Ryanodine receptors contribute to the induction of nociceptive input-evoked long-term potentiation in the rat spinal cord slice

Abstract

Background: Our previous study demonstrated that nitric oxide (NO) contributes to long-term potentiation (LTP) of C-fiber-evoked field potentials by tetanic stimulation of the sciatic nerve in the spinal cord in vivo. Ryanodine receptor (RyR) is a downstream target for NO. The present study further explored the role of RyR in synaptic plasticity of the spinal pain pathway.

Results: By means of field potential recordings in the adult male rat in vivo, we showed that RyR antagonist reduced LTP of C-fiber-evoked responses in the spinal dorsal horn by tetanic stimulation of the sciatic nerve. Using spinal cord slice preparations and field potential recordings from superficial dorsal horn, high frequency stimulation of Lissauer's tract (LT) stably induced LTP of field excitatory postsynaptic potentials (fEPSPs). Perfusion of RyR antagonists blocked the induction of LT stimulation-evoked spinal LTP, while Ins(1,4,5)P3 receptor (IP(3)R) antagonist had no significant effect on LTP induction. Moreover, activation of RyRs by caffeine without high frequency stimulation induced a long-term potentiation in the presence of bicuculline methiodide and strychnine. Further, in patch-clamp recordings from superficial dorsal horn neurons, activation of RyRs resulted in a large increase in the frequency of miniature EPSCs (mEPSCs). Immunohistochemical study showed that RyRs were expressed in the dorsal root ganglion (DRG) neurons. Likewise, calcium imaging in small DRG neurons illustrated that activation of RyRs elevated [Ca(2+)]i in small DRG neurons.

Conclusions: These data indicate that activation of presynaptic RyRs play a crucial role in the induction of LTP in the spinal pain pathway, probably through enhancement of transmitter release.

Figure 1

Involvement of RyR in the induction of LTP of fEPSPs in the spinal dorsal horn in vitro. A, fEPSPs were recorded in the presence of 10 μM bicuculline methiodide and 1 μM strychnine. fEPSPs recorded 10 min before HFS served as control. After HFS, fEPSPs significantly increased (n = 24). The inserted traces were the original recordings (without averaging). a-b corresponds to the time points as indicated. B, The LTP of fEPSPs stably lasted for 3 h (n = 8). C, 20 μM ryanodine applied 1 h before HFS blocked the induction of LTP (P < 0.001) (control n = 7; ryanodine n = 8). The inserted traces were the original recordings. D, 10 μM dantrolene perfused 1 h before HFS blocked the induction of LTP (P < 0.001) (control n = 7; dantrolene n = 7), and this effect was reversible (P < 0.001) (washout n = 3). E, 10 mM dantrolene has no significant effect on the amplitude of fEPSP. Control: the amplitude of fEPSP recorded 5 min before dantrolene application (10 μM); Dantrolene: 30 min after perfusion of dantrolene. Inserts show original traces recorded 5 min before and 30 min after dantrolene. F, 10 μM dantrolene perfused 30 min after HFS had no significant effect on the established LTP (P = 1) (control n = 5; dantrolene n = 6). Data were shown as mean ± s.e.m. in all figures; n: the number of slices.

Figure 2

Involvement of RyR in the induction of LTP of fEPSPs in the spinal dorsal horn in vivo. The spinal LTP was blocked by high concentration of ryanodine (1 mM, 10 μl) intrathecally applied 30 min before tetanic stimulation (P < 0.001, vs. vehicle) (control n = 5; ryanodine n = 6) in the spinal cord in vivo. Representative original recordings (average of six consecutive traces) were shown below from the time points as indicated.

Figure 3

Reversal of L-NAMA-induced inhibition of LTP of fEPSP by cADPR, an endogenous agonist of the RyR. Perfusion with 50 μM L-NAME for 1 h before HFS of LT inhibited the induction of LTP (P < 0.001), which was reversed by co-application of L-NAME and 5 μM cADPR for 1 h before HFS (P < 0.001) (control, n = 5; L-NAME, n = 6; L-NAME + cADPR, n = 5).

Figure 4

No effect of IP3R antagonist on induction of spinal LTP in vitro. A, IP₃R antagonist 2-APB (75 μM) applied for 1 h before HFS of LT had no significant effect either on the induction of LTP of fEPSPs (P = 0.303) (control, n = 7; 2-APB, 45 min from baseline, n = 8), or on the established LTP of fEPSPs (P = 0.173) (control, n = 4; 2-APB, 135 min from baseline, n = 4). B, As a positive control, 2-APB blocked the induction of hippocampal LTP. In the hippocampal slice, 75 μM 2-APB perfused for 1 h before conditioning stimulation (100 Hz for 1 sec, 3 times at 10-sec intervals, mimicking spinal HFS) blocked the induction of hippocampal LTP (control, 30 min after induction of hippocampal LTP in the presence of 10 μM bicuculline methiodide, n = 4; 2-APB, 30 min after induction of hippocampal LTP in the presence of 10 μM bicuculline methiodide and 75 μM 2-APB, n = 5). Error bars represent s.e.m. n: the number of slices. **P < 0.01.

Figure 5

Caffeine-induced LTP of fEPSPs in spinal cord slice. A, After perfusion with 10 mM caffeine for 2 min instantly following washed out, LTP of fEPSPs occurred without conditioning stimulation in the presence of 10 μM bicuculline methiodide and 1 μM strychnine, which lasted for more than 80 min. Inserts show original recordings 5 min before (1) and about 80 min after (2) 10 mM caffeine challenge. Traces were not averaged (n = 13). B, Perfusion with 20 μM ryanodine 1 hour before caffeine challenge blocked the induction of LTP of fEPSPs. Inserts show original recordings without averaging (n = 5).

Figure 6

Expression of ryanodine receptors 1 or 3 (RyR1, RyR3) and their co- localization with IB4 or CGRP in DRG neurons in normal rats. A, Co-localization of RyR1 (red) with IB4 (green) in DRG neurons. B, Co-localization of RyR3 (red) and CGRP (green) in DRG neurons, scale: 50 μm.

Figure 7

Increase in [Ca²⁺]_i by caffeine in isolated DRG neurons. A, Brief puff of 10 mM caffeine for 1 or 2 s increased [Ca²⁺]_i transients in DRG neurons (n = 9). B, Puff of 2 μM ryanodine increased [Ca²⁺]_i transients (n = 9). C, Adding a high concentration of ryanodine (40 μM) to the extracellular solution blocked caffeine-induced increase in [Ca²⁺]_i transients (n = 6). The cell color imaging (a-i) in the right side corresponded to the time points indicated (a-i) in the left side.

Figure 8

Increase in mEPSCs frequency by caffeine in superficial dorsal horn neurons. A, Sample traces of mESPCs recorded from a lamina II neuron in the presence of 0.5 μM TTX before (left) and after bath application of 10 mM caffeine for about 5 min (right). B. A histogram shows the time course of changes in mEPSCs frequency before, after and following washout of 10 mM caffeine in the presence of 0.5 μM TTX in an individual neuron. C and D, The averaged frequency and amplitude for all cells treated with 10 mM caffeine (n = 16; 5 s bins) in the presence of 0.5 μM TTX. Membrane potential was clamped at -70 mV. Error bars represent s.e.m. n: the number of cells.