Osteocalcin-immunoreactive neurons in the vagal and glossopharyngeal sensory ganglia of the rat

Immunohistochemistry for osteocalcin (OC) was performed on the rat vagal and glossopharyngeal sensory ganglia. OC-immunoreactive (IR) neurons were detected in the jugular (10%), petrosal (11%) and nodose ganglia (6%). The cell size analysis demonstrated that OC-IR neurons were predominantly small to medium-sized in the jugular ganglion (mean+/-S.D.=356.3+/-192.2 microm(2), range=86.5-831.5 microm(2)). On the other hand, such neurons were medium-sized to large in the petrosal (mean+/-S.D.=725.6+/-280.7 microm(2), range=124.7-1540.4 microm(2)) and nodose ganglia (mean+/-S.D.=857.5+/-330.2 microm(2), range=367.1-1608.0 microm(2)). In the circumvallate papilla, OC-IR nerve fibers were located in the vicinity of taste buds. Some taste bud cells were also immunoreactive for the calcium-binding protein (CaBP). In the carotid body, however, OC-IR nerve fibers could not be detected. Retrograde tracing with fluorogold revealed that OC-IR nerve fibers in the circumvallate papilla mainly originated from the petrosal ganglion. These findings may suggest that OC-IR petrosal neurons have chemoreceptive function in the tongue.     

Calretinin-containing neurons which co-express parvalbumin and calbindin D-28k in the rat spinal and cranial sensory ganglia; triple immunofluorescence study

The co-expression of calretinin with parvalbumin and calbindin D-28k was examined in the rat cranial and spinal sensory ganglia by triple immunofluorescence method. In the trigeminal and nodose ganglia, 9% and 5% of calretinin-immunoreactive neurons, respectively, also contained both parvalbumin- and calbindin D-28k immunoreactivity. These neurons had large cell bodies. In the trigeminal ganglion, they were restricted to the caudal portion. Such neurons were evenly distributed throughout the nodose ganglion. The co-expression could not be detected in the dorsal root, jugular or petrosal ganglia. Nerve fibers which co-expressed all the three calcium-binding proteins were observed in the inferior alveolar nerve but not the infraorbital nerve or palate. In the periodontal ligament, these nerve fibers formed Ruffini-like endings. These findings suggest that (1) the co-expression in trigeminal neurons is intimately related to their peripheral receptive fields; (2) the three calcium-binding proteins (calretinin, parvalbumin, calbindin D-28k) co-expressed in the trigeminal neurons may have mechanoreceptive function in the periodontal ligament.     

Oral and gastric input to the parabrachial nucleus of the rat

Projections to the parabrachial nucleus (PBN) from the nucleus of the solitary tract (NST) carry afferent signals from both the oral cavity and gastrointestinal tract. Although physiological studies suggest the convergence of oral and gastrointestinal sensory signals in the parabrachial nucleus, anatomical studies have emphasized the segregation of these pathways. To more precisely determine the anatomical relationship between gastric distension and oral afferent representation in PBN, small deposits of two anterograde tracers were made into the NST under physiological guidance in the same rat. Gastric terminations were dense and separate from taste projections in the rostral portion of the external lateral and dorsal lateral subnuclei. Gustatory projections were densest and separate from gastric terminations in the ventral lateral and central medial subnuclei of the caudal waist region, but were intermingled with gastric projections in these subnuclei and the external subnuclei at slightly more rostral levels. Patterns of segregation and overlap often appeared as 'patches' within or across subnuclear boundaries. In a second set of experiments, physiological evidence for overlap in PBN was evaluated from single unit extracellular responses evoked by gastric distension and orosensory (taste and orotactile) stimulation. Neurophysiological recordings verified that a small proportion of single cells within the waist and external subnuclei could be activated by both gastric and orotactile stimulation. The anatomical experiments further revealed intranuclear projections from the caudal NST injections that extended rostrally to sites at which responses to oral stimulation had been recorded. Although existing physiological data suggest such interactions are more limited than those in PBN, these anatomical data suggest that gastric/oral interactions may also exist in the NST.     

Calbindin D28k-containing neurons in the paratrigeminal nucleus receive convergent nociceptive information and project to nucleus of the solitary tract in rat

The paratrigeminal nucleus (PTN) receives orofacial somatic and visceral afferent fibers and contains many calbindin-D28k neurons (CB-containing neurons) that project to nucleus of the solitary tract (NTS). In the present study, retrograde and transganglionic tracing methods combined with immunofluorescence histochemistry and confocal laser scanning microscopy were used. After Fluoro-gold (FG) injection into the unilateral NTS, 74.4% FG-labeled neurons of ipsilateral PTN were double-labeled with CB. Furthermore, 41.0% and 32.5% FG/CB double-labeled neurons co-existed with Fos induced by nociceptive stimulation of the lips and the upper alimentary tract, respectively. In the PTN unilateral to FG injection site, 26.6% CB-LI neurons were double-labeled with PAG, 61.5% and 79.0% CB/PAG double-labeled neurons were triple-labeled with FG and Fos, and 22.9% FG/CB double-labeled neurons were triple-labeled with PAG, 84.3% FG/PAG double-labeled neurons expressed Fos induced by the upper alimentary tract stimulation. In the intact animals, 62.8% CB-LI neurons and 88.3% PAG-LI neurons co-existed with GABA(B)R, respectively. In addition, some terminals from the inferior alveolar nerve (IAN) were closely apposed to CB/Fos double-labeled or CB single-labeled neurons. These results suggested that CB-containing neurons in the PTN receive the nociceptive information converge from the orofacial area and visceral organs, and comprising the glutamatergic excitatory transmission pathway from the PTN to the NTS. This pathway might be modulated by GABA via the GABA(B) receptor.     

VR1-, VRL-1- and P2X3 receptor-immunoreactive innervation of the rat temporomandibular joint

Immunohistochemistry for vanilloid receptor subtype 1 (VR1), vanilloid receptor 1-like receptor (VRL-1) and P2X3 receptor was performed in the rat temporomandibular joint (TMJ). Blood vessels in the articular disk and capsule, the synovial membrane and the fibrous tissue around the condylar process were innervated by VR1- or P2X3 receptor-immunoreactive (ir) nerve fibers. However, VRL-1-immunoreactivity (ir) could not be detected in the TMJ. Retrograde tracing and immunohistochemical methods revealed that 25%, 41% and 52% of TMJ neurons in the trigeminal ganglion (TG) exhibited VR1-, VRL-1- and P2X3 receptor-ir, respectively. VR1-ir TMJ neurons were mostly small to medium-sized, whereas VRL-1- and P2X3 receptor-ir TMJ neurons were predominantly medium-sized to large. In addition, 73%, 28% and 44% of VR1-, VRL-1- and P2X3 receptor-ir TMJ neurons, respectively, coexpressed calcitonin gene-related peptide (CGRP)-ir. The present study suggests that the TMJ has abundant nociceptors which respond to vanilloid compounds, protons, heat and extracellular ATP.     

Endothelins in the cat petrosal ganglion and carotid body: effects and immunolocalization

In response to hypoxia, chemoreceptor cells of the carotid body (CB) release transmitters, which acting on the petrosal ganglion (PG) neuron terminals, increase the chemoafferent discharge. Additionally, vasoactive molecules produced within the CB may modulate hypoxic chemoreception by controlling blood flow and tissue PO2. Endothelin-1 (ET-1) increases basal CB chemosensory discharges in situ, probably due to its vasoconstrictor action. However, the actions of ET-1 on PG neurons or its expression in the PG are not known. Using immunohistochemistry, we found that endothelin-like peptides are expressed in the cat PG and CB under normoxic conditions. Exogenous applications of ET-1 increased the chemosensory activity in the vascularly perfused CB but were ineffective on either the CB or PG superfused preparations, both of which are devoid of vascular control. Thus, our data indicate that the excitatory effect of ET-1 in the carotid chemoreceptor system appears to be mainly due to a vasoconstrictor effect in the CB blood vessels.     

Vesicular glutamate transporter immunoreactivity in the central and peripheral endings of muscle-spindle afferents

Expression of vesicular glutamate transporters (VGLUTs: VGLUT1, VGLUT2 and VGLUT3) in muscle spindle afferents was examined in rats. VGLUT1 immunoreactivity was detected in the sensory endings on the equatorial and juxta-equatarial regions of intrafusal fibers as well as in many axon terminals within lamina IX of the spinal cord. VGLUT1 might be expressed not only in the central axon terminals but also in the peripheral sensory endings of muscle-spindle afferents.     

Differential c-Fos expression in the newborn lamb nucleus tractus solitarius and area postrema following ingestion of colostrum or saline

Visceral stimuli and the gut-brain axis play a crucial role in the control of ingestion even in the neonate. The aim of this study was to assess the neuronal activation in the nucleus tractus solitarius (NTS) and the area postrema (AP) following nutritional and non-nutritional stimulations. Lambs received a single gastric infusion of colostrum or saline at 5% birth weight or were sham infused. Infusion of either liquid led to c-Fos-like immunoreactivity (c-FLI) in the NTS and AP. Differences were observed along the sections of the NTS rostro-caudal axis according to the nature of the stimulation, suggesting a specificity of certain afferents and/or NTS areas for nutritional or non-nutritional signals. In the AP, the neuronal activation induced by colostrum was much higher than that induced by saline. A higher number of TH-immunoreactive cells were activated following colostrum infusion, suggesting a specific involvement of the catecholaminergic pathway in the treatment of meal-related stimuli. In spite of functional convergence, the two medullary structures observed responded differently according to the stimulation, indicating a complementary role in the integration of visceral signals.     

ASIC3-immunoreactive neurons in the rat vagal and glossopharyngeal sensory ganglia

ASIC3-immunoreactivity (ir) was examined in the rat vagal and glossopharyngeal sensory ganglia. In the jugular, petrosal and nodose ganglia, 24.8%, 30.8% and 20.6% of sensory neurons, respectively, were immunoreactive for ASIC3. These neurons were observed throughout the ganglia. A double immunofluorescence method demonstrated that many ASIC3-immunoreactive (ir) neurons co-expressed calcitonin gene-related peptide (CGRP)- or vanilloid receptor subtype 1 (VRL-1)-ir in the jugular (CGRP, 77.8%; VRL-1, 28.0%) and petrosal ganglia (CGRP, 61.7%; VRL-1, 21.5%). In the nodose ganglion, however, such neurons were relatively rare (CGRP, 6.3%; VRL-1, 0.4%). ASIC3-ir neurons were mostly devoid of tyrosine hydroxylase in these ganglia. However, some ASIC3-ir neurons co-expressed calbindin D-28k in the petrosal (5.5%) and nodose ganglia (3.8%). These findings may suggest that ASIC3-containing neurons have a wide variety of sensory modalities in the vagal and glossopharyngeal sensory ganglia.     

Effect of mCOUP-TF1 deficiency on the glossopharyngeal and vagal sensory ganglia

Immunohistochemistry for calcitonin gene-related peptide (CGRP), tyrosine hydroxylase and calbindin D-28k was performed on the glossopharyngeal and vagal ganglia in mCOUP-TFI knockout mice to know the effect of its deficiency on different types of primary sensory neurons. In wild type and heterozygous mice, the glossopharyngeal and vagal ganglia contained abundant CGRP-, tyrosine hydroxylase- and calbindin D-28k-immunoreactive (IR) neurons. In the ganglia of mCOUP-TFI knockout mice, a 38% decrease of CGRP-IR neurons was detected. However, the number of tyrosine hydroxylase- or calbindin D-28k-neurons was not altered by the mCOUP-TFI deficiency. In the tongue of knockout mice, the number of CGRP-IR nerve fibers decreased compared to wild-type and heterozygous mice. The development of CGRP-IR petrosal neurons, which supply innervation of the tongue, may depend on mCOUP-TFI.     

Mice deficient in brain‐derived neurotrophic factor have altered development of gastric vagal sensory innervation

Vagal sensory neurons are dependent on neurotrophins for survival during development. Here, the contribution of brain‐derived neurotrophic factor (BDNF) to survival and other aspects of gastric vagal afferent development was investigated. Post‐mortem anterograde tracing with 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbo‐cyanine perchlorate (DiI) was used to label selectively vagal projections to the stomach on postnatal days (P) 0, 3, 4, and 6 in wild types and heterozygous or homozygous BDNF mutants. Sampling sites distributed throughout the ventral stomach wall were scanned with a confocal microscope, and vagal axon bundles, single axons, putative mechanoreceptor precursors (intraganglionic laminar endings, IGLEs; intramuscular arrays, IMAs), and efferent terminals were quantified. Also, myenteric neurons, which are innervated by IGLEs, were stained with cuprolinic blue and counted. Quantitative comparisons across wild‐type stomach compartments demonstrated that the adult distribution of IMAs was not present at P0 but began to form by P3–6. Among all the quantified elements, at P0, only IGLE density was significantly different in homozygous mutants compared with wild types, exhibiting a 50% reduction. Also, antrum innervation appeared disorganized, and some putative IMA precursors had truncated telodendria. At P3–6, the effect on IGLEs had recovered, the disorganization of antrum innervation had partially recovered, and some IMA telodendria were still truncated. The present results suggest that gastric IGLEs are among the vagal sensory neurons dependent on BDNF for survival or axon guidance. Alternatively, BDNF deficiency may delay gastric IGLE development. Also, BDNF may contribute to IMA differentiation and patterning of antral vagal innervation. J. Comp. Neurol. 518:2934–2951, 2010. © 2010 Wiley‐Liss, Inc.     

Neurofilament immunoreactivity in developing rat autonomic and sensory ganglia

Immunoreactivity to neurofilament (NF) antiserum appears early in the development of both the central and peripheral nervous systems of the rat fetus. In 10 somite embryos, positive cell bodies are present in the ventromedial part of anterior rhombencephalic and mesencephalic neural tube. From there the appearance of immunoreactivity spreads cranially to the prosencephalic anlage before closure of the anterior neuropore and caudally following the sequence of neural tube closure. Immunoreactivity increases rapidly in axon bundles of central and peripheral systems, but in immature cell bodies of sensory ganglia the NF material only forms a ring around the nucleus. At 16 days of gestation, some cell bodies are progressively loaded with NF-immunoreactive material as a thick perinuclear network first and then in more excentrically located aggregates. This category of neurons is mainly observed in the distal part of the trigeminal ganglion, in petrous and nodose ganglia and in cervical dorsal root ganglia. In adult ganglia large cell bodies and some small ones present high NF immunoreactivity. In autonomic cell bodies (in superior cervical ganglion and in parasympathetic cranial ganglia) the immunoreactive material only forms a perinuclear ring slowly transformed into a loose perinuciear meshwork at the end of gestation. Intensely reactive nerve fibers are observed in cranial sensory as well as in sympathetic and parasympathetic ganglia and nerves. No positive cell bodies and only a few NF-immunoreactive nerves are observed in the carotid bodies. The NF immunoreactivity is better visualized on sections of fresh frozen material, treated with acetone, than in fixed specimens.These results are compared to previous observations reported for other species and for developing dorsal root ganglia. This immunostaining may be used to detect differentiation of peripheral sensory and autonomic neurons under experimental conditions. The uneven distribution of NF immunoreactivity in sensory neurons from stage 16 days of gestation as specific for precise subpopulations of neurons is discussed.     

Expression of mRNA for four subtypes of the proteinase-activated receptor in rat dorsal root ganglia

Proteinase-activated receptors (PARs) are members of the superfamily of G-protein coupled receptors that initiate intracellular signaling by the proteolytic activity of extracellular serine proteases. Three member of this family (PAR-1, PAR-3, and PAR-4) are considered thrombin receptors, whereas PAR-2 is activated by trypsin and tryptase. Recently, activation of PAR-2 signal was identified as a pro-inflammatory factor that mediates peripheral sensitization of nociceptors. Activation of PAR-1 in the periphery is also considered to be a neurogenic mediator of inflammation that is involved in peptide release. Here, we investigated the expression of these four members of PARs in the adult rat dorsal root ganglia (DRG) using radioisotope-labeled in situ hybridization histochemistry. We detected mRNA for all subtypes of PARs in the DRG. Histological analysis revealed the specific expression patterns of the PARs. PAR-1, PAR-2, and PAR-3 mRNA was expressed in 29.0+/-4.0%, 16.0+/-3.2%, and 40.9+/-1.3% of DRG neurons, respectively. In contrast, PAR-4 mRNA was mainly observed in non-neuronal cells. A double-labeling study of PARs with NF-200 and alpha calcitonin gene-related peptide (CGRP) also revealed the distinctive expression of PARs mRNA in myelinated or nociceptive neurons. This study shows the precise expression pattern of PARs mRNA in the DRG and indicates that the cells in DRG can receive modulation with different types of proteinase-activated receptors.     

Somatic and visceral primary afferents in the lower thoracic dorsal root ganglia of the cat

Anterograde transport of horseradish peroxidase (HRP) through somatic and visceral nerves was used to estimate the proportions of somatic and visceral dorsal root ganglion (DRG) cells of the lower thoracic ganglia of the cat. A concentrated solution of HRP was applied for at least 5 hours to the central end of the right greater splanchnic nerve and of the left T9-intercostal nerve of adult cats. Some animals remained under chloralose anaesthesia for the duration of the HRP transport time (up to 53 hours) whereas longer HRP application and transport times (4-5 days) were allowed in animals that recovered from barbiturate anaesthesia. Visceral DRG cells were found in approximately equal numbers in all ganglia examined (T7-T11). Population estimates were obtained for the T8 and T9 ganglia where visceral DRG cells were found to be 6.2% (T8) and 5.2% (T9) of the total cell population. In contrast, somatic DRG cells were found in large numbers in the ganglia examined (T8 and T9) where they amounted to over 90% of the cell population. Measurement of cross-sectional areas and estimates of cell diameters of the DRG cells showed greater proportions of large somatic cells (diameter greater than 40 micron) than of large visceral cells. Similar distributions of cell size were found for both somatic and visceral DRG cells with diameters less than 40 micron. These results show that the proportion of visceral afferent fibres in the dorsal roots that mediate the spinal cord projection of the splanchnic nerve is very small. Since viscerosomatic convergence in the thoracic spinal cord is very extensive, the present results suggest considerable divergence of the visceral afferent input to the central nervous system.     

The co-expression of VR1 and VRL-1 in the rat vagal sensory ganglia

Immunohistochemistry for two nociceptive transducers, the vanilloid receptor 1 (VR1) and vanilloid receptor 1-like receptor (VRL-1), was performed on the vagal sensory ganglia. In the jugular ganglion, VR1-immunoreactive (IR) neurons were small to medium-sized (range 49.7–1125.6 μm², mean±S.D. 407.7±219.7 μm²), whereas VRL-1-IR neurons were medium-sized to large (range 223.6–1341.1 μm², mean±S.D. 584.3±253.5 μm²). In the nodose ganglion, VR1- and VRL-1-IR neurons were mostly small to medium-sized (VR1: range 148.5–1464.4 μm², mean±S.D. 554.3±207.4 μm²; VRL-1: range 161.7–1166.2 μm², mean±S.D. 541.9±186.2 μm²). The double immunofluorescence method revealed that co-expression of VR1-immunoreactivity among VRL-1-IR neurons was more abundant in the nodose ganglion (63%) than in the jugular ganglion (4%). The present study suggests that co-expression of VR1 and VRL-1 may be more common in visceral sensory neurons than in somatic sensory neurons.     

The co-expression of VR1 and VRL-1 in the rat vagal sensory ganglia

Immunohistochemistry for two nociceptive transducers, the vanilloid receptor 1 (VR1) and vanilloid receptor 1-like receptor (VRL-1), was performed on the vagal sensory ganglia. In the jugular ganglion, VR1-immunoreactive (IR) neurons were small to medium-sized (range 49.7–1125.6 μm², mean±S.D. 407.7±219.7 μm²), whereas VRL-1-IR neurons were medium-sized to large (range 223.6–1341.1 μm², mean±S.D. 584.3±253.5 μm²). In the nodose ganglion, VR1- and VRL-1-IR neurons were mostly small to medium-sized (VR1: range 148.5–1464.4 μm², mean±S.D. 554.3±207.4 μm²; VRL-1: range 161.7–1166.2 μm², mean±S.D. 541.9±186.2 μm²). The double immunofluorescence method revealed that co-expression of VR1-immunoreactivity among VRL-1-IR neurons was more abundant in the nodose ganglion (63%) than in the jugular ganglion (4%). The present study suggests that co-expression of VR1 and VRL-1 may be more common in visceral sensory neurons than in somatic sensory neurons.     

Coexistence of calbindin D-28k and NADPH-diaphorase in vagal and glossopharyngeal sensory neurons of the rat

The presence and coexistence of calbindin D-28k-immunoreactivity (ir) and nicotinamide adenosine dinucleotide phosphate (NADPH)-diaphorase activity (a marker of neurons that are presumed to convert L-arginine to L-citrulline and nitric oxide) were examined in the glossopharyngeal and vagal sensory ganglia (jugular, petrosal and nodose ganglia) of the rat. Calbindin D-28k-ir nerve cells were found in moderate and large numbers in the petrosal and nodose ganglia, respectively. Some calbindin D-28k-ir nerve cells were also observed in the jugular ganglion. NADPH-diaphorase positive nerve cells were localized to the jugular and nodose ganglia and were rare in the petrosal ganglion. A considerable portion (33–51%) of the NADPH-diaphorase positive neurons in these ganglia colocalized calbindin D-28k-ir. The presence and colocalization of calbindin D-28k-ir and NADPH-diaphorase activity in neurotransmitter-identified subpopulations of visceral sensory neurons were also studied. In all three ganglia, calcitonin gene-related peptide (CGRP)-ir was present in many NADPH-diaphorase positive neurons, a subset of which also contained calbindin D-28k-ir. In the nodose ganglion, many (42%) of tyrosine hydroxylase (TH)-ir neurons also contained NADPH diaphorase activity but did not contain calbindin D-28k-ir. These data are consistent with a potential co-operative role for calbindin D-28k and NADPH-diaphorase in the functions of a subpopulation of vagal and glossopharyngeal sensory neurons.     

Basic and clinical aspects of visceral sensation: transmission in the CNS

Pain and discomfort are the leading cause for consultative visits to gastroenterologists. Acute pain should be considered a symptom of an underlying disease, thereby serving a physiologically important function. However, many patients experience chronic pain in the absence of potentially harmful stimuli or disorders, turning pain into the primary problem rather than a symptom. Vagal and spinal afferents both contribute to the sensory component of the gut-brain axis. Current evidence suggests that they convey different elements of the complex sensory experience. Spinal afferents play a key role in the discriminatory dimension, while vagal input primarily affects the strong emotional and autonomic reactions to noxious visceral stimuli. Drugs, surgical and non-pharmacological treatments can target these pathways and provide therapeutic options for patients with chronic visceral pain syndromes.     

Galanin-like immunoreactivity in capsaicin sensitive sensory neurons and ganglia

In rats treated with capsaicin (CAP) as neonates, galanin-like (GA) immunoreactivity is markedly decreased in the trigeminal ganglion and the dorsal root ganglia as well as in the superficial layers of the dorsal spinal cord (laminae I and II), the substantia gelatinosa, the nucleus and tractus of the spinal trigeminal nerve and the nucleus commissuralis. Since CAP causes selective degeneration of primary sensory neurons of the C-fiber type and type B-cells of sensory ganglia, it is concluded that GA in CAP-sensitive primary sensory neurons represents a novel peptidergic system possibly involved in the transformation or modulation of peripheral nociceptive impulses. This system differs from the CAP-resistant GA-like neurons in other brain areas.