Tetsu Hayakawa

Comparative anatomical study of the tegmentomammillary projections in some mammals: a horseradish peroxidase study.

Mammillary projections from each subnuclei of Gudden s tegmental nuclei were investigated by retrograde transport of horseradish peroxidase (HRP) in the cat, rat, guinea pig, golden hamster and house shrew (Suncus murinus, Insectivora). The dorsal tegmental nucleus of Gudden (TD) is composed of the pars dorsalis (TDD) and the pars ventralis ( TDV ) in the cat, rat, guinea pig, and golden hamster, but the TD of the house shrew can not be divided. The ventral tegmental nucleus of Gudden (TV) is composed of the pars principalis (TVP) and the pars suprafascicularis ( TVS ) in the golden hamster and house shrew, but the TVS is not recognized in the cat, rat and guinea pig. The TDV projects to the lateral mammillary nucleus, and the TVP projects to the medial mammillary nucleus ipsilaterally in the cat, rat, guinea pig and golden hamster. The TVS of the golden hamster projects to the medial mammillary nucleus. The TVP and TVS of the house shrew project to the medial mammillary nucleus, and the TVS also to the lateral mammillary nucleus. In addition, the pars compacta of the nucleus centralis superior projects to the medial mammillary nucleus in the rat, guinea pig and golden hamster. However, the TDD sends no fibers to the mammillary nuclei in these 5 species of mammals.

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.

Neuroanatomical study of afferent projections to the supramammillary nucleus of the rat

We examined the regions projecting to the supramammillary nucleus of the rat with retrograde transport of WGA-HRP and WGA, and anterograde transport of Phaseolus vulgaris leucoagglutinin. The supramammillary nucleus receives major descending afferents from the infralimbic cortex, the dorsal peduncular cortex, the nucleus of the diagonal band of Broca, the medial and lateral preoptic nuclei, bilaterally. The major ascending afferents come from the pars compacta of the nucleus centralis superior, the ventral tegmental nucleus, and the laterodorsal tegmental nucleus. The supramammillary nucleus also receives a few (but distinct) fibers from the anterior and lateral hypothalamic nuclei, the ventral premammillary nucleus, the interpeduncular nucleus, the cuneiform nucleus, the dorsal raphe nucleus, the incertus nucleus, and the C3 region including the prepositus hypoglossi nucleus. All descending fibers run through the medial forebrain bundle. Almost all ascending fibers from the pars compacta of the nucleus centralis superior and the laterodorsal tegmental nucleus run through the mammillary peduncle, and terminate throughout the supramammillary nucleus. A few fibers from the laterodorsal tegmental nucleus and the C3 region run through the fasciculus longitudinalis dorsalis and terminate in the dorsal part of the supramammillary nucleus including the supramammillary decussation.

Immunohistochemical characterization of cardiac vagal preganglionic neurons in the rat.

Cardiac vagal preganglionic neurons (CVN) control cardiac activity by negative chronotropic, dromotropic and inotropic effects. We attempted to characterize the distribution and neuronal properties of the CVN by using double labeling with the retrograde tracer cholera toxin B subunit (CTb) and immunohistochemistry for choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), calcitonin gene-related peptide (CGRP) or nitric oxide synthase (NOS). Injection of CTb into the sinoatrial ganglia resulted in many retrogradely labeled of neurons in the dorsal motor nucleus of the vagus (DMV), the compact (AmC), semicompact (AmS), loose (AmL), external (AmE) formations of the nucleus ambiguus, and the intermediate zone (IZ) between DMV and the nucleus ambiguus. Almost all CTb-labeled neurons showed ChAT immunoreactivity in the DMV, AmC, AmS, AmL and IZ, but most of the CTb-labeled neurons showed no ChAT immunoreactivity in the AmE. Most of the CTb-labeled neurons were double-labeled with CGRP immunoreactivity in the AmC, AmS and AmL, but a few double-labeled neurons were found in the DMV, IZ and AmE. A few CTb-labeled neurons were double-labeled with NOS immunoreactivity only in the DMV. No TH-immunoreactive neurons were found among the CVN. These results indicate that there are four kinds of neurons among the CVN: non-cholinergic CVN in the AmE, cholinergic and CGRP-containing CVN in the AmC, AmS and AmL, and cholinergic or cholinergic and NOS-containing CVN in the DMV.

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.

Brain pericytes serve as microglia-generating multipotent vascular stem cells following ischemic stroke

BackgroundMicroglia are the resident macrophage population of the central nervous system (CNS) and play essential roles, particularly in inflammation-mediated pathological conditions such as ischemic stroke. Increasing evidence shows that the population of vascular cells located around the blood vessels, rather than circulating cells, harbor stem cells and that these resident vascular stem cells (VSCs) are the likely source of some microglia. However, the precise traits and origins of these cells under pathological CNS conditions remain unclear.MethodsIn this study, we used a mouse model of cerebral infarction to investigate whether reactive pericytes (PCs) acquire microglia-producing VSC activity following ischemia.ResultsWe demonstrated the localization of ionized calcium-binding adaptor molecule 1 (Iba1)-expressing microglia to perivascular regions within ischemic areas. These cells expressed platelet-derived growth factor receptor-β (PDGFRβ), a hallmark of vascular PCs. PDGFRβ+ PCs isolated from ischemic, but not non-ischemic, areas expressed stem/undifferentiated cell markers and subsequently differentiated into various cell types, including microglia-like cells with phagocytic capacity.ConclusionsThe study results suggest that vascular PCs acquire multipotent VSC activity under pathological conditions and may thus be a novel source of microglia.

Morphological and immunohistochemical characterization of the trigeminal ganglion neurons innervating the cornea and upper eyelid of the rat.

The cornea is sensitive to nociceptive stimuli and receives dense sensory innervations from the trigeminal ganglion, which also innervates the upper eyelid. We investigated the morphological and immunohistochemical characterization of the trigeminal ganglion neurons innervating the cornea and upper eyelid. We injected the retrograde tracer Fluoro-Gold (FG) into the cornea and the retrograde tracer cholera toxin subunit b (CTb) into the upper eyelid of the same animal. Less than 10% of the FG-labeled neurons were also labeled with CTb. The FG-labeled neurons were small (29.6+/-0.6microm), while the CTb-labeled neurons were large (36.1+/-0.5microm). We also characterized the neurons in the trigeminal ganglion with the retrograde tracer FG following its injection into the cornea or the upper eyelid, and immunohistochemical double-labeling with nociception-related neuronal markers, such as calcitonin gene-related peptides (CGRP), transient receptor potentiated vanilloid 1 (TRPV1), and substance P (SP). About 27% of the neurons innervating the cornea were double-labeled with CGRP, about 23% with TRPV1, and about 8% with SP. About 4% of the neurons innervating the upper eyelid were double-labeled for CGRP, about 11% for TRPV1, and 3% for SP. Thus, the percentages of double-labeled neurons for the neurons innervating the cornea were higher than those for the neurons innervating the upper eyelid. These results indicate that the cornea and the upper eyelid receive innervations mainly from different neurons of the trigeminal ganglia. The cornea is innervated by many characteristic sensory neurons containing nociception-related neuronal markers.

Organization of the habenulo-interpeduncular connections in cats: A horseradish peroxidase study

The afferent fiber connections to the interpeduncular (IP) complex were demonstrated by the retrograde axonal transport of horseradish peroxidase (HRP) in cats. The HRP was injected into each nucleus of the IP complex, that is the central nucleus (IPC), the paramedian nucleus (IPP), the apical nucleus (IPA), and the posterior nucleus (IPN) including the outer division (IPO) and the inner division (IPI), and surrounding areas of the IP complex, using a ventral or dorsal surgical approaches. Most of the labeled neurons were in the medial habenular nucleus (MH) and each of the sub-nuclei of the IP complex was related to a specific part of the MH. Thus, the mediodorsal part of MH projected to the IPC, the medioventral part of MH projected to the IPI, the laterodorsal part of MH projected to the IPA, and the lateroventral part of MH projected to the IPP and the IPO. There were a few labeled cells in the accessory dorsal tegmental nucleus, the nucleus raphe dorsalis (RD), the nucleus centralis superior, the nucleus of the locus coeruleus, the gray matter of the floor of the fourth ventricle, and the nucleus of diagonal band of Broca, but there were no obvious patterns in the projections of these nuclei to the different sub-nuclei of the IP complex. When the area of the HRP injection involved the midbrain reticular formation adjacent to the IP complex and the nucleus reticularis tegmenti pontis (RT) but not the IP complex itself, there were many labeled cells in the lateral habenular nucleus and the medial and lateral mammillary nuclei, but there were no labeled cells in the medial habenular nucleus.

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 study of ascending projections to the lateral mammillary nucleus of the rat.

SummaryWe examined the synaptic organization of ascending projections from the pars ventralis of the dorsal tegmental nucleus of Gudden (TDV) and the laterodorsal tegmental nucleus to the lateral mammillary nucleus (LM). The LM neuropil consists of terminals containing pleomorphic synaptic vesicles and forming symmetric synaptic contact, and terminals containing round synaptic vesicles and forming asymmetric synaptic contact. They make up 63% and 37%, respectively, of all axodendritic terminals. All axosomatic terminals contain pleomorphic vesicles and make symmetric contact. Following injection of WGA-HRP into the TDV, many anterogradely labeled terminals and retrogradely labeled cells are found in the LM. Labeled terminals contact mainly proximal (more than 2 μm diameter) and intermediate (1–2 μm diameter) dendrites. Serial ultrathin sections of the LM show that 55% of axosomatic terminals are labeled anterogradely. Following injection of WGA-HRP into the laterodorsal tegmental nucleus, many anterogradely labeled terminals are found in the LM, but no retrogradely labeled cells are present. Labeled terminals contact mainly distal (less than 1 μm diameter) and intermediate dendrites as well as somata. In the LM neurons, 46% of axosomatic terminals are labeled anterogradely. All labeled terminals from these nuclei contain pleomorphic vesicles and make symmetric synaptic contact. These results indicate that almost all axosomatic terminals come from the TDV and the laterodorsal tegmental nucleus, which send inhibitory inputs to the lateral mammillary nucleus.