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Medullary dorsal horn (MDH), the homolog of spinal dorsal horn, plays essential roles in processing of nociceptive signals from orofacial region toward higher centers, such as the ventral posteromedial thalamic nucleus (VPM) and parafascicular thalamic nucleus (Pf), which belong to the sensory-discriminative and affective aspects of pain transmission systems at the thalamic level, respectively. In the present study, in order to provide morphological evidence for whether neurons in the MDH send collateral projections to the VPM and Pf, a retrograde double tracing method combined with immunofluorescence staining for substance P (SP), SP receptor (SPR) and Fos protein was used. Fluoro-gold (FG) was injected into the VPM and the tetramethylrhodamine-dextran (TMR) was injected into the Pf. The result revealed that both FG- and TMR-labeled projection neurons were observed throughout the entire extent of the MDH, while the FG/TMR double-labeled neurons were mainly located in laminae I and III. It was also found that some of the FG/TMR double-labeled neurons within lamina I expressed SPR and were in close contact with SP-immunoreactive (SP-ir) terminals. After formalin injection into the orofacial region, 41.4% and 34.3% of the FG/TMR double-labeled neurons expressed Fos protein in laminae I and III, respectively. The present results provided morphological evidence for that some SPR-expressing neurons within the MDH send collateral projections to both VPM and Pf and might be involved in sensory-discriminative and affective aspects of acute orofacial nociceptive information transmission., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

In the dorsal facial area (DFA) of the medulla, an activation of either P2 purinergic receptor or nitric oxide synthase (NOS) results in the release of glutamate, leading to an increase in blood flow of the common carotid artery (CCA). It is not known whether activation of the P2 receptor by ATP may mediate activation of NOS/guanylyl cyclase to cause glutamate release and/or whether L-Arg (nitric oxide (NO) precursor) may also cause ATP release from any other neuron, to cause an increase in CCA flow. We demonstrated that microinjections of P2 receptor agonists (ATP, α,β-methylene ATP) or NO precursor (L-arginine) into the DFA increased CCA blood flow. The P2-induced CCA blood flow increase was dose-dependently reduced by pretreatment with NG-nitro-arginine methyl ester (L-NAME, a non-specific NOS inhibitor), 7-nitroindazole (7-NI, a relatively selective neuronal NOS inhibitor) or methylene blue (MB, a guanylyl cyclase inhibitor) but not by that with D-NAME (an isomer of L-NAME) or N5-(1-iminoethyl)-L-ornithine (L-NIO, a potent endothelial NOS inhibitor). Involvement of glutamate release in these responses were substantiated by microdialysis studies, in which perfusions of ATP into the DFA increased the glutamate concentration in dialysates, but co-perfusion of ATP with L-NAME or 7-NI did not. Nevertheless, the arginine-induced CCA blood flow increase was abolished by combined pretreatment of L-NAME and MB, but not affected by pretreatment with a selective P2 receptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). In conclusion, ATP activation of the P2 receptor in the DFA induced activation of neuronal NOS/guanylyl cyclase, which causes glutamate release leading to an increase in CCA blood flow. However, arginine activation of neuronal NOS/guanylyl cyclase, which also caused glutamate release and CCA blood flow increase, did not induce activation of P2 receptors. These findings provide important information for drug design and/or developing therapeutic strategies for the diseases associated with CCA blood flow that supplies intra- and extra-cranial tissues., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

Brain-derived neurotrophic factor (BDNF) is a unique neurotrophin which not only supports the development of neurons but also modulates the synaptic activity in a number of neuronal systems. BDNF is synthesized in neurons, anterogradely transported and released from nerve terminals and exerts acute effects on synaptic transmission in both peripheral and central nervous systems. Previous studies have shown that BDNF is distributed in several groups of neurons in the brain stem which regulate cardiovascular functions. Here we showed that injection of BDNF (40-400 ng/100 nl) into the rostral ventrolateral medulla resulted in a significant increase in arterial blood pressure (Delta35.5+/-3.5 mmHg) in rats. The duration of change in blood pressure was 145+/-40 s with a latency of 3-5 s. There was no significant effect on the heart rate. The injection of glutamate as a positive control also triggered an increase in blood pressure. Injection of phosphate-buffered saline as a control or the same amount of nerve growth factor did not cause significant changes in blood pressure in different preparations. Immunohistochemistry showed that the nerve terminals immunoreactive for BDNF were localized in several brain stem regions and terminate around spinal projection neurons in the rostral ventrolateral medulla. Neurons in the rostral ventrolateral medulla can uptake exogenous BDNF and express the high affinity receptor trkB. From these results we suggest that BNDF in the medulla may play a role in the regulation of blood pressure.

The biological actions of the neuromodulator somatostatin are mediated through a family of G-protein-coupled receptors, of which five members, sst(1-5), have been identified. Although the messenger RNA distribution of the sst(4) receptor has been reported, no information about the distribution of the receptor protein in the central nervous system is available. We have therefore raised a polyclonal peptide antibody against a rat carboxy-terminal sst(4) peptide. The selectivity of the affinity-purified antibody was demonstrated by western blotting of membrane proteins isolated from Chinese hamster ovary-K1 cells expressing the recombinant sst(4) receptor and from the rat hippocampus. This resulted in both cases in the identification of a single band of approximately 42,000 mol. wt. Furthermore, the sst(4) receptor antibody selectively labelled Chinese hamster ovary-K1 cells expressing the recombinant sst(4) receptor in immunocytochemistry. No cross-reactivity was observed with other recombinant somatostatin receptors. Immunohistochemistry on adult rat brain sections showed the sst(4) receptor to have a widespread distribution. This included labelling of cell bodies as well as processes in the cerebral cortex, hippocampus and several nuclei in the brainstem. All signals were absent following antibody preabsorption with the synthetic sst(4) peptide. This study provides the first detailed analysis of the distribution of sst(4) receptor protein in the rat brain.

Regional distribution of gonadotropin-releasing hormone (GnRH)-like-, beta-endorphin (beta-end)-like-, and methionine-enkephalin (met-enk)-like-immunoreactivity was quantified across various regions of the central nervous system (CNS) of male and female goats by using highly specific radioimmunoassays. All the animals were sacrificed during the months of March through June (non-breeding season). Although the distribution of these three neuropeptides was similar to other mammalian species, species-specific gender differences in the levels of neuropeptides were noticed in the goat CNS. Highest levels of GnRH-like immunoreactivities were found in the hypothalamus. The hypothalamus of male goats exhibited significantly higher levels of GnRH-like immunoreactivities compared to female goats. Other regions exhibiting GnRH-like immunoreactivities included olfactory bulbs, preoptic and supraoptic regions, and mamillary bodies. Both beta-end- and met-enk immunoreactivities were detected in all selected regions of goat CNS, but highest levels of these opioid peptide-like immunoreactivities were limited to the forebrain regions of the goat. The supraoptic area of the female goats contained significantly higher levels of beta-end-like immunoreactivities than that of the male goats. Met-enk-peptide-like immunoreactivity also exhibited gender-specific differences in its content in some regions of the CNS. The male goats exhibited significantly higher levels of met-enk-like immunoreactivity in both the striatal and hypothalamic regions of the brain.

The distribution of N-methyl-D-aspartate receptor subunit messenger RNA was examined in the superior olivary complex of the rat using in situ hybridization methods. Non-radioactive methods showed N-methyl-D-aspartate receptor-1 and N-methyl-D-aspartate receptor-2A, -2B and -2C subunit expression to be abundant in principal cells of all major superior olivary complex regions, with N-methyl-D-aspartate receptor-1 expression higher than N-methyl-D-aspartate receptor-2. After auto-radiographic in situ hybridization, counting silver grains over cells was used to quantitate and compare levels of expression of the N-methyl-D-aspartate receptor-2 subunits. N-Methyl-D-aspartate receptor-2A, -2B and -2C expression was detected over cells in the lateral and medial superior olive, the medial, lateral and ventral nucleus of the trapezoid body, and the superior paraolivary nucleus. N-Methyl-D-aspartate receptor-2D expression was only observed in the lateral superior olive, lateral nucleus of the trapezoid body and ventral nucleus of the trapezoid body. Three distinct patterns of labeling were observed. The lateral superior olive and ventral nucleus of the trapezoid body showed higher expression of N-methyl-D-aspartate receptor-2A and -2C than other subunits; the medial nucleus of the trapezoid body showed higher N-methyl-D-aspartate receptor-2B expression than other subunits; and the medial superior olive, superior paraolivary nucleus and ventral nucleus of the trapezoid body showed relatively equivalent expression of N-methyl-D-aspartate receptor-2A, -2B and -2C subunits. N-Methyl-D-aspartate receptor-2D had the lowest expression, with levels greater than background in only the lateral superior olive, ventral nucleus of the trapezoid body and lateral nucleus of the trapezoid body. Immunocytochemistry using antibodies to N-methyl-D-aspartate receptor-2A, -2B and -2C showed immunolabeling consistent with in situ hybridization results. These results show diversity in expression of N-methyl-D-aspartate receptor-2 subunits in different superior olivary complex regions and would predict pharmacological differences.

There appear to be different relationships between mu-opioid receptor densities and the acute and neuroadaptive mu-opioid agonist-induced responses of the multiple opioid neuronal systems, including important pons/medulla circuits. The recent success in creating mu-opioid receptor knockout mice allows studies of mu-opioid agonist-induced pharmacological and physiological effects in animals that express no, one or two copies of the mu-opioid receptor gene. We now report that the binding of mu-opioid receptor ligand, [3H][D-Ala2,NHPhe4,Gly-ol]enkephalin to membrane preparations of the pons/medulla was reduced by half in heterozygous mu-opioid receptor knockout mice and eliminated in homozygous mu-opioid receptor knockout mice. The endogenous mu-opioid agonist peptides endomorphin-1 and -2 activate G-proteins in the pons/medulla from wild-type mice in a concentration-dependent fashion, as assessed using [35S]guanosine-5'-o-(3-thio)triphosphate binding. This stimulation was reduced to half of the wild-type levels in heterozygous mice and eliminated in homozygous knockout mice. The intracerebroventricular injection of either endomorphin-1 or endomorphin-2 produced marked antinociception in the hot-plate and tail-flick tests in wild-type mice. These antinociceptive actions were significantly reduced in heterozygous mu-opioid receptor knockout mice, and virtually abolished in homozygous knockout mice. The mu-opioid receptors are the principal molecular targets for endomorphin-induced G-protein activation in the pons/medulla and the antinociception caused by the intracerebroventricular administration of mu-opioid agonists. These data support the notion that there are limited physiological mu-opioid receptor reserves for inducing G-protein activation in the pons/medulla and for the nociceptive modulation induced by the central administration of endomorphin-1 and -2.

Immunocytochemical methods were used to determine the distribution of calcitonin gene-related peptide (CGRP) and neuropeptide Y (NPY) in the middle region of the adult raccoon cuneate nucleus. Extensive CGRP-immunoreactive fibers and darkly stained punctate structures, thought to be terminals, were concentrated in the dorsal cap and basal region of the middle cuneate nucleus. These regions receive input from the claws and the hairy skin of the paw and forearm. The NPY-immunoreactive fibers and terminals were also found within the dorsal cap of the cuneate nucleus, but were less abundant than CGRP. However, most of the NPY-immunostained fibers and terminals were found in the cluster region of the cuneate nucleus, which receives input from glabrous skin. No CGRP- or NPY-immunoreactive cell bodies were found in the raccoon middle cuneate nucleus. This description of the distribution of CGRP and NPY in the normal animal provides a baseline for future investigations into injury-induced neuropeptide plasticity in the raccoon middle cuneate nucleus.

Vagal afferents originating in abdominal viscera initiate numerous centrally-mediated responses, including behavioural, cardiovascular and hormonal changes associated with satiety, and nausea and vomiting. The present work was undertaken to map the pontomedullary distribution of neurons expressing Fos immunoreactivity following unilateral electrical stimulation of abdominal vagal afferents in conscious unanaesthetized rabbits. After 2 h of stimulation of the anterior trunk of the abdominal vagus nerve (20 Hz, 0.5 mA, 0.5 ms duration, 4.5 min on, 0.5 min off), Fos-positive neurons were found in the area postrema, the nucleus tractus solitarius, the spinal nucleus of the trigeminal nerve, the caudal and the rostral ventrolateral medulla, the locus coeruleus, the subcoeruleus and the lateral parabrachial nucleus. In all these regions, more than 70% of Fos-containing neurons occurred on the ipsilateral side. In control animals only occasional Fos-immunoreactive neurons were observed, usually very faintly labelled. Simultaneous staining for both Fos and tyrosine hydroxylase revealed Fos immunoreactivity in catecholamine neurons, including A1, A2, C1, A5, subcoeruleus and locus coeruleus (A6) groups. Our findings complement functional studies in the rabbit, identifying A1 neurons as part of the central pathway by which afferent abdominal vagal stimulation increases plasma vasopressin, and C1 neurons as part of the central pathway, whereby afferent abdominal vagal stimulation increase arterial pressure.

The midline of the rostral ventral medulla (RVM) is the portion in which many serotonin (5-HT) neurons of the nucleus raphe magnus and the rostral nucleus raphe pallidus are located and where dense catecholaminergic (CA) fibers are distributed. In this study, we investigated the connection between spinally projecting 5-HT neurons and CA fibers in the rat RVM by light and electron microscopic immunocytochemistry. First, light microscopic immunocytochemistry using a triple labeling method revealed that the 5-HT-immunoreactive (IR) neuron containing retrograde tracer from the cervical cord was intimately surrounded by tyrosine hydroxylase (TH)-IR fibers. Second, silver-gold intensified TH-IR axon terminals were found to make synaptic contacts with 5-HT-IR neuronal perikarya and dendrites by double labeling immunoelectron microscopy. These morphological findings suggest that spinally projecting 5-HT neurons, presumed to be involved in pain modulation or sympathetic autonomic control, are directly regulated by CA neurons at the level of the RVM.

Transneuronal tracing techniques were used in order to identify putative spinal interneurons and brainstem sites involved in the control of penile function. Pseudorabies virus was injected into the corpus cavernosus tissue of the penis in rats. After a four day survival period, rats were perfused with fixative and virus-labelled neurons were identified by immunohistochemistry. Postganglionic neurons were retrogradely labelled in the major pelvic ganglia. In the spinal cord, sympathetic and parasympathetic preganglionic neurons were labelled transneuronally. Presumptive interneurons were also labelled in the lower thoracic and lumbosacral spinal cord in locations consistent with what is currently known about such interneurons. In the brainstem, transneuronally labelled neurons were found in the medulla, pons and hypothalamus. Regions consistently labelled included the nucleus paragigantocellularis, parapyramidal reticular formation of the medulla, raphe pallidus, raphe magnus, A5 noradrenergic cell group, Barrington's nucleus and the paraventricular nucleus of the hypothalamus. This study confirmed previous studies from our lab and others concerning the preganglionic and postganglionic neurons innervating the penis. The number, morphology and location of these neurons were consistent with labelling seen following injection of conventional tracers into the penis. The brainstem nuclei labelled in this study were also consistent with what is currently known about the brainstem control of penile function. The labelling appeared to be highly specific, in that descending systems involved in other functions were not labelled. These results provide further evidence that the pseudorabies virus transneuronal tracing technique is a valuable method for identifying neural circuits mediating specific functions.

Neurons in the ventrolateral medulla oblongata of rats, guinea-pigs and cats that contain tyrosine hydroxylase, dopamine-beta-hydroxylase, phenylethanolamine-N-methyltransferase and neuropeptide Y have been demonstrated immunohistochemically in serial coronal sections of tissue taken from the level of the cervical spinal cord to the level of the facial nucleus. The anatomical distribution of these neurons has been described, quantified and reconstructed in three dimensions to compare the neuron populations between species. In all species, between 50 and 90% of immunoreactive neurons lay rostral to the level of the obex. There were no significant differences in the number and distribution of neurons containing catecholamine-synthesizing enzymes between control animals and those pretreated with colchicine, with two exceptions: all dopamine-beta-hydroxylase neurons were weakly immunoreactive without colchicine pretreatment in cats, and pretreatment with colchicine revealed a small rostral group of tyrosine hydroxylase-positive neurons in guinea-pigs. There were remarkable similarities in the rostrocaudal distributions of neurons containing tyrosine hydroxylase, dopamine-beta-hydroxylase and neuropeptide Y in relation to comparable anatomical landmarks across the species. However, the distributions of neurons containing tyrosine hydroxylase. Phenylethanolamine-N-methyltransferase-positive neurons, while densely stained in rats, were only faintly stained in cats and absent in guinea-pigs; the distribution of these neurons was similar to the distribution of neurons containing only tyrosine hydroxylase. The similarity of the distribution of neurons demonstrated using tyrosine hydroxylase, dopamine-beta-hydroxylase and neuropeptide Y immunohistochemistry implies that homologous catecholamine-containing neuron groups do exist in the ventrolateral medulla despite the variation in phenylethanolamine-N-methyltransferase between species. In contrast to the previous classification of neuron groups into A1 and C1 based on the presence or absence of this latter enzyme, the data suggest that a discrete group of tyrosine hydroxylase-immunoreactive neurons, which probably do not contain dopamine-beta-hydroxylase or neuropeptide Y, can be distinguished in the rostral ventrolateral medulla of all species. The absence of detectable dopamine-beta-hydroxylase in this group of neurons suggests that they may not synthesize either adrenaline or noradrenaline.

The distribution and ultrastructure of thyrotropin-releasing hormone-like immunoreactive (TRH-LI) neurons were examined in rabbit medulla oblongata. TRH-LI cell bodies were located in the ventral region of the medulla oblongata: in the paraolivary and parapyramidal regions, regions in and around the pyramidal tract, the dorsolateral region of the lateral reticular nucleus, and the raphe nuclei. The paraolivary and parapyramidal regions contained most of the TRH-LI cell bodies in the medulla oblongata. TRH-LI neurons processes were densely distributed in the dorsal vagal complex and the area postrema. Electron-microscopic immunocytochemical studies revealed TRH-LI neurons at the obex level in the paraolivary region of rabbits. TRH-like immunoreactivity was localized in larger granular vesicles. TRH-LI somata and dendrites received synaptic inputs from both TRH-LI and unlabeled axon terminals. More than half of the TRH-LI axon terminals made synapses with somata or processes of TRH-LI neurons. These observations, together with previous reports that TRH causes respiratory facilitation, suggest that TRH-LI neurons in the paraolivary region in rabbits may be involved in respiratory functions.

The data in the preceding paper [Halliday G. M. and McLachlan E. M. (1991) Neuroscience 43, 531-550] suggest that some neurons in the rostral ventrolateral medulla contain some catecholamine-synthesizing enzymes but may not produce catecholamines. The present study addresses this question directly by comparing the anatomical location and morphology of these neurons with those revealed by formaldehyde-induced fluorescence. Catecholamine-containing somata of rats and guinea-pigs have been demonstrated following FAGLU-perfusion in normal untreated animals, in animals pretreated with pargyline (a monoamine oxidase inhibitor), and in animals pretreated with colchicine (to block axoplasmic transport). The number and location of fluorescent somata in the ventrolateral medulla have been determined in serial coronal sections of tissue from the cervical spinal cord to the level of the facial nucleus. Catecholamine-fluorescent neurons at different levels of the ventrolateral medulla varied in their topography and sensitivity to pharmacological manipulation. However, the rostrocaudal distributions in rats and guinea-pigs were quantitatively remarkably similar implying that homologous groups of catecholamine-containing neurons exist. Comparison between these distributions and those of somata stained immunohistochemically for catecholamine-synthesizing enzymes and neuropeptide Y [Halliday G. M. and McLachlan E. M. (1991) Neuroscience 43, 531-550] revealed that the majority of fluorescent neurons in both species probably contain dopamine-beta-hydroxylase and neuropeptide Y as well as tyrosine hydroxylase. Those neurons lying just caudal to the facial nucleus immunoreactive for tyrosine hydroxylase and phenylethanolamine-N-methyltransferase but not dopamine-beta-hydroxylase and neuropeptide Y also lack catecholamine fluorescence. This rostral group of somata can be identified immunohistochemically in cats. The size and morphology of catecholamine-fluorescent neurons have been analysed in detail, and compared with the same features of the immunohistochemically stained neurons. Three morphological types of catecholamine-containing neurons could be distinguished in material prepared by both techniques from rats and guinea-pigs, and in immunohistochemical material from cats. Rostral tyrosine hydroxylase-positive neurons, which differed morphologically from these three types, were present in all three species. On the basis of anatomical location, neuronal morphology and chemical characteristics, four groups of tyrosine hydroxylase-immunoreactive neurons have been identified in the ventrolateral medulla of rats, guinea-pigs and cats. Only the caudal three of these four groups appear to synthesize catecholamine, probably noradrenaline. From published data it seems likely that these four groups of tyrosine hydroxylase-positive neurons have distinct projections and functions related to cardiovascular and respiratory control.

The immunocytochemical distribution of CLIP (corticotropin-like intermediate lobe peptide) or ACTH(18-39), a small biologically active peptide, was examined in the human brain, using a monoclonal antibody against this peptide. Groups of CLIP-immunoreactive cell bodies, small to medium size and bipolar or triangular in shape, were found in the basal hypothalamus extending from the retrochiasmatic region to the premammillary nuclei area. Immunoreactive fibers with varicosities, terminals and "pipe shape" structures, were distributed within the hypothalamus, limbic structures, the brainstem and spinal cord nuclei, forming a particularly rich network in the hypothalamus, the preoptic area, the septal region, the amygdala and the upper brainstem periaqueductal gray matter. The above neuroanatomical observations confirm and extend previous findings in animals, strengthening even more the possibility that this peptide may be involved in numerous behavioral, autonomic and physiological functions such as regulation of sleep-waking cycle, pain control and respiratory and cardiovascular regulation.

The basal overflow of extracellular endogenous amino acids was measured from the ventrolateral medulla of urethane anaesthetized rats in vivo by microdialysis. Inclusion of a mercury salt, p-chloromercuriphenylsulphonic acid, in the dialysate (Krebs' solution), results in a preferential increase in the overflow of aspartate, glutamate, glycine and GABA. A smaller increase in the overflow of the glutamate precursor and metabolite, glutamine, was also found. There was no significant change in the basal extracellular levels of taurine, asparagine, alanine, serine, ornithine or lysine. Inclusion of a specific GABA uptake inhibitor, nipecotic acid, in the dialysate results in an immediate, dose dependent increase in the overflow of GABA, and to a lesser extent, taurine. Since it is likely that mercury salts increase neurotransmitter release by increasing free intracellular calcium ion concentrations, it is suggested that these results provide further evidence for a physiologically relevant neurotransmitter role for aspartate, glutamate, glycine and GABA in the ventrolateral medulla.