Effects of cevimeline on excitability of parasympathetic preganglionic neurons in the superior salivatory nucleus of rats

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Effects of cevimeline on excitability of parasympathetic preganglionic neurons in the superior salivatory nucleus of rats Yoshihiro Mitoha,b,⁎, Hirotaka Uedac, Hiroyuki Ichikawad, Masako Fujitaa, Motoi Kobashia, Ryuji Matsuoa a

Department of Oral Physiology, Okayama University Graduate School of Medicine and Dentistry and Pharmaceutical Sciences, Okayama 700-8525, Japan Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School, Okayama 700-8525, Japan c Department of Orthodontics, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima 890-8544, Japan d Division of Oral and Craniofacial Anatomy, Tohoku University Graduate School of Dentistry, Sendai 980-8575, Japan b

A R T I C L E I N F O

A B S T R A C T

Keywords: Cevimeline Muscarinic acetylcholine receptor Superior salivatory nucleus Patch-clamp Xerostomia

The superior salivatory nucleus (SSN) contains parasympathetic preganglionic neurons innervating the submandibular and sublingual salivary glands. Cevimeline, a muscarinic acetylcholine receptor (mAChR) agonist, is a sialogogue that possibly stimulates SSN neurons in addition to the salivary glands themselves because it can cross the blood-brain barrier (BBB). In the present study, we examined immunoreactivities for mAChR subtypes in SSN neurons retrogradely labeled with a fluorescent tracer in neonatal rats. Additionally, we examined the effects of cevimeline in labeled SSN neurons of brainstem slices using a whole-cell patch-clamp technique. Mainly M1 and M3 receptors were detected by immunohistochemical staining, with low-level detection of M4 and M5 receptors and absence of M2 receptors. Most (110 of 129) SSN neurons exhibited excitatory responses to application of cevimeline. In responding neurons, voltage-clamp recordings showed that 84% (101/120) of the neurons exhibited inward currents. In the neurons displaying inward currents, the effects of the mAChR antagonists were examined. A mixture of M1 and M3 receptor antagonists most effectively reduced the peak amplitude of inward currents, suggesting that the excitatory effects of cevimeline on SSN neurons were mainly mediated by M1 and M3 receptors. Current-clamp recordings showed that application of cevimeline induced membrane depolarization (9/9 neurons). These results suggest that most SSN neurons are excited by cevimeline via M1 and M3 muscarinic receptors.

1. Introduction The superior salivatory nucleus (SSN), which is located in the lateral reticular formation of the medulla oblongata, contains parasympathetic preganglionic neurons innervating the submandibular and sublingual salivary glands. Our electrophysiological study showed that SSN neurons innervating the salivary glands receive excitatory synaptic inputs via NMDA-type and non-NMDA-type glutamate receptors, as well as inhibitory synaptic inputs via GABAA and glycine receptors in neonatal rats (Ishizuka et al., 2008; Mitoh et al., 2004; Mitoh et al., 2008). These four receptor types are the main constituents for impulse formation. Additionally, our recent immunohistochemical study showed that mAChR subtypes are expressed in adult rat SSN neurons; among five subtypes (M1–M5) (Wess et al., 2007) of mAChRs, M3 receptor immunoreactivity was frequently observed and M2, M4, while M5 receptor immunoreactivities were occasionally observed (Ueda et al.,

2011). Therefore, we hypothesized that cholinergic synaptic inputs via mAChR subtypes modulate the excitability of SSN neurons. Activation of M1, M3, and M5 receptors in postsynaptic membranes indicates excitatory responses (Brown, 2010). Cevimeline, an M1 and M3 receptor agonist, induces excitatory responses, and is currently used for the treatment of xerostomia. Although xerostomia reduces the quality of life because of various disorders in oral function such as taste (Mese and Matsuo, 2007), mastication (Ikebe et al., 2011), swallowing (Kishimoto et al., 2016), and speech (Pauloski et al., 1998), cevimeline and pilocarpine are the only therapeutic agents. Cevimeline was originally developed as an M1 receptor agonist to treat Alzheimer's-type senile dementia and, therefore, can cross the BBB (Fisher et al., 1991; Fisher, 2008). These data suggest that cevimeline also stimulates SSN neurons, which have excitatory mAChRs. Although our preliminary study (Ueda et al., 2009) shows that neonatal rat SSN neurons indicate excitatory responses to cevimeline application, mAChR subtypes

Abbreviations: BBB, blood-brain barrier; FS, fluorescein; mAChR, muscarinic acetylcholine receptor; SSN, superior salivatory nucleus ⁎ Corresponding author at: Okayama University Graduate School of Medicine and Dentistry and Pharmaceutical Sciences, Shikata-cho, Okayama 700-8525, Japan. E-mail addresses: [email protected] (Y. Mitoh), [email protected] (R. Matsuo). http://dx.doi.org/10.1016/j.autneu.2017.05.010 Received 1 March 2017; Received in revised form 4 May 2017; Accepted 24 May 2017 1566-0702/ © 2017 Elsevier B.V. All rights reserved.

Please cite this article as: Mitoh, Y., Autonomic Neuroscience: Basic and Clinical (2017), http://dx.doi.org/10.1016/j.autneu.2017.05.010

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chamber (26-mm diameter; 1.3-ml volume) and perfused with standard aCSF at room temperature at a rate of 2–3 ml/min. For voltage-clamp recording, tetrodotoxin (0.5 μM) was added in standard aCSF. Wholecell recordings were obtained from fluorescence-labeled SSN neurons. Fluorescence images of labeled neurons were identified using a G-2A dichroic mirror, and the surface of the neuron was viewed using Nomarski optics with infrared differential interference contrast videomicroscopy. Current or voltage signals were amplified using a patchclamp amplifier (EPC-8, HEKA Elektronik, Lambrecht/Pfalz, Germany), filtered at 5 kHz (ITC-16, Instrutech, Great Neck, NY), and stored on a personal computer using LabChart software from PowerLab (PowerLab 4/30, AD Instruments, Australia). Recording pipettes were filled with a solution containing (in mM) 140 potassium gluconate, 6 KCl, 0.2 EGTA, 1 MgCl2, 2 Na2ATP, 0.3 NaGTP, 10 HEPES, and 0.1 spermine (adjusted to pH 7.3 with KOH). The tip resistances of the patch pipette were 4–7 MΩ when filled with the recording pipette solution. The liquid junction potential between the patch pipette solution and the aCSF was approximately −10 mV. The actual membrane potential was corrected by this value.

involved in the excitatory effect have not been identified. First, we examined expression of mAChR subtypes in neonatal SSN neurons using immunohistochemistry and compared these results with mAChR subtype expression in adult SSN neurons. Then, we examined the effects of cevimeline on the excitability of neonatal SSN neurons retrogradely labeled with a fluorescent tracer in brainstem slices using a whole-cell patch-clamp technique. 2. Methods The experimental design was approved by the Animal Care and Use Committee in Okayama University. 2.1. Retrograde labeling of SSN neurons Neonatal Wistar rats (7–12-days-old) were used in the present study. As the SSN contains preganglionic neurons innervating some target organs, including the salivary glands, SSN neurons innervating the salivary glands were labeled with a fluorescent dye, as previously described (Ueda et al., 2011). Briefly, 2 days before preparing brain slices, rats were deeply anesthetized with pentobarbital (25 mg/kg, s.c., Dainippon Pharma Co. Ltd., Osaka, Japan) and a dye solution (5%, 0.1–0.2 µl) was injected into the left chorda-lingual nerve, which includes the axonal fibers of SSN neurons. Dextran-fluorescein (FS), anionic, lysine fixable (MW 10,000, Invitrogen, Eugene, OR) and dextranTexas Red-lysine (3,000 MW, Invitrogen, Eugene, OR) were used for immunohistochemistry and electrophysiology, respectively. After recovery from anesthesia, the animals were returned to their mother.

2.5. Drugs Cevimeline was kindly provided by Nippon Kayaku, Co. Ltd. (Tokyo, Japan). Muscarine and atropine were purchased from SigmaAldrich (St. Louis, MO). Pirenzepine, AF-DX 116, DAU 5884 and tropicamide were purchased from Tocris Bioscience (Bristol, UK). Other drugs were purchased from Nacalai Tesque (Kyoto, Japan). Pirenzepine (1 μM), AF-DX 116 (2 μM), DAU 5884 (1 μM), and tropicamide (1 μM) were used as relatively specific antagonists for M1, M2, M3, and M4 receptors, respectively. For M5 receptors, there were no commercially available agonists and antagonists. To identify mAChR subtypes, recorded neurons were pretreated with perfusate that included an antagonist, for > 3 min, and then the test solution, which included the antagonist and cevimeline, was applied. Stimulating solutions were applied locally to the slice with a polyethylene tube (inner diameter, 0.7 mm) whose tip was placed 2–5 mm upstream of the tip of the recording electrode to achieve a faster rise increase in agent concentration and faster exchange of the solution around the recorded cell, relative to conventional bath application. Drug application through the tube was remotely controlled by an electromagnetic valve-based application system (VC-6; Warner Instruments, Hamden, CT) (Kato and Shigetomi, 2001). This application system was appropriate for observation of slow current responses mediated by mAChRs.

2.2. Immunohistochemistry The operated rats (n = 4 rats, 9-days-old) were fixed using 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brainstems were then soaked overnight in phosphate-buffered 20% sucrose solution and 8-μm-thick sections were frozen-sectioned and thaw-mounted onto silane-coated slides. Sections were incubated with primary rabbit anti-mAChR antibody (1:100, Research & Diagnostics Antibodies, Las Vegas, NV) overnight at room temperature, followed by secondary Rhodamine Red-X-conjugated donkey anti-rabbit IgG (1:500, Jackson ImmunoResearch Labs, West Grove, PA) for 2 h at room temperature. The selectivity of the primary antibodies in this study has been demonstrated in previous studies (Ryberg et al., 2008; Tobin et al., 2006). Stained sections were examined with a fluorescence microscope (ECLIPSE 80i, Nikon, Tokyo, Japan), and images were captured with a CCD spot camera.

2.6. Data analysis 2.3. Slice preparation for electrophysiology For concentration-response analysis, data were fitted by the following function;

The present study was performed in neonatal rat SSN neurons (n = 133, 7–12 days old), because it is difficult to perform electrophysiological studies in adult rat SSN neurons. The operated rats were deeply anesthetized with halothane (1–2%) and decapitated. The brain was rapidly removed from the skull and kept for 3 min in sucrose-based extracellular solution (pH 7.4) bubbled with 95% O2 and 5% CO2 at < 4 °C. The sucrose-based extracellular solution contained (in mM) 234 sucrose, 2.5 KCl, 1.25 NaH2PO4, 10 MgSO4, 0.5 CaCl2, 26 NaHCO3, and 11 glucose. Sagittal brainstem slices at 200-μm thickness were obtained using a tissue slicer (LinearSlicer PRO7, Dosaka EM, Kyoto, Japan). The slices were preincubated in standard artificial cerebrospinal fluid (aCSF) (pH 7.4) bubbled with 95% O2 and 5% CO2 at room temperature (24–27 °C) for 1.5–2 h. Standard aCSF contained (in mM) 125 NaCl, 5 KCl, 1.6 MgCl2, 2 CaCl2, 26 NaHCO3, and 10 glucose.

I Imax = [1 + (EC50 [cevimeline]) n]−1 where I is the observed cevimeline-induced inward current, Imax is the maximum value of the current, [cevimeline] is the cevimeline concentration, EC50 is the concentration of cevimeline eliciting 50% of the maximal current, and n is the Hill coefficient. Curves were drawn using MacCurveFit software (Kevin Ranger Software, Mt. Waverley, Victoria, Australia). All data are expressed as the means ± S.E.M. Statistical significance (P < 0.05) was determined by paired t-test or one-way ANOVA. 3. Results 3.1. Immunoreactivities for mAChRs in neonatal rat SSN neurons

2.4. Whole-cell patch-clamp recording Since adult SSN neurons have immunoreactivities for M2–M5 receptors, but not M1 receptors (Ueda et al., 2011), we examined whether

After preincubation, the slices were then transferred to a recording 2

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Fig. 1. Immunofluorescence labeling of muscarinic acetylcholine receptor (mAChR) subtypes in retrogradely labeled SSN neurons from neonatal rats. (A) Representative microphotographs of immunofluorescence for the M1 (a–c), M2 (d–f), M3 (g–i), M4 (j–l), and M5 (m–o) receptors at high magnification. In the vertical panels, “FS-labeled neurons” (a, d, g, j, and m) indicate images of SSN neurons that were retrogradely labeled with fluorescein (FS); “mAChR subtypes” (b, e, h, k, and n) indicate images of M1, M2, M3, M4, and M5 receptor immunoreactivities, respectively; and “merged” (c, f, i, l, and o) indicates superimposed images of FS-labeled neurons and mAChR subtype immunoreactivities. Scale bar = 50 µm. (B) Representative microphotographs of immunofluorescence for the M2 (a) and M3 (b) receptors at low magnification. Scale bar = 100 µm. Panels a–c, d–f, g–i, j–l, and m–o in A, and panels a and b in B show the same fields of view, respectively. All panels in A or B show images at the same magnification, respectively. Arrows indicate mAChR-immunopositive FS-labeled neurons; single arrowheads indicate mAChR-immunopositive non-FS-labeled neurons; and double arrowheads indicate mAChR-immunonegative FS-labeled neurons.

performed on a total number of 129 SSN neurons in voltage-clamp (120 neurons) and current-clamp (9 neurons) modes. Among these 129 neurons, 19 neurons exhibited no response to application of cevimeline. In the voltage-clamp mode, responses to cevimeline application were investigated at a holding potential of − 70 mV. Eighty-four percent (101/120) of tested neurons showed inward postsynaptic currents to cevimeline application (100 or 300 μM). Fig. 2C and D show typical responses to application of cevimeline, and these responses were similar to those obtained after application of muscarine. In responding neurons, the majority (85%, 86/101) of neurons indicated an inward current alone (Fig. 2C), while the minority (15%, 15/101) of neurons indicated a small amplitude of outward current after an inward current (Fig. 2D). Both current responses were completely suppressed by atropine (3 μM). Henceforth, we focused on the effects of cevimeline on postsynaptic membranes of SSN neurons, which displayed inward currents alone. Cevimeline-induced inward currents were produced in a concentrationdependent manner. Fig. 3A shows typical traces of currents after application of cevimeline. The means of peak amplitudes of the currents were plotted against various concentrations (10–1000 μM; 15 s) of cevimeline (Fig. 3B). The values of the EC50 and the Hill coefficient were 45.8 μM and 1.1, respectively, suggesting positive cooperative action.

neonatal rat SSN neurons have mAChR subtypes. A total of 641 FSlabeled SSN neurons obtained from 3 neonatal rats were analyzed. The SSN neurons had immunoreactivities for all mAChR subtypes, with the exception of M2 receptors: M1 (44.7%, 55/123 neurons, Fig. 1Aa–c), M2 (0%, 0/153 neurons, Fig. 1Ad–f), M3 (58.1%, 79/136 neurons, Fig. 1Ag–i), M4 (14.9%, 17/114 neurons, Fig. 1Aj–l), and M5 (10.3%, 12/117 neurons, Fig. 1Am–o). Fig. 1B shows low magnification microphotographs for M2 receptor (absent) and M3 receptor (most abundant) immunoreactivities. The strong immunoreactivities of M3 and M1 receptors implied that neonatal SSN neurons exhibit excitatory responses to application of muscarinic agonists. 3.2. Muscarine- and cevimeline-induced current responses To confirm functional expression of mAChRs in SSN neurons, we first examined responses to application of muscarine, a non-specific agonist of mAChR subtypes in the voltage-clamp mode. The effects of application of muscarine (300 μM, 15 s) were evaluated at a holding potential of − 70 mV (n = 4). Although all tested neurons indicated inward postsynaptic currents (n = 4), 3 of 4 neurons indicated an inward current alone (Fig. 2A), and 1 of 4 neurons also indicated a small amplitude of outward current after an inward current (Fig. 2B). Muscarine-induced inward currents were completely suppressed by atropine (3 μM, Fig. 2A), indicating that mAChRs expressed in postsynaptic membranes of neonatal rat SSN neurons were functional. In experiments assessing responses to cevimeline, recordings were 3

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Fig. 2. Muscarine- and cevimeline-induced current responses in voltage-clamp recording mode. Representative traces in response to 300 μM muscarine (A and B) and 300 μM cevimeline (C and D) application are shown. Under both conditions, SSN neurons exhibited 2 types of current responses: inward current alone (A and C) and mixed inward and outward currents (B and D). The-muscarine-induced current in panel A and the cevimeline-induced current in panels C and D were completely abolished by 3 μM atropine application. The effect of atropine on the muscarine-induced current in panel B was not confirmed.

t = 5.39, P < 0.001, Fig. 4Bc) of the control response, respectively. Meanwhile, residual responses were observed in all recordings, as shown in Fig. 4Ab and d. Then, in the presence of both pirenzepine and DAU 5884 antagonists, the mean peak amplitude of the inward currents was reduced to 18.9% of the control response (n = 8, t = 11.71, *P < 0.001, Fig. 4Be), which was significantly different from responses in the presence of each antagonist alone (F (2, 25) = 4.526, † P < 0.05). On the other hand, the mean peak amplitude of cevimeline-induced inward currents was not affected by AF-DX 116 (104.0% of the control response, n = 8, t = 0.83, P > 0.05, Fig. 4Bb) or tropicamide (97.1% of the control response, n = 8, t = 0.62, P > 0.05, Fig. 4Bd). Furthermore, no significant differences were observed among control groups in Fig. 4Ba–e (F (4, 39) = 0.223, P > 0.05).

3.3. Effects of antagonists for mAChR subtypes on cevimeline-induced inward currents To clarify the mAChR subtypes responsible for cevimeline-induced inward currents, we examined the effects of antagonists for mAChR subtypes (Fig. 4). Since current responses were small (mean peak amplitude < 27 pA, Fig. 4B) after application of cevimeline, we used a relatively high concentration of cevimeline (300 μM) in order to determine the effects of different antagonists easily. Fig. 4A shows example responses obtained from 1 neuron to application of cevimeline (300 μM, 15 s), in the absence and in the presence of the antagonists: pirenzepine (M1, 1 μM), AF-DX 116 (M2, 2 μM), DAU 5884 (M3, 1 μM), and tropicamide (M4, 1 μM). The mean peak amplitudes of the inward currents were significantly reduced by pirenzepine or DAU 5884 to 60.1% (n = 10, t = 6.47, P < 0.001, Fig. 4Ba) or 69.9% (n = 10,

Fig. 3. Concentration-dependent generation of an inward current by cevimeline. (A) Representative traces of inward current in response to cevimeline application (100, 300, 500, and 1000 μM). (B) Concentration-response curve for the relationship between the mean peak amplitude of the inward current and the cevimeline concentration (1, 10, 30, 50, 100, 300, 500, and 1000 μM). Data were fitted with a single logistic function. The number of neurons tested is indicated for each concentration.

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Fig. 4. Effects of specific mAChR antagonists on the cevimeline-induced inward current. (A) Representative traces of cevimeline (300 μM, 30 s)-induced inward current in the absence (a, control) and presence of specific mAChR antagonists (b, 1 μM pirenzepine; c, 2 μM AF-DX 116; d, 1 μM DAU 5884; e, 1 μM tropicamide; and f, 1 μM pirenzepine + 1 μM DAU 5884). The recordings shown in Aa–e were obtained from a single neuron. Usually, 2 test solutions including cevimeline plus antagonist were applied to a neuron after application of the control solution (cevimeline alone). (B) Graphs of the mean peak amplitudes of current induced by cevimeline alone and cevimeline plus an antagonist. The mean peak amplitude of the inward current in response to cevimeline was most strongly attenuated by a combination of pirenzepine and DAU 5884 (*P < 0.001), and the attenuation was significantly greater than that achieved by either pirenzepine or DAU 5884 alone (†P < 0.05). There were no significant differences among the control groups in panel B.

at − 50 mV by DC current injection, persistent action potentials were produced by application of cevimeline (100 μM, 30 s) (4/5 neurons, Fig. 5B). At concentrations > 300 μM cevimeline (n = 3), SSN neurons generated action potentials without DC current injection (data not shown).

3.4. Cevimeline-induced voltage responses To examine the effects of cevimeline on the membrane potentials of SSN neurons, current-clamp recordings were performed (n = 9). The mean resting membrane potentials of SSN neurons were − 62.3 ± 2.0 mV (n = 9). When the membrane potential was held at the resting potential, membrane depolarization, but no action potential, was observed after application of cevimeline (100 μM, 30 s) (9/9 neurons, 7.9 ± 1.1 mV, Fig. 5A). When the membrane potential was held

4. Discussion In the present study, the mAChR subtypes that are involved in the Fig. 5. Cevimeline-induced voltage responses in currentclamp recording mode. (A) Membrane depolarization in response to cevimeline application (100 μM) when neurons were held at a resting potential. (B) Action potentials in response to cevimeline application when held at −50 mV using DC current injection. Cevimeline applied to neurons held at resting potential did not usually produce an action potential.

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required to determine the exact contribution of M5 receptors to generation of cevimeline-induced inward currents and activation of SSN neurons.

excitatory response induced by cevimeline in neonatal rat SSN neuron were examined immunohistochemically and electrophysiologically. The immunohistochemical study demonstrated that SSN neurons primarily express M1 and M3 receptors. In support of these data, the majority of SSN neurons showed inward currents and membrane depolarization after application of cevimeline. The inward currents were largely suppressed by a mixture of antagonists for M1 and M3 receptors, suggesting that the excitatory responses of SSN neurons to cevimeline are mainly mediated by M1 and M3 receptors.

4.3. Possibility of salivary secretion via SSN neurons Since cevimeline excited most SSN neurons, it is possible that cevimeline induces indirect salivary secretion via SSN neurons, in addition to direct salivary secretion by stimulating the salivary glands. Here, we discuss the possibility for an indirect action of cevimeline. First, we compared (1) the cevimeline concentration producing salivary secretion by direct action on the salivary glands, and (2) the concentration that affects SSN neurons. For (1), in the mouse isolated submandibular salivary gland preparations, salivary secretion was investigated when cevimeline solution was perfused via the artery (Kondo et al., 2011), and it was revealed that 3–10 μM results in distinct salivation. For (2), a concentration of > 10 μM resulted in distinct amplitude of inward current, and a concentration of 300 μM resulted in action potentials. Thus, these data imply that a low concentration of cevimeline is unlikely to cause salivation by indirect action on SSN neurons although responsiveness to cevimeline does not seem to be markedly different between acinar cells and SSN neurons. However, in vivo SSN neurons possibly generate action potentials to cevimeline concentrations < 300 μM. Considering that resting saliva is secreted in unanesthetized animals although its neuronal mechanism is unclear, the excitability of SSN neurons increases at least toward action potential generation. We therefore investigated the effect of cevimeline after SSN neurons had been depolarized by DC current injection. At the same concentration (100 μM) of cevimeline, SSN neurons exhibited only depolarization before the current injection (Fig. 5A), but they exhibited action potentials after the current injection (Fig. 5B). Additionally, spontaneously firing SSN neuron was depolarized from − 49 mV to −46 mV by 10 μM cevimeline, and its firing frequency was increased from 2.7 Hz to maximum 4.7 Hz (n = 1, data not shown). Therefore, these data indicate that SSN neurons generate action potentials at lower concentrations of cevimeline, as their depolarization increases. Under in vivo conditions, indirect salivation via SSN neurons may occur. The actual contribution via SSN neurons in salivation by cevimeline needs to be investigated by in vivo experiments. In clinical practice, when we have to discontinue to use cevimeline due to side effects, there are no other drugs to choose because another sialogogue, pilocarpine, is also a muscarinic agonist. In the future, identification of an agent with the ability to cross the BBB and increase SSN neuronal excitability may yield a novel type of sialagogue.

4.1. Expression of mAChR subtypes Integrating the results of the present and previous (Ueda et al., 2011) studies on mAChR subtypes in neonatal and adult rat SSN neurons, the proportion of each subtype detected was markedly different between neonatal and adult neurons: M1 (45:0), M2 (0:40), M3 (60:90), M4 (15:55), and M5 (10:45). Thus, neonatal SSN neurons exhibited a higher proportion of M1 receptors and a lower proportion of M2–M5 receptors compared to adult SSN neurons. The profile of adult SSN neurons was consistent with previous studies, showing very little expression of the M1 receptor and abundant expressions of the M2, M3, and M4 receptors in the lower brainstem of adult rats (Levey et al., 1991; Oki et al., 2005; Yasuda et al., 1993). Developmental changes in mAChR subtype expressions have also been studied in the rat brain using immunoprecipitation (Tice et al., 1996); M1 receptor expression in the cerebellum decreases with age, whereas M2 and M4 receptor expressions in the cortex increase with age. For M5 receptors, expression is very low at 2% throughout the entire brain (Yamada et al., 2003) and no studies have investigated developmental change in M5 receptor to date. Taken together, these findings suggest that M1 receptor expression in SSN neurons decreases while M2, M3, M4, and possibly M5 receptor expression increases with development. 4.2. mAChR subtypes involved in cevimeline-induced excitatory responses Generally, activation of M1, M3, or M5 receptors in the postsynaptic membrane leads to a slow excitatory response via G protein Gq/11 (Brown, 2010; Kuba and Koketsu, 1978). Among these excitatory mAChR subtypes, immunoreactivities for M1 and M3 receptors were mainly detected in neonatal rat SSN neurons. In support of this histochemical result, cevimeline, an agonist for M1 and M3 receptors, induced inward currents in the majority of SSN neurons, and the inward currents were significantly attenuated by M1 or M3 receptor antagonists. In a previous immunohistochemical study, we proposed coexpression of M3 receptors and other receptor subtypes in adult rat SSN neurons (Ueda et al., 2011). In addition, the coexpression of different mAChR subtypes is well documented in both central (Sooksawate and Isa, 2006) and peripheral tissues (Obara et al., 2001). Given that a mixture of M1 and M3 receptor antagonists was most effective for suppressing the current responses, both M1 and M3 receptors appear to be coexpressed and contribute to the effects of cevimeline in neonatal rat SSN neurons. In some SSN neurons, we observed residual responses, which were completely suppressed by atropine (n = 1, data not shown) in the presence of a mixture of antagonists for M1 and M3 receptors (Fig. 4Be). Taken together with detection of immunoreactivity for M5 receptors in the present study, it is conceivable that the residual responses are mediated by M5 receptors. A previous paper showed high homology of the orthosteric site among mAChR subtypes (Jakubík et al., 2014), suggesting that many agonists for mAChR subtypes, including cevimeline, have a high potential for cross-reactivity against other subtypes. In fact, similar reactivities to M1, M3, and M5 receptors against cevimeline have been shown by measuring an increase of intracellular Ca2 + in mAChR subtype-expressing cell lines: the halfmaximal effective concentration for M1, M3, and M5 receptors are 23, 48, and 63 (nM), respectively (Heinrich et al., 2009). Further work is

5. Conclusion The majority of neonatal rat SSN neurons innervating the salivary glands exhibited inward currents and membrane depolarizations after application of cevimeline. Immunohistochemical and pharmacological results indicated that these excitatory responses are mainly mediated by M1 and M3 mAChR subtypes. Cevimeline may, therefore, induce salivary secretion by indirectly acting at the salivary glands via M1 and M3 receptors of SSN neurons, as well as directly acting at the salivary glands. Conflict of interest There are no conflicts of interest to declare. Acknowledgement This study was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grants (Nos. 25462887 and 26500009). 6

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postsynaptic currents of the superior salivatory nucleus innervating the salivary glands and tongue in the rat. Brain Res. 999, 62–72. Mitoh, Y., Funahashi, M., Fujii, A., Fujita, M., Kobashi, M., Matsuo, R., 2008. Development of inhibitory synaptic transmission to the superior salivatory nucleus in rats. Brain Res. 1191, 47–54. Obara, K., Arai, K., Tomita, Y., Hatano, A., Takahashi, K., 2001. G-protein coupled receptor kinase 2 and 3 expression in human detrusor cultured smooth muscle cells. Urol. Res. 29, 325–329. Oki, T., Takagi, Y., Inagaki, S., Taketo, M.M., Manabe, T., Matsui, M., Yamada, S., 2005. Quantitative analysis of binding parameters of [3H]N-methylscopolamine in central nervous system of muscarinic acetylcholine receptor knockout mice. Brain Res. Mol. Brain Res. 133, 6–11. Pauloski, B.R., Rademaker, A.W., Logemann, J.A., Colangelo, L.A., 1998. Speech and swallowing in irradiated and nonirradiated postsurgical oral cancer patients. Otolaryngol. Head Neck Surg. 118, 616–624. Ryberg, A.T., Warfvinge, G., Axelsson, L., Soukup, O., Gotrick, B., Tobin, G., 2008. Expression of muscarinic receptor subtypes in salivary glands of rats, sheep and man. Arch. Oral Biol. 53, 66–74. Sooksawate, T., Isa, T., 2006. Properties of cholinergic responses in neurons in the intermediate grey layer of rat superior colliculus. Eur. J. Neurosci. 24, 3096–3108. Tice, M., Hashemi, T., Taylor, L., McQuade, R.D., 1996. Distribution of muscarinic receptor subtypes in rat brain from postnatal to old age. Brain Res. Dev. Brain Res. 92, 70–76. Tobin, G., Ryberg, A.T., Gentle, S., Edwards, A.V., 2006. Distribution and function of muscarinic receptor subtypes in the ovine submandibular gland. J. Appl. Physiol. (1985) 100, 1215–1223. Ueda, H., Mitoh, Y., Ichikawa, H., Kobashi, M., Yamashiro, T., Matsuo, R., 2009. Cevimeline enhances the excitability of rat superior salivatory neurons. J. Med. Investig. 56, 267–269 (Suppl.). Ueda, H., Mitoh, Y., Fujita, M., Kobashi, M., Yamashiro, T., Sugimoto, T., Ichikawa, H., Matsuo, R., 2011. Muscarinic receptor immunoreactivity in the superior salivatory nucleus neurons innervating the salivary glands of the rat. Neurosci. Lett. 499, 42–46. Wess, J., Eglen, R.M., Gautam, D., 2007. Muscarinic acetylcholine receptors: mutant mice provide new insights for drug developmeny. Nat. Rev. Drug Discov. 6, 721–733. Yamada, M., Basile, A.S., Fedorova, I., Zhang, W., Duttaroy, A., Cui, Y., Lamping, K.G., Faraci, F.M., Deng, C.X., Wess, J., 2003. Novel insights into M5 muscarinic acetylcholine receptor function by the use of gene targeting technology. Life Sci. 74, 345–353. Yasuda, R.P., Ciesla, W., Flores, L.R., Wall, S.J., Li, M., Satkus, S.A., Weisstein, J.S., Spagnola, B.V., Wolfe, B.B., 1993. Development of antisera selective for m4 and m5 muscarinic cholinergic receptors: distribution of m4 and m5 receptors in rat brain. Mol. Pharmacol. 43, 149–157.

References Brown, D.A., 2010. Muscarinic acetylcholine receptors (mAChRs) in the nervous system: some functions and mechanisms. J. Mol. Neurosci. 41, 340–346. Fisher, A., 2008. Cholinergic treatments with emphasis on m1 muscarinic agonists as potential disease-modifying agents for Alzheimer's disease. Neurotherapeutics 5, 433–442. Fisher, A., Brandeis, R., Karton, I., Pittel, Z., Gurwitz, D., Haring, R., Sapir, M., Levy, A., Heldman, E., 1991. ( ± )-Cis-2-methyl-spiro(1,3-oxathiolane-5,3′)quinuclidine, an M1 selective cholinergic agonist, attenuates cognitive dysfunctions in an animal model of Alzheimer's disease. J. Pharmacol. Exp. Ther. 257, 392–403. Heinrich, J.N., Butera, J.A., Carrick, T., Kramer, A., Kowal, D., Lock, T., Marquis, K.L., Pausch, M.H., Popiolek, M., Sun, S.C., Tseng, E., Uveges, A.J., Mayer, S.C., 2009. Pharmacological comparison of muscarinic ligands: historical versus more recent muscarinic M1-preferring receptor agonists. Eur. J. Pharmacol. 605, 53–56. Ikebe, K., Matsuda, K., Kagawa, R., Enoki, K., Yoshida, M., Maeda, Y., Nokubi, T., 2011. Association of masticatory performance with age, gender, number of teeth, occlusal force and salivary flow in Japanese older adults: is ageing a risk factor for masticatory dysfunction? Arch. Oral Biol. 56, 991–996. Ishizuka, K., Oskutyte, D., Satoh, Y., Murakami, T., 2008. Non-NMDA and NMDA receptor agonists induced excitation and their differential effect in activation of superior salivatory nucleus neurons in anaesthetized rats. Auton. Neurosci. 138, 41–49. Jakubík, J., Šantrůčková, E., Randáková, A., Janíčková, H., Zimčík, P., Rudajev, V., Michal, P., El-Fakahany, E.E., Doležal, V., 2014. Outline of therapeutic interventions with muscarinic receptor-mediated transmission. Physiol. Res. 63, 177–189 (Suppl.). Kato, F., Shigetomi, E., 2001. Distinct modulation of evoked and spontaneous EPSCs by purinoceptors in the nucleus tractus solitarii of the rat. J. Physiol. 530, 469–486. Kishimoto, N., Stegaroiu, R., Shibata, S., Ito, K., Inoue, M., Ohuchi, A., 2016. Changes in the oral moisture and the amount of microorganisms in saliva and tongue coating after oral ingestion resumption: a pilot study. Open Dent. J. 10, 79–88. Kondo, Y., Nakamoto, T., Mukaibo, T., Kidokoro, M., Masaki, C., Hosokawa, R., 2011. Cevimeline-induced monophasic salivation from the mouse submandibular gland: decreased Na+ content in saliva results from specific and early activation of Na+/H+ exchange. J. Pharmacol. Exp. Ther. 337, 267–274. Kuba, K., Koketsu, K., 1978. Synaptic events in sympathetic ganglia. Prog. Neurobiol. 11, 77–169. Levey, A.I., Kitt, C.A., Simonds, W.F., Price, D.L., Brann, M.R., 1991. Identification and localization of muscarinic acetylcholine receptor proteins in brain with subtypespecific antibodies. J. Neurosci. 11, 3218–3226. Mese, H., Matsuo, R., 2007. Salivary secretion, taste and hyposalivation. J. Oral Rehabil. 34, 711–723. Mitoh, Y., Funahashi, M., Kobashi, M., Matsuo, R., 2004. Excitatory and inhibitory

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