Substance P-immunoreactive Neurons Research Articles

Distribution, quantification, and characterization of substance P enteric neurons in the submucosal and myenteric plexuses of the porcine colon

The pig is an important translational model for studying intestinal physiology and disorders for its many homologies with humans, including the organization of the enteric nervous system (ENS), the major regulator of gastrointestinal functions. This study focused on the quantification and neurochemical characterization of substance P (SP) neurons in the pig ascending (AC) and descending colon (DC) in wholemount preparations of the inner submucosal plexus (ISP), outer submucosal plexus (OSP), and myenteric plexus (MP). We used antibodies for the pan-neuronal marker HuCD, and choline acetyltransferase (ChAT) and neuronal nitric oxide synthase (nNOS), markers for excitatory and inhibitory transmitters, for multiple labeling immunofluorescence and high-resolution confocal microscopy. The highest density of SP immunoreactive (IR) neurons was in the ISP (222/mm2 in the AC, 166/mm2 in the DC), where they make up about a third of HuCD-IR neurons, compared to the OSP and MP (19–22% and 13–17%, respectively, P < 0.001–0.0001). HuCD/SP/ChAT-IR neurons (up to 23%) were overall more abundant than HuCD/SP/nNOS-IR neurons (< 10%). Most SP-IR neurons contained ChAT-IR (62–85%), whereas 18–38% contained nNOS-IR with the highest peak in the OSP. A subpopulation of SP-IR neurons contains both ChAT- and nNOS-IR with the highest peak in the OSP and ISP of DC (33–36%) and the lowest in the ISP of AC (< 10%, P < 0.001). SP-IR varicose fibers were abundant in the ganglia. This study shows that SP-IR neurons are functionally distinct with variable proportions in different plexuses in the AC and DC reflecting diverse functions of specific colonic regions.

Substance P in Dorsal Root Ganglion Neurons in Young and Adult Rats, after Nociceptive Stimulation during the Neonatal Period.

The nervous system is highly plastic during the neonatal period, being sensitive to noxious stimuli, which may cause short- and long-term pain responsivity changes. Understanding plasticity in peripheral pain pathways is crucial, particularly when the nervous system is still under development and remodeling process. Substance P (SP) is widely used as a marker for peripheral neurons with unmyelinated and small myelinated fibers. We investigated the number of SP immunoreactive neurons in the dorsal root ganglion (DRG) of male and female Wistar rats, 15 and 180 days after nociceptive stimulation during the neonatal period. Right and left 5th lumbar (L5) DRG were incubated in rabbit polyclonal anti-substance P primary followed by biotinylated donkey anti-rabbit secondary antibodies. Reaction was revealed with a nickel-diaminobenzidine solution. Labeled neurons were counted and compared between ages, genders and groups. Gender differences were present in both ages, with the number of SP-positive DRG neurons being larger in 15-days-old males on both sides. After 180 days, males showed a larger number of SP-positive neurons than females only on the nociceptive stimulated side. An increased number of SP-positive neurons in the DRG on the stimulated side was present in females, immediately after nociceptive stimulation, but not after 180 days. In conclusion, neonatal noxious stimulation caused a permanent increase in SP-positive DRG neurons in males that was not observed in females, suggesting that differences in pain processing/responsivity between genders could be related to morphological alterations of the nervous system. Anat Rec, 301:849-861, 2018. © 2017 Wiley Periodicals, Inc.

Acute Colitis Induces Neurokinin 1 Receptor Internalization in the Rat Lumbosacral Spinal Cord

Substance P (SP) and its receptor, the neurokinin 1 receptor (NK1R), play important roles in transmitting and regulating somatosensory nociceptive information. However, their roles in visceral nociceptive transmission and regulation remain to be elucidated. In the previous study, moderate SP immunoreactive (SP-ir) terminals and NK1R-ir neurons were observed in the dorsal commissural nucleus (DCN) of the lumbosacral spinal cord. Thus we hypothesized that the SP-NK1R system is involved in visceral pain transmission and control within the DCN. The acute visceral pain behaviors, the colon histological changes and the temporal and spatial changes of NK1R-ir structures and Fos expression in the neurons of the DCN were observed in rats following lower colon instillation with 5% formalin. The formalin instillation induced significant acute colitis as revealed by the histological changes in the colon. NK1R internalization in the DCN was obvious at 8 min. It reached a peak (75.3%) at 30 min, began to decrease at 90 min (58.1%) and finally reached the minimum (19.7%) at 3 h after instillation. Meanwhile, formalin instillation induced a biphasic visceral pain response as well as a strong expression of Fos protein in the nuclei of neurons in the DCN. Finally, intrathecal treatment with the NK1R antagonist L732138 attenuated the NK1R internalization, Fos expression and visceral nociceptive responses. The present results suggest that the visceral nociceptive information arising from inflamed pelvic organs, such as the lower colon, might be mediated by the NK1R-ir neurons in the DCN of the lumbosacral spinal cord.

Demonstration of intrinsic innervation of the guinea pig upper urinary tract using whole‐mount preparation

The morphology and functional importance of the autonomic nervous system in the upper urinary tract is still not completely understood. Previous histological studies investigating the innervation of the urinary tract have mainly used conventional sections in which the three-dimensional structure of the intramural innervation is difficult to achieve. In contrast, the whole-mount preparation technique is a suitable method for visualizing the distribution of the mesh-like neuronal networks within the urinary tract. The distribution and regional variation of neurofilament (NF), tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), and substance P-immunoreactive (SP-IR) neurons, as well as acetylcholinesterase (AChE) and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d)-positive neurons were investigated using whole-mount preparations of the guinea pig upper urinary tract. Two distinct nervous plexuses were detected within the muscle layers containing NF, TH, ChAT, and SP-IR nerves. AChE-positive nerves were seen in all layers. Only moderate NADPH-d-positive innervation was found. Renal pelvis, upper and lower part of the ureter showed an overall increased innervation compared to the middle portion of the ureter. Ganglia were found at the pelviureteric border displaying NF and TH immunoreactivity. The whole-mount preparation technique provides an elegant method for assessing the three-dimensional architecture of ureteral innervation. The guinea pig upper urinary tract is richly supplied with adrenergic, cholinergic, nitrergic, and sensory nerves which suggest that the autonomous nervous system plays an important role in controlling ureteral motility and blood flow.

Herpes simplex virus type-1 latency-associated transcript-induced immunoreactivity of substance P in trigeminal neurons is reversed by bone morphogenetic protein-7

Herpes simplex virus type-1 (HSV-1) primarily infects mucoepithelial tissues of the eye and the orofacial region. Subsequently, the virus is retrogradely transported through the axons of the trigeminal sensory neurons to their nuclei, where the virus establishes a life-long latent infection. During this latency period, the viral genome is transcriptionally silent except for a single region encoding the latency-associated transcript (LAT). To understand how HSV-1 latency might affect the expression of substance P in sensory neurons, we transfected primary cultures of trigeminal neurons obtained from rat embryos, with LAT expressing plasmids. The expression of LAT increased the percentage of substance P-immunoreactive neurons by two thirds. To examine the effect of bone morphogenetic protein-7 (BMP7) on the LAT-induced increase in substance P expression in trigeminal neurons, cultures transfected with LAT were treated with BMP7. Treatment with BMP7 reversed the effects of LAT on substance P expression in trigeminal neurons. Our data show for the first time that LAT increases substance P expression in trigeminal neurons and BMP7 can reverse these effects of LAT.

Effect of Brn-3a deficiency on primary nociceptors in the trigeminal ganglion

Immunohistochemistry for substance P, somatostatin and vanilloid receptor subtype 1 as well as receptors for somatostatin and opioids was performed on the trigeminal ganglion in wild-type and Brn-3a knockout mice at postnatal day 0. In wild-type mice, the trigeminal ganglion contained abundant substance P-, vanilloid receptor subtype 1-, sst2A receptor- and delta-opioid receptor-immunoreactive neurons, while the ganglion had only a few mu-opioid receptor-immunoreactive neurons. The Brn-3a deficiency had an effect on the cell size but not the number of substance P-immunoreactive neurons. In knockout mice, the proportion of small immunoreactive neurons markedly increased and that of medium- to large-sized immunoreactive ones correspondingly decreased (mean ± S.D. = 54.7 ± 29.1 μm 2, range = 10.9–220.8 μm 2) compared to wild-type mice (mean ± S.D. = 116.6 ± 58.6 μm 2, range = 27.3–400.7 μm 2). As for vanilloid receptor subtype 1-immunoreactive neurons, the number and cell size was barely affected by the deficiency. On the other hand, the loss of Brn-3a caused a decrease in the number of sst2A receptor- or delta-opioid receptor-immunoreactive neurons (more than 95% reduction) and an increase in the number of mu-opioid receptor-immunoreactive neurons (9.3-fold increase). Somatostatin-immunoreactive neurons were not detected in the trigeminal ganglion of wild-type or mutant mice at postnatal day 0. The present study suggests that Brn-3a deficiency may have effects on the survival of trigeminal nociceptors and their expression of some neurochemical substances.

Identification of a G Protein-coupled Receptor Specifically Responsive to β-Alanine

We isolated a cDNA encoding an orphan G protein-coupled receptor, TGR7, which has been recently reported to correspond to MrgD. To search for ligands for TGR7, we screened a series of small molecule compounds by detecting the Ca2+ influx in Chinese hamster ovary cells expressing TGR7. Through this screening, we found that beta-alanine at micromolar doses specifically evoked Ca2+ influx in cells expressing human, rat, or mouse TGR7. A structural analogue, gamma-aminobutyric acid, weakly stimulated cells expressing human or rat TGR7, but another analogue, glycine, did not. In addition, beta-alanine decreased forskolin-stimulated cAMP production in cells expressing TGR7, suggesting that TGR7 couples with G proteins Gq and Gi. In guanosine 5'-O-3-thiotriphosphate binding assays conducted using a membrane fraction of cells expressing TGR7, beta-alanine specifically increased the binding of guanosine 5'-O-3-thiotriphosphate. When a fusion protein composed of TGR7 and green fluorescent protein was expressed in cells, it localized at the plasma membrane but internalized into the cytoplasm after treatment with beta-alanine. In addition, we found that beta-[3H]alanine more efficiently bound to TGR7-expressing cells than to control cells. From these results, we concluded that TGR7 functioned as a specific membrane receptor for beta-alanine. Quantitative PCR analysis revealed that TGR7 mRNA was predominantly expressed in the dorsal root ganglia in rats. By in situ hybridization and immunostaining, we confirmed that TGR7 mRNA was co-expressed in the small diameter neurons with P2X3 and VR1, both in rat and monkey dorsal root ganglia. Our results suggest that TGR7 participates in the modulation of neuropathic pain.

The neural crest and neural crest defects

The neural crest is a fascinating embryonic tissue for more than one reason. In the adult organism it gives rise to an array of distinct cell types and tissues. It is responsible for many birth defects, familial diseases and malignancies, and it is amenable to the elucidation of mechanisms that regulate stem cell differentiation. Subsequent to an epithelial-to-mesenchymal transformation, neural crest cells emigrate from the dorsal aspect of the neural tube into the embryo, stop in different places, and eventually give rise to the autonomic and enteric nervous systems, most primary sensory neurons, endocrine cells, and melanocytes of the skin and internal organs. Furthermore, neural crest cells are involved in the septation of the cardiac outflow tract and they form the cranial mesenchyme, which gives rise to bone, cartilage, and connective tissue of the face and ventral neck. Environmental insults can lead to neural crest defects, including cleft lip/cleft palate and fetal alcohol syndrome. Familial diseases that affect neural crest derivatives include Hischsprung's disease and albinism, whereas well-known neural crest-related malignancies include melanoma, neuroblastoma, neurofibromatosis and pheochromocytoma. Migratory neural crest cells form a heteroge­neous population of cells that includes stem cells, cells with restricted developmental potentials, and cells that are committed to a particular lineage. Growth factors play important roles in the survival, proliferation and differentiation of neural crest cells. In particular, neurotrophin-3 (NTS), the ligand of the tyrosine kinase receptor, TrkC, promotes the survival of proliferating neural crest stem cells. TrkC-deficient mice develop cardiac outflow tract defects that resemble human birth defects, including persistent truncus arteriosus and transposition of the great vessels. In these animals, cardiac neural crest stem cells become fate-restricted precociously. Action of stem cell factor (SCF), the ligand of the tyrosine kinase receptor c-kit, affects multiple systems. Heterozygous c-kit deficient mice, termed 'Dominant spotting' (W), have anemia, are sterile and show changes in coat color (white spotting) due to defects in the hemopoietic system, germ cell line and melanogenesis, respectively. Inactivation of the human c-kit gene causes piebaldism, which is characterized by a white forelock, patchy hypopigmentation of the skin and rare sensoryneural deafness. In the quail neural crest, SCF supports the survival of neural crest stem cells, promotes their dif­ferentiation into small diameter sensory neurons, and, together with a neurotrophin, supports survival of me lanocyte precursors. In c-kit deficient newborn mice, up to one third of substance P-immunoreactive nociceptive sensory neurons are missing, thus confirming across species that SCF signaling is essential for the development of small diameter sensory neurons. In addition, the number of calcitonin gene-related-peptide (CGRP)-immunoreactive putative visceral afferent neurons in the dorsal root ganglion is diminished in these mice. The norepinephrine transporter (NET) is expressed in many embryonic tissues, including premigratory and migratory neural crest cells. Norepinephrine (NE) uptake by neural crest cells promotes their differentiation into noradrenergic neuroblasts in vitro. In contrast, NE uptake inhibitors, such as tricyclic antidepressants and the drug of abuse, cocaine, inhibit noradrenergic differentiation in vitro and in vivo, suggesting that these drugs can be teratogenic. Since NET is expressed in many embryonic tissues, NE transport may have functions also in non-neural cells during embryonic development. In summary, growth factors, alone and synergistically as well as NEplay multiple roles in neural crest development. Biomedical Reviews 2002; 13: 29-37.

Antinociception from lateral hypothalamic stimulation may be mediated by NK 1 receptors in the A7 catecholamine cell group in rat

Stimulation of the lateral hypothalamus (LH) produces antinociception that is modified by intrathecal alpha-adrenergic antagonists. Spinally-projecting noradrenergic neurons in the LH have not been identified, suggesting that the LH may innervate brainstem noradrenergic neurons, such as the A7 catecholamine cell group in the dorsolateral pontine tegmentum, that modify nociception at the level of the spinal cord dorsal horn. Recently we demonstrated in neuroanatomical studies that substance P-immunoreactive neurons in the LH project the A7 area. To identify a functional connection between substance P neurons in the LH and the A7 cell group, the cholinergic agonist carbachol (125 nmol) was microinjected into the LH of female Sprague–Dawley rats and antinociception was obtained on the tail flick or foot withdrawal test. Cobalt chloride (100 nM) was then microinjected near the A7 cell group to block synaptic activation of spinally-projecting A7 neurons, which were identified using tyrosine-hydroxylase immunoreactivity. Within 5 min of the cobalt chloride injection, the antinociceptive effect of carbachol stimulation was blocked. In another set of experiments, the NK 1 receptor antagonist L-703-606 (5 μg) was microinjected near the A7 cell group following LH stimulation with carbachol. L-703-606 also abolished LH-induced antinociception. These results support the conclusion that antinociception produced by activating substance P neurons in the LH is mediated in part by the subsequent activation of spinally-projecting noradrenergic neurons in the A7 cell group.

Microinjection of carbachol in the lateral hypothalamus produces opposing actions on nociception mediated by α 1- and α 2-adrenoceptors

Electrical stimulation of the lateral hypothalamus (LH) produces antinociception partially blocked by intrathecal α-adrenergic antagonists, but the mechanism underlying this effect is not clear. Evidence from immunological studies demonstrates that substance P-immunoreactive neurons in the LH project near the A7 catecholamine cell group, a group of noradrenergic neurons in the pons known to effect antinociception in the spinal cord dorsal horn. Such evidence suggests that LH neurons may activate A7 neurons to produce antinociception. To test this hypothesis, the cholinergic agonist carbachol was microinjected into the LH at doses of 63, 125 and 250 nmol and the resulting effects on tail-flick and nociceptive foot-withdrawal latencies were measured. All three doses significantly increased response latencies on both tests, with the 125-nmol dose providing the optimal effect. Intrathecal injection of the opioid antagonist naltrexone (97 nmol) partially reversed antinociception, but neither the α 2-adrenoceptor antagonist yohimbine nor the α 1-adrenoceptor antagonist WB4101 altered latencies. However, two sequential doses of yohimbine blocked LH-induced antinociception on both tests. In contrast, two sequential doses of WB4101 increased nociceptive responses on both the tail-flick and foot-withdrawal tests. These findings, and those of published reports, suggest that neurons in the LH activate spinally projecting methionine enkephalin neurons, as well as two populations of A7 noradrenergic neurons that exert a bidirectional effect on nociception. One of these populations increases nociception through the action of α 1-adrenoceptors and the other inhibits nociception through the action of α 2-adrenoceptors in the spinal cord dorsal horn.

Characterization of the innervation of the gallbladder mucosa in the Australian brush-tailed possum

Intrinsic and extrinsic nerves innervate the gallbladder epithelium and modulate its functions. Some neurochemicals of this innervation are known in some species, however, the intrinsic and extrinsic origin of this innervation is unclear. Aim: To characterize the neurons projecting to the gallbladder epithelium in the Australian Brush-tailed possum using in vitro retrograde tracing, extrinsic denervation in culture and immunohistochemistry. Methods: Gallbladders were harvested from 9 anesthetized possums. In 6 gallbladders, a fluorescent neural tracer was focally applied to the epithelial surface to identify intrinsic neurons innervating the epithelium. Following organotypic culture, the preparations were dissected and an antiserum against nitric oxide synthase (NOS) was applied. Retrogradely traced and antibody labeled neurons were visualized using fluorescence microscopy and their positions recorded. A further 3 gallbladders were cut into 4 longitudinal segments. Two segments from each gallbladder were fixed immediately and the others placed in organotypic culture for 5 days to achieve extrinsic penervatlon. Multiple-labeling immunohistochemistry for substance P (SP), vasoactive intestinal polypeptide (VlP), calcitonin gene related peptide (CGRP) and tyrosine hydroxylase (TH) were used to characterize the intrinsic and extrinsic innervation of the ganglionated and non-ganglionated sub-epithelial plexus of the gallbladder. Results: Retrogradely traced neurons were found within lOmm of the tracer application site, exclusively within the subepithelial plexus, with 63 _+ 9% being immunoreactive for NOS. The majority of neurons within the sub-epithelial plexus colocalized both NOS and MIP. Some SP immunoreactive neurons were seen. Varicose nerve fibres showing immunoreactivity for NOS, VIP, SP, CGRP and TH were seen within the sub-epithelial plexuses in freshly fixed tissue. Immunoreactivity for CGRP and TH were abolished post-culture, with some reduction in the SP and MIP innervation. Summary: NOS and VlP-colocalizing and SP-immunoreactive neurons of the ganglionated sub-epithelial plexus innervate the gallbladder epithelium, with no innervation from the muscular and serosal plexuses. This is the first demonstration of the neurochemistry and spatial organisation of the intrinsic innervation of the gallbladder epithelium. Sympathetic and extrinsic afferent neurons also directly innervate the epithelium and the ganglionated subepithelial plexus.

Patterns of status epilepticus-induced substance P expression during development

Substance P, which modulates synaptic excitability, can be induced by a variety of stimuli. We studied the expression of hippocampal substance P in rats in using lithium-pilocarpine model of status epilepticus during development. Status epilepticus resulted in an age-specific manner of substance P expression that was anatomically distinctive in hippocampal subfields. Maximal induction of substance P immunoreactivity was seen in the CA1 region of the two-week-old rats, and progressively decreased in the three-, four-week-old rats and adults. Meanwhile, the number of substance P-immunoreactive neurons in the CA3 region and dentate granule cell layer was minimal in the two-week-old animals, but approximated the adult level in the three- and four-week-old rats. No substance P-immunoreactive axon terminals were seen in the strata pyramidale and lucidum in the CA3 region of the two-week-old rats, but they were found to progressively increase in the three-, four-week-old rats and adults. To confirm substance P expression after status epilepticus, we studied the expression of preprotachykinin-A mRNA in the hippocampus of the three-week-old rats by in situ hybridization. Two hours following injection of lithium-pilocarpine, preprotachykinin-A mRNA dramatically increased in the granule cells, as well as in the CA3 and CA1 pyramidal cell layers of the hippocampus. To evaluate the relationship between behavioral seizures and substance P induction, we used the NMDA receptor antagonist MK-801. Injection of MK-801 completely blocked lithium-pilocarpine-induced behavioral seizures and SP induction in the two-week-old rats. These results indicate that seizure activity selectively evokes age-dependent and region-selective expression of substance P.

Morphine treatment induced calcitonin gene-related peptide and substance P increases in cultured dorsal root ganglion neurons

The mechanism of spinal tolerance to the analgesic effects of opiates is unclear at present. We have reported previously that calcitonin gene-related peptide-like immunoreactivity was significantly increased in primary afferents of the spinal dorsal horn during the development of morphine tolerance, suggesting that changes in the level of pain-related neuropeptides in dorsal root ganglion neurons may be involved [Menard D. P. et al. (1996) J. Neurosci. 16, 2342–2351]. In this study, we investigated if in vitro treatment with morphine can mimic the in vivo findings and induce increases in calcitonin gene-related peptide-like immunostaining in cultured dorsal root ganglion neurons from young (three-month-old) and middle-aged (10-month-old) adult rats. Following a repetitive exposure to morphine sulfate (1, 5, 10 μM) for six days, the number of calcitonin gene-related peptide- and substance P-immunoreactive neurons in cultured dorsal root ganglia from three- and 10-month-old rats was significantly increased. A lower concentration (0.5 μM) of morphine induced these increases only in dorsal root ganglion neurons from middle-aged rats. Morphine treatment was also found to increase the number of calcitonin gene-related peptide-immunoreactive neurons possessing multiple, long branches (i.e. with at least one branch >0.5 mm). This apparent increase in the number of calcitonin gene-related peptide- and substance P-immunoreactive neurons observed following morphine treatment was blocked by naloxone, an opiate antagonist, indicating the involvement of genuine opioid receptors. No significant change in the number of neuropeptide Y- or galanin-immunoreactive neurons in cultured dorsal root ganglia was detected following any of these treatments. These data suggest that repeated exposure to morphine rather selectively increases calcitonin gene-related peptide- and substance P-like immunoreactivity in cultured dorsal root ganglion neurons. Moreover, the sensitivity to morphine-induced changes is greater in cultured dorsal root ganglion neurons from 10- compared to three-month-old rats. Hence, cultured dorsal root ganglion neurons can provide a model to investigate the cellular and molecular mechanisms underlying alterations in neuropeptide levels following repeated exposure to opiates and their relevance to the development of opioid tolerance.

Substance P-immunoreactive neurons in the rostromedian area of the midbrain send axons to the lower spinal cord in the chicken

Substance P-immunoreactivity in neurons projecting to the spinal cord was examined using retrograde tract-tracing method combined with immunohistochemical techniques in chickens. Many small substance P-immunoreactive neurons were densely clustered in the midline area in the rostral midbrain, the rostromedian area (80% of the neurons in the rostromedian area). Some of these substance P-immunoreactive neurons in the rostromedian area (about 20% of substance P-immunoreactive neurons) were retrogradely labeled by small injections of wheat germ agglutinin conjugated horseradish peroxidase into the central part of the lumber segments including the intermediomedial nucleus, suggesting the projections from the rostromedian area to the lower spinal preganglionic regions. From the present data and mammalian previous studies, it was suggested that the midline area in the midbrain has fiber connections with the regions related autonomic functions, and all of which exhibit substance P-immunoreactivity.

Substance P-immunoreactive neurons in hamster retinas.

Light-microscopic immunocytochemistry was utilized to localize the different populations of substance P-immunoreactive (SP-IR) neurons in the hamster retina. Based on observation of 2505 SP-IR neurons in transverse sections, 34% were amacrine cells whose pear-shaped or round cell bodies (7-8 microm) were situated in the inner half of the inner nuclear layer (INL) or in the inner plexiform layer (IPL), while 66% of SP-IR somata (6-20 microm) were located in the ganglion cell layer (GCL) which were interpreted to be displaced amacrine cells and retinal ganglion cells (RGCs). At least three types of SP-IR amacrine cells were identified. The SP-IR processes were distributed in strata 1, 3, and 5 with the densest plexus in stratum 5 of the inner plexiform layer. In the wholemounted retina, the SP-IR cells were found to be distributed throughout the entire retina and their mean number was estimated to be 4224 +/- 76. Two experiments were performed to clarify whether any of the SP-IR neurons in the GCL were RGCs. The first experiment demonstrated the presence of SP-IR RGCs by retrogradely labeling the RGCs and subsequently staining the SP-IR cells in the retina using immunocytochemistry. The second experiment identified SP-IR central projections of RGCs to the contralateral dorsal lateral geniculate nucleus. This projection disappeared following removal of the contralateral eye. The number of SP-IR RGCs was estimated following optic nerve section. At 2 months after sectioning the optic nerve, the total number of SP-IR neurons in the GCL reduced from 4224 +/- 76 to a mean of 1192 +/- 139. Assuming that all SP-IR neurons in the GCL which disappeared after nerve section were RGCs, the number of SP-IR RGCs was estimated to be 3032, representing 3-4% of the total RGCs. In summary, findings of the present study provide evidence for the existence of SP-IR RGCs in the hamster retina.

Compartmental expression of trkB receptor protein in the developing striatum

To investigate the role of neurotrophins in the initial formation of striatal patch versus matrix, the spatial and temporal expression of trkB receptors was examined using immunohistochemistry. Polyclonal antibodies, against the C-terminus or the tyrosine kinase domain, revealed trkB-immunoreactive cells and fibers localized to patches beginning on embryonic day 19 in the rat, which co-localized with patchy dopamine fibers, substance P-immunoreactive neurons and glutamate receptors. Patchy striatal trkB expression was maintained after lesioning the nigrostriatal dopamine system. The patchy trkB distribution persisted through postnatal day 14, then became more homogeneous at the same time that nigrostriatal afferents become homogeneous. Later in development, trkB immunoreactivity was most intense in a subpopulation of large striatal cells that were similar in size and frequency to those immunoreactive for choline acetyltransferase. The spatiotemporal expression of trkB receptor in phenotypically distinct striatal patches, as well as evidence that neurotrophins regulate expression of neuronal phenotypic markers during development, may indicate a convergence of neurotrophins and afferent innervation on to future patch cells that may regulate the establishment of striatal compartmentalization.

Control of negative inotropic vagal preganglionic neurons in the dog: synaptic interactions with substance P afferent terminals in the nucleus ambiguus?

Previous research from this laboratory has shown that substance P-immunoreactive (SP) terminals synapse upon negative chronotropic vagal preganglionic neurons (VPNs), but not upon negative dromotropic VPNs, of the ventrolateral nucleus ambiguus (NA-VL). Moreover, SP agonists injected into NA-VL cause bradycardia without decreasing AV conduction. In the current study, we have: (1) defined the electron microscopic characteristics of the SP neurons of NA-VL in dog; and (2) tested the hypothesis that SP nerve terminals synapse upon negative inotropic VPNs of NA-VL, retrogradely labeled from the cranial medial ventricular (CMV) ganglion. Numerous SP terminals and a few SP neurons were observed in the vicinity of retrogradely labeled neurons. SP terminals were observed forming synapses with unlabeled dendrites and with SP dendrites, but never with the retrogradely labeled neurons. Together, these results and earlier findings suggest that SP agonists may be able to induce bradycardia without decreasing AV conduction or ventricular contractility.

Circuitry and role of substance P-immunoreactive neurons in the primate retina

In this paper, we extend our previous light microscopic (LM) study of substance P (SP)-containing amacrine and ganglion cell types of the human retina (Cuenca et al. [1995] J. Comp. Neurol. 356:491-504) to an electron microscopic (EM) and confocal-imaging study in order to reveal synaptic circuitry and putative input and output neurons. SP-immunoreactive (-IR) amacrine cells in primate retina are typically wide-field cells with large cell bodies occurring in normal or displaced positions relative to the inner plexiform layer (IPL). Their main dendrites bear many spines and are monostratified in stratum 3 (S3) of the IPL. Axon-like processes arise from dendrites close to the cell body and run for hundreds of microns at the same level as the dendrites, thus forming a relatively dense plexus in S3 of the IPL. SP-IR axon processes also climb to S1 to surround some amacrine cell bodies, and others pass into the outer plexiform layer (OPL). Still other axons run down to the ganglion cell layer, where they encircle SP-IR ganglion cells and pass on to end in the nerve fiber layer. The SP-IR ganglion cell types have large cell bodies (20-22 microm diameter) and dendrites that costratify in S3 among the SP-IR amacrine cell processes. Double immunostaining and study by confocal microscopy reveals that SP-IR amacrine cells in the monkey colocalize gamma-aminobutyric acid (GABA). Their main plexus of dendrites in S3 of the IPL is skirted on the S2/S3 border by cone bipolar axons that stain for calbindin but intermingles primarily with glycinergic bipolar cell types of S3 and S3-S4. Strongly GABA-IR/weakly glycine-IR amacrine cell bodies, in addition to the SP-IR large-bodied ganglion cell type, are targets of encircling SP-IR axon processes. EM study of the human SP-IR amacrine cell indicates that input synapses to its dendrites are from bipolar cell axons of the S2/S3 border, S3, and the S3/S4 border of the IPL neuropil (33% of the synaptic input) and from amacrine cell processes (67% of the synaptic input). The input amacrine cells are of at least two distinct types based on cytological criteria. Synaptic output from the SP-IR amacrine cell dendrites is to bipolar cell axons as reciprocal synapses (31%), to amacrine cells (40%), and to ganglion cell profiles, primarily in S3 (29%) of the IPL. The SP-IR axons synapse upon SP-IR ganglion cell bodies and axons, upon normally placed and displaced amacrine cell bodies, and upon bipolar cell dendrites in the OPL. In addition, they appear to synapse among themselves. We shall discuss a wiring diagram and the possible role of SP-IR amacrine cells in the primate retina.

Substance P immunoreactive nerve terminals in the dorsolateral nucleus of the tractus solitarius: roles in the baroreceptor reflex

Physiological and light microscopic evidence suggest that substance P (SP) may be a neurotransmitter contained in first-order sensory baroreceptor afferents; however, ultrastructural support for this hypothesis is lacking. We have traced the central projections of the carotid sinus nerve (CSN) in the cat by utilizing the transganglionic transport of horseradish peroxidase (HRP). The dorsolateral subnucleus of the nucleus tractus solitarius (dlNTS) was processed for the histochemical visualization of transganglionically labeled CSN afferents and for the immunocytochemical visualization of SP by dual labeling light and electron microscopic methods. Either HRP or SP was readily identified in single-labeled unmyelinated axons, myelinated axons, and nerve terminals in the dlNTS. SP immunoreactivity was also identified in unmyelinated axons, myelinated axons, and nerve terminals in the dlNTS, which were simultaneously identified as CSN primary afferents. However, only 15% of CSN terminals in the dlNTS were immunoreactive for SP. Therefore, while the ultrastructural data support the hypothesis that SP immunoreactive first-order neurons are involved in the origination of the baroreceptor reflex, they suggest that only a modest part of the total sensory input conveyed from the carotid sinus baroreceptors to the dlNTS is mediated by SP immunoreactive CSN terminals. Five types of axo–axonic synapses were observed in the dlNTS. SP immunoreactive CSN afferents were very rarely involved in these synapses. Furthermore, SP terminals were never observed to form the presynaptic element in an axo–axonic synapse with a CSN afferent. Therefore, SP does not appear to be involved in the modulation of the baroreceptor reflex in the dlNTS.

Central distribution of sensory fibers in the facial nerve: an anatomical and immunohistochemical study.

Wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) injection into the facial nerve of the cat resulted in retrograde labeling in the geniculate and jugular ganglia ipsilaterally. Labeled fibers were found to enter into the brain stem through the intermediate and vagal nerves. These fibers ascended or descended into the dorsal portion of the spinal trigeminal tract and were distributed to the principal sensory nucleus of the trigeminal nerve, marginal layer of the interpolar part of the spinal trigeminal nucleus, nucleus of the solitary tract and ventrolateral portion of the cuneate nucleus. It was of particular interest in the present study that the intensive labeling was present in the medial portion of laminae I-IV of the upper cervical spinal cord. The immunohistochemical study revealed a lot of substance P-immunoreactive neurons in the geniculate and jugular ganglia, and heavy accumulation of immunoreactive fibers in laminae I-II of the upper cervical spinal cord.