Yukio Yajima

(-)-Epigallocatechin gallate protects against NO stress-induced neuronal damage after ischemia by acting as an anti-oxidant.

The present study investigated the effects of (-)-epigallocatechin gallate (EGCG), which is the major component of polyphenol in green tea, on nitric oxide (NO) stress-induced neuronal damage, by monitoring NO mobilizations in the intact rat hippocampus and assaying the viability of cultured rat hippocampal neurons. A 10-min ischemia increased NO (NO(3)(-)/NO(2)(-)) concentrations in the intact rat hippocampus, while EGCG (50 mg/kg i.p.) inhibited the increase by 77% without affecting hippocampal blood flow. The NO donor, sodium nitroprusside (SNP; 50 microM), produced NO (NO(3)(-)/NO(2)(-)), while EGCG inhibited it in a dose-dependent manner at concentrations ranging from 50 to 200 microM. Treatment with SNP (100 microM) reduced the viability of cultured rat hippocampal neurons to 22% of control levels, while EGCG caused it to recover to 51% for 10 microM, 73% for 20 microM, and 70% for 50 microM. Taken together, it appears that EGCG could protect against ischemic neuronal damage by deoxidizing peroxynitrate/peroxynitrite, which is converted to NO radical or hydroxy radical.

The midbrain central gray substance as a highly sensitive neural structure for the production of ultrasonic vocalization in the rat

To test whether there were any functional differences between the central gray substance (PVG) and other neural structures for the production of ultrasonic vocalization in the rat, an electrical stimulation experiment was systematically undertaken in diencephalic and mesencephalic tegmental regions. The sound production sensitivity to electrical stimulation was the highest in PVG. This suggested that the periventricular fiber system of Schützs bundle might be a possible neural structure underlying sound emission in the rat.

Subnuclear distribution of afferents from the oral, pharyngeal and laryngeal regions in the nucleus tractus solitarii of the rat: a study using transganglionic transport of cholera toxin.

The central distributions of afferents from the oral cavity, the pharynx, the larynx and the esophagus to the nucleus tractus solitarii (NTS) were examined by using transganglionic anterograde transport of the cholera toxin B subunit (CT-b). Injections of CT-b into the body of the tongue and the hard palate resulted in heavy labeling of the lateral subnucleus (l-NTS) of the NTS rostral to the area postrema. Injection into the root of the tongue resulted in heavy labeling of the l-NTS, the dorsal half of the medial (m-NTS), the intermediate (im-NTS) and the interstitial (is-NTS) subnuclei rostral to the area postrema. Injections into the soft palate and the pharynx resulted in a similar labeling pattern in the is-NTS, im-NTS and m-NTS to that in the case of the root of the tongue, but this labeling extended rostrocaudally. Heavy labeling of the medial aspect of the l-NTS was found in the case of the soft palate, but the labeling was sparse in the case of the pharynx. Moderate labeling was also found in the commissural subnucleus (co-NTS). Injection into the larynx resulted in labeling of the is-NTS throughout the NTS, and of the rostral half of im-NTS. Injection into the esophagus resulted in heavy labeling of the central subnucleus, and moderate labeling of the co-NTS and the caudal half of im-NTS. A few but consistent anterogradely labeled terminals were found to appose retrogradely labeled small neurons in the rostral tip of the dorsal motor nucleus of vagus in the cases of injections into the root of the tongue, the soft palate, the pharynx, and the larynx. These results have characterized the viscerotopic representation of afferent projections from the oral and the cervical visceral organs to the subnuclei of the NTS.

DIRECT SYNAPTIC PROJECTIONS TO ESOPHAGEAL MOTONEURONS IN THE NUCLEUS AMBIGUUS FROM THE NUCLEUS OF THE SOLITARY TRACT OF THE RAT

Neurons of the nucleus of the solitary tract (NTS) serve as interneurons in swallowing. We investigated the synaptology of the terminals of these neurons and whether they project directly to the esophageal motoneurons in the compact formation of the nucleus ambiguus (AmC). Following wheat germ agglutinin conjugated horseradish peroxidase (WGA‐HRP) injection into the NTS, many anterogradely labeled axodendritic terminals were found in the neuropil of the AmC. The majority of labeled axodendritic terminals (89%) contained round vesicles and made asymmetric synaptic contacts (Grays type I), but a few (11%) contained pleomorphic vesicles and made symmetric synaptic contacts (Grays type II). More than half of the labeled terminals contacted intermediate dendrites (1‐2 μm diameter). There were no retrogradely labeled medium‐sized motoneurons, but there were many retrogradely labeled small neurons having anterogradely labeled axosomatic terminals. A combined retrograde and anterograde transport technique was developed to verify the direct projection from the NTS to the esophageal motoneurons. After the esophageal motoneurons were retrogradely labeled by cholera toxin subunit B conjugated HRP, the injection of WGA‐HRP into the NTS permitted ultrastructural recognition of anterogradely labeled axosomatic terminals contacting directly labeled esophageal motoneurons. Serial sections showed that less than 20% of the axosomatic terminals were labeled in the esophageal motoneurons. They were mostly Grays type I, but a few were Grays type II. In the small neurons, more than 30% of axosomatic terminals were labeled, which were exclusively Grays type I. These results indicate that NTS neurons project directly not only to the esophageal motoneurons, but also to the small neurons which have bidirectional connections with the NTS. J. Comp. Neurol. 381:18‐30, 1997.

Synaptology of the direct projections from the nucleus of the solitary tract to pharyngeal motoneurons in the nucleus ambiguus of the rat

During the pharyngeal phase of the swallowing reflex, the nucleus of the solitary tract (NTS) receives peripheral inputs from the pharynx by means of the glossopharyngeal ganglion and is the location of premotor neurons for the pharyngeal (PH) motoneurons. The semicompact formation of the nucleus ambiguus (AmS) is composed of small and medium‐sized neurons that do not project to the pharynx, and large PH motoneurons. We investigated whether the neurons in the NTS projected directly to the PH motoneurons or to the other kinds of neurons in the AmS by using the electron microscope. When wheat germ agglutinin‐conjugated horseradish peroxidase (WGA‐HRP) was injected into the NTS after cholera toxin subunit B‐conjugated HRP (CT‐HRP) injections into the pharyngeal muscles of male Sprague‐Dawley rats, many nerve terminals anterogradely labeled with WGA‐HRP were found to contact PH motoneurons retrogradely labeled with CT‐HRP. Most of the labeled axodendritic terminals (63%) contained pleomorphic vesicles with symmetric synaptic contacts (Grays type II), and the remaining ones contained round vesicles with asymmetric synaptic contacts (Grays type I). About 14% of the axosomatic terminals on PH motoneuron in a sectional plane were anterogradely labeled, and about 70% of the labeled axosomatic terminals were Grays type II. Observations of serial ultrathin sections revealed that both the small and the medium‐sized neurons received only a few labeled axosomatic terminals that were exclusively Grays type I. These results indicate that the NTS neurons may send mainly inhibitory as well as a few excitatory inputs directly to the PH motoneurons in the AmS. J. Comp. Neurol. 393:391–401, 1998.

ULTRASTRUCTURAL CHARACTERIZATION OF PHARYNGEAL AND ESOPHAGEAL MOTONEURONS IN THE NUCLEUS AMBIGUUS OF THE RAT

The viscerotopic organization of the upper alimentary tract has been established in the nucleus ambiguus, but there is little information about the morphology of the individual neurons innervating the pharynx and esophagus. We studied the ultrastructure of pharyngeal (PH), cervical esophageal (CE), and subdiaphragmatic esophageal (SDE) motoneurons labeled by retrogradely transported wheat germ agglutinin conjugated horseradish peroxidase (WGA‐HRP) in the compact formation of the nucleus ambiguus. WGA‐HRP was injected into the lower pharynx, or the cervical and subdiaphragmatic esophagus of male rats. The retrogradely labeled PH neurons in the rostral portion of the compact formation were large (26.1 × 50.1 μm, 906.7 μm2), polygonal, and contained well‐developed cell organelles with a round nucleus. Subsurface cisterns connected with rough endoplastic reticulum were often present near the postsynaptic membrane. Both CE and SDE neurons in the compact formation were medium‐sized, round or oval, and contained well‐developed cell organelles, although the SDE neuron was significantly larger than the CE neuron (24.9 × 33.6 μm, 593.0 μm2 in the SDE neuron, and 19.5 × 30.2 μm, 440.3 μm2 in the CE neuron).

Identification of ultrasonic vocalization substrates determined by electrical stimulation applied to the medulla oblongata in the rat

The aim of the present investigation was to identify the neural structures, within the rat medulla, that are responsible for rodent ultrasound production. Sound producing substrates were found to be located in the reticular formation and some cranial nerve nuclei as well as several other nuclei situated in the lateral and dorsomedial portions of the medulla. To estimate the degree of involvement in the generation of ultrasound signals, the sound response latencies were measured for each structure. The lateral reticular nucleus and the facial nucleus showed latencies that were significantly shorter than those for other nuclei, and they were assumed to have a primary part in rodent ultrasound production. Audible sounds of considerably longer latencies were produced exclusively by stimulation of the trigeminal spinal tract nucleus. No ultrasounds could be obtained in this region. These results were discussed in terms of innervations of the facial and laryngeal musculature by the specific neural structures. Present results were also discussed with reference to the roles of the bulbar monoaminergic neurons projecting to the spinal cord and the role of ascending nociceptive pathways.

Adenosine triphosphate accelerates recovery from hypoxic/hypoglycemic perturbation of guinea pig hippocampal neurotransmission via a P2 receptor

The present study was designed to assess the effects of adenosine triphosphate (ATP) on hippocampal neurotransmissions under the normal and hypoxic/hypoglycemic conditions. ATP reversely depressed population spikes (PSs), which were monitored in the dentate gyrus of guinea pig hippocampal slices, in a dose-dependent manner at concentrations ranged from 0.1 micro M to 1 mM. A similar depression was obtained with the P(2) receptor agonist, alpha,beta-methylene ATP (alpha,beta-MeATP), and the effect was inhibited by the P(2) receptor antagonists, suramin and PPADS. The inhibitory action of ATP or alpha,beta-MeATP was inhibited by the gamma-aminobutyric acid(A) (GABA(A)) receptor antagonist, bicuculline, but it was not affected by theophylline, a broad inhibitor of adenosine (P(1)) receptors, tetraethylammonium, a broad inhibitor of K(+) channels, or ecto-protein kinase inhibitors. ATP or alpha,beta-MeATP enhanced GABA release from guinea pig hippocampal slices, that was inhibited by deleting extracellular Ca(2+) or in the presence of tetrodotoxin, while ATP had no effect on GABA release from cultured rat hippocampal astrocytes or postsynaptic GABA-gated channel currents in cultured rat hippocampal neurons. Twenty-minutes deprivation of glucose and oxygen from extracellular solution abolished PSs, the amplitude recovering to about 30% of basal levels 50 min after returning to normal conditions. ATP or alpha,beta-MeATP accelerated the recovery after hypoxic/hypoglycemic insult (approximately 80% of basal levels). Adenosine diphosphate and adenosine monophosphate accelerated the recovery, but to a much lesser extent, and adenosine had no effect. The results of the present study thus suggest that ATP inhibits neuronal activity by enhancing neuronal GABA release via a P(2) receptor, perhaps a P2X receptor, thereby protecting against hypoxic/hypoglycemic perturbation of hippocampal neurotransmission.

Ambiguus motoneurons discharging closely associated with ultrasonic vocalization in rats

Unitary discharges closely associated with ultrasound were recorded from the nucleus ambiguus (NA) and adjacent medullary regions in rats. Two main types of units were distinguished: one firing in tonic bursts against little or no background activity prior to ultrasound emission, and the other remaining silent during ultrasound. Tonic burst units in NA are considered as motoneurons which are essential for ultrasound vocalization since lesions including NA completely abolished its production.

SYNAPTOLOGY AND ULTRASTRUCTURAL CHARACTERISTICS OF LARYNGEAL CRICOTHYROID AND POSTERIOR CRICOARYTENOID MOTONEURONS IN THE NUCLEUS AMBIGUUS OF THE RAT

 The laryngeal motoneurons innervating the cricothyroid muscle (CT) are located in the semicompact formation just ventral to the rostral part of the compact formation of the nucleus ambiguus. The motoneurons innervating the posterior cricoarytenoid muscle (PCA) are located in the loose formation. We retrogradely labeled the CT and the PCA motoneurons using cholera toxin subunit B-conjugated horseradish peroxidase, and determined the ultrastructure and synaptic organization of these neurons. The CT and the PCA motoneurons had the appearance of α-motoneurons, i.e., large, oval or polygonal cells containing well-developed organelles and a prominent spherical nucleus. Two kinds of neurons were recognized among the PCA motoneurons. The one (PCA-A) was significantly smaller than the other (PCA-B). The average number of axosomatic terminals in a section was significantly largest in the PCA-B (56.6), smaller in the PCA-A (36.0), and smallest in the CT (32.3) neurons. Most of the axosomatic terminals (64.7%) contained pleomorphic vesicles and made symmetric synaptic contacts (Gray’s type II) with the PCA-A neurons, while more than 60% contained round vesicles with asymmetric synaptic contacts (Gray’s type I) in the CT (69.5%) and the PCA-B (60.6%) neurons. A few terminals associated with subsurface cisterns were present on all laryngeal motoneurons. These results indicated that the CT motoneurons may receive mostly excitatory terminals, whereas the PCA muscle may be regulated by neurons having many inhibitory terminals, and neurons having many excitatory terminals.