Figure 1. The TH⁺ adult DRG neurons are C-LTMRs and form longitudinal lanceolate endings associated with zigzag and awl/auchene hair follicles.

(A-D) Double immunostaining of TH and NFH (A) (n=8), TH and CGRP (B) (n=6), TH and cRet (C) (n=4), and TH and IB4 (D) (n=7) on adult thoracic DRG sections. TH⁺ DRG neurons do not express NFH (<1%) or CGRP (<5%). The majority of TH⁺ neurons express cRet (>90%) but do not bind to the lectin IB4 (<1%).

(E-F) Intracellular recordings of TH⁺ DRG neurons were performed using the ex vivo skin nerve preparation. Small-diameter DRG neurons were randomly selected and recorded (n=21). The recorded neurons were then labeled with Neurobiotin, and co-stained with TH and GFRα2 or TH and IB4. Among the recorded neurons, all of those that exhibited classically defined features of C-LTMRs (n=5; See text) (E) are TH⁺, GFRα2⁺ and IB4^- (F) (arrows).

(G) TH^CreER; Rosa26^IAP mice were treated with 4-HT (1mg per day for three days, from P13 to P15, by oral gavage). Double immunostaining of AP and TH shows that a small number of TH⁺ DRG neurons (~10%) were labeled with AP (n=2).

(H-K) Whole-mount AP staining of thoracic DRGs and trunk hairy skin shows that DRG cell bodies (H) and their peripheral endings in the skin (I) are sparsely labeled. Individual axonal branches arborize and form longitudinal lanceolate endings (J and K) (n=26).

(L) The longitudinal lanceolate endings of the C-LTMRs are similarly labeled in TH^CreER;Rosa26^LSLtdTomato mice, which enables double labeling of different LTMR endings (n=24). Whole-mount immunostaining of the dsRed signal of hairy skin showed that 80% of C-LTMR lanceolate endings are associated with zigzag hair follicles and 20% with awl/auchene hair follicles, while none were observed to associate with guard hair follicles (n=212 hair follicles examined).

Scale bars: 50 μm (A-D) and (G and H); 100 μm (I and J); 20 μm (F) and (K and L).