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Catecholaminergic neurotransmission is normally terminated by rapid re-uptake of the neurotransmitter by a high-affinity Na+/Cl--dependent plasma membrane transporter. Specific transporters have been cloned for both dopamine (DAT) and noradrenaline (NAT) in the rat. While DAT has been studied extensively, NAT expression has received less attention, particularly at the protein level. We used an antibody generated against a 49 residue segment of an extracellular loop region of NAT to study expression of the transporter protein throughout the rat pons and medulla oblongata. NAT was expressed in over 95% of noradrenergic neurones in the A1, A2/area postrema, A5, A6/locus subcoeruleus, and A7 noradrenergic groups. Approximately 10% of C1 adrenergic neurones located in the rostral ventrolateral medulla (RVL) also expressed NAT. Expression of NAT mRNA in bulbospinal C1 cells was confirmed using single-cell reverse transcription polymerase chain reaction (RT-PCR) of acutely isolated RVL neurones. Spinally projecting neurones were identified by retrograde labelling with rhodamine beads, and C1 neurones were identified by RT-PCR using primers specific for tyrosine hydroxylase (TH) or phenylethanolamine N-methyltransferase (PNMT) mRNAs. Thirteen percent of adrenergic bulbospinal neurones tested expressed NAT mRNA. C1 neurones are potentially important in cardiovascular control and blood pressure regulation, and the identification of NAT expression in a sub-population of these neurones provides further evidence for the heterogeneity of this neuronal population., (Copyright 1998 Elsevier Science B.V.)

The polyamines (PAs) are ubiquitous polycationic metabolites in eukaryotic and prokaryotic cells and are believed to be intimately involved in the regulation of DNA, RNA, and protein biosynthesis, the exact function of which remains unclear, mainly because of a lack of knowledge of PA subcellular localization. In this study, using immunoelectron microscopy, we have demonstrated that PAs are predominantly located on free and attached ribosomes of the rough endoplasmic reticulum in the neurons of the lateral reticular nucleus of rat medulla oblongata. The nuclei, axons, and nerve endings were devoid of PA. This suggests that PAs are one of the components of biologically active ribosomes, being closely involved in the translation processes of protein biosynthesis.

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.

An isomer of ouabain, the ouabain-like compound (OI,C), may participate in the regulation of body fluid volume and vascular tone. Forebrain regions, especially the hypothalamus, are reported to be sites of OLC action in the central nervous system. The medulla oblongata is another critical area involved in central cardiovascular regulation. We reported that the microinjection of either monoclonal antibody to ouabain T8B11 or Fab fragment of digoxin-specific antibody into the rostral ventrolateral medulla significantly decreased mean arterial pressure and renal sympathetic nerve activity in anesthetized normotensive rats (TERUYA et al.: J. Clin. Invest. 99: 2791-2798, 1997). Using T8B11, we examined the ouabain-like immunoreactivity in the medulla oblongata of normotensive rats. In periodate-lysine-paraformaldehyde fixed tissues, ouabain-like immunoreactive neurons were detected in the nuclei and regions in the medulla oblongata including the ventrolateral medulla, ventromedial medulla, nucleus ambiguus, caudal raphe nuclei, nucleus of solitary tract, and dorsal motor nucleus of the vagus. When an Fab fragment of digoxin-specific antibody was used as a first antibody, the digoxin-like immunoreactive neurons were distributed in almost the same pattern as those observed with the use of T8B11. In the brain fixed with the "three-step" procedure developed by YAMADA et al. (1987), which was used in a previous ouabain immunohistochemical study of the hypothalamus, ouabain-like immunoreactivity in the medulla oblongata was much weaker in intensity and less restricted in distribution than that in the hypothalamus. These findings suggest that ouabain-like immunoreactivities are present in the medulla oblongata with a manner of distribution different from that seen in the hypothalamus. Some ouabain-immunopositive nuclei and regions in the medulla oblongata, especially the rostral ventrolateral medulla, may be other OLC action sites.

Effects of D(-)2-amino-7-phosphonohepatanoic acid (AP-7), an N-methyl-D-aspartic acid (NMDA) receptor antagonist, administered into rostral ventrolateral medulla (RVLM) on changes in mean arterial pressure (MAP), heart rate (HR), extracellular levels of serotonin (5-HT), dopamine (DA), and norepinephrine (NE) during static muscle contraction were investigated in anesthetized rats. Tibial nerve stimulation-evoked muscle contraction increased MAP and HR by 25+/-3 mmHg and 29+/-4 bpm, respectively. Microdialysis of AP-7 (1 microM) into the RVLM for 30 min attenuated the contraction-evoked cardiovascular responses with similar developed muscle tensions, without baseline HR or blood pressure changes. Administration of AP-7 into the caudal ventrolateral medulla had no effect on MAP or HR responses during contraction. Muscle contraction increased extracellular 5-HT in the RVLM by 144+/-35%, DA by 104+/-15% and NE by 62+/-12%. Perfusion of AP-7 for 30 min into the RVLM attenuated contraction-evoked increases in monoamines, concomitant to attenuating cardiovascular responses. Results demonstrate that NMDA-receptor blockade within the RVLM, but not the CVLM, inhibits cardiovascular responses during muscle contraction. Furthermore, NMDA receptor antagonism within the RVLM results in a decrease of biogenic amine release during muscle contraction, suggesting that extracellular biogenic amine concentrations are modulated by NMDA receptors.

Whole cell patch recordings were made from rostral ventrolateral medullar (RVLM) neurons of brain-stem slices from 8- to 12-day-old rats. By superfusion or pressure ejection to RVLM neurons, 5-hydroxytryptamine (5-HT) elicited three types of membrane potential changes: a slow hyperpolarization (5-HTH), a slow depolarization (5-HTD) and a biphasic response, which persisted in a tetrodotoxin (TTX, 0.3 microM)-containing solution. 5-HTH were accompanied by a decrease of input resistance in the majority of responsive neurons. Hyperpolarization reduced and depolarization increased the 5-HTH; the mean reversal potential was -92.3 mV in 3.1 mM and shifted to -69.3 mV in 7 mM [K+]o. Barium (Ba2+, 0.1 mM) but not tetraethylammonium (TEA, 10 mM) suppressed 5-HTH. The 5-HT1A receptor agonist (+/-)-8-hydroxy-dipropylamino-tetralin (8-OH-DPAT; 5-50 microM) hyperpolarized RVLM neurons. The 5-HT1A antagonist pindobind-5-HT1A (PBD; 1-3 microM) and the 5-HT2/5-HT1 receptor antagonist spiperone (1-10 microM) suppressed 5-HTH and the hyperpolarizing phase of biphasic responses; the 5-HT2 receptor antagonist ketanserin (3 microM) was without significant effect. 5-HTD were associated with an increase or no apparent change of input resistance in RVLM neurons. Hyperpolarization of the membrane decreased or caused no apparent change in 5-HTD. 5-HTD were reduced in an elevated [K+]o (7.0 mM) solution and > 60% in a low Na+ (26 mM) solution and were not significantly changed in a low Cl- (6.7 mM) or Ca(2+)-free/high Mg2+ (10.9 mM) solution. The 5-HT2 receptor agonist alpha-methyl-5-HT (50 microM) depolarized RVLM neurons, and the 5-HT2 antagonist ketanserin (1-10 microM) attenuated the 5-HTD and the depolarizing phase of biphasic responses, whereas the 5-HT1A receptor antagonist PBD (2 microM) was without effect. Inclusion of the hydrolysis resistant guanine nucleotide GDP-beta-S in patch solution significantly reduced the 5-HTH as well as the 5-HTD. The present study shows that, in the immature rat RVLM neurons, 5-HT causes a slow hyperpolarization and depolarization probably by interacting with 5-HT1A and 5-HT2 receptors, which are G-proteins coupled. 5-HTH may involve an increase of an inwardly rectifying K+ conductance, and 5-HTD appear to be caused by a decrease of K+ conductance and/or increase of nonselective cation conductance.

Objective: To demonstrate the presence of adrenomedullin in the central nervous system., Methods: Using immunohistochemical method (ABC) and reverse transcription-polymerase chain reaction (RT-PCR) analysis, the distribution of immunoreactive (IR-) AdM and AdM mRNA was observed in the rat and the human brain., Results: Immunoreactive (IR-) AdM and AdM mRNA were detectable in almost every region of the rat brain examined, including cerebral cortex, paraventricular tissues, hypothalamus, mesencephalon, medulla oblongata and cerebellum. The levels of AdM mRNA in paraventricular tissues and medulla oblongata were higher than those in the other brain regions. AdM mRNA was also detectable in the human brain., Conclusion: The present study shows that AdM is present in the central nervous system, suggesting that AdM might act as a neurotransmitter, neuromodulator or neurohormone in rats or humans.

Immunohistochemical detection of the protein, Fos, was used to identify neurons in the brain activated following a volume load in conscious rabbits with doxorubicin-induced congestive cardiomyopathy. The plasma expander, Haemaccel, was infused intravenously into rabbits for 60 min and significantly increased right atrial pressure, blood pressure and heart rate. The rabbits were perfusion fixed 90 min after the start of the infusion and the distribution of Fos-positive cell nuclei was examined. Compared to control rabbits with heart failure, there was a small significant increase in the number of Fos-positive cell nuclei in the organum vasculosum of the lamina terminalis following volume expansion. In other regions of the brain that were studied in detail, there were no significant increases in Fos production. These included the parvocellular paraventricular nucleus (PVN) of the hypothalamus, the midbrain periaqueductal gray, the nucleus tractus solitarius (NTS), area postrema and the ventrolateral medulla (VLM). In the supraoptic nucleus and the magnocellular PVN, no Fos-positive cell nuclei were present as expected. The median preoptic nucleus, the bed nucleus of the striae terminalis and the diagonal band of Broca contained some Fos but there was no marked difference between volume expanded and control animals. In the anterior cortical and medial subnuclei of the amygdala there was a high concentration of Fos but there was no consistent difference between the two groups. The present findings in heart failure rabbits suggest that most brain regions are not activated sufficiently by the stimulus to elicit Fos expression. The results are in accord with findings showing that sympathetic reflexes initiated by volume expansion are attenuated in heart failure.

The apparent concentration of lithium (Li) in vivo was determined for several regions in the brain and muscle of rats by 7Li NMR imaging at 4.7 T with inclusion of an external standard of known concentration and visibility. The average apparent concentrations were 10.1 mM for muscle, and 4.2-5.3 mM for various brain regions under the dosing conditions used. The results were compared to concentrations determined in vitro by high-resolution 7Li NMR spectroscopy of extracts of brain and muscle tissue from the same rats. The comparison provided estimates of the 7Li NMR visibility of the Li cation in each tissue region. Although there was considerable scatter of the calculated visibilities among the five rats studied, the results suggested essentially full visibility (96%) for Li in muscle, and somewhat reduced visibility (74-93%) in the various brain regions., (Copyright 1998 Academic Press.)

The development of nicotinic acetylcholine receptors (nAChRs) in brains from human fetuses of 4-12 weeks gestational age was studied. The expression of nAChR subunit mRNAs was analyzed using reverse transcriptase-polymerase chain reaction. Expression of alpha 3, alpha 4, alpha 5, alpha 7, beta 2, beta 3 and beta 4 mRNA were all detected in the prenatal spinal cord, medulla oblongata, pons, cerebellum, mesencephalon, subcortical forebrain and cortex during first trimester development. Relative quantification of mRNA showed that the highest levels for alpha 3, alpha 4 and alpha 7 were expressed in the spinal cord, alpha 5 was most abundant in the cortex and beta 3 was highest in the cerebellum. beta 4 seemed to be equally distributed in all regions whereas beta 2 was high in the cortex and cerebellum. A comparison of expression of nAChR subunit mRNAs in the cortex and cerebellum of prenatal and aged (54-81 years) brain showed that mRNA levels for alpha 4, alpha 5, alpha 7, beta 2 and beta 4 were significantly higher in the prenatal cortex and cerebellum than in aged brain, whereas the level of alpha 3 transcript was similar, and beta 3 significantly higher in aged cortex. Specific binding of [3H]-epibatidine to prenatal brain membranes was detected as early as 4-5 weeks of gestation in the spinal cord, medulla oblongata, pons and subcortical forebrain. A positive correlation between gestational age and [3H]-epibatidine and [3H]-cytisine binding was found in several brain regions. The highest specific binding of [3H]-epibatidine and [3H]-cytisine was detected in the spinal cord, pons and medulla oblongata and the lowest in the cortex. Saturation analysis of [3H]-cytisine binding in both prenatal and aged brain were best fit by a model for a single site, whereas binding data for [3H]-epibatidine revealed two classes of binding sites. The early presence of nAChR proteins and gene transcripts shown in the present study suggests an important role for nAChRs in modulating dendritic outgrowth, establishment of neuronal connections and synaptogenesis during development.

1. The respiratory centre of neonatal mice (4 to 12 days old) was isolated in 700 micro(m) thick brainstem slices. Whole-cell K+ currents and single ATP-dependent potassium (KATP) channels were analysed in inspiratory neurones. 2. In cell-attached patches, KATP channels had a conductance of 75 pS and showed inward rectification. Their gating was voltage dependent and channel activity decreased with membrane hyperpolarization. Using Ca2+-containing pipette solutions the measured conductance was lower (50 pS at 1.5 mM Ca2+), indicating tonic inhibition by extracellular Ca2+. 3. KATP channel activity was reversibly potentiated during hypoxia. Maximal effects were attained 3-4 min after oxygen removal from the bath. Hypoxic potentiation of open probability was due to an increase in channel open times and a decrease in channel closed times. 4. In inside-out patches and symmetrical K+ concentrations, channel currents reversed at about 0 mV. Channel activity was blocked by ATP (300-600 microM), glibenclamide (10-70 microM) and tolbutamide (100-300 microM). 5. In the presence of diazoxide (10-60 microM), the activity of KATP channels was increased both in inside-out, outside-out and cell-attached patches. In outside-out patches, that remained within the slice after excision, the activity of KATP channels was enhanced by hypoxia, an effect that could be mediated by a release of endogenous neuromodulators. 6. The whole-cell K+ current (IK) was inactivated at negative membrane potentials, which resembled the voltage dependence of KATP channel gating. After 3-4 min of hypoxia, K+ currents at both hyperpolarizing and depolarizing membrane potentials increased. IK was partially blocked by tolbutamide (100-300 microM) and in its presence, hypoxic potentiation of IK was abolished. 7. We conclude that KATP channels are involved in the hypoxic depression of medullary respiratory activity.

Noradrenergic neurons are implicated in the estrogen-dependent neural regulation of luteinizing hormone secretion in a variety of mammalian species. The current study has used immunocytochemical methods to determine whether estrogen receptors (ER) are expressed within the brainstem of the ewe and to establish their relationship to noradrenergic neurons. Using a monoclonal mouse antiserum directed against the N-terminal of ERa, four distinct populations of ER alpha-immunoreactive cells were identified in ovine medulla and pons. The largest population was found in the superficial laminae of the spinal nucleus of the trigeminal nerve, followed by the nucleus tractus solitarius, lateral area postrema, and ventrolateral medulla. Double-labelling immunocytochemistry using antisera directed against the ER alpha and dopamine-beta-hydroxylase revealed that noradrenergic neurons expressing ER immunoreactivity were only found in ventrolateral medulla (A1 cell group) and nucleus tractus solitarius (A2 cell group). No double-labelled cells were identified in the A5, A6, or A7 noradrenergic cell groups. ERs were expressed with a clear rostrocaudal topography within the A1 and A2 populations, with 80-90% of noradrenergic neurons expressing ERA alpha in the caudalmost medulla as compared with less than 5% rostral to the obex. Our findings demonstrate that, as in the rat, the ovine A1 and A2 neurons express ERs in a defined topographical manner, while, dissimilar to the rat, ER alpha is not synthesized by noradrenergic neurons in the other cell groups. These observations indicate that A1 and A2 noradrenergic neurons in the ovine brainstem are likely to be influenced by circulating estrogens and lay the neuroanatomical foundations for investigating the functional role of these cell populations within the gonadotropin-releasing hormone neuron network of the sheep.

The periaqueductal gray (PAG) and rostral ventromedial medulla (RVM) are important brain stem pain modulating regions. Recent evidence suggests that kappa opioids antagonize the effects of mu opioids in the RVM. However, the anatomical relationship between mu and kappa opioid receptors in PAG and RVM is not well characterized. This study examined relationships between mu and kappa opioid receptor immunoreactivity (IR) and mRNA in PAG and RVM. Brain slices were processed for either immunocytochemistry or in situ hybridization. We found considerable anatomical overlap of mu and kappa opioid IR and mRNA in the RVM and PAG. These results provide an anatomical basis for recent behavioral and electrophysiological findings in RVM, and suggest modulatory interactions between mu and kappa opioids in PAG.

The retrogradely-transported tracer, rhodamine-tagged microspheres, was injected into the pressor region of the rostral ventrolateral medulla (RVLM) to identify paraventricular neurons in the hypothalamus that project to the RVLM. The protein, Fos, was detected immunohistochemically and used to highlight neurons that were activated by a hypotensive haemorrhage. Compared to controls, Fos production was increased by approximately 3-fold in the paraventricular nucleus (P<0.009) and there was a significant increase in the number of retrogradely-labelled cells that expressed Fos. These represented 5% of the retrogradely-labelled cell population. The results suggest that a small subpopulation of PVN neurons projecting to the RVLM are activated by haemorrhage and may be involved in the reflex responses initiated by that stimulus., (Copyright 1998 Elsevier Science B.V.)

The distributions of two alternative splicing variants of metabotropic glutamate receptor mGluR7, mGluR7a and mGluR7b, were examined immunohistochemically in the rat and mouse by using variant-specific antibodies raised against C-terminal portions of rat mGluR7a and human mGluR7b. Many regions throughout the central nervous system (CNS) showed mGluR7-like immunoreactivities (LI). The distribution patterns of mGluR7-LI in the rat were substantially the same as those in the mouse, although some species differences were observed in a few regions. Intense mGluR7a-LI was seen in the main and accessory olfactory bulbs, anterior olfactory nucleus, islands of Calleja, superficial layers of the olfactory tubercle, piriform cortex and entorhinal cortex, periamygdaloid cortex, amygdalohippocampal area, hippocampus, layer I of the neocortical regions, globus pallidus, superficial layers of the superior colliculus, locus coeruleus, and superficial layers of the medullary and spinal dorsal horns. The distribution of mGluR7b was more restricted. It was intense in the islands of Calleja, substantia innominata, hippocampus, ventral pallidum, and globus pallidus. The medial habenular nucleus also showed intense mGluR7a-LI in the rat but not in the mouse. For both mGluR7a- and mGluR7b-LI, localization in the active zones of presynaptic axon terminals was confirmed electron microscopically at synapses of both the asymmetrical and symmetrical types. It is noteworthy that mGluR7a-LI is seen preferentially in relay nuclei of the sensory pathways and that both mGluR7a- and mGluR7b-LI are observed not only in presumed glutamatergic axon terminals, but also in non-glutamatergic axon terminals including presumed inhibitory ones. Thus, mGluR7 may play roles not only as an autoreceptor in glutamatergic axon terminals, but also as a presynaptic heteroreceptor in non-glutamatergic axon terminals in various CNS regions.

We hypothesize that the hiccup reflex is actively inhibited through GABA(B) receptor within central connections of the hiccup reflex arc. Because the hiccup-like reflex can be elicited by electrical stimulation to a limited area within the medullary reticular formation, the hiccup-evoking site (HES), electrical stimulation (50-100 microA, three train pulses at 20 Hz) was delivered to HES by means of a metal electrode containing 1.0 mM baclofen, in anesthetized spontaneously breathing cats. The evoked response was characterized by a brief powerful increase in diaphragmatic activity and a temporal suppression of the posterior cricoarytenoid muscle, laryngeal dilator, which corresponded to the fixed motor pattern of hiccup reflex. The hiccup-like response was rapidly suppressed after microinjection of baclofen (0.1-0.5 nmol) into HES, indicating that HES has GABA(B) receptors. In the other experiments, to histologically examine the inputs to the hiccup reflex arc, unconjugated cholera toxin subunit B (UCTB) was injected into HES. Following injections of UCTB, retrogradely labelled cells were found distributed in various areas of the lower brainstem. Among these areas, the nucleus raphe magnus (RM) is reported to have GABA-containing cells. It is thus hypothesized that RM is most likely to be the source of the GABAergic inhibitory inputs to the hiccup reflex arc.

Inhibition of neurons containing gamma-aminobutyric acid (GABA) may underlie some of the excitatory effects of opioids in the central nervous system (CNS). In the present study, we examined the relationship of the cloned mu- and delta-opioid receptors (MOR1 and DOR1, respectively) to GABAergic neurons in brain and spinal cord. This was done by combining immunofluorescent staining for MOR1 or DOR1 with that for GABA or glutamic acid decarboxylase (GAD); fluorescent retrograde tract-tracing was used in some cases to identify neurons with particular projections. In rats, cells double labeled for GABA and MOR1 were observed in layers II-VI of the parietal cortex and in layers II-IV of the piriform cortex. In the hippocampus, double labeling was observed in the dentate gyrus and in regions CA1 and CA3. Double labeling was very prominent in the striatum and in the reticular nucleus of the thalamus; it was also observed in other portions of the diencephalon. However, double labeling for GABA and MOR1 was never observed in the cerebellar cortex. Cells double labeled for GABA and MOR1 were common in the periaqueductal gray (PAG) and the medial rostral ventral medulla (RVM) of both rats and monkeys, suggesting that involvement of GABAergic neurons with supraspinal opioid antinociception may extend to primates. In the RVM of rats, many of those double-labeled neurons were retrogradely labeled from the dorsal spinal cord. In contrast, double-labeled neurons in the PAG were almost never retrogradely labeled from the RVM. No unequivocal examples of double labeling for DOR1 and GAD were found in any region of the CNS that we examined in either rats or monkeys. However, GABAergic neurons were often apposed by DOR1 immunoreactive varicosities. Our findings suggest that activation of mu-opioid receptors directly modulates the activity of GABAergic neurons throughout the CNS, including neurons involved in the supraspinal component of opioid analgesia. In contrast, delta-opioid receptors appear to be positioned to modulate the activity of GABAergic neurons indirectly.

The distribution of agmatine (decarboxylated arginine) was mapped in the central nervous system (CNS) in the rat. Agmatine-like immunoreactivity was identified by light microscopy, exclusively in the cytoplasm of neuronal perikarya. Immunoreactive neurons were present in the cerebral cortex, predominantly within laminae VI and V and, to a lesser extent, III and mainly in retrosplenial, cingulate, primary somatosensory and auditory cortices, and the subiculum. In the lower brainstem, immunoreactivity was selectively localized to visceral relay nuclei: the nucleus tractus solitarii and pontine parabrachial complex, and periventricular areas including the laterodorsal nucleus, locus coeruleus and dorsal raphe. In the midbrain, immunolabeled cells were concentrated in the ventral tegmental area and periaqueductal gray. In the forebrain, subcortical neurons were labeled predominantly in the preoptic area, amygdala, septum, bed nucleus of the stria terminalis, midline thalamus, and the hypothalamus. Ultrastructural analysis of layer V of the somatosensory cortex demonstrated agmatine-immunoreactivity in neurons, primarily in large dense-core vesicles located in the cytoplasm. Agmatine immunoreactivity was also affiliated with endoplasmic reticulum and the plasmalemma. Cortical neurons and the subiculum were labeled in animals not administered the axonal transport inhibitor, colchicine; thus, may normally contain higher concentrations of the amine than other brain regions. The central distribution of agmatine is consistent with the hypothesis that the amine may be a novel neurotransmitter of neurons involved in behavioral and visceral control., (Copyright 1998 Elsevier Science B.V.)

Acute and chronic angiotensin (Ang) II hypertension are reported to have different mechanisms that involve differential contributions of the peripheral vasculature and the nervous system. Acute Ang II hypertension is mediated primarily by Ang acting at vascular smooth muscle, whereas chronic Ang II hypertension appears to have a neural component. In our experiments, the transition from a peripheral to a neural effect occurs over 10 hr of Ang II infusion in rats. To identify the role of the central nervous system in this transition, we measured Fos immunoreactivity, an indicator of neural activity, in the nucleus of the solitary tract (NTS), caudal ventrolateral medulla (CVL) and rostral ventrolateral medulla (RVL) in normal, sinoaortic denervated (SAD) and sham SAD rats after 2- or 18-hr Ang II infusion (50 ng/kg/min intravenously). Vehicle (5% dextrose) was infused in normal rats as control. Comparable increases in arterial pressure were produced by 2- and 18-hr Ang II infusion in all groups. Fos was increased in the NTS in sham SAD rats by 2- and 18-hr Ang II infusion (P < .05 vs. vehicle control). In the CVL, only 2-hr Ang II infusion was associated with increased Fos in normal and sham SAD rats (P < .05 vs. vehicle control) but not in SAD rats. In the RVL, 18-hr Ang II infusion elevated Fos in all groups (P < .05 vs. vehicle control). Activation of NTS during Ang II infusion is baroreceptor mediated and independent of infusion duration. Acute Ang II infusion produced a baroreceptor-mediated activation of the CVL, a region associated with baroreflex sympathoinhibition. Chronic Ang II infusion produced a baroreceptor-independent activation of the RVL, a brain area associated with sympathoexcitation, suggesting a centrally mediated increase in sympathetic outflow that may be associated with chronically infused Ang II.

In order to study the expression of Fos protein in catecholaminergic neurons in the medullary visceral zone (MVZ), which project to the habenular nucleus (HB), a triple-labeling method combining wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) retrograde tracing with anti-Fos and anti-tyrosine hydroxylase (TH) immunohistochemical staining was used in the rat. WGA-HRP was stereotaxically injected into unilateral HB. Forty-eight hours later, 50 microl of 8% formalin was injected into the foot pad of the right front paw. Two hours after formalin injection, animals were anesthetized and perfused transaortically. Coronal sections (40 micron) were cut from the cervical segment of spinal cord, the medulla oblongata and WGA-HRP injected area with a cryostat. First, sections of the injected area and the medulla oblongata were histochemically processed to demonstrate the presence of retrogradely transported WGA-HRP using the chromogen tetramethylbenzidine (TMB). Then sections of the spinal cord and the medulla oblongata were immunostained with anti-Fos and anti-TH antibodies using the ABC method. Under the light microscope, seven types of variously labeled neurons could be identified in MVZ, namely Fos and TH-immunoreactive (Fos- or TH-IL) neurons, WGA-HRP labeled ones, Fos/HRP, Fos/TH and HRP/TH double-labeled and Fos/HRP/TH triple-labeled cells. The results suggest that some catecholaminergic neurons in MVZ could send projections to HB and this pathway may be involved to relay nociceptive information from spinal cord to brainstem and on to the forebrain.

The ventrolateral portion of the intermediate reticular formation of the medulla (ventrolateral medulla, VLM), including the C1/A1 groups of catecholaminergic neurons, is thought to be involved in control of sympathetic cardiovascular outflow, cardiorespiratory interactions, and reflex control of vasopressin release. As all these functions are affected in patients with multiple systems atrophy (MSA) with autonomic failure, we sought to test the hypothesis that catecholaminergic (tyrosine hydroxylase [TH]-positive) neurons of the VLM are depleted in these patients. Medullas were obtained at autopsy from 4 patients with MSA with prominent autonomic failure and 5 patients with no neurological disease. Patients with MSA had laboratory evidence of severe adrenergic sudomotor and cardiovagal failure. Tissue was immersion fixed in 2% paraformaldehyde at 4 degrees C for 24 hours and cut into 1-cm blocks in the coronal plane from throughout the medulla. Serial 50-microm sections were collected and one section every 300 microm was stained for TH. There was a pronounced depletion of TH neurons in the rostral VLM in all cases of MSA. There was also significant reduction of TH neurons in the caudal VLM in 3 MSA patients compared with 3 control subjects. In 2 MSA cases and in 2 control subjects, the thoracic spinal cord was available for study. There was also depletion of TH fibers and sympathetic preganglionic neurons (SPNs) in the 2 MSA cases examined. Thus, depletion of catecholaminergic neurons in the VLM may provide a substrate for some of the autonomic and endocrine manifestations of MSA.

Imidazoline (I) receptors have been implicated in the regulation of arterial blood pressure and behavior although their distribution in the central nervous system (CNS) remains in question. Presumptive I- receptor sites were detected in the rat central nervous system with a polyclonal antibody to an imidazoline receptor protein (IRP) with binding characteristics of the native receptor. IRP-like immunoreactivity (LI) was detected in neurons and glia by light and electron microscopy. Spinal cord: processes were heavily labeled in superficial laminae I and II of the dorsal horn, lateral-cervical and -spinal nuclei and sympathetic cell column. Medulla: label was concentrated in the area postrema, rostral, subpostremal and central subnuclei of nucleus tractus solitarii, spinal trigeminal nucleus caudalis, and inferior olivary subnuclei. Visceromotor neurons in the dorsal vagal and ambigual nuclei were surrounded by high concentrations of immunoreactive processes. In reticular formation, label was light, though predominant in the intermediate reticular zone and ventrolateral medulla. Pons: label was detected in the neuropil of the periventricular gray, concentrated in the dorsal- and external-lateral subnuclei of lateral parabrachial nucleus, and present intracellularly in the mesencephalic trigeminal nucleus. Midbrain: IRP-LI was most heavily concentrated in the interpeduncular nucleus, nuclei interfascicularis and rostral-linearis, the subcommissural organ, central gray, and in glia surrounding the cerebral aqueduct. Diencephalon: high densities were detected in the medial habenular nucleus, nucleus paraventricularis thalami, other midline-intralaminar thalamic nuclei, the supramammillary and mediobasal hypothalamic nuclei. In the median eminence, immunolabeled processes were restricted to the lamina interna and lateral subependymal zone. Telencephalon: IRP-LI was concentrated in the central amygdaloid nucleus, bed nucleus of stria terminalis and globus pallidus, followed by moderate labeling of the medial amygdaloid nucleus, amygdalostriatal zone and caudoputamen, the hilus of the dentate gyrus, and stratum lacunosum-moleculare of field CA1 of Ammon's horn. The subfornical organ and organum vasculosum lamina terminalis were filled with diffuse granular immunoreactivity. Ultrastructural studies identified IRP-LI within glia and neurons including presynaptic processes. I-receptor(s) localize to a highly restricted network of neurons in the CNS and circumventricular regions lying outside of the blood-brain barrier. Putative imidazoline receptors have a unique distribution pattern, show partial overlap with alpha 2 adrenoreceptors and are heavily represented in sensory processing centers and the visceral nervous system.

We aimed to clarify the topology and immunohistochemistry of CO2/H+-sensitive neurons in the ventral medullary surface (VMS), the central chemoreceptor area in rats. Inhalation of 3 and 7% CO2 in air significantly decreased pH in arterial blood and increased paCO2, which caused hyperpneic and tachypneic responses. Following inhalation of 3 and 7% CO2 in air for 5 min, the density of c-Fos-immunoreactive (IR) neurons increased stepwise not only in the 3rd-5th divisions of the VMS (between the caudal end of the nucleus corporis trapezoidei and the caudal end of the area postrema), but also in the rostroventromedial medulla (RVMM). Following inhalation of 7% CO2 in air for 5 min, glutamate-, glutamic acid decarboxylase (GAD)-, calcineurin- and cAMP-IR neurons were found not only in the VMS, but also in the RVMM. The topology of these neurons was similar to that of the c-Fos-IR neurons. No immunoreactivity was found for serotonin, substance P, somatostatin, cholecystokinin-octapeptide, methionine-enkephalin, choline acetyltransferase, tyrosine hydroxylase, phenylethanolamine N-methyltransferase, NO-synthase, S-100, calbindin-D, calmodulin, or parvalbumin. The densities of c-Fos-, glutamate-, GAD-, calcineurin- and cAMP-IR neurons were almost zero in the 1st division of the VMS, but became higher along the 2nd-4th divisions of the VMS. Regression lines of the density against the 1st-4th divisions of the VMS were significantly linear. These results indicate that H+-sensitive neurons are common in the 4th-5th divisions of the VMS, and that they are glutamatergic, GABAergic, and containing calcineurin and cAMP.

Using the reverse transcription polymerase chain reaction, we detected a single form of phenylethanolamine N-methyltransferase (PNMT) mRNA in hypothalamus and medulla/pons and two forms in cerebellum. These findings indicate that the PNMT gene is expressed in these brain areas and suggest that tissue specific splicing of PNMT mRNA may occur.

Gastric adaptive relaxation is a vago-vagal reflex, probably involving the site of interface of vagal afferents and efferents in the dorsal vagal complex of the medulla. Previous studies have shown that both substance P and nitric oxide in the dorsal vagal complex decrease intragastric pressure. The purpose of this study is, firstly, to localize NK1 tachykinin receptor immunoreactive (ir) staining in the dorsal vagal complex and, secondly, to determine its anatomical relationship to nitrergic cells in the dorsal motor nucleus of the vagus. Sections were stained by avidin-biotin immunocytochemistry using antiserum to NK1 receptor alone or combined with NADPH-diaphorase histochemistry. In the nucleus tractus solitarius, NK1 receptor-ir varicosities were moderately dense in the medial subnucleus, but sparse in the centralis and gelatinosus subnuclei. In the dorsal motor nucleus of the vagus, NK1 receptor-ir staining in cell bodies and fibers was present throughout, with a markedly dense varicose fiber and cell body staining in a lateral column of the rostral portion of the nucleus. NADPH-diaphorase staining is most marked in cell bodies in the same region of the dorsal motor nucleus of the vagus. In dual-stained sections, there was complete overlap of NADPH-diaphorase and NK1 receptor-ir stain; however, the markers were very rarely colocalized within the same vagal motor neurons. Ipsilateral vagotomy almost completely abolished NK1r-ir staining in vagal motor neurons. We conclude that, in the dorsal motor nucleus of the vagus, NK1 receptor is synthesized by a population of vagal motor neurons which are in close anatomical proximity to, but separate from, nitrergic neurons. Based on these observations, substance P-mediated gastric relaxation in this region is unlikely to be via activation of nitrergic vagal preganglionic neurons. In the nucleus tractus solitarius, the NK1 receptor and NADPH-diaphorase stain are not codistributed in subnuclei mediating gastric and esophageal control. Therefore, substance P and nitric oxide may mediate their respective gastrointestinal effects via separate afferent pathways.

The NGF content in each region of the brain of four-week-old rats was ranked in the decreasing order of cerebral cortex, hippocampus, cerebellum, midbrain/diencephalon, and pons/medulla oblongata, and the NGF concentration, in the decreasing order of hippocampus, cerebral cortex, cerebellum, midbrain/diencephalon, and pons/medulla oblongata in both AFD and SFD groups. The NGF content and concentration in the cerebral cortex were about the same value at each age between those in the AFD and SFD groups. Those in the hippocampus were a little higher in the SFD group than in the AFD group at the ages of three and four weeks, unlike those in the other regions, where the values for the cerebellum, midbrain/diencephalon and pons/medulla oblongata tended to be somewhat higher in the AFD group than in the SFD group. The NGF concentrations in the hippocampus and cerebral cortex increased with growth: the concentration in the hippocampus at four weeks of age was about 4-fold of that at one week in the AFD group and about 5.7-fold of that at one week in the SFD group; and likewise the concentration in the cerebral cortex at four weeks of age was about 5.3-fold in the AFD group and about 7-fold in the SFD group. The NGF concentrations in the cerebellum decreased, and those in midbrain/diencephalon and pons/medulla oblongata hardly changed with growth in either AFD or SFD group. From these results NGF may have stronger implications for the neuronal growth in the hippocampus compared with those in the lower brain regions of the SFD rats.

[125I]beta-endorphin bound to high affinity (Kd = 0.25 nM) receptors in the caudal dorsomedial medulla of rats with a Bmax of 97 fmol/mg protein. The relative potency for displacement of [125I]beta-endorphin binding was: beta-endorphin(1-31) > beta-endorphin(1-27) > DAMGO > naloxone > N-acetyl-beta-endorphin(1-31) > U50488 > DPDPE. The Bmax for [3H]DAMGO binding was 81 fmol/mg protein, indicating that most [125I]beta-endorphin binding corresponds to mu-opioid receptors. [3H]DAMGO binding was not influenced by lesioning noradrenergic nerve terminals in the caudal dorsomedial medulla. Our findings indicate that beta-endorphin interacts primarily with mu-opioid receptors in the caudal dorsomedial medulla. These receptors are not affected by noradrenergic denervation.

Estrogen plays a profound role in regulating the structure and function of many neuronal systems in the adult rat brain. The actions of estrogen were thought to be mediated by a single nuclear estrogen receptor (ER) until the recent cloning of a novel ER (ER-beta). To ascertain which ER is involved in the regulation of different brain regions, the present study compared the distribution of the classical (ER-alpha) and novel (ER-beta) forms of ER mRNA-expressing neurons in the central nervous system (CNS) of the rat with in situ hybridization histochemistry. Female rat brain, spinal cord, and eyes were frozen, and cryostat sections were collected on slides, hybridized with [35S]-labeled antisense riboprobes complimentary to ER-alpha or ER-beta mRNA, stringently washed, and opposed to emulsion. The results of these studies revealed the presence of ER-alpha and ER-beta mRNA throughout the rostral-caudal extent of the brain and spinal cord. Neurons of the olfactory bulb, supraoptic, paraventricular, suprachiasmatic, and tuberal hypothalamic nuclei, zona incerta, ventral tegmental area, cerebellum (Purkinje cells), laminae III-V, VIII, and IX of the spinal cord, and pineal gland contained exclusively ER-beta mRNA. In contrast, only ER-alpha hybridization signal was seen in the ventromedial hypothalamic nucleus and subfornical organ. Perikarya in other brain regions, including the bed nucleus of the stria terminalis, medial and cortical amygdaloid nuclei, preoptic area, lateral habenula, periaqueductal gray, parabrachial nucleus, locus ceruleus, nucleus of the solitary tract, spinal trigeminal nucleus and superficial laminae of the spinal cord, contained both forms of ER mRNA. Although the cerebral cortex and hippocampus contained both ER mRNAs, the hybridization signal for ER-alpha mRNA was very weak compared with ER-beta mRNA. The results of these in situ hybridization studies provide detailed information about the distribution of ER-alpha and ER-beta mRNAs in the rat CNS. In addition, this comparative study provides evidence that the region-specific expression of ER-alpha, ER-beta, or both may be important in determining the physiological responses of neuronal populations to estrogen action.

An in vitro autoradiographic technique has recently been developed to visualize receptor-activated G-proteins by using agonist-stimulated [35S]guanylyl-5'-O-(gamma-thio)-triphosphate ([35S]GTPgammaS) binding in the presence of excess guanosine 5'-diphosphate. This technique was used to localize opioid-activated G-proteins in guinea pig brain, a species that contains the three major types of opioid receptors. This study used selective mu, delta, and kappa opioid agonists as well as nociceptin or orphanin FQ (N/OFQ) peptide, an endogenous ligand for an orphan opioid receptor-like (ORL1) receptor, to stimulate [35S]GTPgammaS binding in guinea pig brain sections. Opioid receptor specificity was confirmed by blocking agonist-stimulated [35S] GTPgammaS binding with the appropriate antagonists. In general, the distribution of agonist-stimulated [35S]GTPgammaS binding correlated with previous reports of receptor binding autoradiography, although quantitative differences suggest regional variations in receptor coupling efficiency. Mu, delta, and kappa opioid-stimulated [35S]GTPgammaS binding was found in the caudate-putamen, nucleus accumbens, amygdala, and hypothalamus. Mu-stimulated [35S]GTPgammaS binding predominated in the hypothalamus, amygdala, and brainstem, whereas kappa-stimulated [35S]GTPgammaS binding was particularly high in the substantia nigra and cortex and was moderate in the cerebellum. N/OFQ-stimulated [35S] GTPgammaS binding was highest in the cortex, hippocampus, and hypothalamus and exhibited a unique anatomical distribution compared with opioid-stimulated [35S]GTPgammaS binding. The present study extends previous reports on opioid and ORL1 receptor localization by anatomically demonstrating functional activity produced by mu, delta, and kappa opioid and ORL1 receptor activation of G-proteins.

Central administration of corticotropin-releasing hormone (CRH) induces immediate-early gene (IEG) expression (c-fos and NGFI-B) in forebrain structures in a pattern similar to that observed following restraint stress. Lactating rats display modified neuroendocrine and behavioural responses to stress which have been hypothesized to be at least partially mediated through changes within the circuitry converging on the PVN, including CRH activated pathways. Quantitative measures of regional expression of c-fos and NGFI-B mRNA representative of two classical intracellular pathways, were used to define modification of the circuitry involved in the altered response to central CRH in the lactating female. Compared to saline controls, virgin female rats injected with 5 micrograms CRH i.c.v. displayed significantly increased immediate-early gene expression in the hypothalamic paraventricular nucleus (PVN), arcuate nucleus, lateral septum, bed nucleus of the stria terminalis, central, medial and cortical nuclei of the amygdala, and all subfields of the hippocampal formation. In lactating rats treated with CRH there was a significant increase in c-fos gene expression in the CeA and in the hippocampal subfields CA1, CA4 and dentate gyrus but not in the other areas examined. The i.c.v. administration of CRH significantly increased NGFI-B expression in the PVN, arcuate nucleus, medial amygdala and all hippocampal subfields of virgin rats. Lactating rats treated with CRH failed to show a significant increase in NGFI-B expression in the PVN, median eminence, arcuate nucleus, medial amygdala, CA2 and CA3 subfields of the hippocampus. These results further suggest that changes in specific neural circuits might at least partially underlie the modified responses to CRH and perhaps to stress in the lactating female.

Catecholamines are a class of neurotransmitters involved in central nervous system autonomic control. Both acute and chronic hypoxia create alterations in ventilation and blood pressure via catecholamine release, although the mechanisms of these alterations are unknown. The enzymes tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) catalyze the rate-limiting step in the catecholamine pathway and production of epinephrine, respectively. Both have been colocalized with Fos protein in metabolic mapping studies of the O2-chemosensory pathway of adult and early postnatal rat. Thus, catecholamines are putative neurotransmitters in a subset of second and higher order respiratory neurons. To characterize the effects of prenatal hypoxia on subsequent TH and PNMT gene and protein expression, pregnant rats were placed in moderate hypoxia (10% O2) from gestational d 18 until birth. Northern and Western analyses of dorsal (catecholaminergic/adrenergic cell group 2) and ventral (catecholaminergic/adrenergic cell group 1) medullary tissue of postnatal (P) age P0, P3, P7, P10, and P14 pups were then done to examine changes in TH and PNMT mRNA and protein compared with normoxia-reared controls. Compared with controls, pups exposed to maternal hypoxia during pregnancy had lower levels of TH mRNA and protein at birth in dorsal medulla and higher levels of TH mRNA the first postnatal week in the ventral medulla. Pups that had been hypoxic in utero showed significantly lower levels of PNMT protein during the second postnatal week in dorsal medulla than did controls. Prenatal hypoxia-induced changes in levels of enzymes responsible for catecholamine synthesis may later be manifest as developmental deficiencies in neuronal function. This may compromise responses to acute hypoxic challenges during early postnatal life and contribute to autonomic nervous system disorders of the newborn such as apnea and sudden infant death syndrome.

An ectomesenchymoma was found in a 62-day-old female calf, and examined immunohistochemically. The neoplastic cells were characterized by positive immunostaining for neurofilaments, glial fibrillary acidic protein, alpha smooth muscle actin, myoglobin, and keratin. This tumour, which displayed neurocytic, astrocytic, and myoblastic differentiation, may have arisen from pluripotential ectomesenchyme. Brain tumours showing multidirectional phenotypic expression presumably occur mainly in fetuses or calves.

The role of P2 receptors in controlling hypoglossal motoneuron (XII MN) output was examined (1) electrophysiologically, via application of ATP to the hypoglossal nucleus of rhythmically active mouse medullary slices and anesthetized adult rats; (2) immunohistochemically, using an antiserum against the P2X2 receptor subunit; and (3) using PCR to identify expression of P2X2 receptor subunits in micropunches of tissue taken from the XII motor nucleus. Application of ATP to the hypoglossal nucleus of mouse medullary slices and anesthetized rats produced a suramin-sensitive excitation of hypoglossal nerve activity. Additional in vitro effects included potentiation of inspiratory hypoglossal nerve output via a suramin- and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS)-sensitive mechanism, XII MN depolarization via activation of a suramin-sensitive inward current, decreased neuronal input resistance, and a slow-onset theophylline-sensitive reduction of inspiratory output likely resulting from hydrolysis of extracellular ATP to adenosine and activation of P1 receptors. Immunohistochemically, P2X2 receptors were detected in inspiratory XII MNs that were labeled with Lucifer yellow. These data, combined with identification of mRNA for three P2X2 receptor subunit isoforms within the hypoglossal nucleus (two of which have not been localized previously in brain) and the previous demonstration that P2X receptors are ubiquitously expressed in cranial and spinal motoneuron pools, support not only a role of P2 receptors in modulating inspiratory hypoglossal activity but a general role of P2 receptors in modulating motor outflow from the CNS.

Spinally projecting neurons of the ventromedial medulla (VMM) compose an important efferent pathway for the modulation of nociception. These neurons receive a substantial gamma-aminobutyric acid (GABA)-ergic input, but the GABA receptor that mediates this input is unknown. This study examined the distribution of GABA(A) receptor alpha1 and alpha3 subunits in serotonergic and nonserotonergic neurons of the VMM that project to the dorsal horn in the rat. A pledget of Gelfoam soaked in Fluoro-Gold was placed at the thoracolumbar junction of the spinal cord to label spinally projecting neurons. Alternate sections of the medulla were then incubated with a mixture of antisera to either serotonin and the alpha1 subunit, or to serotonin and the alpha3 subunit of the GABA(A) receptor. Nearly 30% of spinally projecting neurons in the VMM were immunoreactive for the alpha1 subunit. A similar percentage of spinally projecting neurons in the VMM were immunoreactive for the alpha3 subunit, although diffuse cellular labeling combined with intense staining of processes in the neuropil precluded a rigorous semi-quantitative estimation of this population. No alpha1-subunit-immunoreactive neurons colocalized serotonin. In contrast, serotonergic neurons were immunoreactive for the alpha3 subunit. However, these double-labeled neurons were a modest percentage of the serotonergic population. A small percentage of spinally projecting serotonergic neurons was immunoreactive for the alpha3 subunit. These results suggest that significant numbers of spinally projecting serotonergic and nonserotonergic neurons of the VMM possess GABA(A) receptors that differ in their respective subunit compositions and that both classes of neurons may mediate the antinociception produced by the microinjection of GABA(A) receptor antagonists in the VMM.

To reveal neurones in the cat medulla oblongata involved in carotid baroreceptor/chemoreceptor reflexes, the distribution of c-Fos oncoprotein immunoreactivity was studied following electrical stimulation of the right carotid sinus nerve. The neurochemistry of the activated neurones was investigated using antisera to tyrosine hydroxylase, neuropeptide Y, somatostatin, and glutamate. Nitric oxide containing neurones were identified using antiserum to nitric oxide synthase (NOS) and by the histochemical localization of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase. Following sinus nerve stimulation numerous c-Fos-IR cells were detected both ipsilaterally and contralaterally in the nucleus tractus solitarii, the area postrema and throughout the ventrolateral medulla. Dual labelling studies revealed that 3.3% of c-Fos-immunoreactive cells in the nucleus tractus solitarii were also immunoreactive for tyrosine hydroxylase. The double labelled cells were scattered within the medial and ventrolateral subnuclei, predominantly rostral to obex. A higher proportion (10.3%) of c-Fos-IR cells in the ventrolateral medulla also showed tyrosine hydroxylase immunoreactivity. Caudal to obex, these were scattered in the reticular formation between the spinal trigeminal nucleus and the lateral reticular nucleus, while more rostrally they were found within the lateral reticular nucleus, the nucleus ambiguus and the lateral tegmental field. Cells expressing c-fos and reactive for glutamate, neuropeptide Y or NADPH-diaphorase (or NOS) were only rarely seen, and co-localization of c-Fos and somatostatin immunoreactivities was not seen. These results suggest that of the neurones forming pathways within the medulla activated on carotid sinus nerve stimulation, presumably mediating baro- and chemoreceptor reflexes, relatively few utilize catecholamines, glutamate, neuropeptide Y or nitric oxide as their transmitter substance.

Knowledge of the distribution of kinin receptors in the human brain will aid our understanding of the role of kinins in neurophysiology. Furthermore, induction of the kinin B1 receptor may be important in the pathogenesis of neural diseases. Using polyclonal antibodies directed to specific regions of the B1 and B2 kinin receptors and standard immunolabelling techniques, we report on the localisation of these receptors on neurones in specific areas of the human brain. B2 bradykinin receptors are present in neurones of the brain stem, basal nuclei, cerebral cortex, thalamus and hypothalamus. B2 immunolabelling was also observed in the endothelial lining of the superior sagittal dural sinus and ependyma of the lateral and third ventricles. B1 kinin receptors have been localised on neurones of the thalamus, spinal cord and hypothalamus. Although binding of labelled bradykinin to neuronal membranes has been demonstrated, this is the first conclusive evidence for the existence of immunoreactive B1, and further confirmation of B2 receptors on human neurones.

The supratentorial cerebral dura of the albino rat is equipped with a rich sensory innervation including nociceptive axons and their terminals, which display intense calcitonin gene-related peptide (CGRP) immunoreactivity both in the connective tissue and around blood vessels. Stereotactic electrical stimulation of the trigeminal (Gasserian) ganglion, regarded as an experimental migraine model, induces marked increase and disintegration of club-like perivascular CGRP-immunopositive nerve endings in the dura. Intravenous administration of sumatriptan, prior to electrical stimulation, prevents disintegration of perivascular terminals and induces accumulation of CGRP in terminal and preterminal portions of peripheral sensory axons. Consequently, immunopositive terminals and varicosities increase in size; accumulation of axoplasmic organelles results in a "hollow" appearance of many varicosities. Since sumatriptan exerts its anti-migraine effect by virtue of its agonist action on 5-HT1D receptors, we suggest that sumatriptan prevents the release of CGRP from dural perivascular terminals by an action at 5-HT1D receptors. In the caudal trigeminal nucleus electrical stimulation of the trigeminal ganglion induces, in interneurons, increased expression of the oncoprotein c-fos which is not prevented by intravenous application of sumatriptan. Disparate findings regarding this effect are partly due to the fact that sumatriptan very poorly passes the blood-brain barrier and partly to different experimental paradigms used by different authors.

1. The density of barosensitive neurons in the medulla was examined in spontaneously hypertensive rats (SHR) and in normotensive Wistar-Kyoto (WKY) rats. In control experiments, rats were sham-operated, while in test experiments arterial baroreceptors were stimulated by pressor responses to i.v. administration of phenylephrine and the density of c-Fos-labelled neurons was immunocytologically examined. 2. In both control and test experiments, c-Fos-labelled neurons were distributed in cardiovascular control sites: the nucleus tractus solitarii (NTS) and the caudal and rostral ventrolateral medullas (CVLM/RVLM). 3. In both WKY rats and in SHR, the total density of labelled neurons in test experiments was significantly higher than in control experiments. 4. In control experiments, no significant difference was found in the distribution and density of labelled neurons in the NTS and in the CVLM/RVLM between rats and SHR. 5. In test experiments, no significant difference was found in the distribution and density of labelled neurons in the NTS between WKY rats and SHR. 6. In test experiments in SHR, the density of labelled neurons in the CVLM just caudal to the obex level was significantly higher than that in WKY rats, whereas the density of labelled neurons in WKY rats in the RVLM just rostral to the obex level was significantly higher than that in SHR. 7. These results indicate that stimulation of the arterial baroreceptor induces strain-specific differences in the density of barosensitive neurons in the CVLM/RVLM near the obex level.

It is known that the nucleus of the solitary tract (NTS) and the ventrolateral medulla (VLM) project to the central nucleus of the amygdala (Ce), conveying visceral information. Conversely, the Ce sends projections to the NTS and the VLM. To understand better the role of catecholamine and gamma-aminobutyric acid (GABA) in these reciprocal connections, experiments were performed by combining lectin-conjugated horseradish peroxidase (WGA-HRP) anterograde and retrograde transport with preembedding immunocytochemistry to detect tyrosine hydroxylase (TH), and postembedding immunocytochemistry to detect GABA. The light microscopic study suggested that the majority of neurons in the NTS and the VLM projecting to the Ce were TH immunoreactive (TH-IR). Most of them were located at the level of the obex. Under the electron microscope, the GABAergic and non-GABAergic terminals were found to form synaptic contacts with the TH-(IR) or Ce-projecting or TH-IR/Ce-projecting double-labelled neurons of the NTS and VLM. The GABAergic terminals mostly formed symmetrical synaptic contacts with the postsynaptic structure in which perikarya (14-19%), dendrites (79-84%), and spines (2%) were observed. Approximately 94% of the axon terminals in the NTS and 90% of those in the VLM arising from the Ce were GABAergic and appeared not to form synaptic contacts with the TH-IR or Ce-projecting neurons in these regions. The present results demonstrated that the catecholaminergic neurons of the NTS and VLM projecting to the Ce receive an extensive GABAergic innervation and that the amygdala projection to the medulla is mostly GABAergic.

Activation of serotonin-1A receptors (5-HT(1A)R) in the medulla oblongata lowers sympathetic nerve discharge and blood pressure. Binding sites for 5-HT(1A)R ligands are present in ventral medullary nuclei [e.g., rostral ventrolateral medulla (RVLM), raphe pallidus (RPa), and parapyramidal region (PPR)] that project to sympathetic preganglionic neurons in the intermediolateral cell column (IML). However, the projections and the neurochemical contents of the ventral medullary neurons that are likely to be involved in the hypotensive actions of 5-HT(1A) agonists are unclear. Using a sheep antibody to a fragment of the third intracellular loop of the 5-HT(1A)R, we localized 5-HT(1A)R immunoreactivity (ir) to IML-projecting neurons that were retrogradely labeled with rhodamine beads injected into the IML of adult male rats. The percentages of IML-projecting neurons containing 5-HT(1A)R-ir were 49% in RPa, 34% in PPR, and 44% in RVLM. Using multiple-immunofluorescence labeling, we also demonstrated 5-HT(1A)R-ir in serotonergic (5-HT) and in catecholaminergic (tyrosine hydroxylase; TH-ir) neurons of the ventral medulla. The percentages of 5-HT-ir neurons containing 5-HT(1A)R-ir were 28% in RPa, 18% in PPR, and 31% in raphe obscurus. In addition, 5-HT(1A)R-ir was present in 14% of TH-ir neurons of the RVLM. Moreover, some IML-projecting neurons in the PPR and RPa were doubly immunolabeled for 5-HT(1A)R-ir and 5-HT, and some IML-projecting neurons in the RVLM were doubly immunolabeled for 5-HT(1A)R-ir and TH-ir. These data provide anatomical evidence for the presence of 5-HT(1A)R on serotonergic and catecholaminergic bulbospinal neurons and for their potential role in directly modifying the activity of these ventral medullary neurons.

The present study provides a detailed comparative description in the adult rat brain of areas that express mRNAs coding for the fibroblast growth factor subtype receptors 1-3 (FGFR1-3). One observation in this analysis was a widespread expression in the brain of all three FGFR mRNAs, according to the following rank order: FGFR1, diencephalon < telencephalon < mesencephalon and metencephalon < myelencephalon; FGFR2 and FGFR3, telencephalon < diencephalon < mesencephalon and metencephalon < myelencephalon. Another observation was an apparent cellular specificity in their basal expression. Thus, the FGFR1 mRNA was expressed mainly in large and weakly stained cells, whereas FGFR2 transcripts were expressed primarily in small and strongly stained cells and in cells of brain regions devoid of neuronal cells, such as the white matter. FGFR3 mRNA was always detected in small and strongly stained cells with scattered distribution and was not expressed in the white matter. However, FGFR2 mRNA was weakly expressed also in large cells localized in some nuclei of the lower brainstem, in the diagonal band, and in the septum. Furthermore, in the medial habenula and in the nuclei of the pons, there exists a high density of cells expressing both FGFR1 and FGFR2 (60-100%). With neurotoxic lesions involving 6-hydroxydopamine microinjections in the substantia nigra, reactive glial cells in the lesioned area and surrounding the cannula tract showed an increase in the expression of both FGFR1 and FGFR2 mRNAs, whereas no increased expression was found for FGFR3 mRNA. Taken together, these findings showed that these three FGF receptors exist in all subtypes of cells of each brain region. Their apparent cellular specificity suggests that these receptor subtypes can have a differential trophic role in the brain, reflecting the various biological activities shown by the ligands of the FGF family.

Excitatory purinoceptors of P2x-type have long been known to exist on smooth muscle cells, but recently it has been shown that they are also involved in synaptic transmission in the CNS. We have used a P2x-specific agonist, alpha,beta-methylene-ATP, as a 3H-labelled radioligand, to study the distribution and characteristics of P2x receptor-binding sites in the spinal cord and medulla oblongata. Using auto-radiographic techniques, [3H]alpha,beta-methylene-ATP binding was found throughout the grey matter of the spinal cord with small areas of above-average density of binding sites in the marginal zone and substantia gelatinosa at all spinal levels and in the central grey matter of the thoracic spinal cord. In the medulla, [3H]alpha,beta-methylene-ATP binding was found to be strong in all cranial nerve nuclei, particularly those known to receive primary sensory fibres. We have found that the binding of [3H]alpha,beta-methylene-ATP in the spinal cord and medulla was inhibited by a broad-spectrum P2-receptor antagonist suramin (IC50 approximately 27 microM). This is in accordance with the data obtained previously in the forebrain and cerebellum. There was, however, no inhibition of [3H]alpha,beta-methylene-ATP binding by another close analogue of alpha,beta-methylene-ATP and P2x ligand beta,gamma-methylene-ATP (10 microM). The latter result is discussed in terms of possible involvement of Ca2+ in the binding of [3H]alpha,beta-methylene-ATP to P2x receptors in the CNS.

Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) are endogenous ligands that have greater affinity and selectivity for the mu-opiate receptor than any other known mammalian peptide. A polyclonal antiserum, screened for specificity to endomorphin-2 by immunodot-blot assay and preabsorption controls, was used for localization of this peptide. Immunocytochemistry performed on the brainstem, spinal cord, and sensory ganglia of rats by the avidin-biotin-peroxidase method revealed a continuous dense aggregation of endomorphin-2-like immunoreactive varicose fibers in the superficial laminae of the dorsal horn of the medulla and spinal cord. Immunoreactive fibers were detected in the dorsal root as well as within the dorsal root ganglia. The results suggest that endomorphin-2 is synthesized in primary sensory neurons in ganglia, transported to the superficial dorsal horn, and released near neurons expressing mu receptors. Its distribution appears to represent a functional unit likely to be associated with modulation of nociceptive stimuli.

Neurotensin is widely located in neurons of the central and peripheral nervous systems among mammalian species. To obtain a comparative evaluation, we examined the distribution of neurotensin-containing cell bodies and fibers in the central nervous system of the pigeon and the chicken. The pattern of localization of neurotensin immunoreactivity was similar in the two species. Abundant accumulations of neurotensin-containing cell bodies were found in the dorsolateral corticoid area, the piriform cortex, the parahippocampal area, the medial part of the frontal neostriatum, the lateral part of the caudal neostriatum, nucleus accumbens, the bed nucleus of the stria terminalis, ventral paleostriatum, the preoptic area, the ventromedial hypothalamic nucleus, the inferior hypothalamic nucleus, the infundibular hypothalamic nucleus, and the mammillary nuclei. Extremely dense networks of neurotensin-containing fibers were found in the pallial commissure, the lateral septal nucleus, the preoptic area, the periventricular gray around the third ventricle, the dorsalis hypothalamic area, the hypothalamic nuclei, the parabrachial nucleus, the locus ceruleus, and the dorsal vagal complex. Major differences of immunoreactivity between the two species were as follows. 1) The chicken neurohypophysis contained an extremely large accumulation of immunoreactive fibers, but there were few in the median eminence. The reverse was found in the pigeon. 2) The optic tectum in the pigeon contained immunoreactive cells and fibers in layers 2 and 4, but no immunoreactivity was seen in the chicken optic tectum. 3) The cerebellar cortex in the pigeon contained a small number of immunoreactive fibers, whereas that in the chicken did not. 4) The pigeon spinal cord contained immunoreactive neurons in the subependymal layer, but the chicken spinal cord did not. Our observations suggest the presence of a very wide network of neurotensin-containing neurons in the avian brain and spinal cord, which is also the case in mammals.

The present series of experiments were designed to examine a potential role for central descending pain facilitatory systems in mediating secondary hyperalgesia produced by topical application of mustard oil and measuring the nociceptive tail-flick reflex in awake rats. Topical application of mustard oil (100%) to the lateral surface of the hind leg produced a facilitation of the tail-flick reflex that was significantly reduced in spinal transected animals. Mustard oil hyperalgesia was also inhibited in animals that had received electrolytic lesions in the rostral ventromedial medulla (RVM). Intrathecal (i.t.) administration of the non-selective cholecystokinin (CCK) receptor antagonist proglumide (10 micrograms) prior to mustard oil application completely blocked both the lesser and greater hyperalgesic responses observed in spinal transected and normal animals, respectively, and produced an inhibition of the tail-flick reflex in normal animals. Administration of the selective CCKB receptor antagonist L-365260 i.t. dose-dependently inhibited mustard oil hyperalgesia (ID50 = 364 ng) at doses approximately 5-fold less than the CCKA receptor antagonist devazepide (ID50 = 1760 ng). Similar to spinal proglumide, microinjection of the neurotensin antagonist SR48692 (3.5 micrograms) into the RVM blocked mustard oil hyperalgesia and inhibited the tail-flick reflex. These data suggest that secondary hyperalgesia produced by mustard oil is mediated largely by a central, centrifugal descending pain facilitatory system which involves neurotensin in the RVM and spinal CCK (via CCKB receptors). The inhibition of the tail-flick reflex produced by mustard oil following spinal or supraspinal administration of receptor antagonists suggests concurrent activation of central descending facilitatory and inhibitory systems.

The distribution of GABAergic and glycinergic premotor neurons projecting to the trigeminal motor nucleus (Vm) was examined in the lower brainstem of the rat by a double labeling method combining retrograde axonal tracing with immunofluorescence histochemistry. After injection of the fluorescent retrograde tracer, tetramethylrhodamine dextran amine (TRDA), into the Vm unilaterally, neurons labeled with TRDA were seen ipsilaterally in the mesencephalic trigeminal nucleus, and bilaterally in the parabrachial region, the supratrigeminal and intertrigeminal regions, the reticular formation just medial to the Vm, the principal sensory and spinal trigeminal nuclei, the pontine and medullary reticular formation, especially the parvicellular part of the medullary reticular formation, the alpha part of the gigantocellular reticular nucleus, and the medullary raphe nuclei. Some of these neurons labeled with TRDA were found to display glutamic acid decarboxylase (the enzyme involved in GABA synthesis)-like or glycine-like immunoreactivity. Such double-labeled neurons were seen mainly in the supratrigeminal region, the reticular region adjacent to the medial border of the Vm, and the dorsal part of the lateral reticular formation of the medulla oblongata; a number of them were further scattered in the intertrigeminal region, the alpha part of the gigantocellular reticular nucleus, the nucleus raphe magnus, the principal sensory trigeminal nucleus, and the interpolar subnucleus of the spinal trigeminal nucleus. These neurons were considered to be inhibitory (GABAergic or glycinergic) neurons sending their axons to motoneurons in the Vm, or to local interneurons within and around the Vm.

Receptors for the neuropeptide, neurotensin, were localized by immunohistochemistry in the rat brain by using an antibody raised against a sequence of the third intracellular loop of the cloned high affinity receptor. Selective receptor immunostaining was observed throughout the brain and brainstem. This immunostaining was totally prevented by preadsorbing the antibody with the immunogenic peptide. The regional distribution of the immunoreactivity conformed for the most part to that of [3H]- or [125I]-neurotensin binding sites previously identified by autoradiography. Thus, the highest levels of immunostaining were observed in the islands of Calleja, diagonal band of Broca, magnocellular preoptic nucleus, pre- and parasubiculum, suprachiasmatic nucleus, anterodorsal nucleus of the thalamus, substantia nigra, ventral tegmental area, pontine nuclei and dorsal motor nucleus of the vagus, all of which had previously been documented to contain high densities of neurotensin binding sites. There were, however, a number of regions reportedly endowed with neurotensin binding sites, including the central amygdaloid nucleus, periaqueductal gray, outer layer of the superior colliculus and dorsal tegmental nucleus, which showed no or divergent patterns of immunostaining, suggesting that they might be expressing a molecularly distinct form of the receptor. At the cellular level, neurotensin receptor immunoreactivity was predominantly associated with perikarya and dendrites in some regions (e.g., in the basal forebrain, ventral midbrain, pons and rostral medulla) and with axons and axon terminals in others (e.g., in the lateral septum, bed nucleus of the stria terminalis, neostriatum, paraventricular nucleus of the thalamus and nucleus of the solitary tract). These data indicate that neurotensin may act both post- and presynaptically in the central nervous system and confirm that some of its effects are exerted on projection neurons. There were also areas, such as the cerebral cortex, nucleus accumbens and para- and periventricular nucleus of the hypothalamus, which contained both immunoreactive perikarya/dendrites and axon terminals, consistent with either a joint association of the receptor with afferent and efferent elements or its presence on interneurons. Taken together, these results also suggest that the neurotensin high affinity receptor protein is associated with a neuronal population that is more extensive than originally surmised from in situ hybridization studies.

Using an antiserum raised against Lys- gamma 2-melanocyte-stimulating hormone (Lys- gamma 2-MSH), with a high specificity for this peptide and its des-Lys derivative, gamma 2-MSH, we found Lys- gamma 2-MSH-like immunoreactivity to have a widespread distribution in the rat brain. In colchicine-treated rats, groups of immunopositive cell bodies were found in the intermediate and anterior lobes of the pituitary gland, in the hypothalamic arcuate nucleus and in the commissural part of the nucleus of the solitary tract (NTS). Immunopositive fibers were found to originate from the latter two cell body regions. The distribution of these fibers was similar to that of the pro-opiomelanocortin containing cell bodies and projections as it has been described previously. Immunopositive terminals were found in brain region containing neurons which have been shown to express mRNA for melanocortin receptors, though the distribution of Lys-gamma 2-MSH-like immunoreactivity is considerably more widespread than that of mRNA for the 'gamma-MSH receptor' (the melanocortin MC3 receptor), which has been reported to be mainly expressed in the hypothalamus. In the periphery Lys-gamma 2-MSH immunoreactivity was localized in the adrenal medulla and in neuronal fibers and varicosities in the heart. The vascular system, the bronchi and kidney were immunonegative. The occurrence of Lys-gamma 2-MSH immunoreactivity in many of the brain regions which are involved in cardiovascular regulation offers leads for further studies on the putative role of gamma-MSHs in cardiovascular control. The occurrence in the rat heart of Lys-gamma 2-MSH-containing fibers suggests a role of the gamma-MSHs in cardiac function.

In this study, we analyzed immunohistochemically the distribution of the A subtype of alpha 2-adrenergic receptor (alpha 2A-AR) in the rat central nervous system using light level immunohistochemistry. By using affinity-purified antisera, we found perikaryal labeling was diffuse and/or punctate; immunoreactive puncta were heterogeneous in size and number in a region-specific manner. Dense deposits of immunoreaction product were found associated with neuropil also, particularly in the lateral parabrachial nucleus, locus coeruleus, lateral septum, diagonal band, stratum lacunosum-moleculare of CA1, and various nuclei of the amygdala and extended amygdala. Prominently immunoreactive olfactory structures include the anterior olfactory nucleus and the granular layer of the olfactory bulb. The cortex was generally light to moderately labeled with greater immunoreactivity in the cingulate and insular cortices. alpha 2A-AR-like immunoreactivity was intense in the basal forebrain and continuous from the nucleus accumbens through the substantia innominata and fundus of the striatum. Most immunoreactivity in the diencephalon was restricted to the hypothalamus with light to moderate labeling in the thalamus. Generally light immunoreactivity was observed in midbrain structures. In the pons and medulla, both perikaryal and neuropil labeling were observed. Together with the accompanying paper describing the neural distribution of alpha 2C-AR-like immunoreactivity, our results provide an extensive immunohistochemical cartography of alpha 2-ARs in the adult rat central nervous system.

The localization of glucose transporters (GLUTs) was examined in various regions of the rat brain. The mRNA of GLUT1 and GLUT3 were found ubiquitously in every brain region (cortex, hippocampus, midbrain, striatum, hypothalamus, medulla oblongata and cerebellum). The mRNA and protein of GLUT4, an insulin-regulatable glucose transporter in peripheral tissues, were also identified, particularly abundantly in the cerebellum. In situ hybridization analysis revealed that GLUT4 mRNA was present in some discrete cells, such as Purkinje cells in the cerebellum, the vestibular nucleus in the medulla oblongata and also in ependymal cells along the cerebral ventricles. The GLUT4 mRNA level in the cerebellum changed little in fasted or experimentally induced diabetic rats while those in adipose tissues decreased much. The results suggest that insulin-sensitive glucose uptake may occur in some specific cells of the brain but is regulated in a different manner from those in peripheral cells.