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Pain-related synaptic plasticity in spinal dorsal horn neurons: role of CGRP

Pain-related synaptic plasticity in spinal dorsal horn neurons: role of CGRP Background: The synaptic and cellular mechanisms of pain-related central sensitization in the spinal cord are not fully understood yet. Calcitonin gene-related peptide (CGRP) has been identified as an important molecule in spinal nociceptive processing and ensuing behavioral responses, but its contribution to synaptic plasticity, cellular mechanisms and site of action in the spinal cord remain to be determined. Here we address the role of CGRP in synaptic plasticity in the spinal dorsal horn in a model of arthritic pain. Results: Whole-cell current- and voltage-clamp recordings were made from substantia gelatinosa (SG) neurons in spinal cord slices from control rats and arthritic rats (> 6 h postinjection of kaolin/ carrageenan into the knee). Monosynaptic excitatory postsynaptic currents (EPSCs) were evoked by electrical stimulation of afferents in the dorsal root near the dorsal root entry zone. Neurons in slices from arthritic rats showed increased synaptic transmission and excitability compared to controls. A selective CGRP1 receptor antagonist (CGRP8-37) reversed synaptic plasticity in neurons from arthritic rats but had no significant effect on normal transmission. CGRP facilitated synaptic transmission in the arthritis pain model more strongly than under normal conditions where both facilitatory and inhibitory effects were observed. CGRP also increased neuronal excitability. Miniature EPSC analysis suggested a post- rather than pre-synaptic mechanism of CGRP action. Conclusion: This study is the first to show synaptic plasticity in the spinal dorsal horn in a model of arthritic pain that involves a postsynaptic action of CGRP on SG neurons. sensitization, the relative contribution of pre- and postsy- Background Inflammatory processes in peripheral tissues lead to cen- naptic mechanisms and of peripheral and supraspinal fac- tral sensitization in the spinal cord, which contributes to tors are not entirely clear. The superficial dorsal horn of hyperalgesia and allodynia typically associated with the spinal cord, particularly substantia gelatinosa (SG), is inflammatory pain. Although evidence suggests that plas- a major projection site of small-diameter afferent nerve tic changes in the spinal dorsal horn account for central fibers that predominantly transmit nociceptive signals Page 1 of 12 (page number not for citation purposes) Molecular Pain 2006, 2:31 http://www.molecularpain.com/content/2/1/31 [1,2]. SG neurons also receive descending inputs from the The source of CGRP in the spinal cord dorsal horn is pri- brainstem [1,3]. Therefore, in addition to intraspinal neu- mary afferents. CGRP coexists with substance P in small- roplastic changes, peripheral as well as supraspinal factors diameter afferent fibers, and CGRP containing terminals may contribute to central sensitization. and CGRP receptors are found in the dorsal horn, includ- ing SG [30-33]. CGRP is released in the spinal dorsal horn Pain-related neuroplastic changes in central nervous sys- by noxious stimulation and peripheral inflammation tem (CNS) structures can be shown definitively by the such as the K/C arthritis [26,34,35]. Peripheral inflamma- electrophysiological analysis of synaptic transmission and tion also leads to changes in CGRP binding sites in the neuronal excitability in spinal cord or brain slice prepara- dorsal horn [32,36]. tions obtained from animals in which an experimental pain state has been induced [4-7]. The slice preparation Spinal application of CGRP facilitates nociceptive behav- allows the analysis of pain-related plasticity because it is ior [24,37,38] and sensitizes the responses of dorsal horn disconnected from the site of peripheral injury (inflam- neurons to innocuous and noxious peripheral stimula- mation) and from other CNS areas, be it supraspinal sites tion [28,29,38,39] and to intraspinally administered exci- (spinal cord slice) or spinal cord (brain slices). Therefore, tatory amino acids [23] and substance P [39]. In a slice changes measured in the slice preparation are maintained preparation, CGRP produced a slow depolarization and independently of continuous inputs to the area of interest. enhanced excitability of dorsal horn neurons; the effect on Accordingly, changes of synaptic circuitry in SG neurons evoked synaptic transmission was not studied [40]. Con- were shown in slices from animals with complete Fre- versely, block of spinal CGRP receptors with an antagonist und's adjuvant induced hindpaw inflammation [4,5,8,9] (CGRP8-37) or antiserum induced antinociception in ani- and synaptic plasticity was demonstrated in amygdala mal models of inflammatory [25,41,42,42-44] or central neurons from animals with knee joint arthritis [7,10,11]. neuropathic pain [45]. CGRP8-37 also inhibited the responses of spinal dorsal horn neurons to transdermial The kaolin and carrageenan (K/C) induced knee joint electrical stimulation of the hindpaw [46] and to noxious arthritis is a well established model of inflammatory pain. mechanical stimulation of the knee joint [29]. CGRP8-37 Electrophysiological, pharmacological, neurochemical prevented or reversed central sensitization of dorsal horn and behavioral studies have used this model to analyze neurons in the arthritis and capsaicin pain models pain mechanisms at different levels of the nervous system [28,29]. Arthritic CGRP knockout mice showed reduced and showed the sensitization of primary afferent nerve nociceptive behavioral responses [47]. fibers, spinal dorsal horn neurons and neurons in the cen- tral nucleus of the amygdala (CeA) [12-17]. Using slice Although it is widely accepted that CGRP plays an impor- preparations, synaptic plasticity was demonstrated in the tant role in the modulation of spinal nociceptive process- CeA, but not yet in the spinal cord, in the K/C arthritis ing, the cellular mechanisms and pre- or post-synaptic pain model. sites of action through which CGRP contributes to central sensitization remain to be determined. The present study The purpose of this study was to compare synaptic trans- addressed the role of CGRP in synaptic plasticity in the mission and neuronal excitability in SG neurons in spinal superficial dorsal horn in vitro in a model of arthritic pain cord slices from normal and from arthritic animals using induced in vivo. Our data show for the first time synaptic patch-clamp recordings. Another goal was to determine plasticity and increased excitability of SG neurons in the the role of calcitonin gene-related peptide (CGRP) in K/C arthritis pain model. A CGRP receptor antagonist pain-related spinal plasticity since CGRP has emerged as inhibits synaptic plasticity whereas CGRP itself facilitates an important molecule at different levels of the pain neu- synaptic transmission through a postsynaptic mechanism raxis in the arthritis pain model. that involves direct membrane effects on SG neurons. CGRP is a 37 amino acid peptide that activates adenylyl Results cyclase and protein kinase A through G-protein-coupled Whole-cell patch-clamp recordings of SG neurons were receptors, including the CGRP1 receptor for which selec- made in spinal cord slices from normal naïve rats (n = 31 tive antagonists are available [18-21]. CGRP is involved in neurons) and rats with a knee joint arthritis induced 6 h peripheral and spinal pain mechanisms [22-29]. We before slices were obtained (n = 25 neurons). The record- showed recently that CGRP also plays an important role ing sites were always visually verified to be in the central in the transmission of nociceptive information to the part of the gray translucent region forming lamina II. All amygdala through the spino-parabrachio-amygdaloid SG neurons in this study showed monosynaptic responses pathway [10]. (excitatory postsynaptic currents, EPSCs) to electrical stimulation of afferent fibers in the dorsal root (DR) near the dorsal root entry zone (DREZ). EPSCs were judged to Page 2 of 12 (page number not for citation purposes) Molecular Pain 2006, 2:31 http://www.molecularpain.com/content/2/1/31 be monosynaptic on the basis of stable latencies of the trol neurons (n = 17), i.e., action potential firing occurred EPSC peak amplitude (coefficient of variation < 2%, at more hyperpolarized membrane potentials (Fig. 2D; P [48,49]). Calculated from latency and distance between < 0.01, unpaired t-test). stimulation and recording sites, the conduction velocities (CV) ranged from 0.15 to 0.85 m/s (mean 0.46 ± 0.03 m/ Inhibition of pain-related synaptic plasticity by a CGRP1 s), which is in the range of rodent C-fibers [48,49]. No dif- receptor antagonist (CGRP8-37) ference in resting transmembrane potential (RMP) and CGRP8-37 (1 μM; 10 min) inhibited synaptic transmis- input resistance (Ri) was detected between neurons from sion in SG neurons in slices from arthritic animals but had normal rats (RMP = -58.7 ± 1.7 mV; Ri = 211.8 ± 16.6 MΩ) and from arthritic rats (RMP = -57.0 ± 1.8 mV; Ri = 205.3 ± 13.7 MΩ). Synaptic plasticity in SG neurons in the arthritis pain model Input-output functions of monosynaptic inputs to SG neurons increased in the arthritis pain model (Figure 1). Monosynaptic EPSCs with progressively larger amplitudes were evoked by electrical DR/DREZ stimulation with increasing intensities. Compared with control SG neurons from normal animals, synaptic transmission was signifi- cantly enhanced in SG neurons recorded in slices from arthritic rats. Input-output relationships were obtained by measuring EPSC peak amplitude (pA) as a function of afferent fiber stimulus intensity (μA) for each neuron (see individual examples of an SG neuron in a slice from a nor- mal animal [Fig. 1A] and in an SG neuron from an arthritic animal [Fig. 1B]). In arthritis, evoked monosyn- aptic EPSCs had larger amplitudes, but EPSC threshold was unchanged. The input-output relationships of SG neurons from control rats (n = 16) and SG neurons from arthritic rats (n = 9) were significantly different (Fig. 1C; P < 0.0001, F = 58.45, two-way ANOVA). These data 1,207 show enhanced synaptic transmission at first-order syn- apses on SG neurons in the arthritis pain model. Enhanced synaptic transmission in the reduced slice prep- aration indicates synaptic plasticity because the arthritis pain-related changes are maintained, at least in part, inde- pendently of peripheral and supraspinal mechanisms. Syna arth Figure 1 ritis p ptic tr aansmission in model in SG neurons is enhanced in the Synaptic transmission in SG neurons is enhanced in Increased excitability of SG neurons in the arthritis pain the arthritis pain model. A,B, Whole-cell voltage-clamp model recordings of monosynaptic EPSCs evoked with increasing Compared with control neurons, neurons from arthritic stimulus intensities in an SG neuron in a spinal cord slice from a normal animal and in an SG neuron in a slice from an rats had a lower threshold and higher rate of action poten- arthritic animal (obtained 6 h post-induction of arthritis). tial firing generated by direct depolarization of the cell via Evoked monosynaptic EPSCs had larger amplitudes in arthri- the recording electrode in current-clamp mode (Figure 2). tis than under control conditions. Square wave electrical Input-output functions of neuronal excitability were stimuli of 150 μs duration were delivered at a frequency < obtained by measuring the number of action potentials 0.25 Hz. Stimulus intensity was increased from 0–800 μA. (Hz) evoked by depolarizing current pulses of increasing Each trace is the average of 3–4 EPSCs. Neurons were held magnitude (0 to 200 pA; see individual examples in Fig. at -60 mV. C, Input-output relationships of monosynaptic 2A and 2B). Input-output functions of SG neurons from EPSC peak amplitudes (pA) evoked in SG neurons from nor- arthritic animals (n = 13) were significantly increased mal rats (n = 16) and from arthritic rats (n = 9) were signifi- compared to control neurons from normal animals (n = cantly different. * P < 0.05, ** P < 0.01 (two-way ANOVA 25; Fig. 2C; P < 0.001; F = 12.77, two-way ANOVA). followed by Bonferroni posttests). Data are given as the 1,180 means ± SEM. The threshold for evoking action potentials was lower in SG neurons from arthritic animals (n = 11) than in con- Page 3 of 12 (page number not for citation purposes) Molecular Pain 2006, 2:31 http://www.molecularpain.com/content/2/1/31 Synaptic facilitation by CGRP is enhanced in the arthritis pain model CGRP enhanced synaptic transmission in a concentra- tion-dependent fashion (Figure 4). Individual examples show that CGRP (10 nM; 10 min) increased monosynap- tic EPSCs in an SG neuron from an arthritic rat (Fig. 4B) more strongly than in an SG neuron from a normal rat (Fig. 4A). Concentration-response data (Fig. 4C) show that the maximum facilitatory effect (efficacy) of CGRP was significantly (P < 0.01, F = 9.58, two-way ANOVA) 1,26 greater in SG neurons from arthritic rats (n = 16) com- pared to control neurons from normal animals (n = 10). The potency of CGRP was comparable under normal con- ditions (EC = 2.0 nM) and in arthritis (EC = 1.4 nM). 50 50 It should be noted that CGRP attenuated synaptic trans- mission in 6 of 16 SG neurons from normal animals but not in any SG neuron from arthritic animals. Postsynaptic effects of CGRP To determine the site of action of CGRP we used well- established electrophysiological methods, including the analysis of miniature EPSCs (mEPSCs) (Figure 5) and Increased excitability of SG ne mo Figure 2 del urons in the arthritis pain neuronal excitability (Figure 6). Presynaptic changes at Increased excitability of SG neurons in the arthritis the transmitter release site affect mEPSC frequency, pain model. Increased action potential firing rates and decreased thresholds for action potentials were recorded in whereas changes at the postsynaptic membrane alter SG neurons in slices from arthritic rats compared to con- mEPSC amplitude (quantal size) [50,51]. CGRP (10 nM; trols. A, B, Current-clamp recordings of action potentials 10 min) increased the amplitude of mEPSCs in TTX (1 (spikes) generated by direct intracellular injections of depo- μM)-containing ACSF without affecting their frequency, larizing current pulses of increasing magnitude (0 to 200 pA; suggesting a post- rather than pre-synaptic site of action 500 ms) in an SG neuron from a normal animal (A) and in an (Figure 5). This postsynaptic effect is illustrated in the cur- SG neuron from an arthritic animal (B). C, Analysis of the rent traces recorded in voltage-clamp mode in an individ- input-output relationships shows significantly increased spike ual SG neuron (Fig. 5A). Normalized cumulative frequency in arthritis (n = 13 neurons) compared to control distribution analysis of mEPSC amplitude and frequency (n = 25 neurons; P < 0.05; two-way ANOVA followed by shows that CGRP caused a significant shift toward higher Bonferroni posttests). D, Significantly decreased spike thresholds (membrane potentials at which action potential amplitude in this neuron (see Fig. 5B and 5C) and also firing started) were recorded in SG neurons in arthritis (n = increased mean mEPSC amplitude in the sample of neu- 11) compared to control neurons (n = 17; P < 0.01; unpaired rons (Fig. 5B, inset; P < 0.05, paired t-test, n = 5) but had t-test). * P < 0.05, ** P < 0.01. no effect on the interevent interval (frequency) distribu- tion (Fig. 5C, inset). no significant effect on normal synaptic transmission CGRP increased neuronal excitability (Figure 6). Action (Figure 3). Individual examples show that CGRP8-37 potentials were evoked in current-clamp mode by direct clearly inhibited monosynaptic EPSCs recorded in an SG depolarizing current injections (500 ms) of increasing neuron in a slice from an arthritic rat (Fig. 3B) but had lit- magnitude (0 to 250 pA) through the patch electrode (Fig. tle effect in an SG neuron in a slice from a normal rat (Fig. 6A and 6B). Input-output functions of neuronal excitabil- 3A). In the sample of SG neurons from arthritic rats (n = ity were obtained by averaging the frequency of action 5), CGRP8-37 inhibited synaptic strength (measured as potentials (spikes) evoked at each current intensity (Fig. peak amplitudes, Fig. 3C) and total charge (measured as 6C). CGRP (10 nM; 10 min) increased the input-output area under the curve, Fig. 3D) significantly (P < 0.01, function significantly (n = 5; P < 0.001; F = 12.77, 1,180 paired t-test), but had no significant effect on synaptic two-way ANOVA) while lowering the threshold for action transmission in SG neurons from normal rats (n = 7). potential generation to more hyperpolarized membrane These data suggest that CGRP1 receptors are endog- potentials (Fig. 6D; P < 0.01, paired t-test). In the presence enously activated to facilitate synaptic transmission in the of TTX (1 μM) CGRP (10 nM) also induced an inward arthritic pain model. Next, we determined the effect and membrane current that was significantly larger (P < 0.01, site of action of the receptor ligand CGRP. unpaired t-test) in SG neurons from arthritic rats (27.3 ± Page 4 of 12 (page number not for citation purposes) Molecular Pain 2006, 2:31 http://www.molecularpain.com/content/2/1/31 Figure 3 CGRP8-37 inhibits pain-related synaptic plasticity but has no significant effect on normal synaptic transmission CGRP8-37 inhibits pain-related synaptic plasticity but has no significant effect on normal synaptic transmis- sion. A, B, CGRP8-37 (1 μM) inhibited monosynaptic EPSCs recorded in an SG neuron in a slice from an arthritic rat (B) but not in another SG neuron in a slice from a normal rat (A). Each trace is the average of 8–10 monosynaptic EPSCs. C, D, CGRP8-37 (1 μM) significantly inhibited the EPSC peak amplitude (C), a measure of synaptic strength, and area under the curve (total charge, D) in SG neurons in slices from arthritic rats (P < 0.01, paired t-test, n = 5) but not in control neurons (n = 7) from normal rats. Analysis of raw data (pA, pC) is shown on the left; normalized data (% of predrug values) are shown on the right in C and D. Voltage-clamp recordings were made at -60 mV. CGRP8-37 was applied by superfusion of the slice in ACSF for 10–12 min. ** P < 0.01 (paired t-test). Page 5 of 12 (page number not for citation purposes) Molecular Pain 2006, 2:31 http://www.molecularpain.com/content/2/1/31 E mo Figure 4 nhanced syn del aptic facilitation by CGRP in the arthritis pain Miniature EPSC (mEPSC) pre-synaptic effects of CGRP Figure 5 analysis indicates post- rather than Enhanced synaptic facilitation by CGRP in the arthri- Miniature EPSC (mEPSC) analysis indicates post- tis pain model. A, B, Whole-cell voltage-clamp recordings rather than pre-synaptic effects of CGRP. A, Original of monosynaptic EPSCs in an SG neuron in a slice from a current traces of mEPSC recorded in an individual SG neu- normal animal (A) and in another SG neuron in a slice from ron in the presence of TTX (1 μM) show that CGRP (10 nM; an arthritic animal (B, 6 h postinduction of arthritis). CGRP 10 min) increases amplitude but not frequency of mEPSCs. (10 nM) potentiated synaptic transmission more strongly in B, C: Normalized cumulative distribution analysis of mEPSC arthritis than under normal conditions. Square wave electri- amplitude and frequency in the same neuron as in 5A shows cal stimuli of 150 μs duration were delivered at a frequency < that CGRP caused a significant shift toward higher amplitude 0.25 Hz. Each trace is the average of 8–10 EPSCs. C, Con- (B, P < 0.001, Kolmogorov-Smirnov test) but had no effect centration-response data show that the maximum effect (effi- on the interevent interval (frequency) distribution (C). In the cacy) of CGRP was significantly greater in SG neurons from sample of neurons (n = 5) CGRP selectively increased mean arthritic rats (n = 16) compared to control neurons from mEPSC amplitude (P < 0.05, paired t-test) but not mEPSC normal animals (n = 10). Peak EPSC amplitudes during each frequency (see bar histograms in B, C). Symbols and error concentration of CGRP were averaged and expressed as per- bars represent mean ± SEM. Neurons were recorded in volt- cent of predrug (baseline) control (100%). Sigmoid curves age-clamp at -60 mV. * P < 0.05. were fitted to the data using the following formula for nonlin- ear regression (GraphPad Prism 3.0; Y = A+(B-A)/[1+(10C/ 10X)D], where A = bottom plateau, B = top plateau, C = log(EC50), D = slope coefficient. Symbols show mean ± SEM. 3.1 pA, n = 5) than in SG neurons from normal rats (12.4 Neurons were held at -60 mV. CGRP was applied by super- ± 2.9 pA, n = 5). These data suggest a direct postsynaptic fusion of the slice in ACSF for 10 min. * P < 0.05 (two-way effect on membrane properties. ANOVA followed by Bonferroni posttests). Discussion The key findings of this study are as follows. Synaptic transmission and neuronal excitability in SG neurons are increased in slices from arthritic rats compared to control Page 6 of 12 (page number not for citation purposes) Molecular Pain 2006, 2:31 http://www.molecularpain.com/content/2/1/31 CGRP increase Figure 6 s neuronal excitability and induces direct membrane currents CGRP increases neuronal excitability and induces direct membrane currents. A, B, Current-clamp recordings of action potentials (spikes) generated in an SG neuron by direct intracellular injections of depolarizing current pulses of increas- ing magnitude (0 to 250 pA; 500 ms) before (A) and during CGRP (10 nM, B). C, CGRP increased input-output function by sig- nificantly increasing spike frequency (n = 5 neurons; P < 0.05–0.01, two-way ANOVA followed by Bonferroni posttests). For the measurement of action potential firing in current-clamp, neurons were recorded at -60 mV. D, CGRP (10 nM) also decreased spike thresholds (membrane potentials at which action potential firing started) significantly (n = 5; P < 0.01, paired t- test). * P < 0.05, ** P < 0.01. neurons from normal rats. These data suggest plastic and supraspinal influences. Blockade of CGRP receptors changes in the arthritis pain model that are maintained in inhibits synaptic plasticity in SG neurons from arthritic the reduced slice preparation independently of peripheral animals, suggesting the contribution of endogenously Page 7 of 12 (page number not for citation purposes) Molecular Pain 2006, 2:31 http://www.molecularpain.com/content/2/1/31 activated CGRP receptors. CGRP facilitates synaptic trans- changes rather than variability of the experimental condi- mission and increases neuronal excitability through a tions. postsynaptic site of action. The present study also offers a mechanism for these plastic This study focused on synaptic transmission of afferent changes: enhanced function of CGRP1 receptors. A widely information to SG neurons. According to conventional used selective CGRP1 receptor antagonist (CGRP8-37) criteria such as stable latencies of the EPSC peak [48,49], [18,20,21] inhibited synaptic plasticity but had little effect synaptic responses were considered monosynaptic and on normal transmission. These findings are consistent had a latency that indicated a slow conduction velocity with in vivo data showing that blockade of spinal CGRP (CV) of the responsible afferents in the range of rodent C- receptors inhibits sensitization of dorsal horn neurons in fibers [48,49,52]. However, a note of caution should be pain models [28,29] and nociceptive behavior added. Since we were not able to preserve long dorsal [25,25,41,42,42-45] in pain models. Here we show for the roots in the majority of the experiments but rather stimu- first time that the DR-SG circuitry is one site of action of lated the DR stump near the DREZ, we can not be sure that CGRP receptors to modulate synaptic transmission. CGRP the calculated CV at the central terminal accurately reflects containing terminals and CGRP receptors are present in the CV in the axon of the peripheral fiber. Still, it is evi- the dorsal horn, including SG [30-33]. CGRP is released in dent that afferent input to SG neurons from small diame- the dorsal horn in the K/C arthritis model [34]. ter fibers but not fast conducting A-beta fibers was studied here. Further, the SG neurons included in this study ful- Our data with exogenously administered CGRP further filled the criteria for central SG neurons with monosynap- indicate a post- rather than pre-synaptic site of action at tic C-fiber input as described in detail by others [48,49]. the DR-SG synapse and a direct membrane effect on SG neurons. This mechanism of action could explain the Our data show for the first time changes of synaptic trans- CGRP-induced sensitization of dorsal horn neurons in mission in small diameter fibers to SG neurons in a model vivo [23,28,29,38,39,39] and facilitation of nociceptive of arthritic pain. Previously, the slice preparation had behavior [24,37,38]. CGRP has been shown before to been used to determine changes in transmission to SC depolarize and increase excitability of dorsal horn neu- neurons in an inflammatory pain model induced by intra- rons in current-clamp [40]. Our simultaneous recording plantar complete Freund's adjuvant 48 h or 7–10 d before and analysis of evoked synaptic transmission, miniature slices were obtained [4,5,9]. In these studies, changes in EPSCs and membrane currents in voltage-clamp and synaptic transmission had been observed. They included excitability in current-clamp show a direct facilitatory a lower threshold for evoking EPSCs in SG neurons, a rel- action of CGRP on SG neurons to increase their respon- ative increase in the C-fiber versus A-delta fiber evoked siveness to afferent input and their output (action poten- EPSC amplitude, and an increased percentage of SG neu- tial generation). The enhanced CGRP function in the rons receiving mono- or polysynaptic A-beta input [4,5,9]. arthritis pain model could involve a change in the cou- pling to downstream effector systems such as kinases and The present study extends these observations in several ion channels as well as increased receptor expression or ways. We used a different pain model, the K/C induced affinity. The effects of peripheral inflammation on CGRP arthritis that produces electrophysiological and behavio- binding sites in the dorsal horn have been reported to be ral changes in vivo with a well defined and highly repro- somewhat inconsistent in that a mixture of up- and down- ducible time course [10,11,16,17]. This allowed us to regulation was found [32,36]. The inhibitory effect of select a constant time point (6 h postinduction of arthri- CGRP on normal synaptic transmission observed in some tis) to study changes in the slice preparation. Arthritis neurons could reflect an action on the recently described pain-related changes reach a plateau 6 h postinduction inhibitory projection islet cells on central SG neurons and persist at that level for days. Another novel aspect of [48]. our studies is the analysis of complete input-output func- tions of the DR-SG synapse and of neuronal excitability of SG neurons with C-fiber input (such as those selected in SG neurons. Our data show synaptic plasticity combined the present study) have been shown to excite monosynap- with excitability changes in the arthritis pain model. There tically SG neurons with A-delta input. These neurons then was no significant change of the threshold for evoking excite monosynaptically lamina I neurons [49], some of EPSCs suggesting that the stimulation and recording con- which project rostrally to form the spino-parabrachio- ditions were indeed comparable in slices from normal amygdaloid pathway [49,53,54]. This pathway is highly and arthritic animals. The fact that differences of synaptic peptidergic and utilizes CGRP to transmit information to transmission between normal and arthritic conditions the amygdala [10] were observed at different stimulus intensities along the input-output relationships further suggests functional Page 8 of 12 (page number not for citation purposes) Molecular Pain 2006, 2:31 http://www.molecularpain.com/content/2/1/31 Electrophysiology Conclusion This study is the first to show synaptic plasticity in the spi- Whole-cell current- and voltage-clamp recordings were nal dorsal horn (SG) in a model of arthritic pain. Synaptic made from substantia gelatinosa (SG) neurons in trans- plasticity involves CGRP1 receptor activation. CGRP acts verse lumbar spinal cord slices (500 μm) from normal rats postsynaptically to increase the input and output func- (controls) and arthritic rats (16–21 day old), using DIC- tions of SG neurons. enhanced infrared video-microscopy for visualization or the "blind" patch technique as in our previous studies [7,10,11]. Patch electrodes were made from borosilicate Methods Male Sprague Dawley rats (16–21 d) were housed in a glass capillaries (1.5 mm outer diameter, 1.12 mm inner temperature controlled room and maintained on a 12 h diameter; Drummond, Broomall, PA) pulled on a Flam- day/night cycle. Water and food were available ad libitum. ing-Brown micropipette puller (P-80/PC; Sutter Instru- Electrophysiological data were obtained from untreated ments, Novato, CA). Patch electrodes had tip resistances normal rats and rats with monoarthritis in the knee (6 h of 4–6 MΩ. The following internal solution was used after induction). All experimental procedures were (compounds in mM): K-Gluconate (122), NaCl (5), (0.3), EGTA (1), HEPES (10), Na ATP (5), Na GTP approved by the Institutional Animal Care and Use Com- CaCl 2 2 3 mittee (IACUC) at the University of Texas Medical Branch (0.4), and MgCl (2); pH 7.3; 300 mOsm. (UTMB) and conform to the guidelines of the Interna- tional Association for the Study of Pain (IASP) and of the Recording electrodes were positioned in the center of the National Institutes of Health (NIH). SG under visual control. The boundaries of the SG are eas- ily discerned under light microscopy. After tight (> 1 GΩ) Arthritis pain model seals were formed and the whole-cell configuration was In the group of arthritic rats, arthritis was induced in the obtained, neurons were included in the sample if the rest- left knee joint as previously described [7,10,11]. A kaolin ing membrane potential was more negative than -50 mV suspension (4%, 100 μl) was injected into the left knee and action potentials overshooting 0 mV were evoked by joint cavity through the patellar ligament. After repetitive direct depolarizing current injection through the record- flexions and extensions of the knee for 15 minutes, a car- ing electrode. Data acquisition and analysis of voltage and rageenan solution (2%, 100 μl) was injected into the knee current signals were done using a dual 4-pole Bessel filter joint cavity, and the leg was flexed and extended for (Warner Instrument Corp., Hamden, CT), low-noise Dig- another 5 minutes. Spinal cord slices were obtained 6 h idata 1322 interface (Axon Instruments, Foster City, CA), after arthritis induction. Axoclamp-2B or Axopatch 200 B amplifiers (Axon Instr.), Pentium PC, and pCLAMP8 and pCLAMP9 software Spinal cord slice preparation (Axon Inst.). Recordings were made at -60 mV. Series Transverse spinal cord slices were prepared using a modi- resistance was at least one order of magnitude less than fied version of the technique established by E.R. Perl's input resistance and was continuously monitored group [48,49]. Rats were deeply anesthetized with pento- throughout the experiment. barbital (50 mg/kg, i.p.). After a lumbosacral laminec- tomy the spinal cord with associated dorsal roots on one Monosynaptic excitatory postsynaptic currents (EPSCs) side was quickly removed and placed in ice-cold, sucrose- were evoked by stimulation of the DR with a suction elec- substituted, artificial cerebrospinal fluid (sucrose ACSF) trode or, in the majority of experiments, by focal stimula- containing (in mM): sucrose (234), KCl (3.6), CaCl tion of the DR stump near the DREZ with a concentric (2.5), MgCl (1.2), NaH PO (1.2), NaHCO (25), and bipolar electrode. Electrical stimuli (150 μs square-wave 2 2 5 3 glucose (12); equilibrated to pH 7.4 with a mixture of pulses) were delivered at frequencies below 0.25 Hz. 95% O and 5% CO . A vibrotome (Camden Instruments, Input-output relationships were obtained by increasing 2 2 London, UK) was used to prepare transverse (500 μm the stimulus intensity in 50 or 100 μA steps. For the eval- thick) slices from the lumbar spinal cord. Spinal cord uation of a drug effect on synaptically evoked responses, slices were maintained at room temperature (21°C) for at the stimulus intensity was adjusted to 80% of the intensity least 1 h in standard ACSF containing (in mM): NaCl required for orthodromic spike generation. EPSCs were (117), KCl (4.7), NaH PO (1.2), CaCl (2.5), MgCl judged to be monosynaptic on the basis of stable latencies 2 4 2 2 (1.2), NaHCO3 (25), glucose (11); equilibrated to pH 7.4 of the EPSC peak amplitude (coefficient of variation < 2% with 95% O /5% CO . A single slice was then transferred [48,49]). CV was estimated from latency of the evoked 2 2 to the recording chamber and submerged in ACSF, which EPSC peak and the conduction distance between stimula- superfused the slice at ~5 ml/min. tion and recording sites. The following parameters were recorded to measure arthritis-related or drug-induced changes. Peak amplitude Page 9 of 12 (page number not for citation purposes) Molecular Pain 2006, 2:31 http://www.molecularpain.com/content/2/1/31 and area under the curve (AUC) of monosynaptic EPSCs DREZ, dorsal root entry zone (typically the mean of 8–10 consecutive EPSCs) were measured in voltage-clamp to determine synaptic strength EPSC, excitatory postsynaptic current and total charge, respectively. Frequency and amplitude of miniature EPSCs (mEPSCs, recorded in 1 μM TTX) were K/C, kaolin and carrageenan determined from 1 min recording periods (voltage- clamp) using the Mini Analysis Program 6.0.3 (Synap- Ri, input resistance tosoft Inc., Decatur, GA). Input-out functions of excitabil- ity were calculated from the number of evoked action RMP, resting transmembrane potential potentials (spikes) evoked by direct intracellular injec- tions of depolarizing currents (500 ms; increments of 50 SG, substantia gelatinosa pA). Competing interests Drugs The author(s) declare that they have no competing inter- CGRP (receptor agonist) and CGRP8-37 (selective CGRP1 ests. receptor antagonist) [18,20,21] were dissolved in ACSF on the day of the experiment and applied to the spinal Authors' contributions cord slice by gravity-driven superfusion in the ACSF for 10 GCB carried out the majority of the experiments. VN, JSH min at a rate of 5 ml/min. Solution flow into the recording and HA performed additional experiments. GCB, JSH, YF, chamber (1 ml volume) was controlled with a three-way HA and VN performed the data analysis. VN and WDW stopcock. Duration of drug application and concentra- conceptualized the project and formulated the hypothe- tions were selected based on our previous studies [10]. sis. VN designed and directed the experiments and wrote the manuscript. Data analysis and statistics All averaged values are given as the mean ± SEM. Statisti- Acknowledgements This work was supported by NIH grants NS38261 and NS11255. cal significance was accepted at the level of P < 0.05. Input-output functions and concentration-response rela- References tionships were compared using a two-way ANOVA with 1. Willis WD, Coggeshall RE: Sensory mechanisms of the spinal cord 3rd Bonferroni posttests. EC values were calculated from sig- edition. New York, Plenum; 2004:1-962. moid curves fitted to the cumulative concentration- 2. 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Pain-related synaptic plasticity in spinal dorsal horn neurons: role of CGRP

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Copyright © 2006 by Bird et al; licensee BioMed Central Ltd.
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Medicine & Public Health; Pain Medicine; Molecular Medicine; Neurobiology
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Abstract

Background: The synaptic and cellular mechanisms of pain-related central sensitization in the spinal cord are not fully understood yet. Calcitonin gene-related peptide (CGRP) has been identified as an important molecule in spinal nociceptive processing and ensuing behavioral responses, but its contribution to synaptic plasticity, cellular mechanisms and site of action in the spinal cord remain to be determined. Here we address the role of CGRP in synaptic plasticity in the spinal dorsal horn in a model of arthritic pain. Results: Whole-cell current- and voltage-clamp recordings were made from substantia gelatinosa (SG) neurons in spinal cord slices from control rats and arthritic rats (> 6 h postinjection of kaolin/ carrageenan into the knee). Monosynaptic excitatory postsynaptic currents (EPSCs) were evoked by electrical stimulation of afferents in the dorsal root near the dorsal root entry zone. Neurons in slices from arthritic rats showed increased synaptic transmission and excitability compared to controls. A selective CGRP1 receptor antagonist (CGRP8-37) reversed synaptic plasticity in neurons from arthritic rats but had no significant effect on normal transmission. CGRP facilitated synaptic transmission in the arthritis pain model more strongly than under normal conditions where both facilitatory and inhibitory effects were observed. CGRP also increased neuronal excitability. Miniature EPSC analysis suggested a post- rather than pre-synaptic mechanism of CGRP action. Conclusion: This study is the first to show synaptic plasticity in the spinal dorsal horn in a model of arthritic pain that involves a postsynaptic action of CGRP on SG neurons. sensitization, the relative contribution of pre- and postsy- Background Inflammatory processes in peripheral tissues lead to cen- naptic mechanisms and of peripheral and supraspinal fac- tral sensitization in the spinal cord, which contributes to tors are not entirely clear. The superficial dorsal horn of hyperalgesia and allodynia typically associated with the spinal cord, particularly substantia gelatinosa (SG), is inflammatory pain. Although evidence suggests that plas- a major projection site of small-diameter afferent nerve tic changes in the spinal dorsal horn account for central fibers that predominantly transmit nociceptive signals Page 1 of 12 (page number not for citation purposes) Molecular Pain 2006, 2:31 http://www.molecularpain.com/content/2/1/31 [1,2]. SG neurons also receive descending inputs from the The source of CGRP in the spinal cord dorsal horn is pri- brainstem [1,3]. Therefore, in addition to intraspinal neu- mary afferents. CGRP coexists with substance P in small- roplastic changes, peripheral as well as supraspinal factors diameter afferent fibers, and CGRP containing terminals may contribute to central sensitization. and CGRP receptors are found in the dorsal horn, includ- ing SG [30-33]. CGRP is released in the spinal dorsal horn Pain-related neuroplastic changes in central nervous sys- by noxious stimulation and peripheral inflammation tem (CNS) structures can be shown definitively by the such as the K/C arthritis [26,34,35]. Peripheral inflamma- electrophysiological analysis of synaptic transmission and tion also leads to changes in CGRP binding sites in the neuronal excitability in spinal cord or brain slice prepara- dorsal horn [32,36]. tions obtained from animals in which an experimental pain state has been induced [4-7]. The slice preparation Spinal application of CGRP facilitates nociceptive behav- allows the analysis of pain-related plasticity because it is ior [24,37,38] and sensitizes the responses of dorsal horn disconnected from the site of peripheral injury (inflam- neurons to innocuous and noxious peripheral stimula- mation) and from other CNS areas, be it supraspinal sites tion [28,29,38,39] and to intraspinally administered exci- (spinal cord slice) or spinal cord (brain slices). Therefore, tatory amino acids [23] and substance P [39]. In a slice changes measured in the slice preparation are maintained preparation, CGRP produced a slow depolarization and independently of continuous inputs to the area of interest. enhanced excitability of dorsal horn neurons; the effect on Accordingly, changes of synaptic circuitry in SG neurons evoked synaptic transmission was not studied [40]. Con- were shown in slices from animals with complete Fre- versely, block of spinal CGRP receptors with an antagonist und's adjuvant induced hindpaw inflammation [4,5,8,9] (CGRP8-37) or antiserum induced antinociception in ani- and synaptic plasticity was demonstrated in amygdala mal models of inflammatory [25,41,42,42-44] or central neurons from animals with knee joint arthritis [7,10,11]. neuropathic pain [45]. CGRP8-37 also inhibited the responses of spinal dorsal horn neurons to transdermial The kaolin and carrageenan (K/C) induced knee joint electrical stimulation of the hindpaw [46] and to noxious arthritis is a well established model of inflammatory pain. mechanical stimulation of the knee joint [29]. CGRP8-37 Electrophysiological, pharmacological, neurochemical prevented or reversed central sensitization of dorsal horn and behavioral studies have used this model to analyze neurons in the arthritis and capsaicin pain models pain mechanisms at different levels of the nervous system [28,29]. Arthritic CGRP knockout mice showed reduced and showed the sensitization of primary afferent nerve nociceptive behavioral responses [47]. fibers, spinal dorsal horn neurons and neurons in the cen- tral nucleus of the amygdala (CeA) [12-17]. Using slice Although it is widely accepted that CGRP plays an impor- preparations, synaptic plasticity was demonstrated in the tant role in the modulation of spinal nociceptive process- CeA, but not yet in the spinal cord, in the K/C arthritis ing, the cellular mechanisms and pre- or post-synaptic pain model. sites of action through which CGRP contributes to central sensitization remain to be determined. The present study The purpose of this study was to compare synaptic trans- addressed the role of CGRP in synaptic plasticity in the mission and neuronal excitability in SG neurons in spinal superficial dorsal horn in vitro in a model of arthritic pain cord slices from normal and from arthritic animals using induced in vivo. Our data show for the first time synaptic patch-clamp recordings. Another goal was to determine plasticity and increased excitability of SG neurons in the the role of calcitonin gene-related peptide (CGRP) in K/C arthritis pain model. A CGRP receptor antagonist pain-related spinal plasticity since CGRP has emerged as inhibits synaptic plasticity whereas CGRP itself facilitates an important molecule at different levels of the pain neu- synaptic transmission through a postsynaptic mechanism raxis in the arthritis pain model. that involves direct membrane effects on SG neurons. CGRP is a 37 amino acid peptide that activates adenylyl Results cyclase and protein kinase A through G-protein-coupled Whole-cell patch-clamp recordings of SG neurons were receptors, including the CGRP1 receptor for which selec- made in spinal cord slices from normal naïve rats (n = 31 tive antagonists are available [18-21]. CGRP is involved in neurons) and rats with a knee joint arthritis induced 6 h peripheral and spinal pain mechanisms [22-29]. We before slices were obtained (n = 25 neurons). The record- showed recently that CGRP also plays an important role ing sites were always visually verified to be in the central in the transmission of nociceptive information to the part of the gray translucent region forming lamina II. All amygdala through the spino-parabrachio-amygdaloid SG neurons in this study showed monosynaptic responses pathway [10]. (excitatory postsynaptic currents, EPSCs) to electrical stimulation of afferent fibers in the dorsal root (DR) near the dorsal root entry zone (DREZ). EPSCs were judged to Page 2 of 12 (page number not for citation purposes) Molecular Pain 2006, 2:31 http://www.molecularpain.com/content/2/1/31 be monosynaptic on the basis of stable latencies of the trol neurons (n = 17), i.e., action potential firing occurred EPSC peak amplitude (coefficient of variation < 2%, at more hyperpolarized membrane potentials (Fig. 2D; P [48,49]). Calculated from latency and distance between < 0.01, unpaired t-test). stimulation and recording sites, the conduction velocities (CV) ranged from 0.15 to 0.85 m/s (mean 0.46 ± 0.03 m/ Inhibition of pain-related synaptic plasticity by a CGRP1 s), which is in the range of rodent C-fibers [48,49]. No dif- receptor antagonist (CGRP8-37) ference in resting transmembrane potential (RMP) and CGRP8-37 (1 μM; 10 min) inhibited synaptic transmis- input resistance (Ri) was detected between neurons from sion in SG neurons in slices from arthritic animals but had normal rats (RMP = -58.7 ± 1.7 mV; Ri = 211.8 ± 16.6 MΩ) and from arthritic rats (RMP = -57.0 ± 1.8 mV; Ri = 205.3 ± 13.7 MΩ). Synaptic plasticity in SG neurons in the arthritis pain model Input-output functions of monosynaptic inputs to SG neurons increased in the arthritis pain model (Figure 1). Monosynaptic EPSCs with progressively larger amplitudes were evoked by electrical DR/DREZ stimulation with increasing intensities. Compared with control SG neurons from normal animals, synaptic transmission was signifi- cantly enhanced in SG neurons recorded in slices from arthritic rats. Input-output relationships were obtained by measuring EPSC peak amplitude (pA) as a function of afferent fiber stimulus intensity (μA) for each neuron (see individual examples of an SG neuron in a slice from a nor- mal animal [Fig. 1A] and in an SG neuron from an arthritic animal [Fig. 1B]). In arthritis, evoked monosyn- aptic EPSCs had larger amplitudes, but EPSC threshold was unchanged. The input-output relationships of SG neurons from control rats (n = 16) and SG neurons from arthritic rats (n = 9) were significantly different (Fig. 1C; P < 0.0001, F = 58.45, two-way ANOVA). These data 1,207 show enhanced synaptic transmission at first-order syn- apses on SG neurons in the arthritis pain model. Enhanced synaptic transmission in the reduced slice prep- aration indicates synaptic plasticity because the arthritis pain-related changes are maintained, at least in part, inde- pendently of peripheral and supraspinal mechanisms. Syna arth Figure 1 ritis p ptic tr aansmission in model in SG neurons is enhanced in the Synaptic transmission in SG neurons is enhanced in Increased excitability of SG neurons in the arthritis pain the arthritis pain model. A,B, Whole-cell voltage-clamp model recordings of monosynaptic EPSCs evoked with increasing Compared with control neurons, neurons from arthritic stimulus intensities in an SG neuron in a spinal cord slice from a normal animal and in an SG neuron in a slice from an rats had a lower threshold and higher rate of action poten- arthritic animal (obtained 6 h post-induction of arthritis). tial firing generated by direct depolarization of the cell via Evoked monosynaptic EPSCs had larger amplitudes in arthri- the recording electrode in current-clamp mode (Figure 2). tis than under control conditions. Square wave electrical Input-output functions of neuronal excitability were stimuli of 150 μs duration were delivered at a frequency < obtained by measuring the number of action potentials 0.25 Hz. Stimulus intensity was increased from 0–800 μA. (Hz) evoked by depolarizing current pulses of increasing Each trace is the average of 3–4 EPSCs. Neurons were held magnitude (0 to 200 pA; see individual examples in Fig. at -60 mV. C, Input-output relationships of monosynaptic 2A and 2B). Input-output functions of SG neurons from EPSC peak amplitudes (pA) evoked in SG neurons from nor- arthritic animals (n = 13) were significantly increased mal rats (n = 16) and from arthritic rats (n = 9) were signifi- compared to control neurons from normal animals (n = cantly different. * P < 0.05, ** P < 0.01 (two-way ANOVA 25; Fig. 2C; P < 0.001; F = 12.77, two-way ANOVA). followed by Bonferroni posttests). Data are given as the 1,180 means ± SEM. The threshold for evoking action potentials was lower in SG neurons from arthritic animals (n = 11) than in con- Page 3 of 12 (page number not for citation purposes) Molecular Pain 2006, 2:31 http://www.molecularpain.com/content/2/1/31 Synaptic facilitation by CGRP is enhanced in the arthritis pain model CGRP enhanced synaptic transmission in a concentra- tion-dependent fashion (Figure 4). Individual examples show that CGRP (10 nM; 10 min) increased monosynap- tic EPSCs in an SG neuron from an arthritic rat (Fig. 4B) more strongly than in an SG neuron from a normal rat (Fig. 4A). Concentration-response data (Fig. 4C) show that the maximum facilitatory effect (efficacy) of CGRP was significantly (P < 0.01, F = 9.58, two-way ANOVA) 1,26 greater in SG neurons from arthritic rats (n = 16) com- pared to control neurons from normal animals (n = 10). The potency of CGRP was comparable under normal con- ditions (EC = 2.0 nM) and in arthritis (EC = 1.4 nM). 50 50 It should be noted that CGRP attenuated synaptic trans- mission in 6 of 16 SG neurons from normal animals but not in any SG neuron from arthritic animals. Postsynaptic effects of CGRP To determine the site of action of CGRP we used well- established electrophysiological methods, including the analysis of miniature EPSCs (mEPSCs) (Figure 5) and Increased excitability of SG ne mo Figure 2 del urons in the arthritis pain neuronal excitability (Figure 6). Presynaptic changes at Increased excitability of SG neurons in the arthritis the transmitter release site affect mEPSC frequency, pain model. Increased action potential firing rates and decreased thresholds for action potentials were recorded in whereas changes at the postsynaptic membrane alter SG neurons in slices from arthritic rats compared to con- mEPSC amplitude (quantal size) [50,51]. CGRP (10 nM; trols. A, B, Current-clamp recordings of action potentials 10 min) increased the amplitude of mEPSCs in TTX (1 (spikes) generated by direct intracellular injections of depo- μM)-containing ACSF without affecting their frequency, larizing current pulses of increasing magnitude (0 to 200 pA; suggesting a post- rather than pre-synaptic site of action 500 ms) in an SG neuron from a normal animal (A) and in an (Figure 5). This postsynaptic effect is illustrated in the cur- SG neuron from an arthritic animal (B). C, Analysis of the rent traces recorded in voltage-clamp mode in an individ- input-output relationships shows significantly increased spike ual SG neuron (Fig. 5A). Normalized cumulative frequency in arthritis (n = 13 neurons) compared to control distribution analysis of mEPSC amplitude and frequency (n = 25 neurons; P < 0.05; two-way ANOVA followed by shows that CGRP caused a significant shift toward higher Bonferroni posttests). D, Significantly decreased spike thresholds (membrane potentials at which action potential amplitude in this neuron (see Fig. 5B and 5C) and also firing started) were recorded in SG neurons in arthritis (n = increased mean mEPSC amplitude in the sample of neu- 11) compared to control neurons (n = 17; P < 0.01; unpaired rons (Fig. 5B, inset; P < 0.05, paired t-test, n = 5) but had t-test). * P < 0.05, ** P < 0.01. no effect on the interevent interval (frequency) distribu- tion (Fig. 5C, inset). no significant effect on normal synaptic transmission CGRP increased neuronal excitability (Figure 6). Action (Figure 3). Individual examples show that CGRP8-37 potentials were evoked in current-clamp mode by direct clearly inhibited monosynaptic EPSCs recorded in an SG depolarizing current injections (500 ms) of increasing neuron in a slice from an arthritic rat (Fig. 3B) but had lit- magnitude (0 to 250 pA) through the patch electrode (Fig. tle effect in an SG neuron in a slice from a normal rat (Fig. 6A and 6B). Input-output functions of neuronal excitabil- 3A). In the sample of SG neurons from arthritic rats (n = ity were obtained by averaging the frequency of action 5), CGRP8-37 inhibited synaptic strength (measured as potentials (spikes) evoked at each current intensity (Fig. peak amplitudes, Fig. 3C) and total charge (measured as 6C). CGRP (10 nM; 10 min) increased the input-output area under the curve, Fig. 3D) significantly (P < 0.01, function significantly (n = 5; P < 0.001; F = 12.77, 1,180 paired t-test), but had no significant effect on synaptic two-way ANOVA) while lowering the threshold for action transmission in SG neurons from normal rats (n = 7). potential generation to more hyperpolarized membrane These data suggest that CGRP1 receptors are endog- potentials (Fig. 6D; P < 0.01, paired t-test). In the presence enously activated to facilitate synaptic transmission in the of TTX (1 μM) CGRP (10 nM) also induced an inward arthritic pain model. Next, we determined the effect and membrane current that was significantly larger (P < 0.01, site of action of the receptor ligand CGRP. unpaired t-test) in SG neurons from arthritic rats (27.3 ± Page 4 of 12 (page number not for citation purposes) Molecular Pain 2006, 2:31 http://www.molecularpain.com/content/2/1/31 Figure 3 CGRP8-37 inhibits pain-related synaptic plasticity but has no significant effect on normal synaptic transmission CGRP8-37 inhibits pain-related synaptic plasticity but has no significant effect on normal synaptic transmis- sion. A, B, CGRP8-37 (1 μM) inhibited monosynaptic EPSCs recorded in an SG neuron in a slice from an arthritic rat (B) but not in another SG neuron in a slice from a normal rat (A). Each trace is the average of 8–10 monosynaptic EPSCs. C, D, CGRP8-37 (1 μM) significantly inhibited the EPSC peak amplitude (C), a measure of synaptic strength, and area under the curve (total charge, D) in SG neurons in slices from arthritic rats (P < 0.01, paired t-test, n = 5) but not in control neurons (n = 7) from normal rats. Analysis of raw data (pA, pC) is shown on the left; normalized data (% of predrug values) are shown on the right in C and D. Voltage-clamp recordings were made at -60 mV. CGRP8-37 was applied by superfusion of the slice in ACSF for 10–12 min. ** P < 0.01 (paired t-test). Page 5 of 12 (page number not for citation purposes) Molecular Pain 2006, 2:31 http://www.molecularpain.com/content/2/1/31 E mo Figure 4 nhanced syn del aptic facilitation by CGRP in the arthritis pain Miniature EPSC (mEPSC) pre-synaptic effects of CGRP Figure 5 analysis indicates post- rather than Enhanced synaptic facilitation by CGRP in the arthri- Miniature EPSC (mEPSC) analysis indicates post- tis pain model. A, B, Whole-cell voltage-clamp recordings rather than pre-synaptic effects of CGRP. A, Original of monosynaptic EPSCs in an SG neuron in a slice from a current traces of mEPSC recorded in an individual SG neu- normal animal (A) and in another SG neuron in a slice from ron in the presence of TTX (1 μM) show that CGRP (10 nM; an arthritic animal (B, 6 h postinduction of arthritis). CGRP 10 min) increases amplitude but not frequency of mEPSCs. (10 nM) potentiated synaptic transmission more strongly in B, C: Normalized cumulative distribution analysis of mEPSC arthritis than under normal conditions. Square wave electri- amplitude and frequency in the same neuron as in 5A shows cal stimuli of 150 μs duration were delivered at a frequency < that CGRP caused a significant shift toward higher amplitude 0.25 Hz. Each trace is the average of 8–10 EPSCs. C, Con- (B, P < 0.001, Kolmogorov-Smirnov test) but had no effect centration-response data show that the maximum effect (effi- on the interevent interval (frequency) distribution (C). In the cacy) of CGRP was significantly greater in SG neurons from sample of neurons (n = 5) CGRP selectively increased mean arthritic rats (n = 16) compared to control neurons from mEPSC amplitude (P < 0.05, paired t-test) but not mEPSC normal animals (n = 10). Peak EPSC amplitudes during each frequency (see bar histograms in B, C). Symbols and error concentration of CGRP were averaged and expressed as per- bars represent mean ± SEM. Neurons were recorded in volt- cent of predrug (baseline) control (100%). Sigmoid curves age-clamp at -60 mV. * P < 0.05. were fitted to the data using the following formula for nonlin- ear regression (GraphPad Prism 3.0; Y = A+(B-A)/[1+(10C/ 10X)D], where A = bottom plateau, B = top plateau, C = log(EC50), D = slope coefficient. Symbols show mean ± SEM. 3.1 pA, n = 5) than in SG neurons from normal rats (12.4 Neurons were held at -60 mV. CGRP was applied by super- ± 2.9 pA, n = 5). These data suggest a direct postsynaptic fusion of the slice in ACSF for 10 min. * P < 0.05 (two-way effect on membrane properties. ANOVA followed by Bonferroni posttests). Discussion The key findings of this study are as follows. Synaptic transmission and neuronal excitability in SG neurons are increased in slices from arthritic rats compared to control Page 6 of 12 (page number not for citation purposes) Molecular Pain 2006, 2:31 http://www.molecularpain.com/content/2/1/31 CGRP increase Figure 6 s neuronal excitability and induces direct membrane currents CGRP increases neuronal excitability and induces direct membrane currents. A, B, Current-clamp recordings of action potentials (spikes) generated in an SG neuron by direct intracellular injections of depolarizing current pulses of increas- ing magnitude (0 to 250 pA; 500 ms) before (A) and during CGRP (10 nM, B). C, CGRP increased input-output function by sig- nificantly increasing spike frequency (n = 5 neurons; P < 0.05–0.01, two-way ANOVA followed by Bonferroni posttests). For the measurement of action potential firing in current-clamp, neurons were recorded at -60 mV. D, CGRP (10 nM) also decreased spike thresholds (membrane potentials at which action potential firing started) significantly (n = 5; P < 0.01, paired t- test). * P < 0.05, ** P < 0.01. neurons from normal rats. These data suggest plastic and supraspinal influences. Blockade of CGRP receptors changes in the arthritis pain model that are maintained in inhibits synaptic plasticity in SG neurons from arthritic the reduced slice preparation independently of peripheral animals, suggesting the contribution of endogenously Page 7 of 12 (page number not for citation purposes) Molecular Pain 2006, 2:31 http://www.molecularpain.com/content/2/1/31 activated CGRP receptors. CGRP facilitates synaptic trans- changes rather than variability of the experimental condi- mission and increases neuronal excitability through a tions. postsynaptic site of action. The present study also offers a mechanism for these plastic This study focused on synaptic transmission of afferent changes: enhanced function of CGRP1 receptors. A widely information to SG neurons. According to conventional used selective CGRP1 receptor antagonist (CGRP8-37) criteria such as stable latencies of the EPSC peak [48,49], [18,20,21] inhibited synaptic plasticity but had little effect synaptic responses were considered monosynaptic and on normal transmission. These findings are consistent had a latency that indicated a slow conduction velocity with in vivo data showing that blockade of spinal CGRP (CV) of the responsible afferents in the range of rodent C- receptors inhibits sensitization of dorsal horn neurons in fibers [48,49,52]. However, a note of caution should be pain models [28,29] and nociceptive behavior added. Since we were not able to preserve long dorsal [25,25,41,42,42-45] in pain models. Here we show for the roots in the majority of the experiments but rather stimu- first time that the DR-SG circuitry is one site of action of lated the DR stump near the DREZ, we can not be sure that CGRP receptors to modulate synaptic transmission. CGRP the calculated CV at the central terminal accurately reflects containing terminals and CGRP receptors are present in the CV in the axon of the peripheral fiber. Still, it is evi- the dorsal horn, including SG [30-33]. CGRP is released in dent that afferent input to SG neurons from small diame- the dorsal horn in the K/C arthritis model [34]. ter fibers but not fast conducting A-beta fibers was studied here. Further, the SG neurons included in this study ful- Our data with exogenously administered CGRP further filled the criteria for central SG neurons with monosynap- indicate a post- rather than pre-synaptic site of action at tic C-fiber input as described in detail by others [48,49]. the DR-SG synapse and a direct membrane effect on SG neurons. This mechanism of action could explain the Our data show for the first time changes of synaptic trans- CGRP-induced sensitization of dorsal horn neurons in mission in small diameter fibers to SG neurons in a model vivo [23,28,29,38,39,39] and facilitation of nociceptive of arthritic pain. Previously, the slice preparation had behavior [24,37,38]. CGRP has been shown before to been used to determine changes in transmission to SC depolarize and increase excitability of dorsal horn neu- neurons in an inflammatory pain model induced by intra- rons in current-clamp [40]. Our simultaneous recording plantar complete Freund's adjuvant 48 h or 7–10 d before and analysis of evoked synaptic transmission, miniature slices were obtained [4,5,9]. In these studies, changes in EPSCs and membrane currents in voltage-clamp and synaptic transmission had been observed. They included excitability in current-clamp show a direct facilitatory a lower threshold for evoking EPSCs in SG neurons, a rel- action of CGRP on SG neurons to increase their respon- ative increase in the C-fiber versus A-delta fiber evoked siveness to afferent input and their output (action poten- EPSC amplitude, and an increased percentage of SG neu- tial generation). The enhanced CGRP function in the rons receiving mono- or polysynaptic A-beta input [4,5,9]. arthritis pain model could involve a change in the cou- pling to downstream effector systems such as kinases and The present study extends these observations in several ion channels as well as increased receptor expression or ways. We used a different pain model, the K/C induced affinity. The effects of peripheral inflammation on CGRP arthritis that produces electrophysiological and behavio- binding sites in the dorsal horn have been reported to be ral changes in vivo with a well defined and highly repro- somewhat inconsistent in that a mixture of up- and down- ducible time course [10,11,16,17]. This allowed us to regulation was found [32,36]. The inhibitory effect of select a constant time point (6 h postinduction of arthri- CGRP on normal synaptic transmission observed in some tis) to study changes in the slice preparation. Arthritis neurons could reflect an action on the recently described pain-related changes reach a plateau 6 h postinduction inhibitory projection islet cells on central SG neurons and persist at that level for days. Another novel aspect of [48]. our studies is the analysis of complete input-output func- tions of the DR-SG synapse and of neuronal excitability of SG neurons with C-fiber input (such as those selected in SG neurons. Our data show synaptic plasticity combined the present study) have been shown to excite monosynap- with excitability changes in the arthritis pain model. There tically SG neurons with A-delta input. These neurons then was no significant change of the threshold for evoking excite monosynaptically lamina I neurons [49], some of EPSCs suggesting that the stimulation and recording con- which project rostrally to form the spino-parabrachio- ditions were indeed comparable in slices from normal amygdaloid pathway [49,53,54]. This pathway is highly and arthritic animals. The fact that differences of synaptic peptidergic and utilizes CGRP to transmit information to transmission between normal and arthritic conditions the amygdala [10] were observed at different stimulus intensities along the input-output relationships further suggests functional Page 8 of 12 (page number not for citation purposes) Molecular Pain 2006, 2:31 http://www.molecularpain.com/content/2/1/31 Electrophysiology Conclusion This study is the first to show synaptic plasticity in the spi- Whole-cell current- and voltage-clamp recordings were nal dorsal horn (SG) in a model of arthritic pain. Synaptic made from substantia gelatinosa (SG) neurons in trans- plasticity involves CGRP1 receptor activation. CGRP acts verse lumbar spinal cord slices (500 μm) from normal rats postsynaptically to increase the input and output func- (controls) and arthritic rats (16–21 day old), using DIC- tions of SG neurons. enhanced infrared video-microscopy for visualization or the "blind" patch technique as in our previous studies [7,10,11]. Patch electrodes were made from borosilicate Methods Male Sprague Dawley rats (16–21 d) were housed in a glass capillaries (1.5 mm outer diameter, 1.12 mm inner temperature controlled room and maintained on a 12 h diameter; Drummond, Broomall, PA) pulled on a Flam- day/night cycle. Water and food were available ad libitum. ing-Brown micropipette puller (P-80/PC; Sutter Instru- Electrophysiological data were obtained from untreated ments, Novato, CA). Patch electrodes had tip resistances normal rats and rats with monoarthritis in the knee (6 h of 4–6 MΩ. The following internal solution was used after induction). All experimental procedures were (compounds in mM): K-Gluconate (122), NaCl (5), (0.3), EGTA (1), HEPES (10), Na ATP (5), Na GTP approved by the Institutional Animal Care and Use Com- CaCl 2 2 3 mittee (IACUC) at the University of Texas Medical Branch (0.4), and MgCl (2); pH 7.3; 300 mOsm. (UTMB) and conform to the guidelines of the Interna- tional Association for the Study of Pain (IASP) and of the Recording electrodes were positioned in the center of the National Institutes of Health (NIH). SG under visual control. The boundaries of the SG are eas- ily discerned under light microscopy. After tight (> 1 GΩ) Arthritis pain model seals were formed and the whole-cell configuration was In the group of arthritic rats, arthritis was induced in the obtained, neurons were included in the sample if the rest- left knee joint as previously described [7,10,11]. A kaolin ing membrane potential was more negative than -50 mV suspension (4%, 100 μl) was injected into the left knee and action potentials overshooting 0 mV were evoked by joint cavity through the patellar ligament. After repetitive direct depolarizing current injection through the record- flexions and extensions of the knee for 15 minutes, a car- ing electrode. Data acquisition and analysis of voltage and rageenan solution (2%, 100 μl) was injected into the knee current signals were done using a dual 4-pole Bessel filter joint cavity, and the leg was flexed and extended for (Warner Instrument Corp., Hamden, CT), low-noise Dig- another 5 minutes. Spinal cord slices were obtained 6 h idata 1322 interface (Axon Instruments, Foster City, CA), after arthritis induction. Axoclamp-2B or Axopatch 200 B amplifiers (Axon Instr.), Pentium PC, and pCLAMP8 and pCLAMP9 software Spinal cord slice preparation (Axon Inst.). Recordings were made at -60 mV. Series Transverse spinal cord slices were prepared using a modi- resistance was at least one order of magnitude less than fied version of the technique established by E.R. Perl's input resistance and was continuously monitored group [48,49]. Rats were deeply anesthetized with pento- throughout the experiment. barbital (50 mg/kg, i.p.). After a lumbosacral laminec- tomy the spinal cord with associated dorsal roots on one Monosynaptic excitatory postsynaptic currents (EPSCs) side was quickly removed and placed in ice-cold, sucrose- were evoked by stimulation of the DR with a suction elec- substituted, artificial cerebrospinal fluid (sucrose ACSF) trode or, in the majority of experiments, by focal stimula- containing (in mM): sucrose (234), KCl (3.6), CaCl tion of the DR stump near the DREZ with a concentric (2.5), MgCl (1.2), NaH PO (1.2), NaHCO (25), and bipolar electrode. Electrical stimuli (150 μs square-wave 2 2 5 3 glucose (12); equilibrated to pH 7.4 with a mixture of pulses) were delivered at frequencies below 0.25 Hz. 95% O and 5% CO . A vibrotome (Camden Instruments, Input-output relationships were obtained by increasing 2 2 London, UK) was used to prepare transverse (500 μm the stimulus intensity in 50 or 100 μA steps. For the eval- thick) slices from the lumbar spinal cord. Spinal cord uation of a drug effect on synaptically evoked responses, slices were maintained at room temperature (21°C) for at the stimulus intensity was adjusted to 80% of the intensity least 1 h in standard ACSF containing (in mM): NaCl required for orthodromic spike generation. EPSCs were (117), KCl (4.7), NaH PO (1.2), CaCl (2.5), MgCl judged to be monosynaptic on the basis of stable latencies 2 4 2 2 (1.2), NaHCO3 (25), glucose (11); equilibrated to pH 7.4 of the EPSC peak amplitude (coefficient of variation < 2% with 95% O /5% CO . A single slice was then transferred [48,49]). CV was estimated from latency of the evoked 2 2 to the recording chamber and submerged in ACSF, which EPSC peak and the conduction distance between stimula- superfused the slice at ~5 ml/min. tion and recording sites. The following parameters were recorded to measure arthritis-related or drug-induced changes. Peak amplitude Page 9 of 12 (page number not for citation purposes) Molecular Pain 2006, 2:31 http://www.molecularpain.com/content/2/1/31 and area under the curve (AUC) of monosynaptic EPSCs DREZ, dorsal root entry zone (typically the mean of 8–10 consecutive EPSCs) were measured in voltage-clamp to determine synaptic strength EPSC, excitatory postsynaptic current and total charge, respectively. Frequency and amplitude of miniature EPSCs (mEPSCs, recorded in 1 μM TTX) were K/C, kaolin and carrageenan determined from 1 min recording periods (voltage- clamp) using the Mini Analysis Program 6.0.3 (Synap- Ri, input resistance tosoft Inc., Decatur, GA). Input-out functions of excitabil- ity were calculated from the number of evoked action RMP, resting transmembrane potential potentials (spikes) evoked by direct intracellular injec- tions of depolarizing currents (500 ms; increments of 50 SG, substantia gelatinosa pA). Competing interests Drugs The author(s) declare that they have no competing inter- CGRP (receptor agonist) and CGRP8-37 (selective CGRP1 ests. receptor antagonist) [18,20,21] were dissolved in ACSF on the day of the experiment and applied to the spinal Authors' contributions cord slice by gravity-driven superfusion in the ACSF for 10 GCB carried out the majority of the experiments. VN, JSH min at a rate of 5 ml/min. Solution flow into the recording and HA performed additional experiments. GCB, JSH, YF, chamber (1 ml volume) was controlled with a three-way HA and VN performed the data analysis. VN and WDW stopcock. Duration of drug application and concentra- conceptualized the project and formulated the hypothe- tions were selected based on our previous studies [10]. sis. VN designed and directed the experiments and wrote the manuscript. Data analysis and statistics All averaged values are given as the mean ± SEM. Statisti- Acknowledgements This work was supported by NIH grants NS38261 and NS11255. cal significance was accepted at the level of P < 0.05. Input-output functions and concentration-response rela- References tionships were compared using a two-way ANOVA with 1. Willis WD, Coggeshall RE: Sensory mechanisms of the spinal cord 3rd Bonferroni posttests. EC values were calculated from sig- edition. New York, Plenum; 2004:1-962. moid curves fitted to the cumulative concentration- 2. 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Molecular Pain – Springer Journals

Published: Sep 26, 2006

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