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. 2010 Dec;151(3):633-643.
doi: 10.1016/j.pain.2010.08.030. Epub 2010 Sep 29.

Persistent inflammation alters the density and distribution of voltage-activated calcium channels in subpopulations of rat cutaneous DRG neurons

Shao-Gang Lu  1 , Xiu-Lin Zhang, David Z Luo, Michael S Gold
Affiliations

Persistent inflammation alters the density and distribution of voltage-activated calcium channels in subpopulations of rat cutaneous DRG neurons

Shao-Gang Lu et al. Pain. 2010 Dec.

Abstract

The impact of persistent inflammation on voltage-activated Ca(2+) channels in cutaneous DRG neurons from adult rats was assessed with whole cell patch clamp techniques, sqRT-PCR and Western blot analysis. Inflammation was induced with a subcutaneous injection of complete Freund's adjuvant (CFA). DiI was used to identify DRG neurons innervating the site of inflammation. Three days after CFA injection, high threshold Ca(2+) current (HVA) density was significantly reduced in small and medium, but not large diameter neurons, reflecting a decrease in N-, L- and P/Q-type currents. This decrease in HVA current was associated with an increase in mRNA encoding the α2δ1-subunit complex, but no detectable change in N-type subunit (Ca(V)2.2) mRNA. An increase in both α2δ1 and Ca(V)2.2 protein was detected in the central nerves arising from L4 and L5 ganglia ipsilateral to the site of inflammation. In current clamp experiments on small and medium diameter cutaneous DRG neurons from naïve rats, blocking ∼40% of HVA current with Cd(2+) (5μM), had opposite effects on subpopulations of cutaneous DRG neurons (increasing excitability and action potential duration in some and decreasing excitability in others). The alterations in the density and distribution of voltage-activated Ca(2+) channels in subpopulations of cutaneous DRG neurons that develop following CFA injection should contribute to changes in sensory transmission observed in the presence of inflammation.

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Figures

Figure 1
Figure 1
Voltage gated Ca2+ currents in cutaneous DRG neurons. A. High threshold (HVA) current in a typical small diameter cutaneous afferent had a threshold for activation ~−20 mV and demonstrated little inactivation over a 60 ms voltage step. The protocol used to evoked current is shown below current traces. B. Pooled (mean ± SEM) current-voltage (I–V) data from small (n = 33), medium (n = 26) and large (n = 14) diameter cutaneous DRG neurons from control ganglia. Current evoked in each neuron was normalized to the membrane capacitance. C. Pooled conductance-voltage (G–V) data generated from instantaneous I–V data (i.e., tail currents) from the same neurons plotted in B.
Figure 2
Figure 2
The impact of inflammation on membrane capacitance and capsaicin evoked currents in cutaneous DRG neurons. A. Histograms of membrane capacitance for control (CTRL, n = 73) and inflamed (CFA, n = 63) DRG neurons. Bin size is 6 pF. To facilitate comparing the two histograms, they have been plotted together and normalized with respect to the total number of neurons studied in each group. B. Typical capsaicin (CAP, 500 nM) evoked responses from a 30 μm diameter control (a) and 31 μm diameter inflamed (b) cutaneous afferent. The holding potential for both current traces was −60 mV. C. Pooled peak CAP evoked current from control (CTRL, n = 26) and inflamed (CFA, n = 22) neurons. * is p < 0.05 (Students t test).
Figure 3
Figure 3
The impact of inflammation on HVA currents in cutaneous DRG neurons. Pooled I–V data for small (S), medium (M) and large (L) diameter cutaneous DRG neurons normalized to cell body capacitance are plotted in panels A, B and C, respectively. Current density in small and medium, but not large diameter neurons from control (CTRL) rats is significantly larger than that in neurons from inflamed (CFA) rats. Normalized G–V data corresponding to the I–V data plotted in panels A, B and C are plotted in panels D, E and F. There was no detectable influence of inflammation on the voltage-dependence of activation in these subpopulations of cutaneous DRG neurons. The number of neurons in each group is indicated in parentheses.
Figure 4
Figure 4
The impact of inflammation on subpopulations of cutaneous DRG neurons defined by capsaicin (CAP) sensitivity. Peak inward HVA current density at 0 mV in CAP sensitive (CAP+) and CAP insensitive (CAP-) cutaneous DRG neurons from control (CTRL) and inflamed (CFA) ganglia. Current density was significantly (p 0.05) between CAP+ (n = 48) and CAP– (n = 61) neurons with respect to current density, nor was there an interaction (p > 0.05) between the impact of inflammation and CAP sensitivity.
Figure 5
Figure 5
Inflammation decreased multiple types of HVA current in cutaneous DRG neurons. HVA current subtypes were isolated pharmacologically. A. HVA current evoked in typical cutaneous afferent from a naïve rat before (Baseline) and after sequential application of nifedipine (Nif, 10 μM) to block L-type currents, ω-conotoxin GVIA (CTx, 200 nM) to block N-type currents, ω-agatoxin IVA (AgTx, 200 nM) to block P/Q-type currents, and cadmium (Cd2+, 50 μM) to block residual current (R). B. Diary plot of data from afferent shown in A. Pooled data from small (C) and medium (D) diameter neurons from inflamed rats analyzed as a percent change in current subtype density from that obtained in control neurons.
Figure 6
Figure 6
The impact of a decrease in HVA-current on the excitability of cutaneous DRG neurons. Blocking ~40% of HVA current resulted in an increase in the excitability of some small and medium diameter neurons (A, B and C) and a decrease in the excitability of others (D, E, and F). Depolarizing current injection in the form of a “ramp and hold” stimulus was used to assess excitability before (black traces) and after (grey traces) the application of Cd2+ (5 μM; A and D). A change in current threshold, action potential threshold, or the number of evoked spikes greater than 2 standard deviations from the baseline response was considered a response to Cd2+. The response to suprathreshold stimuli delivered in the form of a “square wave” depolarizing current injection (750 ms) at 1x, 2x and 3x rheobase was increased in neurons excited by Cd2+ (B, n = 7) and decreased in those inhibited by Cd2+ (E, n = 8). Changes in excitability were associated with consistent and significant changes in the action potential waveform in neurons excited by Cd2+ (C), while changes in those inhibited by Cd2+ (F) were more variable; voltage traces in C and F were typical with baseline traces in black and those obtained in the presence of Cd2+ in grey. Insets to each voltage trace are the afterhyperpolarizations on an expanded scale where the action potential is cut off to facilitate inspection of the AHP.
Figure 7
Figure 7
The impact of inflammation of Ca2+ channel subunit expression levels. SYBR green was used for semi-quantitative RT-PCR analysis of CaV2.2 (A) and α2δ1 (B) subunit expression levels. GAPDH was used as an internal comparator where the ΔΔCT method was used to determine the fold change in subunit expression in ganglia contralateral (Contra) and ispilateral (Ipsi) to the site of inflammation, relative to that in naïve ganglia (n = 4). L4 and L5 ganglia on each side were pooled so that the “n” for each group is an individual rat. While more inflamed ganglia were processed, data were still analyzed with a paired t-test, where * is p < 0.05.
Figure 8
Figure 8
The impact of inflammation on the Ca2+ channel subunit levels in the central nerves from L4 and L5 ganglia. Total protein was extracted from the central nerves of L4 and L5 ganglia from naïve rats and from the side contralateral (Contra) and ipsilateral (Ipsi) to the site of inflammation. Band intensity for channel subunits was normalized to that of the loading control (GAPDH). A sample of protein from rat brain was used to normalize data between blots. Pooled data revealed an increase in both CaV2.2 (A) and α2δ1 (B) in nerves ispilateral to the site of inflammation (p < 0.05, one-way ANOVA).
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