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. Author manuscript; available in PMC: 2023 Jun 12.
Published in final edited form as: Tex J Micros. 2021;52(1):8–13.

EXPRESSION OF SEROTONIN RECEPTOR SUBTYPE 3A (5HT3A) ON RAT TRIGEMINAL SENSORY NEURONS

SUKHBIR KAUR 1, ANGELA LOPEZ-RAMIREZ 1, TAYLOR M HICKMAN 1, MICHAEL P HUNTER 1, DAYNA L AVERITT 1,*
PMCID: PMC10259189  NIHMSID: NIHMS1904575  PMID: 37309469

Abstract

Serotonin (5-hydroxytryptamine, 5HT) is a neurotransmitter and proinflammatory mediator found largely in the peripheral nervous system where it can initiate pain signaling. 5HT binds a variety of 5HT receptors on sensory nerve endings specialized in detecting noxious stimuli, termed nociceptors. A subset of sensory neurons involved in pain signaling express the transient receptor potential vanilloid 1 ion channel (TRPV1), a pain generator. 5HT can both directly activate sensory neurons and sensitize TRPV1 leading to enhanced nociceptor sensitivity (peripheral sensitization). Previous studies in male rats reported that the 5HT receptor subtype 3A (5HT3A) and TRPV1 are co-expressed on sensory neurons, but it is unknown if 5HT3A and TRPV1 are co-expressed on female sensory neurons. Given that craniofacial pain disorders occur at a 2-3x greater prevalence in women, examining pain mechanisms in female trigeminal sensory neurons that innervate the craniofacial region is critical to advancing craniofacial pain management in women. Here we examined whether (1) 5HT acting via the 5HT3A receptor produces sexually dimorphic orofacial pain behaviors in rats and (2) whether 5HT3A receptor mRNA is expressed in trigeminal sensory neurons, including the TRPV1-expressing subpopulation, and increase pain signaling. We report that 5HT evokes pain behaviors in male and female rats, which was not significantly reduced by antagonism of 5HT3A. We performed in situ hybridization to label 5HT3A and TRPV1 mRNA in trigeminal sensory neurons and found distinct cell populations with either 5HT3A mRNA or TRPV1 mRNA in males and females. Further, 5HT3A antagonism failed to reduce pain signaling in cultured trigeminal sensory neurons. These data suggest that the 5HT3A subtype on trigeminal sensory neurons innervating the orofacial soft tissues does not play a significant role in sexually dimorphic craniofacial pain disorders. As previous studies have reported that granisetron reduces masseter muscle pain, 5HT3 may play a role in sex differences in myofascial pain disorders but not in other craniofacial pain disorders.

Keywords: pain, serotonin, 5HT3A receptor, TRPV1 ion channel, RNAScope©

Introduction

Trigeminal pain disorders, like migraine and temporomandibular joint disorder, disproportionately affect women with migraine being 2-3 times more prevalent in women (Berkley, 1997; LeResche et al., 2003). More than half of these women report menstrual-associated migraine in their reproductive years (Granella et al., 1993). Thus, gonadal hormone fluctuations [mainly estrogen (E2)] may be a possible explanation underlying these sex differences in trigeminal pain disorders. In addition, these disorders activate the immune system that release a milieu of pro-inflammatory mediators that can further modulate trigeminal pain signaling. One such proinflammatory and pronociceptive mediator released in the periphery is serotonin (5HT). 5HT is a neurotransmitter that is also a known peripheral algogen released by cells of the immune system and can act via a variety of excitatory ionotropic and metabotropic 5HT receptors to sensitize peripheral sensory neurons (meaning lower their activation threshold). Of the seven known 5HT receptors (5HT1-7), 5HT3A is an ionotropic receptor that belongs to the nicotinic acetylcholine superfamily of ion channels, whose activation results in the flow of sodium and potassium ions leading to an excitatory current in the sensory neuron (Maricq et al., 1991; Thompson & Lummis, 2007).

5HT is also known to sensitize a cation channel highly expressed in a subpopulation of trigeminal sensory neurons, the transient receptor potential vanilloid 1 (TRPV1) ion channel. TRPV1 is a thermosensor expressed in small- to medium-sized sensory neurons in the trigeminal ganglia that also acts as a pain generator (Loyd et al., 2013). Activation of TRPV1 by heat (>42°C), capsaicin (the ‘spicy’ chemical in chili peppers), and protons results in an influx of calcium (Ca+2) ions into the sensory neuron. The calcium influx results in the release of additional proinflammatory molecules, largely substance P and calcitonin gene related peptide (CGRP) (Kaur et al., 2018; Vay et al., 2012). 5HT can act through 5HT receptors to sensitize TRPV1 resulting in an increased Ca+2 influx and CGRP release (Loyd et al., 2013). We have previously reported that during periods of hormonal fluctuation, 5HT can evoke greater pain behaviors in female rats during the proestrus and estrus phases of the estrous cycle, characterized by rapid fluctuations in estrogen levels. During proestrus, estrogen rapidly rises and peaks and during estrus estrogen rapidly declines. Moreover, treatment with the 5HT2A receptor antagonist, M100907, can attenuate these pain behaviors in both male and female rats suggesting an important role of excitatory 5HT receptors in pain processing (Kaur et al., 2018).

Of the known excitatory 5HT receptors, 5HT2A and 5HT3A have been shown to co-express with TRPV1 in male trigeminal sensory neurons, which provides an anatomical substrate for enhancing pain signaling in the neurons (Kaur et al., 2018). Studies have also reported that blocking the 5HT3A receptor in the central nervous system reduces the sensitization of TRPV1, thus implicating an interaction between 5HT3A and TRPV1 in pain signaling (Kim et al., 2014). Further, 5HT3A antagonists are used as potent antiemetic drugs to block the release of 5HT in the gastrointestinal tract and reduce visceral pain (Theriot et al., 2020). Given that craniofacial pain disorders occur at a 2-3x greater prevalence in women, examining pain mechanisms in female trigeminal sensory neurons that innervate the craniofacial region is critical to advancing craniofacial pain management in women. Here we examined whether (1) 5HT acting via the 5HT3A receptor produces sexually dimorphic orofacial pain behaviors in rats and (2) whether 5HT3A receptor mRNA is expressed in trigeminal sensory neurons, including the TRPV1-expressing subpopulation, and increase pain signaling.

Materials and Methods

Subjects:

A total of 16 adult male and 31 adult female Sprague–Dawley rats (200–300g; Charles River Laboratories, Wilmington, MA) were used in the experiments. Rats were separated by sex and pair-housed in a 12:12-h light: dark cycle with ad libitum food and water access. All studies were approved by the Texas Woman’s University Institutional Animal Care and Use Committee and conform to federal guidelines and guidelines of the Committee for Research and Ethical Issues of the International Association for the Study of Pain. This study was conducted in strict compliance with the Animal Welfare Act, implementing Animal Welfare Regulations, and the principles of the Guide for the Care and Use of Laboratory Animals.

Vaginal cytology:

Vaginal lavages were performed between 9:00 AM to 11:00 AM at 24-h intervals beginning 2 weeks (at least two consecutive cycles; 10 days) before testing to confirm that all female rats were cycling normally. Daily records were maintained on the stages of their cycle through the day of experimental testing and tissue collection. Proestrus was identified as a predominance of nucleated epithelial cells and estrus was identified as a predominance of cornified epithelial cells. Diestrus 1 (or metestrus) was differentiated from diestrus 2 (or diestrus) by the presence of leukocytes (Becker et al., 2005; Loyd & Murphy, 2008; McLean et al., 2012).

Ovariectomy:

A subset of female rats were deeply gas anesthetized (3% induction; 2.5% maintenance) by inhalation of Isothesia (isoflurane, USP, Henry Schein Animal Health, Dublin, OH) and a single incision was made across the abdomen. The abdominal muscle was opened and the ovary bundles were ligated with 4-O silk sutures, excised, and removed. The fascia was closed with 5-O silk suture and the skin was closed with Vicryl sutures to prevent wicking. Rats were allowed 2 weeks for recovery and ovarian hormone dissipation. Trigeminal ganglia were removed from these animals following the 2-week recovery period using methodology described under the cell culture methodology section.

Behavior Testing:

Square-shaped plexiglass boxes (30 x 30 x 30 cm) with mirrored sides were used to observe orofacial nocifensive (pain-provoked) behaviors. Rats were acclimated to the behavior testing apparatus 24 hours prior to testing. On the day of testing, rats were placed in the individual boxes immediately post-injection and nocifensive behavior was recorded with a video camera for a 30-min time period. The videos were manually quantified using iMovie software (Apple Inc., Mac OS) by counting the number of forelimb swipes over the injection site in 6 min bouts over an 18 min period and reported as a measure of spontaneous nocifensive behavior. Data was counted by an independent observer blind to the experimental condition.

Adult intact male and cycling female rats in either proestrus or estrus received an unilateral intradermal injection of the selective 5HT3A antagonist granisetron (1 μM; 0.1 nmol / 100 μL; Sigma-Aldrich) (Loyd, Chen, et al., 2012) or saline vehicle control (0.9%; 100 μL) into the vibrissal pad (n = 7 - 8 per sex and per treatment group). Fifteen-minutes after the pre-treatment, female rats received an injection of 5HT (3 μg / 50 μL) and the male rats received an injection of 5HT combined with capsaicin (3 μg 5HT + 1 μg CAP / 50 μL) at the same site. The male rats received the addition of capsaicin because our previous studies indicate 3 μg 5HT does not induce orofacial nocifensive behavior, while in females 3 μg 5HT produces significant orofacial nocifensive behavior. Immediately following the 5HT injection, orofacial nocifensive behavior were recorded and counted as described above.

In situ hybridization:

Trigeminal ganglia were bilaterally extracted from males and cycling female rats two weeks following orofacial behavior testing and immediately frozen on dry ice. The tissues were stored at −80° until processing. The ganglia (n=2 / animal) were embedded in Tissue-Tek Optimal Cutting Temperature (O.C.T) compound (Sakura Finetek USA) and cut into 30 μm sections on a Leica Cryostat CM3050 at −20°. The slides were then stored at −80° until further processing. For in situ hybridization, the RNAScope© Fluorescent Multiplex Assay was performed according to the manufacturer’s specifications (ACD Biotechne) with optimizations performed for TG tissue. Briefly, slides were fixed in 4% paraformaldehyde, sequentially dehydrated in 50% ethanol, 70% ethanol, and 100% ethanol and stored at −20° overnight. The next day, slides were air-dried and incubated at 60° and a hydrophobic barrier was drawn using an ImmEdge Hydrophobic Barrier Pen (Vector Laboratories). Following a hydrogen peroxide and protease IV treatment, the slides were treated with either experimental probes (50:1 dilution; Table 1) or control probes and incubated at 40° for 2 hours. Slides were washed in buffer and amplification steps were performed with AMP-1, AMP-2, and AMP-3 (AMP indicates proprietary signal amplification molecules; ACD Biotechne), then slides were sequentially treated with OPAL fluorophores (Table 1; Akoya Biosciences). Excess liquid was carefully drained from the slides and 1 - 2 drops of Prolong Gold antifade mounting medium (Fisher Scientific) and slides were cover slipped and air-dried overnight at room temperature. The slides were imaged using a Zeiss LSM 900 confocal microscope. Zeiss ZEN software was used to view and capture the images at 40X. The staining intensity was filtered by applying a laser power based on the control slides (Laser power - 570nm: 0.2%, 520nm: 1%) to remove low intensity pixels that represent nonspecific/background staining. All images were saved in tiff format for presentation.

Table 1:

Specifications of the RNAScope™ Fluorescent Multiplex Assay for in situ hybridization.

Probe Target Channel Fluorophore Image Color Working Dilution
Htr-3a Serotonin receptor subtype 3A (5HT3A) mRNA C1 OPAL 570 Magenta puncta 1:1500
Trpv1 Transient Receptor Potential Vanilloid 1 ion channel (TRPV1) mRNA C2 OPAL 520 Green puncta 1:1000
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Primary culture of trigeminal ganglia neurons:

Trigeminal ganglia (n=4 rats per 24-well plate run in triplicate) were extracted from adult ovariectomized female rats (~200 g) immediately following decapitation under brief gas anesthesia (3% isoflurane). Primary neuron cultures were prepared using previously described methods (Loyd et al., 2011). Briefly, trigeminal ganglia were suspended in Hank’s balanced salt solution (HBSS) on ice and gently washed three times. After dissociation with collagenase (5%, Worthington Biochemical Corp) and trypsin (1%, Sigma-Aldrich) at 37 °C, the cells were suspended in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) containing 10% fetal bovine serum, 1X glutamine (Gibco), 1X penicillin-streptomycin (Gibco), nerve growth factor (100 ng/mL; Harlan, Indianapolis, IN), and treated with mitotic inhibitors 5-fluoro-2’-deoxyuridine (3 μg/mL; Invitrogen) and uridine (7 μL/mL; Sigma-Aldrich). Cells were then lightly dissociated using a 20-gauge followed by a 23-gauge needle and then applied to 24-well poly-D-lysine-coated plates (Corning) and maintained in an incubator at 37 °C and 5% CO2.

CGRP Release Assay:

Primary cultures of trigeminal sensory neurons were grown and maintained for 5 days prior to running the CGRP release assay. The assay was performed using a protocol previously described (Loyd et al., 2011). Briefly, cultures were washed twice with 300 μL HBSS. Cells were then incubated in HBSS for 15 minutes and supernatant was collected for measurement of basal CGRP release from the neurons. The same cultures were then pretreated row-wise with either granisetron (100 nM) or 17β-estradiol (E2; 50 nM), followed by 5HT (100 μM) for 15 minutes and supernatant was collected for measurement of treatment-evoked CGRP release. Then cells in all the rows were stimulated with capsaicin (50 nM) to trigger CGRP release and supernatant was collected for measurement of the effects of treatment on capsaicin-evoked CGRP release. CGRP in the superfusate was detected using a rat-specific CGRP ELISA (Cayman Chemical) and quantified using a Biotek ELx808 absorbance reader (Biotek). All experiments were conducted in duplicate with n = 6 wells per treatment group for a total of approximately 12 wells per group.

Data analysis:

All data were analyzed and graphed with GraphPad Prism software version 9.0.0 (GraphPad, San Diego, CA). Orofacial nocifensive behavior and CGRP release data were expressed as mean ± standard error of the mean (SEM) and were analyzed by two-way ANOVA. Grubb’s test was used to exclude a single outlier within an experimental group if present and Bonferroni’s correction was used to calculate a priori pairwise comparisons.

Results

Granisetron does not significantly attenuate 5HT-evoked orofacial nocifensive behaviors in male or female rats

Since 5HT3A is expressed in the trigeminal ganglia and is an excitatory ion channel, we first determined whether 5HT evokes nocifensive behaviors via the 5HT3A receptor in male and female rats. 5HT alone evoked significant orofacial pain behaviors (saline pre-treatment group) within 10 minutes of 5HT injection in female rats and within 10 minutes of injection of 5HT with capsaicin (5HT+CAP) in male rats [p < 0.05]. Local pre-treatment with the selective 5HT3A receptor antagonist, granisetron, 15 minutes prior to either 5HT injection in females or 5HT+CAP injection in males did not significantly attenuate orofacial nocifensive behaviors (Figure 1A and 1B) [p>0.05].

Figure 1.

Figure 1.

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Effects of granisetron on female and male rats. Bar graphs illustrate the effects of saline pre-treatment (open bars) and granisetron pre-treatment (closed bars) on serotonin (5HT) alone in female rats (A) or 5HT with capsaicin (5HT+CAP) in male rats (B) on orofacial nocifensive behaviors recorded as the number of forelimb swipes from 0-18 minutes following injection into the vibrissal (cheek) pad. Asterisks indicate significant effect of 5HT on pain behaviors compared to vehicle treatment (p<0.05). There was no significant effect of granisetron on pain behaviors (p>0.05).

5HT3A receptor mRNA and TRPV1 ion channel mRNA are co-expressed in male and female trigeminal sensory neurons

We then performed in situ hybridization to determine whether 5HT3A receptor mRNA co-expresses with TRPV1 ion channel mRNA in the trigeminal sensory neurons of the trigeminal ganglia of male and female rats. We report 5HT3A mRNA expression in distinct small and medium diameter neurons in the trigeminal sensory neurons of the trigeminal ganglia of males and cycling female rats (Figure 2; magenta fluorescent puncta). TRPV1 ion channel mRNA was also observed in small and medium diameter neurons in the trigeminal sensory neurons of the trigeminal ganglia of males and cycling female rats (green fluorescent puncta). In both males and females, and across the estrous cycle, there was a subset of the sensory neuron population that coexpressed 5HT3A receptor mRNA and TRPV1 ion channel mRNA (Figure 2; white fluorescent puncta).

Figure 2:

Figure 2:

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Expression of 5HT3A receptor and TRPV1 ion channel in trigeminal ganglia. Representative images of the expression of 5HT3A receptor mRNA (magenta) and TRPV1 ion channel mRNA (green) in the trigeminal ganglia of male and cycling female rats across the three stages of the estrous cycle [diestrus, proestrus (P), and estrus (E)]. The first column illustrates TRPV1 ion channel mRNA expression, the second column illustrates 5HT3A receptor mRNA expression, and the third column is an overlay of 5HT3A receptor mRNA and TRPV1 ion channel mRNA. Arrows indicate sensory neurons co-expressing 5HT3A receptor mRNA and TRPV1 ion channel mRNA (white).

Granisetron does not significantly attenuate proinflammatory peptidergic activity in cultured trigeminal sensory neurons

As the proinflammatory peptide CGRP is released from sensory neurons when TRPV1 is activated, quantification of CGRP is a measure of nociceptive peptidergic activity in sensory neurons. Here we tested whether blocking the 5HT3A receptor attenuates 5HT- and/or capsaicin-evoked CGRP release from cultured trigeminal sensory neurons extracted from ovariectomized female rats.

When the neurons were treated with 5HT, E2, or granisetron alone (pretreatment) there was no effect on CGRP release (Figure 3) [p>0.05]. Capsaicin evoked significant CGRP release that was enhanced by 5HT and E2 (Figure 3 grey bars) [p<0.05]. CGRP release was not attenuated by pretreatment with the 5HT3A antagonist granisetron [p>0.05].

Figure 3:

Figure 3:

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Effects of granisetron on cGRP release. Primary cultures pretreated with either the selective 5HT3 receptor antagonist granisetron (black pretreatment bar) or vehicle (grey pretreatment bar) prior to treatment with serotonin (5HT) and 17β-estradiol (E2) and stimulation with the TRPV1 agonist capsaicin. Asterisks indicate significant effect of 5HT+E2 on CGRP release as compared to vehicle (p<0.05). There was no significant effect of granisetron on the enhanced CGRP release (p>0.05).

Discussion

Trigeminal pain disorders are more prevalent and severe in duration and intensity in women than men. Understanding the role of excitatory 5HT receptors in peripheral pain processing is important to the development of more effective, and even sex-specific, therapeutics. We have previously reported that 5HT-evoked pain behaviors are increased during phases of the rodent estrous cycle when gonadal hormones are greatly fluctuating and that blocking the Gq-coupled 5HT2A receptor attenuates E2-enhanced pain behaviors (Kaur et al., 2018). In this study we report that (1) blocking the 5HT3A receptor does not attenuate 5HT-evoked orofacial nocifensive pain behaviors, (2) 5HT3A mRNA co-expresses with TRPV1 on male and female trigeminal sensory neurons, and (3) blocking the 5HT3A receptor does not attenuate capsaicin-evoked CGRP release from cultured trigeminal sensory neurons.

Previous studies have reported that blocking the excitatory 5HT2A and 5HT3A receptors attenuates pain signaling in male cultured trigeminal sensory neurons (Loyd, Chen, et al., 2012; Loyd et al., 2013; Loyd et al., 2011). In support, 5HT2 and 5HT4 receptor antagonists attenuate 5HT-potentiated current in mouse DRG neurons (Sugiura et al., 2004). In the present study, we report that orofacial pain behaviors were not attenuated in the presence of granisetron. These data indicate that the ionotropic 5HT3 receptor is not necessary for 5HT-evoked pain in the vibrissal pad. It is likely that the 5HT3 receptor is involved in generating pain in male and female rats, but antagonism of only the 5HT3 receptor is not sufficient to reduce orofacial pain in male or female rats. Rather, the involvement of the other excitatory G-protein coupled 5HT receptors are key to orofacial pain, such as the 5HT2A receptor. Interestingly, 5HT3 receptors are upregulated in the masseter muscle of women with myofascial pain compared to healthy controls indicating that 5HT3 receptors may be more involved in pain processing in pathological trigeminal nociceptors innervating painful muscle rather than other trigeminal nociceptors (Christidis et al., 2014). In support, granisetron significantly reduces muscle pain, but not thermal pain, in men and women (Ernberg et al., 2020; Louca et al., 2013).

Studies have also reported that 5HT1A, 5HT1D, 5HT2A and 5HT3A receptors co-localize with TRPV1 on male trigeminal sensory neurons (Loyd et al., 2011). To date, no studies had examined whether 5HT receptors are co-expressed with the TRVPV1 ion channel in female trigeminal sensory neurons. In the present study, we report that 5HT3A mRNA co-expresses with TRPV1 ion channels on male and female trigeminal sensory neurons. The soma of nociceptors housed in the trigeminal ganglia are typically small-to medium-sized in diameter (~15-35 μm). 5HT3A receptor mRNA and TRPV1 ion channel mRNA were expressed in the small- to medium-sized sensory neuron population. As the TRPV1 ion channel in a major pain generator, the population of sensory neurons that express TRPV1 are classified as nociceptors, though not all nociceptors express TRPV1 ion channels. Thus, our data provide evidence that 5HT3A receptors are expressed by a subpopulation of nociceptors in the trigeminal ganglia. Regions with white fluorescent puncta indicating a cell expressing both TRPV1 mRNA and 5HT3A receptor mRNA was observed in both sexes and across the four stages of estrous cycle. While 5HT3A receptors appear to be localized on nociceptors in the trigeminal ganglia, our behavioral data indicate that blocking only the peripheral 5HT3A receptors is not sufficient to reduce orofacial pain. We are currently examining whether 5HT2A receptor mRNA is co-expressed in the TRPV1 population of trigeminal nociceptors and whether blocking peripheral 5HT2A receptors is sufficient to reduce orofacial pain in female rats.

CGRP release is a measure of nociceptive peptidergic activity in sensory neurons and studies have correlated high CGRP release to worsened migraine symptoms in women (Goadsby et al., 1990; Hansen et al., 2010). Moreover, studies have reported that 5HT enhances capsaicin-evoked CGRP release from rat sensory neurons (Loyd et al. 2011) and female human tooth pulp (Loyd, Sun, et al., 2012). In concurrence with the in vivo arm of this study, our in vitro examination of the effects of granisetron on capsaicin-evoked CGRP release found that blocking the 5HT3 receptor does not alter 5HT-evoked pain signaling.

Conclusion

Overall, the present study indicates that, while the ionotropic 5HT3 receptor may be involved in 5HT-evoked orofacial pain in female rats, selective antagonism of the 5HT3 receptor with granisetron is not sufficient to reduce 5HT-evoked orofacial pain and thus not involved in the sexually dimorphic effects of peripheral 5HT on orofacial pain. Thus, it is more likely that a metabotropic 5HT receptor needs to be blocked to reduce 5HT-evoked orofacial pain in female rats. Further, this study is the first to report that 5HT3A receptor mRNA is co-expressed with TRPV1 ion channel mRNA on both male and female trigeminal sensory neurons. Our data leads us to speculate that drugs targeting the peripheral 5HT3 receptor may not be sufficient to reduce trigeminal pain conditions not of muscle origin, such as migraine, in both men and women. Our current studies are now focused on the metabotropic 5HT receptors known to be localized to the trigeminal ganglia to determine whether targeting a different excitatory 5HT receptor may reduce orofacial pain.

Funding

This work was funded by a Texas Society of Microscopy Small Grant awarded to SK, a Texas Woman’s University Research Enhancement Grant to DLA, and a National Institute of Dental and Craniofacial Research grant DE026749 to DLA.

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