3.1 Peripheral inflammation increases AMPAR-mediated Ca2+-permeability in dorsal horn neurons
Kainate-induced cobalt uptake has been used to visualize Ca2+
-permeable AMPARs in dorsal horn neurons [18
]. To determine whether CFA-induced peripheral inflammation alters the expression of spinal Ca2+
-permeable AMPARs, we analyzed kainate-induced cobalt uptake in spinal cord transverse slices derived from adult rats (25–35 days) 24 h after saline or CFA injection. Although the dorsal horns of adult saline-treated rats contained fewer cobalt-positive cells than those of younger naïve rats (P6-14) [18
], their patterns of cobalt uptake were similar (). Cobalt-positive cells were predominantly observed in laminae I and II; few were distributed in laminae III–VII (). To assess whether this pattern reflected selective cobalt uptake through AMPARs, we pre-incubated the slices with GYKI 52466, CNQX, or APV. AMPAR antagonists, CNQX and GYKI 52466 entirely abolished kainate-induced cobalt uptake in the slices, whereas NMDAR antagonist APV had no effect (). To determine whether kainate-induced cobalt uptake occurred in neurons rather than in other cell types, we incubated the sections with a primary antibody against neuronal marker NeuN after kainate-induced cobalt uptake. In the dorsal horn, most cells that took up cobalt (90 ± 7%, n = 4) were positive for NeuN (). We found that CFA (but not saline) injection significantly increased cobalt uptake loading in dorsal horn on the ipsilateral, but not contralateral, side (). After 24 h, the number of cobalt-positive cells was 2.77-fold greater (p
< 0.01, n = 4) in laminae I–II and 3.73-fold greater (p
< 0.01, n = 4) in laminae III VII of CFA-injected rats than in those regions of saline-treated rats (). However, some NeuN-labeled cells in the superficial and deep dorsal horn were still not cobalt-positive (), indicating that this group of dorsal horn neurons fails to alter AMPAR Ca2+
permeability in response to peripheral inflammatory stimulation.
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 (more ...)
3.2 Peripheral inflammation augments extrasynaptic AMPAR-mediated currents and [Ca2+]i transients in “tonic” but not in “transient” lamina II neurons
The fact that a CFA-induced increase in cobalt uptake was observed predominantly in neuronal bodies of dorsal horn suggests that Ca2+-permeable AMPARs are upregulated in dorsal horn neurons during CFA-induced peripheral inflammation. By simultaneously recording AMPA-induced membrane current and associated [Ca2+]i changes in soma and dendrites of superficial lamina II neurons of the spinal L4–5 dorsal horn, we directly evaluated upregulation of AMPARs and their Ca2+ permeability 24 h after saline or CFA injection. According to intrinsic firing properties during sustained membrane depolarization, lamina II neurons were predominantly divided into two groups: “tonic” and “transient” (). Tonic neurons (n = 35) were defined as those able to support continued discharge of action potentials during 1-s depolarizing inward current and an increased frequency of discharge with increasing current intensity (). Transient neurons (n = 28) were those that exhibited a strong adaptation by generating short bursts of spikes or just a single spike regardless of depolarizing current intensity (). Tonic and transient neurons demonstrated similar changes in [Ca2+]i after depolarizing inward currents (), indicating that the contribution of voltage-gated Ca2+ channels to spike generation was similar in the two groups.
To activate the total pool of AMPARs and mimic excessive glutamate release from presynaptic neurons and glia during dorsal horn injuries or inflammation [2
], we bath applied AMPA to the slices. Bath administration of AMPA (5 μM, 60 s) evoked an inward current at a holding potential of −60 mV in both tonic and transient neurons (, bottom trace). It was characterized by a slow rising phase and desensitization to a plateau level. This current is predominantly mediated by extrasynaptic AMPARs due to their relative abundance compared to synaptic ones in the neuronal plasma membrane [3
]. No significant difference was observed between the amplitudes of the AMPA-induced currents in the tonic (−204 ± 18 pA, n = 35) and transient (−173 ± 19 pA, n = 28) neurons (; p
= 0.24). The current activation was associated with a synchronous rise in [Ca2+
in both the soma and dendrites in all examined neurons (, upper traces). A typical calcium response to AMPA application consisted of a fast initial transient rise in [Ca2+
followed by a slow decay to the baseline within several minutes. A comparison of [Ca2+
transient amplitudes, expressed as an increase in the ratio of fura-2 fluorescence at 340 and 380 nm, ΔR (see Methods), showed no significant differences in the amplitudes between tonic and transient groups of neurons. Average amplitudes of AMPA-induced [Ca2+
transients were 0.47 ± 0.10 (n = 21, tonic) vs 0.48 ± 0.09 (n = 22, transient; p
> 0.1) for soma and 0.60 ± 0.12 (n = 14, tonic) vs 0.52 ± 0.09 (n = 18, transient; p
> 0.1) for dendrites (). These findings indicate that the two groups of neurons have similar levels of AMPAR expression.
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 (more ...)
The observed AMPA-induced currents and [Ca2+]i transients were mediated by AMPAR activation, as NBQX, a non-selective AMPAR antagonist, and GYKI 52466, a selective AMPAR antagonist, significantly inhibited the currents. NBQX inhibited AMPA-induced current amplitudes by 97 ± 9% (n = 3, p < 0.0001) and 98 ± 11% (n = 3, p < 0.0001) in tonic and transient neurons, respectively. GYKI 52466 inhibited the amplitudes by 75 ± 15% (n = 5, p < 0.05) and by 86 ± 15% (n = 7, p < 0.01) in the tonic and transient neurons, respectively. NBQX inhibited somatic and dendritic [Ca2+]i transients in tonic neurons by 93 ± 20% and 88 ± 12%, respectively (n = 3, p < 0.05) and somatic [Ca2+]i transients in transient neurons by 92 ± 17% (n = 3, p < 0.05). GYKI 52466 inhibited somatic and dendritic [Ca2+]i transients in tonic neurons by 94 ± 20% (n = 5, p < 0.05) and 92 ± 18% (n = 5, p < 0.01), respectively, and inhibited somatic and dendritic [Ca2+]i transients of the transient group by 83 ± 15% (n = 3, p < 0.05) and 92 ± 15% (n = 3, p < 0.05), respectively.
CFA-induced inflammation significantly increased AMPA-induced currents and associated [Ca2+
transients in tonically firing group of neurons (). The amplitude of AMPA-induced inward current was increased by 125 ± 13% (n = 20; p
< 0.001) 24 h post-CFA compared to post-saline (). The increase in AMPA-induced current was associated with an augmentation in amplitudes of somatic and dendritic [Ca2+
transients. Twenty-four hours after CFA injection, the amplitude of [Ca2+
transients increased by 92 ± 11% and 96 ± 17% in soma and dendrites, respectively (n = 11; p
< 0.05; average amplitude in soma: 0.47 ± 0.10 post-saline vs 0.91 ± 0.10 post-CFA; average amplitude in dendrites: 0.60 ± 0.12 post-saline vs 1.17 ± 0.21 post-CFA; ). At the same time, we found no significant differences between the tonic neurons from the saline-and CFA-treated animals in electrophysiological properties such as input resistance (saline: 612 ± 111 MΩ, n = 25; CFA: 604 ± 75 MΩ, n = 25. p
> 0.5), capacitance (saline: 22 ± 1 pF, n = 26; CFA: 24 ± 1 pF, n = 30. p
= 0.2) as well as a series resistance (saline: 28 ± 2 MΩ, n = 25; CFA: 30 ± 2 MΩ, n = 25. p
= 0.4). These findings clearly indicate a marked increase in functional expression of AMPARs in the dendrites and soma of tonically firing SG neurons during the maintenance of persistent inflammation. The data from our laboratory [41
] and those of others [28
] have demonstrated that CFA-induced inflammatory input does not significantly alter the amplitude of synaptically evoked AMPAR-mediated excitatory postsynaptic currents (eEPSCs) in the superficial dorsal horn neurons at 24 h post-CFA. Thus, the increase in AMPA-induced currents in the tonically firing SG neurons that we observed after inflammation can be attributed to upregulation of an extrasynaptic pool of AMPARs. In addition, a marked increase in the amplitudes of [Ca2+
transients suggests that this upregulation of AMPA-induced current is accounted for by an increase in the number of extrasynaptic Ca2+
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 (more ...)
Interestingly, CFA-induced inflammation did not produce significant changes in AMPAR-mediated currents and associated somatic and dendritic [Ca2+]i transients in transient neurons. The amplitudes of AMPA-induced currents were 173 ± 19 pA in the saline-treated group (n = 28) and 150 ± 17 pA in the CFA-treated group (n = 21; p > 0.37; ). The amplitudes of AMPA-induced [Ca2+]i transients in soma were 0.48 ± 0.09 (n = 21) and 0.47 ± 0.08 (n = 8) in the saline- and CFA-treated groups, respectively (p > 0.9; ). The amplitudes of AMPA-induced [Ca2+]i transients in dendrites were 0.52 ± 0.09 (n = 18) and 0.59 ± 0.1 (n = 6) in the saline- and CFA-treated groups, respectively (p > 0.6; ). Additionally, inflammation did not significantly change series resistance (saline: 27 ± 1 MΩ, n = 38; CFA: 29 ± 1 MΩ, n = 32. p = 0.2), input resistance (saline: 682 ± 92 MΩ, n = 38; CFA: 663 ± 91 MΩ, n = 37. p > 0.5), or capacitance (saline: 17 ± 2 pF, n = 38; CFA: 19 ± 1 pF, n = 37. p = 0.5) in the transient neurons. These results indicate that the total pool of extrasynaptic and synaptic AMPARs in dendrites and somata was not changed in the transient group of SG neurons during maintenance of persistent inflammation.
3.3 Peripheral inflammation increases the proportion of Ca2+-permeable AMPARs in the total pool of extrasynaptic AMPARs
Persistent inflammation changes a proportion of Ca2+
-permeable AMPARs to Ca2+
-impermeable AMPARs at synapses in dorsal horn neurons during the maintenance period of CFA-induced inflammatory pain [28
]. To establish whether inflammation also changes this parameter for extrasynaptic populations of AMPARs, we first examined the effect of selective Ca2+
-permeable AMPAR blocker on AMPA-induced currents. IEM-1460, a polyamine derivative, rapidly and reversibly blocks Ca2+
-permeable AMPARs [10
]. In the saline-treated rats, IEM-1460 (40 μM) produced a small, insignificant effect on amplitudes of AMPA-induced currents when it was applied to slices before AMPA (5–7 min, , left graph) or when it was applied during a steady-state period of AMPA-induced current (, right graph). In tonic neurons from the saline-treated group, the inhibitory effect of IEM-1460 on AMPA-induced current amplitude was small and insignificant (7 ± 2%, n = 7; p
> 0.45; ), indicating that Ca2+
-permeable extrasynaptic AMPARs only slightly contribute to the total AMPA-induced current under normal conditions. On the contrary, sensitivity of synaptic AMPAR-mediated eEPSCs in SG neurons to Ca2+
-permeable AMPAR blockers was substantially greater (~23% inhibition [41
]), further indicating that synaptic AMPARs do not contribute substantially to the generation of AMPA-induced currents. In contrast, in the CFA-treated group, the inhibitory effect of IEM-1460 on AMPA-induced current amplitude was substantially enhanced in the tonic neurons (26 ± 5%; n = 10; p
< 0.01, ). This finding suggests that persistent peripheral inflammation not only increases the number of Ca2+
-permeable AMPARs at extrasynaptic sites in the tonic SG neurons but also increases their proportion in the total pool of extrasynaptic AMPARs. IEM-1460 had no significant effect on the amplitude of AMPA-induced current in the transient neurons from saline- or CFA-treated groups (saline: 7 ± 3%, n = 5, CFA: 15 ± 5%, n = 5, p
= 0.22, ).
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 (more ...)
The proportion of extrasynaptic Ca2+
-permeable AMPARs in the somatic and dendritic pools was also examined based on the unique rectification properties of AMPAR-mediated currents. To obtain an I-V
relationship of the AMPA-induced currents, we held neurons at −70 mV and ramped every 5 s initially to +50 mV and then to −70 mV before and during bath application of AMPA (, upper panel). To isolate an AMPAR-mediated component of a current, we subtracted the ramp currents recorded before AMPA application from those recorded during the agonist application (see Methods for details). In the tonic neurons, the I-V
curves showed weak rectification at positive membrane potentials in the saline-treated group that differed from the strong rectification of synaptic eEPSCs [41
]. However, a significant inward rectification was observed in the CFA-treated group (). To estimate the rectification of AMPA-induced currents, we calculated a rectification index expressed as a ratio of the current amplitudes at positive and negative membrane potentials (RI+30/−50
). RI was 0.74 ± 0.07 (n = 11) in the saline-treated group and 0.27 ± 0.05 (n = 12; p
< 0.001, ) in the CFA-treated group. In contrast, RI for synaptic AMPAR-mediated eEPSCs was 0.26 and 0.18 in SG neurons from saline-treated and CFA-inflamed rats, respectively [41
], further confirming that synaptic and extrasynaptic AMPA receptors have different properties.
CFA-induced inward rectification of AMPA-induced currents could be reversed by IEM-1460 (). The RI value was significantly increased to 0.91 ± 0.05 after IEM-1460 treatment (n = 5; p < 0.001, ). These findings further indicate an increased proportion of extrasynaptic Ca2+-permeable AMPARs in dorsal horn neurons during the maintenance of peripheral inflammation.
3.4 Peripheral inflammation induces GluR1 membrane insertion at extrasynaptic sites of dorsal horn neurons
To further validate whether extrasynaptic Ca2+
-permeable AMPARs are increased in plasma membrane of dorsal horn neurons under persistent inflammatory conditions, we used a combined approach of post-embedding immunogold labeling with electron microscopy (EM) to compare ultrastructural distribution of GluR1 and GluR2 in superficial dorsal horn 24 h after saline (n = 2) or CFA (n = 2) injection. We focused on these two subunits because they are more abundant in dorsal horn than are GluR3 and GluR4 [9
]. We counted the immunogold-labeled particles at synapses, in extrasynaptic membranes, and in cytoplasm in both saline- and CFA-treated groups. Consistent with previous reports [41
], we observed GluR1 and GluR2 immunogold labeling in postsynaptic membranes, cytoplasm, and extrasynaptic membranes in the saline-treated group (). CFA injection produced a tendency toward an increase in the number of GluR1-labeled particles in extrasynaptic membranes and a decrease in synaptic membranes and cytoplasm (). The ratio of the number of GluR1-labeled particles in the CFA-treated group to the number in the saline-treated group was 0.62 at synapses, 2.54 at extrasynaptic membranes, and 0.79 in the cytoplasm (n = 57; ). In agreement with our previous report [41
], CFA injection led to a decrease in the number of GluR2-labled particles at synapses and a tendency toward an increase in the cytoplasm (). The ratio of the number of GluR2-labeled particles in the CFA-treated group to the number in the saline-treated group was 0.61 at synapses, 1.05 at extrasynaptic membranes, and 1.2 in the cytoplasm (n = 47; ). These findings indicate that peripheral inflammation might induce GluR1 membrane insertion at extrasynaptic membranes and GluR2 internalization at synapses in superficial dorsal horn.
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 (more ...)
EM immunogold study of low-density extrasynaptic receptors has spatial and sample size limitations. To further confirm our observation, we used a surface biotinylation expression assay and synaptosomal fractionation approach to compare the expression of GluR1 in surface plasma membranes and in synaptic membranes derived from the dorsal horn. For the surface biotinylation expression assay, live slices were prepared from the ipsilateral L4–5
dorsal horn 24 h after saline (n = 4) or CFA (n = 4) injection. The surface receptors were labeled with biotin and then precipitated [41
]. The amount of surface GluR1 was 33% greater in the CFA-treated group than in the saline-treated group (p
< 0.05) (). In control experiments, β-actin, an intracellular protein, could not be precipitated by biotin (). Using differential centrifugation [49
], we collected synaptosomal fractions that contained abundant synaptic receptors from the ipsilateral L4–5
dorsal horn tissues 24 h after saline (n = 4) or CFA (n = 4) injection. Western blot analysis showed that the level of GluR1 in the synaptosomal fraction of the CFA-treated group was similar to that of the saline-treated group (p
> 0.05; ). Taken together, our findings suggest that the amount of GluR1 in extrasynaptic membranes is increased in dorsal horn under persistent inflammatory conditions.
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. (more ...)