Excitatory Effects of Calcitonin Gene-Related Peptide (CGRP) on Superficial Sp5C Neurons in Mouse Medullary Slices

Abstract

The neuromodulator calcitonin gene-related peptide (CGRP) is known to facilitate nociceptive transmission in the superficial laminae of the spinal trigeminal nucleus caudalis (Sp5C). The central effects of CGRP in the Sp5C are very likely to contribute to the activation of central nociceptive pathways leading to attacks of severe headaches like migraine. To examine the potential impacts of CGRP on laminae I/II neurons at cellular and synaptic levels, we performed whole-cell patch-clamp recordings in juvenile mouse brainstem slices. First, we tested the effect of CGRP on cell excitability, focusing on neurons with tonically firing action potentials upon depolarizing current injection. CGRP (100 nM) enhanced tonic discharges together with membrane depolarization, an excitatory effect that was significantly reduced when the fast synaptic transmissions were pharmacologically blocked. However, CGRP at 500 nM was capable of exciting the functionally isolated cells, in a nifedipine-sensitive manner, indicating its direct effect on membrane intrinsic properties. In voltage-clamped cells, 100 nM CGRP effectively increased the frequency of excitatory synaptic inputs, suggesting its preferential presynaptic effect. Both CGRP-induced changes in cell excitability and synaptic drives were prevented by the CGRP receptor inhibitor BIBN 4096BS. Our data provide evidence that CGRP increases neuronal activity in Sp5C superficial laminae by dose-dependently promoting excitatory synaptic drive and directly enhancing cell intrinsic properties. We propose that the combination of such pre- and postsynaptic actions of CGRP might underlie its facilitation in nociceptive transmission in situations like migraine with elevated CGRP levels.

Keywords: calcitonin gene-related peptide; cell excitability; excitatory postsynaptic currents; migraine; spinal trigeminal nucleus caudalis.

Figure 1

Firing patterns of Sp5C laminae I/II cells. (A) Bright field micrographs illustrate a typical freshly prepared transverse slice (350 µm thickness; left), and examples of biocytin-filled neurons in lamina II (middle) and in lamina I (right, with soma location indicated by an asterisk on the left, and axon pointed by arrow). AP, area postrema; cc, central canal; IO, inferior olivary complex; NA, nucleus ambiguous; sptV, spinal tract of the trigeminal nerve; XII, hypoglossal nucleus. (B) Five classes of Sp5C laminae I/II cells are distinguished by their properties of action potential discharge to depolarizing currents. Cells were recorded under whole-cell current-clamp mode, with membrane potential initially held at −70 mV (by current injection). Pattern of spiking was determined by their responses (top three panels) to the serial of current injection (bottom panels). The initial burst-like spiking (arrow) was enlarged on the right. (C) Distribution of spiking patterns of Sp5C cells recorded in the absence and in the presence of inhibitory and excitatory synaptic blockers. Kynurenic acid (KA, 2 mM) was used to block fast glutamatergic synaptic transmission, and picrotoxin (PTX, 100 µM) plus strychnine (10 µM) were used to block fast GABAergic and glycinergic synaptic transmissions, respectively.

Figure 2

Calcitonin gene-related peptide (CGRP) excites tonic-firing cells in Sp5C laminae I/II. Depolarizing current (35–100 pA) was adjusted individually to evoke 4–10 APs per pulse (500 ms) in each cell before CGRP application, with membrane potential set at −70 mV. (A) Original traces taken before, during and after CGRP (100 nM for 5 min) illustrate the reversible enhancement of AP firing in a lamina I cell. (B) Representative traces from another cell show how the CGRP receptor antagonist BIBN 4096BS (10 µM) prevents the excitatory effect of CGRP. (C,D) Histograms summarize CGRP-induced changes in AP discharges (C) and time to first AP (D) in the absence and in the presence of BIBN 4096BS. Numbers in columns indicate sample size. * p < 0.05; ** p < 0.01.

Figure 3

CGRP enhances Sp5C cell intrinsic excitability. Action potentials of Sp5C laminae I/II cells were evoked in the presence of kynurenic acid (KA), picrotoxin (PTX) and strychnine to block fast synaptic transmissions. (A,B) Overlapped traces from two cells illustrate the concentration-dependent effect of CGRP application on the evoked APs. Dashed line indicates the initial potential of −70 mV. (C) Superimposed traces are the 1st APs of the cell in B, with enlarged time scale to illustrate the CGRP-induced reduction in AP threshold and afterhyperpolarization (AHP). (D–H) Histograms characterize the impacts of CGRP on AP discharges (D) and time to evoke 1st AP (E), AP threshold (F), maximal rising slope of AP (indicative of available sodium channels; (G) and AHP (H). (I) Summary of CGRP-induced membrane depolarization depicted further by a voltage trace above the histogram. * p < 0.05; ** p < 0.01.

Figure 4

Involvement of L-type calcium channels in the excitatory effect of CGRP. All recordings were performed in the cocktail of KA, PTX and strychnine to block fast synaptic transmissions. (A) Voltage traces were collected from a Sp5C cell in laminae II before and during CGRP (500 nM) application, in the presence of the L-type calcium channel blocker nifedipine (10 µM). (B,C) Histograms summarize that nifedipine blocks the responses of Sp5C cells to high concentration of CGRP (500 nM), manifested in AP discharges and time to evoke 1st AP (B), voltage threshold for AP and afterhyperpolarization (C).

Figure 5

Calcitonin gene-related peptide (CGRP) facilitates excitatory synaptic drive onto Sp5C laminae I/II cells. Spontaneously occurring excitatory postsynaptic currents (spEPSCs) were monitored under whole-cell voltage-clamp mode at −70 mV, in the presence of antagonists for GABA_ARs and GlyRs. (A) Raw traces of spEPSCs from a lamina I cell were collected before CGRP application (control), 5 min in CGRP (100 nM) and 10 min after wash. Superimposed traces on the right represent the averaged events from the cell before (black trace) and during drug application (red trace). (B) Histograms summarize the effects of acutely applied CGRP on the frequency and the averaged peak amplitude of spEPSCs. Inset in up-right corner with the normalized changes in spEPSC frequency further reinforces the dose-dependent effect of CGRP. (C) Typical responses of spEPSCs to CGRP (100 nM) in the presence of a potent non-peptide antagonist BIBN 4096BS (10 µM, 20 min). Kynurenic acid (KA, 2 mM) was applied at the end of this experiment to verify the glutamatergic origin of spEPSCs. (D) Summary of the dampening effects of CGRP receptor antagonists on spEPSCs in Sp5C I/II cells. * p < 0.05; ** p < 0.01.

Figure 6

Immunohistochemically processed sections showing the outer laminae of the mouse dorsolateral Sp5C. Immunofluorescence of CGRP receptor components receptor activity modifying protein 1 (RAMP1) (A,D,E) and calcitonin-like receptor (CLR) (B,G–I) in the spinal trigeminal tract (sptV) and lamina I/II of the mouse Sp5C ((A–C,G) dorsolateral, (D,I) ventrolateral, (E,H) lateral); (C,F) are control stainings without first antibody but incubated with the second antibody Cy3; blue is nucleus staining (DAPI). The dotted lines in (D,G) show the approximate border between sptV and lamina (I). Arrows point to cell bodies that are closely approached by RAMP1/CLR immuno-positive nerve fibres, the arrowhead in G shows CLR immuno-positive fibres accompanying a penetrating medullary blood vessel, and the arrowhead in the control staining F points to an unspecific immunofluorescence in the wall of a blood vessel; magnification bars 200 µm in (A–C) and 50 µm in (D–I). No CGRP receptor component immunofluorescence of cell bodies is visible.