Gate control of sensory neurotransmission in peripheral ganglia by proprioceptive sensory neurons

Figures

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  Figure 1. Loss of parvalbumin (PV) mRNA and protein in the DRG of PVDTA mice. (A) Breeding strategy for PVDTA mice. The red arrowheads denote loxP sites. The dorsal root ganglion (DRG) neurons of PVDTA progeny express the red fluorescent reporter tdTomato in PV−, Advil+ neurons. (B) qRT-PCR showing PV mRNA expression in the DRG of PVDTA mice is significantly reduced compared to littermate controls, relative to GAPDH expression. (C) Representative DRG tissue sections stained with anti-PV antibody. TdTomato represents the advillin-expressing neuronal population. Scale bar = 500 µm. (D) Quantification of PV+ neurons in the DRG of littermate and PVDTA animals, expressed as a percentage of the total number of DRG neurons (n = 3 per group). Data are mean ± SEM with unpaired Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

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  Figure 2. L4 DRG neurons show enhanced activity with noxious peripheral stimulation in PVDTA mice, as do wide dynamic range neurons in the dorsal horn. (A) Representative images of in vivo calcium imaging of DRG neurons expressing GCaMP3, and their response to 150 g/cm2 prod or 55°C applied to ipsilateral hind paw. Scale bar = 100 µm. (B) Heat map demonstrating responses to mechanical prod and 55°C heat stimulation. (C) Peak fluorescence of DRG neurons in littermate (n = 263 cells) and PVDTA mice (n = 179 cells). The latter exhibit increased calcium transients evoked by 150 g/cm2 prod, 55°C and 0°C. (D) In vivo recordings of wide dynamic range (WDR) neurons in the deep dorsal horn (n = 45 littermate cells and n = 17 PVDTA cells) demonstrate that PVDTA cells show a 2-fold increase in action potentials fired to brush stimuli and an increase to cotton swab and vibration. They also demonstrate hyperexcitability to noxious mechanical and thermal stimulation. Data are mean ± SEM. Statistical analysis was performed using the multiple unpaired Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

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  Figure 3. Acute thermal and mechanical pain thresholds are reduced and spontaneous inflammatory pain behaviour augmented in PVDTA mice. (A) Cotton swab test shows small differences between PVDTA mice and controls (P = 0.08, Mann-Whitney test). Mechanical pain thresholds are lower in PVDTA mice assessed using (B) manual up/down von Frey and (C) electronic von Frey assays, as are thermal pain thresholds using the (D) hot plate and (E) dry ice assays. (F) The Randall-Selitto assay demonstrates noxious mechanical hyperalgesia in PVDTA animals. Data are mean ± SEM. using unpaired Student’s t-test (littermate n = 11–14; PVDTA n = 10–11). [G(i)] PVDTA mice demonstrate enhanced spontaneous pain measured with hind paw lifts following intraplantar (i.pl.) administration of 20 µl 500 mM PGE2. [G(ii)] Paw lifts are the mean number of contralateral paw lifts subtracted from ipsilateral paw lifts in animals administered i.pl. PGE2. [H(i)] The experiments described in G(i) were repeated with 200 µM Bicuculline co-administered with i.pl. PGE2. Littermates also demonstrate enhanced spontaneous pain, comparable to PVDTA animals. [H(ii)] The mean number of contralateral paw lifts subtracted from ipsilateral…