Our previous studies showed that an arousal/yawning response was evoked by microinjection of several agents (L-glutamate, cyanide, histamine etc) into the paraventricular nucleus (PVN) in an anesthetized, spontaneously breathing rat. In this study we examined involvement of CRF neurons in the arousal/yawning response by double-immunohistochemistry against c-fos and CRF. The activating site within the brain during the arousal/yawning response was observed by c-fos expression. The experiment was applied to two groups of anesthetized rats. In the experimental group frequent yawns were induced by injecting L-glutamate into the PVN, while in the control group no injection was made. In the experimental group, c-fos/CRF double-labelled neurons were found in the PVN, especially in the dorsal area of the medial parvocellular subdivision and the rostral area of anterior parvocellular subdivision. We therefore conclude that CRF neurons in the PVN are involved in the arousal/yawning response. Hemispheric asymmetry of c-fos expression was also found in the cerebral cortex and the amygdala of the experimental group. These characteristics were not observed in the control group. [Jpn J Physiol 54 Suppl:S196 (2004)]

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We have previously demonstrated that both nNOS activity and its protein amount were increased specifically in the brainstem of hypertensive Dahl salt-sensitive (DS) rats compared to that of normotensive DS rats. Immunohistochemical study revealed that the number of nNOS containing neurons (nNOS neurons) in the brainstem was greater in hypertensive DS rats than in normotensive DS rats. In this study, we specified the localization of nNOS neurons in the hypothalamus although the overall nNOS activity in the diencepharons has found not to be significantly different between the two groups. The DS rats were divided into two groups: one fed 8% NaCl food (hypertensive DS rats) and the other fed 0.4% NaCl food (normotensive DS rats). Immunohistochemical study showed that nNOS neurons localized in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) within the diencephalons of both hypertensive and normotensive DS rats. The number of nNOS neurons in the SON was greater in hypertensive DS rats that in normotensive DS rats, while it was no significant difference in the number of nNOS neurons in the PVN between the two rat groups. These results indicated that the nNOS neuronal system in the diencephalons was partly amplified in hypertensive Dahl S rats. [Jpn J Physiol 54 Suppl:S196 (2004)]

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To investigate the role of lymphocytes and cytokines in neuritogenesis of sympathetic neurons induced by lymphoid tissues, we performed immunochemical studies in spleen of wild-type and C.B-17 SCID (T and B cell deficient) mice, using antibodies against tyrosine hydroxylase (TH) and nerve growth factor (NGF). In SCID mice, TH-positive fibers were significantly sparse in parenchymal fields of lymphocytes, but not altered around vasculature in white pulp, compared to wild-type mice. On the contrary, NGF-positive cells were found to be abundant in the lymphoid fields of SCID mice. Reconstitution with wild-type splenic lymphocytes significantly increased TH-positive fibers in the splenic lymphoid fields of SCID mice 3 weeks after the lymphocyte injection. ELISA showed that the splenic level of IL-1 was significantly more in SCID mice than wild-type mice. The IL-6 level was not significantly different between these mice. These studies suggest that lymphocytes are essential to promote neuritogenesis of sympathetic neurons in lymphoid tissues, which may be mediated by a cytokine different from NGF, IL-1 or IL-6. [Jpn J Physiol 54 Suppl:S196 (2004)]

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The rostral ventromedial medulla (RVMM) sympathetically regulates thermogenesis in brown adipose tissue (BAT) and constriction of skin blood vessels. However, the sympathetic-related neurons in the RVMM remain to be identified. First, we found that vesicular glutamate transporter 3 (VGLUT3) -immunoreactive neurons in the rostral raphe pallidus nucleus and raphe magnus nucleus expressed Fos in response to both fever-inducing stimulus (central injection of prostaglandin E₂) and cold-exposure (4°C, 4 hours) in rats. Most of the VGLUT3-expressing neurons were negative for serotonin immunoreactivity, and many of them were labeled trans-synaptically with pseudorabies virus inoculated into rat interscapular BAT or tail. Furthermore, we found that the VGLUT3-positive neurons directly project onto sympathetic preganglionic neurons in the intermediolateral cell column (IML) of the spinal cord; and that VGLUT3-positive axon terminals in the IML formed asymmetric synapses, suggesting the excitatory characteristics of the synapses. These results suggest that the rostral medullary raphe regions contain glutamatergic neurons that could be categorized as a novel group of sympathetic premotor neurons involved in fever induction and thermoregulation. [Jpn J Physiol 54 Suppl:S196 (2004)]

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It is well known that neuropeptide Y (NPY), a potent appetite enhancer, controls gastric motility. The administrations of NPY into the dorsal motor nucleus of the vagus facilitated antral motility via Y1 receptors. The intracerebroventricular injections of NPY delayed gastric emptying via Y2 receptors. Thus, NPY is involved in the central control of gastric motor functions. However, relationship between NPY and gastric reservoir functions has not been clarified. Our previous research revealed that intracisternal administrations of orexin-A facilitated distal stomach motility and relaxed the proximal stomach. Therefore, NPY may also induce relaxation of the proximal stomach to facilitate reservoir function. To demonstrate it, the effects of the intracisternal administration of NPY on the proximal stomach motility were examined in urethane-chloralose anaesthetized rats. The intracisternal administration of NPY (3 nmol) induced relaxation of the proximal stomach lasting for more than 30 min. The intragastric pressure decreased, dose-dependently. The administration of [Leu31, Pro34]NPY (a Y1 receptor agonist) induced relaxation more potent than NPY. The administration of NPY13-36 (a Y2 receptor agonist), did not induce any response in the proximal stomach. Thus, NPY induced relaxation of the proximal stomach via Y1 receptors in the CNS. [Jpn J Physiol 54 Suppl:S197 (2004)]

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Barrington's nucleus is a central component of the micturition circuit. Mainly two types of neurones have been identified: one type of neurones increases their firing during micturition contraction, and the other type decreases their firing during this phase (inverse neurones). The present study aims to elucidate a role of the inverse neurone in the regulation of the bladder contractility. [Methods] Single neurones in and around Barrington's nucleus were investigated with glass microelectrodes in anaesthetized cats. Projections of descending axons to the spinal cord were identified antidromically by stimulating the upper lumbar, mid lumbar, and/or lumbosacral levels. [Results] Inverse neurones displayed tonic firing when the bladder relaxes. The firing of most of the neurones began to decrease after the onset of micturition contraction. During micturition contraction, the majority of inverse neurones were completely silent, and the remaining neurones displayed weak tonic firing. When the bladder began to relax, firing rates increased again toward the previous steady level. But this firing increase occurred after the onset of the bladder relaxation. Most of the neurones were not activated antidromically from the spinal cord even when strong stimulus (more than 1 mA) was applied. In conclusion, the firing property (firing increase occurs after the onset of the bladder relaxation), as well as the scarceness of spinal cord-projecting neurones suggested that the inverse neurone in Barrington's nucleus does not trigger bladder relaxation. [Jpn J Physiol 54 Suppl:S197 (2004)]

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It has been reported in field of aromatherapy that fragrance of lavender reveals a sedative effect.This study deals with effects of olfactory stimulation with fragrance of lavender oil on autonomic nerve activity in anesthetized rats. Under urethane anesthesia, fragrance stimulation with lavender oil was given for 10 min. As the control, vapor of distilled water was applied for 10 min. The efferent nerve activity was recorded from sympathetic branch of WAT of epididymis, BAT, adrenal medulla, spleen and muscular sympathetics in the gastrocnemic region. Nerve activity was also recorded from gastric, thymic, pancreatic and celiac branch of vagus nerve. Results: The fragrance of lavenderfacilitated activities of vagal branches as well as muscular sympathetics, and inhibited sympathetic nerve activities in WAT, BAT, adrenal medulla and spleen. Stimulation of water vapor was without effect. These observations suggest that fragrance of lavender activates gastrointestinal function, increase in blood stream in muscular region, assilative body function and may improve immune function through activation of vagus nerve activity to thymus and suppression of sympathetic nerve activity to spleen. [Jpn J Physiol 54 Suppl:S197 (2004)]

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In previous study, we indicated that intraventricular administration of a dibenzoxazepine derived agent (DBZ-D) acts to enhance both the responses of regional cerebral blood flow and of the neurons in the nucleus basalis of Meynert (NbM) following electrical stimuli of the saphenous nerve. Glutamate (GLU) decarboxylase immunoreactivity is also identified in axonal terminals throughout the NbM neurons. In this study, therefore, it was examined if the direct application of DBZ-D may influence on the GLU-induced responses in the NbM neuron. Acetylcholine (Ach)-induced responses were also tested. All neurons examined (N=31) were antidromically identified from 18 Wistar male rat by cortical stimulation and also confirmed to be excited by electrical stimulation of the saphenous nerve under urethane anesthesia (1.1g/kg b.w.). Mean antidromic latency was 3.1+0.3 (1.2-10.1) msec.
GLU and Ach applied iontophoretically, had made an excitation of the firing discharge of all and 25 neurons tested, respectively. Simultaneous direct administration of DBZ-D enhanced the GLU-induced responses significantly (p<0.05), otherwise did not the Ach-induced responses at all.
These data suggest that 1) noxious stimuli cause an increased cortical blood flow via glutaminergic inputs and 2) that the intraventricular DBZ-D may act on the NbM neuron to modulate the glutaminergic synaptic inputs for increasing cortical blood flow following the neural stimulation in the rat. [Jpn J Physiol 54 Suppl:S197 (2004)]

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Motilin, a 22 amino acid peptide, is reported to be a key factor in controlling the interdigestive migrating contractions of the gastrointestinal tract. Erythromycin (EM) and its derivatives are known as nonpeptide motilin agonists. In fact, they induce gastrointestinal contractions similar to those observed during the interdigestive migrating contractions. Although their actions on either nerve tissues or gut smooth muscles are reported, mechanisms and sites of their actions have not been fully understood. The present electrophysiological experiments were made to examine actions of EM as well as motilin in the myenteric plexus of the guinea-pig ileum removed after 18 h-fasting. Intracellular recordings were made from myenteric neurons using glass microelectrodes, and focal electrical stimulation was given to evoke fast EPSPs characteristic of S neurons. EM and motilin (up to 1 μM) applied by superfusion caused membrane depolarizations in a proportion of S neurons and in a few AH neurons. EM inhibited fast EPSPs in amplitude; this inhibition of fast EPSPs was often accompanied with EM-induced depolarizations. When the EM depolarizations were eliminated manually by passing inward currents, fast EPSPs were confirmed to be inhibited in the presence of EM. It should be added that motilin also depressed fast EPSPs without changing postsynaptic nicotinic sensitivity. It was concluded that EM might inhibit synaptic transmission in the myenteric plexus possibly by acting presynaptically as a motilin agonist. [Jpn J Physiol 54 Suppl:S198 (2004)]

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The effect of corticotrophin-releasing factor (CRF) on oxytocin (OT) mRNA expressing neurons in the rat paraventricular nucleus (PVN) was examined using whole-cell patch-clamp recordings and single-cell reverse transcription-polymerase chain reaction (SC-RT-PCR) technique. Under current-clamp, CRF (10 nM to 600 nM) resulted in increased basal firing rate and depolarization while decreased the amplitude of the afterhyperpolarization (AHP) in a dose-dependent manner. CRF-induced depolarization was unaffected by co-perfusion with 0.5 μM tetrodotoxin (TTX) + 10 μM CNQX + 10 μM bicuculline. Under voltage-clamp, 100-600 nM CRF significant reduced the apamin-sensitive slow Ca²⁺-dependent K⁺ current (I_AHP), while increased the ZD-7288-sensitive hyperpolarization-activated cation current (I_H). Extracellular application of 1 μM α-helical CRF-(9-14) (α-h CRF), a selective CRF receptors antagonist, completely blocked the CRF-induced effects in both current- and voltage-clamp. And the SC-RT-PCR analysis indicated that all the OT mRNA-expressing neurons co-expressed CRF receptor 1 mRNA and CRF receptor 2 mRNA. These results suggest that OT mRNA expressing neurons co-express CRF receptors mRNA, and CRF combined to CRF receptors, depolarizes these neurons via inhibits I_AHP and enhances I_H channel activities. [Jpn J Physiol 54 Suppl:S198 (2004)]

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Neuropeptide W(NPW) is a novel hypothalamic peptide that activates the orphan G protein-coupled receptors,GPR7 and GPR8.Two endogenous molecular forms of NPW that consist of 23- and 30-amino acid residues were identified. Although intracerebroventricular (i.c.v) administration of NPW is known to suppress spontaneouse-feeding at dark phase and fasting-induced food intake and to decrease body weight and plasma growth hormone, but to increase prolactin and corticosterone, little is known about its effect on other physiological functions. We examined the effects of i.c.v. administration of NPW30(0.3 and 3nmol) on mean arterial pressure (MAP),heart rate (HR),and plasma norepinephrine and adrenaline in conscious rats. NPW30 (3nmol) provoked increases in MAP (85.12±3.16 to 106.26±2.66 mm Hg) and HR (305.75±13.76 to 428.45±26.82 beats/min) and plasma norepinephrine and adrenaline. Intravenously administered NPW30 (3nmol) had no significant effects on MAP and HR. These results indicate that central NPW30 regulates sympathetic nervous outflow and affects cardiovascular function. [Jpn J Physiol 54 Suppl:S198 (2004)]

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Hypotension is one of the most common complications in dental practice. Some stressors (eg, pain, anxiety, tension) are considered to be the cause. Trigeminal stimulation plays an important role and baroreflex seems to participate in this complication. The effect of electrical stimulation of both the aortic depressor nerve (ADN) and infraorbital nerve (ION) on blood pressure and renal sympathetic nervous activities (RSNA) was examined in anesthetized rabbits. Under uretane and α-chloralose anesthesia, animals were ventilated artificially. Arterial pressure was monitored from the femoral artery. Bipolar electrodes were placed to stimulate ADN and ION. Electrical stimulation of the ION was performed by square impulses with a duration of 0.25 ms. The frequencies of stimuli were 5 and 25 Hz, and the strengths were 10 V. Stimulation of the ADN was 0.25 ms in duration, 10 V in strength and 25 Hz in frequency. The renal sympathetic nerve was exposed and isolated. RSNA was recorded from an intact branch of the renal nerve placed on bipolar recording electrodes.RSNA decreased when blood pressure fell after stimulation of ADN and ION. Hypotension and decrease of RSNA evoked with simultaneous stimuli of the ADN and ION lasted grater than that evoked with individual stimulation of the ADN or ION. This result suggested that simultaneous stimulaion of aoritc and trigeminal nerve lowered blood pressure further and it contributed to the occurrence of hypotension in dental practice. [Jpn J Physiol 54 Suppl:S198 (2004)]

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The effect of stresscopin (SCP) on oxytocin (OT) mRNA expressing neurons in the rat paraventricular nucleus (PVN) was examined using whole-cell patch-clamp recordings and single-cell reverse transcription-polymerase chain reaction (SC-RT-PCR) technique. Under current-clamp, SCP (10 nM to 600 nM) resulted in decreased basal firing rate and hyperpolarization in a dose-dependent manner. SCP -induced hyperpolarization was unaffected by co-perfusion with 0.5 μM tetrodotoxin (TTX) + 10 μM CNQX + 10 μM bicuculline. Under voltage-clamp, 300 nM SCP increased the inwardly rectifying K⁺ current. Extracellular application of 200 μM Ba²⁺, a blocker of G protein-activated, inwardly rectifying potassium (GIRK) channels, completely blocked the effects of SCP on OT mRNA expressing neurons in voltage-clamp. Extracellular application of 1 μM α-helical CRF-(9-14) (α-h CRF), a corticotrophin-releasing factor (CRF) receptors antagonist, completely blocked the SCP-induced effects in both current- and voltage-clamp. And the SC-RT-PCR analysis indicated that all OT mRNA-expressing neurons co-expressed CRF receptor 1 and receptor 2 mRNA. These results suggest that OT neurons co-express CRF receptors, and SCP inhibits to OT neurons through activation of GIRK current activities. [Jpn J Physiol 54 Suppl:S199 (2004)]

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The functional significance of the sympathetic nerves is still unclear since no sympathetically mediated vasoconstrictor responses have been found to occur reflexly in orofacial tissues in response to a variety of stimuli. We studied the modulating effect of ongoing sympathetic activation on the vasoconstrictor response elicited by brief stimulation of the cervical superior sympathetic trunk (CST) and on that sometimes elicited by electrical stimulation of the peripheral end of the inferior alveolar nerve (IAN). The blood flow increase (antidromic vasodilatation) elicited by inferior alveolar nerve (IAN) stimulation was not affected by ongoing repetitive CST stimulation over the frequency range examined (0.2ms, 10 Hz), although reflex parasympathetic vasodilatation was attenuated. The vasoconstrictor responses elicited by IAN stimulation in some preparations were reduced in a frequency dependent manner (at 0.2ms, 1 Hz) during ongoing CST stimulation (and replaced by vasodilator responses). The vasoconstrictor response evoked directly by brief CST stimulation was attenuated, but not transformed to a vasodilator response, by ongoing CST stimulation. Thus in the cat lower lip 1) sympathetic stimulation attenuated one type of vasodilator response (parasympathetic-mediated vasodilatation), but not another (antidromic vasodilatation), and 2) ongoing sympathetic (CST) stimulation at low frequencies (<1 Hz) prevented further sympathetic-mediated vasoconstriction. [Jpn J Physiol 54 Suppl:S199 (2004)]

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Injection of acetylcholine into the subfornical organ (SFO) produces water intake and alterations in blood pressure. It has been suggested that the nicotinic activation of SFO neurons is related to the cadiovascular response while muscarinic activation is involved in drinking behavior as well as the cardiovascular response. If this is so, some of the neurons in the SFO should be preferentially sensitive to muscarine and others preferentially sensitive to nicotine. To investigate this possibility, we applied muscarine and nicotine to SFO neurons of rat brain slices and compared the responses using patch clamp recordings. 12 (46%) out of 21 neurons were depolarized more by muscarine than by nicotine while 9 neurons (35%) were depolarized more by nicotine than by muscarine at 10 μM in the whole-cell current-clamp mode. To elucidate the responsiveness of SFO neurons to the cholinergic agonists further, 30 neurons were tested in the whole-cell voltage-clamp mode. Muscarine and nicotine induced inward currents in the SFO neurons. At 10 μM, 23 (77%) of the 30 neurons tested were responsive to muscarine and/or nicotine. The amplitudes of the peak currents in response to muscarine were larger than those to nicotine in 13 (43%) and the responses to nicotine were greater than those to muscarine in 10 (33%). This suggests that preferential cholinergic activation through either muscarinic or nicotinic receptors in rat SFO expresses different participations in physiological functions such as drinking behavior and cadiovascular regulation. [Jpn J Physiol 54 Suppl:S199 (2004)]

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The area postrema (AP) is one of the circumventricular organs, which is located on the dorsal surface of the medulla oblongata at the caudal end of the forth ventricle. In our previous studies, we showed that AP neurons could be divided into three subpopulations, such as cells displaying the hyperpolarization-activated cation current (Ih), cells displaying the fast transient outward current (fast Ito) and cells displaying the slow transient outward current (slow Ito). In this study, we investigated morphological properties of each subpopulation of AP neurons. Slices containing cells recorded with Neurobiotin-containing electrodes were fixed in 4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer (pH 7.4) from overnight to 5 days. After rinses with phosphate-buffered saline (PBS, pH 7.4), these slices were treated with 0.1% H₂O₂ for 20 minutes and Triton-X100 (0.4 - 0.5% in PBS) for 2 to 3 hours. They were rinsed in PBS and then incubated in the Vectastain ABC Reagent (Vector Laboratories Inc. Burlingame, CA) in PBS for 2 to 3 hours. After rinses with PBS, slices were reacted with diaminobenzidine (DAB) and H₂O₂ (0.003%) in PBS to visualize the injected cells. The morphology of the successfully stained cells was identified on light microscopic observation. AP neurons were found to be small cell size, i.e. cell diameter around 10-15 μm. Cells displaying the slow Ito typically showed a larger diameter than other subpopulations. The difference in the cell size was consistent with the difference in the electrical capacitance. [Jpn J Physiol 54 Suppl:S199 (2004)]

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We have previously reported that intravenous (i.v.) administration and intracerebroventricular administration of clonidine, a lipophilic α₂-adrenoceptor agonist, significantly inhibited colonic motility in rats, but did not inhibit colonic motility in vitro. This inhibitory effect was significantly antagonized by pre-administraion of yohimbine, an α₂-adrenoceptor antagonist, but not by pre-administration of prazosin, an α₁-adrenoceptor antagonist. We presume that the mechanism of the inhibitory effect of clonidine on the colonic motility might involve in an indirect action mediated by activation of α₂-adrenoceptors at sites outside the colon. Since clonidine could cross the blood-brain barrier, it is unclear whether clonidine inhibits colonic motility through activation of central or peripheral α₂-adrenoceptors. The aim of this study was to investigate the site of α₂-adrenoceptors involved in the inhibitory effect on colonic motility in rats. The hydrophilic α₂-adrenoceptor agonist apraclonidine which could not easily cross the blood-brain barrier was used. Intravenous administration of apraclonidine was found to suppress the colonic motility with a dose-dependent manner. The inhibitory effect induced by apraclonidine (i.v.) on colonic motility was significantly antagonized by pre-administration of yohimbine. These results suggest that the suppression of colonic motility by α₂-adrenoceptor agonist involves in activation of peripheral α₂-adrenoceptors, but not at the site of colon. [Jpn J Physiol 54 Suppl:S200 (2004)]

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To evaluate biomedical effects of the motion in the video image evoked by camera works, the motion components in the video image should be extracted, and their effects on physical parameters should be tested independently. As the first step of this effort, eye movement evoked by viewing a CG movie composed of a series of the motion vector, which were extracted from a real image, was compared with that evoked by viewing the original real image. The CG movie was composed of a randomly distributed dots, which were provided with a series of global vectors calculated from the movement of the corresponding points of the real image, and was presented on a 80 inch screen by liquid crystalline projectors. Eye movement was measured by using an infrared computer-assisted eye/pupil meter. 4 subjects were given informed consents. Eye movements evoked by the composite motion random dots had slow movement components, which were well comparable with those evoked by the real image, while fast components were quite different. [Jpn J Physiol 54 Suppl:S200 (2004)]

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Present study was undertaken to investigate responses of ovarian blood flow (OBF) to electro-acupuncture (EA) stimulation at different frequencies (2 and 80 Hz) and intensities (1.5, 3 and 6 mA) in anaesthetised rats. OBF was measured on the surface of the left ovary using laser Doppler flowmetry. Acupuncture needles with a diameter of 0.3 mm were inserted bilaterally into the abdominal and the hindlimb muscles and connected to an electrical stimulator. Both low (2 Hz)- and high (80 Hz) -frequency EA at 1.5 mA and high-frequency EA at 3 mA had no effect on OBF. Low-frequency EA at 3 and 6 mA elicited significant increases in OBF. In contrast, high-frequency EA with an intensity of 6 mA evoked significant decreases in OBF, followed by decreases in MAP. After severance of the ovarian sympathetic nerves, the increases in the OBF responses to low-frequency EA at 3 and 6 mA were totally abolished, and the responses at 6 mA showed a tendency to decrease, probably because of concomitant decreases in MAP. The decreased OBF and MAP responses to high-frequency EA at 6 mA remained after the ovarian sympathectomy, and the difference in the responses before and after ovarian sympathectomy was not significant. The present study showed that low-frequency EA stimulation increases OBF as a reflex response via the ovarian sympathetic nerves, whereas high-frequency EA stimulation decreases OBF as a passive response following systemic circulatory changes. [Jpn J Physiol 54 Suppl:S200 (2004)]

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Orexins (orexin-A: ORX-A and orexin-B: ORX-B) are neuropeptides which regulate feeding and energy homeostasis. Our previous experiments with intracelluar recordings from myenteric plexus neurons and with intestinal contraction mesurements by Magnus method suggested that ORX-A contributed in the regulation of gut motility via enteric nervous system. Moreover, it was reported that ORX-A affected neuronal activity and synaptic transmission in the submucosal plexus. However, actions of ORXs on mucosal functions are not yet known. The present study investigated actions of ORXs on mucosal transport functions by measuring transmural short-circuit currents (Isc) using Ussing chamber technique in the guinea-pig small intestine. When ORX-A (10 nM to 100 nM) was applied to mucosal side of intesitinal submucosa-mucosa preparations made by removing muscularis and serosa, Isc was not changed. But when ORX-A was applied to submucosal side, Isc was transiently increased. This Isc change was inhibited by submucosal TTX. ORX-B (10 nM to 100 nM) induced smaller TTX-sensitive changes in Isc than ORX-A, when applied to submucosal side. These results suggest that ORXs stimulates mucosal transport by activating submucosal plexus neurons. Immunohistochemical localization of ORX receptors were also demonstrated using the same preparations. [Jpn J Physiol 54 Suppl:S200 (2004)]

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To evaluate responses of the autonomic nervous system (ANS) in rats, we tried to develop some simple non-invasive tests like as clinically used for patients.
Under Nembutal anesthesia, skin blood flows (SBFs) were measured bilaterally in the plantar surface of the rat hind paw by laser Doppler flowmetry. Simultaneously, ECG, a respiration and the rectal temperature were also monitored. These experiments were carried out under the constant environment (room temperature: 23°C, humidity: 59%). Following stimuli were applied: i) somatic noxious stimuli (pricking of the cornea, pinching of the tail and mechanical pressure to the gastrocnemius muscle), ii) cooling of the tail with ethyl chloride, iii) ANS function tests clinically utilized in human (Valsalva-like maneuver, Aschner reflex, and head-up and -down tilt). In addition, the influence of body temperature (BT) to ANS functions was examined at different BTs by cooling or heating the animal body. Power spectral analysis of variabilities of SBFs and heart rate, and cross-spectral analysis between them showed the details of the changes of ANS functions. In some experiments, β-adrenergic and muscarinic blockers were applied to assess the degree of the involvement of the sympathetic and parasympathetic nervous system.
It is suggested that these non-invasive tests in rats are available as simple and reliable ANS function tests. These tests may be useful for animal models with pathological condition such as one with chronic pain.
[Jpn J Physiol 54 Suppl:S201 (2004)]

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The effects of cigarette smoking on cardiovascular outputs (blood pressure and pulse) and properties (Na+, total protein and amylase activity, pH, volume and viscosity) of saliva were examined in smokers. As a control, drawing of a cigarette filter was done instead of cigarette smoking. Saliva samples were collected by expectorating into a cylinder for 2 min, every 10 min. Immediately after expectoration, the volume, viscosity, pH and concentration of Na+ of the saliva were determined. The viscosity was measured by NEVA (New Equipment for Viscosity Assessment; Ishikawa Iron Works Corporation, Kitakyushu) meter. The collected saliva was maintained on ice, and then concentrations of protein and amylase activity were also measured. The blood pressure and pulse were slightly decreased in drawing a filter. Smoking increased blood pressure and pulse and volume of saliva, comparing with those in the control. We are going to investigate the effects of drawing a filter on properties of saliva in smokers and non-smokers. [Jpn J Physiol 54 Suppl:S201 (2004)]

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The effects of mechanical sensory stimulation (by pinching, brushing or pressing) of either a hindpaw or a hind leg on ovarian blood flow measured by laser Doppler flowmetry were studied in anesthetized non-pregnant adult female rats. Pinching of a hindpaw and pressing of a hind leg were effective stimuli for the ovarian blood flow. Pinching of a hindpaw increased blood pressure and ovarian sympathetic nerve activity, and decreased ovarian blood flow. The decrease response in ovarian blood flow following pinching of a hindpaw was reversed to the increase response after surgical severance of ovarian sympathetic nerves. Pressing of a hind leg decreased blood pressure, ovarian sympathetic nerve activity and ovarian blood flow. The response of decrease in ovarian blood flow following pressing of a hind leg was slightly augmented after severance of ovarian sympathetic nerves. Electrical stimulation of the distal part of the severed splanchnic nerve at the left side decreased the left ovarian blood flow, and this response was abolished by i.v. injection of phentolamine, the α-adrenoceptor antagonist. The present results demonstrate that ovarian blood flow increase or decrease passively to responses of an increase or a decrease in systemic arterial blood pressure following mechanical sensory stimulation of a hindlimb, and these passive responses of ovarian blood flow are modulated by reflexive activity of ovarian sympathetic vasoconstrictor nerves to attenuate blood pressure-dependent passive changes in ovarian blood flow. [Jpn J Physiol 54 Suppl:S201 (2004)]

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We have investigated the characteristics of peripheral orexin system in guinea-pig intestine by means of intracellular recording, Magnus method, Ussing method and fluorescent-immunohistochemistry. This time, enteric orexin system in the rat intestine was investigated to compare with that of guinea-pig, because rats are more commonly used in sleep researches. The guinea-pig intestine indicated twitch contraction after transmural electrical nerve stimulation (duration: 1-3 ms, 5-10 pulses/20 s, 10 Hz, voltage: 40 v), while rat intestine showed weak oscillatory responses to strong electrical stimuli (duration: 10-50 ms, 5-10 pulses/20 s, 10 Hz, voltage: 40 v). Orexin dose-dependently (10-300 nM) augmented the nerve stimulation-induced twitch response in the guinea-pig intestine. On the other hand, orexin dose-dependently increased resting tension of the rat intestine (3-100 nM), without affecting nerve stimulation-induced responses. The actions of orexin A were stronger than that of orexin B in guinea-pig intestine, while orexin B was stronger in the rat intestine. The oral part of rat ileum was more sensitive than the lower part of the ileum. This specie dependency of orexin response may reflect food difference and it may affect the sleep-awake cycle of the animals. [Jpn J Physiol 54 Suppl:S201 (2004)]

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[Introduction] Despite accumulated numerous knowledge as to human muscle sympathetic nerve activity (SNA) measured by microneurography, whether sympathetic tone of muscle SNA parallels that of other SNAs, in particular renal and cardiac SNAs, remains unknown. [Methods] In urethane (250mg/ml) and α-chloralose anesthetized, vagotomized and aortic-denervated rabbits, we recorded muscle SNA from tibial nerve using microneurography, together with renal and cardiac SNAs. We stepwise changed sympathetic tone of each SNA by isolating the carotid sinuses and increasing intra-carotid sinus pressure (CSP) stepwise from 40 to 160 mmHg with an increase of 20-mmHg. Each pressure step was maintained for 60 s. We averaged each SNA over 1 minute, and calculated the correlation coefficient (r) between muscle and renal or cardiac SNA. Additionally, we compared static characteristics of baroreflex control of SNA between them. [Results] Muscle SNA averaged over 1 minute correlated strongly both with renal (r2 =0.96) and cardiac SNAs (r2 =0.96). [Conclusion] Sympathetic tone of muscle SNA parallels that of renal and cardiac SNAs in response to baroreceptor pressure change. [Jpn J Physiol 54 Suppl:S202 (2004)]

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The muscarinic agonists pilocarpine and cevimeline are used as sialogogues in treating dry mouth. These drugs also induce drinking behavior though muscarinic receptors in the central nervous system. Pilocarpine and cevimeline were applied by intracerebroventricular (ICV) injection in rats and water intake was measured. By the ICV injection of pilocarpine, water intake was increased in a dose-dependent manner at 0.03-100 nmol. But abnormal behaviors appeared in the concentration of more than 100 nmol. The volume of water intake was 10.8 ± 2.6ml at 100 nmol. By the ICV injection of cevimeline, water intake was also increased in a dose-dependent manner at 10-1000 nmol (2.3 ± 2.0ml at 1000 nmol), but induced drinking was reduced by the over concentration of 3000 nmol. The water intake by the injection of 0.3 nmol pilocarpine and 300 nmol cevimeline were nearly equal (2.3 ± 0.9 ml and 2.1 ± 0.6 ml, for one hour, recepectively). Hence, we compared with time courses of the water intake at these concentration of drugs. By pilocarpine, the peak of water intake appeared at 5 min and finished at 10 min. However, by cevimeline, the peak of water intake appeared at 10 min and the drinking behavior continued for 30 min. The time courses were similar to those of salivary secretion in previous reports (Omori Y. et al. 2003). The results suggest that pilocarpine induces an acute and strong dipsogenic response, but cevimeline induces a long-lasting and weak dipsogenic response. [Jpn J Physiol 54 Suppl:S202 (2004)]

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To examine the effects of ischemia on cardiac and skeletal muscle sympathetic nerve activity, we used microdialysis technique on the hearts and skeletal muscle of anesthetized rabbits and measured dialysate norepinephrine (NE) and lactate (Lac) during ischemic skeletal muscle and myocardium. We applied microdialysis technique to the left ventricular myocardium and acute myocardial ischemia was induced by a 60-min occlusion of coronary artery. The dialysate NE and Lac levels were served as indices of NE release and myocardial ischemia. In separate rabbit, we applied microdialysis to adductor muscle and acute muscle ischemia was induced by 60-min occlusion of iliac artery. The basal myocardial dialysate NE and Lac levels were 48±6, 0.52±0.08 pg/ml. During ischemia the increase in myocardial dialysate NE(1062±252 at 0-15, 21099±3049 at 15-30, 36566±4466 at 30-45, 33058±3717 pg/ml at 45-60min) accompanied with the increase in dialysate Lac (1.87±0.36, 2.75±0.42, 2.34±0.26, 2.03±031 pg/ml). On the other hand, occlusion of iliac artery increased in skeletal muscle dialysate Lac(0.51±0.03 at basal, 0.89±0.09 at 0-15, 1.00±0.02 at 15-30, 1.21±0.09 at 30-45, 1.39±0.06 pg/ml at 45-60min) but decreased in skeletal muscle dialysate NE (19.3±2.2 at basal, 16.1±4.5, 9.4±3.4, 3.2±1.3, 2.3±0.4 pg/ml). Both occlusion of coronary and iliac artery induced increases in dialysate Lac in myocardium and skeletal muscle. However, ischemia induced differential NE responses in the myocardium and skeletal muscle. [Jpn J Physiol 54 Suppl:S202 (2004)]

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The sympathetic nervous system has been purported to respond in a massive and generalized manner. However, in the present study, we demonstrated that intravenous administration of 2DG resulted in regional differences in sympathetic nerve activity in conscious rats. Wistar rats were instrumented chronically with electrodes for the measurements of renal (RSNA) and lumber (LSNA) sympathetic nerve activity, electroencephalogram (EEG), electrocardiogram and electromyogram; and catheters for systemic arterial and central venous pressures. At least 2 days after surgery, the changes in RSNA and LSMA were measured simultaneously and continuously before and after intravenous administration of the 2DG (750mg/kg) which resulted in an immediate reduction in both RSNA and LSNA; however, RSNA increased while LSNA remained lower thereafter. The time course of changes in the theta component (6-9 Hz) of the EEG showed a similar pattern with those changes in the RSNA. Heart rate decreased in a sustained fashion to 2DG. The results demonstrate a case in which the sympathetic nervous system responds in a non-uniform and differential manner. Moreover, it is suggested that the RSNA seemed to be functionally related to theta activity of the EEG. [Jpn J Physiol 54 Suppl:S202 (2004)]

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A large number of viable genetically modified mice are now available that can be used to elucidate the role of the deleted, mutated, or overexpressed genesfor regulaton of physiological functions. To assess functional changes induced by genetic modification, many physiological parameters have been studied. However, few attempts have been made in mice to measure sympathetic nerve activity which play a critical role for regulation of many physiological functions including cardiovascular control, energy metabolism, and the immune system. In the present study we have established the methods for measuring renal sympathetic nerve activity in freely moving mice. The left kidney was exposed retroperitoneally through a left flank incisionand the sympathetic nerves running on or beside the renal artery were identified with aid of a dissecting microscope. The two tips of the stainless steel electrodes were attached to the nerve. The renal sympathetic nerve activitywas recorded successfully without contamination by external noise such as that from the electrocardiogram and/or electromyogram. Nerve recoding continued over 3-4 days after surgery. .The renal sympathetic nerve activity was increased by ~240% during movement relative to the quiet awake state and was suppressed completely by administration of hexamethonium. Thus, this method for measurement of the RSNA could be useful for study of sympathetic nerve activity in genetically modified mice. [Jpn J Physiol 54 Suppl:S203 (2004)]

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The superior salivatory (SS) nucleus consists of a group of parasympathetic preganglionic neurons which innervate targets such as the submandibular and sublingual salivary glands, and anterior region of the tongue. We studied the properties of the synaptic inputs to SS neurons supplying the different targets in neonatal Wistar rats (4-8 days old). The SS neurons supplying the salivary glands and tongue were labeled by retrograde axonal transport of a fluorescent dye. Using a whole-cell patch-clamp technique, the EPSCs and IPSCs were recorded from labeled cells in brainstem slices (200 μm). Whole-cell currents were evoked by electrical stimulation of tissue surrounding the cell. To optimize the positioning the stimulating electrode, dendrite morphology of the SS neurons was examined by Neurobiotin. SS neurons have 3-5 primary dendrites that extend 200-780 μm in neurons supplying the salivary glands and 120-530 μm in neurons supplying the tongue. Robust currents were usually obtained at a few points 100-400 μm from the soma. EPSCs were mediated by NMDA and non-NMDA glutamate receptors. IPSCs were mediated by GABA_A, and glycine receptors. In SS neurons for the salivary glands, the ratio of the NMDA component to total EPSCs was larger than that of non-NMDA component, and the ratio of the GABA_A component to the total IPSCs was larger than that of the glycine component. The characteristics of the excitatory and inhibitory inputs may be involved in determining the firing properties of the SS neurons supplying the salivary glands and tongue. [Jpn J Physiol 54 Suppl:S203 (2004)]

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Electrical impedance method has been used for measurements of various biological parameters in a noninvasive manner. In the present study, the relationship between the thoracic impedance and the thoracic air volume was quantitatively analyzed in human using four-electrode system. A constant current excited from an oscillator (500 or 50kHz) was injected between a pair of electrodes on the bilateral thorax walls. The impedance between another pair of electrodes was determined assuming that the voltage drop between those two electrodes is linear to the variations of the corresponding impedance change. The impedance was stably recorded without high-pass filtering when the postural changes of the examinees in a sitting position were minimum. The thoracic impedance thus measured contained components attributable to the variation of air volume whose frequency was normally far below 1Hz and blood volume whose frequency was around 1.3Hz. The ration between these two components largely depended on the positions of the electrodes. Basal thoracic air volume was changed intensively to various given levels. Then lung volume was further changed within a given range by inspiring or expiring air from a thin polyethylene bag with a given volume (500~4000 ml). The impedance change thus recorded varied linearly to the thoracic air volume when the four electrodes were set within restricted areas. The advantage and disadvantage in the assessment of thoracic air volume using electrical impedance method will be discussed. [Jpn J Physiol 54 Suppl:S203 (2004)]

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Autonomic nervous system (ANS) plays a crucial role for the viability of animals by regulating physiological activity of organs. Since ANS is located in the brainstem, the most ancient part of the brain, it has been a general understanding that ANS originated early in vertebrate evolution or even earlier. However, no attempts have been made to determine how far we can trace the ANS back in evolution. In this presentation, based upon a series of physiological studies carried out in Hydra, we present a hypothesis that the ANS originated in its primitive form very early in metazoan evolution, as early as in phylum Cnidaria or in Plathyhelminths which had gastrovascular cavity. [Jpn J Physiol 54 Suppl:S203 (2004)]

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The amplitude of the startle reflex is elevated when elicited in the presence of a cue previously paired with footshock, fear potentiated startle. This suggests a possibility of evaluation of the other emotion such as pleasure and aversive. Our objective is to study a modulation effect in rat startle response by reward-related and aversive-related conditioned stimuli (CS). Male albino Wistar rats, weighing 300-350g, were used. Oral cannuellae was implanted in each animal oral, and the other terminal of the tube was fixed on the head. Startle stimulus was a 40 msec burst of white noise. The CS of 3.2 sec duration was produced by 3 LED (420 Lux). The unconditioned stimulus was a shock generated by scramble shocker (0.2, 0.4, 0.6 mA in strength and 0.2 sec duration), 10% glucose solution or 5% saline solution. The startle response was increased by conditioning of aversive stimuli. The startle amplitude was 518±593 (relative units, n=3) in aversive CS, compared with 348±345 of tone alone. However, the response was not changed by reward conditioning. The startle amplitude was 106±136 (relative units, n=8) in aversive CS, compared with 120±139 of tone alone. The startle amplitude was enhanced proportionally by CS corresponded to electrical current intensity in fear conditioning. However, the startle amplitude showed an inverse U-shaped responses in discrimination learning of CS associated with different UCS intensity. These suggest that startle response might depend on quantity and quality of emotion. [Jpn J Physiol 54 Suppl:S204 (2004)]

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Objective: Free-radicals have been implicated in aging, mutagenesis, inflammation, and other pathological conditions. We conducted a study to clarify the relation between psychological eustress of pleasant feeling and free radical-scavenging cappacity (FRSC) in saliva. Methods: Saliva was collected from 27 healthy volunteers. FRSC before, during, and after watching a cheerful comical video for 30 minutes were measured by using 1,1-diphenyl-2-picrylhydrazyl. Results: The median values of FRSC (μmol/ml) before, at 10, 20 min during, after watching the video were 54.5, 66.8, 66.6, and 69.4, respectively. FRSC values obtained after watching the video were significantly higher than those before watching it (p<0.001). When the FRSC before watching was taken as 1, the value for the group that felt Very good, Good, or Ordinary + Dull while watching the video for 30 min was 1.38, 1.20 or 0.98, respectively (p<0.01). Conclusion: Watching a comical video enhanced the FRSC in saliva; and, in addition, a pleasant feeling boosted it even more. [Jpn J Physiol 54 Suppl:S204 (2004)]

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Our previous study showed that the hardness of food could work as a conditioned stimulus (CS) for the conditioned food aversion (Sako et al., 2001). In the present study, behavioral characteristics of the rats aversive-conditioned to the food with qualities of taste and hardness were investigated. For the CS, 4 types of pellets, such as hard and sweet (HS), soft and sweet (SS), hard and none taste (HN) and soft and none taste (SN), were used. All pellets contained same nutrition, but sweet pellets contained 5% sodium saccharin additionally. When the rats were aversive-conditioned to HS or SS with 0.15M LiCl i. p. (2% BW), they avoided all of foods with sweet quality even if their hardness were not same as CS. On the other hand the rats aversive-conditioned to HN or SN avoided CS, but they did not avoid other foods with sweet taste, even if they had same hardness of CS. These results suggest that rats acquired the conditioned food aversion give taste priority over hardness on their food choice. [Jpn J Physiol 54 Suppl:S204 (2004)]

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We chose the conditioned taste aversion (CTA) paradigm, a model of emotional learning, in order to examine the effect of modification of gravitational force on acquisition and retention of emotional learning and memory. We used centrifugal machine for rodents to add the gravitational force and 3D clinostat to cancel the gravitational force. The centrifugation of 1G or low gravitational environment was applied between 0.1% sodium saccharin as a conditioned stimulus (CS) and an i.p. injection of 0.15 M LiCl as an unconditioned stimulus (US). Under the 1G condition, both acquisition and retention was suppressed. When 1G condition was employed instead of LiCl as US, the CTA was established, indicating that the malaise induced by 1G cancelled that by LiCl. Although low gravitational condition did not serve as US, it suppressed both acquisition and retention. Furthermore, after CTA acquisition with low gravitation, expression of an immediately early gene, c-Fos was greatly suppressed in the external lateral subnucleus of parabrachial nucleus and in several midline-intralaminar thalamic nuclei, which strongly appeared under control condition. These results suggest that low gravitational force prevents CTA memory formation through suppression of association process between CS and US in medial thalamic pathway to limbic system as well as gustatory pathways of brain stem. [Jpn J Physiol 54 Suppl:S205 (2004)]

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We examined voluntary wheel activity, body weight, food intake and water drink throughout the life of 36 female virgin Wistar rats fed ad libitum. We also determined the duration from menarche to menopause. The animals were housed in a steel cage with a wheel for voluntary exercise. Twelve rats were ovariectomized at 60 days of age. The mean life span of the intact rats was 755 +/- 191 days. The mean age at menarche and menopause was 45 +/- 13 and 403+/- 56 days, respectively. Both parameters were changed in a particular manner with aging, which was characterized by a numerical formula. Voluntary wheel activity throughout the life span was characterized by increasing and decreasing phases with a peak at 90 days of age. Body weight reached a maximum value (400 g) at 450 days and was characterized by an initial steep increase before menarche, a stagnated phase during estrogen secretion, and a slight temporal increase after menopause. Food and water intake reached a minimum at 450 days. Power spectral analysis confirmed that the menstrual cycle lasted for 4.39 +/- 0.53 days from menarche to menopause. The coefficient of variation of voluntary wheel activity revealed prominent increases in individual differences after menopause. A longer duration between menarche and menopause was significantly related to an elongated life span. [Jpn J Physiol 54 Suppl:S205 (2004)]

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A wireless recording system was developed to examine the electroencephalogram (EEG) activity in unrestrained piglets. The equipment was composed of a miniature preamplifier and an FM transmitter, and was driven by batteries. The signal from the telemetry equipment was detected by antennas on the ceiling of the Faraday chamber, and then filtered and, magnified with a conventional amplifier. Under general anesthesia, silver-silver chloride electrodes were implanted onto the dura matter and were fixed by dental cement with the preamplifier and transmitter on the surface of the skull of the male Landrace piglets. Stainless-steel wire electrodes were sewn into the upper eye-lids to detect their movement. After the piglets recovered from surgery, the EEG and eye-lid movement were recorded in the unrestrained condition while they were lying down in the daytime. Off-line, power spectral analysis of the EEG activity using a Fast Fourier Transformation algorithm indicated that slow waves with frequencies from 0.5 to 3.9 Hz were often dominant in the EEGs of these animals without the eye-lid movements. In contrast, the slow waves diminished when eye-lid movements were recorded. [Jpn J Physiol 54 Suppl:S205 (2004)]

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The subfornical organ (SFO) is involved in the modulation of sodium and water intake in the rat. To clarify whether serotonergic mechanisms in the SFO participate in the modulatory system, experiments were carried out to investigate the effects of sodium and water intake on serotonin (5-hydroxytryptamine, 5-HT) release in the subfornical organ (SFO) in freely moving rats. The intake of 0.3 M NaCl and water was induced by subcutaneous injections of the diuretic furosemide (FURO, 10 mg/kg) and the angiotensin converting enzyme inhibitor captopril (CAP, 5 mg/kg), and extracellular concentrations of 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the region of the SFO were measured using intracerebral microdialysis techniques. The combined treatment with FURO and CAP produced significant decreases in the 5-HT and 5-HIAA levels in the SFO area. The 0.3 M NaCl and water intake caused by the treatment significantly increased the 5-HT and 5-HIAA concentrations in the SFO area compared with the basal levels. No significant changes in the 5-HT and 5-HIAA levels elicited by either the FURO and CAP treatment or water and NaCl intake were observed in the sites away from the SFO. These results suggest that the serotonergic mechanism in the SFO area may be important for the control of sodium appetite and thirst. [Jpn J Physiol 54 Suppl:S205 (2004)]

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The present study was done to investigate whether 0.3 M NaCl and water intake alters the release of serotonin (5-hydoxytryptamine, 5-HT) in the region of the lateral parabrachial nucleus (LPBN) in freely moving rats. The intake of 0.3 M NaCl and water was induced by subcutaneous administrations of the diuretic furosemide (FURO, 10 mg/kg) and the angiotensin converting enzyme inhibitor captopril (CAP, 5 mg/kg), and extracellular concentrations of 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were measured using in vivo microdialysis methods. The intake of 0.3 M NaCl and water significantly increased the 5-HT and 5-HIAA levels in the LPBN area. The combined treatment with FURO and CAP caused significant decreases in the 5-HT and 5-HIAA concentrations in the LPBN area under the condition that 0.3 M NaCl and water are not available for drinking. These results suggest that the serotonergic system in the LPBN area may play an important role in the modulation of sodium appetite and thirst. [Jpn J Physiol 54 Suppl:S206 (2004)]

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This study investigated the relation between the mechanisms controlling heart rate and respiratory movement in carp. An electrocardiogram (ECG) and electromyogram (EMG) of the lower jaw for detection of the respiratory movements (mouth and opercular movements) were recorded simultaneously by the radiotelemetry system (DSI, USA). Adult carp were reared in individual tanks under a 12h/12h light-dark cycle, and the water temperature was kept at 25°C. In carp, both the heart rate (HR) and respiratory rate (RR) were irregular compared to the HR and RR in mammals. The HR was usually within a range of about 40 to 120 /min and the HR was greater or less than the RR. In some cases, both were equal, and both parameters were almost synchronized. When 2 or 3 respiratory movements grouped and were followed by pauses of respiratory movement, the R-R intervals of the ECG during the grouping respiratory movements were shorter than those during the pauses. The carp were stressed by tapping on the tank wall or by switching off the room light. These stresses transiently decreased both the HR and RR. After intraperitoneal administration of atropine sulfate (1mg/kg), the HR increased and became regular, and stress responses of the HR disappeared. These results suggest that the mechanisms controlling the heart rate and respiratory movement were closely related. And the vagal nerve plays a major role in controlling the HR mechanism in carp. [Jpn J Physiol 54 Suppl:S206 (2004)]

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Galanin-like peptide (GALP), isolated recently from the porcine hypothalamus, is expressed predominantly in the arcuate nucleus (ARC), a feeding-controlling center. GALP is a peptide of 60 amino acids, in which residues (9-21) are identical to galanin (1-13). GALP shows a high affinity for galanin-receptor 2 (GalR2) than for GalR1. Therefore GALP is thought to be the endogenous ligand for GalR2. It is reported that intracerebroventricular (icv) injection of GALP stimulates food intake in rats. Functional relation between GALP and other feeding-regulatory peptides is not obvious. To determine which region in the hypothalamus is activated by GALP, we injected GALP into the nuclei and assessed orexigenic effects of GALP. GALP at 0.03 nmol injected into the dorso median nucleus (DMN) remarkably increased food intake, while that into the lateral hypothalamic area and the paraventricular nucleus had weaker or no effects. Following the study, we examined that which neurons are stimulated by icv GALP injection. GALP (0.3 nmol) icv injection expressed c-fos in the DMN, in which about 40% of NPY-containing neurons in the DMN co-expressed c-fos. GALP icv injection after BIBP3226 (Y1 receptor antagonist) icv injection did not stimulate food intake. These data suggest that an increase of food intake caused by icv GALP injection is mediated at least partly by an activation of NPY-containing neurons in the DMN. [Jpn J Physiol 54 Suppl:S206 (2004)]

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Although the accumulated evidences show orexin plays important roles in the narcolepsy, there are few reports indicating the orexinergic regulation on the neurotransmitters' releases related to the symptoms of narcolepsy. The present study was designed to elucidate a physiological change of orexin across sleep cycles and also a possible mechanism of how orexins affect vigilance states and behavior. Since a lack of orexins results in the excessive day time sleepiness (EDS) and the loss of muscle tone (cataplexy), orexin may alter the levels of neurotransmitters in the center of desynchronizing EEG activity in the hypothalamus and of regulating motor activity in the brainstem. Thus, we examined the release of histamine in the frontal cortex and norepinephrine (NE) in the thalamus of the rat after an intravenous injection of orexin-A.Intraveous injection of orexin-A (1 nmol, 2 nmol and 10 nmol) elicited the release of NE from the thalamus significantly. On the contrary, only 10 nmol of orexin-A elicted the release of histamine significantly in the frontal cortex. These results suggested that the increase in NE release may reflect the EMG activity and the increase in histamine release may reflect the EEG activity. The difference of the optimal concentration to affect on EMG and EEG may explain why the beginning of EDS is usually followed cataplexy. [Jpn J Physiol 54 Suppl:S206 (2004)]

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We devised an apparatus (CMP) to keep watch behavior from food-acquisition to feeding termination in mice. The system compelled them to climb a cylindrical wire net of 180 cm high in order to acquire food. CMP characterized feeding behavior when being fed and starved. Such characters were assumed to be derived from changes in blood glucose levels. Thus effects of supplying glucose were examined on the feeding behavior.C57 BL/6J mice were introduced into CMP at 8 wk old. Acclimating 28-35 days, behavior was measured for 2 hr from beginning dark period under fed and starved conditions. Administered were glucose (2 g/kg bw, i.p., GL), 2-deoxy-D-glucose (500 mg/kg bw, i.p., 2-DG) or same volume of saline. GL decreased food consumption while 2-DG increased it. GL gave no changes in food-acquiring behavior under both fed and starved conditions. 2-DG suppressed the behavior under only the starved condition. Thus blood glucose and 2-DG might affect feeding behavior. But the effects were different between food consumption and food-acquiring behavior. [Jpn J Physiol 54 Suppl:S207 (2004)]

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The molecular clockwork that cause the fundamental feedback loops to stably oscillate with a ~24 hr-periodicity remain unclear. Serum-shocked fibroblasts were used as a cellular clock model. Circadian changes in the subcellular localization and phosphorylation of BMAL1 protein in these cells were assessed by immunocytochemistry and immunoblotting. A significant time lag between Bmal1 transcription and the cytoplasmic/nuclear accumulation of BMAL1 was observed. After its nuclear accumulation, BMAL1 accumulated in the cytoplasm again, mainly by nucleoexport, before the increase of Bmal1 transcripts. Nuclear accumulation of BMAL1 matched nuclear accumulation of CLOCK and the peak of Per1 transcription. Nuclear BMAL1 was gradually phosphorylated and then dephosphorylated in a temporally regulated manner, although cytoplasmic BMAL1 was not. In serum-shocked mCry1/mCry2 (CRY)-deficient fibroblasts, which lack a functional clock, both the cytoplasmic and nuclear BMAL1 were only present as hyperphosphorylated forms and their circadian nucleocytoplasmic shuttling was absent. Similar to the fibroblast, circadian regulated nucleocytoplasmic shuttling of BMAL1:CLOCK were also observed in the SCN. We propose that the nucleocytoplasmic shuttling and phosphorylation states of BMAL1 are regulated by circadian clock, and that this temporally regulated and time-delayed nuclear entry of BMAL1 is important in the maintenance of a stably oscillating clock. [Jpn J Physiol 54 Suppl:S207 (2004)]

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In order to know whether or not the clock genes in the hippocampus are involved in prolonged hyperphagia produced by mild restraint, the expressions of clock genes, Per1 and Dec1, and Neuropeptide Y (NPY) gene in the rat hippocampus were examined by mean of in situ hybridization. Immediately after weaning, 6-8 female rats were restrained together in a small pouch for 30 min in the morning every second day for 4 weeks. Body weight as well as food and water intake were measured every day during and for 4 weeks after the stressor application. The rats were killed by decapitation 2, 4 and 6 weeks after weaning. The brain was frozen, cut in a frontal plane including the hippocampus and subjected to in situ hybridization. The hybridization signals were quantified by MCID. As a result, Per1 was expressed in all regions of the hippocampus, whereas Dec1 expression was restricted to CA1. NPY gene was expressed in CA1 and the dentate gyrus (DG). In the control rats, Per1 expression showed a significant day-night difference in DG, but not in other regions of the hippocampus. Restraint significantly increased Per1 expression in all regions of the hippocampus at 2, 4 and 6 weeks. On the other hand, Dec1 expression in CA1 was significantly suppressed by restraint during the entire period of experiment. Restraint has no effect on NPY expression in the hippocampus. These findings suggest possible roles of Per1 and Dec1 in the hippocampus in the stress response and/or prolonged hyperphagia. [Jpn J Physiol 54 Suppl:S207 (2004)]

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Three male subjects spent 2 or 4 weeks together in an isolated facility with dim light condition (150 lx). In total, 11 subjects participated in this experiment. The daily life in the isolated facility was strictly scheduled. The subjects were divided into two groups; one group performed physical exercise for ca. 1 hour after lunch and the other group spent quietly. Sleep electroencephalography (EEG) and plasma melatonin rhythm were measured every second weeks. As a result, the plasma melatonin rhythms in the subjects without exercise showed significant phase-delay shifts 2 weeks after the start of isolation. In these subjects, the nocturnal sleep was deteriorated more than a little. For instance, the sleep latency was lengthened, the frequency of midnight wake was increased and the score of sleep satisfaction was decreased. By contrast, the peak phases of plasma melatonin rhythm were essentially unchanged with physical exercise. In these subjects, sleep was kept almost intact. These findings firstly indicate that a light-dark cycle with dim light (150 lx) is unable to keep the human melatonin rhythm entrained, and secondly confirmed that physical exercise accelerated entrainment of the human circadian pacemaker. Deterioration of sleep EEG seems to be a result of internal dissociation between the circadian pacemaker and the scheduled sleep time. [Jpn J Physiol 54 Suppl:S207 (2004)]

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Lactoferrin (LF) is known as an iron-binding glycoprotein. We have shown that orally administered bovine-LF (bLF) was transported into cerebrospinal fluid via blood circulation in neonatal and weaned piglets or calves, however its physiological effects in the central nervous system (CNS) were still unclear. In this study, a thermogenetic effect of bLF under stressful condition was investigated in adult rats. Male Wistar-Imamichi strain rats (8 weeks) were exposed to cold stress (7°C) combined with restraint stress. Rectal temperature (RT) was recorded up to 4h, and blood sample was collected at 30 min and 2h of exposing to the stresses. Catecholamine concentration in plasma was measured by HPLC-ECD system. bLF (100 mg/kg) was administered by intraperitoneal injection or feeding of supplemented diet with 1% bLF. Restraint stress induced an increasing of RT at room temperature in both of saline and bLF injected rats. Interestingly, bLF caused a marked increasing of RT even under the combination of cold and restraint stresses, although the RT slightly decreased in the saline injected rat. Similar changes in the RT were also confirmed in the 1% bLF supplemented diet-fed rats. Plasma noradrenaline concentration increased under all stressful conditions, and bLF enhanced NA release in the restraint rat at room temperature. These results suggest that bLF enhanced thermogenesis under the stressful conditions. Moreover, this effect of bLF was possibly related with the sympathetic nervous activities in rats. [Jpn J Physiol 54 Suppl:S208 (2004)]

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We previously examined the effect of voluntary abdominal breathing (VAB) related to Zen Meditation on EEG, and found that the spectral power in high-frequency alpha band (10-13Hz) increased during the eyes-open condition as well as the eyes-closed condition. Because the urinary 5-HT level after the VAB increased, we hypothesized that the activity of the 5-HT neuron located in the brain stem may be involved in modulating the EEG power during VAB. In the present study, we performed topographic analysis of the EEG power during VAB in an eyes-closed condition by using a brain mapping technique. Although the spectral power of low-frequency alpha band (8-10Hz) appeared over the occipital and the parietal area promptly after closing the eyes, it gradually decreased during VAB. The high-frequency alpha power began to increase ~5 minutes after VAB and its topographic distribution extended from the occipital to the central area. It further extended from the occipital to the frontal area 10 minutes after VAB; this extended distribution was sustained until the completion of VAB. The urinary and/or blood 5-HT levels tended to increase after VAB. These results support our hypothesis that the activity of 5-HT neurons located in the brain stem may be involved in modulating the EEG power during VAB. [Jpn J Physiol 54 Suppl:S208 (2004)]

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The response of the neuronal firing activity in and around the suprachiasmatic nucleus (SCN) to the retinal illumination was investigated with extracellular single unit recording in anaesthetized mice during daytime and nighttime. Retinal illumination during nighttime increased firing frequency of most SCN neurons with spontaneous firing. When animal was exposed to light during daytime, spontaneous firing rate in the SCN increased in 33% of total recording neurons and not changed in 67% of total neurons. Spontaneous firing decreased in a part of SCN neurons (11%) after retinal illumination. Outside the SCN, the light-suppressed neurons were observed more frequently than light-activated neurons and there was no difference in the proportion of the types of light response between daytime and nighttime. In light-activated cells in the SCN at Zeitgeber time 16, light intensity threshold for activation was <10¹¹ photons cm⁻² s⁻¹ and the maximum response was evoked at 10¹⁴ photons cm⁻² s⁻¹, which was comparable to those in photic resetting of behavioral rhythms. These results suggest that the photic entrainment of locomotor activity rhythms are closely associated with the light-induced changes in firing frequency in the SCN cells in mice. [Jpn J Physiol 54 Suppl:S208 (2004)]

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