Peptide 19 in the rat vagal and glossopharyngeal sensory ganglia

Peptide 19 (PEP 19) is a 7.6-kDa polypeptide which binds to calmodulin and inhibits calcium-calmodulin signaling. In this study, PEP 19-immunoreactivity (PEP 19-IR) was examined in the rat vagal and glossopharyngeal sensory ganglia. Twenty-nine percent, 59%, and 41% of sensory neurons contained PEP 19-IR in the jugular, petrosal, and nodose ganglia, respectively. These neurons were of various sizes (jugular, mean +/- SD = 635.8 +/- 392.6 microm2, range = 105.9-1695.9 microm2; petrosal, mean +/- SD = 370.9 +/- 228.5 microm2, range = 57.7-1662.7 microm2; nodose, mean +/- SD = 380.5 +/- 157 microm2, range = 87.5-950.4 microm2) and scattered throughout these ganglia. Double immunofluorescence method revealed that PEP 19-IR neurons which had parvalbumin-IR were rare in the ganglia (jugular, 4%; petrosal, 10%; nodose, 8%). PEP 19-IR neurons which contained calbindin D-28k were abundant in the petrosal (20%) and nodose (22%) ganglia but not in the jugular ganglion (8%). Retrograde tracing method indicated that many PEP 19-IR neurons projected to the circumvallate papilla and soft palate. In the soft palate, taste buds were innervated by PEP 19-IR nerve fibers. The present study suggests that PEP 19-IR neurons include chemoreceptors in the vagal and glossopharyngeal sensory ganglia.     

Substance P content in cultured neonatal rat vagal sensory neurons: the effect of nerve growth factor

To begin to study the factors regulating the synthesis and release of substance P (SP) in the sensory vagus nerve, cultures of neonatal rat nodose ganglia were developed. In microexplant cultures, obtained from small fragments of nodose ganglia, SP was present in low amounts: after 3 weeks, 141 +/- 36 pg per well, 10 ganglia equivalents per well. To enhance neuron survival, nodose ganglia were enzymatically dissociated using neutral protease. Estimated survival at 5 days was 20-30%, with 800-1200 surviving neurons per plated ganglion, and decreased slowly thereafter. Specific SP immunostaining was present in 10-20% of neurons, mostly of small diameter (18-22 micron). SP content was low for 5 days then rose progressively after 14 days to 80-150 pg per plated ganglion. The addition of nerve growth factor (NGF, 100 ng/ml) to the culture medium did not alter neuron survival. However, SP content was doubled in the presence of NGF, or fell rapidly to one-half control levels following its withdrawal: e.g. following 12 days in culture with NGF 1185 +/- 176 pg/well vs NGF withdrawn day 8-12, 592 +/- 118 pg/well, mean +/- S.D., P less than 0.01. Somatostatin, present in one-sixth the amount of SP, was unaltered by NGF. In subsequent studies, plating of neurons onto previously dissociated rat atriacytes increased survival by 50% but did not alter SP content per surviving neurons. These studies demonstrate that SP is present in dissociated cultures of rat vagal sensory neurons; the quantities and estimated net synthesis rate correspond to previous observations in vivo. The studies also demonstrate that SP content but not neuron survival are regulated by NGF in nodose ganglion neurons. This model may prove valuable for the study of SP and other sensory neuropeptides in this important class of visceral afferent neurons.     

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.     

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.     

Localization of NADPH-diaphorase-containing neurons in sensory ganglia of the rat

The presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity was studied histochemically in the sensory ganglia of the rat. Supraspinally, the trigeminal ganglion possessed only a few cells positively stained for NADPH-diaphorase, while a large number of positive neurons was found in the nodose ganglion. In the dorsal root ganglia, the distribution of positive cells showed a peculiar pattern in relation to spinal levels. Very minor populations (less than 2% of the total ganglionic cells) exhibited positive reaction in ganglia at levels ranging from the first cervical (C1) to fourth thoracic (T4) and from the second lumber (L2) through the entire sacral levels. In the middle to lower thoracic levels (from T5 to L1), however, abundant diaphorase-positive cells were observed. From these positive neurons it was possible to trace intensely stained nerve fibers. In the lower thoracic level, for example, dense positive fibers were seen in the ramus communicans. Retrograde tracing studies revealed that diaphorase-containing neurons in the lower thoracic level project at least partly to the gastric wall and the celiac ganglion. These results indicate that the diaphorase-positive ganglionic neurons in the thoracicolumbar levels may carry autonomic visceral afferent information. Double staining with NADPH-diaphorase histochemistry and peptide immunohistochemistry revealed that NADPH-diaphorase colocalizes with calcitonin gene-related peptide and substance P in many of these visceral afferent neurons.     

Substance P expresses in intrapancreatic ganglia of the rats

Substance P (SP) is an important neuropeptide regulating the functions of the pancreas and might play a vital role in the onset of diabetes. SP has been shown to express in nerve fibers of the pancreas, but whether SP exists in the intrapancreatic ganglia, which are components of a complex autonomous nervous system to co-ordinate the functions of the pancreas, is unsure. The aim of this study was to systematically investigate rat intrapancreatic ganglia and the expression of SP in intrapancreatic ganglia. After surgical denervation of posterior branches of bilateral T₅–L₂ dorsal root ganglia, SP expression remained > 60% in the pancreas. Moreover, after high doses of capsaicin were injected to destroy sensory nerves, the remainder of SP protein levels was still > 50% in the pancreas. In the intrapancreatic ganglia, for anti-SP polyclonal antibody, the immunoreactivity to SP was found in 34.0 ± 1.2% of PGP 9.5-positive intrapancreatic neurons (47 ganglia from 13 rats); for anti-SP monoclonal antibody, the immunoreactivity to SP was found in 26.6 ± 1.0% of PGP 9.5-positive intrapancreatic neurons (18 ganglia from 6 rats). In summary, the present molecular and immunocytochemistry studies showed that there existed intrinsic SP expression in the neurons of intrapancreatic ganglia, which constituted around half of SP origins in the pancreas suggesting that intrapancreatic ganglia might play an important role on mechanisms involving SP.     

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.     

A topographic map of sensory cell terminal arborizations in the cricket CNS: Correlation with birthday and position in a sensory array

The development of a group of club-shaped sensilla called clavate hairs, located on the cerci of the cricket (Acheta domesticus), was examined morphologically. The clavate hairs are located on the base of the cercus and are thought to inform the animal of its orientation with respect to gravity. There are two groups of clavate hairs distinguished from one another by the orientation of their sockets: a dorso-medial group whose sockets are oriented perpendicular to the long axis of the cercus and a ventro-medial group inclined 45–60° away from the perpendicular. The ventro-medial group consists of a series of rows of sensilla running parallel to the long axis of the cercus. By examining a cast-off exoskeleton in the scanning electron microscope and comparing it with newly developed cuticle of the subsequent instar (Fig. 3), we showed how receptors were added to the ventro-medial array of clavate hairs. The first ventral hair (#10, Fig.1) appeared in the second instar. Three more hairs were added in the third instar: two (#11 and #12) proximal to hair 10 forming the first row and one (#20) medial to 11 and initiating the second row. After the third instar one hair was usually added proximal to each row each time the specimen molted. Because of the regular positioning of hairs and their orderly addition to the array, it is possible to identify uniquely all of the hairs in the three largest rows of ventral hairs (Fig. 4). We developed a simple method for staining the neuron associated with each hair. A hair was injured by cutting off its tip. A bubble of cobaltous acetate was then placed on the hair for 18–20 hours and only the neuron associated with the injured hair took up the stain. The synaptic terminal arborizations of identified neurons examined in this manner were unique and reproducible from specimen to specimen (Fig. 6). Furthermore, there is a topographic order to the terminal arborizations. Within one row the oldest neurons project furthest into the nervous system and arborize over the greatest area, whereas younger neurons arborize in more restricted areas near the entrance of the cercal nerve. Thus it was concluded that birthday was corretated with the morphology of the synaptic arborization. By staining neurons that were the same age but located in different rows, we determined that birthday was not the only variable influencing the morphology of the terminal arbors. The terminal arbors of neurons 11 and 20, both of which first appear in the third instar, were very different from one another. Thus another variable, presumably position on the body surface, was also correlated with the morphology of a neuron's terminal arborization. We concluded from these results that position on the cercus as well as birthday is encoded in the developmental program of these neurons and that the morphology of their terminal arborizations is a joint function of these two variables.     

Substance P post-synaptically potentiates glutamate-induced currents in dorsal vagal neurons

We examined the post-synaptic actions of glutamate, N-methyl-d-aspartate (NMDA) and substance P on dorsal vagal neurons, using the patch-clamp technique on brainstem slices of young rats. The vagal neurons were identified electrically and histologically. All vagal neurons responded to glutamate and NDMA and about 30% to substance P, with dose-dependent inward currents. The glutamate-induced currents were blocked partially by either CPP (3((R)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid) or CNQX (6-cyano-7-nitro-quinoxaline-2,3-dione), indicating that these currents resulted from the activation of at least two types of glutamate receptors: NMDA receptors and AMPA/kainate receptors. The NK1 receptor-selective antagonist, RP67580, blocked substance P-induced currents, suggesting that NK1 receptors do coexist with NMDA receptors and AMPA/Kainate receptors. Substance P potentiated the effects of glutamate. This potentiation lasted 10–20 min and was blocked by CPP and by RP67580, but not by CNQX, demonstrating that the increase in glutamate-induced currents resulted from the interaction between NK1 receptors and NMDA channels. These results provided the first evidence that the receptors for substance P and glutamate coexist on dorsal vagal neurons and interact with each other to modulate visceral efferent functions.     

Evidence of substance P immunoreactive neurons in dorsal root ganglia and vagal ganglia projecting to the guinea pig pylorus

The origin of extrinsic substance P fibers in the guinea pig pyloric wall was investigated by combining retrograde axonal tracing and indirect immunofluorescence techniques. After injection of Fast Blue into the pyloric wall labeled cells were found in the T7-T9 dorsal root ganglia and the nodose and jugular ganglia. About 60% of the labeled cells in the dorsal root ganglia contained substance P-like immunoreactivity. After local application of colchicine, a few substance P positive cells were observed in the nodose and jugular ganglia, some of which also contained Fast Blue.     

Substance P released from sensory nerve endings influences tear secretion and goblet cell function in the rat

The aim of this study was to present morphological and functional evidence to evaluate whether tear secretion is influenced by neuropeptides released from sensory nerve endings of the conjunctiva. Following unilateral electrical stimulation of the trigeminal ganglion, tears were collected at both sides and assessed for volume and protein concentration; as well as gel electrophoresis and luminol chemiluminescence with immunostaining to immunoglobulin A and lysozyme measurements. Goblet cell density (goblet cells/100 basal cells) was recorded during histopathological examination of removed lids. Rats were pretreated with atropine to block parasympathetic; guanethidine to block sympathetic neuronal pathways; or hexamethonium to block synaptic transmission in ganglia. Capsaicin was used to deplete neurotransmitters from sensory nerve endings or SR140333 to block substance P tachykinin NK1 receptor mediated responses. Effects of inadequate electrode position or incidental lesion of trigeminal ganglion were examined by placing the electrode in false position, or no stimulation at a correct position. Electrical stimulation resulted in 380% increase of tear secretion (p < 0.001) and 30% decrease of goblet cell density (p < 0.001) on the the stimulated side compared to the unstimulated side. Atropine, guanethidine and hexamethonium pretreatments had no effect (p > 0.05), but capsaicin and SR140333 inhibited the effect of stimulation (by 96% and 72%, respectively, p < 0.001). Inadequate stimulation did not increase the tear secretion (p < 0.05). Protein concentration decreased, whilst tear volume and total secreted protein increased (p < 0.005) after stimulation. Electrophoresis showed no difference in protein pattern between stimulated and control side and analysis of equivalent amount of tear protein with luminol chemiluminescence indicated no difference in immunoglobulin A and lysozyme ratio following stimulation (p>0.05). We conclude that antidromic electrical activation of conjunctival sensory nerve endings significantly increases water, mucus and protein phases of tear. It is suggested that the sensory neuropeptide substance P plays a pivotal role in this neurogenic regulatory mechanism.     

Substance P-containing sensory neurons in the rat dorsal root ganglia innervate the adrenal medulla.

The adrenal medulla is innervated by both cholinergic and substance P (SP)-containing fibres via the splanchnic nerve. SP has been shown to modulate catecholamine (CA) secretion in isolated chromaffin cells and in the perfused rat adrenal gland, however, the origin of SP-containing fibres is not known. In the present study, we have combined the techniques of SP immunohistochemistry and retrograde tracing with Fast blue injected into the left adrenal medulla of the rat in order to study whether SP-containing sensory neurons in the dorsal root ganglia innervate the adrenal medulla. The results showed that there were on average 281 +/- 31 SP-like immunoreactive cells in each left dorsal root ganglion, T3-T13 (range, 234 +/- 19 in T4 to 372 +/- 43 in T13, n = 8). The average total number of Fast blue-labelled cells (T3-T13) in 8 experiments was 172 +/- 26, distributed normally about a peak at T8 (33.8 +/- 6.3 cells) and T9 (33.3 +/- 6.8 cells) with the least at T3 (1.5 +/- 0.8) and T13 (5.2 +/- 2.0). No Fast blue-labelled cells were found in the right DRG. In the left DRG, the average number of cells exhibiting both SP and Fast blue labelled cells were distributed from T7 to T9. These results demonstrate that SP-containing sensory neurons in the dorsal root ganglia provide an ipsilateral innervation of the adrenal medulla in rats.     

Substance P in the cerebral vasculature: depletion by capsaicin suggests a sensory role

Substance P-like immunoreactivity (SPI) was investigated in the chicken retina by means of the indirect immunofluorescence method with flat-mounts. SPI cells were located mostly in the peripheral retinal regions, while in the central region, none or only a few cells were seen. Based upon their 3-dimensional profiles, SPI cells can be divided into two types: one type belongs to the stratified amacrine cells of the first sublayer and the other to those of the third to fifth sublayer.     

Substance P immunoreactivity in the superior cervical ganglia of normotensive and genetically hypertensive rats

Substance P-like immunoreactivity (SPI) was investigated in the superior cervical ganglion of normotensive and genetically hypertensive (GH) Otago Wistar rats aged 1, 2, 8-10 and 50-60 weeks, by used of an indirect immunoperoxidase method. SPI was not seen in neuronal cell bodies but a subpopulation of ganglion cells was supplied by SP-positive terminals which closely invested the cell surface. This subpopulation showed no particular topographical distribution. The number of SP-positive terminal varicosities per unit area was several times higher in GH rats than in normotensive rats at all ages over 2-60 weeks. The proportion of neurons supplied by SP-positive terminals (sampled in 8-10 week-old rats) was also greater in GH than in normotensive rats. Decentralization of the ganglion or chronic capsaicin treatment removed all SP-immunoreactive terminals around the cell bodies, indicating that the SP-positive terminals are collaterals of thoracic sensory afferents. As SP has been reported to have an excitatory effect in sympathetic ganglia, intraganglionic release of SP might contribute to the development of hypertension in the GH strain.     

Substance P-like-immunoreactive sensory neurons in dorsal root ganglia of the chick embryo: ontogenesis and influence of peripheral targets.

The expression of substance P (SP) was studied in sensory neurons of developing chick lumbosacral dorsal root ganglia (DRG) by using a mixture of periodic acid, lysine and paraformaldehyde as fixative and a monoclonal antibody for SP-like immunostaining. The first SP-like-immunoreactive DRG cells appeared first at E5, then rapidly increased in number to reach a peak (88% of ganglion cells) at E8, and finally declined (59% at E12, 51% after hatching). The fall of the SP-like-positive DRG cells resulted from two concomitant events affecting a subset of small B-neurons: a loss of neuronal SP-like immunoreactivity and cell death. After one hindlimb resection at an early (E6) or late (E12) stage of development (that is before or after establishment of peripheral connections), the DRG were examined 6 days later. In both cases, a drastic neuronal death occurred in the ispilateral DRG. However, the resection at E6 did not change the percentage of SP-like-positive neurons, while the resection at E12 severely reduced the proportion of SP-like-immunoreactive DRG cells (25%). In conclusion, connections established between DRG and peripheral target tissues not only promote the survival of sensory neurons, but also control the maintenance of SP-like-expression. Factors issued from innervated targets such as NGF would support the survival of SP-expressing DRG cells and enhance their SP content while other factors present in skeletal muscle or skin would hinder SP expression and therefore lower SP levels in a subset of primary sensory neurons.     

Substance P in the rat parotid gland: evidence for a dual origin from the otic and trigeminal ganglia

The substance P innervation of the rat parotid was investigated using retrograde tracing with the fluorescent dye True Blue, in combination with the immunohistochemical localization of substance P. Retrogradely-labelled cells were seen in the otic, trigeminal, superior cervical and nodose ganglia. A proportion of the True Blue-labelled cells in the otic and trigeminal ganglia also contained substance P-like immunoreactivity providing direct evidence for a dual origin of substance P in the rat parotid. In addition, these results imply that in the rat otic ganglion substance P and acetylcholine coexist.