Morphology of P2X3‐immunoreactive nerve endings in the rat tracheal mucosa

Nerve endings with immunoreactivity for the P2X3 purinoreceptor (P2X3) in the rat tracheal mucosa were examined by immunohistochemistry of whole‐mount preparations with confocal scanning laser microscopy. P2X3 immunoreactivity was observed in ramified endings distributed in the whole length of the trachea. The myelinated parent axons of P2X3‐immunoreactive nerve endings ramified into several branches that extended two‐dimensionally in every direction at the interface between the epithelial layer and lamina propria. The axonal branches of P2X3‐immunoreactive endings branched off many twigs located just beneath the epithelium, and continued to intraepithelial axon terminals. The axon terminals of P2X3‐immunoreactive endings were beaded, rounded, or club‐like in shape and terminated between tracheal epithelial cells. Flat axon terminals sometimes partly ensheathed neuroendocrine cells with immunoreactivity for SNAP25 or CGRP. Some axons and axon terminals with P2X3 immunoreactivity were immunoreactive for P2X2, while some terminals were immunoreactive for vGLUT2. Furthermore, a retrograde tracing method using fast blue (FB) revealed that 88.4% of FB‐labeled cells with P2X3 immunoreactivity originated from the nodose ganglion. In conclusion, P2X3‐immunoreactive nerve endings in the rat tracheal mucosa have unique morphological characteristics, and these endings may be rapidly adapting receptors and/or irritant receptors that are activated by mucosal irritant stimuli.     

Innervation of the syrinx of the zebra finch (Taeniopygia guttata)

In songbirds, the learning and maintenance of song is dependent on auditory feedback, but little is known about the presence or role of other forms of sensory feedback. Here, we studied the innervation of the avian vocal organ, the syrinx, in the zebra finch. Using a combination of immunohistochemistry, immunofluorescence and neural tracing with subunit B of cholera toxin (CTB), we analysed the peripheral and central endings of the branch of the hypoglossal nerve that supplies the syrinx, the tracheosyringeal nerve. In the syringeal muscles, we show the presence of numerous choline acetyl transferase‐like immunoreactive en plaque motor endplates and substance P‐like immunoreactive, thin and varicose free nerve endings. Substance P‐like immunoreactive free nerve endings were also present in the luminal syringeal tissues, especially in the luminal epithelium of the trachea and pessulus. Also, by a combination of immunofluorescence and transganglionic tracing following injections of CTB in the tracheosyringeal nerve, we identified as central targets of the syringeal receptors the caudolateral part of the interpolaris subnucleus of the descending trigeminal tract, a caudolateral region of the nucleus tractus solitarius, and a lateral band of the principal sensory trigeminal nucleus. Further studies are required to determine the sensory modalities of these receptors and the connections of their specific synaptic targets.     

Correlation between adenosine triphosphate levels, dopamine release and electrical activity in the carotid body: Support for the metabolic hypothesis of chemoreception

An unsolved issue for the arterial chemoreceptors is the mechanism by which hypoxia and other natural stimuli lead to an increase of activity in the carotid sinus nerve. According to the 'metabolic hypothesis', the hypoxic activation of the carotid body (CB) is mediated by a decrease of the ATP levels in the type I cells, which then release a neurotransmitter capable of exciting the sensory nerve endings. Using an in vitro preparation of cat CB, we report that ATP levels in the CB do in fact decrease when the organs are exposed to moderate, short lasting hypoxia (5 min 20% O2). Additionally, we found that decreases in ATP levels induced by 2-deoxyglucose (2 mM) or sodium cyanide (0.1 mM) are closely correlated with dopamine release from type I cells and electrical activity in the carotid sinus nerve elicited by these agents. The possible cause-effect relationship of these events is discussed.     

Immunoelectron microscopy of AMPA receptor subunits reveals three types of putative glutamatergic synapse in the rat vestibular end organs

To characterize the synapses between hair cells and afferent nerve endings in the rat vestibular end organs, the ultrastructural localization of AMPA receptor subunits (GluR1-4) was examined by postembedding immunogold cytochemistry. Immunoreactivities for GluR2/3 and GluR4 were associated with the synapses between type I hair cells and the surrounding chaliceal nerve endings and with the bouton type nerve endings contacting type II hair cells. There was no detectable immunoreactivity for GluR1. A third type of immunoreactive synapse was found between the outer face of chalices and type II hair cells. While the linear densities of gold particles (particles per micrometer postsynaptic specialization) of bouton type endings and chaliceal nerve endings were the same, the former type of ending showed larger postsynaptic specializations and, hence, a higher number of receptor molecules. These data indicate that there are three types of putative glutamatergic synapse in the vestibular end organ.     

Identification of spinal afferent nerve endings in the colonic mucosa and submucosa that communicate directly with the spinal cord: The gut–brain axis

The major sensory nerve pathway between the colon and central nervous system (spinal cord and brain) that underlies the gut–brain axis, is via spinal afferent neurons, with cell bodies in dorsal root ganglia (DRG). Our aim was to identify the sensory nerve endings in the colon that arise from single colorectal-projecting DRG neurons. C57BL/6 mice were anesthetized and lumbosacral L6-S1 DRG injected with dextran biotin. Mice recovered for 7 days. The whole colon was then removed and stained to visualize single axons and nerve endings immunoreactive to calcitonin gene-related peptide (CGRP). Single axons arising from DRG were identified in the distal colon and their morphological features and CGRP immunoreactivity characterized. After entering the colon, single axons ramified rostrally or caudally along many rows of myenteric ganglia with little circumferential displacement, giving off varicose endings in multiple ganglia. Nerve endings arising from two classes of colorectal-projecting DRG neuron were identified. One class was peptidergic neurons that had nerve endings in circular muscle, myenteric ganglia, and submucosa. Another class of nonpeptidergic neurons innervated mucosal crypts, myenteric ganglia, and submucosa. Different morphological types of nerve endings which innervate different anatomical layers of colon can arise from the same axon and sensory neuron in DRG. These findings suggest single peptidergic and nonpeptidergic sensory neurons in DRG are potentially capable of detecting sensory stimuli from different anatomical layers of the colon, via different types of nerve endings.     

The distribution of P2X3 purine receptor subunits in the guinea pig enteric nervous system

Adenosine 5'-triphosphate (ATP) excites 70-90% of enteric neurons through P2X type purine receptors, and is likely to be an enteric neurotransmitter. Recent studies indicate that the P2X2 subunit is expressed by specific subgroups of enteric neurons, and that there are enteric neurons that are responsive to ATP but lack this subunit. In the present work, we have investigated whether the P2X3 subunit is similarly localised to specific subgroups of neurons, and whether these are different from the P2X2 subunit-expressing neurons. The P2X3 subunit was localised by immunohistochemistry to nerve cells of the myenteric ganglia of the stomach, small and large intestines, and nerve cells of the submucosal ganglia in the small and large intestines of the guinea pig. All immunoreactivity was absorbed with the P2X3 receptor peptide against which the antiserum was raised. In myenteric ganglia of the ileum, P2X3 receptor immunoreactivity was in calretinin, enkephalin and nitric oxide synthase (NOS)-immunoreactive neurons. In submucosal ganglia, all calretinin-immunoreactive nerve cells were P2X3 receptor immunoreactive. In the submucosal ganglia of the ileum, 13 +/- 3% of neuropeptide Y (NPY)-immunoreactive neurons were also P2X3 receptor immunoreactive, whereas in the distal colon, almost all NPY-expressing nerve cells were P2X3 receptor immunoreactive. The localisation of the P2X3 subunit was largely distinct from that of the P2X2 subunit, although both subunits occur in some NOS neurons, where P2X2 and P2X3 subunits may form heteromeric receptors. Unlike the P2X2 subunit, the P2X3 subunit is not expressed in intrinsic sensory neurons in the ileum. It is concluded that the P2X3 receptor subunit is expressed in specific functional groups of neurons; the major types are excitatory and inhibitory muscle motor neurons, ascending interneurons and cholinergic secretomotor neurons.     

Presence of synapsin I in afferent and efferent nerve endings of vestibular sensory epithelia

The presence and localization of synapsin I were investigated in the cat vestibular epithelium, using a rabbit anti-synapsin I antibody. The staining was performed by immunofluorescence or by a peroxidase—anti-peroxidase technique. A strong immunoreactivity was observed with both methods. It appeared as spherical patches distributed in the lower part of the epithelium. This distribution pattern is very similar to that of the efferent synaptic endings which are filled with microvesicles. Elongated immunoreactive segments were also observed around some hair cells. They suggest the presence of synapsin I in the afferent sensory endings contacting the hair cells. This immunoreactivity occurs in early postnatal and adult stages.     

Cat carotid bodies reinnervated by normal or foreign nerves

The carotid body of the cat was reinnervated with either its native nerve, the carotid sinus nerve (CSN, re-anastomosis), or a foreign nerve, the lingual branch of the IXth cranial nerve (LN, cross-anastomosis). In both types of preparations, regenerating axons from the LN or CSN readily penetrated carotid body parenchymal tissue, as demonstrated by axoplasmic transport of radiolabeled material from the petrosal (sensory) ganglion. Electron microscopy revealed nearly normal fiber invasion into lobules of glomus (type I) and sustentacular (type II) cells following reinnervation by either the foreign or native nerve. However, while the regenerated CSN fibers formed a normal complement of specialized axon terminals in contact with type I cells, the incidence of such terminals in LN reinnervated carotid bodies was reduced by over 90% (2-19 months survival time). This low incidence of specialized LN endings was correlated with reductions in the magnitude of the chemosensory discharge elicited in these preparations by asphyxia, NaCN or acetylcholine. These data suggest that chemosensitivity depends upon intimate association between glomus cells and afferent nerve endings; and that the ability to form such contacts may reside in particular axons whose incidence is higher in the CSN than in the LN.     

Distribution of transient receptor potential channels in the rat carotid chemosensory pathway

Glomus cells in the carotid body respond to decreases in oxygen tension of the blood and transmit this sensory information in the carotid sinus nerve to the brain via neurons in the petrosal ganglion. G-protein-coupled membrane receptors linked to phospholipase C may play an important role in this response through the activation of the cation channels formed by the transient receptor potential (TRP) proteins. In the present study, expression of TRPC proteins in the rat carotid body and petrosal ganglion was examined using immunohistochemical techniques. TRPC3, TRPC4, TRPC5, TRPC6, and TRPC7 were present in neurons throughout the ganglion. TRPC1 was expressed in only 28% of petrosal neurons, and of this population, 45% were tyrosine hydroxylase (TH)-positive, accounting for essentially all the TH-expressing neurons in the ganglion. Because TH-positive neurons project to the carotid body, this result suggests that TRPC1 is selectively associated with the chemosensory pathway. Confocal images through the carotid body showed that TRPC1/3/4/5/6 proteins localize to the carotid sinus nerve fibers, some of which were immunoreactive to an anti-neurofilament (NF) antibody cocktail. TRPC1 and TRPC3 were present in both NF-positive and NF-negative fibers, whereas TPRC4, TRPC5, and TRPC6 expression was primarily localized to NF-negative fibers. Only TRPC1 and TRPC4 were localized in the afferent nerve terminals that encircle individual glomus cells. TRPC7 was not expressed in sensory fibers. All the TRPC proteins studied were present in type I glomus cells. Although their role as receptor-activated cation channels in the chemosensory pathway is yet to be established, the presence of TRPC channels in glomus cells and sensory nerves of the carotid body suggests a role in facilitating and/or sustaining the hypoxic response.     

TASK-1, TASK-2, TASK-3 and TRAAK immunoreactivities in the rat carotid body

In the present study, we investigated the immunohistochemical localization of two-pore K⁺-channels TASK-1, TASK-2, TASK-3 and TRAAK in the rat carotid body. Type I cells were positive for TASK-1, TASK-2, TASK-3 and TRAAK. Intrinsic nerve cell bodies were also strongly positive for TASK-1, TASK-2 and TRAAK, but negative for TASK-3. In addition, some type II cells, Schwann cells in the nerve bundles and fibroblast between type I cell clusters were also immunostained for TASK-1. Smooth muscle cells of the carotid body artery were intensely positive for TASK-3. Our results indicate that TASK-1 immunoreactivity was ubiquitously distributed in many cell types and immunoreactivities for TASK-2, TASK-3 and TRAAK were cell type specific distribution patterns in the rat carotid body.     

Identifying unique subtypes of spinal afferent nerve endings within the urinary bladder of mice

Spinal afferent neurons are responsible for the transduction and transmission of noxious (painful) stimuli and innocuous stimuli that do not reach conscious sensations from visceral organs to the central nervous system. Although the location of the nerve cell bodies of spinal afferents is well known to reside in dorsal root ganglia (DRG), the morphology and location of peripheral nerve endings of spinal afferents that transduce sensory stimuli into action potentials is poorly understood. The individual nerve endings of spinal afferents that innervate the urinary bladder have never been unequivocally identified in any species. We used an anterograde tracing technique developed in our laboratory to selectively label only spinal afferents. Mice were anesthetized and unilateral injections of dextran‐amine made into lumbosacral DRGs (L5‐S2). Seven to nine days postsurgery, mice were euthanized, the urinary bladder removed, then fresh‐fixed and stained for immunoreactivity to calcitonin‐gene‐related‐peptide (CGRP). Four distinct morphological types of spinal afferent ending in the bladder were identified. Three types existed in the detrusor muscle and one major type in the sub‐urothelium and urothelium. Most nerve endings were located in detrusor muscle where the three types could be identified as having: “branching”, “simple”, or “complex” morphology. The majority of spinal afferent nerve endings were CGRP‐immunoreactive. Single spinal afferent axons bifurcated many times upon entering the bladder and developed varicosities along their axon terminal endings. We present the first morphological identification of spinal afferent nerve endings in the mammalian urinary bladder.     

Effects of 2-deoxy-d-glucose on in vitro cat carotid body

The process of chemosensory transduction in the arterial chemoreceptors is not well understood. According to the metabolic hypothesis of chemoreception, a decrease in arterial pO2 will produce a decrease in the adenosine triphosphate (ATP) content in the chemosensory type I cells, leading to release of a neurotransmitter and increased sensory neural activity. There is a paucity of direct experimental support for this hypothesis, and in the present work, we have tested the postulates of the metabolic hypothesis in an in vitro preparation of cat carotid body using 2-deoxy-D-glucose as an ATP-depleting agent. This preparation, when superfused with Tyrode containing 5 mM Na+-pyruvate instead of glucose, responds normally to hypoxia, low pH and acetylcholine, and maintains normal ATP levels. Under these conditions, 2-deoxy-D-glucose is a chemostimulant, i.e. electrical activity in the carotid sinus nerve is increased, with a threshold concentration of 0.25 mM and a maximum response at about 2-4 mM. These concentrations of 2-deoxyglucose evoke a dose-dependent release of [3H]dopamine (synthesized from [3H]tyrosine) from the carotid bodies which parallels the electrical activity. The 2-deoxy-D-glucose-evoked release and electrical activity is dependent on the presence of extracellular Ca2+. These same concentrations of 2-deoxy-D-glucose significantly reduce the ATP content of the carotid bodies. The events postulated by the metabolic hypothesis, i.e. decrease in ATP content, release of a putative neurotransmitter and activation of the sensory nerve endings are found to occur simultaneously. A possible cause-effect relationship between these three events is discussed.     

Morphology,innervation, and peripheral sensory cells of the siphon of aplysia californica

The siphon of Aplysia californica has several functions, including involvement in respiration, excretion, and defensive inking. It also provides sensory input for defensive withdrawals that have been studied extensively to examine mechanisms that underlie learning. To better understand the neuronal bases of these functions, we used immunohistochemistry to catalogue peripheral cell types and innervation of the siphon in stage 12 juveniles (chosen to allow observation of tissues in whole‐mounts). We found that the siphon nerve splits into three major branches, leading ultimately to a two‐part FMRFamide‐immunoreactive plexus and an apparently separate tyrosine hydroxylase–immunoreactive plexus. Putative sensory neurons included four distinct types of tubulin‐immunoreactive bipolar cells (one likely also tyrosine hydroxylase immunoreactive) that bore ciliated dendrites penetrating the epithelium. A fifth bipolar neuron type (tubulin‐ and FMRFamide‐immunoreactive) occurred deeper in the tissue, associated with part of the FMRFamide‐immunoreactive plexus. Our observations emphasize the structural complexity of the peripheral nervous system of the siphon, and the importance of direct tests of the various components to better understand the functioning of the entire organ, including its role in defensive withdrawal responses. J. Comp. Neurol. 523:2409–2425, 2015. © 2015 Wiley Periodicals, Inc.     

Microglia release ATP by exocytosis

Microglia survey the brain environment by sensing several types of diffusible molecules, among which extracellular nucleotides released/leaked from damaged cells have central roles. Microglia sense ATP or other nucleotides by multiple P2 receptors, after which they change into several different phenotypes. However, so far, it is largely unknown whether microglia themselves release ATP and, if so, by what mechanism. Here we show that exocytosis is the mechanism by which microglia release ATP. When we stimulated microglia with ionomycin, they released ATP and the release was dependent on Ca²⁺, vesicular H⁺‐ATPase, or SNAREs but independent of connexin/pannexin hemichannels. VNUT was found to be expressed in microglia and exhibited no colocalization with lysosome. We also visualized the exocytosis of ATP by a quinacrine‐based fluorescent time‐lapse imaging. Moreover, we found that lipopolysaccharide increased the ionomycin‐induced release of ATP, which was dependent on the increase in VNUT. Taken together, our data suggested that exocytosis is the mechanism of ATP release from microglia. When activated, they would release ATP by increasing VNUT‐dependent exocytotic mechanisms. GLIA 2013;61:1320–1330     

Immunolocalization of VR1 and VRL1 in rat larynx

Immunoreactivity for vanilloid receptor subtype 1 (VR1) and its analogue vanilloid receptor-like protein 1 (VRL1) were examined in combination with immunoreactivity for substance P (SP) and calcitonin gene-related peptide (CGRP) in the rat larynx. VR1 and VRL1 immunoreactivity were observed in the intraepithelial free nerve endings, subepithelial nerve plexus and laryngeal epithelial cells. Most of VR1 immunoreactive nerves were also immunoreactive for SP or CGRP. VR1 immunoreactive intraepithelial nerve endings may be laryngeal nociceptors.     

Mechanisms for termination of the action of dopamine in carotid body chemoreceptors

The possible presence of a high affinity uptake mechanism for dopamine and the metabolism of this amine was investigated in the rabbit carotid body. The type I cells of this sensory organ contain high levels of dopamine and share with sympathetic nerve endings (which possess a high affinity uptake mechanism for catecholamines) the properties of being presynaptic catecholaminergic elements, but also have in common with chromaffin cells from intact adrenal medulla (which lack a high affinity catecholamine uptake) a similar embryological origin and ultrastructural appearance. Our experiments revealed only a low affinity uptake process (Km = 6.76 X 10(-4) M [3H]dopamine; Vmax = 1.84 X 10(-9) mol [3H]dopamine/mg protein/min) in the rabbit carotid body. In agreement with our kinetic findings, the uptake of [3H]dopamine was found to be independent of the Na+ concentration in the incubation media. The efflux from the tissue of incorporated [3H]dopamine was very fast (88% wash-out with a half-time of 4 min). DOPAC was the principal catabolite of dopamine in the carotid body, and the organ exhibited a very low capacity for norepinephrine synthesis. Neither chronic carotid sinus nerve section nor chronic sympathectomy modified the uptake of [3H]dopamine, suggesting that the lack of expression of a high affinity uptake was not a consequence of trophic suppression by the neural innervation of the organ. We conclude that overflow, or wash-out, of released dopamine may be quantitatively the most important mechanism for the inactivation of this putative neurotransmitter in the rabbit carotid body.     

Ultrastructural evaluation of calcitonin gene-related peptide immunoreactivity in the human cochlea and vestibular endorgans

Calcitonin gene-related peptide (CGRP) is a neuropeptide widely distributed in the peripheral and central nervous system. Demonstrated in the efferent systems of the mammalian cochlea and vestibule, immunoreactive patterns of CGRP may vary by species. There is, however, no information in the literature investigating CGRP localization in the human cochlea. In the present study, the ultrastructural localization of CGRP immunoreactivity was evaluated in the human inner ear with immunoelectron microscopy. It was found that, in human cochlea, CGRP immunoreactivity was located in unmyelinated nerve fibres of the spiral lamina, inner spiral fibres beneath inner hair cells, tunnel spiral fibres, tunnel crossing fibres and outer radial fibres. In endorgans of human vestibule, CGRP immunoreactivity was located in vesiculated nerve fibres and bouton-type nerve terminals which were seen to contact afferent nerve chalices surrounding type I sensory cells and afferent nerve fibres, or to form an en passant contact with afferent dendrites. CGRP immunoreactivity appeared to be confined to efferent systems in all cases. This study presents evidence that CGRP could serve a role in neurotransmission or neuroregulation in both cochlear and vestibular efferent systems of human.     

Sushi repeat‐containing protein X‐linked 2: A novel phylogenetically conserved hypothalamo‐pituitary protein

Sushi repeat‐containing protein X‐linked 2 (SRPX2) is a novel protein associated with language development, synaptic plasticity, tissue remodeling, and angiogenesis. We investigated the expression and spatial localization of SRPX2 in normal mouse, rat, monkey, and human brain using in situ hybridization and immunohistochemistry. Antibody specificity was determined using in vitro siRNA based silencing of SRPX2. Cell type‐specific expression was verified by double‐labeling with oxytocin or vasopressin. Western blot was used to detect SRPX2 protein in rat and human plasma and cerebrospinal fluid. Unexpectedly, SRPX2 mRNA expression levels were strikingly higher in the hypothalamus as compared to the cortex. All SRPX2 immunoreactive (ir) neurons were localized in the hypothalamic paraventricular, periventricular, and supraoptic nuclei in mouse, rat, monkey, and human brain. SRPX2 colocalized with vasopressin or oxytocin in paraventricular and supraoptic neurons. Hypothalamic SRPX2‐ir positive neurons gave origin to dense projections traveling ventrally and caudally toward the hypophysis. Intense axonal varicosities and terminal arborizations were identified in the rat and human neurohypophysis. SRPX2‐ir cells were also found in the adenohypophysis. Light SRPX2‐ir projections were observed in the dorsal and ventral raphe, locus coeruleus, and the nucleus of the solitary tract in mouse, rat and monkey. SRPX2 protein was also detected in plasma and CSF. Our data revealed intense phylogenetically conserved expression of SRPX2 protein in distinct hypothalamic nuclei and the hypophysis, suggesting its active role in the hypothalamo‐pituitary axis. The presence of SRPX2 protein in the plasma and CSF suggests that some of its functions depend on secretion into body fluids.     

P2X3 receptor in injured human sensory neurons

The ATP-gated cation channel P2X3 is expressed selectively by rat sensory neurones, and may play a role in nociception by binding ATP released from damaged or inflamed tissues. However, the distribution of this channel in human sensory neurons is not known. Using a specific antibody, we have demonstrated intense P2X3 immunoreactivity within a subset (60%) of small/medium diameter sensory neurones and fine nerve fibres in intact post-mortem human dorsal root ganglia (DRG). Co-localization studies showed < 15% overlap with the trkA immunostaining in DRG, indicating that P2X3 was expressed predominantly in sensory neurons that are also isolectin B4 positive. There was a significant decrease in numbers of P2X3-like immunoreactive neurons in human DRG after central axotomy (to 36%), similar to the decrease in rat DRG after peripheral axotomy. However, Western blotting demonstrated a specific 66 kDa band in human DRG and peripheral organs, including intestine, where histochemistry showed P2X3 immunoreactivity in myenteric plexus neurons. Thus P2X3 antagonists may be analgesic, but are unlikely to have a selective effect on pain in humans.