Hitoshi Bamba

Cytochemical study of prolactin-releasing peptide (PrRP) in the rat brain.

Strong positive signals for PrRP mRNA and PrRP-like immunoreactivity (PrRP-LI) were detected in the nucleus of the solitary tract and ventral and lateral reticular formation of the caudal medulla oblongata. Weak mRNA signals and immunoreactivity were seen scattered from the hypothalamic dorsomedial nucleus (DMH) to ventromedial nucleus (VMH). Nerve processes and terminals with PrRP-LI were detected from the septal region to the diencephalon. These nerve processes were also clearly visible around capillary walls and in the vicinity of the ependymal cells of the third and lateral ventricles. These observations suggested that PrRP might be secreted into the systemic circulation and cerebrospinal fluid and may play functional roles other than in the release of prolactin from the anterior pituitary.

Synergistic effect between proteasome and autophagosome in the clearance of polyubiquitinated TDP-43

Cytoplasmic aggregates of ubiquitinated TAR DNA‐binding protein 43 (TDP‐43) are a pathological hallmark of amyotrophic lateral sclerosis (ALS). However, the mechanism of TDP‐43 polyubiquitination remains elusive. We investigated the effect of nuclear exclusion of TDP‐43 on aggregate formation and fragmentation, using TDP‐43 expression constructs for WT or mutant TDP‐43 with a modified nuclear localizing signal (LQ‐NLS). Overexpression of the LQ‐NLS mutant alone induced no detectable cytoplasmic aggregates during a 72‐hr period. Polyubiquitination of both WT TDP‐43 and the LQ‐NLS mutant was similar in total cell lysates exposed to the proteasome inhibitor lactacystin. However, analysis of subcellular fractions demonstrated a higher concentration of polyubiquitinated TDP‐43 in the nuclear fraction than in the cytosol for WT, and vice versa for the LQ‐NLS mutant. Polyubiquitin‐charged WT and mutant TDP‐43 were highly concentrated in the membrane/microsome fraction, which was also positive for the autophagosome marker LC3. In addition, the autophagy inhibitor 3‐methyladenine (3MA) blocked degradation of both TDP‐43 types, whereas lactacystin was minimally restorative. Furthermore, lactacystin plus 3MA induced prominent cytoplasmic aggregates. We also demonstrated mediation of TDP‐43 polyubiquitination by lysine 48 of ubiquitin, indicating a degradation signal in both TDP‐43 types. This is the first report delineating the distribution of polyubiquitinated TDP‐43 and the degradation pathway of TDP‐43 and clarifying the crucial role of autophagosomes in TDP‐43 clearance. We also demonstrate that nuclear exclusion itself is not an immediate trigger for ALS pathology. Further clarification of the mechanism of polyubiquitination of TDP‐43 and the role of autophagosomes may help in understanding and treating ALS.

Axonal ligation induces transient redistribution of TDP-43 in brainstem motor neurons.

Nuclear exclusion of TAR DNA binding protein 43 (TDP-43) and formation of cytosolic aggregates are a pathological characteristic of amyotrophic lateral sclerosis (ALS). However, the molecular basis of the aberrant distribution of TDP-43 remains elusive. Here, we show evidence that axonal ligation induced transient nuclear exclusion and peripheral accumulation of TDP-43, without apparent cytosolic aggregates in hypoglossal neurons in mice. Immunohistochemistry showed marked loss of nuclear TDP-43 7-14 days after ligation, which was accompanied by reduction of choline acetyltransferase (ChAT). TDP-43 staining was restored in the nucleus on day 28 exclusively in the neurons with normalized ChAT expression. We also showed that importin beta, which was shown to mediate nuclear transport of TDP-43 was downregulated transiently by nerve ligation. The analysis of the peripheral nerves proximal to the ligation revealed that TDP-43 markedly accumulated with a concomitant decrease in active autophagosome. Moreover, we showed that TDP-43 was present in the microsome fraction containing endoplasmic reticulum (ER) or autophagosomes in the brainstem section, indicating that TDP-43 is axonally transported with vesicles. These results indicate that axonal damage is associated with redistribution of TDP-43 through the combination of defective axonal autophagy periphery and the impaired nuclear transport system in the soma. Moreover, it was also shown that transient redistribution of TDP-43 does not prevent motor neurons from axonal regeneration. Therefore, our data suggest that the subcellular distribution of TDP-43 correlates to the innervation status of motor neurons, which may be governed by unidentified cause of ALS.

Vocal Fold Paralysis as a Sign of Chest Diseases: A 15-year Retrospective Study

BackgroundVocal fold paralysis (VFP) is sometimes the only sign of chest diseases. However, some patients with VFP due to chest diseases are not diagnosed correctly at the first examination, which may leave the patients untreated for a long time. Depending on the situation, chest x-ray is not enough for detecting the primary lesion. The objective of this study was to discuss the diagnostic procedure for VFP based on the retrospective analysis of the cases.MethodsA total of 42 patients (29 males and 13 females) with VFP due to chest disease examined at the Department of Otolaryngology of Kyoto Prefectural University of Medicine between 1988 and 2002 were reviewed retrospectively.ResultsOf the primary chest diseases, lung cancer (15 cases) was the most common, followed by thoracic aortic aneurysm (TAA) (9 cases), metastatic tumor from other regions (6 cases), pulmonary and mediastinal tuberculosis (TB) (5 cases), and esophageal cancer (4 cases). While the primary lesions were easily detected with chest x-ray in most of the cases, some lesions in the aortopulmonary window were difficult to detect. Contrast-enhanced computed tomography (CT) was useful to detect any mass in this region.ConclusionsIn the diagnosis of VFP due to chest diseases, chest x-ray was useful but not always enough for detecting the primary lesion. Necessity of further examinations including contrast-enhanced chest CT must be kept in mind for the cases with negative chest radiographs.

Localization of Fibroblast Growth Factor-1 in Cholinergic Neurons Innervating the Rat Larynx

Cholinergic neurons in the dorsal motor nucleus of the vagus (DMNV) are particularly vulnerable to laryngeal nerve damage, possibly because they lack fibroblast growth factor-1 (FGF1). To test this hypothesis, we investigated the localization of FGF1 in cholinergic neurons innervating the rat larynx by immunohistochemistry using central-type antibodies to choline acetyltransferase (cChAT) and peripheral type (pChAT) antibodies, as well as tracer experiments. In the DMNV, only 9% of cChAT-positive neurons contained FGF1, and 71% of FGF1-positive neurons colocalized with cChAT. In the nucleus ambiguus, 100% of cChAT-positive neurons were FGF1 positive. In the intralaryngeal ganglia, all ganglionic neurons contained both pChAT and FGF1. In the nodose ganglia, 66% of pChAT-positive neurons were also positive for FGF1, and 90% of FGF1-positive ganglionic cells displayed pChAT immunoreactivity. Neuronal tracing using cholera toxin B subunit (CTb) demonstrated that cholinergic neurons sending their axons from the DMNV and nucleus ambiguus to the superior laryngeal nerve were FGF1 negative and FGF1 positive, respectively. In the nodose ganglia, some FGF1-positive cells were labeled with CTb. The results indicate that for innervation of the rat larynx, FGF1 is localized to motor neurons, postganglionic parasympathetic neurons, and sensory neurons, but expression is very low in preganglionic parasympathetic cholinergic neurons.

Distribution of vanilloid receptors in the rat laryngeal innervation.

Objective Capsaicin is known to selectively activate nociceptic sensory neurons through vanilloid receptors. In this study we investigated the distribution of vanilloid receptor subtype 1 (VR1) and vanilloid receptor-like protein 1 (VRL-1) in the rat larynx. Material and Methods The distributions of VR1 and VRL-1 were determined immunohistochemically. The colocalization of vanilloid receptors with common choline acetyltransferase (cChAT), vasoactive intestinal polypeptide (VIP), substance P (SP) and neuronal nitric oxide synthase (nNOS) was also studied using an immunohistochemical double-labeling technique. Results VRL-1-positive fibers were detected in the laryngeal epithelium and lamina propria. VR1-positive nerve fibers were seen in the lamina propria but not in the mucosal epithelium. VR1- and VRL-1-positive cells were distributed in the intralaryngeal ganglia and colocalization of capsaicin receptors with VIP, nNOS and cChAT was seen. Conclusion These findings suggest that these capsaicin receptors participate in the parasympathetic innervation as well as in nociception of the rat larynx.

Axotomy alters alternative splicing of choline acetyltransferase in the rat dorsal motor nucleus of the vagus nerve

Choline acetyltransferase of the peripheral type (pChAT) is a splice variant that lacks exons 6–9 of the common‐type ChAT (cChAT); the role of pChAT remains unknown. We investigated the expression of pChAT and cChAT after axotomy to try to elucidate its function. In the dorsal motor nucleus of the vagus nerve (DMNV), nucleus ambiguus (NA), and hypoglossal nucleus (HN) of control rats, we observed neural expression of cChAT but no pChAT‐positive neurons. Following nerve transection, we clearly detected pChAT‐labeled neurons in the DMNV and weakly labeled neurons in the NA, but pChAT was not seen in the HN. In the DMNV, the mean number of cChAT‐positive neurons decreased rapidly to 40.5% of control at 3 days post transection, and to 5.0% of control after 7 days. The number of cChAT‐positive neurons then gradually increased and reached a plateau of about 25% of control value at 28 days post transection. pChAT‐positive neurons did not appear until 7 days after transection. On the same day, pChAT mRNA was detected in the DMNV neurons by reverse transcription‐polymerase chain reaction (RT‐PCR) by using laser capture microdissection. The number of pChAT‐positive neurons gradually decreased, and only 10% of the cholinergic neurons retained pChAT expression 56 days post transection. Double‐immunofluorescence analysis showed that some of the DMNV neurons expressed both cChAT and pChAT upon recovery from axotomy. These results suggest that the expression of pChAT is associated with the regenerative or degenerative processes of motoneurons especially for general visceral efferents. J. Comp. Neurol. 513:237–248, 2009.

Neurotransmitters and neuromodulators involved in laryngeal innervation

The distribution and role of neurotransmitters and neuromodulators in laryngeal innervation are reviewed, and our recent findings regarding the nitrergic innervation of the larynx are demonstrated for the better understanding of the complexity of the laryngeal innervation system. Noradrenergic innervation of the larynx was studied with fluorescence histochemistry and electron microscopy after application of 5-hydroxydopamine. These studies confirmed the existence of noradrenergic innervation for the submucosal glands and blood vessels, and the origin and course of noradrenergic nerve fibers contained in the laryngeal nerves and their destinations in the larynx. Cholinergic innervation of the larynx has not been clarified in detail. Many kinds of neuropeptides have been demonstrated to be involved in laryngeal innervation. Vasoactive intestinal polypeptide originating from intralaryngeal ganglionic neurons participates in laryngeal vasodilation and reduction of laryngeal seromucous secretion. Neuropeptide Y nerve fibers are few in the larynx, and most originate from the superior cervical ganglion. They are distributed around the large or medium-sized blood vessels, especially arteries. They are also associated with excretory structures. Substance P was the first neuropeptide found to be a sensory neurotransmitter in the laryngeal afferent system. It is also involved in regulation of laryngeal blood flow and secretion. Calcitonin gene—related peptide is associated with the sensory, autonomic, and motor innervation of the larynx. The majority of enkephalin nerve fibers are located close to excretory structures, although no information on the physiological significance of enkephalin is available. In addition to the above neuropeptides, the peptides histidine isoleucine, histidine methionine, and helospectin have been shown to exist in the larynx. The nitrergic innervation of the larynx has been recently studied with NADPH-diaphorase histochemistry and immunohistochemistry using antiserum against nitric oxide synthase. Nitric oxide originates from the neurons in the intralaryngeal ganglia and is believed to modulate blood flow and secretion of the larynx. It controls the laryngeal exocrine secretion in cooperation with intrinsic vasoactive intestinal polypeptide and/or extrinsic calcitonin gene—related peptide. Nitric oxide from the nodose ganglion may modulate nociception of the larynx. The existence of nitrergic neurons located in the intrinsic laryngeal muscles has been demonstrated. Many of them are bipolar or pseudounipolar, so they might be sensory in nature. The effect of injury of the recurrent laryngeal nerve on the induction of nitric oxide synthase in the laryngeal motoneurons is also discussed.

A Case of Vocal Cord Cyst With Cryptococcal Infection

t c c A c F ryptococcus neoformans is an opportunistic fungus that is found abundantly in the excreta of pigeons. The lung s the most common site of infection. Cryptococcosis of the larynx is very rare. The first case f laryngeal cryptococcosis was reported by Reese and olclasure in 1975. Only 7 cases have been reported in the nglish literature previously. However, this is the 1st report of cryptococcal infection f the vocal cord cyst in the world.

Capsaicin receptor expression in rat laryngeal innervation.

Capsaicin elicits a sensation of burning pain by selectively activating sensory neurons that convey information about noxious stimuli to the central nervous system. Vanilloid receptor subtype 1 (VR1) and the vanilloid receptor-like protein 1 (VRL-1) are activated, not only by capsaicin, but also by noxious heat and protons, and it has been suggested that they are polymodal nociceptors. We investigated the expression of VR1 and VRL-1 in the rat larynx and nodose ganglion using VR1 and VRL-1 immunohistochemical analysis with visualization by diaminobenzidine reaction. Fibers positive for VRL-1 were detected in the laryngeal epithelium and lamina propria. Cells positive for VRL-1 were distributed in the intralaryngeal ganglia. Half of the neurons in the nodose ganglion had VR-1 immunoreactivity, and almost 10% of the nodose ganglion neurons were positive for VRL-1. These findings suggest that these capsaicin receptors play an important role in the nociception of the laryngeal innervation.