Group I metabotropic glutamate receptors control metaplasticity of spinal cord learning through a protein kinase C-dependent mechanism

Abstract

Neurons within the spinal cord can support several forms of plasticity, including response-outcome (instrumental) learning. After a complete spinal transection, experimental subjects are capable of learning to hold the hindlimb in a flexed position (response) if shock (outcome) is delivered to the tibialis anterior muscle when the limb is extended. This response-contingent shock produces a robust learning that is mediated by ionotropic glutamate receptors (iGluRs). Exposure to nociceptive stimuli that are independent of limb position (e.g., uncontrollable shock; peripheral inflammation) produces a long-term (>24 h) inhibition of spinal learning. This inhibition of plasticity in spinal learning is itself a form of plasticity that requires iGluR activation and protein synthesis. Plasticity of plasticity (metaplasticity) in the CNS has been linked to group I metabotropic glutamate receptors (subtypes mGluR1 and mGluR5) and activation of protein kinase C (PKC). The present study explores the role of mGluRs and PKC in the metaplastic inhibition of spinal cord learning using a combination of behavioral, pharmacological, and biochemical techniques. Activation of group I mGluRs was found to be both necessary and sufficient for metaplastic inhibition of spinal learning. PKC was activated by stimuli that inhibit spinal learning, and inhibiting PKC activity restored the capacity for spinal learning. Finally, a PKC inhibitor blocked the metaplastic inhibition of spinal learning produced by a group I mGluR agonist. The data strongly suggest that group I mGluRs control metaplasticity of spinal learning through a PKC-dependent mechanism, providing a potential therapeutic target for promoting use-dependent plasticity after spinal cord injury.

Figure 1.

Group I mGluRs are necessary for metaplastic inhibition of spinal learning. Unshocked subjects demonstrated typical spinal learning, increasing flexion duration over the 30 min testing interval (A, F). Brief exposure to uncontrollable shock produced a deficit in spinal learning that was apparent at test at 24 h (A, F). Antagonists to the group I mGluRs, mGluR1 (CPCCOEt in 100% DMSO) (A–E) or GluR5 (MPEP in 0.9% saline) (F–J) restored spinal learning in dose-dependent manner. A–D and F–I depict performance over time; E and J depict group means. The shaded region represents SEM over time; error bars represent SEM for group means (n = 6 subjects/group for CPCCOEt; n = 8 subjects/group for MPEP). *p < 0.05 from unshocked.

Figure 2.

mGluR1 but not mGluR5 antagonism acutely facilitates acquisition of spinal learning. The mGluR1 antagonist CPCCOEt (in 100% DMSO) increased the rate of spinal learning when subjects were tested immediately after drug exposure (A). This effect was most pronounced in the first 5 min of testing, a time point that is associated with molecular plasticity in spinal learning (Gómez-Pinilla et al., 2007). The mGluR5 antagonist MPEP (in 0.9% saline) did not improve spinal learning (B). The shaded region represents SEM over time (n = 8 subjects/group).

Figure 3.

A group I mGluR agonist induces lasting metaplastic inhibition of spinal learning. Subjects that were given DHPG (in 0.9% saline) had impaired spinal learning at test at 24 h, as evident by impaired performance over time (A) and group means (B). The shaded region represents SEM over time; error bars represent SEM for group means (n = 16 subjects/dose). *p < 0.05 from vehicle.

Figure 4.

Activation of PKC in the spinal cord by uncontrollable shock. ELISA revealed that uncontrollable shock produced a significant increase in PKC activity that reached statistical significance by 1 h after shock exposure. Error bars represent SEM for group means (n = 4 subjects/group). *p < 0.05.

Figure 5.

PKC activation is necessary for metaplastic inhibition of spinal learning. Uncontrollable shock produced a metaplastic inhibition of spinal learning at testing at 24 h (A, E). Spinal learning was restored by two mechanistically distinct PKC inhibitors BIM (A–D) and chelerythrine (E–G) delivered in 100 and 10% DMSO, respectively. Group means are depicted in D and G. The shaded region represents SEM over time; error bars represent SEM for group means (n = 8 subjects/group). *p < 0.05 from all groups except for shocked subjects in middle dose; **p < 0.05 from all other groups.

Figure 6.

PKC activation does not affect acquisition of spinal learning. The rate of acquisition of spinal learning was not significantly affected by the PKC inhibitor BIM (in 100% DMSO). The shaded region represents SEM over time (n = 8 subjects/group).

Figure 7.

Group I mGluRs inhibit spinal learning via a PKC-dependent mechanism. Replicating previous findings (Fig. 3), the group I mGluR agonist DHPG was found to induce metaplastic inhibition of spinal learning at test at 24 h (A, B). Delivery of the PKC inhibitor BIM (in 100% DMSO) before DHPG (in 0.9% saline) blocked this effect, restoring the capacity for spinal learning (B, C). To achieve a balanced design, all subjects received both vehicles. The shaded region represents SEM over time; error bars represent SEM for group means (n = 10 subjects/group).

Figure 8.

A proposed cellular/molecular model of the plasticity (A) and inhibitory metaplasticity (B) of spinal cord learning. Previous work has revealed that iGluR activation is necessary for spinal learning and that molecules that facilitate iGluR-mediated plasticity, such as BDNF and CaMKII, also facilitate spinal learning (for details, see text). Together, these data suggesting that the pattern of glutamate release during response-contingent shock induces iGluR-mediated plasticity, which supports learning (A). However, iGluRs are also implicated in inhibition of spinal learning by uncontrollable nociceptive stimulation. Based on the present results, we propose that high levels of glutamate produced by uncontrollable shock activate mGluRs, resulting in PKC activation and long-term alteration of iGluR function resulting in a spinal learning deficit (B).