Inflammation alters trafficking of extrasynaptic AMPA receptors in tonically firing lamina II neurons of the rat spinal dorsal horn

Abstract

Peripheral inflammation alters AMPA receptor (AMPAR) subunit trafficking and increases AMPAR Ca(2+) permeability at synapses of spinal dorsal horn neurons. However, it is unclear whether AMPAR trafficking at extrasynaptic sites of these neurons also changes under persistent inflammatory pain conditions. Using patch-clamp recording combined with Ca(2+) imaging and cobalt staining, we found that, under normal conditions, an extrasynaptic pool of AMPARs in rat substantia gelatinosa (SG) neurons of spinal dorsal horn predominantly consists of GluR2-containing Ca(2+)-impermeable receptors. Maintenance of complete Freund's adjuvant (CFA)-induced inflammation was associated with a marked enhancement of AMPA-induced currents and [Ca(2+)](i) transients in SG neurons, while, as we previously showed, the amplitude of synaptically evoked AMPAR-mediated currents was not changed 24 h after CFA. These findings indicate that extrasynaptic AMPARs are upregulated and their Ca(2+) permeability increases dramatically. This increase occurred in SG neurons characterized by intrinsic tonic firing properties, but not in those exhibited strong adaptation. This increase was also accompanied by an inward rectification of AMPA-induced currents and enhancement of sensitivity to a highly selective Ca(2+)-permeable AMPAR blocker, IEM-1460. Electron microcopy and biochemical assays additionally showed an increase in the amount of GluR1 at extrasynaptic membranes in dorsal horn neurons 24h post-CFA. Taken together, our findings indicate that CFA-induced inflammation increases functional expression and proportion of extrasynaptic GluR1-containing Ca(2+)-permeable AMPARs in tonically firing excitatory dorsal horn neurons, suggesting that the altered extrasynaptic AMPAR trafficking might participate in the maintenance of persistent inflammatory pain.

Fig. 1

Increased kainate-induced cobalt loading in the dorsal horn after CFA injection. (A) Representative examples of kainate-induced cobalt loading in live spinal cord slices in the absence or presence of the NMDA receptor antagonist APV (500 μM) or AMPAR antagonists CNQX (250 μM) and GYKI 52466 (250 μM). (B) The neuronal marker NeuN overlaps with cobalt-uptake in the superficial dorsal horn. (C) CFA (but not saline) injection increased cobalt uptake in dorsal horn neurons on the ipsilateral, but not contralateral, side. (D) Statistical summary of the number of cobalt-positive dorsal horn neurons in laminae I-II (top graph) and laminae III-VII (bottom graph) 24 h after saline and CFA.

Fig. 2

Sustained membrane depolarization revealed two groups of SG neurons exhibiting different discharge patterns. (A) Transmitted light image of patch electrode position (square) in the SG of a transverse dorsal horn slice; scale bar = 200 μm. (B) A fluorescent image of a neuron loaded with fura-2 (200 μM); scale bar = 20 μm. (C, D) Current-clamp recordings of typical firing patterns for tonic (C) and transient (D) groups of neurons in response to three different intensities of depolarizing currents shown at the bottom of each panel. (E, F) Recordings of depolarizing current-induced changes in the cytosolic free calcium concentration ([Ca²⁺]_i) in the soma (black traces) and dendrites (grey traces) in tonic (E) and transient (F) groups of neurons.

Fig. 3

AMPAR-mediated currents and [Ca²⁺]_i transients are similar in tonic and transient neurons of naive rats. (A) Representative traces of a somatic membrane current (bottom trace) and associated [Ca²⁺]_i transients (upper traces) recorded from the soma (black trace) and dendrites (grey trace) in tonic neurons during AMPA bath application (5 μM, 60 s). (B, C) Pre-application of AMPAR antagonists NBQX (30 μM, B) and GYKI 52466 (100 μM, C) abolished AMPA-induced current (lower traces) and associated somatic (upper black traces) and dendritic (upper grey traces) [Ca²⁺]_i transients. (D) Pooled results demonstrate that AMPA-induced current amplitudes (left graph) and [Ca²⁺]_i transients (center and right graphs) recorded from soma and dendrites of different groups of SG neurons are similar. ** p < 0.001, *** p < 0.0001 versus the control; NS, not significant.

Fig. 4

AMPAR-mediated currents and associated [Ca²⁺]_i transients are markedly potentiated in tonic but not in transient SG neurons during persistent inflammation. (A) Representative examples of AMPA-induced currents (lower traces) and [Ca²⁺]_i transients (upper traces) in soma (black traces) and dendrites (grey traces) in tonic neurons 24 h after saline (control) or CFA. (B, C) Scatter dot plots illustrate a spread in extrasynaptic AMPAR-mediated currents in tonic (B) and transient (C) neurons 24 h after saline or CFA. (D) A statistical summary of current amplitudes (left graph) and [Ca²⁺]_i transients (right two graphs) in soma and dendrites of different groups of SG neurons 24 h post-saline and post-CFA. * p < 0.05, ** p < 0.001, *** p < 0.0001 versus the saline-treated group; ^# p < 0.05, ^## p < 0.001 versus the transient SG neurons; NS, not significant.

Fig. 5

Persistent peripheral inflammation increases the proportion of Ca²⁺-permeable AMPARs in the extrasynaptic plasma membrane of tonic SG neurons. (A) A selective blocker of Ca²⁺-permeable AMPARs, IEM-1460 (40 μM), substantially inhibited AMPA-induced currents in tonic neurons of CFA-treated but not of saline-treated rats. Left, an overlay of AMPA-induced currents recorded in the absence (black traces) and presence (pre-incubation for 5 min; grey traces) of IEM-1460 24 h after saline or CFA. Right, IEM-1460 was bath applied during a steady-state phase of AMPA-induced current. Dotted lines represent exponential fitting of the currents; dotted arrow indicates the value of IEM-1460 inhibition. (B) A statistical summary of IEM-1460 inhibition of extrasynaptic AMPARs in tonic and transient SG neurons. *** p < 0.0001 versus the saline-treated group. (C) The top panel illustrates the protocol for reconstruction of the I-V relationship from ramp recordings. The bottom panel shows I-V curves obtained in tonic neurons at 24 h post-saline or post-CFA. Note that IEM-1460 reverses the rectification of AMPA-induced currents recorded from neurons of CFA-treated rats. (D, E) The scatter plot illustrates the spread in rectification index (RI = I_+30mV/I_−50mV) (D), and the bar graph shows the statistical summary for RI (E) in tonic neurons 24 h post-saline and post-CFA before (black) and after (grey) IEM-1460 application. *** p < 0.0001 versus the saline-treated group, ^## p < 0.001 versus the CFA-treated group.

Fig. 6

Ultrastructural distribution of GluR1 and GluR2 in the superficial dorsal horn 24 h after saline (left) or CFA (right) injection. (A) Representative micrographs of postembedding immunogold labeling for GluR1 (5 nm) and GluR2 (15 nm). In these representative images, the synapses are marked by the presence of GluR2; GluR1 is more prevalent at synapses in the saline-treated group. In the CFA-treated group, GluR1 is evident in the extrasynaptic membrane (asterisk). PSD, postsynaptic density; pre, presynaptic terminal; scale bar = 100 nm. (B) The left bar graph shows the number of GluR1- and GluR2-labeled immunogold particles at synapses (Syn), at extrasynaptic membranes (Extra), and in cytoplasm (Cyto) of superficial dorsal horn neurons 24 h after CFA or saline injection. The right bar graph shows ratios of the number of GluR1- and GluR2-labeled particles in the CFA-treated group to those in the saline-treated group at synapses, extrasynaptic membranes, and cytoplasm of superficial dorsal horn neurons 24 h after injections.

Fig. 7

GluR1 membrane insertion in dorsal horn neurons 24 h after CFA injection. (A) Surface expression of GluR1 in dorsal horn neurons 24 h after CFA or saline injection. Top, representative Western blot; bottom, statistical summary of the densitometric analysis. The level of sample loaded for the total (T) expression was 10% of that for the biotinylated surface (S) expression. * p < 0.05 versus the saline-treated group. β-actin, an unbiotinylated intracellular protein, was used as a control. (B) Expression of GluR1 in the synaptosomal fraction from dorsal horn 1 day after CFA or saline injection. Top, representative Western blot; bottom, statistical summary of the densitometric analysis. N-cadherin, a membrane marker, was used as a control.