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

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.

Figure 1

Synaptic transmission in SG neurons is enhanced in the arthritis pain model. A,B, Whole-cell voltage-clamp recordings of monosynaptic EPSCs evoked with increasing 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 arthritic animal (obtained 6 h post-induction of arthritis). Evoked monosynaptic EPSCs had larger amplitudes in arthritis than under control conditions. Square wave electrical stimuli of 150 μs duration were delivered at a frequency < 0.25 Hz. Stimulus intensity was increased from 0–800 μA. Each trace is the average of 3–4 EPSCs. Neurons were held at -60 mV. C, Input-output relationships of monosynaptic EPSC peak amplitudes (pA) evoked in SG neurons from normal rats (n = 16) and from arthritic rats (n = 9) were significantly different. * P < 0.05, ** P < 0.01 (two-way ANOVA followed by Bonferroni posttests). Data are given as the means ± SEM.

Figure 2

Increased excitability of SG neurons in the arthritis pain model. Increased action potential firing rates and decreased thresholds for action potentials were recorded in SG neurons in slices from arthritic rats compared to controls. A, B, Current-clamp recordings of action potentials (spikes) generated by direct intracellular injections of depolarizing current pulses of increasing magnitude (0 to 200 pA; 500 ms) in an SG neuron from a normal animal (A) and in an SG neuron from an arthritic animal (B). C, Analysis of the input-output relationships shows significantly increased spike frequency in arthritis (n = 13 neurons) compared to control (n = 25 neurons; P < 0.05; two-way ANOVA followed by Bonferroni posttests). D, Significantly decreased spike thresholds (membrane potentials at which action potential firing started) were recorded in SG neurons in arthritis (n = 11) compared to control neurons (n = 17; P < 0.01; unpaired t-test). * P < 0.05, ** P < 0.01.

Figure 3

CGRP8-37 inhibits pain-related synaptic plasticity but has no significant effect on normal synaptic transmission. 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).

Figure 4

Enhanced synaptic facilitation by CGRP in the arthritis pain model. A, B, Whole-cell voltage-clamp recordings of monosynaptic EPSCs in an SG neuron in a slice from a normal animal (A) and in another SG neuron in a slice from an arthritic animal (B, 6 h postinduction of arthritis). CGRP (10 nM) potentiated synaptic transmission more strongly in arthritis than under normal conditions. Square wave electrical stimuli of 150 μs duration were delivered at a frequency < 0.25 Hz. Each trace is the average of 8–10 EPSCs. C, Concentration-response data show that the maximum effect (efficacy) of CGRP was significantly greater in SG neurons from arthritic rats (n = 16) compared to control neurons from normal animals (n = 10). Peak EPSC amplitudes during each concentration of CGRP were averaged and expressed as percent of predrug (baseline) control (100%). Sigmoid curves were fitted to the data using the following formula for nonlinear 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. Neurons were held at -60 mV. CGRP was applied by superfusion of the slice in ACSF for 10 min. * P < 0.05 (two-way ANOVA followed by Bonferroni posttests).

Figure 5

Miniature EPSC (mEPSC) analysis indicates post- rather than pre-synaptic effects of CGRP. A, Original current traces of mEPSC recorded in an individual SG neuron in the presence of TTX (1 μM) show that CGRP (10 nM; 10 min) increases amplitude but not frequency of mEPSCs. B, C: Normalized cumulative distribution analysis of mEPSC amplitude and frequency in the same neuron as in 5A shows that CGRP caused a significant shift toward higher amplitude (B, P < 0.001, Kolmogorov-Smirnov test) but had no effect on the interevent interval (frequency) distribution (C). In the sample of neurons (n = 5) CGRP selectively increased mean mEPSC amplitude (P < 0.05, paired t-test) but not mEPSC frequency (see bar histograms in B, C). Symbols and error bars represent mean ± SEM. Neurons were recorded in voltage-clamp at -60 mV. * P < 0.05.

Figure 6

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 increasing magnitude (0 to 250 pA; 500 ms) before (A) and during CGRP (10 nM, B). C, CGRP increased input-output function by significantly 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.