The glutamatergic nature of TRPV1-expressing neurons in the spinal dorsal horn

Abstract

The transient receptor potential vanilloid receptor 1 (TRPV1) is expressed on primary afferent terminals and spinal dorsal horn neurons. However, the neurochemical phenotypes and functions of TRPV1-expressing post-synaptic neurons in the spinal cord are not clear. In this study, we tested the hypothesis that TRPV1-expressing dorsal horn neurons are glutamatergic. Immunocytochemical labeling revealed that TRPV1 and vesicular glutamate transporter-2 were colocalized in dorsal horn neurons and their terminals in the rat spinal cord. Resiniferatoxin (RTX) treatment or dorsal rhizotomy ablated TRPV1-expressing primary afferents but did not affect TRPV1- and vesicular glutamate transporter-2-expressing dorsal horn neurons. Capsaicin significantly increased the frequency of glutamatergic spontaneous excitatory post-synaptic currents and miniature excitatory post-synaptic currents in almost all the lamina II neurons tested in control rats. In RTX-treated or dorsal rhizotomized rats, capsaicin still increased the frequency of spontaneous excitatory post-synaptic currents and miniature excitatory post-synaptic currents in the majority of neurons examined, and this effect was abolished by a TRPV1 blocker or by non-NMDA receptor antagonist. In RTX-treated or in dorsal rhizotomized rats, capsaicin also produced an inward current in a subpopulation of lamina II neurons. However, capsaicin had no effect on GABAergic and glycinergic spontaneous inhibitory post-synaptic currents of lamina II neurons in RTX-treated or dorsal rhizotomized rats. Collectively, our study provides new histological and functional evidence that TRPV1-expressing dorsal horn neurons in the spinal cord are glutamatergic and that they mediate excitatory synaptic transmission. This finding is important to our understanding of the circuitry and phenotypes of intrinsic dorsal horn neurons in the spinal cord.

Fig. 1

Confocal images showing the effect of RTX on TRPV1- and IB₄-positive dorsal root ganglion (DRG) neurons and afferent terminals in the spinal cord. A, representative confocal images showing TRPV1-immunoreactive (green) and IB₄-positive (red) DRG neurons from one vehicle-treated and one RTX-treated rat. B, confocal images showing TRPV1-immunoreactive (green) and IB₄ (red)-positive afferent terminals in the spinal dorsal horn of one vehicle-treated and one RTX-treated rat. Colocalization of TRPV1 immunoreactivity and IB₄ labeling is indicated in yellow when two images are digitally merged. All images are single confocal optical sections.

Fig. 2

Confocal images showing the presence of TRPV1- and VGluT₂-immunoreactive neurons and terminals in the superficial spinal dorsal horn in RTX-treated and dorsal rhizotomized rats. A, representative low- and high-magnification confocal images showing VGluT₂ (green)- and TRPV1 (red)-immunoreactive neurons and terminals in laminas I and II from one vehicle-treated control rat. B, representative confocal images showing VGluT₂ (green)- and TRPV1 (red)-immunoreactive neurons and terminals in laminas I and II from one RTX-treated and one dorsal rhizotomized rat. Colocalization of TRPV1 and VGluT₂ is indicated in yellow when two images are digitally merged. All images are single confocal optical sections.

Fig. 3

Effect of capsaicin on the frequency and amplitude of sEPSCs of lamina II neurons in vehicle-treated rats. A, original traces show sEPSCs during control and application of 2 µM capsaicin in an amplitude increase type neuron. Cumulative distributions of the inter-event interval and amplitude of sEPSCs of the same neuron during control and application of 2 µM capsaicin. B, summary data show the effect of capsaicin on the frequency (n = 12) and amplitude (n = 11) of sEPSCs in lamina II neurons of vehicle-treated rats. * P < 0.05 compared with the control.

Fig. 4

Effect of capsaicin and capsazepine on the frequency and amplitude of sEPSCs of lamina II neurons in RTX-treated rats. A, original traces show sEPSCs during control and application of 2 µM capsaicin in an amplitude no-change type neuron. Note that 10 µM CNQX abolished the sEPSCs in both amplitude no-change and amplitude increase type neurons. Cumulative distributions of the inter-event interval and amplitude of sEPSCs of the same neuron as that in panel A during control and application of capsaicin. B, summary data show the differential effect of capsaicin on the frequency and amplitude of sEPSCs of 23 lamina II neurons in RTX-treated rats. C, summary data show the effect of capsaicin on sEPSCs of 8 lamina II neurons in the presence of 20 µM capsazepine in RTX-treated rats. * P < 0.05 compared with the control.

Fig. 5

Effect of capsaicin on the frequency and amplitude of sEPSCs of lamina II neurons in sham-operated and dorsal rhizotomized rats. A, summary data show the effect of capsaicin on the frequency and amplitude of sEPSCs of lamina II neurons in sham-operated rats. B, group data show the differential effect of capsaicin on sEPSCs of 36 lamina II neurons in dorsal rhizotomized rats. C, summary data show results for 9 lamina II neurons in the presence of 20 µM capsazepine in dorsal rhizotomized rats. * P < 0.05 compared with the control.

Fig. 6

Effect of capsaicin on the frequency and amplitude of mEPSCs of lamina II neurons in vehicle- and RTX-treated rats. A, original traces show mEPSCs during control and application of 2 µM capsaicin in an amplitude increase type neuron in one vehicle-treated rat. Cumulative distributions of the inter-event interval and amplitude of mEPSCs of the same neuron. B, summary data show the effect of capsaicin on the frequency (n=16) and amplitude (n=15) of mEPSCs of lamina II neurons in vehicle-treated rats. C, original traces show mEPSCs during control and application of 2 µM capsaicin in an amplitude no-change type neuron. Cumulative distributions of the inter-event interval and amplitude of mEPSCs of the same neuron. D, group data show the differential effect of capsaicin on mEPSCs of 33 lamina II neurons in RTX-treated rats. * P < 0.05 compared with the control.

Fig. 7

Effect of capsaicin on the frequency and amplitude of mEPSCs of lamina II neurons in sham-operated rats and those subjected to dorsal rhizotomy. A, group data show the effect of capsaicin on the frequency and amplitude of sEPSCs of lamina II neurons in sham-operated rats. B, summary data show the differential effect of capsaicin on the frequency and amplitude of mEPSCs of 17 lamina II neurons in dorsal rhizotomized rats. * P < 0.05 compared with the control.

Fig. 8

Capsaicin-induced currents in a subpopulation of spinal lamina II neurons of RTX-treated or dorsal rhizotomized rats. A, original records show an inward current in response to application of 2 µM capsaicin in an EPSC amplitude-increase type of lamina II neuron of one RTX-treated rat. B, original recordings show an inward current during capsaicin application in an EPSC amplitude-increase type of lamina II neuron of one dorsal rhizotomized rat. C, summary data show the peak amplitude of inward currents produced by capsaicin application in lamina II neurons of RTX-treated (n = 4 neurons) and dorsal rhizotomized (n = 7 neurons) rats.

Fig. 9

Effect of capsaicin on the frequency and amplitude of sIPSCs of lamina II neurons in RTX-treated and dorsal rhizotomized rats. A: original traces show sIPSCs of a lamina II neuron during control and application of 2 µM capsaicin in one dorsal rhizotomized rat. Note that bath application of 50 µM picrotoxin abolished sIPSCs. B and C: summary data show the lack of effect of capsaicin on the sIPSCs of lamina II neurons in RTX-treated or dorsal rhizotomized rats.

Fig. 10

Comparison of the effects of oxotremorine and capsaicin on the frequency and amplitude of sIPSCs of lamina II neurons in RTX-treated rats. A: original traces show sIPSCs of a lamina II neuron during control and during bath application of 3 µM oxotremorine (Oxo) or 2 µM capsaicin (Caps) in one neuron of an RTX-treated rat. B: summary data showing that oxotremorine, but not capsaicin, potentiated the frequency and amplitude of sIPSCs of 11 lamina II neurons in RTX-treated rats. * P < 0.05 compared with the control.