PKCα is required for inflammation-induced trafficking of extrasynaptic AMPA receptors in tonically firing lamina II dorsal horn neurons during the maintenance of persistent inflammatory pain

Abstract

Persistent inflammation promotes internalization of synaptic GluR2-containing, Ca(2+)-impermeable AMPA receptors (AMPARs) and insertion of GluR1-containing, Ca(2+)-permeable AMPARs at extrasynaptic sites in dorsal horn neurons. Previously we have shown that internalization of synaptic GluR2-containing AMPARs requires activation of spinal cord protein kinase C alpha (PKCα), but molecular mechanisms that underlie altered trafficking of extrasynaptic AMPARs are unclear. Here, using antisense (AS) oligodeoxynucleotides (ODN) that specifically knock down PKCα, we found that a decrease in dorsal horn PKCα expression prevents complete Freund's adjuvant (CFA)-induced increase in functional expression of extrasynaptic Ca(2+)-permeable AMPARs in substantia gelatinosa (SG) neurons of the rat spinal cord. Augmented AMPA-induced currents and associated [Ca(2+)](i) transients were abolished, and the current rectification 1 day post-CFA was reversed. These changes were observed specifically in SG neurons characterized by intrinsic tonic firing properties, but not in those that exhibited strong adaptation. Finally, dorsal horn PKCα knockdown produced an antinociceptive effect on CFA-induced thermal and mechanical hypersensitivity during the maintenance period of inflammatory pain, indicating a role for PKCα in persistent inflammatory pain maintenance. Our results indicate that inflammation-induced trafficking of extrasynaptic Ca(2+)-permeable AMPARs in tonically firing SG neurons depends on PKCα, and that this PKCα-dependent trafficking may contribute to persistent inflammatory pain maintenance.

Perspective: This study shows that PKCα knockdown blocks inflammation-induced upregulation of extrasynaptic Ca(2+)-permeable AMPARs in dorsal horn neurons and produces an antinociceptive effect during the maintenance period of inflammatory pain. These findings have potential implications for use of PKCα gene-silencing therapy to prevent and/or treat persistent inflammatory pain.

Fig. 1. Effect of PKCα AS ODN on PKCα protein expression in spinal cord

Intrathecal injection of 10 µg PKCα AS ODN, but not saline or 10 µg MS ODN, significantly reduced expression of PKCα, but not PKCγ, in the dorsal horn of spinal cord lumbar enlargement segments compared to that in naïve rats. Top: Representative Western blots. Bottom: Statistical summary of the densitometric analysis expressed relative to naïve rats after normalization to corresponding β-actin.

Fig. 2. Knockdown of dorsal horn PKCα attenuates CFA-induced augmentation of AMPA-induced current and [Ca²⁺]_i transients in tonically firing SG neurons

(A) Top: a fluorescent image of SG neuron loaded with fura-2 (200 µM); scale bar = 20 µm. Bottom: typical firing pattern for tonic neurons in response to sustained depolarizing current. (B) Representative examples of a somatic membrane current (bottom trace) and associated [Ca²⁺]_i transients (upper traces), recorded from the soma (black trace) and dendrites (blue trace), in tonic neurons during AMPA bath application (5 µM, 60 s) in the AS- (right) and MS ODNs-treated (left) groups 1 d post-CFA. (C) Statistical summary of the amplitudes of AMPA-induced current (left graph) and associated [Ca²⁺]_i transients in soma and dendrites (right graph) of tonic SG neurons from the saline-, AS- and MS ODNs-treated groups 1 d after saline or CFA injection. ^* p < 0.05, ^*** p < 0.001. AS, antisense; MS, missense.

Fig.3. Effect of dorsal horn PKCα knockdown on the I-V relationship of AMPARs-mediated currents in tonically firing SG neurons

(A) I-V curves obtained in tonic SG neurons of the AS- and MS ODNs-treated groups 1d post-saline. Insert illustrates the protocol for reconstruction of the I-V relationship from ramp recordings. (B) The scatter plot illustrates a spread in rectification index (RI = I_+30mV/I_−50mV) in tonic SG neurons after AS ODN or MS ODN treatment for 4 days. (C–D) I-V curves (C) and a scatter plot of RI (D) in tonic SG neurons from the AS- and MS ODN-treated groups 1d after CFA injection. (E) A statistical summary for RI in tonic neurons from the AS ODN- and MS ODN-treated groups 1 d post-saline and post-CFA before (black graph) and after (grey) an application of selective blocker of Ca²⁺-permeable AMPARs, IEM-1460 (40 µM). ^*** p < 0.001 versus the saline-injected group, ^#p < 0.05, ^### p < 0.001 versus the CFA-injected group.

Fig. 4. Dorsal horn PKCα knockdown reduces CFA-induced peripheral thermal and mechanical hypersensitivities and maintenance of inflammatory pain

(A) Intrathecal injection of 10 µg PKCα AS ODN, but not saline or 10 µg MS ODN, significantly attenuated the CFA-induced decrease in paw withdrawal latency (PWL) in response to thermal stimulation. (B–D) Effect of PKCα AS ODN or MS ODN treatment on CFA-induced increase in the paw withdrawal frequency (PWF) in response to 2 g (B), 4 g (C), and 8 g (D) von Frey filaments at different time points after CFA injection. * p < 0.05, ** p < 0.01 vs the corresponding time point in the saline-treated CFA-inflamed group. AS, antisense; MS, missense.