The Effects of Mutual Interaction of Orexin-A and Glucagon-Like Peptide-1 on Reflex Swallowing Induced by SLN Afferents in Rats.

(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.

Electrophysiological study on sensory nerve activity from the submandibular salivary gland in rats.

To evaluate the role of afferent information from the salivary gland, we analyzed the neural activity of the sensory nerve innervating the submandibular gland in anesthetized rats. The sensory nerves running through the parasympathetic nerve supply responded to mechanical pressure applied to the surface of the main duct and the body of the gland, whilst those in the sympathetic nerve supply responded only to the body of the gland. The sensory nerves in the sympathetic and parasympathetic nerve routes responded to pressure in the duct system produced by a retrograde injection of saline into the main duct. The threshold pressure for production of afferent discharges was higher than the maximum secretory pressure evoked by electrical stimulation of the parasympathetic secretory nerve. The retrograde ductal injection of drugs related to the inflammatory process (capsaicin and bradykinin) evoked intense multi-unit discharges in the sensory nerves of both routes. The sensory nerve in the sympathetic route was responsive to ligation of the artery to the gland. These results suggest that sensory nerves in the sympathetic and parasympathetic routes mainly conduct noxious information, and that those in the sympathetic route are responsive to ischemia and may control blood flow of the gland.

Central glucagon like peptide-1 inhibits reflex swallowing elicited by the superior laryngeal nerve via caudal brainstem in the rat.

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.

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

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.

Role of the lateral hypothalamus in submandibular salivary secretion during feeding in rats.

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.

Central orexin inhibits reflex swallowing elicited by the superior laryngeal nerve via caudal brainstem in the rat.

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.

Differential involvement of two cortical masticatory areas in submandibular salivary secretion in rats.

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.

Muscarinic receptor immunoreactivity in the superior salivatory nucleus neurons innervating the salivary glands of the rat.

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.

Central ghrelin inhibits reflex swallowing elicited by activation of the superior laryngeal nerve in the rat.

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.

Immunohistochemical study on the distribution and origin of GABAergic nerve terminals in the superior salivatory nucleus.

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.

Postnatal development of inhibitory synaptic transmission to superior salivatory neurons in rats.

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.

Cevimeline enhances the excitability of rat superior salivatory neurons.

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.

Fourth ventricular administration of ghrelin induces relaxation of the proximal stomach in the rat.

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.

Development of inhibitory synaptic transmission to the superior salivatory nucleus in rats.

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.

Purinergic modulation of area postrema neuronal excitability in rat brain slices.

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.

Central neuropeptide Y induces proximal stomach relaxation via Y1 receptors in the dorsal vagal complex of the rat.

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.

Variety of morphological and electrophysiological properties of area postrema neurons in adult rat brain slices.

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.