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A new follicular cyst was reported. The lower part of the cyst wall was composed of both basophilic and shadow cells as seen in pilomatricoma, whereas the upper part of the wall consisted of clear cells. Our case apparently derives from hair matrix and outer root sheath. [ABSTRACT FROM AUTHOR]

Objective: Saliva serves multiple important functions crucial for maintaining a healthy oral and systemic environment. Among them, the pH buffering effect, which is primarily mediated by bicarbonate ions, helps maintain oral homeostasis by neutralizing acidity from ingested foods. Therefore, higher buffering capacity, reflecting the ability to neutralize oral acidity, may influence taste sensitivity, especially for sour taste since it involves sensing H + ions. This study aims to explore the relationship between salivary buffering capacity and taste sensitivities to the five basic tastes in healthy adult humans., Design: Eighty seven healthy adult students participated in this study. Resting saliva volume was measured using the spitting method. The liquid colorimetric test was used to assess salivary buffering capacity. The whole-mouth taste testing method was employed to determine the recognition threshold for each tastant (NaCl, sucrose, citric acid, quinine-HCl, monosodium glutamate)., Results: Taste recognition thresholds for sour taste as well as sweet, salty, and bitter tastes showed no correlation with salivary buffering capacity. Interestingly, a negative relationship was observed between recognition threshold for umami taste and salivary buffering capacity. Furthermore, a positive correlation between salivary buffering capacity and resting saliva volume was observed., Conclusions: Salivary buffering capacity primarily influences sensitivity to umami taste, but not sour and other tastes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Taste buds contain 2 types of GABA-producing cells: sour-responsive Type III cells and glial-like Type I cells. The physiological role of GABA, released by Type III cells is not fully understood. Here, we investigated the role of GABA released from Type III cells using transgenic mice lacking the expression of GAD67 in taste bud cells (Gad67-cKO mice). Immunohistochemical experiments confirmed the absence of GAD67 in Type III cells of Gad67-cKO mice. Furthermore, no difference was observed in the expression and localization of cell type markers, ectonucleoside triphosphate diphosphohydrolase 2 (ENTPD2), gustducin, and carbonic anhydrase 4 (CA4) in taste buds between wild-type (WT) and Gad67-cKO mice. Short-term lick tests demonstrated that both WT and Gad67-cKO mice exhibited normal licking behaviors to each of the five basic tastants. Gustatory nerve recordings from the chorda tympani nerve demonstrated that both WT and Gad67-cKO mice similarly responded to five basic tastants when they were applied individually. However, gustatory nerve responses to sweet-sour mixtures were significantly smaller than the sum of responses to each tastant in WT mice but not in Gad67-cKO mice. In summary, elimination of GABA signalling by sour-responsive Type III taste cells eliminates the inhibitory cell-cell interactions seen with application of sour-sweet mixtures., (© 2024. The Author(s).)

Expression of alpha-synuclein (Syn), a presynaptic neuronal protein, was immunohistochemically examined in intact rat submandibular, sublingual, and lingual glands. The submandibular gland contained abundant periductal Syn-immunoreactive (-ir) nerve fibers. Abundant Syn-ir varicosities were present in acini of the sublingual and serous lingual glands. By confocal laser scanning microscopy, Syn-ir nerve fibers around smooth muscle actin (SMA)-ir cells alone were infrequent; however, those around aquaporin-5 (AQP5)-ir cells alone and both SMA- and AQP5-ir cells were abundant in the sublingual and serous lingual glands. SMA-ir cells were occasionally immunoreactive for toll-like receptor 4, a Syn receptor. Syn-ir nerve fibers contained tyrosine hydroxylase (TH) in the submandibular gland and choline acetyltransferase (ChAT) in all examined salivary glands. In the superior cervical (SCG), submandibular, and intralingual ganglia, sympathetic and parasympathetic neurons co-expressed Syn with TH and ChAT, respectively. SCG neurons innervating the submandibular gland contained mostly Syn. In the thoracic spinal cord, 14.7% of ChAT-ir preganglionic sympathetic neurons co-expressed Syn. In the superior salivatory nucleus, preganglionic parasympathetic neurons projecting to the lingual nerve co-expressed Syn and ChAT. The present findings indicate that released Syn acts on myoepithelial cells. Syn in pre- and post-ganglionic neurons may regulate neurotransmitter release and salivary volume and composition., (© 2024 American Association for Anatomy.)

Cephalic-phase insulin release (CPIR) occurs before blood glucose increases after a meal. Although glucose is the most plausible cue to induce CPIR, peripheral sensory systems involved are not fully elucidated. We therefore examined roles of sweet sensing by a T1R3-dependent taste receptor and sugar sensing by oral glucose transporters in the oropharyngeal region in inducing CPIR. Spontaneous oral ingestion of glucose significantly increased plasma insulin 5 min later in wild-type (C57BL/6) and T1R3-knockout mice, but intragastric infusion did not. Oral treatment of glucose transporter inhibitors phlorizin and phloretin significantly reduced CPIR after spontaneous oral ingestion. In addition, a rapid increase in plasma insulin was significantly smaller in WT mice with spontaneous oral ingestion of nonmetabolizable glucose analog than in WT mice with spontaneous oral ingestion of glucose. Taken together, the T1R3-dependent receptor is not required for CPIR, but oral glucose transporters greatly contribute to induction of CPIR by sugars., (© 2023. The Author(s).)

Fermented milk is consumed worldwide because of its nutritious and healthful qualities. Although it is somewhat sour, causing some to dislike it, few studies have examined taste aspects of its ingredients. Wild-type mice and T1R3-GFP-KO mice lacking sweet/umami receptors were tested with various taste components (sucrose, galactose, lactose, galacto-oligosaccharides, fructo-oligosaccharides, l- and d-lactic acid) using 48 h two-bottle tests and short-term lick tests. d-lactic acid levels were measured after the ingestion of d- or; l-lactic acid or water to evaluate d-lactic acidosis. In wild-type mice, for the sweet ingredients the number of licks increased in a concentration-dependent manner, but avoidance was observed at higher concentrations in 48 h two-bottle tests; the sour ingredients d- and l-lactic acid showed concentration-dependent decreases in preference in both short- and long-term tests. In 48 h two-bottle tests comparing d- and l-lactic acid, wild-type but not T1R3-GFP-KO mice showed higher drinking rates for l-lactic acid. d-lactic acidosis did not occur and thus did not contribute to this preference. These results suggest that intake in short-term lick tests varied by preference for each ingredient, whereas intake variation in long-term lick tests reflects postingestive effects. l-lactic acid may have some palatable taste in addition to sour taste.

Orexin (OX), which regulates sleep and wakefulness and feeding behaviors has 2 isoforms, orexin-A and -B (OXA and OXB). In this study, the distribution of OXA and OXB was examined in the rat superior salivatory nucleus (SSN) using retrograde tracing and immunohistochemical and methods. OXA- and OXB-immunoreactive (-ir) nerve fibers were seen throughout the SSN. These nerve fibers surrounded SSN neurons retrogradely labeled with Fast blue (FB) from the corda-lingual nerve. FB-positive neurons had pericellular OXA- (47.5%) and OXB-ir (49.0%) nerve fibers. Immunohistochemistry for OX receptors also demonstrated the presence of OX1R and OX2R in FB-positive SSN neurons. The majority of FB-positive SSN neurons contained OX1R- (69.7%) or OX2R-immunoreactivity (57.8%). These neurons had small and medium-sized cell bodies. In addition, half of FB-positive SSN neurons which were immunoreactive for OX1R (47.0%) and OX2R (52.2%) had pericellular OXA- and OXB-ir nerve fibers, respectively. Co-expression of OX1R- and OX2R was common in FB-positive SSN neurons. The present study suggests a possibility that OXs regulate the activity of SSN neurons through OX receptors., Competing Interests: Declaration of competing interest The authors do not have any conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

(1) Background: Our previous studies revealed that orexin-A, an appetite-increasing peptide, suppressed reflex swallowing via the commissural part of the nucleus tractus solitarius (cNTS), and that glucagon-like peptide-1 (GLP-1), an appetite-reducing peptide, also suppressed reflex swallowing via the medial nucleus of the NTS (mNTS). In this study, we examined the mutual interaction between orexin-A and GLP-1 in reflex swallowing. (2) Methods: Sprague-Dawley rats under urethane-chloralose anesthesia were used. Swallowing was induced by electrical stimulation of the superior laryngeal nerve (SLN) and was identified by the electromyographic (EMG) signals obtained from the mylohyoid muscle. (3) Results: The injection of GLP-1 (20 pmol) into the mNTS reduced the swallowing frequency and extended the latency of the first swallow. These suppressive effects of GLP-1 were not observed after the fourth ventricular administration of orexin-A. After the injection of an orexin-1 receptor antagonist (SB334867) into the cNTS, an ineffective dose of GLP-1 (6 pmol) into the mNTS suppressed reflex swallowing. Similarly, the suppressive effects of orexin-A (1 nmol) were not observed after the injection of GLP-1 (6 pmol) into the mNTS. After the administration of a GLP-1 receptor antagonist (exendin-4(5-39)), an ineffective dose of orexin-A (0.3 nmol) suppressed reflex swallowing. (4) Conclusions: The presence of reciprocal inhibitory connections between GLP-1 receptive neurons and orexin-A receptive neurons in the NTS was strongly suggested.

Bitter taste receptors TAS2Rs detect noxious compounds in the oral cavity. Recent heterologous expression studies reported that some compounds function as antagonists for human TAS2Rs. For examples, amino acid derivatives such as γ-aminobutyric acid (GABA) and Nα,Nα-bis(carboxymethyl)-L-Lysine (BCML) blocked responses to quinine mediated by human TAS2R4. Probenecid inhibited responses to phenylthiocarbamide mediated by human TAS2R38. In this study, we investigated the effects of these human bitter receptor antagonists on behavioral lick responses of mice to elucidate whether these compounds also function as bitter taste blockers. In short-term (10 s) lick tests, concentration-dependent lick responses to bitter compounds (quinine-HCl, denatonium and phenylthiourea) were not affected by the addition of GABA or BCML. Probenecid reduced aversive lick responses to denatonium and phenylthiourea but not to quinine-HCl. In addition, taste cell responses to phenylthiourea were inhibited by probenecid. These results suggest some bitter antagonists of human TAS2Rs can work for bitter sense of mouse., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The effects of glucagon like peptide-1 (GLP-1) on reflex swallowing were examined using anaesthetized rats. GLP-1 was injected into the dorsal vagal complex (DVC) using glass micropipettes. Swallowing was induced by repeated electrical stimulation of the central cut end of the superior laryngeal nerve (SLN) and was identified by the electromyogram lead penetrated in the mylohyoide muscle through bipolar electrodes. Microinjection of GLP-1 into the medial DVC (M-DVC) increased the frequency of swallowing during the electrical stimulation of the SLN and extended the latency of the first swallowing. Microinjection of GLP-1 into the lateral DVC (L-DVC) did not change the frequency of swallowing or the latency of the first swallowing. Neither the injection of vehicle into the M-DVC nor L-DVC affected swallowing frequency. Pre-injection of exendin (5-39), a GLP-1 receptor antagonist, attenuated the degree of suppression of swallowing frequency induced by the administration of GLP-1 in addition to shortening the latency of the first swallowing. To identify the effective site of GLP-1, lesion experiments were performed. Electrical lesion of the commissural part of the NTS (cNTS) and the vacuum removal of the area postrema (AP) did not affect the inhibition of reflex swallowing induced by the injection of GLP-1 into the M-DVC. Electrical lesion of the medial nucleus of the NTS (mNTS) and its vicinity abolished the inhibitory effects of swallowing induced by the injection of GLP-1. These results suggest that GLP-1 inhibits reflex swallowing via the mNTS in the dorsal medulla., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Aflatoxin (AFT) contamination is frequent in foods grown in tropical regions, including rice. Although AFTs are generally not found in temperate-region foods, global warming has affected typical temperate-region climates, potentially permitting the contamination of foods with AFT-producing Aspergillus flavus (A. flavus). Here we investigated the AFT production in rice during storage under natural climate conditions in Japan. We examined AFTs in brown rice and rough rice artificially contaminated with A. flavus for 1 year in Japan, and we subjected AFTs in white rice to the same treatment in airtight containers and examined the samples in warm and cold seasons, simulating the storage of white rice in general households. In the brown rice, AFTs increased after 2 months (March) and peaked after 9 months (October). The AFT contamination in the rough rice was minimal. After the polishing and cooking of the brown rice, AFTs were undetectable. In the white rice stored in airtight containers, AFTs increased after 1 month (August) and peaked after 2 months (September). Minimal AFTs were detected in the cold season. Thus, AFT contamination in rice may occur in temperate regions following A. flavus contamination. The storage of rice as rough rice could provide be useful for avoiding AFT contamination.

To evaluate the role of the lateral hypothalamic area (LH) in the masticatory-salivary reflex, we investigated submandibular salivary secretion and the electromyographic (EMG) activity of the jaw-closer masseter muscle in sham-operated rats and rats with unilateral LH lesions. One week prior to surgery and recording, the rats were given daily experience of eating pellets; powder; or hard, medium or soft mash, all of which were composed of laboratory chow. Salivary secretion was induced during eating and grooming behavior. During eating, the powdered food induced the highest salivary flow rate, and the soft (wet) mash induced the lowest salivary flow rate. Conversely, the amount of food consumed (dry weight) was greatest when soft mash was provided and lowest when the powder or pellets (a dry diet) were provided. The EMG activity of the masseter muscle during eating was greatest during consumption of the pellets and weakest during consumption of the powder. LH lesions that were ipsilateral to the examined submandibular gland reduced salivary secretion to about 20-30% of the control value, whereas contralateral LH lesions reduced it to about 40-50% of the control value. Neither masseter muscle EMG activity nor food consumption was markedly affected by the presence of an LH lesion. These results suggest that the texture of food, especially its water content, affects the flow rate of saliva and that the LH is heavily involved in the masticatory-salivary reflex., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We examined the effects of orexins on the reflex swallowing using anesthetized rats. Orexins were administered into the fourth ventricle. Swallowing was induced by repeated electrical stimulation of the central cut end of the superior laryngeal nerve (SLN) and was identified by the electromyogram lead penetrated the mylohyoid muscle through bipolar electrodes. The frequency of swallowing during the electrical stimulation of the SLN decreased after the administration of orexin-A in a dose-dependent manner. The latency of the first swallowing tended to be extended after the administration of orexin-A. The administration of orexin-B did not affect swallowing frequency. Pre-administration of SB334867, an orexin-1 receptor antagonist, attenuated the degree of inhibition of swallowing frequency induced by the administration of orexin-A. To identify the effective site of orexin-A, the effect of a microinjection of orexin-A into the dorsal vagal complex (DVC) was evaluated. Orexin-A was injected into one of the lateral DVC, the intermediate DVC, or the medial DVC. Microinjection of orexin-A into the medial DVC but not the other two sites decreased swallowing frequency. Pre-injection of SB334867 into the medial DVC disrupted the inhibitory response induced by fourth ventricular administration of orexin-A. The electrical lesion of the commissural part of the NTS, but not ablation of the AP, abolished the inhibition of reflex swallowing induced by fourth ventricular administration of orexin-A. These results suggest that orexin-A inhibits reflex swallowing via orexin-1 receptors situated in the commissural part of the NTS and/or its vicinity., (Copyright © 2014 Elsevier Inc. All rights reserved.)

To evaluate the role of the masticatory area in the cerebral cortex in the masticatory-salivary reflex, we investigated submandibular salivary secretion, jaw-movement trajectory and electromyographic activity of the jaw-opener (digastric) and jaw-closer (masseter) muscles evoked by repetitive electrical stimulation of the cortical masticatory area in anesthetized rats. Rats have two cortical masticatory areas: the anterior area (A-area) in the orofacial motor cortex, and the posterior area (P-area) in the insular cortex. Our defined P-area extended more caudally than the previous reported one. P-area stimulation induced vigorous salivary secretion (about 20 µl/min) and rhythmical jaw movements (3-4 Hz) resembling masticatory movements. Salivary flow persisted even after minimizing jaw movements by curarization. A-area stimulation induced small and fast rhythmical jaw movements (6-8 Hz) resembling licking of solutions, but not salivary secretion. These findings suggest that P-area controls salivary secretion as well as mastication, and may be involved in the masticatory-salivary reflex., (© 2013 Published by Elsevier B.V.)

The superior salivatory nucleus (SSN) contains preganglionic parasympathetic neurons to the submandibular and sublingual salivary glands. Cevimeline, a muscarinic acetylcholine receptor agonist, stimulates the salivary glands and is presently used as sialogogue in the treatment of dry mouth. Since cevimeline passes through the blood-brain barrier, it is also able to act on muscarinic acetylcholine receptors in the central nervous system. Our preliminary experiment using the whole-cell patch-clamp technique has shown that cevimeline excites SSN neurons in rat brain slices, suggesting that SSN neurons have muscarinic acetylcholine receptors; however, it is unclear which subtypes of muscarinic acetylcholine receptors exist in SSN neurons. In the present study, we investigated immunohistochemically muscarinic acetylcholine receptor subtypes, M1 receptor (M1R), M2R, M3R, M4R, and M5R in SSN neurons. SSN neurons innervating the salivary glands, retrogradely labeled with a fluorescent tracer from the chorda-lingual nerve, mostly expressed M3R immunoreactivity (-ir) (92.3%) but not M1R-ir. About half of such SSN neurons also showed M2R- (40.1%), M4R- (54.0%) and M5R-ir (46.0%); therefore, it is probable that SSN neurons co-express M3R-ir with at least two of the other muscarinic receptor subtypes. This is the first report to show that SSN neurons contain muscarinic acetylcholine receptors., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The effect of ghrelin on rhythmic reflex swallowing was examined in urethane-chloralose anesthetized rats. Swallowing was monitored by recording electromyographic activities of the suprahyoid muscle. Fourth ventricular administration of ghrelin decreased swallowing frequency during electrical stimulation of the central cut end of the superior laryngeal nerve (SLN stimulation). A significant decrease in swallowing frequency was observed after ghrelin administration at doses of 3, 10, 30 and 100 pmol. The administration of ghrelin with growth hormone secretagogue receptor antagonist ([D-Lys(3)] GHRP-6) did not change swallowing frequency during SLN stimulation. Neither mean blood pressure nor heart rate changed after the administration of 10 pmol ghrelin. Bilateral vagotomy did not disrupt the ghrelin response. These observations indicate that the ghrelin response does not depend on either cardiovascular or abdominal responses. Microinjection of ghrelin (0.3 pmol) into the vicinity of the solitary tract inhibited swallowing induced by SLN stimulation. Fourth ventricular administration of orexin-A (3 nmol) also inhibited reflex swallowing elicited by SLN stimulation. These results suggest that ghrelin and other orexigenic peptides inhibit reflex swallowing by modifying neural activities of the dorsal medulla where the swallowing center is housed., (Copyright 2009 Elsevier B.V. All rights reserved.)

The effects of fourth ventricular administration of ghrelin on motility of the proximal stomach were examined in anesthetized rats. Intragastric pressure (IGP) was measured using a balloon situated in the proximal part of the stomach. Administration of ghrelin into the fourth ventricle induced relaxation of the proximal stomach in a dose-dependent manner. Significant reduction of IGP was observed at doses of 3, 10, or 30 pmol. The administration of ghrelin (10 or 30 pmol) with growth hormone secretagogue receptor (GHS-R) antagonist ([D-Lys3] GHRP-6; 1 nmol) into the fourth ventricle did not induce a significant change in IGP. The sole administration of [D-Lys3] GHRP-6 also did not induce a significant change in IGP. Bilateral sectioning of the vagi at the cervical level abolished the relaxation induced by the administration of ghrelin (10 or 30 pmol) into the fourth ventricle, suggesting that relaxation induced by ghrelin is mediated by vagal preganglionic neurons. Microinjections of ghrelin (200 fmol) into the caudal part of the dorsal vagal complex (DVC) induced obvious relaxation of the proximal stomach. Similar injections into the intermediate part of the DVC did not induce significant change. Dose-response analyses revealed that the microinjection of 2 fmol of ghrelin into the caudal DVC significantly reduced IGP. These results revealed that ghrelin induced relaxation in the proximal stomach via GHS-R situated in the caudal DVC.

Cevimeline, a therapeutic drug for xerostomia, is an agonist of muscarinic acetylcholine receptors (mAChRs), and directly stimulates the peripheral mAChRs of the salivary glands. Since cevimeline is distributed in the brain after its oral administration, it is possible that it affects the central nervous system. However, it is unknown how cevimeline affects the superior salivatory (SS) neurons, which control submandibular salivation. In the present study, we examined the effects of cevimeline on the SS neurons using the whole-cell patch-clamp technique in brain slices. In Wistar rats (6-10 days), the SS neurons were retrogradely labeled by Texas Red applied to the chorda-lingual nerve. Two days after injection, whole-cell recordings were obtained from the labeled cells, and miniature excitatory postsynaptic currents (mEPSCs) were examined. Cevimeline induced the inward currents dose-dependently and increased the frequency of mEPSCs. Therefore, it is suggested that cevimeline enhances the excitability via post- and presynaptic muscarinic receptors in the rat SS neurons. In conclusion, cevimeline may enhance the excitability of the SS neurons.

The primary parasympathetic center of the submandibular and sublingual salivary glands is the superior salivatory (SS) nucleus, and its neurons receive excitatory (glutamatergic) and inhibitory (GABAergic and glycinergic) synaptic transmissions in rats. In the present study, we focused on the postnatal development of inhibitory transmission to SS neurons. Gramicidin-perforated whole-cell patch-clamp recordings were performed in rat brainstem slices on postnatal day 2 (P2)-P14. Developmental changes in the intracellular Cl(-) concentration ([Cl(-)](in)) were examined based on the reversal potentials of total inhibitory postsynaptic currents (GABAergic plus glycinergic), which were evoked by electrical stimulation near the recording neuron. The [Cl(-)](in) in the P8-P14 group was significantly lower than in the P2-P7 group. The effect of GABA application at the resting potentials changed from depolarization to hyperpolarization around P8, suggesting that SS neurons acquired mature inhibitory systems around P8. The period at which GABA responses change from excitatory to inhibitory in SS neurons was discussed compared with those of the forebrain, brainstem, and spinal neurons.

The superior salivatory nucleus (SSN) is the primary parasympathetic center controlling submandibular salivatory secretion. Our previous electrophysiological study revealed that many SSN neurons receive GABAergic and glycinergic synaptic inputs. In the present study, we examined the distribution of GABAergic and glycinergic nerve terminals, GABA(A) receptors in the SSN, and the origin of GABAergic nerve terminals innervating the SSN. Glutamic acid decarboxylase (GAD) and glycine transporter 2 (GLYT2) were used as markers of GABAergic and glycinergic nerve terminals, respectively. GAD- and GLYT2-positive nerve terminals and GABA(A) receptors were examined immunohistochemically in SSN neurons labeled by the retrograde axonal transport of FastBlue (FB) injected into the chorda-lingual nerve. The SSN neurons abundantly contained GAD-positive nerve terminals and GABA(A) receptors, suggesting that SSN neurons undergo strong GABAergic inhibition. The origin of GABAergic terminals was examined in neurons labeled by the retrograde transport of FluoroGold (FG) injected into the SSN. GAD was used as a marker of GABAergic neurons. Numerous FG-labeled neurons were found in the forebrain and brainstem. However, in FG-labeled neurons, GAD-positive neurons were occasionally observed in the reticular formation of the brainstem. These findings suggest that SSN neurons mainly receive GABAergic projections from the reticular formation.

The primary parasympathetic center of the submandibular and sublingual salivary glands is the superior salivatory (SS) nucleus, neurons of which receive excitatory (glutamatergic) and inhibitory (GABAergic and glycinergic) synaptic transmissions in rats. In the present study, to examine postnatal neural development, we focused on inhibitory transmission to the SS neurons in neonatal rats from postnatal day 2 (P2) to P14. Conventional and gramicidin-perforated whole-cell patch-clamp techniques were applied to the neurons in brainstem slices. The decay time constants of GABAergic and glycinergic postsynaptic currents (PSCs) consisted of fast (tau(fast)) and slow (tau(slow)) components. Both tau(fast) and tau(slow) of PSC components tended to become faster with development. The equilibrium potential of Cl(-) (E(Cl-)) was estimated from the reversal potentials of total PSCs (GABAergic plus glycinergic). The E(Cl-) in the P8-P14 group was significantly more negative than E(Cl-) in the P2-P7 group. Exogenous GABA application at the resting potentials produced depolarization in 83% of SS neurons at P2-P7 and accompanied the action potential in some neurons. In contrast, at P8-P14, GABA evoked hyperpolarization in 78% of SS neurons; therefore, SS neurons did not acquire mature inhibitory systems until P14. The development of SS neurons is discussed as compared with the development of peripheral salivary gland tissue and brainstem neurons that participate in oral motor and sensory functions.

ATP has been shown to excite neurons in various regions of the central nervous system. Whereas immunohistochemical studies show P2X receptors in the area postrema, the responsiveness of area postrema neurons to extracellular ATP has not been studied. To investigate the effects of purinoceptor activation on area postrema neuronal excitability, we performed whole-cell recordings from area postrema neurons in rat brain slices. Most area postrema neurons responded to ATP application, and most responses were excitatory. Voltage-clamp recordings showed three different types of response: (1) a postsynaptic or extrasynaptic excitatory response (inward currents; n=26/51 cells), (2) a presynaptic excitatory response (increased frequency of miniature excitatory postsynaptic currents with only a small direct postsynaptic current; n=24/51 cells, or (3) a postsynaptic inhibitory response (outward current; n=1/51). The excitatory responses were found in both of the two major electrophysiological cell classes, i.e. cells displaying I(h) and cells not displaying I(h), while the inhibitory responses were found in only cells not displaying I(h). Current-clamp recordings showed ATP-induced depolarization (n=13/15) or hyperpolarization (n=2/15) of membrane potential that modulated the frequency of action potentials. In the presence of CNQX, mEPSCs were abolished and bath-applied ATP did not generate mEPSCs, indicating that glutamate release was facilitated by the activation of presynaptically located ATP receptors. Our pharmacological results from studies with ATP, alphabetame-ATP, betame-ATP and PPADS indicate that the post- and/or extrasynaptic responses are most likely mediated by P2X(7) receptors and/or receptors composed of P2X(2) and P2X(5) subunits. We conclude that half of the presynaptic responses are most likely mediated by P2X(7) receptors and/or receptors composed of P2X(2) and P2X(5) subunits while the others also contain P2X(1) subunits. It is well known that P2X(7) subunit forms only homomultimeric P2X receptors. Finally, the present study suggests that purinoceptor activation may contribute to the control of several autonomic functions by area postrema neurons.

Effects of neuropeptide Y (NPY) on motility of the proximal stomach was examined in anesthetized rats. Intragastric pressure was measured using a balloon situated in the proximal part of the stomach. Administration of NPY into the fourth ventricle induced relaxation of the proximal stomach in a dose-dependent manner. Administration of an Y1 receptor (Y1R) agonist [Leu31, Pro34]NPY induced a larger relaxation than NPY. The administration of an Y2 receptor agonist (NPY 13-36) did not induce significant changes in motility. Microinjections of [Leu31, Pro34]NPY into the caudal part of the dorsal vagal complex (DVC) induced relaxation of the proximal stomach. In contrast, similar injections into the intermediate part of the DVC increased IGP of the proximal stomach. Administration of NPY into the fourth ventricle did not induce relaxation after bilateral injections of the Y1R antagonist (1229U91) into the caudal DVC. These results indicate that NPY induces relaxation in the proximal stomach via Y1Rs situated in the DVC. Because bilateral vagotomy below the diaphragm abolished the relaxation induced by the administration of NPY into the fourth ventricle, relaxation induced by NPY is probably mediated by vagal preganglionic neurons. Intravenous injection of atropine methyl nitrate reduced relaxation induced by administration of NPY. Therefore, relaxation induced by NPY is likely mediated by peripheral cholinergic neurons.

Whole-cell recordings were performed to examine the morphological properties of electrophysiologically classified area postrema (AP) neurons in rat brain slices. Using electrophysiological criteria, AP neurons were subdivided into three groups: (1) cells displaying both the hyperpolarization-activated cation current (I(h)) and the fast transient outward current (fast I(to)); (2) cells displaying only the fast I(to); (3) cells displaying only the slow I(to). All AP neurons had a single axon that was distinctly thinner than the cells' dendrites. No systematic differences, across groups, in the orientation of dendrites or axons were identified. Mean values of cell size and capacitance of neurons from group 3 were significantly larger than those of the other groups. Interestingly, a number of cells from groups 1 and 3 but not group 2 were found to extend their dendrites into the nucleus tractus solitarius (NTS), suggesting that AP neurons could receive vagal afferent inputs at their dendritic termini within the NTS. Although the AP has been implicated to contain uniformly shaped neurons, this study indicates the presence of significantly different subpopulations of AP neurons, which were characterized not only electrophysiologically but also morphologically.

The origin of sensory nerves that innervate the submandibular salivary gland was investigated in the rat. After application of wheat germ agglutinin-horseradish peroxidase to the cut endings of the sympathetic and parasympathetic nerve branches at the hilus of the gland, labeled cells were mainly found in the dorsal root ganglia and the trigeminal ganglion, respectively. The labeled neurons in these ganglia were of various sizes compared to unlabeled neurons, suggesting that the sensory nerves of the gland conduct various modalities of sensory information.

We investigated the functions of nicotinic receptor activation on area postrema neurons by making whole-cell recordings in rat brainstem slices. Excitatory responses to nicotine application were found in approximately 78% (35/45) of all cells tested. Responsive cells included both the cells that display the hyperpolarization-activated cation current (I(h)) and cells that do not display I(h). An inhibitory effect of nicotine was never seen. Current-clamp recordings showed the nicotine-induced depolarization of a cell's membrane potential that could be sufficient to cause spontaneous firing. In voltage-clamp recordings, many cells showed nicotine-induced inward currents (18.3+/-3.2 pA, n=6) that persisted during pharmacological blockade of synaptic transmission (e.g., zero [Ca(2+)](out) and 5 mM [Mg(2+)](out), n=6/8). Other two cells, however, showed increases in the frequency of excitatory postsynaptic currents (EPSCs), which were blocked by CNQX (n=2/8). We analyzed miniature EPSCs (mEPSCs) recorded from cells that showed no inward currents but marked increases in the frequency of mEPSCs (0.8+/-0.2 to 4.8+/-1.7 Hz, n=4) during nicotine application. Nicotine augmented mEPSC amplitude (n=4); however, amplitude distribution was not significantly changed in two of four cells tested. We conclude that nicotinic receptors in the rat area postrema can excite cells via (1) a direct post- and/or extrasynaptic mechanism; and (2) an indirect enhancement of glutamate release.

Area postrema neurons mediate various autonomic responses, including emesis. We examined the effects of propofol, a widely used anesthetic with antiemetic properties, on the hyperpolarization-activated cation current (Ih) in rat area postrema neurons using a slice patch-clamp technique. Although propofol suppressed Ih of area postrema neurons in a dose-dependent manner that was similar to what we observed for the hippocampal CA1 neurons, the IC50 for Ih in area postrema neurons (38 microM) was more than six times less than that found for hippocampal CA1 neurons (235 microM). We conclude that rat area postrema neurons are exquisitely sensitive to propofol. Given that reductions of Ih are associated with decreased excitability in neurons, we believe that the known antiemetic effects of propofol anesthesia are at least partly a result of a direct action on area postrema neurons to lower their excitability.

The excitatory and inhibitory synaptic inputs to parasympathetic preganglionic neurons in the superior salivatory (SS) nucleus were investigated in brain slices of neonatal (4-8 days old) rat using the whole-cell patch-clamp technique. The SS neurons innervating the submandibular and sublingual salivary glands and innervating the lingual artery in the anterior region of the tongue were identified by retrograde transport of a fluorescent tracer. Whole-cell currents were evoked by electrical stimulation of tissue surrounding the cell. These evoked postsynaptic currents were completely abolished by antagonists for N-methyl-D-aspartate (NMDA) glutamate, non-NMDA glutamate, gamma-aminobutyric acid type A (GABAA), and glycine receptors, suggesting that SS neurons receive glutamatergic excitatory, and GABAergic and glycinergic inhibitory synaptic inputs. In SS neurons for the salivary glands, the ratio of the NMDA component to the total excitatory postsynaptic current (EPSC) was larger than that of the non-NMDA component. This profile was reversed in the SS neurons for the tongue. In SS neurons for the salivary glands, the ratio of the GABAA component to the total IPSC was larger than the ratio of the glycine component to total inhibitory postsynaptic current (IPSC). The decay time constants of the GABAA component were slower than those for glycine. These characteristics of the excitatory and inhibitory inputs may be involved in determining the firing properties of the SS neurons innervating the salivary glands and the tongue.

To clarify the functional properties of the hyperpolarization-activated cation current (Ih) as a pacemaker current in area postrema neurons, whole-cell recordings were made in visually identified cells in rat brain slices. The activation of Ih was identified in approximately 62 % of area postrema neurons tested. The cells displaying Ih showed a depolarizing "sag" in responses to hyperpolarizing current injection in current-clamp mode. The reversal potential for the Ih was -36 mV, and this was shown to depend on the external concentration of Na+ and K+ ions. Extracellular Cs+ ions (2 mM) and ZD7288 (100 microM), a potent selective Ih channel antagonist, blocked Ih and induced a membrane potential hyperpolarization, suggesting the sustained activation of Ih near the resting potential and a contribution from Ih to membrane potential maintenance at more depolarized levels. In contrast, extracellular Ba2+ ions caused a depolarization of the membrane potential, suggesting the blockade of inward rectifier K+ currents. ZD7288 decreased the spontaneous discharge rate by prolonging the slow depolarization between two spikes, with minimal effect on the amplitude of the afterhyperpolarization or action potential waveforms. Ih stabilized the latency of rebound action potentials. Ih was weakly activated by external 8-bromoadenosine 3',5' cyclic monophosphate (1 mM) or forskolin (50-100 microM), indicating that the Ih channel subtypes in area postrema cells could be modulated by intracellular cAMP. Our findings indicate that Ih contributes to the subthreshold membrane and firing properties of rat area postrema neurons and may regulate their resting membrane potential and firing patterns.

Intracellular and field potential recordings were taken from the hippocampal CA1 and CA3 neurons in rat brain slices to investigate the effects of 2,6 di-isopropylphenol (propofol) on the neuronal excitability during GABA(A)-C1 channel blockade by picrotoxin (100 microM). Propofol produced a membrane hyperpolarization and an inhibition of the magnitude of the 'voltage sag' that was mediated by the activation of a hyperpolarization-activated inward current (I(H)). Propofol (>100 microM) decreased the spontaneous discharge rate of epileptiform burst responses in CA1 neurons up to 38+/-6% of the control level. Propofol also markedly reduced the duration of both spontaneous and evoked epileptiform burst responses. A propofol-induced decrease in the spontaneous discharge rate in CA3 neurons was coincident with that in CA1 neurons. The effects of propofol on the membrane potential and spontaneous discharge rate but not on the duration of burst responses were duplicated by ZD7288 (potent selective antagonist for I(H) channels), indicating that the blockade of I(H) significantly contributes to reduction of cell's excitability. The present study suggests that various actions including suppressive effects on I(H) contribute to the anesthetic and anti-convulsant properties of propofol.

Nonadrenergic noncholinergic (NANC) nerves are known to be nitrergic and to have an important role in the regulation of gastrointestinal motility and function. Cardiac antiarrhythmic therapy in humans is accompanied by a high incidence of gastrointestinal side-effects. We investigated the effect of mexiletine, a class Ib antiarrhythmic drug, on NANC lower oesophageal sphincter relaxation. Mexiletine concentration dependently inhibited the NANC relaxation induced by 30 mM KCl (EC(50)=4.4 x 10(-6) M); the production of 3',5'-cyclic guanosine monophosphate (cGMP) after KCl stimulation was concentration dependently decreased. The relaxation induced by the exogenous nitric oxide (NO) donor, diethylamine NONOate (10(-5) M), was not inhibited by mexiletine, and the cGMP production after diethylamine NONOate application was not altered. Mexiletine did not alter the activity of NO synthase. These findings suggest that mexiletine inhibits NANC relaxation via NO-cGMP pathway modulation, possibly by inhibiting myenteric nitrergic neurotransmission in the lower oesophageal sphincter in rabbits.

We found coexistence of the transient outward potassium current (I(TO)) and the hyperpolarization-activated inward current (I(H)) in 26 of 82 area postrema neurons tested using the whole-cell patch-clamp technique in rat brain slices. Cells displaying both the I(TO) and the I(H) typically showed "voltage sag" and "rebound potentials" in response to hyperpolarizing current injection and repetitive firing with strong adaptation was seen with depolarizing current injection. When cells were held at membrane potentials more negative than the resting level (e.g., -85mV), the afterhyperpolarization was enhanced. Voltage clamp recordings were performed to examine the characteristics of I(TO) and I(H) in and the contributions of these currents to the electroresponsiveness of area postrema cells. We show, in this study, the voltage-dependent properties of I(H) and I(TO), and how these currents modulate the intrinsic membrane properties of area postrema cells. We discuss the functional significance of the specific subset of area postrema neurons whose cells have both I(H) and I(TO) channels.

We investigated the electrophysiological properties of the area postrema neurons in acutely prepared rat brain slices using the whole-cell patch-clamp technique. Two different types of transient outward potassium current (I(to)), fast and slow, were found in the area postrema. Both the decay time constant and rise time were significantly faster in the fast I(to) than in the slow I(to). Both current-clamp and voltage-clamp recordings revealed that the activation of fast and slow I(to) contributes to generation of the different spiking patterns, late spiking and interrupted spiking, respectively. The activation and inactivation of both I(to) were strongly voltage-dependent. Curve fitting by the Boltzmann equation revealed no significant difference in the activation and inactivation curves for each I(to) except that the slope factor of inactivation was larger for fast I(to). Both I(to) were suppressed dose-dependently by application of 4-aminopyridine. Each spiking pattern was enhanced when cells were held at a more hyperpolarized membrane potential, i.e. a longer latency of the first spike or longer interspike interval between the first and second spikes. The voltage-dependent modulation of the spiking pattern was consistent with the voltage-dependent activation of I(to). The present study shows significant subdivisions of the area postrema neurons distinguished by a difference in the kinetics of I(to) and spiking patterns. We discuss the role of I(to) as the ionic current underlying neuronal excitability.

In frogs, the glossopharyngeal nerve (GL) innervates taste receptors on almost the entire tongue. The mandibular branch (MBF) and palatine branch (PN) of the facial nerve innervate taste receptors on a very small area at the base of the tongue and on the palate, respectively. In the present study, effects of amiloride, an epithelial sodium channel blocker, on the tonic responses of the GL, MBF and PN in frogs to NaCl, LiCl, KCl and CaCl(2) were investigated. In three nerves, amiloride at 0.5 mM, a relatively high concentration, did not affect the responses to 0.15 (concentration just above threshold)-0.5 M NaCl, 0.5 M LiCl and 0.3 M KCl, whereas it almost completely inhibited the response to 1.0 mM CaCl(2). Amiloride may exert an inhibitory action on the response to CaCl(2) by a competitive antagonism between Ca(2+) and a monovalent cation of amiloride, because the response to Ca(2+) is competitively inhibited by other cations such as Na(+) and Mg(2+). The lack of inhibitory effect of amiloride on the responses in the GL, MBF and PN to NaCl suggests that amiloride-sensitive sodium channels in the apical membrane of taste receptor cells are not involved in sodium taste transduction in frogs.

Background: Nonadrenergic noncholinergic (NANC) nerves have important roles in the regulation of the lower esophageal sphincter (LES) motility and function. The effects of thiopental, ketamine, and midazolam on NANC LES relaxation were investigated., Methods: The isometric tension of circular muscle strips from Japanese White rabbits was examined. The NANC relaxation was induced by KCl (30 mM) in the presence of atropine (3 x 10(-6) M) and guanethidine (3 x 10(-6) M). The modifications of the NANC and sodium nitroprusside (SNP; 10(-5) M)-induced relaxation by the anesthetics were examined. The content of 3',5'-cyclic guanosine monophosphate (cGMP) was measured by radioimmunoassay., Results: The KCl-induced relaxation was abolished by pretreating with tetrodotoxin (10(-6) M). The NANC relaxation was inhibited in the presence of N(G)-nitro-L-arginine (L-NNA; 3 x 10(-5) M), methylene blue (10(-6) M), apamin (10(-7) M), and glibenclamide (10(-5) M). The SNP-induced relaxation was inhibited by methylene blue but was not affected by tetrodotoxin, L-NNA, apamin, or glibenclamide. Ketamine (EC50 = 8.8 x 10(-5) M) and midazolam (EC50 = 4.8 x 10(-6) M) suppressed the NANC response in a concentration-dependent manner, leaving SNP-induced response unchanged. Thiopental altered neither of the relaxations. cGMP content was decreased in the presence of ketamine and midazolam., Conclusion: The NANC relaxation was mediated by nitric oxide and by low-conductance calcium- and adenosine triphosphate-sensitive potassium channels of smooth muscle. The modulation of the nitric oxide-cGMP pathway was related, at least in part, to the inhibitory actions of ketamine and midazolam on the NANC LES relaxation.

Ever since the development of human monoclonal antibody CLN-IgG in 1982, we anticipated the identification of the antigen that is recognized by this antibody. Despite its scarce expression on the cell surface, susceptibility to proteolytic enzymes and adherence to experimental equipment, we finally succeeded in determining the antigen moiety that is recognized by this antibody by means of CLN-IgG conjugated column affinity chromatography, two-dimensional electrophoresis, MALDI-TOF/MS and use of glioblastoma cell line U-251MG. The antigen was found to be vimentin, a cytoskeletal protein, and we succeeded in determining a 79 amino acids sequence of the epitope which turned out to comprise a part of the c2 (coil 2 of the central rod) domain of vimentin.

Metal bite-raising splints of 0.5 mm thickness were attached to the upper molar teeth on both sides of the jaw in rabbits. The effects of these splints on masticatory behaviour during the chewing of soft food (bread) by freely moving rabbits were investigated. We recorded electromyograms (EMGs) of the masseter and digastric muscles. The animals exhibited prolongation of the chewing cycle, decreased EMG activity of the masseter muscle and increased EMG activity of the digastric muscle during chewing after introduction of the bite-raising splints. The effects of the splints on the activities of masticatory muscles were abolished by bilateral sectioning of the maxillary and inferior alveolar nerves. It seems likely that afferents from oral sensory receptors were responsible for the changes in masticatory behaviour after the introduction of the occlusal splint.

Bioavailability of a newly-synthesized and chemically-stable 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G) as a vitamin C supplement was investigated in rats and guinea pigs. Oral administration of AA-2G to the animals resulted in an increase of serum ascorbic acid (AA) levels. However, in these sera the intact form was not detectable by the high performance liquid chromatography (HPLC) method, indicating its hydrolysis through the process of absorption. After an intravenous injection of AA-2G, the intact form diminished rapidly from the serum, followed by prolonged and marked elevation of serum AA levels. Various tissue homogenates from rats and guinea pigs were examined for their releasing activity of AA from AA-2G. High activity was observed in kidney, small intestine and serum of rats and in small intestine and kidney of guinea pigs. These hydrolytic activities were completely inhibited by castanospermine, a specific alpha-glucosidase inhibitor, suggesting the participation of alpha-glucosidase in the in vivo hydrolysis of AA-2G. AA-2G was found to exhibit obvious therapeutic effect in scorbutic guinea pigs by its repeated oral administrations. These results indicate that AA-2G is a readily available source of vitamin C activity in vivo.

A sensitive enzyme immunoassay for OPC-7251, a novel pyridone carboxylic acid antimicrobial agent, was developed and applied for the determination of human plasma levels. OPC-7251 was coupled to bovine serum albumin through a formation of N-hydroxysuccinimide ester. By immunization of rabbits, highly specific antiserum was raised. Using the antiserum and beta-D-galactosidase-labeled hapten, the homologous assay system allowed the detection of 2 pg of this compound. Plasma samples were precisely analyzed down to the minimum value of 200 pg/ml after heat treatment. The system was further validated by the recovery test and correlation with the HPLC analyses. Percutaneous application of 10 g of 1% OPC-7251 cream to healthy volunteers resulted in the peak plasma value of 1.6 ng/ml about 8 hours after dosing, indicating extremely low absorption efficiency through a transdermal system.