Introduction
Patients with migraine usually experience not only headache but also balance problems and dizziness, i.e. motion sickness and recurrent vertigo [
1,
2]. With headache frequency increased, the incidence of dizziness and associated handicap significantly elevated [
2]. Of note, up to 85% of patients with chronic migraine (CM) reported vestibular symptoms [
2]. Unfortunately, most migraine patients with vestibular symptoms do not respond well to treatment with traditional vestibular suppressants [
3]. Despite this, it is not yet understood whether vestibular symptoms arise through overlapping neurobiology or independent mechanisms of migraine. Consequently, the application of migraine-specific treatments remains limited in this population [
4]. Central sensitization of trigeminovascular neurons in the trigeminal nucleus caudalis (TNC) is a leading culprit that contributes to sensory hypersensitivity in migraine [
5], while the enhancement of synaptic transmission is the neural basis of central sensitization in rat models of CM [
6,
7]. Our previous study demonstrated that central sensitization in the vestibular nucleus (VN) was the potential mechanism for vestibular symptoms in migraine, and vestibular dysfunction was significantly related to neuronal activation in VN [
8]. Thus, effectively suppressing neuronal activation via modulating synaptic transmission efficiency in VN might be a promising approach in the treatment of vestibular symptoms in migraine patients.
The neuropeptide calcitonin gene-related peptide (CGRP) has been demonstrated to play an essential role in facilitating pain transmission [
9]. CGRP is a 37-amino acid neuropeptide, and produced mainly by neurons [
10]. CGRP mediates its function primarily by activating CGRP1 receptor, containing calcitonin receptor-like receptor (CLR), a G protein-coupled receptor (GPCR), together with receptor activity modifying protein 1 (RAMP1), a single transmembrane protein which is required for functional expression of CLR at the cell surface [
11,
12]. Prior study found that the expression level of CGRP in VN increased significantly in a rat model of motion sickness, whilst anisodamine could markedly suppress the CGRP expression in VN and reverse motion sickness-related behaviors [
13]. Our previous study showed similar results in CM rats, and blunting CGRP synthesis in VN caused a marked reversal of nitroglycerin (NTG)-induced vestibular dysfunction [
8]. To date, clinical trials targeting CGRP and its receptors witness a great success in migraine treatment [
14]. However, whether CGRP and its receptors are viable therapeutic targets in migraine patients with vestibular symptoms is uncertain, and whether CGRP antibodies and CGRP1 receptor antagonists can alleviate vestibular symptoms in migraine patients requires further investigation.
CGRP has been demonstrated to produce long-term potentiation (LTP) in the insular cortex and anterior cingulate cortex [
15]. This phenomenon could be significantly blocked by specific CGRP1 receptor complex antagonists, CGRP
8 − 37 or BIBN4096BS, which further achieving analgesic effects [
15,
16]. Besides that, these two antagonists could also reverse pain-related synaptic function in the amygdala of arthritic rats, as evidenced by the reduced neuronal excitability and decreased amplitude of miniature excitatory postsynaptic currents (mEPSCs) [
17], pointing that CGRP and CGRP1 receptor signaling might modulate synaptic function in CM. Although CGRP1 receptor is generally considered a Gα
s-coupled GPCR, it is becoming clear that it also couples to additional G proteins and other proteins [
12,
18]. In vitro studies showed that CGRP activated protein kinase C (PKC) and extracellular signal regulated kinase (ERK) in HEK293 cells, and pre-incubation spinal cord slices with specific ERK or PKC inhibitor significantly shortened CGRP-stimulated firing time of lamina I neurons [
19]. Moreover, cAMP response element-binding protein (CREB), one of the major downstream mediators of ERK, has been considered as an important indicator of central sensitization in CM rats [
20,
21]. Downregulation of phosphorylated CREB at Serine 133 site (p-CREB-S133) was significantly associated with the reduction of neuronal activation in TNC after CM [
21]. These data suggests that CGRP and CGRP1 receptor modulate synaptic transmission in CM rats might through PKC/ERK/CREB signaling pathway.
Based on the above-mentioned evidence, we hypothesized that CGRP1 receptor antagonist BIBN4096BS would reduce neuronal activation in VN and alleviate vestibular dysfunction in rat models of CM and that these beneficial effects might be mediated by PKC/ERK/CREB signaling. Through these attempts, we explore the therapeutic efficacy of CGRP1 receptor antagonist in a CM model as a preclinical approach for potential clinical translation in migraine patients with vestibular symptoms.
Discussion
Multiple lines of evidence show that migraine and vestibular symptoms are frequently encountered clinically [
2,
4,
37]. The rate of disability is high, and there is currently a lack of effective prophylactic treatment [
38]. The exact pathophysiology of vestibular symptoms in migraine is incompletely understood but could include anatomical pathways and neurochemical modulation between TNC and VN [
8,
39]. CGRP or CGRP1 receptor antibodies and antagonists have proved to appreciably alleviate headache severity and frequency in both of episodic and chronic migraine patients [
9]. Our previous study found that increased expression of CGRP was positively associated with neuronal activation in VN, as well as vestibular dysfunction after CM [
8]. To date, whether CGRP1 receptor involves in the development of vestibular sensitization, and how CGRP regulates neuronal activation in VN after CM remains largely unknown. Thus, the present study was to investigate whether CGRP1 receptor contributes to vestibular dysfunction after CM by regulating synaptic transmission in VN.
Our results showed that CGRP and two major components of its receptor, CLR and RAMP1, were significantly elevated in VN after CM. CLR and RAMP1 were primarily co-expressed with neurons in VN. Multiple treatments with CGRP1 receptor selective antagonist, BIBN4096BS, alleviated mechanical allodynia, thermal hyperalgesia and vestibular dysfunction induced by intermittent administration of NTG, and this process was associated with the downregulation of synaptic associated proteins, restoration of aberrant morphological characteristics of synapses, and suppression of neuronal activation in VN after CM. Inhibiting CGRP1 receptor with BIBN4096BS decreased the overexpression of PKC, and phosphorylation of ERK and CREB-S133 in VN after CM. Taken together, our data suggests the therapeutic potentials of BIBN4096BS to restore vestibular dysfunction in patients with CM.
Consistent with our previous study, we established a reliable CM model that had vestibular dysfunction via systematic NTG administration [
8]. Each NTG administration caused several behavior signs of allodynia and hyperalgesia, mimicking the acute attack of migraine. While repeated NTG administration induced the pronounced and sustained allodynia and hyperalgesia as found in most of CM patients [
10,
32]. Unlike the lowered pain thresholds, significant vestibular dysfunction was not observed after the initial NTG injection, and vestibular function was exaggerated after the third NTG injection. These phenomena were consistent with those reported in clinical-based studies: the development of vestibular symptoms, including motion intolerance, often lagged several years behind headache in migraine patients; patients with chronic migraine experienced more frequent vestibular symptoms than patients with episodic migraine [
2,
40,
41]. Additionally, migraine is more prevalent in women than in men, which are mainly due to the role of estrogen [
42,
43]. In fact, the regulation of pain by estrogen is very complicated. The level and stability of estrogen can affect its modulation of pain, including aggravating or relieving [
44]. The main trigger factors of menstrual-related migraine seem to be the withdrawal of estrogen, and reducing the magnitude of decline in estrogen concentration can prevent menstrual-related migraine [
43,
45]. Studies have found that endogenous changes in the concentration of estrogen and progesterone in premenstrual syndrome or the use of exogenous hormones (such as oral contraceptives) may cause vertigo [
46]. In order to avoid the influence of estrogen, we chose male mice to establish the model. Given the significant sex differences in the prevalence of migraine, the sex dimorphism of CGRP1 receptor involvement in pathogenesis of migraine-related dizziness requires further exploration.
A plethora of studies have demonstrated the critical role of CGRP in the trigeminovascular system that are involved in migraine pathology [
47]. Abundant expression of CGRP in laminae I and II of the TNC has been reported as an important indicator for central sensitization of CM, which is responsible for the development of spontaneous cephalic cutaneous and extracephalic allodynia [
5,
21]. Consistently, this study also observed the elevated expression of CGRP in TNC and VN after CM. In addition, reducing CGRP expression in VN attenuated excitability of neurons and reversed the vestibular dysfunction induced by NTG administration [
8]. These data indicated that elevated CGRP level in VN might be an index to vestibular sensitization in CM rats. The various effects of CGRP act primarily through CGRP1 receptor, which comprising CLR and RAMP1 [
47,
48]. In preclinical animal models and clinical trials, “gepants” are proved to have high affinity for CGRP1 receptor [
48]. To make it easier to translate our findings for clinical application, the present study applied BIBN4096BS (namely: olcegepant) as the treatment regimen [
48,
49]. However, another GPCR, the calcitonin receptor (CTR), which is closely-related to CLR, together with RAMP1 (namely: AMY
1 receptor) recently has been reported to have high affinity for both of CGRP and amylin [
48]. Additionally, adrenomedullin, an amylin analogue, is demonstrated to effectively provoke migraine-like headache in migraine patients [
50]. Less is known about the nociceptive role of AMY
1 receptor in migraine pathology and the clinical significance of the interaction between CGRP and AMY
1 receptor. Thus, we selected CGRP1 receptor as the primary receptor for the following studies.
CGRP1 receptor are broadly expressed in the mammalian CNS [
5,
51]. Considering the limited passage of the gepants (2%) across the blood–brain barrier, BIBN4096BS was administrated via intracerebroventricular route to explore the central action of CGRP1 receptor [
47]. We found that antagonizing CGRP1 receptor significantly abolished behavioral signs of mechanical allodynia, thermal hyperalgesia and vestibular dysfunction in CM rats, and these were closely related with the neuronal activation in VN. But how CGRP modulates the neuronal activation in VN after CM is still unclear. According to the immunofluorescence staining, CLR and RAMP1 were both primarily co-expressed with neurons, implying that CGRP might exert its effect mainly through the neuronal CGRP1 receptor. In vivo and in vitro studies demonstrated that exogenous CGRP can enhance the neural synaptic transmission in spinal cord, anterior cingulate cortex and insular cortex, and that could be notably blocked by BIBN4096BS [
15,
16,
52,
53]. Therefore, we proposed that CGRP could modulate the synaptic transmission efficiency via CGRP1 receptor in VN after CM. We first examined the expression of several synaptic associated proteins, including PSD95, Syp and Syt-1, in VN after CM. PSD95 is a structural protein located on the postsynaptic membrane, implicated in synaptic maturation, strengthening, and plasticity [
54]. Syp, a major integral membrane protein of synaptic vesicles, is necessary for vesicle formation and exocytosis, which can regulate the efficiency of synaptic transmission by influencing the synaptic structure and the release of neurotransmitters [
55,
56]. Syt-1, acting as a Ca
2+ sensor at neuronal synapses, is a marker of synaptic transmission, which synchronizes neurotransmitter release with Ca
2+ influx during action potential firing and mediates ultrafast exocytosis of synaptic vesicles [
57,
58]. Our study found that the expression of synaptic associated proteins was significantly increased after CM, while multiple treatments of BIBN4096BS markedly downregulated their expression. Moreover, we detected the synaptic ultrastructure and the density of neuronal dendritic spines of VN neurons in CM rats, which are closely related to synaptic transmission efficiency, to further confirm that CGRP/CGRP1 receptor involves in the regulation of synaptic transmission in VN after CM.
We further explored downstream orchestrators of CGRP1 receptor inhibition to determine potential mechanisms for neuronal activation. Activation of the glutamate N-methyl-D-aspartate (NMDA) receptors is a hallmark to the development and maintenance of central sensitization in chronic pain, including migraine [
10]. Incubation with CGRP or activation of CGRP1 receptor significantly promotes NMDA receptors-mediated excitatory potentials in central amygdala [
10,
59]. The downstream activation of PKC induces phosphorylation of NMDA receptors and increases calcium inputs which further enhances the excitability of dorsal horn neuron, thus maintaining a state of central sensitization [
10,
19]. Similar to the previous results, we found that the expression levels of PKC, phosphorylated ERK and CREB-S133 were significantly increased in CM rats. The effects of NTG administration on PKC expression and phosphorylation of ERK and CREB-S133 were reversed by BIBN4096BS. Additionally, inhibition of PKC with CHE reduced the overexpression of synaptic associated proteins, p-ERK and p-CREB-S133 caused by CM, and inhibited the activation of neurons in the VN of CM rats. These results suggested that CGRP may improve synaptic transmission and neuron activation of VN through CGRP1 receptor/PKC/ERK/CREB in CM rats.
In neuronal circuits, the vast majority of excitatory synaptic transmission occurs at the postsynaptic dendritic spines [
60]. In our studies, we found more abundant dendritic spines in CM group compared to saline group. And multiple BIBN4096BS treatments significantly decreased the density of dendritic spines in VN. Protein kinase C (PKC)-dependent mechanisms can promote synaptic function in the mature brain. Studies found that the activation of protein kinase C (PKC) ε and α has been linked to synaptogenesis. And PKCε/α activator bryostatin can increase dendritic spine density of pyramidal neurons in hippocampal CA1 region of aged rats [
61]. In mature mouse hippocampal neuron cultures, PKC activator bryostatin increased the proportion of mushroom dendritic spines in total spine density, enhanced synaptic transmission [
62]. As well, atypical protein kinase C (aPKC), a subgroup of PKC, can promote increased dendritic spine density in hippocampal neurons through p190 RhoGAP and RhoA pathway [
63,
64]. In our research, we found that inhibiting PKC with chelerythrine chloride (CHE) ameliorated the overexpression of synapse-associated proteins. Therefore, we speculate that the activation of PKC pathway can also cause an increase in the density of neuronal dendritic spines at the VN site. More direct evidence is needed in our future studies.
According to previous research, under physiological conditions, the GABAergic neurons that project to the vestibular nucleus are mainly located in spinal trigeminal nucleus orails (Sp5O) and a small amount of spinal trigeminal nucleus interpolaris (Sp5I), and the glutamatergic neurons that project to VN are mainly located at Sp5O, Sp5I and spinal trigeminal nucleus candalis (Sp5C or TNC). GABA immunoreactive neurons account for about 15% of trigeminal vestibular neurons, and glutamatergic neurons account for about 37% of the trigeminal vestibular neurons [
39]. Our previous study found that CM induced neuronal activation in the TNC and VN, and TNC-projecting VN neurons were activated after CM [
8]. As well, glutamatergic neurons of VN, the excitatory neurons, were recently considered to act as the fundamental role in controlling posturo-locomotor behaviors [
30]. However, we currently have no direct evidence for the type of neurons activated at the VN site in CM rats. We will explore it more deeply in future research.
There are several limitations in this study. Although CGRP1 receptor is mainly expressed in neurons of VN areas, we cannot completely exclude that the neuroprotective effects exerted by CGRP1 receptor inhibition may involve glia in CM rats. Also, we are focusing on PKC/ERK/CREB signaling, but other mechanisms require further investigation. Then, we didn’t perform the basal vestibular function test before each NTG administration. Although we speculate that vestibular dysfunction in migraine patients may be a progressive rather than acute process, the data of the vestibular function test before each NTG administration may help us understand the development of the disease comprehensively. Our research ignored the study of three synaptic associated proteins in the synaptic subcellular fractions, and we will pay attention to this part in the future research. In our study, BIBN4096BS and protein kinase C (PKC) inhibitor chelerythrine chloride (CHE) were administered intracerebroventricularly, rather than local microinjection into the VN. Therefore, we cannot completely rule out effects of these drugs on other brain regions. Last, we only explored the effects of morphological alternations on synaptic transmission. Future studies on electrophysiology are needed to examine the effects on synaptic function.
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