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.

Low expression of FGF1 (fibroblast growth factor-1) in rat parasympathetic preganglionic neurons.

Fibroblast growth factor-1 (FGF1), a member of the FGF family of growth factors, is localized in cholinergic neurons where it has trophic activity. We recently reported that cholinergic neurons in the dorsal motor nucleus of the vagus (DMNV) contain little FGF1, raising the possibility that FGF1 is not localized to parasympathetic preganglionic cholinergic neurons. To clarify this issue, we investigated the co-localization of FGF1 with cholinergic neuron markers in the Edinger-Westphal nucleus (EWN), salivatory nucleus, DMNV, and sacral parasympathetic nucleus by double immunofluorescence using antibodies to FGF1 and choline acetyltransferase (ChAT). The neurons in the EWN were devoid of FGF1. In the salivatory nucleus, 13% of ChAT-positive neurons were also positive for FGF1. In the DMNV, only 8% of ChAT-positive neurons contained FGF1, and in the sacral parasympathetic nucleus, 18% of ChAT-positive neurons were FGF1-positive. We also confirmed that a large number of ChAT-positive motor neurons in the oculomotor nucleus, facial nucleus, hypoglossal nucleus, and spinal motor neurons contained FGF1. The results confirmed that parasympathetic preganglionic neurons are largely devoid of FGF1, which is a unique feature among cholinergic neurons.

Immunohistochemical study of TAFII250 in the rat laryngeal nervous system.

The cause of spasmodic dysphonia, a dystonic disorder of the larynx, remains unclear. Recently, TAFII250, TATA-box binding protein associated factor, was suggested to be involved in dystonia parkinsonism. There is a possibility that TAFII250 is involved in spasmodic dysphonia, but little information is available about the expression of TAFII250 in the laryngeal nervous system. In this study, we investigated the localization of TAFII250 protein in the rat laryngeal nervous system by immunohistochemistry. TAFII250-immunoreactivity was detected in the nodose ganglion and superior cervical ganglion. In these nuclei, TAFII250 was localized in the nucleus of NeuroTrace-positive neurons but not in GFAP-positive glial cells. No positive cells were detected in the motor and parasympathetic nervous system. TAFII250-immunoreactivity was sustained between 3 and 7 days after vagotomy, but at 14 days expression was down-regulated in the distal part of the nodose ganglion. These findings suggest that TAFII250 plays an important role in the laryngeal innervation of the sensory and sympathetic nervous systems.

Localization of heme oxygenase-2 in the canine larynx.

The localization of heme oxygenase-2 (HO-2) in the larynx of the dog was investigated using immunohistochemistry. HO-2-positive cells were seen among neurons in intralaryngeal ganglia. Nerve fibers positive to HO-2 immunohistochemistry were seen surrounding laryngeal glands and arterioles and also in the adventitia of arterioles. HO-2-positive fibers were also seen running parallel to the mucosa in the lamina propria but no positive fibers were seen in the epithelium. Some of the intramuscular neurons found in the intrinsic laryngeal muscles were HO-2-positive, although no positive motor fibers were seen, and the neuromuscular junctions were also HO-2-negative. The results implicate the participation of HO-2-in the parasympathetic innervation of the larynx.

[Meta-analysis of pharmacotherapies for allergic rhinitis].

We performed meta-analysis using the data in literatures of the clinical study related to pharmacotherapies for allergic rhinitis in Japan as evidences. We extracted double-blind studies which used first-generation antihistamines, early-stage second-generation antihistamines, late-stage second-generation antihistamines and arachidonic acid metabolite-receptor antagonists as investigational drugs. In meta-analysis of first-generation antihistamines and early-stage second-generation antihistamines, significant differences between them were detected in final overall improvement and usefulness. In meta-analysis of early-stage second-generation antihistamines and late-stage second-generation antihistamines, significant differences between them were detected in usefulness and sleepiness as an adverse effect. In meta-analysis of late-stage second-generation antihistamines and arachidonic acid metabolite-receptor antagonists, significant differences between them were detected in final overall improvement and usefulness. These results indicate a historical trend in the development of drugs including measures to deal with sleepiness as an adverse effect. The arachidonic acid metabolite antagonists appeared to be promising among the oral drugs for allergic rhinitis, although data related to the arachidonic acid metabolite antagonists are still few and further collection of them is necessary.

[Value of upper gastrointestinal endoscopic examination of head and neck cancer patients].

Between January 1995 and March 1999, we performed the upper gastrointestinal endoscopic examinations on 287 patients with head and neck cancers and detected 23 cases (8%) of esophageal cancer and 8 cases (2.8%) of gastric cancer, showing how frequently esophageal cancer occurs in head and neck cancer. The esophageal cancer involved the oral cavity in 8 cases (9.5%), the oropharynx in 3 cases (8.6%), the hypopharynx in 10 cases (19.6%), and the larynx in 2 cases (2%). Esophageal cancer occurred most frequently in hypopharyngeal cancer, particularly the pyriform sinus type and the postcricoid type. We conclude that upper gastrointestinal endoscopic examination, including Lugol staining, is necessary in head and neck cancer patients.

[Simple evaluation of CT findings in the paranasal sinuses for chronic sinusitis].

The CT scores and scoring for improvement based on them, which we proposed previously, is a simple and highly reproducible method of evaluation of sinus units before and after an operation for chronic sinusitis. We compared this evaluation method with the results of quantitative assay and showed its advantages and disadvantages. The subjects were 258 sinuses in patients who underwent endonasal sinus surgery (ESS) in the department of otolaryngology, Kyoto Prefectural University of Medicine Hospital from April 1996 to April 1997. The subjects were evaluated according to the following 4 grades negligible shadow in the paranasal sinus CT scored 0, less than 50% shadow scored 1, more than 50% of shadow scored 2, and mostly filled with shadow scored 3. Furthermore, the preoperative and postoperative CT scores were compared and the rate of improvement was rated in the following 3 grades: score 0 for unchanged or aggravated subjects, score 1 for subjects showing improved CT score by 1 grade, and score 2 for those showing improved CT score by 2 grades or a postoperative CT score of 0. Quantitative image analysis was input into a personal computer and the ratio occupied by the shadow was calculated, as the shadow ratio. While some discrepancies were seen in parts in the comparison of the quantitative image analysis and CT scores as the former captures minute shadows, a positive correlation was obtained overall. Attention is needed to accurately evaluate small paranasal sinuses such as the frontal sinus, and small amounts of shadow, which are areas where errors may occur. A satisfactory correlation was obtained between the score for the improvement rate and the difference in the shadow ratios before and after surgery. The CT scores and the scores for the improvement rate showed no difference from the results of other evaluation methods reported in the past, and evaluation of similar precision was possible. It was thought that this simple evaluation method of CT findings in the paranasal sinuses, which we examined in the present study, was quite useful as a simple stage-classification method that could be utilized in everyday practice considering its facility, reproducibility and satisfactory precision.

Involvement of carbon monoxide in the innervation of the canine cervical esophagus and trachea.

We investigated the involvement of carbon monoxide (CO) in the innervation of the canine cervical esophagus and trachea by means of immunohistochemistry using an antiserum against heme oxygenase-2 (HO-2). We detected HO-2-immunoreactive nerve fibers around the blood vessels and submucosal glands of the esophagus and trachea. We found HO-2-immunoreactive neurons in ganglia in the trachea and in the myenteric plexus of the esophagus. These results suggest that CO is involved as a neurotransmitter in the innervation of the canine esophagus and trachea.

Induction of nitric oxide synthase activity in nucleus ambiguus motoneurons after injury to the rat recurrent laryngeal nerve.

Recent studies have implicated nitric oxide (NO) in neuronal degeneration and plasticity in the motor nervous system. In the present study, we investigated the induction of nitric oxide synthase (NOS) in the motoneurons in the nucleus ambiguus (NA) after injury to the rat recurrent laryngeal nerve (RLN) using nicotinamide-adenine-dinucleotide-phosphate-diaphorase (NADPH-d) histochemistry. NADPH-d reactivity was clearly induced in motoneurons in the ipsilateral NA after transection or avulsion of the RLN, compared with control animals. This finding suggests that NO may play an important role in the pathogenesis of RLN paralysis. Another interesting finding in the present study was the induction of NADPH-d reactivity in nerve terminals of the NA after RLN injury. This finding suggests that RLN injury has some effect on nitrergic input to the NA and a direct effect on the motoneurons.

Functional morphology of the suprachiasmatic nucleus.

In mammals, the biological clock (circadian oscillator) is situated in the suprachiasmatic nucleus (SCN), a small bilaterally paired structure just above the optic chiasm. Circadian rhythms of sleep-wakefulness and hormone release disappear when the SCN is destroyed, and transplantation of fetal or neonatal SCN into an arrhythmic host restores rhythmicity. There are several kinds of peptide-synthesizing neurons in the SCN, with vasoactive intestinal peptide, arginine vasopressin, and somatostatine neurons being most prominent. Those peptides and their mRNA show diurnal rhythmicity and may or may not be affected by light stimuli. Major neuronal inputs from retinal ganglion cells as well as other inputs such as those from the lateral geniculate nucleus and raphe nucleus are very important for entrainment and shift of circadian rhythms. In this review, we describe morphological and functional interactions between neurons and glial elements and their development. We also consider the expression of immediate-early genes in the SCN after light stimulation during subjective night and their role in the mechanism of signal transduction. The reciprocal interaction between the SCN and melatonin, which is synthesized in the pineal body under the influence of polysynaptic inputs from the SCN, is also considered. Finally, morphological and functional characteristics of clock genes, particularly mPers, which are considered to promote circadian rhythm, are reviewed.

Neurochemical substances in neurons of the canine intrinsic laryngeal muscles.

The localization of vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), catecholamines (CA) and carbon monoxide (CO) in neurons of the intrinsic laryngeal muscles of the dog was investigated using immunohistochemistry. Cells with VIP-like immunoreactivity were found between the striated muscle fibres of the cricothyroid and posterior cricoarytenoid muscles, some aggregated into a ganglion and some solitary. Neurons with immunoreactivity to haem oxygenase-2 (HO-2), an immunohistochemical marker for CO, were also seen. VIP-or HO-2-negative cells also existed in various numbers in the intramuscular ganglia. No CGRP-like immunoreactive neurons were found although CGRP-like immunoreactive varicose fibres were seen in the intramuscular ganglia. No neurons or fibres immunoreactive to tyrosine hydroxylase (TH), the key enzyme in synthesis of CA, were detected. The exact function of these intramuscular ganglia is yet to be clarified, but they may be sensory neurons.

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.

Calcitonin gene-related peptide-like immunoreactive motoneurons innervating the canine intrinsic laryngeal muscles.

The percentage of calcitonin gene-related peptide (CGRP)-immunoreactive motoneurons in the nucleus ambiguus innervating the intrinsic laryngeal muscles of colchicine-treated dogs was examined by using cholera toxin B subunit-gold (CTBG) as a retrograde tracer and by immunohistochemistry. Neurons that were labeled with CTBG from the cricothyroid muscle were located in the ventromedial division of the rostral part of the nucleus ambiguus, and the ratio of CGRP-positive neurons was 93.0%. Neurons labeled with CTBG from the thyroarytenoid muscle were located in the dorsal division of the medial part of the nucleus ambiguus, and the percentage of neurons with CGRP was 71.4%. Neurons labeled with CTBG from the posterior cricoarytenoid muscle were located in the ventral division of the medial part of the nucleus ambiguus, and the percentage of CGRP-positive neurons was 85.5%. These findings suggest that the innervation and/or the neurotrophic mechanism involving CGRP for each intrinsic laryngeal muscle is different.

Neuron-glia interaction in the suprachiasmatic nucleus: a double labeling light and electron microscopic immunocytochemical study in the rat.

The morphological interactions between astroglial and neuronal elements were elucidated in the rat suprachiasmatic nucleus (SCN) by light and electron microscopic immunocytochemistry using antibodies against glial fibrillary acidic protein (GFAP), vasoactive intestinal peptide (VIP) and arginine-vasopressin (AVP). Throughout the SCN, particularly in its ventral portion, GFAP-like-immunoreactive (GFAP-LI) astroglial elements were found. These astrocytes displaying GFAP-like immunoreactivity occasionally contained fairly well-developed organelles. Some of these astrocytes were found as satellite cells in close contact with non-immunoreactive neuronal perikarya and processes. Around the neurons, GFAP-LI astroglial processes were also observed to cover some portions of presynaptic and postsynaptic elements. In addition, these astroglial elements were seen between two neuronal somata and pericytes of blood capillaries as glial endfeet. By double labeling immunoelectron microscopy using antibodies against GFAP/VIP and GFAP/AVP, some portions of VIP-like-immunoreactive or AVP-like-immunoreactive neuronal somata and processes were found to be engulfed by GFAP-LI astroglial processes. The possible functional roles of the morphological interactions between astroglial and neuronal elements are discussed.

Nitric oxide synthase and NADPH-diaphorase in neurons of the rat, dog and guinea pig nodose ganglia.

Localization of nitric oxide synthase (NOS) in the nodose ganglia of the dog, rat and guinea pig was investigated. A double-staining technique of NOS immunohistochemistry and NADPH-diaphorase (NADPHd) histochemistry was used; then the ratio of NADPHd-positive and NOS-positive cells to the total cells was calculated. The distribution of positive cells within the canine nodose ganglion was also investigated. NADPHd-positive neurons were detected in all the ganglia. Three intensities of reactivity to NADPHd histochemistry (strong, weak or negative) were detected in the neurons of all three species. There were more cells that stained strongly for NADPHd in the rat, but fewer in the dog and guinea pig, indicating that a species difference may exist. NADPHd-positive neurons were less abundant in the rostral third of the canine nodose ganglion than in the middle or caudal thirds. NADPHd reactivity was completely co-localized to the cells that demonstrated neuronal NOS immunoreactivity in the canine nodose ganglion. Thus, NADPHd histochemical reactivity may be a reliable marker of NOS in the nodose ganglion.

Direct retinal projections to GRP neurons in the suprachiasmatic nucleus of the rat.

The retinal projections to gastrin-releasing peptide (GRP)-expressing neurons in the rat suprachiasmatic nucleus (SCN) were investigated by double immunofluorescence and immunoelectron microscopy. Optic nerve terminals labeled by cholera toxin B subunit (CTb) which was transported from the retinal ganglion cells were intermingled with GRP-immunoreactive cell bodies and processes in the ventrolateral portion of the SCN. Ultrastructural analysis revealed that CTb-immunoreactive retinal terminals made synaptic contacts with GRP-immunoreactive dendritic processes. These results demonstrated that photic information is directly input from the optic nerve to GRP neurons in the SCN and these GRP neurons may be involved in circadian entrainment by light.

Coexistence of calcitonin gene-related peptide and NADPH-diaphorase in the canine superior cervical ganglion.

By means of double staining technique of NADPH-diaphorase (NADPH-d) histochemistry and calcitonin gene-related peptide (CGRP) immunohistochemistry, we investigated the coexistence of NADPH-d reactivity and CGRP immunoreactivity in the canine superior cervical ganglion (SCG). Most of NADPH-d reactivity and CGRP immunoreactivity were coexisted in the principal postganglionic neurons. These neurons were distributed throughout the ganglion without specific localization. The present findings suggest the intimate role of CGRP and nitric oxide in postganglionic neurons of the canine SCG.

Induction of NGFI-A gene expression in the rat suprachiasmatic nucleus by photic stimulation.

Photic induction of NGFI-A gene expression was investigated in the rat suprachiasmatic nucleus (SCN) using in situ hybridization histochemistry. Following light exposure for 30 min, NGFI-A mRNA appeared in the ventral portion of the rostral SCN, in the ventrolateral and in part of the dorsomedial portion at the middle level, and in the lateral portion of the caudal SCN. The distribution of NGFI-A mRNA was wider than that of c-fos mRNA which was confined to the ventrolateral portion at the middle level of the SCN. By double labeling in situ hybridization, approximately half of NGFI-A mRNA-positive cells in the SCN were shown to coexpress vasoactive intestinal peptide (VIP) mRNA, while 16% of cells positive for c-fos mRNA coexpressed VIP mRNA. These findings indicate that the broadness of NGFI-A mRNA and c-fos mRNA expression after photic stimulation are different. NGFI-A gene induced in these cells of the SCN including VIP neurons may be involved in circadian entrainment by light.