-TDT, WGA and TRPV1, CGRP or Calbindin.E. Representative immunostaining of 5HT3A-GFP, WGA and TRPV1, CGRP or Calbindin. Arrows indicate colocalization of WGA and reporter gene.

Fig 2. Expression profile of lingual neurons with single-cell PCR.

Retrogradely labeled tongue-innervating TG neurons were manually picked to perform single-cell PCR of target genes. Gene expression was calculated for relative mRNA expression for each cell for each gene. a. Data are presented as heatmap for log2 values of the relative mRNA expression for each gene for each cell. UBB was used as an internal control. b. Number of neurons expressing each gene in large, medium and small cells were counted and presented as percentage of neurons expressing the target gene within each size group.

Fig 3. Intensity of expression of target genes based on sensory neuronal cell size.

Relative mRNA expression values from single-cell PCR for each gene are presented as median +/- 95% confidence interval in large, medium and small neurons. Expression intensities for each gene was compared using parametric one-way ANOVA with Bonferroni post-hoc test, if the variances in means were not significant. If significant, non-parametric Kruskal-Wallis with Bonferroni post hoc test was used. p<0.05 was considered significant.

Fig 4. Identification of subgroups and overlapping markers in lingual sensory neurons.

a. Heat map representing number of neurons expressing each gene in large, medium and small neurons. b. List of major markers expressed, overalapping genes in large medium and small neurons. Subgroups identified from Usoskin et al and Patil et al studies that correspond to the major markers is also listed.