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. Author manuscript; available in PMC: 2026 Apr 29.
Published in final edited form as: Neuroscience. 2025 Nov 7;591:40–51. doi: 10.1016/j.neuroscience.2025.11.004

Calcitonin Gene-Related Peptide Differentially Modulates Intracellular Calcium Levels in Response to Depolarizing Stimuli in Trigeminal Ganglion Neurons and Glial Cells

Nicole M Nalley 1, Paul L Durham 1
PMCID: PMC12799331  NIHMSID: NIHMS2122761  PMID: 41207466

Abstract

Elevated levels of calcitonin gene-related peptide (CGRP) are implicated in migraine pathology, but its effects on the excitability state of trigeminal ganglion Aδ and C-fiber neurons and glia have not been fully investigated. The goal of this study was to determine changes mediated by CGRP on intracellular calcium levels in trigeminal neurons and glia in response to depolarizing stimulation. Intracellular calcium levels were determined in Aδ and C-fiber neurons of primary trigeminal ganglion cultures obtained from neonatal Sprague Dawley rats using Fura-2 and fluorescent microscopy. Cells were left untreated or preincubated for 2 hours with CGRP, then incubated with the depolarizing stimuli KCl or ATP. Data analysis was performed using Olympus CellSens Dimension software and JASP. CGRP greatly increased the calcium amplitude in response to 60 mM KCl and 100 μM ATP in Aδ neurons, while causing a smaller, similar response in C-fiber neurons. CGRP also increased the percentage of Aδ neurons responsive to 60 mM KCl and enhanced the magnitude of the calcium response. In glial cells, CGRP increased the magnitude of the 60 mM KCl-mediated response. However, CGRP suppressed the stimulatory calcium response to 15 mM KCl in neurons and glial cells and differentially modulated the calcium response to 30 mM KCl in Aδ and C-fiber neurons. These results provide evidence of a novel role of CGRP to regulate the excitability state of Aδ and C-fiber neurons and glial cells implicated in pain signaling in migraine differently in response to the strength of the depolarizing stimulus.

Keywords: Primary cultures, ATP, KCl, Fura-2, Migraine, Ion channels

Graphical Abstract

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Introduction

Migraine is one of the most prevalent, debilitating pain disorders and is commonly associated with chronic pain (Stovner et al., 2007). Migraine is characterized by severe headache attacks that can last a few hours to a few days with increased sensitivity to light, touch, smell, noise, and hyperexcitability of the brain (Aurora & Wilkinson, 2007; Burstein et al., 2015). Migraine pathology is known to involve sensitization and activation of the trigeminal ganglion nerves that provide sensory innervation to the head and face (Chichorro et al., 2017; List & Jensen, 2017; Romero-Reyes & Uyanik, 2014; Sessle, 2011). The trigeminal ganglion is a cluster of sensory nerve cell bodies outside the central nervous system that relays nociceptive information to the spinal trigeminal nucleus (Burstein & Jakubowski, 2005; Buzzi, 2001; Chichorro et al., 2017; Shankland, 2000). The ganglion consists of 4 cell types including Aδ and C-fiber nociceptive pseudounipolar neurons and the associated glia, satellite glia cells and Schwann cells (Messlinger et al., 2020). Aδ neurons are myelinated neurons with a diameter of ~ 35–50 μm that function to protect and minimize tissue injury and damage by rapidly transmitting short, sharp nociceptive signals. C-fiber neurons are unmyelinated neurons with a smaller diameter of ~ 20–30 μm and are responsible for transmitting longer duration nociceptive signals associated with persistent burning or dull pain and itch. Satellite glia cells, which are closely associated with Aδ and C-fiber neuronal cell bodies and form a functional unit, are responsible for communicating with neurons, providing nutrients, releasing cytokines, and modulating the excitability and activation threshold of neurons (Durham & Garrett, 2010; Goto et al., 2017; Qarot et al., 2024; Takeda et al., 2009). Schwann cells myelinate the axonal processes projecting Aδ neurons while Remak Schwann cells ensheathe C-fiber axons (Jessen & Mirsky, 2016; Messlinger et al., 2020).

Upon activation of trigeminal ganglion neurons, the neuropeptide calcitonin gene-related peptide (CGRP) is released in peripheral tissues, the ganglion, and in the spinal trigeminal nucleus (Cornelison et al., 2016; Durham, 2016; Edvinsson & Goadsby, 1995; Edvinsson et al., 2018). Elevated levels of CGRP correlate with pain levels experienced in migraine and promote neurogenic inflammation and pain (Cohen et al., 2022; Iyengar et al., 2017). The cellular effects of CGRP are mediated by the CGRP receptor, which is a G-protein coupled receptor that has three components: receptor activity-modifying protein (RAMP1); calcitonin receptor-like receptor (CLR); and receptor component receptor (RCP) (Iyengar et al., 2017). The RAMP1 subunit is critical for receptor function since it defines the relative potency of ligands and directly binds CGRP. Upon CGRP binding to RAMP1, the receptor couples to the Gs protein subunit that stimulates adenylate cyclase leading to elevated levels of cAMP and stimulation of the protein kinase A (PKA) pathway. The CGRP receptor has also been shown to couple to other G proteins and other downstream signaling proteins including protein kinase C and MAP kinases (Cady et al., 2011; Fabbretti et al., 2006; Sun et al., 2004; Vause & Durham, 2009, 2010). Activation of the PKA, PKC, and ERK pathways modulates the activity of ion channels in the plasma membrane and hence function to regulate the excitability state of trigeminal neurons (Sun et al., 2004).

The activity of ion channels associated with neurons and glia controls the membrane potential and their excitability state (Grider et al., 2025). Voltage-gated ion channels are opened in response to a change in membrane voltage (Alexander et al., 2023) from the resting membrane potential maintained by potassium channels (Kawamoto et al., 2012). Calcium channels are opened or closed in response to depolarizing stimuli (Grider et al., 2025) and increased intracellular calcium levels are known to regulate neurotransmission, membrane excitability, enzyme activation, gene expression, and many other cellular processes. Calcium influx is regulated through multiple voltage-gated calcium channels including L, N, and P/Q (Zhang et al., 2025). Elevated levels of extracellular potassium chloride (KCl), which occurs during an inflammatory response and is implicated in migraine pathology, causes neurons to depolarize and release pro-inflammatory molecules including CGRP (Rienecker et al., 2020). ATP is another stimulus associated with migraine pathogenesis since activation of ATP-sensitive ion channels are reported to function as a migraine trigger in susceptible individuals (Al-Karagholi et al., 2021). Elevated levels of ATP mediate depolarization, release of neurotransmitters and CGRP, and enhance gene transcription via an influx of calcium through the ligand-gated P2X receptors (Giniatullin & Nistri, 2023; Kawamoto et al., 2012; Khakh & North, 2012). In the trigeminal ganglion, P2X receptors are expressed by nociceptive Aδ and C-fiber neurons with the P2X3 receptor subunit primarily present on sensory neurons that release CGRP (Giniatullin & Nistri, 2023).

In this study, changes in intracellular calcium levels in response to KCl and ATP in primary mixed cultures of trigeminal ganglia were determined using the ratiometric fluorescent dye Fura-2 AM ester with fluorescent microscopy. Results from this study provide evidence of a novel role of CGRP to differentially modulate the calcium response in neuronal and glial cells to varying KCl concentrations and potentiate the stimulatory effect of ATP in Aδ neurons.

Experimental procedures

Animals

The animal protocol used in this study was approved by the Institutional Animal Care and Use Committee at Missouri State University and conducted in accordance with the Animal Welfare Act, National Institutes of Health guidelines, and ARRIVE guidelines. Adult pregnant female Sprague-Dawley rats were purchased from Missouri State University’s internal breeding colonies (Springfield, MO). Animals were housed in clean, plastic cages with unlimited access to food and water. The holding room was kept at a constant temperature of 22–24°C, with a 12-hour light/dark cycle.

Establishing primary mixed cultures of trigeminal ganglia

Primary mixed cultures of trigeminal neurons and glia were established essentially as described in a recent protocol (Antonopoulos et al., 2024). Trigeminal ganglia were harvested from male and female 3–5-day old Sprague Dawley rat pups and placed in 4°C L-15 media buffered with HEPES to a pH of 7.4 (Sigma Aldrich, St. Louis, MO), which will be referred to as plating media, during dissections. Following dissections, tissues were placed in 10 mL of a room temperature enzyme digestive solution containing 10 mg/mL Dispase II (Sigma Aldrich) and RQ1 DNase, (Promega, Madison, WI). The tissues were rotated at 15 RPM in a 37°C incubator for 30 minutes and then centrifuged at 500 RPM for 2–3 minutes to pellet the tissues. The supernatant was discarded, and tissues resuspended in 5 mL of plating media. Mechanical trituration was used to dissociate cells and obtain a mostly single-cell population. The supernatant containing cells was transferred to a sterile tube and the dissociation step repeated. Following centrifugation, the pellet was resuspended in 10 mL of plating media and large tissues removed using a sterile glass Pasteur pipet. The final 10 mL of supernatant containing cells was centrifuged for 3 minutes at 1500 RPM to pellet the dissociated neurons and glia. Cells were then resuspended in a room temperature solution containing 90% fetal bovine serum (FBS, Atlanta Biologicals, Norcross, GA) and 10% dimethyl sulfoxide (DMSO, Sigma Aldrich), and 500 μL aliquots distributed into cryogenic vials for long-term storage in liquid nitrogen.

To establish mixed cultures from the cryopreserved trigeminal ganglion cells, a vial was removed from the cryogenic Dewar and thawed by adding 10 mL of room temperature plating media. The cells were centrifuged for 3 minutes at 1500 RPM. Following centrifugation, the supernatant was discarded and the pellet containing cells resuspended in an appropriate plating volume of TG complete media. TG complete media consists of L-15 medium containing 10% FBS (Atlanta Biologicals), 50 mM glucose (Sigma Aldrich), 250 mM ascorbic acid (Sigma Aldrich), 8 mM glutathione (Sigma Aldrich), 2 mM glutamine (Sigma Aldrich), 10 ng/mL mouse 2.5 S nerve growth factor (Alomone Laboratories, Jerusalem, Israel), an antibiotic mixture of penicillin (100 units/mL) and streptomycin (100 μg/mL, Sigma Aldrich), and the antimycotic amphotericin B (2.5 mg/mL, Sigma Aldrich). Cells were plated on 12 mm poly-L-lysine coated glass coverslips (Electron Microscopy Sciences, Hatfield, PA) at a density of ~2 ganglia (~34,000 cells) per 24-well plate (Greiner Bio-One, Germany) in 500 μL of TG complete medium per well.

Characterization of cryopreserved cultures

Cell types were identified in 2-day old mixed cultures of trigeminal ganglia based on their unique morphology seen in 200x differential interphase contrast (DIC) images as previously described (Antonopoulos et al., 2024). In the same cultures, DAPI mounting medium (ab104139, Abcam, Cambridge, UK) was added to stain individual cell nuclei and preserve the fluorescent signal. Multiple images were acquired using a Zeiss Axiocam mRm camera (Carl Zeiss, Thornwood, NY) mounted on a Zeiss Imager Z1 fluorescent microscope.

Immunocytochemistry

Cellular changes in protein expression were investigated using immunocytochemistry as previously described (Antonopoulos et al., 2024). Primary cultures of cryopreserved trigeminal ganglion were incubated at 37°C for two days after plating. Cells were fixed using 4% paraformaldehyde diluted in phosphate buffered saline (PBS, pH 7.4) for 15 minutes. After fixing, cells were blocked and permeabilized using a PBS solution containing 0.1% Triton X-100 (Sigma Aldrich) and 5% donkey serum (Jackson ImmunoResearch Laboratories, West Grove, PA) for 20 minutes. Cells were then incubated at 4°C overnight in a humidified chamber with primary antibodies that specifically bound the CGRP subunit protein RAMP1 (Alomone Labs, ARR021AN402), which was diluted (1:5000) in PBS containing 1% donkey serum. The following day, cells were incubated for 1 hour at room temperature with Alexa-Fluor conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) diluted at 1:200 in PBS. As a control, some slides were incubated with only the Alexa-Flour conjugated secondary antibodies.

Coverslips were rinsed with PBS then placed on Diamond White Glass positively charged glass microscope slides (Globe scientific, Mahwah, NJ). DAPI mounting medium (Abcam) was added to each coverslip and a rectangular glass coverslip was gently placed over the slide and secured with a clear coat of nail polish. Slides were stored at 4°C until time for imaging. Multiple images of each coverslip were captured at 200x magnification within one week of immunostaining using a Zeiss Axiocam mRm camera mounted on a Zeiss Images Z1 fluorescent microscope.

Calcium imaging

Changes in intracellular calcium levels in three-day old cultures of trigeminal ganglion neurons and glial cells were determined using fluorescent microscopy and Fura-2 essentially as described (Antonopoulos et al., 2024; Durham & Masterson, 2013; Masterson & Durham, 2010; Vause et al., 2007). The ratiometric fluorescent dye Fura-2 AM ester (ThermoFisher Scientific, stored at −20°C) at a stock solution concentration of 1 mM was added to each well as a 1:1000 dilution to achieve a final concentration of 1 μM in HEPES-buffered saline (HBS, pH 7.4) solution. The cells were incubated at 37°C for 30 minutes to facilitate uptake and cleavage of the AM ester to maintain the Fura-2 dye in the cell. The HBS incubation solution is comprised of 22.5 mM HEPES (Sigma Aldrich), 135 mM NaCl (Sigma Aldrich), 3.5 mM KCl (PR1MA, Fenton, MO), 1 mM MgCl2 (Sigma Aldrich), 2.5 mM CaCl2 dihydrate (Sigma Aldrich), 3.3 mM glucose (PR1MA) and 0.1% BSA (Sigma Aldrich) in sterile cell water. Following incubation, cells were rinsed with HBS three times then incubated for an additional 30 minutes at 37°C. To perform calcium imaging, a coverslip containing cells was removed from the 24 well plate and placed in the microscope stage chamber containing 1 mL of HBS and then an additional 1 mL of HBS was added to the chamber for a total volume of 2 mL of HBS. An Olympus IX83 microscope and Olympus CellSens Dimension software version 4.1 (Evident Scientific, Tokyo, Japan) was used to capture a 200X image of the cells to establish a reference image. After the reference image was captured and dye loading confirmed, baseline calcium levels were measured every 20 seconds for 2 minutes. Following that initial 2 minutes, readings were briefly paused to allow manual addition of a 3 M stock solution of KCl to achieve a final concentration of 60 mM, 30 mM, 15 mM, or a 5 mM stock solution of ATP (Sigma Aldrich) to achieve a final concentration of 100 μM. Simultaneously with the addition of KCl or ATP to the media, data collection was resumed, and readings were taken every second for 20 seconds, then every 20 seconds for an additional 160 seconds. To investigate the effect of CGRP, 2 hours prior to initiation of calcium imaging, CGRP was added to the cell media of separate coverslips to achieve a final concentration of 1 μM. A 100 μM stock solution of the neuropeptide CGRP (TOCRIS Bioscience, Bristol, UK) was prepared in sterile cell water, and stored in aliquots at −20°C.

For analysis, regions of interest (ROI) were manually drawn around the cell body of all Aδ and C-fiber neurons, five satellite glia cells, and five Schwann cells in the field of view prior to any stimulation. During analysis, a scale bar was used to identify Aδ and C-fiber neuronal cells. Cells with a cell body diameter greater than 30 μm were labeled as Aδ neurons while neurons with a cell body less than 30 μm were labeled as C-fiber neurons. The calcium response as measured by the 340/380 ratio, which corresponds to the ratio of bound vs unbound intracellular calcium, was determined for each cell (Fig. 1). A positive response in neurons was regarded as an increase in calcium levels by at least 0.1 relative light units within ten seconds post stimulation and for glia if there was a 0.1 unit increase after 20 seconds post stimulation. These cells were considered responsive. Cells that exhibited a high baseline level of 0.4 relative light units above the average baseline value were not included in the analysis. Similarly, cells in which calcium levels did not decrease following their peak, but rather exhibited a sustained elevated response, were considered outliers and were not included in the analysis. Cells that did not exhibit an increase of more than 0.1 relative light units above baseline were considered unresponsive and were not included in the analysis. The remaining cells after removing sustained elevated responsive cells and unresponsive cells were labeled as transient elevated responsive cells. The average transient change in intracellular calcium response for cells was calculated to generate the graphs and data are presented as relative calcium levels based on the 340/380 Fura-2 ratio. To investigate the magnitude of the response, the area under the curve (AUC) was measured using NIH ImageJ 1.54d for each graph produced by the average of the transient elevated responsive cells from each coverslip. The amplitude was measured in individual cells by comparing the peak values of all transient elevated responsive cells. The average percentage of responsive, unresponsive, transient elevated responsive cells, and sustained elevated responsive cells for each coverslip were compared between the control and CGRP groups. Each condition was performed in triplicate in a minimum of 4 independent experiments.

Figure 1. Summary of analyzed parameters of intracellular calcium levels monitored by changes in Fura-2 in neurons and glia.

Figure 1.

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A) Representative images are provided to show the relative level of intracellular calcium in unstimulated cells and cells 40 seconds post stimulation with 60 mM KCl. Enlarged images of boxed regions highlight the morphology and the difference in intracellular calcium levels before and after stimulus in each cell type. Graphs represent the average change in relative calcium levels of KCl-stimulated cultures. The color bar correlates to the ratio of 340/380nm. B) Characterization of calcium waveforms included measurements of amplitude and area under the curve (AUC). A cell’s response to stimuli was characterized as responsive (< 0.1 unit increase from baseline) or unresponsive. Responsive cells were split into two categories: transient elevation and sustained elevation.

Statistical analysis

An ANOVA was performed using JASP 0.18.3 to compare the means for amplitude, area under the curve, percentage of unresponsive cells, percentage of cells with a sustained elevated response, and percentage of cells exhibiting a transient elevated responsive in cultures stimulated with KCl or ATP and cultures incubated with CGRP prior to addition of KCl or ATP. A p value was considered statistically significant if less than 0.05. Cells exhibiting high baseline, a sustained response, or unresponsive to KCl stimulation were considered as outliers and were not included in the data set for statistical analysis since these characteristics reflect an altered state of neuronal excitability, which may cause the cell to respond abnormally depending on the cell type and type of stimulus (Kawamoto et al., 2012; Rienecker et al., 2020).

Results

Characterization of cryopreserved cultures

DIC microscopy was used to identify the four main cell types in the trigeminal ganglion based on their unique cellular and nuclear morphology (Fig. 2A). Aδ neurons were identified by their large, round cell body with a diameter ranging between 35–50 μm. C-fiber neurons were identified by their smaller, round cell body with a diameter between 20–30 μm. Satellite glia cells were characterized by their small, elongated flat cell body, round nucleus, and extension of multiple processes while Schwann cells were identified by their bipolar morphology, elongated nucleus, and extended processes.

Figure 2.

Figure 2.

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RAMP1 is Expressed in Neurons and Glia of Primary Trigeminal Ganglion Mixed Cultures. A. Differential interference contrast (DIC) and DAPI-stained merged images are shown. Enlarged images below highlight the unique morphology and presence of the four major cell types: Aδ and C-fibers neurons, satellite glial cells, and Schwann cells. B. Established cultures were co-stained with an antibody to the CGRP receptor RAMP1 and the nuclear dye DAPI. RAMP1 was abundantly expressed in the cell body and processes of Aδ and C-fiber neurons, while lower levels were observed in satellite glia, and Schwann cells. Enlarged images of boxed neuronal (blue) and glial (orange) regions highlight the morphology and presence of the four major cell types. Arrows identify the different cell types including Aδ neuron (blue), C-fiber neuron (red), satellite glial cell (yellow) and Schwann cells (green).

Immunocytochemistry

To determine which cells expressed the CGRP receptor component RAMP1, primary cultures of trigeminal ganglia were immunostained using an antibody specific for RAMP1 and co-stained with the nuclear dye DAPI (Fig. 2B). RAMP1 expression was observed in all four cell types: Aδ and C-fiber neurons, satellite glia, and Schwann cells. Based on the immunostaining intensity, RAMP1 was most abundantly expressed in the cell body of Aδ and C-fiber neurons and could be observed in axonal processes. No specific cellular staining was observed in the absence of primary antibodies (data not shown).

CGRP potentiation of the calcium response to 60 mM KCl

Initially, changes in intracellular calcium levels in primary trigeminal ganglion cultures were determined in neuronal and glial cells in response to the addition of 60 mM KCl using the ratiometric dye Fura-2. Under naive conditions, addition of 60 mM KCl resulted in a rapid, transient increase in the relative level of intracellular calcium in Aδ neurons from an average baseline 340/380 ratio of 0.26 +/− 0.01 (Fig. 3A; 106 total cells). There were 5 cells exhibiting a high baseline and were not included in the analysis. The average peak amplitude was 0.55 +/− 0.02 and the average AUC was 5681 +/− 604. The average percentage of responsive cells was 62.4 % +/− 6.5 (66 cells) and average percentage of unresponsive cells was 37.6 % +/− 6.5 (40 cells). The average percentage of transient elevated responsive cells was 39.2 % +/− 6.5 (45 cells) and average percentage of sustained elevated responsive cells was 20.2 % +/− 6.5 (16 cells).

Figure 3.

Figure 3.

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CGRP Potentiates the Stimulatory Response in Trigeminal Ganglion Neurons and Glia to 60 mM KCl. The average calcium levels based on the ratio of bound (340 nm) versus unbound intracellular calcium (380 nm) in cells treated with only 60 mM KCl (solid line) or pretreated with CGRP 2 hours prior to KCl stimulation (dashed line) are shown in Aδ neurons (A), C-fiber neurons (B), satellite glial cells (C), and Schwann cells (D). Error bars are shown for the SEM at peak amplitude and at 40 second intervals. N = 8 in triplicate.

A rapid and transient increase in the 340/380 ratio was also observed in C-fiber neurons from an average baseline ratio of 0.25 +/− 0.01 (Fig. 3B; 129 total cells). There were 6 cells that exhibited a high baseline. The average peak amplitude was 0.56 +/− 0.02 and average AUC of 5519 +/− 636. The average percentage of responsive cells was 67.5 % +/− 6.4 (84 cells) and average percentage of unresponsive cells was 32.5 % +/− 6.4 (45 cells). The average percentage of transient elevated responsive cells was 51.1 % +/− 5.9 (64 cells) and average percentage of sustained elevated responsive cells was 12.5 % +/− 4.1 (14 cells).

Similar to the response observed in neuronal cells, satellite glia exhibited a transient increase in the F340/380 ratio from an average baseline ratio of 0.29 +/− 0.01 (Fig. 3C; 114 total cells). There were 4 cells that exhibited a high baseline. The average peak amplitude was 0.60 +/− 0.02 and average AUC was 3898 +/− 405. The average percentage of responsive cells was 72.9 % +/− 6.5 (82 cells) and average percentage of unresponsive cells was 27.1 % +/− 6.5 (32 cells). The average percentage of transient elevated responsive cells was 61.1 % +/− 7.5 (64 cells) and average percentage of sustained elevated responsive cells was 12.7 % +/− 4.5 (14 cells).

A transient increase in the F340/380 ratio was observed in Schwann cells from an average baseline ratio of 0.28 +/− 0.01 (Fig. 3D; 117 total cells). There was 1 cell that exhibited a high baseline. The average peak amplitude was 0.58 +/− 0.02 and average AUC was 3104 +/− 326. The average percentage of responsive cells was 75.2 % +/− 6.1 (88 cells) and average percentage of unresponsive cells was 24.8 % +/− 6.1 (29 cells). The average percentage of cells exhibiting a transient elevated response was 54.6 % +/− 5.9 (64 cells) and average percentage of sustained elevated responsive cells was 19.75 % +/− 7.2 (23 cells).

CGRP significantly potentiated the calcium response in cultures stimulated with 60 mM KCl in all four cell types (Fig. 3). The mean calcium response for Aδ neurons changed from an average baseline ratio of 0.28 +/− 0.02 to 0.70 +/− 0.02 (74 total cells), while in C-fiber neurons the average changed from 0.24 +/− 0.01 to 0.67 +/− 0.03 (130 total cells). The calcium response in satellite glial cells changed from 0.30 +/− 0.01 to 0.61 +/− 0.02 (114 total cells) while Schwann cells changed from 0.28 +/− 0.01 to 0.60 +/− 0.02 (115 total cells). Both types of neurons exhibited a significant increase in the average amplitude of the calcium response with a maximum of 0.70 +/− 0.04 for Aδ neurons (p = 0.001) and 0.67 +/− 0.03 for C-fiber neurons (p = 0.005). CGRP stimulated a significant increase in the AUC for satellite glial cells (6227 +/− 667, p = 0.012) and Schwann cells (5084 +/− 557, p= 0.020). There was also a significant increase in the average percentage of transient elevated responsive Aδ neurons (59.78 % +/− 7.4; 43 cells; p = 0.031). While not statistically significant, the AUC and percentage of responsive Aδ neurons was increased (p = 0.059 and p = 0.055 respectively) while the percentage of unresponsive Aδ neurons decreased (p = 0.054).

CGRP differentially modulates the calcium response to 30 mM KCl

Under naïve conditions, the mean calcium response in cells stimulated with 30 mM KCl resulted in a rapid, transient increase in the F340/380 ratio in Aδ neurons from a baseline ratio of 0.26 +/− 0.02 (Fig. 4A; 57 total cells). There were 5 cells that exhibited a high baseline. The average peak amplitude was 0.49 +/− 0.02 and average AUC was 2485 +/− 348. The average percentage of responsive cells was 75.3 % +/− 9.7 (45 cells) and average percentage of unresponsive cells was 24.7 % +/− 9.7 (12 cells). The average percentage of transient elevated responsive cells was 60.4 % +/− 8.5 (36 cells) and average percentage of sustained elevated responsive cells was 5.2 % +/− 3.5 (4 cells).

Figure 4.

Figure 4.

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CGRP Modulates the Stimulatory Response to 30 mM KCl in Trigeminal Ganglion Neurons. The average calcium levels based on the ratio of bound (340 nm) versus unbound intracellular calcium (380 nm) in cells treated with only 30 mM KCl (solid line) or pretreated with CGRP 2 hours prior to KCl stimulation (dashed line) are shown in Aδ neurons (A), C-fiber neurons (B), satellite glial cells (C), and Schwann cells (D). Error bars are shown for the SEM at peak amplitude and at 40 second intervals. N = 4 in triplicate.

An increase in the F340/380 ratio was similarly observed in C-fiber neurons from a baseline ratio of 0.24 +/− 0.01 (Fig. 4B; 97 total cells). There were 2 cells that exhibited high baseline. The average peak amplitude was 0.53 +/− 0.02 and average AUC was 3588 +/− 509. The average percentage of responsive cells was 59.0 % +/− 9.3 (61 cells) and average percentage of unresponsive cells was 41.0 % +/− 9.3 (36 cells). The average percentage of transient elevated responsive cells was 51.7 % +/− 10.1 (50 cells) and average percentage of sustained elevated responsive cells was 6.3 % +/− 4.0 (9 cells).

A transient increase in the F340/380 ratio was observed in satellite glia from a baseline ratio of 0.29 +/− 0.01 (Fig. 4C; 58 total cells). There were no cells exhibiting a high baseline. The average peak amplitude was 0.63 +/− 0.02 and average AUC was 2443 +/− 430. The average percentage of responsive cells was 74.6 % +/− 8.1 (44 cells) and average percentage of unresponsive cells was 25.4 % +/− 9.1 (14 cells). The average percentage of transient elevated responsive cells was 74.6 % +/− 8.1 (44 cells) and average percentage of sustained elevated responsive cells was 0.0 % +/− 0.0 (0 cells).

Similarly, Schwann cells exhibited an increase in the F340/380 ratio from a baseline ratio of 0.28 +/− 0.01 (Fig. 4D; 60 total cells) in response to 30 mM KCl. There were no cells exhibiting a high baseline. The average peak amplitude was 0.59 +/− 0.02 and average AUC was 2221 +/− 408. The average percentage of responsive cells was 68.3 % +/− 10.7 (42 cells) and average percentage of unresponsive cells was 31.7 % +/− 10.7 (18 cells). The average percentage of transient elevated responsive cells was 66.6 % +/− 10.4 (41 cells) and average percentage of sustained elevated responsive cells was only 1.7 % +/− 1.7 (1 cell).

As seen in Figure 5, CGRP differentially modulated the mean calcium response, increasing the response in Aδ neurons (0.25 +/− 0.02 to 0.51 +/− 0.03; 46 total cells) while suppressing the response in C-fiber neurons (0.23 +/− 0.01 to 0.43 +/− 0.02; 86 total cells). Specifically, CGRP incubation mediated a significant increase in average AUC (3848 +/− 443; p = 0.044) in Aδ neurons but caused a decrease in the average amplitude in C-fiber neurons (0.43 +/− 0.02; p = 0.002). In contrast, CGRP did not modulate the observed calcium responses to 30 mM KCl in satellite glia (56 total cells) or Schwann cells (56 total cells).

Figure 5.

Figure 5.

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CGRP Suppresses the Stimulatory Response to 15 mM KCl in Trigeminal Ganglion Neurons and Glia. The average calcium levels based on the ratio of bound (340 nm) versus unbound intracellular calcium (380 nm) in cells treated with only 15 mM KCl (solid line) or pretreated with CGRP 2 hours prior to KCl stimulation (dashed line) are shown in Aδ neurons (A), C-fiber neurons (B), satellite glial cells (C), and Schwann cells (D). Error bars are shown for the SEM at peak amplitude and at 40 second intervals. N = 4 in triplicate.

CGRP suppresses the calcium response to 15 mM KCl

Under naïve conditions, the mean calcium response in cells stimulated with 15 mM KCl resulted in a rapid and transient increase in the F340/380 ratio in Aδ neurons from a baseline ratio of 0.29 +/− 0.02 (Fig. 5A; 33 total cells). There were 7 cells that exhibited a high baseline. The average peak amplitude was 0.61 +/− 0.03 and average AUC was 5866 +/− 1007. The average percentage of responsive cells was 89.8 % +/− 5.1 (30 cells) and average percentage of unresponsive cells was 10.2 % +/− 5.2 (3 cells). The average percentage of transient elevated responsive cells was 64.8 % +/− 10.5 (21 cells) and average percentage of sustained elevated responsive cells was 15.7 % +/− 10.4 (5 cells).

Similar to Aδ neurons, 15 mM KCl caused an increase in the F340/380 ratio in C-fiber neurons from a baseline ratio of 0.24 +/− 0.02 (Fig. 5B; 40 total cells). There were 7 cells that exhibited a high baseline. The average peak amplitude was 0.57 +/− 0.03 and average AUC was 4675 +/− 1102. The average percentage of responsive cells was 91.9 % +/− 4.3 (37 cells) and average percentage of unresponsive cells was only 8.1 % +/− 4.3 (3 cells). The average percentage of transiently elevated responsive cells was 59.1 % +/− 8.5 (24 cells) and average percentage of sustained elevated responsive cells was 15.7 % +/− 8.6 (6 cells).

In agreement with the calcium response seen with higher concentrations of KCl, satellite glia exhibited an increase in the F340/380 ratio from a baseline ratio of 0.30 +/− 0.01 (Fig. 5C; 47 total cells). There were 4 cells that exhibited a high baseline. The average peak amplitude was 0.57 +/− 0.02 and average AUC was 5096 +/− 1013. The average percentage of responsive cells was 87.4 % +/− 4.6 (41 cells) and average percentage of unresponsive cells was 12.6 % +/− 4.6 (6 cells). The average percentage of transiently elevated responsive cells was 54.8 % +/− 10.2 (26 cells) and average percentage of sustained elevated responsive cells was 24.1 % +/− 8.7 (11 cells).

Similar to satellite glia, Schwann cells exhibited an increase in the F340/380 ratio from a baseline ratio of 0.30 +/− 0.02 (Fig. 5D; 38 total cells) in response to 15 mM KCl. There were 2 cells that exhibited a high baseline. The average peak amplitude was 0.60 +/− 0.04 and average AUC was 4655 +/− 1058. The average percentage of responsive cells was 89.0 % +/− 6.1 (33 cells) and average percentage of unresponsive was 11.0 % +/− 6.1 (5 cells). The average percentage of transiently elevated responsive cells was 51.5 % +/− 13.8 (19 cells) and average percentage of sustained elevated responsive cells was 32.5 % +/− 12.7 (12 cells).

In contrast to the potentiating effect of CGRP to 60 mM, as seen in Figure 6, CGRP suppressed the calcium response in Aδ neurons (0.23 +/− 0.02 to 0.47 +/− 0.04; 46 total cells), satellite glia (0.28 +/− 0.01 to 0.56 +/− 0.02; 63 total cells), and Schwann cells (0.25 +/− 0.02 to 0.57 +/− 0.03; 62 total cells). The average amplitude of Aδ neurons decreased (0.50 +/− 0.03; p = 0.031), as well as the average AUC of both types of glia cells (satellite glia: 2600 +/− 397; p = 0.030; Schwann cells: 2149 +/− 412; p = 0.036). In addition, the average percentage of sustained elevated responsive glia was significantly decreased in satellite glia (7.3 % +/− 5.9; p = 0.04; 4 cells) and Schwann cells (5.8 % +/− 5.8; p = 0.008; 3 cells).

Figure 6.

Figure 6.

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CGRP Potentiates the Stimulatory Response to 100 μM ATP in Trigeminal Ganglion Aδ Fiber Neurons. The average calcium levels based on the ratio of bound (340 nm) versus unbound intracellular calcium (380 nm) in cells treated with only 100 μM ATP (solid line) or pretreated with CGRP 2 hours prior to ATP stimulation (dashed line) are shown in Aδ neurons (A), C-fiber neurons (B), satellite glial cells (C), and Schwann cells (D). Error bars are shown for the SEM at peak amplitude and at 40 second intervals. N = 4 in triplicate.

CGRP potentiates the calcium response to ATP

Under naïve conditions, the mean calcium response in cells stimulated with 100 μM ATP resulted in a rapid and transient increase in the F340/380 ratio in Aδ neurons from a baseline ratio of 0.27 +/− 0.02 (Fig. 6A; 37 total cells). There was one cell that exhibited a high baseline. The average peak amplitude was 0.41 +/− 0.03 and average AUC was 1488 +/− 342. The average percentage of responsive was 42.5 % +/− 11.2 (14 cells) and average percentage of unresponsive cells was 57.5 % +/− 11.2 (23 cells). The average percentage of transient elevated cells was 23.9 % +/− 9.4 (7 cells) and average percentage of sustained elevated responsive cells was 16.9 % +/− 8.0 (6 cells).

An increase in the F340/380 ratio was also observed in C-fiber neurons from a baseline ratio of 0.28 +/− 0.01 (Fig. 6B; 65 total cells). There were no cells that exhibited a high baseline. The average peak amplitude was 0.45 +/− 0.04 and average AUC was 473 +/− 91.9. The average percentage of responsive cells was 19.5% +/− 9.6 (11 cells) and average percentage of unresponsive cells was 80.5 % +/− 9.6 (54 cells). The average percentage of transient elevated responsive cells was 16.6 % +/− 9.6 (8 cells) and average percentage of sustained elevated responsive cells was 2.9 % +/− 2.0 (3 cells).

Similar to both neuronal cell types, satellite glia exhibited an increase in the F340/380 ratio from a baseline ratio of 0.30 +/− 0.01 (Fig. 6C; 47 total cells). There was 1 cell that exhibited a high baseline. The average peak amplitude was 0.55 +/− 0.02 and average AUC was 2512 +/− 185. The average percentage of responsive cells was 76.0 % +/− 8.6 (44 cells) and average percentage of unresponsive cells was 25.4 % +/− 9.1 (15 cells). The average percentage of transiently elevated responsive cells was 74.6 % +/− 9.1 (43 cells) and average percentage of sustained elevated responsive cells was 0.0 % +/− 0.0 (0 cells).

Schwann cells were also found to exhibit an increase in the F340/380 ratio from a baseline ratio of 0.25 +/− 0.01 (Fig. 6D; 59 total cells). There was 1 cell that exhibited a high baseline. The average peak amplitude was 0.55 +/− 0.01 and average AUC was 2756 +/− 294. The average percentage of responsive cells was 72.3 % +/− 8.7 (43 cells) and average percentage of unresponsive cells was 27.7 % +/− 8.7 (16 cells). The average percentage of transient elevated responsive cells was 70.9 % +/− 8.9 (42 cells) and average percentage of sustained elevated responsive cells was 0.0 % +/− 0.0 (0 cells).

As seen in Figure 6, CGRP potentiated the calcium response in Aδ neurons (0.25 +/− 0.02 to 0.58 +/− 0.02; 32 total cells) but did not modulate the response of C-fiber neurons (69 total cells) or glia cells (satellite glia: 48 total cells; Schwann cells: 47 total cells) to 15 mM KCl. The average peak calcium amplitude of the Aδ neurons was significantly increased in response to CGRP (0.64 +/− 0.10; p = 0.004). A summary of all the significant changes in the intracellular calcium responses meditated by CGRP are highlighted in Table 1.

Table 1.

Summary of Significant Changes in Calcium Response Mediated by CGRP. Individual p values are shown for each condition. Upward arrows are associated with a significant increase in calcium response while downward arrows are associated with a significant decrease in intracellular response. See text for explanation of parameters.

60 mM KCI Amplitude AUC Responsive Unresponsive Transient Sustained
Aδ fiber ↑ 0.001*** ↑ 0.059 ↑ 0.055 ↓ 0.054 ↑ 0.031* 0.808
C- Fiber ↑ 0.005** 0.590 0.997 0.86 0.588 0.598
SGC 0.363 ↑ 0.012* 0.335 0.962 0.962 0.877
Schwann 0.092 ↑ 0.020* 0.292 0.292 0.92 0.272
30 mM KCI Amplitude AJC Responsive Unresponsive Transient Sustained
Aδ fiber 0.170 ↑ 0.044* 0.949 0.804 0.59 0.524
C- Fiber ↓ 0.002** 0.775 0.516 0.541 0.312 0.549
SGC 0.510 0.496 0.753 0.753 0.312 0.549
Schwann 0.803 0.569 0.457 0.451 0.350 1.00
15mM KCI Amplitude AUC Responsive Unresponsive Transient Sustained
Aδ fiber ↓ 0.031* 0.243 0.177 0.177 0.752 0.182
C- Fiber 0.499 0.896 0.690 0.690 0.490 0.448
SGC 0.673 ↓ 0.030* 0.972 0.972 0.444 ↓ 0.043*
Schwann 0.844 ↓ 0.036* 0.292 0.475 0.475 ↓ 0.008**
100 μM ATP Amplitude AUC Responsive Unresponsive Transient Sustained
Aδ fiber ↑ 0.004** 0.908 0.711 0.703 0.888 0.313
C- Fiber 0.681 0.857 0.999 0.999 0.554 0.678
SGC 0.887 0.497 0.167 0.198 0.236 1.00
Schwann 0.399 0.483 0.398 0.334 0.396 1.00
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DISCUSSION

The method used in this study to investigate changes in intracellular calcium levels mediated by CGRP involved establishing mixed cultures of cryopreserved neurons and glial cells from trigeminal ganglia of male and female neonatal rats as recently described (Antonopoulos et al., 2024). The rationale for using mixed cultures is that inclusion of all four cell types more closely mimics the in vivo situation with respect to cell ratios but not tissue morphology, which is a limitation of our study. The concentrations of KCl used in our study are in agreement with results from a human study (Mayevsky et al., 1996), human slices (Petzold et al., 2005), and preclinical models (Charles & Brennan, 2009; Somjen, 2001) involving cortical spreading depression that reported extracellular levels in the range of 10–80 mM. Under these culture conditions, 60 mM KCl caused a transient robust increase in the level of intracellular calcium in both Aδ and C-fiber neurons. The addition of 60 mM KCl to culture medium is used as a common experimental tool to induce depolarization and calcium influx into cells via L-type, N-type, P/Q-type, and NMDA receptor channels, leading to a measurable increase in intracellular calcium levels (Bellamy et al., 2006; Rienecker et al., 2020). Furthermore, addition of 60 mM KCl to culture media induces functional cellular responses including changes in gene expression and a significant increase in the release of CGRP from trigeminal neurons (Durham & Cady, 2004). Similarly, both 30 mM and 15 mM KCl were sufficient to cause a large increase in intracellular calcium in trigeminal neurons that was similar in amplitude and duration to that observed with 60 mM KCl and agrees with prior results using enriched neuronal cultures (Masterson & Durham, 2010). These KCl-mediated calcium changes occurred even though satellite glial cells were present in the cultures. In the trigeminal ganglion, satellite glial cells are the primary regulators of extracellular potassium ion homeostasis and under normal physiological conditions remove excessive potassium ions to modulate neuronal function and response to inflammatory conditions (Dublin & Hanani, 2007; Hanani, 2005). Based on our findings, the Aδ and C-fiber neurons in our mixed trigeminal ganglion cultures are equally responsive to lower concentrations of KCl with 15 mM KCl being sufficient to cause a similar elevation in intracellular calcium as mediated by 60 mM KCl, a stimulus known to cause neuronal activation (Rienecker et al., 2020).

Although satellite glial cells and Schwann cells are known to modulate the excitability state of trigeminal neurons (Dublin & Hanani, 2007; Durham & Garrett, 2010; Hanani, 2005; Jessen & Mirsky, 2016; Messlinger et al., 2020), the effects of different concentrations of extracellular KCl levels on trigeminal glial cells has not been investigated. Each of the KCl concentrations caused an elevation of intracellular calcium in satellite glia and Schwann cells in a manner similar in amplitude and magnitude to the response observed in Aδ and C-fiber neurons. The importance of satellite glial cells to modulate the excitability state of trigeminal nociceptive neurons in preclinical models of migraine is well established (Capuano et al., 2009; Ceruti et al., 2011; Cieslak et al., 2015; Durham & Garrett, 2009; Messlinger et al., 2020; Spray & Hanani, 2019). Stimulation of peripheral nociceptors has been reported to enhance neuron-glia signaling via paracrine and gap junctions to establish an inflammatory loop in the trigeminal ganglion that promotes and sustains peripheral sensitization, a key feature of migraine pathology (Bernstein & Burstein, 2012). Further, in another study, dysfunctional trigeminal satellite glial cells were reported to contribute to cranial muscle tenderness in craniofacial pain conditions that include migraine (Laursen et al., 2014). Our finding that satellite glial cells are depolarized by KCl agrees with the reported intracellular calcium signaling in neurons and satellite glia in response to chemical, mechanical, and electrical stimulation that causes release of ATP and the excitatory neurotransmitter glutamate (Cho et al., 2023; da Silva et al., 2015; Laursen et al., 2014; Suadicani et al., 2010). This type of calcium signaling may be involved in cross-excitation in the trigeminal ganglion such that activation in one branch can lead to sensitization in the other branches (Spray & Hanani, 2019; Thalakoti et al., 2007). Similarly, an important role of activation of Schwann cells in migraine pathology was demonstrated in a study that provided evidence of a CGRP-mediated neuronal/Schwann cell pathway that promoted sustained allodynia (De Logu et al., 2022). With respect to migraine pathology, an influx of extracellular calcium induced by elevated KCl levels would lead to activation of the satellite glia and Schwann cells to facilitate synthesis and release of pro-inflammatory mediators including cytokines, chemokines, nitric oxide, and glutamate (Durham, 2016; Messlinger et al., 2020). Elevation in the extracellular concentrations of those mediators could promote and sustain an inflammatory loop and peripheral sensitization of trigeminal Aδ and C-fiber neurons and thus contribute to migraine pathology.

A novel finding of this study was the ability of CGRP to differentially modulate the calcium response in trigeminal neurons and glia to varying concentrations of extracellular KCl. Elevated levels of CGRP are implicated in the pathology of migraine. Based on preclinical studies, activation of Aδ and C-fiber neurons and the subsequent release of CGRP promotes neurogenic inflammation and peripheral sensitization (Cohen et al., 2022; Iyengar et al., 2017). Under our culture condition, RAMP1, which is the ligand-binding subunit of the CGRP receptor, was expressed on all four cell types, albeit with much higher levels of expression in Aδ and C-fibers neurons when compared to glial cells (Benemei et al., 2009; Durham, 2006; Storer et al., 2004). The finding that RAMP1 is expressed on all four cell types with greater expression in neurons agrees with results from prior published in vivo and in vitro studies (Iyengar et al., 2017; Messlinger et al., 2020). In this study, CGRP potentiated the stimulatory effects of 60 mM KCl on the amplitude and magnitude of the calcium response in Aδ and C-fiber neurons, satellite glial and Schwann cells. In addition, the percentage of Aδ neurons, but not C-fiber neurons, exhibiting a transient elevation in intracellular calcium was also significantly increased in the presence of CGRP. With respect to migraine pathology, a slow wave of neuronal and glial cell depolarization and hyperpolarization termed cortical spreading depression leads to a large increase in the extracellular concentration of KCl (Charles & Baca, 2013; Takizawa et al., 2020). Physiologically relevant levels of extracellular K+ in the mM range (25–80 mM) have been reported in a human study during head injury (Mayevsky et al., 1996), human brain slices (Petzold et al., 2005), and preclinical models (Charles & Brennan, 2009; Somjen, 2001). Thus, in migraine with aura, CGRP could potentiate the depolarizing effect of KCl on trigeminal Aδ and C-fiber nociceptive neurons that innervate the meninges and the glial cells within the ganglion to promote peripheral sensitization and enhanced pain signaling characteristic of the headache phase of migraine (Blumenfeld et al., 2021; Geppetti et al., 2005; Messina et al., 2023).

A somewhat unexpected finding from our study was CGRP suppression of the neuronal calcium response to 15 mM KCl and a mixed modulatory response to 30 mM KCl. In cultures stimulated with 30 mM KCl, the amplitude of the calcium signal was significantly suppressed in C-fiber neurons, but the magnitude of the calcium response was significantly enhanced in Aδ neurons. It is plausible that the differential modulation seen with CGRP may be serving a protective role by enhancing the excitability state of rapid responding Aδ neurons that function to prevent or minimize tissue injury while inhibiting prolonged activation of the longer duration pain signaling C-fiber neurons. However, in cultures stimulated with 15 mM KCl, CGRP suppressed the amplitude of the calcium response in Aδ neurons, but CGRP did not alter the amplitude or magnitude of the response of C-fiber neurons to 15 mM KCl. This is the first evidence to our knowledge to demonstrate that CGRP can differentially modulate the excitability state of trigeminal neurons based on the extracellular level of KCl (potassium ions). Although elevated levels of KCl and CGRP are implicated in the phenomenon known as cortical spreading depression within the meninges during migraine with aura (Brennan et al., 2007; Charles & Baca, 2013), it is not known whether there is a possible direct interaction. Our findings are suggestive that CGRP may be protective when the depolarization state is mild, which may correspond to the premonitory or prodrome phase of migraine (Blumenfeld et al., 2021). However, if the depolarization signal continues to increase because of higher extracellular potassium ion levels as occurs during cortical spreading depression, the role of CGRP may switch to potentiate the excitation state of trigeminal neurons leading to the headache phase and more severe migraine pain. Furthermore, these data support the clinical recommendation to treat a migraine attack with CGRP-targeting drugs when the pain is mild to moderate, which correlates with increasing levels of CGRP, but not in the prodrome phase of migraine prior to trigeminal nerve activation (Cohen et al., 2022; Ornello et al., 2025). To our knowledge, this is the first evidence to support the notion that CGRP may not always function in a pro-inflammatory manner. Rather, we propose that CGRP may serve a novel protective role under low inflammatory conditions that would promote initial sensitization of Aδ and C-fiber neurons during the premonitory or prodrome phase of migraine.

Although the CGRP receptor protein subunit RAMP1 expression in satellite glia and Schwann cells was much less than that observed in trigeminal neurons, CGRP potentiated the stimulatory effect of 60 mM KCl on the amplitude and magnitude of the intracellular calcium response in trigeminal glial cells. The ability of CGRP to modulate a cellular response in trigeminal glia demonstrates that these cells express functional CGRP receptors, which agrees with results from previous studies (Cady et al., 2011; Li et al., 2008; Vause & Durham, 2009, 2010). In those published studies, CGRP was shown to stimulate iNOS gene expression and nitric oxide production via activation of MAPK pathways, stimulate cytokine expression and release, and induce expression of a diverse array of proteins involved in the MAP kinase signaling pathway. The increased amplitude and magnitude observed in glial cells could facilitate the release of pro-inflammatory mediators such as cytokines, chemokines, and nitric oxide that are known to cause sensitization and activation of trigeminal neurons (Durham, 2016). The calcium influx likely would also lead to activation of pro-inflammatory signaling pathways including MAP kinases that are also activated by CGRP (Messlinger et al., 2020), and this may provide a possible mechanism that enhances the neuronal response to KCl. Similar to the less robust effect of CGRP on neuronal activity, no significant changes in calcium signaling were observed in either glial cell type in mixed cultures in response to 30 mM KCl. In contrast to the response to 60 mM KCl, the magnitude and percentage of glia exhibiting a sustained calcium response in cultures stimulated with 15 mM KCl was significantly suppressed in satellite glia and Schwann cells, which are implicated in peripheral sensitization of trigeminal neurons (De Logu et al., 2022; Messlinger et al., 2020). The CGRP-mediated suppression of the 15 mM KCl response in glia is likely to involve, at least in part, upregulation of the inward rectifying potassium channel Kir 4.1 expressed by trigeminal ganglion satellite glial cells (Olsen & Sontheimer, 2008; Takeda et al., 2015; Vit et al., 2008). The Kir 4.1 channel functions to modulate the concentration of extracellular potassium ions via its ability to take up potassium ions and thus contributes to the modulation of neuronal excitability in the surrounding environment. This type of differential modulation of trigeminal ganglia glia has not been reported and represents a novel function of CGRP as a glial cell modulator and provides evidence to support the notion that CGRP may not always function as a pro-inflammatory neuropeptide. To our knowledge, this is the first evidence the CGRP can differentially modulate the glial cell calcium response to varying KCl concentrations and hence, this dual role may be important in regulating the excitability state of trigeminal nociceptive neurons during different migraine phases.

In migraine with aura, cortical spreading depression leads to release of protons, potassium ions (K+), nitric oxide, prostaglandins, and ATP in cerebrospinal fluid (Charles & Baca, 2013; Takizawa et al., 2020). Elevated levels of ATP can cause sensitization and depolarization of nociceptive neurons and mediate activation of glial cells to sustain the inflammatory response (Bland-Ward & Humphrey, 2000; Goto et al., 2017; Hamilton & McMahon, 2000). Trigeminal Aδ and C-fiber neurons predominantly express the ligand-gated calcium channel ATP receptor P2X3 while satellite glia and Schwann cells express higher levels of P2X7 (Giniatullin & Nistri, 2023; Staikopoulos et al., 2007). Activation of the P2X3 receptor is implicated in migraine pathology since ATP-induction of this receptor can mediate sensitization and at higher concentrations directly stimulate trigeminal nociceptive neurons (Giniatullin & Nistri, 2023; Giniatullin et al., 2008). In our study, the addition of 100 μM ATP to the mixed neuronal-glia cell cultures caused a robust transient increase in intracellular calcium in both types of neurons and glia. However, CGRP only potentiated the ATP-mediated calcium response in Aδ neurons, which upon activation facilitates a rapid response to protect tissues and cells from further injury and promote healing and homeostasis. Our finding that CGRP can enhance the stimulatory nociceptive and inflammatory effect of extracellular ATP agrees with the proposed synergistic relationship of CGRP and ATP in migraine pathology to mediate and sustain sensitization and activation of trigeminal nociceptive neurons (Giniatullin & Nistri, 2023). Thus, stimulated CGRP secretion from trigeminal neurons would enhance and prolong the inflammatory and nociceptive response during the headache phase of migraine in response to ATP released during cortical spreading depression.

In summary, results from this study have provided novel insight into how CGRP functions to differentially modulate the excitability state of trigeminal ganglion neurons and glia in response to higher and lower KCl levels. These findings identify a not yet identified potential protective function of CGRP and thus CGRP may play an important role in the initial stages of inflammation to suppress neuronal depolarization and inhibit further release of CGRP and other pro-inflammatory mediators from trigeminal neurons to control the response. However, once the level of inflammatory signals reaches a critical threshold, CGRP would function to potentiate the response by enhancing the amplitude and magnitude of the calcium signal especially in Aδ neurons. In the context of migraine pathology, this enhanced CGRP-mediated response in Aδ neurons to KCl and ATP would facilitate severe pain and avoidance behaviors to protect the nervous system during a migraine attack. Elevated CGRP levels would also cause enhanced activation of C-fiber neurons responsible for the sustained pain associated with migraine, which can last from hours to days. CGRP was also found to modulate the response to KCl in trigeminal glial cells implicated in peripheral sensitization associated with migraine pathology. In future studies, it would be of interest to utilize CGRP receptor antagonists to better understand the cellular mechanisms mediating the differential response to CGRP on trigeminal neurons and glia and utilize molecular markers such as NF200 or peripherin to help confirm neuron subtypes. As with any in vitro model, we acknowledge potential limitations of our study including the use of neonatal animals, the parameters used for inclusion/exclusion criteria in the calcium analysis, and identification of neuronal cell types based on size of cell body. In conclusion, our findings provide evidence that CGRP should be considered as an important neuron-glia modulator that can exert inhibitory or stimulatory effects depending on the level of the inflammatory stimulus to promote a cellular response. Future in vivo studies are needed to determine whether CGRP functions in this dual role in preclinical cortical spreading depression models of migraine.

Highlights.

CGRP potentiated calcium response to 60 mM KCl in trigeminal ganglia neurons and glia

CGRP differentially modulates neuronal response to 30 mM KCl in neurons and glia

CGRP suppressed calcium response to 15 mM KCl in trigeminal neurons and glia

CGRP potentiated stimulatory effect of 100 μM ATP in trigeminal ganglia Aδ neurons

Acknowledgments:

The authors would like to thank Angela Goerndt for their technical assistance in the care and maintenance of the animals used in this study and Mikayla Scharnhorst and Donovan Aardema Faigh for preparation of cryopreserved cells. Funding: This work was supported by the National Institutes of Health [NCCIH 1R15AT012501].

Abbreviations:

CGRP

calcitonin gene-related peptide

KCL

potassium chloride

ATP

adenosine triphosphate

Footnotes

Declaration of interest

Declaration of interest: none.

Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work the author(s) used Google and PubMed to identify relevant published studies and EndNote as a reference management tool. After using these tools/services, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

Informed Consent Statement: Not applicable

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Data Availability Statement:

Data will be provided upon request.

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