Mitsuhiko Miura

The comparison of effects of various anesthetics on expression of Fos protein in the rat brain

The purpose of this study was to compare effects of various anesthetic drugs on expression of Fos protein in the supraspinal neurons of rats. Optimum anesthesia was maintained under monitoring of EEG. Degree of Fos expression was evaluated by counts of Fos-immunoreactive (ir) neurons. Urethane induced the largest amount of Fos-ir neurons (n = 8439), while fentanyl/midazolam the smallest (n = 112). alpha-Chloralose, halothane, pentobarbital sodium and urethane/alpha-chloralose induced amounts in between. The results indicate that fentanyl/midazolam is the most recommendable anesthetic drug in the Fos expression study.

Projections of supraspinal structures to the phrenic motor nucleus in cats studied by a horseradish peroxidase microinjection method

The activity of phrenic motor neurons is influenced by the cardiovascular control system of the supraspinal structures. In order to obtain the basic data for analyzing the anatomical relations between the cardiovascular and the pulmonary control system, supraspinal structures projecting to the phrenic motor nucleus of the cat spinal cord were studied using a horseradish peroxidase method. A double-barrel coaxial electrode was employed. To determine the site of the phrenic motor neurons, the inner barrel electrode, filled with 3 M NaCl solution, was used for recording the activity of these neurons. The outer barrel electrode, filled with a 20% HRP solution, was used for injecting HRP iontophoretically into the phrenic motor nucleus. Out of 13 experiments, 5 showed that the HRP-injection sites were centered in and almost confined to the phrenic motor nucleus. Some 1798 HRP-labeled cells were thus identified in the selected 5 experiments. They were distributed in the medulla oblongata (93.5%), pons (6.0%) and midbrain (0.5%). The majority were concentrated in the nucleus para-ambiguus (48.9%), nucleus tractus solitarii (21.5%) and in or around the nucleus retrofacialis (9.8%). A few labeled cells were scattered throughout the nucleus raphe (1.1%) and the parabrachial and Koelliker-Fuse nuclei (0.3%), suggesting that these nuclei may be, if any, only minor sources of input to the phrenic motor nucleus.

Neuronal expression of Fos protein in the rat brain after baroreceptor stimulation

The purpose of this study was to identify the CNS neurons that express Fos protein after repeated activation of the baroreceptor reflex. This was done in Wistar rats anesthetized with urethane and alpha-chloralose with careful physiological controls. The intact control rat showed few Fos-immunoreactive (ir) neurons, whereas the anesthetized control rat showed many Fos-ir neurons in the CNS from the medulla oblongata to the forebrain. After repeated stimulation of baroreceptors by pressor responses to phenylephrine (dose), we counted the amounts of Fos-ir neurons (response). The correlation coefficient of the dose-response relationship was high, and significant only in the medial part of the nucleus tractus solitarii (NTS) in the medulla and periaqueductal gray (PAG) in the midbrain, whereas it was comparatively high but insignificant in the commissure and lateral parts of the NTS, caudal and rostral ventrolateral medulla, periambiguus nucleus, dorsal and ventral medullary reticular nuclei, lateral parabrachial nucleus, paraventricular nucleus thalamus, and dorsomedial nucleus hypothalamus. No significant correlation was found in the humoral control nuclei in the preoptico-hypothalamic structure. Fos expression was never detected in the sensory neurons in the ganglia petrosum and nodosum, and in the sympathetic preganglionic neurons in the intermediolateral nucleus of the thoracic spinal cord. This study shows that Fos expression in the CNS neurons is induced not only by baroreceptor stimulation but also by anesthesia and/or sham-operation, and that Fos expression in the NTSm and PAG neurons faithfully responds to baroreceptor stimulation.

Circulatory and respiratory responses to glutamate stimulation of the lateral parabrachial nucleus of the cat

Electrical stimulation of the dorsal part of the lateral parabrachial nucleus (Pbl) induced a pressor response associated with a brief hyperpneic response, whereas glutamate stimulation induced no response, suggesting that the response to electrical stimulation may be due to excitation of passing fibers. Electrical stimulation of the lateral part of the Pbl induced a pressor and hyperpneic response, while glutamate stimulation induced either a pressor-depressor or single depressor response associated with a hyperpneic and polypneic response, suggesting that the response to electrical stimulation of the lateral part may be due to excitation of cell bodies. WGA-HRP study clarified that the cells in the region around the ventrolateral edge of the brachium conjunctivum project to the cells in the C1 area and subfacial cell group of the rostral ventrolateral medulla (RVLM), suggesting that the cells in this region may send a message to the sympathetic premotor and respiratory premotor cells in the RVLM. Inversely, the cells in the C1 area and the subfacial cell group project to the lateral part of the Pbl, suggesting mutual innervation between the Pbl and RVLM.

Projections of upper structure to the spinal cardioacceleratory center in cats: an HRP study using a new microinjection method

We studied, with the horseradish peroxidase (HRP) method, the supraspinal structure projections to the cardioacceleratory center in the intermediolateral nucleus (ILN) of T3-4 segments of the cat spinal cord. A fiber-filled double-barrel coaxial electrode was devised. The inner barrel electrode, filled with 3 M NaCl solution, was used for stimulating intraspinal structures to determine the cardioacceleratory center. The outer barrel electrode with an Agar stop, filled with a 20% HRP solution, was used for injecting HRP by pulses of positive electric current. In every experiment, HRP was injected into 3 points within the cardioacceleratory center at intervals of 1 mm. Out of 36 experiments, 10 showed that the HRP injection sites were centered in and almost confined to the ILN, and the population of the HRP-labeled cells was not less than 50. Some 1146 HRP-labeled cells were thus identified. They were distributed in the medulla oblongata (72.1%), pons (10.2%), midbrain (8.5%) and hypothalamus (9.2%). They were concentrated in the medullary reticular formation (37.8%), median raphe (26.9%) and pontine reticular formation (10.2%). Contrary to expectation, the HRP-labeled cells were few in the nucleus tractus solitarii (3.7%), paraventricular hypothalamic area (3.3%) and nucleus locus coeruleus (none).

Functional subdivisions of the nucleus tractus solitarii of the rat as determined by circulatory and respiratory responses to electrical stimulation of the nucleus

We have recently identified two functional subdivisions of the nucleus tractus solitarii (NTS) in the cat: the pressor and apneustic (inspiratory) response zone, and the depressor and apneic (hypopneic) response zone. The presence of such functional subdivisions in the NTS of the rat was explored. The circulatory and respiratory responses to electrical stimulation of a small part of the NTS were surveyed in all regions of the NTS in rats anesthetized with urethane (500 mg/kg, i.p.). A set of depressor, bradycardiac and apneic responses was elicited in the dorsomedial portions, whereas a pressor response with or without an apneic (hypopneic) response was elicited in the ventral and lateral portions. The presence of a difference in the functional subdivisions between spontaneously hypertensive rats (SHR) and normotensive rats (WKY) was also explored. The SHR group showed significantly larger pressor responses than the WKY group (P less than 0.001), despite its higher basal level of arterial blood pressure, but significantly fewer bradycardiac responses (P less than 0.001). This suggests the SHR group is more sensitive in terms of sympathetic vasomotor activity but less sensitive in parasympathetic cardioinhibitory activity. There was no significant difference in the distribution of the pressor and depressor response zones between the two strains.

Distribution of glutamate- and GABA-immunoreactive neurons projecting to the cardioacceleratory center of the intermediolateral nucleus of the thoracic cord of SHR and WKY rats : a double-labeling study

We aimed at (1) determining the distribution of glutamate (Glu)- and gamma-aminobutyric acid (GABA)-containing neurons in the brainstem with projections to the cardioacceleratory sympathetic preganglionic neurons in the intermediolateral nucleus (IML) of the upper thoracic cord and (2) determining whether such afferent projections in spontaneously hypertensive rats (SHR) differ from those of control Wistar-Kyoto (WKY) rats. We used a combination of electrophysiological methods to determine the site of HRP injection in the spinal cord and double-labeling methods for plotting the distribution of Glu- and GABA-immunoreactive neurons with projections to this site. HRP/Glu-labeled neurons (possibly glutamatergic) and HRP/GABA-labeled neurons (possibly GABAergic) were detected in 27% and 7% of the total HRP-labeled neurons of the central autonomic nuclei of 3 SHR rats and 3 WKY rats. HRP/Glu-labeled neurons were distributed predominantly ipsilaterally in 20 nuclei of the medulla oblongata, pons and hypothalamus, while HRP/GABA-labeled neurons were distributed in 7 nuclei of the medulla oblongata. No significant differences were found between the average percentages of HRP/Glu-labeled and HRP/GABA-labeled neurons in SHR and WKY rats. These findings indicate that: (1) the Glu-containing neurons represent a greater proportion than the GABA-containing neurons, (2) the proportions of these neurons appear to be similar in WKY and SHR rats and (3) generation of inbred tachycardia and hypertension in SHR rats can not be attributed to the topological and quantitative differences in the distribution of the glutamatergic and GABAergic neurons in the central autonomic nuclei.

Sites of origin and termination of gastric vagus preganglionic neurons: an HRP study in the rat

Using the horseradish peroxidase (HRP) method, we studied the relation between sites and forms of gastric vagus preganglionic neurons in the rat and gastric regions on which they project. Fifty microliters of a physiological saline solution containing 3 mg of HRP was injected into 6 different gastric regions: cardia, lesser curvature, pyloric antrum, greatger curvature, ventral corpus and dorsal corpus. After a survival period of 24h, the medulla oblongata was serially sectioned and processed for histochemical demonstration of HRP. In the medulla oblongata, HRP-labeled cells were distributed in 2 regions: (1) in the regions within the dorsal vagus nucleus (DVN); (2) in the regions within the ambiguous complex. The labeled cells in the DVN were observed in every experiment, while ones in the ambiguous complex were only seen in the cardia and lesser curvature experiments. In the ventral corpus experiment the labeled cells were observed dominantly on the left side and in the dorsal corpus experiment dominantly on the right side. The majority of the labeled cells in the ambiguous complex were medium-sized and multipolar in shape, while the majority of ones in the DVN were medium-sized and fusiform in shape.

Distribution and projection of the medullary cardiovascular control neurons containing glutamate, glutamic acid decarboxylase, tyrosine hydroxylase and phenylethanolamine N-methyltransferase in rats

This study was aimed at showing the distribution and projection of the medullary cardiovascular control neurons that contain a standard neurotransmitter or a related enzyme in the rat. A small amount of HRP was injected into either the depressor area of the caudal ventrolateral medulla (D-CVLM) or the pressor area of the rostral ventrolateral medulla (P-RVLM). Using an immunohistochemical method, we identified HRP-labelled neurons which were stained with antiserum to glutamate (Glu), glutamic acid decarboxylase (GAD), tyrosine hydroxylase (TH) or phenylethanolamine N-methyltransferase (PNMT). Our findings are summarized as follows. (1) The Glu-containing neurons in the nucleus tractus solitararii (NTS) project to the D-CVLM (n = 279, 100% assumed as a standard value) and P-RVLM (n = 225, 81% against the standard), indicating divergent excitatory projection. (2) The GAD-containing neurons in the NTS (n = 74, 27% against the standard) project to the P-RVLM, indicating the convergent inhibitory projection. (3) The projections of the TH-containing neurons from the NTS (n = 19, 7% against the standard) and CVLM (n = 4, 1% against the standard) to the P-RVLM are weaker than those of the GAD-containing neurons, suggesting that the catecholaminergic neurons play a minor role in inhibition of the sympathetic activity of the P-RVLM neurons. These results suggest that the glutamatergic NTS neurons excite both the P-RVLM and D-CVLM neurons, and the gamma-aminobutyric acid (GABA)ergic NTS and CVLM neurons inhibit the sympathetic activity of the P-RVLM neurons.

Evidence that glutamate is the transmitter mediating respiratory drive from medullary premotor neurons to phrenic motoneurons: A double labeling study in the rat

Glutamate immunoreactivity was found in 81-84% of the bulbospinal neurons projecting to the phrenic motoneuron pool, which were located in the para-ambiguous nucleus and the ventral vicinity of the retrofacial nucleus. Their glutamate contents were identified by a double labeling technique in combination with the retrograde transport of horseradish peroxidase and immunocytochemistry of the glutamate. The remaining 16-19% of the bulbospinal neurons projecting to the phrenic motoneuron pool did not contain glutamate immunoreactivity. Thus it appears that glutamate may be a major transmitter of the inspiratory drive from the medullary respiratory neurons to the phrenic motoneurons.