CGRP enhanced excitatory synaptic transmission in the IC
Our recent study showed that CGRP increased the excitatory synaptic transmission in the ACC . Here we wanted to see if similar effect would be found in the IC, which also plays important roles in pain perception and chronic pain. We recorded EPSCs at pyramidal neurons of layer II/III in the agranular and dysgranular insular cortex in the presence of a GABAA receptor antagonist, PTX (100 μM). The holding potential was -70 mV and local stimulation was given at layer V in the IC. The schematic diagram and representative recording diagram were shown as Fig. 1A. After achieving the stable baseline recording in response to paired-pulse stimulation (50 ms interpulse interval) for at least 10 min, the CGRP (10 nM) were applied. As shown in Fig. 1B and 1C, amplitudes of EPSCs were significantly increased after applied the 10 nM CGRP (131.6 ± 10.3% of baseline, p<0.01 as compared with baseline, one-way ANOVA, n=9 neurons/6 mice). The potentiation induced by CGRP was long-lasting, and persisted during the washout period for at least 30 min (168.8 ± 23.1% of baseline, p<0.01 compared with baseline, one-way ANOVA, n=9 neurons/6 mice, filled circles, Fig. 1C). The effect of CGRP was dose-related. At a dose of 1 nM, CGRP failed to induce any significant potentiation (106.6 ± 10.8% of baseline, F (2, 27) =2.4, p=0.1, one-way ANOVA, n=6 neurons/4 mice, open circles, Fig. 1C). Furthermore, there was no further potentiation after the washout of CGRP (102.1 ± 7.1% of baseline, F (2, 27) =2.4, p=0.1, one-way ANOVA, n=6 neurons/4 mice, open circles, Fig. 1C).
Thepotentiation induced by CGRP is NMDAR independent
Since the activation of NMDARs is crucial for most forms of LTP, we would like to test if CGRP induced potentiation is NMDAR dependent. In this set of experiments, an NMDAR antagonist, AP5 (50 μM) was present in the bath solution throughout the experiments. Bath application of CGRP caused similar amount of potentiation as those without AP5 (see above) (CGRP: 132.9 ± 12.5% of baseline, washout: 140.7 ± 21.3% of baseline, p<0.01 and p<0.01 compared with baseline, respectively; one-way ANOVA, n=8 neurons/5 mice, Fig. 2).
CGRP1 receptor is involved in the potentiation induced by CGRP
Among several receptors for CGRP, CGRP1 receptor is the dominant type that distributes in the CNS [3, 4]. We next tested the role of CGRP1 receptor in this CGRP induced potentiation. Two different antagonists, peptide CGRP8-37 and non-peptide antagonist BIBN 4096, were used. As shown in Fig. 3, after application of CGRP8-37 or BIBN 4096, 10 nM CGRP produced potentiation was significantly reduced (CGRP8-37: F (2, 27) =0.3, p=0.7, one-way ANOVA, n=7 neurons/5 mice, Fig. 3A; BIBN 4096: F (2, 27) =0.5, p=0.6, one-way ANOVA, n=8 neurons/4 mice, Fig. 3B). These results indicated that CGRP1 mediated CGRP induced potentiation in the IC.
Effects on paired-pulse ratio by CGRP
Next, we would like to determine if CGRP may produce potentiation by enhancing the release of transmitters. Paired-pulse responses to a paired stimulation at 50 ms interval were collected. We calculated PPR before and after CGRP application. As shown in Fig. 4A and 4B, sample traces and pooled data showed that PPR was significantly reduced after applied CGRP and the reduction was long-lasting during the washout period (baseline: 1.6 ± 0.2, CGRP: 1.4 ± 0.1, washout: 1.4 ± 0.2, p<0.001 and p<0.001 compared with baseline, respectively; one-way ANOVA, n=9 neurons/6 mice). Furthermore, in experiments with the CGRP8-37 or BIBN 4096, the reduction of PPR were blocked (CGRP8-37: F (2, 27) =0.5, p=0.6, one-way ANOVA, n=7 neurons/5 mice, Fig. 4C left; BIBN 4096: F (2, 27) =0.6, p=0.5, one-way ANOVA, n=8 neurons/4 mice, Fig. 4C right). These data suggested that CGRP may produce its effect by affecting the release of glutamate, although we cannot completely rule out possible postsynaptic effects as well.
Effect of CGRP on sEPSCs
Spontaneous events are thought to be the results of the presynaptic action potential evoked neurotransmitter vesicles release from the readily releasable pool . The effects of CGRP on sEPSCs recorded from the pyramidal neurons of the IC were examined. As shown in Fig. 5, the frequency of sEPSCs was significantly increased with the bath application of 10 nM CGRP (2.3 ± 0.4 Hz vs. 3.3 ± 0.6 Hz, t= -2.8, p<0.05, paired t test, n=9 neurons/6 mice). A cumulative fraction plot showed that the inter-event interval was reduced during CGRP application (Fig. 5B left). While, the amplitude of sEPSCs was not significantly affected (6.6 ± 0.7 pA vs. 7.5 ± 0.7 pA, t=-1.3, p=0.2, paired t test, n=9 neurons/6 mice, Fig. 5B and C right). Next, we examined if the effects of CGRP can be blocked by CGRP1 receptor antagonists. We found that both CGRP8-37 and BIBN 4096 blocked the effects of CGRP on sEPSCs (CGRP8-37: frequency: 3.0 ± 0.5 Hz vs. 3.1 ± 0.6 Hz, t=-0.6, p=0.5, paired t test, n=7 neurons/4 mice; amplitude: 10.9 ±1.0 pA vs. 10.7±0.8 pA; t=0.7, p=0.5, paired t test, n=7 neurons/4 mice; BIBN 4096: frequency: 2.9 ± 0.4 Hz vs. 3.1 ± 0.4 Hz, t=-1.9, p=0.1, paired t test, n=8 neurons/4 mice; amplitude: 10.0 ± 1.0 pA vs. 10.1 ± 1.0 pA, t=-1.3, p=0.2, paired t test, n=8 neurons/4 mice). These results suggested that CGRP effects maybe mainly presynaptic and needs CGRP1 receptors.
Effect of CGRP on mEPSCs
Unlike the sEPSCs, miniature synaptic transmission is resulted from neurotransmitter release independent of action potential, which occurs randomly in the absence of stimuli. We also recorded mEPSCs in the IC in the presence of 1 μM TTX to further determine the role of presynaptic mechanisms of CGRP induced potentiation. It found that the frequency of mEPSCs were significantly increased after perfusing 10 nM CGRP (1.2 ± 0.2 Hz vs. 2.3 ± 0.4 Hz, t=-4.7, p<0.01, paired t test, n=9 neurons/5 mice, Fig. 6). Besides, a cumulative fraction plot showed a decrease of inter-event-interval during CGRP application (Fig. 6B). As to the amplitude of mEPSCs, no significant changes were observed (8.9 ± 0.4 pA vs. 8.9 ± 0.5 pA, t=0.2, p=0.8, paired t test, n=9 neurons/5 mice, Fig. 6B and C right). We also found that the effect of CGRP on mEPSCs was blocked in the presence of CGRP8-37 or BIBN 4096 (CGRP8-37: frequency: 2.2 ± 0.3 Hz vs. 2.3 ± 0.3 Hz, t=-1.1, p=0.3, paired t test, n=7 neurons/4 mice; amplitude: 8.8 ± 0.2 pA vs. 8.8 ± 0.1 pA, t=0.3, p=0.8, paired t test, n=7 neurons/4 mice; BIBN 4096: frequency: 2.2 ± 0.4 Hz vs. 2.3 ± 0.5 Hz, t= -1.4, p=0.2, paired t test, n=6 neurons/4 mice; amplitude: 9.7 ± 0.4 pA vs. 9.5 ± 0.4 pA, t= 1.9, p=0.1, paired t test, n=6 neurons/4 mice). These results demonstrated that CGRP enhanced excitatory synaptic transmission via increasing the probability of presynaptic neurotransmitter release in the IC and CGRP1 receptors are important for this process.
AC1-PKA signal pathways were required for CGRP induced potentiation
The primary signal transduction pathway for the CGRP receptor is mediated by Gαs, which activates AC, leading to the production of cyclic adenosine monophosphate (cAMP) and activation of protein kinase A (PKA) . Consistently, our previous study showed that in the ACC, the CGRP induced potentiation did need AC1 and PKA . Here we tried to determine if this signal pathway is also required in the IC. Firstly, a selective AC1 inhibitor, NB001 (50 μM)  was bathed during the baseline and CGRP periods. The results showed that NB001 completely blocked the effect of CGRP (F (2, 27) =0.2, p=0.7, one-way ANOVA, n=7 neurons/5 mice, Fig. 7A). Furthermore, a PKA inhibitor, KT5720 (1 μM) was found to attenuate CGRP produced effects (F (2, 27) =2.5, p=0.1, one-way ANOVA, n=7 neurons/4 mice, Fig. 7B). Our previous studies in the IC as well as ACC found that the same dose of inhibitor NB001 or KT5720 did not significantly affect baseline excitatory transmission [29, 31, 32].