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INTRODUCTION
The trigeminal nociceptive system is complex. Physicians are responsible for managing disorders related to pain perceived in the head, face, and neck region [1]. The primary relay center for processing nociceptive signals from the orofacial region is the trigeminal subnucleus caudalis (Vc), often known as the medullary dorsal horn upon account of its structural resemblance to the spinal dorsal horn [2,3]. The substantia gelatinosa (SG; lamina II), a sensory nucleus in the spinal cord, was named by Rolando for its translucent, gelatinous appearance [4]. SG neurons play a key role in processing incoming painful stimuli and relaying this information to ascending neurons [5]. Specifically, SG neurons in Vc are essential for transmitting orofacial nociceptive signals via thin myelinated Aδ and unmyelinated C primary afferent fibers to higher brain regions [6].
Citronellol (β-Citronellol; CT; C10H20O: Fig. 1) is a monoterpene alcohol found in the essential oil of several types of plants, such as Cymbopogon citratus, which is widely used in traditional medicine and food [7]. In recent years, researchers have shown interest in this compound with the aim of shedding light on its empirical application based on scientific evidence. Various studies have reported medicinal benefits of CT, particularly its antimicrobial, antifungal, antihypertensive, vasodilatory, antioxidant, and anti-inflammatory properties [7-9].
In addition, it has been reported by Brito et al. [9] in 2012 that CT can reduce nociception induced by acetic acid, formalin, and hot plate test. This effect is reverted by naloxone, indicating that CT likely impacts opioid receptors [9]. Without lowering the number of mononuclear cells, CT can decrease total leukocytes, neutrophils, and TNF-α in pleurisy caused by carrageenan [9]. Then, Brito et al. [10] in 2013 demonstrated that CT could decrease nociceptive behavior and activate the expression of Fos protein in the olfactory bulb, piriform cortex, retrosplenial cortex, and periaqueductal grey, indicating its action on the descending pain pathway in orofacial pain induced by formalin, capsaicin, and glutamate [10]. Although CT has been shown to have an analgesic effect on orofacial pain, the underlying mechanism involved in its effect remains largely unexplored. Based on this background, this study was conducted to investigate effects of CT on SG neurons of the Vc using the whole-cell patch-clamp technique.
METHODS
Animals
All animal experiments were approved by the Institutional Animal Care and Use Committee of Jeonbuk National University (JBNU 2023-0122) and were conducted in accordance with the guidelines for the Care and Use of Laboratory Animals. Immature ICR mice postnatal (7–19 days) were utilized. They were housed in plastic cages at 22oC ± 2oC on a standard under 12-h light, 12-h dark cycles with free access to food and water ad libitum.
Brain slice preparation
The experimental procedure used to prepare brain slices was the same as shown in our previous studies [11,12]. Briefly, brains of mice were rapidly harvested through the decapitation process between 10:00 AM and 12:00 PM (Universal Time Coordinate +9:00). They were then immersed in ice-cold, oxygenated (95% O2 and 5% CO2), artificial cerebrospinal fluid (ACSF) including: 126 mM NaCl, 2.5 mM KCl, 2.4 mM CaCl2, 1.2 mM MgCl2, 11 mM D-glucose, 1.4 mM NaH2PO4, and 25 mM NaHCO3 (pH 7.3–7.4). Brains were fixed to an agar block and then cut into 190- to 220-μm-thick coronal slices consisting of the rostral part of Vc using a vibratome (Leica VT1200S; Leica Biosystems). Brain slices were then transferred to an oxygenated ACSF solution under conditions of 31oC for at least one hour before electrophysiological recording processes.
Electrophysiological experiments
Each slice immersed in ACSF was placed in a recording chamber and covered with mesh. Oxygenated ACSF was supplied at 4–5 ml/min for slices. Using an upright microscope (BX51W1; Olympus) equipped with infrared-differential interference contrast optics, individual slices were visualized. The SG (lamina II) of the Vc was identified as a translucent band along the lateral edge of the coronal slice, merely medial to the spinal trigeminal tract.
The electrophysiological experiments were conducted via a high-chloride pipette solution composed of 140 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, 4 mM Mg-ATP, and 10 mM EGTA, with the pH adjusted to 7.3 using KOH. This solution was utilized to record chloride current at a holding potential of –60 mV.
Patch pipettes were obtained from borosilicate glass capillaries (PG 52151-4; WPI) using a Flaming/Brown puller (P-97; Sutter Instruments Co.). Tip resistances of recording electrodes were between 4 and 6 MΩ. After establishing a giga-ohm seal on SG neurons, whole-cell recordings were initiated by applying a gentle negative pressure to rupture the membrane patch. Signals were amplified using an Axopatch 200B amplifier (Molecular Devices), filtered at 1 kHz, and digitized at 1 kHz using an Axon Digidata 1322A (Molecular Devices). All experiments were conducted at room temperature.
Chemicals
CT was purchased from Sigma-Aldrich. The compound was stocked at a concentration of 300 mM in dimethyl sulfoxide (DMSO). The maximum DMSO concentration was less than 0.33%, which had no effect on membrane currents of SG neurons. Tetrodotoxin citrate (TTX), 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX), and D-2-amino-5-phosphonopentanoic acid (AP5) were purchased from Tocris Bioscience. Other chemicals, including picrotoxin, strychnine hydrochloride (strychnine), glycine, GABA, and components of ACSF, were purchased from Sigma-Aldrich. All drugs were diluted these stock solutions with ACSF to achieve their desired working concentrations.
Data and statistical analysis
CT was confirmed to respond to a minimum of 20 pA in whole-cell voltage-clamp mode, thereby minimizing the possibility of measurement error. For statistical analysis, the means of two or more experimental groups were compared using Student’s t-test or one-way analysis of variance (ANOVA) as appropriate. Statistical analyses and plotting of traces were conducted using Origin 8 software (OriginLab Corp.) and pClamp 10.6 software (Molecular Devices). Numerical data are expressed as the mean ± standard error of the mean (SEM). Differences were considered statistically significant when the p-value was less than 0.05. Levels of statistical significance were indicated as follows: ns (not significant), * (p < 0.05), ** (p < 0.01), *** (p < 0.001), and **** (p < 0.0001). The "n" value in the figures represents the number of SG neurons analyzed.
RESULTS
CT induces repeatable inward currents on SG neurons
To confirm the effect of CT, various concentrations (ranging from 10 µM to 300 µM) were bath applied to SG neurons in a voltage clamp mode. Fig. 2A and B clearly show that CT has a concentration-dependent action, with an increase in mean inward current (pA) corresponding to an increase in CT concentration (µM). The following pA values were recorded: 10 µM, 2.54 ± 0.82 pA; 30 µM, 3.39 ± 0.56 pA; 100 µM, 6.93 ± 1.24 pA; 300 µM, 32.42 ± 5.404 pA. A line graph was used to compare mean inward currents between different concentrations of CT. A one-way ANOVA revealed a significant effect of CT concentration on inward currents (F = 36.68, p < 0.0001). Tukey’s post-hoc test indicated a significant difference between 300 µM and 10 µM CT groups (n = 5 per group, p < 0.0001). These responses that differed with CT concentration showed that CT could directly affect postsynaptic positions of SG neurons.
To investigate whether SG neurons exhibited desensitization after repeated exposure, repeated applications of 300 µM CT were performed using a high-chloride pipette solution. CT-induced inward currents of 12 neurons had no significant diffenrence between first and second applications as shown in Fig. 2C. The mean relative amplitude (± SEM) of the second CT applications compared to the first application was 1.11 ± 0.06 (n = 12; Fig. 2D). These data imply that the SG neurons of the Vc are not desensitized by repeated applications of CT.
Direct action of CT on the SG neurons of the Vc
TTX is recognized as a prominent compound in research focused on understanding mechanisms of neurotransmitter release and interactions with the postsynaptic membrane [13,14]. Therefore, CT was used in the presence of TTX to investigate whether CT could affect SG neurons by modulating action potential generation. CT-induced inward currents of 12 neurons were preserved in the presence of 0.5 µM TTX (Fig. 3A). No significant difference was observed in the mean relative amplitude of CT with and without TTX. The mean relative current induced by CT with TTX was 0.99 ± 0.08 (n = 12), which was not significantly different from that induced by CT alone (p > 0.05) as described in Fig. 3B.
Additionally, to evaluate the degree of effect on ionotropic glutamate receptors, CT was applied together after a 3-min pretreatment with a cocktail of AP5 (an N-methyl-d-aspartate receptor antagonist) and CNQX (a non- N-methyl-d-aspartate glutamate receptor antagonist). As observed in Fig. 4A, CT-induced inward currents of 8 tested neurons were similar between the two cases. Specifically, the mean relative amplitude in the presence of 20 µM AP5 and 10 µM CNQX was 1.05 ± 0.08, which was not significantly different from that in the absence of AP5 and CNQX (n = 8, p > 0.05) as presented in Fig. 4B. These results imply that CT can directly act on postsynaptic SG neurons of Vc. Moreover, CT-induced action is not mediated by the activation of ionotropic glutamate receptors.
CT activates GABAA receptor and/or glycine receptor
Picrotoxin, a non-competitive antagonist of GABAA receptors, can block chloride ion movement through GABAA receptor [15]. In this section, 50 µM picrotoxin was utilized with CT to determine whether the CT-induced currents was GABAA receptor-mediated. As shown in Fig. 5A, the application of picrotoxin significantly reduced CT-induced currents compared to the control condition without picrotoxin in the same neurons. As shown in Fig. 5B, the mean relative amplitude decreased to 0.29 ± 0.06 when picrotoxin was applied. This decrease was significant compared to CT alone (n = 9, p < 0.0001).
Strychnine, a competitive antagonist of glycine receptors, has been widely used in experimental settings to examine the role of glycine receptors and their interaction with other neurotransmitter systems [16]. Similar to the effect observed with picrotoxin, when 2 µM strychnine was co-applied with CT, CT-induced currents were significantly suppressed as shown in Fig. 6A. The histogram in Fig. 6B illustrates a significant reduction in mean relative amplitude to 0.33 ± 0.06 in the presence of strychnine compared to CT alone (n = 8, p < 0.0001). Furthermore, CT-induced inward currents were almost blocked in the presence of picrotoxin and strychnine (Fig. 7A). The mean relative amplitude was decreased to 0.15 ± 0.02 in the presence of picrotoxin and strychnine (n = 11, p < 0.0001; Fig. 7B). These results indicate that CT-induced effects are mediated by activation of GABAA and/or glycine receptors.
CT enhanced both GABA-induced and glycine-induced responses
Inhibiting neurotransmission, including two main neurotransmitters GABA and glycine, is crucial for maintaining the balance of neurons. Under the condition of a high chloride pipette solution, both GABA and glycine induced inward currents in the voltage-clamp mode. A low concentration of CT (100 µM) failed to elicit measurable inward membrane currents. Therefore, a combination of a low CT (100 µM) concentration with GABA and glycine was used to investigate the relationship between the low CT and responses caused by GABA and glycine. As shown in Fig. 8A and B, the low CT concentration enhanced the inward currents induced by GABA and glycine. The mean relative amplitude by co-application of GABA or glycine and low CT was increased to 1.60 ± 0.20 (n = 8, p < 0.05; Fig. 8C) or 1.67 ± 0.166 (n = 8, p < 0.01; Fig. 8D) compared to those by only GABA or only glycine, respectively. These results indicate that CT has potentiation effects on both GABA- and glycine-induced responses of SG neurons of the Vc.
DISCUSSION
In summary, CT-induced inward currents were not sensitive to TTX, CNQX, and AP5, but were inhibited by picrotoxin and strychnine. Additionally, CT enhanced both GABA- and glycine-mediated responses. These results suggest that CT mimics actions of glycine and GABA on the SG neuron of Vc in mice.
Natural products have been used to cure a wide range of diseases for thousands of years. They might also be created as new orofacial antinociception treatment targets. Essential oils of medicinal plants are abundant in secondary metabolites, primarily serving as a defense mechanism for plants. Terpenes composed of isoprene units constitute a significant portion of secondary metabolites present in plants and have several pharmacological effects as described in the literature. For example, our recently reported findings suggested that monoterpenes such as linalool, citral, and borneol have antinociceptive effects and may be potential alternative treatments for orofacial pain [17-19]. As with the monoterpenes mentioned above, CT also has the effect of reducing orofacial antinociception. Brito et al. [10] showed that CT could significantly reduce pain at doses of 25, 50, and 100 mg/kg in formalin, capsaicin, and glutamate tests. Immunofluorescence analysis revealed that CT activated key areas of the central nervous system (CNS), including the olfactory bulb, piriform cortex, retrosplenial cortex, and periaqueductal gray. These findings provide first-time evidence that CT can attenuate orofacial pain, at least in part, through activation of CNS regions, particularly the retrosplenial cortex and periaqueductal gray [10].
This study focused on SG neurons in the spinal cord's dorsal horn (lamina II), which has been suggested as the crucial gate for modulating nociception information [20,21]. The dorsal horn's laminae I-III contain an abundance of inhibitory interneurons that can regulate sensory information before it is transmitted to other regions of the brain and spinal cord. Most neurons in layers I-III are contain glycine and GABA. These interneurons account for approximately 30% of laminae I-II neurons and 40% of laminae III neurons [20,22]. GABAergic and glycinergic neurotransmitters, which are main inhibitory neurotransmitters in the CNS, are responsible for pain perception and mediation. Lack of GABA activity may contribute to increased pain sensitivity in neuropathic pain [23]. One study with normal rats showed that spinal injection of GABAA or GABAB receptor antagonists caused tactile allodynia and thermal hyperalgesia, which could be reversed with exogenous GABA receptor agonists. Isoguvacine, a spinal GABAA receptor agonist, has been suggested as a potential treatment for neuropathic pain because of its unique anti-hyperalgesia and antiallodynic properties [24]. Although not many studies have examined the relationship between CT antinociception and GABA receptor modulation, one study has shown that CT can regulate of GABAergic transmission by guarding against pentylenetetrazol and picrotoxin-induced convulsions [25].
Our study demonstrated that a low concentration (100 µM) of CT could significantly enhance inward currents of SG neurons of the Vc induced by GABA and glycine, emphasizing CT’s role as a modulator of inhibitory neurotransmission. This agreed with an electrophysiological study on Xenopus oocytes, which showed that CT could enhance responses of GABAA receptor at low GABA concentrations (10 and 30 µM) [26]. This enhancement was attributed to CT’s binding to a potentiation site on the GABAA receptor, increasing its sensitivity to GABA. Building on these findings, our results indicate that CT’s potentiation effects extend beyond GABA to also enhance glycine-induced responses, suggesting a broad-spectrum role for CT in modulating inhibitory signaling. By amplifying effects of two major inhibitory neurotransmitters, CT appears to act as a key enhancer of inhibitory synaptic transmission. This effect could strengthen the inhibitory tone, potentially contributing to the fine-tuning of neural circuit activity and the prevention of excessive excitation. These findings highlight a conserved mechanism of CT action across different receptor systems, suggesting that its role as an enhancer of inhibitory neurotransmission is fundamental. Further studies are needed to explore molecular interactions underlying these effects and to assess implications of CT’s modulation for both normal neural function and potential therapeutic applications in conditions involving disrupted inhibitory signaling.
Glutamate and glutamate receptors are present in areas of the brain, spinal cord, and periphery involved in pain sensation and transmission. Glutamate has potential to stimulate primary afferent nociceptors after its release from inflamed or injured tissues. N-methyl-d-aspartate (NMDA) receptor antagonist ketamine injection into the temporomandibular joint (TMJ) can induce considerable attenuation of glutamate-induced TMJ pain [27]. Likewise, CT treatment can reduce glutamate-induced nociception through its interaction with the glutamatergic system [10]. In addition, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors belong to the ionotropic glutamate receptor family that can respond to glutamate. These receptors are responsible for most of the fastest excitatory neurotransmission. They play important roles in the brain's vital functions [28]. However, excessive AMPA receptors can cause neurological disorders [29]. To treat various chronic neurological disorders, many studies have investigated the ability or potential of AMPA receptor antagonists to reduce excitotoxicity [28,30].
In this study, CT did not affect on ionotropic glutamate receptors, specifically AMPA and NMDA receptors. This suggests that CT might not modulate excitatory neurotransmission under conditions tested. However, previous studies that reported the effect of CT on ionotropic glutamate receptors were in contrast to our findings. For example, a study using whole-cell patch-clamp electrophysiology in HEK293 cells demonstrated significant inhibition of all AMPA receptor subunits, including GluA1, GluA2, GluA1/2, and GluA2/3 [31]. This inhibition was characterized by a reduction in peak current amplitude and a slower rate of desensitization and deactivation, indicating potential neuroprotective effects by limiting excessive AMPA receptor activation [31]. These findings suggest that CT might have therapeutic potential for neurological disorders associated with AMPA receptor overactivation, such as epilepsy and Alzheimer’s disease [31]. Discrepancies between our results and previous studies might be attributed to differences in experimental conditions. For instance, the use of SG neurons in our study, which could more closely mimic physiological conditions compared to HEK293 cells in prior research, might have contributed to the variation. Additionally, factors such as CT concentration, receptor subtype expression, and the presence of other modulatory co-factors might have influenced the outcomes. These contrasting findings underscore the complexity of CT's interaction with glutamate receptors and highlight the need for further investigation. A better understanding of the precise mechanisms and conditions under which CT affects excitatory neurotransmission could provide valuable insights into its therapeutic potential for neurological disorders [31].
While the present findings clearly demonstrated modulatory effects of CT on SG neurons, a notable limitation was the lack of neuronal subtype classification during electrophysiological recordings. To overcome this limitation, future research should incorporate cell-type identification methods to further elucidate specific neural circuits and mechanisms underlying CT’s action. Furthermore, to more definitively clarify the role of CT in orofacial pain modulation, additional in vitro and in vivo studies employing animal models of orofacial pain are required to determine whether CT exerts antinociceptive effects at both central and peripheral levels, and whether CT administration reduces pain-related behaviors.
In conclusion, we demonstrate that CT can directly activate GABAA receptor or glycine receptor and enhance GABA and glycine-mediated actions on SG neurons of the Vc in mice. This suggests that CT may serve an agent concerning to inhibitory synaptic transmission of the CNS. Thus, CT might be a promising material for orofacial pain management.
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