Facilitation of synaptic transmission and pain responses by CGRP in the amygdala of normal rats

Abstract

Calcitonin gene-related peptide (CGRP) plays an important role in peripheral and central sensitization. CGRP also is a key molecule in the spino-parabrachial-amygdaloid pain pathway. Blockade of CGRP1 receptors in the spinal cord or in the amygdala has antinociceptive effects in different pain models. Here we studied the electrophysiological mechanisms of behavioral effects of CGRP in the amygdala in normal animals without tissue injury.Whole-cell patch-clamp recordings of neurons in the latero-capsular division of the central nucleus of the amygdala (CeLC) in rat brain slices showed that CGRP (100 nM) increased excitatory postsynaptic currents (EPSCs) at the parabrachio-amygdaloid (PB-CeLC) synapse, the exclusive source of CGRP in the amygdala. Consistent with a postsynaptic mechanism of action, CGRP increased amplitude, but not frequency, of miniature EPSCs and did not affect paired-pulse facilitation. CGRP also increased neuronal excitability. CGRP-induced synaptic facilitation was reversed by an NMDA receptor antagonist (AP5, 50 microM) or a PKA inhibitor (KT5720, 1 microM), but not by a PKC inhibitor (GF109203X, 1 microM). Stereotaxic administration of CGRP (10 microM, concentration in microdialysis probe) into the CeLC by microdialysis in awake rats increased audible and ultrasonic vocalizations and decreased hindlimb withdrawal thresholds. Behavioral effects of CGRP were largely blocked by KT5720 (100 microM) but not by GF109203X (100 microM).The results show that CGRP in the amygdala exacerbates nocifensive and affective behavioral responses in normal animals through PKA- and NMDA receptor-dependent postsynaptic facilitation. Thus, increased CGRP levels in the amygdala might trigger pain in the absence of tissue injury.

Figure 1

CGRP enhances synaptic transmission in the CeLC in slices from normal animals. (A) Monosynaptic EPSCs evoked at the PB-CeLC synapse with increasing stimulus intensities before and during CGRP (100 nM, 12 min). Individual traces are the average of 10 EPSCs. (B) CGRP (100 nM, 10-14 min) increased input-output function of the PB-CeLC synapse significantly (n = 10, P < 0.0001, F_1,198 = 67.97, two-way ANOVA). Input-output curves were generated by plotting peak EPSC amplitude (pA) as a function of afferent fiber volley stimulus intensity (μA). (C) Synaptic facilitation by CGRP was blocked by co-administration of a CGRP1 receptor antagonist (CGRP8-37, 1 μM). Individual traces are the average of 8-10 EPSCs. (D) Cumulative concentration-response relationship of CGRP effects on synaptic transmission at the PB-CeLC synapse (n = 15). Peak amplitudes of monosynaptic EPSCs were averaged for each concentration of CGRP and expressed as percent of predrug control (set to 100%). Concentration-response curve was obtained by non-linear regression analysis using the formula y = A+(B-A)/[1+(10^C/10^X)^D], where A is the bottom plateau, B top plateau, C = log(IC₅₀), and D is the slope coefficient (GraphPad Prism software). CGRP8-37 (1 μM, n = 6) blocked the effect of CGRP (100 nM). CeLC neurons were recorded at -60 mV in slices from naïve untreated animals. Symbols and error bars represent mean ± SEM. *, **, *** P < 0.05-0.001 (Bonferroni posttests).

Figure 2

Post- rather than pre-synaptic effects of CGRP. (A, B) Paired-pulse ratio (PPR), a measure of presynaptic mechanisms, was not affected by CGRP (100 nM, 12 min). (A) Current traces (average of 8-10 EPSCs) recorded in an individual CeLC. Inter-stimulus interval, 50 ms. (B) CGRP had no significant effect on PPR in the sample of neurons (n = 12, P > 0.05, F_1,110 = 0.24, two-way ANOVA). (C) Original current traces of mEPSCs recorded in an individual CeLC neuron in the presence of TTX (1 μM). CGRP (100 nM, 12 min) increased amplitude, but not frequency, of mEPSCs. (D) Cumulative distribution analysis of mEPSCs amplitude and frequency. CGRP (100 nM, 12 min) caused a significant shift toward larger amplitudes (n = 4, P < 0.001, Kolmogorov-Smirnov test) but had no effect on inter-event interval distribution. CGRP selectively increased mean mEPSC amplitude (P < 0.05, paired t-test) but not mean frequency (n = 4; see bar histograms showing data normalized to predrug control). (E) Number of action potentials evoked in a CeLC neuron by direct intracellular injections of depolarizing current pulses (500 ms) of increasing magnitude (lower traces) increased during superfusion of CGRP (100 nM, 12 min; upper traces). (F) CGRP increased input-output functions significantly (n = 11, P < 0.0001, F_1,156 = 82.12, two-way ANOVA). Recordings were made in slices from naïve (untreated) animals. Neurons were recorded at -60 mV. Symbols and error bars represent mean ± SEM. * P < 0.05 (paired-test).

Figure 3

Inhibition of PKA, but not PKC, blocks synaptic effects of CGRP. (A) Original recordings of monosynaptic EPSCs (average of 10 EPSCs) evoked at the PB-CeLC synapse. Facilitatory effects of CGRP (100 nM) were blocked by co-administration of a PKA inhibitor (KT5720, 1 μM). (B) Summary of time course data for the sample of CeLC neurons (n = 7) show the inhibitory effects of KT5720 were reversible after washout. Peak amplitudes of EPSCs recorded during drug application were expressed as percent of predrug control values (set to 100%). (C) Individual traces (average of 8-10) of monosynaptic EPSCs show that the facilitatory effects of CGRP (100 nM) were not blocked by co-administration of a PKC inhibitor (GF109203x, 1 μM). (D) Summary of time course data for the sample of CeLC neurons show that the effects of CGRP did not desensitize during drug application for 30 min (n = 6; display as in (B)). Symbols and error bars represent mean ± SEM. * P < 0.05; n.s. (not significant), P > 0.05 (paired t-test, comparing the last measurement before and during application of cAMP-RP or KT5720). Statistical analysis was performed on raw data.

Figure 4

NMDA receptor antagonist blocks CGRP effects. (A) Original recordings of monosynaptic EPSCs (average of 8-10 EPSCs) evoked at the PB-CeLC synapse in the presence of AP5 (50 μM). CGRP (100 nM) had no effect. (B) Normalized data for the sample of CeLC neurons (n = 4). Peak amplitudes of EPSCs recorded during drug application were expressed as percent of predrug control values (set to 100%). (C) Individual traces (average of 8-10) of monosynaptic EPSCs show that the facilitatory effects of CGRP (100 nM) were blocked by co-administration of AP5 (50 μM). (D) Normalized data for the sample of CeLC neurons (n = 4; display as in (B)). Bar histograms show mean ± SEM. n.s. (not significant), P > 0.05 (paired t-test), ** P < 0.01 (ANOVA with Bonferroni posttests). Statistical analysis was performed on raw data.

Figure 5

Inhibition of PKA, but not PKC, blocks behavioral effects of CGRP. Vocalizations and hindlimb withdrawal thresholds were measured in awake rats before and during application of CGRP into the CeLC. Audible (A) and ultrasonic (B) vocalizations in response to brief (15 s) compression of the knee with innocuous (500 g/30 mm²) or noxious (2000 g/30 mm²) intensity (see Methods) were measured for 1 min starting with the onset of the stimulus (see Methods). No vocalizations were detected in a control period of 5-10 min before stimulation. Administration of CGRP (10 μM, concentration in microdialysis probe; 15 min) into the CeLC evoked or increased vocalizations of naïve rats (n = 5 in each group). Co-administration of a PKA inhibitor (KT5720, 100 μM, n = 5) reversed the effects of CGRP; a PKC inhibitor (GF109203x, 100 μM, n = 5) had no significant effect. (C) Thresholds of hindlimb withdrawal reflexes measured by compressing the knee with a calibrated forceps (see Methods) were decreased by CGRP administered into the CeLC (10 μM, concentration in microdialysis probe; 15-20 min; n = 5 in each group). Co-administration of KT5720 (100 μM, n = 5) partially reversed the effects of CGRP. GF109203x (100 μM, n = 5) had no significant effect. Bar histograms and error bars represent mean ± SE. * P < 0.05, ** P < 0.01 (ANOVA with Bonferroni posttests, compared to predrug control). ^# P < 0.05 (ANOVA with Bonferroni posttests, compared to CGRP).

Figure 6

Histological verification of drug application sites. Diagrams adapted from [49] show coronal sections through the right hemisphere at different levels posterior to bregma (-1.88 and -2.12). Next to each diagram is shown in detail the CeA and its subdivisions, the medial (CeM), lateral (CeL) and latero-capsular (CeLC) part. Each symbol indicates the location of the tip of one microdialysis probe. The boundaries of the different amygdala nuclei are easily identified under the microscope (see Figure 1 in [30]). Calibration bars for diagrams are 1 mm.