Figure 4. Relationship between skin target identity and Aβ-LTMR brainstem synapses over development.

A. (left) AAV virus injection for skin type-specific labeling of Aβ-LTMR central projections with synaptophysin-tdTomato or synaptophysin-mScarlet, hereafter referred to as synaptophysin-FP (fluorescent protein). The GN and CN were sectioned in their entirety and stained for fluorescent proteins and synaptic markers. (right) The number of FP⁺ pre-synaptic boutons and axons ascending the dorsal column were identified in each sample. Representative example of labeled boutons apposed to the excitatory postsynaptic marker Homer1 in the GN (top). Both hairy and glabrous skin-innervating Aβ-LTMRs form syn-FP⁺ boutons apposed to Homer1⁺ puncta in the GN or CN. Representative example of labeled axons (arrows) ascending the dorsal column, in a transverse section of the cervical spinal cord (bottom). Arrows point to individual axons.

B-C. Quantification of the average number of FP⁺ pre-synaptic boutons formed by Aβ-LTMRs innervating different skin targets. Each data point represents one animal; the number of axons identified in each animal is shown in parentheses.

B. Adult forepaw glabrous skin-innervating Aβ-LTMRs form more synapses than forepaw hairy skin-innervating Aβ-LTMRs in the CN (*p<0.05, Mann-Whitney U test).

C. At P14, hindpaw glabrous skin-innervating Aβ-LTMRs and hindlimb hairy skin-innervating Aβ-LTMRs form similar numbers of pre-synaptic boutons in the GN. At adult time points, paw glabrous skin Aβ-LTMRs form significantly more pre-synaptic boutons in the GN than paw or trunk hairy skin Aβ-LTMRs (p<0.01 Kruskal-Wallis one-way ANOVA, *p<0.01, Mann-Whitney U test with Bonferroni correction for multiple comparisons).

D. Summary of skin type-dependent synaptic refinement in Aβ-LTMRs.