Akinori Takanaga

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.

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.

Distribution and ultrastructure of dopaminergic neurons in the dorsal motor nucleus of the vagus projecting to the stomach of the rat.

Almost all parasympathetic preganglionic motor neurons contain acetylcholine, whereas quite a few motor neurons in the dorsal motor nucleus of the vagus (DMV) contain dopamine. We determined the distribution and ultrastructure of these dopaminergic neurons with double-labeling immunohistochemistry for tyrosine hydroxylase (TH) and the retrograde tracer cholera toxin subunit b (CTb) following its injection into the stomach. A few TH-immunoreactive (TH-ir) neurons were found in the rostral half of the DMV, while a moderate number of these neurons were found in the caudal half. Most of the TH-ir neurons (78.4%) were double-labeled for CTb in the half of the DMV caudal to the area postrema, but only a few TH-ir neurons (5.5%) were double-labeled in the rostral half. About 20% of gastric motor neurons showed TH-immunoreactivity in the caudal half of the DMV, but only 0.3% were TH-ir in the rostral half. In all gastric motor neurons, 8.1% were double-labeled for TH. The ultrastructure of the TH-ir neurons in the caudal DMV was determined with immuno-gold-silver labeling. The TH-ir neurons were small (20.4 x 12.4 microm), round or oval, and contained numerous mitochondria, many free ribosomes, several Golgi apparatuses, a round nucleus and a few Nissl bodies. The average number of axosomatic terminals per section was 4.0. More than half of them contained round synaptic vesicles and made asymmetric synaptic contacts (Grays type I). Most of the axodendritic terminals contacting TH-ir dendrites were Grays type I (90%), but a few contained pleomorphic vesicles and made symmetric synaptic contacts (Grays type II).

Monosynaptic inputs from the nucleus tractus solitarii to the laryngeal motoneurons in the nucleus ambiguus of the rat.

The cricothyroid (CT) and the posterior cricoarytenoid (PCA) muscles in the larynx are activated by the laryngeal motoneurons located within the nucleus ambiguus; these motoneurons receive the laryngeal sensory information from the nucleus tractus solitarii (NTS) during respiration and swallowing. We investigated whether the neurons in the NTS projected directly to the laryngeal motoneurons, and what is the synaptic organization of their nerve terminals on the laryngeal motoneurons 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) was injected into the CT muscle or the PCA muscle, the anterogradely WGA-HRP-labeled terminals from the NTS were found to directly contact the retrogradely CT-HRP-labeled dendrites and soma of both the CT and the PCA motoneurons. The labeled NTS terminals comprised about 4% of the axosomatic terminals in a section through the CT motoneurons, and about 9% on both the small (PCA-A) and the large (PCA-B) PCA motoneurons. The number of labeled axosomatic terminals containing round vesicles and making asymmetric synaptic contacts (Gray’s type I) was almost equal to that of the labeled terminals containing pleomorphic vesicles and making symmetric synaptic contacts (Gray’s type II) on the CT motoneurons. The labeled axosomatic terminals were mostly Gray’s type II on the PCA-A motoneurons, while the majority of them were Gray’s type I on the PCA-B motoneurons. These results indicate that the laryngeal CT and PCA motoneurons receive a few direct excitatory and inhibitory inputs from the neurons in the NTS.

α-Synuclein-positive structures induced in leupeptin-infused rats

Abnormal accumulation of alpha-synuclein is regarded as a key pathological step in a wide range of neurodegenerative processes, not only in Parkinsons disease (PD) and dementia with Lewy bodies (DLB) but also in multiple-system atrophy (MSA). Nevertheless, the mechanism of alpha-synuclein accumulation remains unclear. Leupeptin, a protease inhibitor, has been known to cause various neuropathological changes in vivo resembling those of aging or neurodegenerative processes in the human brain, including the accumulation of neuronal processes and neuronal cytoskeletal abnormalities leading to neurofibrillary tangle (NFT)-like formations. In the present study, we administered leupeptin into the rat ventricle and found that alpha-synuclein-positive structures appeared widely in the neuronal tissue, mainly in neuronal processes of the fimbria and alveus. Immunoelectron microscopic study revealed that alpha-synuclein immunoreactivity was located in the swollen axons of the fimbria and alveus, especially in the dilated presynaptic terminals. In addition colocalization of alpha-synuclein with ubiquitin was rarely observed in confocal laser-scan image. This is the first report of experimentally induced in vivo accumulation of alpha-synuclein in non-transgenic rodent brain injected with a well-characterized protease inhibitor by an infusion pump. The present finding suggests that the local accumulation of alpha-synuclein might be induced by the impaired metabolism of alpha-synuclein, which are likely related to lysosomal or ubiquitin-independent proteasomal systems.

The intrinsic origin of nitric oxide synthase immunoreactive nerve fibers in the right atrium of the guinea pig.

We previously reported three kinds of nitric oxide synthase-immunoreactive (NOS-ir) axons in the guinea pig heart: the sparse fiber network covering the right atrium, the basket-like endings around intracardiac neurons, and the axons in the septal region. The sparse NOS-ir nerve fiber network in the right atrium remained after vagotomy and has been suggested to be originated from intrinsic cardiac ganglia. Using Chorera toxin B as a retrograde tracer, we determined a part of them were derived from cardiac ganglionic neurons located in the area near the vena cavae.

Ultrastructure of the rostral ventral respiratory group neurons in the ventrolateral medulla of the rat.

The neurons in the ventrolateral medulla that project to the spinal cord are called the rostral ventral respiratory group (rVRG) because they activate spinal respiratory motor neurons. We retrogradely labeled rVRG neurons with Fluoro-Gold (FG) injections into the fourth cervical spinal cord segment to determine their distribution. The rostral half of the rVRG was located in the area ventral to the semicompact formation of the nucleus ambiguus (AmS). A cluster of the neurons moved dorsally and intermingled with the palatopharyngeal motor neurons at the caudal end of the AmS. The caudal half of the rVRG was located in the area including the loose formation of the nucleus ambiguus caudal to the AmS. We also labeled the rVRG neurons retrogradely with wheat germ agglutinin-horseradish peroxidase (WGA-HRP) to determine their ultrastructural characteristics. The neurons of the rVRG were medium to large (38.1 x 22.1 microm), oval or ellipsoid in shape, and had a dark cytoplasm containing numerous free ribosomes, rough endoplasmic reticulum (rER), mitochondria, Golgi apparatuses, lipofuscin granules and a round nucleus with an invaginated nuclear membrane. The average number of axosomatic terminals in a profile was 33.2. The number of axosomatic terminals containing round vesicles and making asymmetric synaptic contacts (Grays type I) was almost equal to those containing pleomorphic vesicles and making symmetric synaptic contacts (Grays type II). The axodendritic terminals were large (1.55 microm), and about 60% of them were Grays type I. The rVRG neurons have ultrastructural characteristics, which are different from the palatopharyngeal motor neurons or the prorpiobulbar neurons.

A novel monoclonal antibody recognizes lysosome-like structures and reflects regional and age-related differences in the rat dentate gyrus

The granule cells (GCs) of dentate gyrus exhibit regionally specific morphology, and continue to be born and to develop well into adult life. We used a novel monoclonal antibody, MAb2G7, elicited by immunization of a mouse with a microsome fraction of the hippocampus, to evaluate regional and age-related differences in GCs immunohistochemically. Weak cytoplasmic reactions were observed in many neurons, but intense MAb2G7-positive dots were observed only in GCs. Using electron microscopy, we observed that these dots were localized in the internal droplets of secondary lysosome-like structures in GCs. The MAb2G7-positive granules were quantitatively analyzed in young adult and middle-aged rats. Larger numbers of reactive granules were observed in the infrapyramidal blade (IPB) than in the suprapyramidal blade (SPB) and the numbers of positive granules were proportionally reduced in the two areas in middle-aged rats. The changes in the MAb2G7 immunoreactivity may reflect different activation or neurogeneration of GCs in the IPB versus the SPB, and in middle-aged versus young adult rats.