Immunohistochemical analysis and distribution of epithelial mast cells in the rat larynx and trachea.

Mast cells (MCs) in rat airways have been classified into two subtypes: epithelial MCs and connective tissue MCs (CTMCs). However, the immunohistochemical characteristics, cellular morphology, and distribution of epithelial MCs in the upper airways remain unclear. The present study investigated the morphological characteristics and distribution of epithelial MCs using 5-hydroxytryptamine (5-HT) and other immunohistochemical markers in sectioned or whole-mount preparations of the rat larynx and trachea. A double immunofluorescence analysis revealed the colocalization of 5-HT immunoreactivity with c-kit, a stem cell factor receptor commonly used as a MC marker, in both epithelial MCs and CTMCs. Dopa decarboxylase, an enzyme involved in 5-HT synthesis, was detected in both subtypes, suggesting their ability to synthesize and release 5-HT. Tryptase and histidine decarboxylase (a biosynthetic enzyme of histamine), which are well-known mediators of MCs, were exclusive to CTMCs. Epithelial MCs were pleomorphic with long cytoplasmic processes, whereas CTMCs were round and lacked cytoplasmic processes. The density of epithelial MCs was significantly higher in the glottis and cranial part of the trachea than in the epiglottis and other parts of the trachea. The present results showed that the morphology and immunohistochemical characteristics of epithelial MCs were different from those of CTMCs in the rat larynx and trachea, and variform epithelial MCs were predominantly located at the entrance of the upper airways. Epithelial MCs may release 5-HT to regulate innate immune responses by modulating epithelial cell functions at the entrance gate of the upper airways.

Type 2 vomeronasal receptor expression in the olfactory organ of African lungfish, Protopterus annectens.

The olfactory organ of tetrapods, with few exceptions, comprises the main and accessory organs: olfactory epithelium (OE) and vomeronasal organ (VNO). Unlike tetrapods, teleost fish lack a VNO. However, lungfish, a type of sarcopterygian fish closely related to tetrapods, possesses a lamellar OE similar to the OE of teleosts and a recess epithelium (RecE) resembling the amphibian VNO. The RecE has been hypothesized as a primordial VNO. Olfactory receptors in tetrapods are distinctively expressed in the OE and VNO. For instance, type 2 vomeronasal receptors (V2Rs) in Xenopus are categorized into those exclusively expressed in the OE and those solely expressed in the VNO. It remains unclear whether V2Rs are differentially expressed between the lamellar OE and RecE in lungfish. This study investigated V2R expression in the lamellar OE and RecE of the African lungfish, Protopterus annectens. P. annectens V2Rs were categorized into three groups: those exclusively expressed in the lamellar OE, those exclusively expressed in the RecE, and those expressed in both the lamellar OE and RecE. V2Rs exclusively expressed in the RecE and those expressed in both the lamellar OE and RecE formed a distinct clade in the phylogenetic tree, whereas others were solely expressed in the lamellar OE. These findings suggest that lungfish V2R expression represents an intermediate stage toward complete segregation between V2Rs expressed in the OE and those expressed in the VNO.

In situ hybridization analysis of olfactory receptor expression in the sea turtle olfactory organ.

The olfactory organ of turtles consists of an upper chamber epithelium (UCE) with associated glands, and a lower chamber epithelium (LCE) devoid of glands. The UCE and LCE are referred to as the air-nose and the water-nose, respectively, because the UCE is thought to detect airborne odorants, while the LCE detects waterborne odorants. However, it is not clear how the two are used in the olfactory organ. Odorant receptors (ORs) are the major olfactory receptors in turtles; they are classified as class I and II ORs, distinguished by their primary structure. Class I ORs are suggested to be receptive to water-soluble ligands and class II ORs to volatile ligands. This study analyzed the expression of class I and II ORs in hatchlings of the green sea turtle, Chelonia mydas, through in situ hybridization, to determine the localization of OR-expressing cells in the olfactory organ. Class I OR-expressing cells were distributed mainly in the LCE, implying that the LCE is receptive to waterborne odorants. Class II OR-expressing cells were distributed in both the UCE and LCE, implying that the entire olfactory organ is receptive to airborne odorants. The widespread expression of class II ORs may increase opportunities for sea turtles to sense airborne odorants.

Morphological Analysis of the Olfactory System of the Pig-Nosed Turtle, Carettochelys insculpta.

The turtle olfactory organ consists of the upper (UCE) and lower (LCE) chamber epithelium, projecting to the ventral and dorsal parts of the olfactory bulbs, respectively. The UCE is associated with glands, contains ciliated olfactory receptor neurons, and is assumed to detect odorants primarily in air, while the LCE is devoid of glands, contains microvillous olfactory receptor neurons, and is assumed to detect odorants primarily in water. Examining the olfactory system of the pig-nosed turtle, Carettochelys insculpta, this study found that both the upper and lower chambers of the nasal cavity were lined with sensory epithelium devoid of associated glands and contained ciliated olfactory receptor neurons. Moreover, the olfactory bulbs were not divided into dorsal and ventral parts. These results suggest that the olfactory system of the pig-nosed turtle is a single system specialized for detecting odorants in water.

Immunohistochemical distribution of proteins involved in glutamate release in subepithelial sensory nerve endings of rat epiglottis.

To elucidate the efferent functions of sensory nerve endings, the distribution of calretinin and vesicular glutamate transporter 1 (VGLUT1) in laryngeal laminar nerve endings and the immunohistochemical distribution of proteins associated with synaptic vesicle release, i.e., t-SNARE (SNAP25 and syntaxin 1), v-SNARE (VAMP1 and VAMP2), synaptotagmin 1 (Syt1), bassoon, and piccolo, were examined. Subepithelial laminar nerve endings immunoreactive for Na+-K+-ATPase α3-subunit (NKAα3) were largely distributed in the whole-mount preparation of the epiglottic mucosa, and several endings were also immunoreactive for calretinin. VGLUT1 immunoreactivity was observed within terminal part near the outline of the small processes of NKAα3-immunoreactive nerve ending. SNAP25, syntaxin 1, and VAMP1 immunoreactivities were detected in terminal parts of calretinin-immunoreactive endings, whereas VAMP2 immunoreactivity was only observed in a few terminals. Terminal parts immunoreactive for calretinin and/or VGLUT1 also exhibited immunoreactivities for Syt1, Ca2+ sensor for membrane trafficking, and for bassoon and piccolo, presynaptic scaffold proteins. The presence of vesicular release-related proteins, including SNARE proteins, in the terminals of laryngeal laminar endings indicate that intrinsic glutamate modulates their afferent activity in an autocrine-like manner.

Morphology and chemical characteristics of taste buds associated with P2X3-immunoreactive afferent nerve endings in the rat incisive papilla.

The present study investigated the cellular components and afferent innervations of taste buds in the rat incisive papilla by immunohistochemistry using confocal scanning laser microscopy. Taste buds containing guanine nucleotide-binding protein G(t), subunit α3 (GNAT3)-imunoreactive cells were densely distributed in the lateral wall of incisive papilla forming the opening of nasoincisor ducts. GNAT3-immunoreactive cells in the taste buds were slender in shape and the tips of apical processes gathered at one point at the surface of the epithelium. The number of taste buds was 56.8 ± 4.5 in the incisive papilla. The incisive taste buds also contained ectonucleoside triphosphate diphosphohydrolase 2-immunoreactive cells and synaptotagmin-1-immunoreactive cells in addition to GNAT3-immunoreactive cells. Furthermore, GNAT3-immunoreactive cells were immunoreactive to taste transduction molecules such as phospholipase C, ß2-subunit, and inositol 1,4,5-trisphosphate receptor, type 3. P2X3-immunoreactive subepithelial nerve fibers intruded into the taste buds and terminated with hederiform or calix-like nerve endings attached to GNAT3-immunoreactive cells and synaptosomal-associated protein, 25 kDa-immunoreactive cells. Some P2X3-immunoreactive endings were also weakly immunoreactive for P2X2. Furthermore, a retrograde tracing method using fast blue dye indicated that most of the P2X3-immunoreactive nerve endings originated from the geniculate ganglia (GG) of the facial nerve. These results suggest that incisive taste buds are morphologically and cellularly homologous to lingual taste buds and are innervated by P2X3-immunoreactive nerve endings derived from the GG. The incisive papilla may be the palatal taste papilla that transmits chemosensory information in the oral cavity to the GG via P2X3-immunoreactive afferent nerve endings.

MRI grading of spinal stenosis is not associated with the severity of low back pain in patients with lumbar spinal stenosis.

BACKGROUND: Although lumbar spinal stenosis (LSS) often coexists with other degenerative conditions, few studies have fully assessed possible contributing factors for low back pain (LBP) in patients with LSS. The purpose of this study was to identify factors associated with the severity of LBP in patients with LSS. METHODS: The patients with neurogenic claudication caused by LSS, which was confirmed by magnetic resonance imaging (MRI) were included in this cross-sectional study. Data included ratings of LBP, buttock and leg pain, and numbness on a numerical rating scale (NRS), 36-item Short-Form Survey (SF-36) scores, muscle mass measured by bioelectrical impedance analysis, and radiographic measurements including lumbopelvic alignment and slippage. The severity of LSS, endplate defects, Modic endplate changes, intervertebral disc degeneration, and facet joint osteoarthritis were evaluated on MRI. Spearman correlation and multivariate linear regression analyses were used to examine the factors associated with the severity of LBP (NRS score). RESULTS: A total of 293 patients (135 male and 158 female, average age 72.6 years) were analyzed. LBP was moderately correlated with buttock and leg pain, and buttock and leg numbness. Significant but weak correlations were observed between LBP and body mass index, appendicular and trunk muscle mass, all domains of SF-36, pelvic tilt, total number of endplate defects and Modic endplate changes, and summary score of disc degeneration grading, but not severity or number of spinal stenoses. In the multivariate regression analysis, age, female sex, trunk muscle mass, diabetes, NRS buttock and leg pain, NRS buttock and leg numbness, SF-36 vitality, pelvic tilt, and total number of endplate defects were associated with the severity of LBP. CONCLUSIONS: Trunk muscle mass, lumbopelvic alignment, and endplate defects, but not severity of stenosis are partly associated with severity of LBP, but buttock and leg pain and buttock and leg numbness have strongest relationships with LBP in patients with LSS.

Supervised physical therapy versus surgery for patients with lumbar spinal stenosis: a propensity score-matched analysis.

BACKGROUND: Previous studies comparing surgical with nonsurgical treatment for lumbar spinal stenosis (LSS) reported that surgery is superior to nonsurgical treatments, but intensive and adequate volume of physical therapy were rarely performed. The purpose of this study was to compare the 1-year follow-up outcomes of patients with LSS treated with supervised physical therapy or surgery using propensity score-matched analysis. METHODS: A total of 224 patients with LSS who received supervised physical therapy (n = 38) or surgery (n = 186) were included, of which 66 were matched on baseline demographics, radiological findings, and patient-reported outcomes. The physical therapy group received supervised physical therapy twice weekly for 6 weeks. The physical therapy sessions included manual therapy, individually tailored exercises, cycling, and body-weight supported treadmill walking. The surgery group underwent decompression surgery with or without spinal fusion. A propensity score analysis was performed using a one-to-one nearest neighbor approach. RESULTS: The surgery group showed greater improvements in Zurich claudication questionnaire symptom severity and physical function, SF-36 physical functioning, bodily pain, and mental health, but had more severe stenosis and symptoms and mental health problems than the physical therapy group at baseline (P <  0.05). After propensity score matching, there were no significant differences in baseline characteristics, and all clinical outcomes at 1 year, except for a higher percentage of responders achieving minimum clinically important difference in the role-emotional subscale of SF-36 in the surgery group (P <  0.05). CONCLUSIONS: When baseline characteristics were considered, supervised physical therapy yielded similar effects to lumbar surgery. These results suggest that supervised physical therapy is preferred over surgery as first-choice treatment, to prevent complications and to minimize health care costs, especially in mild to moderate cases of LSS.

GluN2A- and GluN2B-immunoreactive type I cells attached to vesicular glutamate transporter 2-immunoreactive afferent nerve terminals of the rat carotid body.

We previously reported the immunoreactivity for the vesicular glutamate transporter 2 (VGLUT2) in afferent nerve terminals attached to chemoreceptor type I cells of the carotid body (CB), suggesting that glutamate is released from afferent terminals to stimulate these cells. In the present study, we examined the immunoreactivity for the glutamate-binding subunits of N-methyl-D-aspartate (NMDA) receptors, GluN2A and GluN2B in the rat CB, and the immunohistochemical relationships between these subunits and VGLUT2. Immunoreactivities for GluN2A and GluN2B were predominant in a subpopulation of tyrosine hydroxylase-immunoreactive type I cells rather than those of dopamine beta-hydroxylase-immunoreactive cells. Punctate VGLUT2-immunoreactive products were attached to GluN2A- and GluN2B-immunoreactive type I cells. Bassoon-immunoreactive products were localized between VGLUT2-immunoreactive puncta and type I cells immunoreactive for GluN2A and GluN2B. These results suggest that afferent nerve terminals release glutamate by exocytosis to modulate chemosensory activity of a subpopulation of type I cells via GluN2A- and GluN2B subunits-containing NMDA receptors.

Morphology of GNAT3-immunoreactive chemosensory cells in the nasal cavity and pharynx of the rat.

Solitary chemosensory cells and chemosensory cell clusters are distributed in the pharynx and larynx. In the present study, the morphology and reflexogenic function of solitary chemosensory cells and chemosensory cell clusters in the nasal cavity and pharynx were examined using immunofluorescence for GNAT3 and electrophysiology. In the nasal cavity, GNAT3-immunoreactive solitary chemosensory cells were widely distributed in the nasal mucosa, particularly in the cranial region near the nostrils. Solitary chemosensory cells were also observed in the nasopharynx. Solitary chemosensory cells in the nasopharyngeal cavity were barrel like or slender in shape with long lateral processes within the epithelial layer to attach surrounding ciliated epithelial cells. Chemosensory cell clusters containing GNAT3-immunoreactive cells were also detected in the pharynx. GNAT3-immunoreactive cells gathered with SNAP25-immunoreactive cells in chemosensory clusters. GNAT3-immunoreactive chemosensory cells were in close contact with a few SP- or CGRP-immunoreactive nerve endings. In the pharynx, GNAT3-immunoreactive chemosensory cells were also attached to P2X3-immunoreactive nerve endings. Physiologically, the perfusion of 10 mM quinine hydrochloride (QHCl) solution induced ventilatory depression. The QHCl-induced reflex was diminished by bilateral section of the glossopharyngeal nerve, suggesting autonomic reflex were evoked by chemosensory cells in pharynx but not in nasal mucosa. The present results indicate that complex shape of nasopharyngeal solitary chemosensory cells may contribute to intercellular communication, and pharyngeal chemosensory cells may play a role in respiratory depression.

Supervised physical therapy versus unsupervised exercise for patients with lumbar spinal stenosis: 1-year follow-up of a randomized controlled trial.

OBJECTIVE: To compare the 1-year outcomes of patients with lumbar spinal stenosis treated with supervised physical therapy or unsupervised exercise. DESIGN: A single-center randomized controlled trial with concealed allocation, blinded assessor and intention-to-treat analysis. SETTING: Spine care center. SUBJECTS: A total of 86 patients presenting with symptoms of neurogenic claudication caused by lumbar spinal stenosis. INTERVENTIONS: The physical therapy group received supervised physical therapy sessions twice a week for 6 weeks and home exercise program. The home exercise group received 6-week home exercise program only. MAIN MEASURES: The primary outcome was symptom severity on the Zurich claudication questionnaire at 1 year. Secondary outcomes included physical function, pain, health-related quality of life and the surgery rate after 1 year. RESULTS: At 1 year, more patients in the physical therapy group than in the home exercise group achieved minimum clinically important differences in Zurich claudication questionnaire symptom severity (60.5% vs 32.6%; adjusted odds ratio [AOR] 4.3, [95% CI [1.5-12.3], P = 0.01); Zurich claudication questionnaire physical function (55.8% vs 32.6%; AOR 3.0 [1.1-8.1], P = 0.03); SF-36 bodily pain (48.8% vs 25.6%; AOR 2.8 [1.1-7.3], P = 0.03), and SF-36 general health (20.9% vs 7.0%; AOR 6.1 [1.1-33.0], P = 0.04). The surgery rate at 1 year was lower in the physical therapy than in the home exercise group (7.0% vs 23.3%; AOR 0.2 [0.04-0.9] P = 0.04). CONCLUSIONS: Supervised physical therapy produced greater improvements in symptom severity and physical function than unsupervised exercise and was associated with lower likelihood of receiving surgery within 1 year.

[Constrictive Pericarditis with Multiple Organ Failure after Coronary Artery Bypass Grafting].

A 67-year-old male patient, who had undergone coronary artery bypass grafting (CABG) 16 years before, developed congestive heart failure 5 years after surgery. Three years later, he developed repeated heart failure, sepsis by methicillin-resistant Staphylococcus aureus (MRSA), renal failure, repeated thrombophlebitis on his right leg and atrial fibrillation. He also suffered from clouding of consciousness and flapping tremor caused by hyperammonemia. The three bypass grafts showed normal flow, but the pericardium was severely thickened. Therefore, pericardiotomy was performed via median sternotomy with additional left thoracotomy without using cardiopulmonary bypass. Although, he developed MRSA mediastinitis after surgery, he recovered after a month of continuous negative pressure wound therapy. His liver function and septic conditions gradually recovered. No recurrence of heart failure has been observed for 8 years since his second surgery.

Distribution and morphology of baroreceptors in the rat carotid sinus as revealed by immunohistochemistry for P2X3 purinoceptors.

The morphological characteristics of baroreceptors in the rat carotid sinus were reevaluated by whole-mount preparations with immunohistochemistry for P2X3 purinoceptors using confocal scanning laser microscopy. Immunoreactive nerve endings for P2X3 were distributed in the internal carotid artery proximal to the carotid bifurcation, particularly in the region opposite the carotid body. Some pre-terminal axons in nerve endings were ensheathed by myelin sheaths immunoreactive for myelin basic protein. Pre-terminal axons ramified into several branches that extended two-dimensionally in every direction. The axon terminals of P2X3-immunoreactive nerve endings were flat and leaf-like in shape, and extended hederiform- or knob-like protrusions in the adventitial layer. Some axons and axon terminals with P2X3 immunoreactivity were also immunoreactive for P2X2, and axon terminals were closely surrounded by terminal Schwann cells with S100 or S100B immunoreactivity. These results revealed the detailed morphology of P2X3-immunoreactive nerve endings and suggested that these endings respond to a mechanical deformation of the carotid sinus wall with their flat leaf-like terminals.

Morphology of GNAT3-immunoreactive chemosensory cells in the rat larynx.

The upper airways play important roles in respiratory defensive reflexes. Although solitary chemosensory cells and chemosensory cell clusters have been reported in the laryngeal mucosa of mammalian species, the distribution and cellular morphology of chemosensory cells remain unclear. In the present study, the distribution and morphology of solitary chemosensory cells and chemosensory cell clusters were examined by immunofluorescence for GNAT3 on whole-mount preparations of the rat laryngeal mucosa. Electrophysiological experiments were performed to analyze the respiratory reflexes evoked by bitter stimuli to the laryngeal cavity. In the whole area of the laryngeal mucosa, the numbers of GNAT3-immunoreactive solitary chemosensory cells and chemosensory clusters were 421.0 ± 20.3 and 62.7 ± 6.9, respectively. GNAT3-immunoreactive solitary chemosensory cells were mainly distributed in the mucosa overlying epiglottic and arytenoid cartilage, and chemosensory clusters were mainly distributed on the edge of the epiglottis and aryepiglottic fold. GNAT3-immunoreactive solitary chemosensory cells were slender with elongated processes or had a flask-like/columnar shape. The number of GNAT3-immunoreactive cells in chemosensory clusters was 6.1 ± 0.4, ranging between 2 and 14 cells. GNAT3-immunoreactive cells in the cluster were variform and the tips of apical processes gathered at one point at the surface of the epithelium. The tips of apical cytoplasmic processes in solitary chemosensory cells and cells in the cluster were immunoreactive for espin, and faced the laryngeal cavity. Physiological experiments showed that the application of 10 mm quinine hydrochloride to the laryngeal cavity decreased respiratory frequency. The present results revealed the chemosensory field of the larynx and the morphological characteristics of the laryngeal chemosensory system for respiratory depression.

Phylogeographic Analysis of Madagascan Goats Using mtDNA Control Region and SRY Gene Sequences.

In the present study, we estimated the genetic diversity and relationships, as well as the propagation routes, of Madagascan goats using mtDNA control region and SRY gene sequences. The mtDNA sequences of 40 Madagascan goats revealed 10 haplotypes and a quite low nucleotide diversity (0.0014), suggesting a founder and/or bottleneck effect resulting from goat migration to Madagascar island. The analysis of sequences identical to Madagascan haplotypes indicated close genetic relationships between goats from Madagascar and Africa. Sequence analysis of the SRY gene in 40 male Madagascan goats revealed two haplotypes: Y1A (45%) and Y2A (55%). The paternal result indicated genetic influences from Africa, South Asia, and the Near East proximal to Madagascar. The analyses of the mtDNA control region and SRY gene sequences suggested a genetic relationship between Africa and Madagascar. Moreover, SRY sequences indicated influences from South Asia and the Near East. These phylogenetic results provide important genetic information for elucidating the propagation routes of Madagascan goats.

A comparative study of 2-year follow-up outcomes in lumbar spinal stenosis patients treated with physical therapy alone and those with surgical intervention after less successful physical therapy.

BACKGROUND: The efficacy of physical therapy for patients with lumbar spinal stenosis (LSS) has been reported only for the short term, and few reports have compared outcomes of surgical treatment with nonsurgical treatment after physical therapy. The purpose of this study was to assess 2-year outcomes of LSS patients treated with surgery or under follow-up observation after physical therapy for 6 weeks. METHODS: Patients presenting with neurogenic claudication, radiologically-confirmed central LSS affecting both legs and refractory symptoms to pharmacotherapy of more than 3 months were enrolled. Patients were treated with manual therapy, stretching and strengthening exercises, and body weight-supported treadmill walking once a week for 6 weeks. Clinical outcomes were measured using the Zurich Claudication Questionnaire (ZCQ), visual analog scale of low back pain, leg pain, and numbness, the Japanese Orthopedic Association Back Pain Evaluation Questionnaire and the SF-36. Two years after physical therapy, patients were classified into the observation group (Group I) or the surgery group (Group II), whose patients failed to respond to physical therapy and wanted to undergo surgery. RESULTS: Thirty-eight patients were enrolled; 28 had complete data at 2 years: 21 and 7 in Groups I and II, respectively. Group II had a higher body mass index (BMI) than Group I. There were no significant differences in clinical outcomes at baseline. Six weeks after physical therapy, Group I had significantly better outcomes for symptom severity and physical function on the ZCQ subscales, physical functioning and bodily pain on the SF-36 subscales. These outcomes in Group I were maintained or improved and did not differ significantly between groups at 2-years. However, the physical function on the ZCQ subscales was improved in Group II more than those in Group I (mean difference -0.6; 95% CI: -1.2 to -0.03, P < 0.05) at 2 years. CONCLUSIONS: At 2 years, the outcomes except for the change in physical function score in the ZCQ subscale did not differ significantly between patients who had undergone surgery and those who avoided surgery.

Design of potent dipeptidyl peptidase IV (DPP-4) inhibitors by employing a strategy to form a salt bridge with Lys554.

We report a design strategy to obtain potent DPP-4 inhibitors by incorporating salt bridge formation with Lys554 in the S1' pocket. By applying the strategy to the previously identified templates, quinoline 4 and pyridines 16a, 16b, and 17 have been identified as subnanomolar or nanomolar inhibitors of human DPP-4. Docking studies suggested that a hydrophobic interaction with Tyr547 as well as the salt bridge interaction is important for the extremely high potency. The design strategy would be useful to explore a novel design for DPP-4 inhibitors having a distinct structure with a unique binding mode.

Stimulation of dopamine D2-like receptors in the lumbosacral defaecation centre causes propulsive colorectal contractions in rats.

KEY POINTS: The pathophysiological roles of the CNS in bowel dysfunction in patients with irritable bowel syndrome and Parkinson's disease remain obscure. In the present study, we demonstrate that dopamine in the lumbosacral defaecation centre causes strong propulsive motility of the colorectum. The effect of dopamine is a result of activation of sacral parasympathetic preganglionic neurons via D2-like dopamine receptors. Considering that dopamine is a neurotransmitter of descending pain inhibitory pathways, our results highlight the novel concept that descending pain inhibitory pathways control not only pain, but also the defaecation reflex. In addition, severe constipation in patients with Parkinson's disease can be explained by reduced parasympathetic outflow as a result of a loss of the effect of dopaminergic neurons. ABSTRACT: We have recently demonstrated that intrathecally injected noradrenaline caused propulsive contractions of the colorectum. We hypothesized that descending pain inhibitory pathways control not only pain, but also the defaecation reflex. Because dopamine is one of the major neurotransmitters of descending pain inhibitory pathways in the spinal cord, we examined the effects of the intrathecal application of dopamine to the spinal defaecation centre on colorectal motility. Colorectal intraluminal pressure and expelled volume were recorded in vivo in anaesthetized rats. Slice patch clamp and immunohistochemistry were used to confirm the existence of dopamine-sensitive neurons in the sacral parasympathetic nuclei. Intrathecal application of dopamine into the L6-S1 spinal cord, where the lumbosacral defaecation centre is located, caused propulsive contractions of the colorectum. Inactivation of spinal neurons using TTX blocked the effect of dopamine. Although thoracic spinal transection had no effect on the enhancement of colorectal motility by intrathecal dopamine, the severing of the pelvic nerves abolished the enhanced motility. Pharmacological experiments revealed that the effect of dopamine is mediated primarily by D2-like dopamine receptors. Neurons labelled with retrograde dye injected at the colorectum showed an inward current in response to dopamine in slice patch clamp recordings. Furthermore, immunohistochemical analysis revealed that neurons immunoreactive to choline acetyltransferase express D2-like dopamine receptors. Taken together, our findings demonstrate that dopamine activates sacral parasympathetic preganglionic neurons via D2-like dopamine receptors and causes propulsive motility of the colorectum in rats. The present study supports the hypothesis that descending pain inhibitory pathways regulate defaecation reflexes.

Morphology of P2X3-immunoreactive nerve endings in the rat laryngeal mucosa.

The morphological characteristics of P2X3-immunoreactive nerve endings in the laryngeal mucosa were herein examined using immunohistochemistry with confocal laser microscopy. Ramified intraepithelial nerve endings immunoreactive to P2X3 were distributed in the epiglottis and arytenoid region. The axon terminals of P2X3-immunoreactive ramified endings were beaded or flat in shape. These endings were also immunoreactive to P2X2 and not identical to the nerve endings immunoreactive to Na(+)-K(+)-ATPase α3-subunit, substance P (SP), and calcitonin gene-related peptide (CGRP). P2X3-immunoreactive axon terminals were also immunoreactive to vGLUT1, vGLUT2, and vGLUT3. In addition to ramified endings, P2X3-immunoreactive nerve endings were associated with α-gustducin-immunoreactive solitary chemosensory cells and/or SNAP25-immunoreactive neuroendocrine cells. Furthermore, P2X3-immunoreactive nerve endings were also observed in the taste bud-like chemosensory cell clusters of the stratified squamous epithelium covering epiglottic and arytenoid cartilage. The P2X3-immunoreactive nerve endings that associated with sensory and/or endocrine cells and chemosensory cell clusters were also immunoreactive to P2X2, vGLUT1, vGLUT2, and vGLUT3, but not to SP or CGRP. In conclusion, P2X3-immunoreactive nerve endings may be classified into two types, i.e., intraepithelial ramified nerve endings and nerve endings associated with chemosensory cells and neuroendocrine cells.

Three-dimensional architectures of P2X2-/P2X3-immunoreactive afferent nerve terminals in the rat carotid body as revealed by confocal laser scanning microscopy.

We investigated the three-dimensional architectures of P2X2-/P2X3-immunoreactive nerve terminals in the rat carotid body using immunohistochemistry with confocal laser microscopy. Nerve endings immunoreactive for P2X2 and P2X3 were associated with clusters of type I cells, whereas some nerve endings were sparsely distributed in a few clusters. Most nerve endings surrounding type I cells were hederiform in shape and extended several flattened axon terminals, which were polygonal or pleomorphic in shape and contained P2X2-/P2X3-immunoreactive products. Three-dimensional reconstruction views revealed that some flattened nerve endings with P2X3 immunoreactivity formed arborized, sac- or goblet-like terminal structures and were attached to type I cells immunoreactive for tyrosine hydroxylase (TH). However, P2X3-immunoreactive axon terminals were sparsely distributed in type I cells immunoreactive for dopamine beta-hydroxylase. Multi-immunolabeling for P2X2, S100, and TH revealed that P2X2-immunoreactive axon terminals were attached to TH-immunoreactive type I cells on the inside of type II cells with S100 immunoreactivity. These results revealed the detailed morphology of P2X2-/P2X3-immunoreactive nerve terminals and suggest that sensory nerve endings may integrate chemosensory signals from clustered type I cells with their variform nerve terminals.