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Sections of the medulla oblongata from the brains of sheep were examined for prion protein (PrP) by immunohistochemistry. On the basis of the morphology and neuroanatomical distribution of the deposits, distinct disease-associated patterns of PrP deposition were identified in scrapie-affected sheep, suggesting at least four distinct phenotypes of scrapie. In addition, clearly defined patterns of PrP deposition, readily distinguished from the disease-associated PrP deposits, were identified in some normal sheep from scrapie-free flocks. In five sheep, believed to be preclinically affected by scrapie, PrP deposition of a disease-specific type but of restricted distribution was identified, demonstrating the sensitivity of the technique for the diagnosis of scrapie. The neuroanatomical distribution of these early PrP deposits suggest that the route of entry of the scrapie agent into the brain is via parasympathetic motor neurons in the vagus nerve which innervate the gastrointestinal tract.

Recent data suggest that there is interaction between peripheral angiotensin II and nitric oxide. However, sparse information is available on the mutual interaction of these two compounds in the brain. The potential intercourse of nitric oxide with brain neuropeptides needs to be substantiated by assessing its local production and gene expression of the synthesizing enzymes involved. The aim of the present study was to evaluate whether the gene expression of brain nitric oxide synthase (bNOS) is related to the sites of gene expression of different components of the rat brain renin angiotensin system (renin, angiotensin converting enzyme (ACE) or angiotensin receptors of AT1 and AT2 subtypes). The levels of corresponding mRNAs were measured and correlated in nine structures of adult rat brain (hippocampus, amygdala, septum, thalamus, hypothalamus, cortex, pons, medulla and cerebellum). As was expected, positive correlation was observed between renin and angiotensin-converting enzyme mRNAs. Moreover, a significant correlation was found between brain NO synthase and AT1 receptor mRNAs, but not with mRNA of the AT2 receptor, ACE and renin. Parallel distribution of mRNAs coding for bNOS and AT1 receptors in several rat brain structures suggests a possible interaction between brain angiotensin 11 and nitric oxide, which remains to be definitely demonstrated by other approaches.

DNA contents, ranging from 4C to more than 500C, were determined by a quantitative microfluorimetric assay in supramedullary neuron nuclei of the pufferfish Diodon holacanthus. The distribution of C values after ethidium bromide staining indicates an inter- and intra-individual variation in DNA contents which do not correspond to duplications of the total genome, suggesting that DNA replication is specific for particular genes (endoreplication). Moreover, the DNA content appears to be correlated with nuclear size. A relationship between the DNA amounts and the presence of AT- and GC-rich sequences has been shown. This work demonstrates, for the first time, DNA endoreplication in a specific neuronal type in vertebrates.

The ultrastructure and synaptic relationships of the angiotensin II-containing neurons in the area postrema of the rat were studied by immunocytochemistry using the avidin-biotin-complex-DAB method, and also using silver-gold intensification following the DAB reaction. At the light microscopic level, the angiotensin II-like immunoreactive neurons were observed within the area postrema, especially in the upper region. At the electron microscopic level, the angiotensin II-like immunoreactive cell bodies were observed as having a round, unindented nucleus. The nuclei of these neurons were not immunostained. The angiotensin II-like immunoreactive axon terminals often contained a few dense core vesicles in addition to many small clear synaptic vesicles. Numerous axon terminals were found to make synapses on immunonegative dendrites; they were also found to make synapses on angiotensin II-like immunoreactive dendrites. Many angiotensin II-like immunoreactive dendrites received synapses from immunonegative axon terminals. Although angiotensin II-like immunoreactive cell bodies were sometimes postsynaptic to immunoreactive axon terminals, they did not receive synapses from immunonegative axon terminals. These results provide solid morphological evidence of AP endogenous angiotensin II and confirm that in spite of circulating angiotensin II, the local neurons in the AP may also play an important role in angiotensin II-induced cardiovascular regulation., (Copyright 2000 Wiley-Liss, Inc.)

Regional distribution of prolactin-releasing peptide (PrRP) in the human brain was studied by radioimmunoassay. The antiserum raised against human PrRP-31 in a rabbit was used in the assay, which showed 100% cross reaction with PrRP-20 and no significant cross reaction with other peptides. The highest concentrations of immunoreactive-PrRP were found in hypothalamus (912 +/- 519 fmol/g wet weight, n = 6, mean +/- SEM), followed by medulla oblongata (496 +/- 136 fmol/g wet weight) and thalamus (307 +/- 117 fmol/g wet weight). On the other hand, immunoreactive-PrRP was not detected in frontal lobe or temporal lobe (<50 fmol/g wet weight). Sephadex G50 column chromatography of the immunoreactive-PrRP in the hypothalamus and medulla oblongata showed three immunoreactive peaks; one peak eluting in the position of PrRP-20, one eluting in the position of PrRP-31 and one eluting earlier. Reverse phase high-performance liquid chromatography (HPLC) of these brain tissue extracts showed a peak eluting in the position of PrRP-20 and PrRP-31. The present study has shown for the first time the presence of immunoreactive-PrRP in the human brain. The immunoreactive-PrRP levels in the human hypothalamus were, however, lower than the levels of other neuropeptides with prolactin-releasing activity, such as thyrotropin-releasing hormone and vasoactive intestinal polypeptide.

The distribution and ascending projections to the hypothalamic paraventricular nucleus of phenylethanolamine N-methyltransferase (PNMT)-immunoreactive perikaria were studied in adult pigeons using a combination of retrograde transport of Fluorogold injected into the paraventricular nucleus, and double immunohistochemical procedures for PNMT, tyrosine hydroxylase and neuropeptide Y. PNMT-immunoreactive cell bodies were found in the subtrigeminal reticular nucleus of the ventrolateral medulla and in the nucleus of the solitary tract, mainly in the subnuclei: medialis superficialis, pars posterior, and medialis ventralis, pars posterior. PNMT-immunoreactive perikaria were also tyrosine hydroxylase immunoreactive, and are located within the rostral tyrosine hydroxylase immunoreactive cell groups of these areas. No perikaria double-labeled for neuropeptide Y and PNMT were found. Retrograde labeled cell bodies were observed in the subtrigeminal reticular nucleus and in the nucleus of the solitary tract. PNMT-immunoreactive retrogradely labeled cells were mainly observed in the subtrigeminal reticular nucleus. These data suggest the presence in the pigeon of medullary adrenergic cell groups partially comparable to mammalian C1 and C2 groups. Comparison of these results with data previously obtained in amphibians and reptiles suggests that the presence of a hypothalamically-projecting C1-like group might be a plesiomorphic medullary attribute in amniotes, whereas the variable presence of C2 and C3-like groups, as well as the content of NPY in the putative adrenergic perikaria, seem to be species-specific.

gamma-Aminobutyric acid (GABA)ergic and glycinergic neurons were examined light- and electron-microscopically in laminae I and II of the medullary dorsal horn (MDH, i.e. spinal trigeminal nucleus caudalis in the rat). The majority of GABA- and glycine (Gly)-immunoreactive (-ir) neurons showed both GABA- and Gly-immunoreactivities (-IRs). Noxious stimulation (subcutaneous injection of formalin into perioral regions) induced Fos-IR in some of GABA- and Gly-ir neurons. GABA- and Gly-ir neuronal profiles were postsynaptic to substance P-ir axon terminals. These results suggest that nociceptive information being carried by primary afferent SP-fibers may be relayed directly to GABAergic and glycinergic neurons in laminae I and II of the MDH.

1. In SUNCUS: murinus the ultrapotent capsaicin analogue resiniferatoxin (RTX) induced an emetic response in the dose range 1 - 1000 microg kg(-1), s.c. The latency was inversely related to dose and ranged from 41.2+/-4.4 min. (1 microg kg(-1), s.c.) to 2.7+/-0.6 min. (1000 microg kg(-1), s.c.). 2. The emetic response to RTX (10 or 100 microg kg(-1), s.c.) was blocked or markedly reduced by pre-treatment with RTX (100 microg kg(-1), s.c.), 8-OH-DPAT (100 microg kg(-1), s.c.), morphine (2 mg kg(-1), s.c.), neonatal capsaicin (100 mg kg(-1), s.c.) and the NK(1) receptor antagonist CP-99,994 (10 - 20 mg kg(-1), s.c.) but not by the 5-HT(3) receptor antagonist tropisetron (200 microg kg(-1), s.c.). 3. RTX (100 microg kg(-1), s.c.) induced c-fos-like immunoreactivity in the area postrema and parts of the nucleus tractus solitarius. This pattern is consistent with the proposal that the emetic effect is mediated via one or both of these structures and an involvement of substance P is discussed. 4. RTX (10 and 100 microg kg(-1), s.c.) had broad-spectrum antiemetic effects in Suncus as indicated by its ability to block or markedly reduce the emetic response to motion (1 Hz, 4 cm lateral, 10 min.), cisplatin (20 mg kg(-1), i.p.), intragastric copper sulphate (40 mg kg(-1), p.o.), nicotine (10 mg kg(-1), s.c.) and RTX (100 microg kg(-1), s.c.) itself. 5. It is proposed that the site of the anti-emetic effect is in the nucleus tractus solitarius and mechanisms involving the modulation of substance P release are discussed. 6. The general utility of SUNCUS: for investigations of vanilloid receptors is reviewed in the light of the exquisite sensitivity of the emetic reflex in this species to resiniferatoxin.

Under anesthesia, inactivation of the retrotrapezoid nucleus (RTN) region markedly inhibits breathing and chemoreception. In conscious rats, we dialyzed muscimol for 30 min to inhibit neurons of the RTN region reversibly. Dialysis of artificial cerebrospinal fluid had no effect. Muscimol (1 or 10 mM) significantly decreased tidal volume (VT) (by 16-17%) within 15 min. VT remained decreased for 50 min or more, with recovery by 90 min. Ventilation (VE) decreased significantly (by 15-20%) within 15 min and then returned to baseline within 40 min as a result of an increase in frequency. This, we suggest, is a compensatory physiological response to the reduced VT. Oxygen consumption was unchanged. In response to 7% CO(2) in the 1 mM group, absolute VE and change in VE were significantly reduced (by 19-22%). In the 10 mM group, the response to dialysis included a time-related increase in frequency and decrease in body temperature, which may reflect greater spread of muscimol. In the awake rat, the RTN region provides a portion of the tonic drive to breathe, as well as a portion of the response to hypercapnia.

Immunohistochemical localization of the substance P receptor (SPR) was examined in the developing rat medulla oblongata, with special reference to the development of substance P (SP)-immunoreactive neurons which form the medullary raphe nuclei. During development, SPR immunoreactivity was detected in cells lying lateral to the medullary midline from embryonic day 13 (E13) to postnatal day 5 (P5). The SPR-positive cell bodies were located close to the fourth ventricle, and bore long processes extending to the ventral pial surface. This SPR immunoreactivity co-localized with staining for monoclonal antibody 1D11, a specific marker of immature astrocytes. Substance P (SP)-immunoreactive neurons were first detected at E14 in the ventrolateral part of the medulla. By E16 their number had increased and they were arrayed in two rows closely parallel to the SPR-immunoreactive processes of non-neuronal cells. By P1, two separate SP-immunoreactive cell clusters could be recognized at the midline, representing dorsally the nascent raphe pallidus and ventrally the raphe obscurus. In addition, many SP-immunoreactive fibers traveled rostrocaudally in the medulla oblongata, juxtaposed to the midline sheets of SPR-immunoreactive long processes. SPR-immunoreactive processes at the midline were also immunoreactive for S-100, a glia-specific calcium-binding protein that is known to promote axonal growth of raphe neurons. These results suggest that SPR-expressing immature glial cells at the medullary midline are involved in the development of SP-immunoreactive raphe neurons, both in the formation of the medullary raphe nuclei and in axon guidance and growth.

Altered thyroid statuses are associated with autonomic disorders. Thyrotropin-releasing hormone (TRH) synthesized in medullary raphe pallidus (Rpa), raphe obscurus (Rob) and the parapyramidal regions (PPR) regulates vagal and sympathetic preganglionic motoneurons. Hypothyroidism increased TRH gene expression and c-Fos immunoreactivity (IR) in these nuclei. Whether these increases represent a direct action of thyroid hormone was studied by detecting the presence of thyroid hormone receptor beta2 (TRbeta2) in pro-TRH-synthesizing neurons in the Rpa, Rob and the PPR using immunohistochemistry with specific TRbeta2 antiserum and in situ hybridization with digoxigenin-labeled pro-TRH cRNA probe. TRbeta2 IR was widely distributed throughout the medulla and primarily localized within the cell nuclei. Particularly intense immunostaining was presented in the Rpa, Rob and the PPR neurons. The combination of immunohistochemistry with in situ hybridization revealed that all pro-TRH mRNA-positive neurons in these ventral medullary nuclei were also TRbeta2 IR positive. The numbers of TRbeta2 IR-positive neurons in each nucleus were identical in both euthyroid rats and hypothyroid rats induced by 6-n-propyl-2-thiouracil in drinking water for 4 weeks. The finding that TRbeta2 localized in pro-TRH-synthesizing neurons in the ventral medullary nuclei provides an anatomical substrate for a direct thyroid hormone action on these neurons in the regulation of TRH gene expression, which may contribute to the altered autonomic activity in different thyroid statuses.

We examined the effects of acute and chronic psychogenic stress on the activation pattern of enkephalin-containing perikarya in the rat ventrolateral medulla. Rats allocated to the chronic stress groups were subjected to 90 min of immobilization for 10 days. On the 11th day, the chronically stressed rats were exposed to homotypic (90-min immobilization) or to heterotypic but still psychogenic (90-min immobilization coupled to air jet stress) stress. The acute stress group was subjected once to an acute 90-min immobilization. For each group, the rats were anesthetized either before stress (time 0) or 90, 180, and 270 min after the onset of stress. Brain sections were then processed using immunocytochemistry (Fos protein) followed by radioactive in situ hybridization histochemistry (enkephalin mRNA). Following immobilization, the acute group displayed a marked increase in the number of activated enkephalin-containing perikarya within the paragigantocellularis and lateral reticular nuclei. This level of activation was sustained up to 180 min following the onset of the immobilization stress and had returned to baseline levels by 270 min from the initiation of the stress. However, this stress-induced activation of enkephalin-containing perikarya of the ventrolateral medulla was not seen following either homotypic or heterotypic stress in the chronically stressed group. These results provide evidence that enkephalin-containing perikarya of the ventrolateral medulla may constitute a potential circuit through which they regulate some aspect of the stress responses. Conversely, this enkephalinergic influence from the ventrolateral medulla was shown to be absent following chronic stress exposure. This would suggest a decrease in enkephalin inhibitory input originating from the ventrolateral medulla, thereby allowing a neuroendocrine and/or autonomic response to chronic stress.

Although the distribution of cholinergic cells is remarkably similar across the vertebrate species, no data are available on more primitive species, such as cartilaginous fishes. To extend the evolutionary analysis of the cholinergic systems, we studied the distribution of cholinergic neurons in the brain and rostral spinal cord of Scyliorhinus canicula by immunocytochemistry using an antibody against the enzyme choline acetyltransferase (ChAT). Western blot analysis of brain extracts of dogfish, sturgeon, trout, and rat showed that this antibody recognized similar bands in the four species. Putative cholinergic neurons were observed in most brain regions, including the telencephalon, diencephalon, cerebellum, and brainstem. In the retrobulbar region and superficial dorsal pallium of the telencephalon, numerous small pallial cells were ChAT-like immunoreactive. In addition, tufted cells of the olfactory bulb and some cells in the lateral pallium showed faint immunoreactivity. In the preoptic-hypothalamic region, ChAT-immunoreactive (ChAT-ir) cells were found in the preoptic nucleus, the vascular organ of the terminal lamina, and a small population in the caudal tuber. In the epithalamus, the pineal photoreceptors were intensely positive. Many cells of the habenula were faintly ChAT-ir, but the neuropil of the interpeduncular nucleus showed intense ChAT immunoreactivity. In the pretectal region, ChAT-ir cells were observed only in the superficial pretectal nucleus. In the brainstem, the somatomotor and branchiomotor nuclei, the octavolateral efferent nucleus, and a cell group just rostral to the Edinger-Westphal (EW) nucleus contained ChAT-ir neurons. In addition, the trigeminal mesencephalic nucleus, the nucleus G of the isthmus, some locus coeruleus cells, and some cell populations of the vestibular nuclei and of the electroreceptive nucleus of the octavolateral region exhibited ChAT immunoreactivity. In the reticular areas of the brainstem, the nucleus of the medial longitudinal fascicle, many reticular neurons of the rhombencephalon, and cells of the nucleus of the lateral funiculus were immunoreactive to this antibody. In the cerebellum, Golgi cells of the granule cell layer and some cells of the cerebellar nucleus were also ChAT-ir. In the rostral spinal cord, ChAT immunoreactivity was observed in cells of the motor column, the dorsal horn, the marginal nucleus (a putative stretch-receptor organ), and in interstitial cells of the ventral funiculus. These results demonstrate for the first time that cholinergic neurons are distributed widely in the central nervous system of elasmobranchs and that their cholinergic systems have evolved several characteristics that are unique to this group., (Copyright 2000 Wiley-Liss, Inc.)

In the mouse medulla oblongata, we characterized binding properties and functional responses of two recognition sites for imidazoline compounds: I(1)-imidazoline and alpha(2)-adrenergic receptors. The mouse medulla expresses a higher density of I(1)-receptors than in the rat, whereas alpha(2)-receptor densities were similar between the two species. In anesthetized, ventilated and paralyzed mice, we tested the hypotensive actions of the I(1)/alpha(2) agonist moxonidine, determined its central site of its actions, and the relative roles of I(1) and alpha(2)-receptors. Experiments were performed in C(57)Bl(6) wild type and alpha(2A)-adrenergic receptor deficient mice. In both types of mice, neuronal activation within the rostral ventrolateral medulla (RVLM) region by glutamate microinjection elicited increases in arterial pressure. Moxonidine (0.5 nmol/site/10 nl) microinjected bilaterally into this vasopressor region decreased arterial pressure by 30% and heart rate by 11% in wild type mice. Efaroxan, the I(1)/alpha(2) antagonist (0.4 nmol) when microinjected into the RVLM elevated blood pressure itself and abolished the action of moxonidine, whereas alpha(2)-blockade with SK&F 86466 had no significant effect on blood pressure and did not attenuate moxonidine's effect. To more definitively test the role of alpha(2)-adrenergic receptors in the action of moxonidine, moxonidine was microinjected into the RVLM of alpha(2A)-adrenergic deficient mice. The decreases in arterial pressure were nearly identical to those of wild type mice, whereas bradycardia was attenuated. Thus, in the mouse moxonidine acts within the RVLM region to lower arterial pressure mainly through the I(1)-imidazoline receptor independent of alpha(2)-adrenergic receptors.

The constitutive expression of heat shock protein 108 (HSP108) mRNA is mapped in a normal chicken central nervous system using in situ hybridization technique. HSP108 mRNAs were found to be mainly localized in the small neuroglial cells of various regions of the brain, although some neuronal cells also showed positive signals. This tendency is observed to be more marked in the cerebellum; HSP108 signals were not found in the Purkinje cells, but in Bergmann glial cells and oligodendrocytes. Although neuronal cells in the deep cerebellar nuclei and the molecular layer showed occasional HSP108 signals, the expression pattern of HSP108 mRNA is different from homologous HSP90 that is mostly expressed in neurons, but rather similar to that of TfBP immunoreactivity, a new member of the HSP108 family. The constitutive neuroglial localization of HSP108 could suggest that HSP108 may play an important role in the normal metabolism of neuroglial cells in the chicken brain.

Moxonidine is an antihypertensive drug that lowers sympathetic vasomotor tone by stimulating either alpha2-adrenergic (alpha2-AR) or imidazoline I1 receptors within the rostral ventrolateral medulla (RVL). In this study, we investigated the effects of moxonidine (10 microM) on RVL neurons in brain stem slices of neonatal rats. We recorded mainly from retrogradely labeled RVL bulbospinal neurons (putative presympathetic neurons) except for some extracellular recordings. Prazosin was used to block alpha1-adrenoceptors. Moxonidine inhibited the extracellularly recorded discharges of all spontaneously active RVL neurons tested (bulbospinal and unidentified). This effect was reversed or blocked by the selective alpha2-AR antagonist SKF 86466 (10 microM). In contrast, the I1 imidazoline ligand AGN 192403 (10 microM) had no effect on the spontaneous activity. In whole cell recordings (holding potential -70 mV), moxonidine produced a small and variable outward current (mean 7 pA). This current was observed in both tyrosine hydroxylase-immunoreactive and other bulbospinal neurons and was blocked by SKF 86466. Excitatory postsynaptic currents (EPSCs) evoked by focal electrical stimulation were isolated by incubation with gabazine and strychnine, and inhibitory postsynaptic currents (IPSCs) were isolated with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Moxonidine reduced the amplitude of the evoked EPSCs (EC(50) = 1 microM; 53% inhibition at 10 microM) but not their decay time constant (5.6 ms). The effect of moxonidine on EPSCs persisted in barium (300 microM) and was reduced approximately 80% by SKF 86466. Moxonidine also reduced the amplitude of evoked IPSCs by 63%. In conclusion, moxonidine inhibits putative RVL presympathetic neurons both presynaptically and postsynaptically. All observed effects in the present study are consistent with an alpha2-AR agonist activity of moxonidine.

As members of the chromogranin family, chromogranin A, chromogranin B, and secretogranin II are acidic proteins found in large, dense core vesicles. They are endoproteolytically processed to smaller peptides and released after neuronal stimulation. Using immunocytochemistry, this study closely examines chromogranin B-like immunoreactivity within the human brainstem and then takes a comparative view of co-distribution patterns by chromogranin B, chromogranin A, and secretogranin II. We used an antiserum raised against a synthetic peptide (PE-11) present in the chromogranin B molecule. Secretogranin II was localized with an antiserum against secretoneurin, a 33 amino acid peptide, found within the secretogranin II precursor. Like chromogranin A and secretoneurin, chromogranin B is expressed through all levels of the human brainstem. Chromogranin B was exclusively detected in neuronal structures. The medial part of the substantia nigra pars reticulata, the nucleus interpeduncularis, the area of the central gray, and the raphe complex displayed a high density of PE-11-like immunoreactivity. Furthermore, a prominent staining was found in the medial, dorsal and gelatinous subnuclei of the solitary tract and the dorsal motor nucleus of vagus. The substantia gelatinosa of the caudal trigeminal nucleus and spinal cord were also very strongly PE-11-immunopositive. In conclusion, chromogranin B and secretogranin II showed similar distributions while neuronal localization typically differed from chromogranin A aside from a few exceptions. These findings may provide a framework for future research in revealing a functional role of chromogranin peptides in the human brainstem.

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.

Injection of the excitatory amino acid (EAA) antagonist kynurenic acid (KYN) into the rostral ventrolateral medulla (RVLM) of anesthetized rats has no effect on arterial pressure. However, we recently reported that after inhibition of the caudal ventrolateral medulla, injection of KYN into the RVLM decreased arterial pressure to the same level as produced by complete inhibition of the RVLM. We have suggested that these results reflect tonically active EAA-mediated inputs to the RVLM producing both direct excitation of RVLM vasomotor neurons and indirect inhibition of these neurons. On the basis of this model, we hypothesize that the balance between these EAA-driven direct excitatory and indirect inhibitory influences on the RVLM may be altered in models of experimental hypertension. To begin to test this hypothesis, the effects of injecting KYN into the RVLM of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) were compared. In chloralose-anesthetized WKY, bilateral injection of KYN into the RVLM did not alter arterial pressure, whereas similar injections in SHR reduced mean arterial pressure by approximately 40 mm Hg. After inhibition of the caudal ventrolateral medulla, which similarly increased arterial pressure in both strains, injection of KYN into the RVLM reduced mean arterial pressure to the same level as produced by autonomic blockade. These results suggest that the balance of excitatory and inhibitory influences on RVLM vasomotor neurons driven by tonically active EAA-mediated inputs to the RVLM is disrupted in SHR and may contribute to the hypertension in SHR.

Disease-specific PrP (PrP(Sc)) is at least part of the infectious particle (prion) causing bovine spongiform encephalopathy (BSE) or scrapie in sheep. Digestion with protease allows a distinction between normal PrP (PrP(C)) and PrP(Sc) i.e. PrP(C) is completely digested while PrP(Sc) is cleaved at the N-terminus leading to a fragment of reduced molecular weight (PrP 27-30). Detection of this fragment by Western blotting has been described more than a decade ago for rodent PrP. We have now optimized the technique in order to allow rapid analysis of hundreds of samples per day. Here we report the application of this technique to the analysis of 3000 regularly slaughtered cattle from Swiss abattoirs. For comparison all the animals were subsequently examined by classical methods (i.e. histology and immunohistochemistry). All but one animal were negative for BSE by all methods. The Western blot positive animal was confirmed to be a BSE case and the carcass was removed from the food chain. We conclude that it is feasible to examine slaughtered cattle on a routine basis without causing delays to the meat processing industry.

The appearance of a new variant of CJD (vCJD) in young patients has caused considerable public concern and there is evidence that this novel disease is caused by the same agent as BSE. BSE is a prion disease that became epidemic in the UK, with a peak incidence in January 1993. New test systems should aim to identify BSE-infected cattle early in the incubation period. We compared the established histological and immunohistochemical methods and the Western blot method used by Prionics with the PET blot method that detects prion PrP(Sc) deposits in formalin-fixed and paraffin-embedded tissue. Investigating the obex region with the PET blot, all BSE cases were detectable and no false positive cases occurred. From the Swiss culling program, five clinically healthy cattle out of 1761 were identified as incubating BSE. With the PET blot method four of them showed the same PrP(Sc) deposition pattern that was seen in clinical BSE, though less conspicuous. In one of the five cases, PrP(Sc) was restricted to two brain stem nuclei, a pattern that was reported to be the first manifestation of PrP(Sc) deposits in the brain after peripheral infection and one that occurs after half of the incubation time. In this case, histology and Western blot were negative.

The expression of the immediate early gene, c-fos, was used to determine the distribution of brainstem neurons activated by stimulation of the distal hypoglossal nerve (XIIn) trunk. The traditional view of the XIIn is one of purely motor function; however, stimulation of XIIn excites neurons in the trigeminal spinal nucleus. The rationale for this study was to use c-fos expression as a marker for postsynaptic activity to define the pattern of brainstem neurons excited by XIIn stimulation. It was further hypothesized that if the afferent fibers that course within XIIn supply deep lingual tissues, then c-fos expression after direct stimulation of XIIn should display a pattern similar to that seen after chemical irritant stimulation of the deep tongue muscle. In barbiturate-anesthetized male rats electrical stimulation of XIIn produced a significant increase in Fos-positive neurons in the dorsal paratrigeminal nucleus (dPa5) and laminae I-II of caudal subnucleus caudalis (Vc) and upper cervical dorsal horn. Mustard oil injection into the deep tongue muscle also produced an increase in c-fos expression in dPa5; however, the highest density of expression occurred in laminae I-II at the dorsomedial aspect of rostral Vc. Both electrical stimulation of XIIn and mustard oil stimulation of the deep tongue increased c-fos expression in the caudal ventrolateral medulla, an autonomic relay nucleus. These results suggest that one site of innervation for afferent fibers that travel within the distal trunk of XIIn is to supply the deep tongue muscle and to terminate in the dPa5. A second group of postsynaptic neurons activated only by XIIn stimulation was located in lamina I-II in caudal portions of Vc and upper cervical dorsal horn, a laminar distribution consistent with a role for XIIn afferents in sensory or autonomic aspects of lingual function.

In the present study, we investigated the expression of N-methyl-D-aspartate (NMDA) receptor subunits in the parabrachial/Kölliker-Fuse complex (PB/KF), the intertrigeminal region, the ventrolateral pons, the nucleus of the solitary tract, the ventrolateral medulla with the ambiguus nucleus, and the caudal spinal trigeminal nucleus, which are presumably involved in mediating the autonomic responses to nasotrigeminal stimulation (diving response). Our immunocytochemical data demonstrate that the majority of neurons in the respective nuclei stain for the NR1 subunit, which is a mandatory component of all NMDA receptors. NR1 immunoreactivity was found mainly on neuronal cell bodies and primary dendrites. The ubiquitous expression of the NR1 subunit was confirmed by in situ hybridization, revealing a strong NR1 mRNA signal over neurons in all nuclei investigated. Among the NR2A-D subunits, the strongest expression was observed for the NR2D transcript, both in the PB/KF and in the brainstem. For the PB/KF, we found in addition a moderate expression of NR2A mRNA in the internal lateral PB and of NR2B mRNA in the external lateral PB. The remaining PB nuclei and the KF were essentially devoid of NR2A-C transcripts. For the nucleus of the solitary tract and in the spinal trigeminal nucleus, we found, in addition to the strong NR2D mRNA signal, moderate expression of the NR2A-C transcripts. In the ventrolateral medulla, a moderate signal was seen for NR2C transcript, whereas signals for the NR2A and -B subunits were negligible. Our data suggest that, in PB/KF and pontomedullary brainstem nuclei involved in mediating the diving response, glutamatergic neurotransmission is apparently mediated through a specific type of NMDA receptor channels, consisting essentially of NR1 and NR2D subunits., (Copyright 1999 Wiley-Liss, Inc.)

The objective of this study was to characterize the projection pattern and electrophysiological properties of the rostral medullary adrenergic neurons (C(1)) that express neuropeptide Y (NPY) mRNA in rat. NPY mRNA was found in a variable fraction of tyrosine hydroxylase immunoreactive (TH-IR) neurons depending on the medullary level. By retrograde labeling (Fast Blue, FluoroGold), NPY mRNA was detected in virtually all C(1) cells (96%) and C(3) cells (100%) with hypothalamic projections but in only 9% of C(1) cells and 58% of C(3) cells projecting to thoracic segment 3 (T(3)) or T(6) of the spinal cord. To identify the electrophysiological properties of the C(1) cells that express NPY mRNA, we recorded from baroinhibited neurons within the C(1) region of the ventrolateral medulla (RVLM) and tested for projections to segment T(3), the hypothalamus, or both. By using the juxtacellular method, we labeled these cells with biotinamide and determined whether the recorded neurons were TH-IR and contained NPY mRNA. At rostral levels (Bregma -11.8 mm), barosensitive neurons had a wide range of conduction velocities (0.4-6.0 m/second) and discharge rates (2-28 spikes/second). Most projected to T(3) only (27 of 31 cells), and 4 projected to both the hypothalamus and the spinal cord. Most of the baroinhibited cells with spinal projections but with no hypothalamic projections had TH-IR but no NPY mRNA (11 of 17 cells). Only 1 cell had both (1 of 17 cells), and 5 cells had neither (5 of 17 cells). Both TH-IR and NPY mRNA were found in neurons with dual projections (2 of 2 cells). At level Bregma -12.5 mm, baroinhibited neurons had projections to the hypothalamus only (13 of 13 cells) and had unmyelinated axons and a low discharge rate. Four of five neurons contained both TH-IR and NPY mRNA, and 1 neuron contained neither. In short, NPY is expressed mostly by C(1) cells with projection to the hypothalamus. NPY-positive C(1) neurons are barosensitive, have unmyelinated axons, and have a very low rate of discharge. Most bulbospinal C(1) cells with a putative sympathoexcitatory role do not make NPY., (Copyright 1999 Wiley-Liss, Inc.)

The Kir6.1/uK(ATP)-1, subunit of ATP-sensitive K(+) channels (K(ATP)), was localized in adult rat brain by in situ hybridization and immunohistochemistry. The mRNA of this subunit was ubiquitously expressed in various neurons and nuclei of the adult rat brain. Interestingly, Kir6.1/uK(ATP)-1 mRNA was also expressed in glial cells. Kir6.1/uK(ATP)-1 protein staining gave a dispersed array of fine dots throughout all neurons and glial cells examined. Under electron microscope, the immunoreactive products were specifically restricted to the mitochondria. The present study indicates that this K(ATP) subunit is localized in the mitochondria and may play a fundamental role in vital brain function.

The effects of neonatal handling on the opioid dynorphin peptides in the brain and pituitary gland of Sprague-Dawley rats were investigated. Ten weeks after the neonatal handling, handled rats had higher tissue levels of dynorphin A and B in the hypothalamus, pituitary gland and striatum and slightly higher dynorphin B levels in the hippocampus, medulla oblongata and midbrain as compared with non-handled controls. The results indicate a persistent upregulation of the dynorphin system in certain brain areas after neonatal handling, which could contribute to the behavioural changes in these rats observed later in life. Observation in the open field and the elevated plus-maze tests confirmed behavioural effects of neonatal handling, i.e. showing that handled rats exhibit attenuated fearfulness in novel environments as compared with non-handled rats., (Copyright 1999 Harcourt Publishers Ltd.)

The conversion of a ubiquitous cellular protein (PrP(C)), an isoform of the prion protein (PrP), to the pathology-associated isoform PrP(Sc) is one of the hallmarks of transmissible spongiform encephalopathies such as bovine spongiform encephalopathy (BSE). Accumulation of PrP(Sc) has been used to diagnose BSE. Here we describe a quantitative enzyme-linked immunosorbent assay (ELISA) that involves antibodies against epitopes within the protease-resistant core of the PrP molecule to measure the amount of PrP in brain tissues from animals with BSE and normal controls. In native tissue preparations, little difference was found between the two groups. However, following treatment of the tissue with heat and guanidine thiocyanate (Gh treatment), the ELISA discriminated BSE-specific PrP(Sc) from PrP(C) in bovine brain homogenates. PrP(Sc) was identified by Western blot, centrifugation, and protease digestion experiments. It was thought that folding or complexing of PrP(Sc) is most probably reversed by the Gh treatment, making hidden antigenic sites accessible. The digestion experiments also showed that protease-resistant PrP in BSE is more difficult to detect than that in hamster scrapie. While the concentration of PrP(C) in cattle is similar to that in hamsters, PrP(Sc) sparse in comparison. The detection of PrP(Sc) by a simple physicochemical treatment without the need for protease digestion, as described in this study, could be applied to develop a diagnostic assay to screen large numbers of samples.

The distribution of parvalbumin (PV) immunoreactivity in the tench brain was examined by using the avidin-biotin-peroxidase immunocytochemical method. This protein was detected in neuronal populations throughout all main divisions of the tench brain. In the telencephalic hemispheres, PV-immunopositive neurons were distributed in both the dorsal and ventral areas, being more abundant in the area ventralis telencephali, nucleus ventralis. In the diencephalon, the scarce distribution of PV-containing cells followed a rostrocaudal gradient, and the most evident staining was observed in the nucleus periventricularis tuberculi posterioris and in a few nuclei of the area praetectalis. In the mesencephalon, abundant PV-immunoreactive elements were found in the tectum opticum, torus semicircularis, and tegmentum. In the tectum opticum, PV-immunoreactivity presented a laminar distribution. Three PV-containing neuronal populations were described in the torus semicircularis, whereas in the tegmentum, the PV staining was mainly located in the nucleus tegmentalis rostralis and in the nucleus nervi oculomotorii. In the metencephalon, Purkinje cells were PV-immunopositive in the valvula cerebelli, lobus caudalis cerebelli, and in the corpus cerebelli. In the myelencephalon, PV immunoreactivity was abundant in the nucleus lateralis valvulae, in the nucleus nervi trochlearis, nucleus nervi trigemini, nucleus nervi abducentis, nucleus nervi glossopharyngei, and in the formatio reticularis. Mauthner cells were also PV immunostained. By contrast to other vertebrate groups, only a restricted population of PV-containing neurons was GABA-immunoreactive in the tench, demonstrating that this calcium-binding protein cannot be considered a marker for GABAergic elements in the teleost brain. This study demonstrates a low phylogenetic conservation of the distribution of PV comparing teleosts and tetrapods., (Copyright 1999 Wiley-Liss, Inc.)

Preembedding immunoperoxidase staining methods were used to permit ultrastructural analyses of the distribution in rat brain and pituitary of the corticotropin-releasing factor-binding protein (CRF-BP), a moiety distinct from CRF receptors, but which is nonetheless capable of binding the peptide and reversibly neutralizing its biological actions. In anterior pituitary, CRF-BP immunoreactivity (ir) was detected in corticotropelike cells, with reaction product associated principally with secondary lysosomes and multivesicular bodies and not at all with secretory granules. In brain, marked regional differences in the subcellular pattern of CRF-BP staining were evident. In isocortex, where BP/peptide colocalization is rare, BP-ir was distributed in cells and processes in a manner similar to that of a prototypic neuropeptide, including in terminals commonly engaging in synaptic contacts with unlabeled dendritic profiles. In the bed nucleus of the stria terminalis, a site that contains overlapping accumulations of CRF-BP-ir projections and CRF-ir perikarya, BP staining was restricted to vesicle-laden varicosities that rarely engaged in synaptic contacts with somatic or dendritic elements but were frequently apposed to unlabeled axon varicosities and terminals. In the ventromedial medulla, a site of partial CRF/BP overlap, most cells displayed a subcellular localization CRF-BP-ir like that seen in cortex, whereas in others the distribution shared similarities with that observed in pituitary. The results suggest that the function of the CRF-BP may differ in different cellular contexts. In cellular targets of CRF or in neurons in which peptide and BP coexist, the CRF-BP may play a role in processing and degradation of CRF and/or ligand-receptor complexes. In other areas of the central nervous system, the BP seems positioned to serve as a transmitter/modulator at conventional synapses or as an autocrine or paracrine modulator of local CRF effects.

We examined whether modulation of cardiovascular responses by administering 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, an AMPA-receptor antagonist) into the rostral (RVLM) or caudal (CVLM) ventrolateral medulla are mediated via changes in extracellular levels of glutamate. Microdialysis probes were inserted bilaterally into the RVLM or the CVLM. For the RVLM experiments (n=8), muscle contraction for 2 min increased mean arterial pressure (MAP) and heart rate (HR) by 18+/-3 mmHg and 24+/-5 bpm, respectively. Extracellular glutamate concentrations increased from 1.5+/-0.3 to 4.3+/-0.9 ng/5 microl during the contraction. Microdialysis of CNQX (1.0 microM) for 30 min into the RVLM attenuated the increases in MAP, HR, and glutamate concentration in response to a muscle contraction (8+/-2 mmHg, 11+/-3 bpm, and 2.2+/-0.7 ng/5 microl, respectively). Developed tensions did not change during contractions before and after CNQX. Microdialysis of CNQX into the CVLM (n=8) potentiated the contraction-evoked responses in MAP (19+/-3 vs. 34+/-3 mmHg) and HR (25+/-4 vs. 49+/-5 bpm) without a change in developed tension. Following CNQX perfusion into the CVLM, the levels of extracellular glutamate in the CVLM were also augmented during the contraction. Results suggests that AMPA-receptors within the RVLM and CVLM differentially modulate cardiovascular responses during static muscle contraction via increasing and decreasing, respectively, extracellular glutamate concentrations.

Substance P (SP) is a member of the tachykinin family of bioactive peptides and has highest affinity for the NK-1 receptor. We have developed the non-peptide compound HSP-117 as a selective antagonist of the NK-1 receptor. Binding of 3H-SP to the membranes of IM-9 cells was inhibited by the antagonists HSP-117 and CP-99,994, the inhibitory activity of HSP-117 being 50-fold that of CP-99,994. The SP-induced firing responses of single neuron activity in slices of the nucleus tractus solitarius of ferrets were inhibited by 10 microM HSP-117. Intracerebroventricular injection of HSP-117 significantly inhibited retching and vomiting induced by copper sulphate and morphine and the inhibitory effect of HSP-117 on emesis was greater than that of CP-99,994. Moreover, emesis induced by copper sulphate and morphine were inhibited by the microinjection of HSP-117 and CP-99,994 into the area postrema and by lesion of the area postrema. These results indicate that HSP-117 is a potent anti-emetic agent, blocking NK-1 receptors in the area postrema and that NK-1 receptors in the area postrema play an important role in emesis induced by broad-spectrum emetic stimuli.

Opioid receptors, besides mediating the effects of analgesic compounds, are involved in drug addiction. Although a large amount of work has been done studying these receptors in mammals, little information has been obtained from nonmammalian vertebrates. We have studied the regional distribution in the central nervous system (CNS) of the zebrafish of the recently cloned delta-opioid receptor homologue ZFOR1 using nonradioactive in situ hybridization. Our findings show that different nuclei within the main subdivisions of the brain displayed specific mRNA signal. The expression is widespread throughout the brain, but only specific cells within each nucleus displayed ZFOR1. Stained cells were abundant in the telencephalon, both in the olfactory bulb and telencephalic hemispheres, and in the diencephalon, where expression was observed in all the different subdivisions. In the mesencephalon, expression of ZFOR1 was abundant in the periventricular layer of the optic tectum. In the cerebellum, expression of ZFOR1 was detected in valvula cerebelli, corpus cerebelli, and lobus vestibulolateralis in both granule and Purkinje cells. In the myelencephalon, cells expressing ZFOR1 were also distributed in the octavolateralis area, the reticular formation, and the raphe nuclei, among others. Also, ZFOR1 was detected in cells of the dorsal and ventral horn of the spinal cord. This work presents the first detailed distribution of a delta-opioid receptor in the CNS of zebrafish. Distribution of ZFOR1 expression is compared with that of the delta-opioid receptor described in mammals., (Copyright 1999 Wiley-Liss, Inc.)

The area postrema, which lacks a blood-brain barrier, was examined for the presence of prolactin receptors, which would render it a potential site for vascular prolactin to directly interact with neuronal elements. Using an in vitro autoradiographic technique, frozen sections of New Zealand white rabbit medulla were incubated with radiolabelled ovine prolactin alone (total binding) or radiolabelled ovine prolactin in the presence of excess unlabelled ovine prolactin (non-specific binding). The specificity of the binding was also assessed using excess unlabelled human prolactin or ovine LH. While excess unlabelled ovine and human prolactin caused a statistically significant reduction in radio labeled prolactin binding, unlabelled LH was without effect. Results reveal the presence of specific prolactin binding sites within the area postrema, a previously unknown prolactin target area of the CNS., (Copyright 1999 Elsevier Science B.V.)

Bilateral blockade of angiotensin (Ang) receptors in the rostral ventrolateral medulla (RVLM) causes a profound fall in arterial pressure. In this study, we tested whether this effect is due to an interaction between Ang receptors and either glutamatergic or gamma-aminobutyric acidergic (GABAergic) synaptic inputs to RVLM sympathoexcitatory neurons. In urethane-anaesthetised rats, bilateral microinjections of the Ang receptor antagonists [Sar1,Thr8]Ang II or [Sar1,Ile8]Ang II into the RVLM pressor region caused large decreases in arterial pressure, heart rate and renal sympathetic nerve activity (RSNA). These responses were not significantly altered following bilateral microinjections into the RVLM of the glutamate receptor antagonist kynurenic acid (4.5 nmol). Furthermore, bilateral injections of kynurenic acid plus the GABA(A) receptor antagonist bicuculline (200 pmol) into the RVLM increased the baseline arterial pressure and RSNA, but did not alter the percentage decreases in these variables evoked by bilateral microinjections of [Sar1,Ile8]Ang II. However, the level of arterial pressure and RSNA following bilateral injections of kynurenic acid, bicuculline and [Sar1,Ile8]Ang II were similar to the levels before injection of any of these compounds. The effectiveness of the microinjections of kynurenic acid and bicuculline into the RVLM was demonstrated by the observation that they virtually abolished the somato-sympathoexcitatory and baroreceptor-sympathoinhibitory reflexes, which are mediated by glutamatergic and GABAergic synapses, respectively, in the RVLM. These results indicate that (1) blockade of Ang receptors greatly reduces the firing rate of RVLM sympathoexcitatory neurons via a mechanism that is independent of glutamatergic or GABAergic neurotransmission, and (2) in the absence of inputs mediated by ionotropic glutamate, GABA(A) and Ang receptors, there are other mechanisms which generate a level of tonic activity in RVLM sympathoexcitatory neurons sufficient to maintain a normal level of sympathetic vasomotor activity.

We performed anatomical and physiological studies to determine the site and actions of sulfated cholecystokinin octapeptide (CCK8-S) on breathing. Peptide locations were determined by combined immunodetection of CCK8-S- containing synaptic varicosities and retrograde labeling of medullary neurons projecting to the ventral respiratory group. Retrogradely labeled neurons and CCK8-S immunolabeled varicosities overlapped within the nuclei of the solitary tract, ventral respiratory group, and the Kolliker-Fuse nucleus. Additional CCK8-S immunoreactive terminals were located in the rostroventrolateral medullary reticular nucleus, lateral paragigantocellular reticular nucleus, and the caudal pontine reticular nucleus. The respiratory effects of CCK8-S, which binds to CCK(A) and CCK(B) receptors, were examined by intravenous injection in adult rats and by bath application in the in vitro neonatal rat brainstem - spinal cord preparation. CCK8-S produced an increase in the mean amplitude of diaphragmatic electromyogram (EMG) of 28 +/- 35% (SD) and a decrease in mean respiratory interval of 13 +/- 4% in vivo. In vitro, CCK8-S significantly increased inspiratory duration and decreased respiratory interval, primarily by shortening expiratory duration. CCK8-unsulfated, a specific agonist for CCK(B) receptors, did not produce these effects. CCK8-S effects in the in vitro preparation were partially blocked by the CCK receptor antagonist lorglumide (final bath concentration 600 nM). These results suggest that CCK8-S modulates the respiratory rhythm via CCK(A) receptors within one or more medullary or pontine respiratory groups in both neonatal and adult rats.

Exposure to stress is known to induce widespread changes in the central nervous system (CNS) involving multiple neuropeptides. The neuropeptide galanin has been implicated in the central response to different stressors; however, the role of galanin in the response to restraint stress has not been reported. Therefore, this study utilised in situ hybridisation histochemistry to observe the effects of acute and chronic restraint stress on preprogalanin (preproGAL) mRNA expression in the CNS of normotensive (Wistar Kyoto; WKY) and Spontaneously Hypertensive (SHR) rats. Rats were exposed to 1 h of restraint for 0 (control), 1, 3, 5, or 10 consecutive days, and central preproGAL mRNA expression following these restraint periods was compared between strains. Significant differences in the basal expression of preproGAL mRNA were detected, with expression decreased by approximately 50% in the supraoptic nucleus (SON; P<0. 01) and increased by approximately 100% in the rostral ventrolateral medulla (RVLM; P<0.05) of SHR when compared to WKY. Following acute restraint (1 session), preproGAL mRNA expression was significantly increased by approximately 135% in the central nucleus of the amygdala (CeA; P<0.05) in WKY. In SHR, significant increases of up to 300% were observed in the CeA (P<0.01) and SON (P<0.05) following chronic restraint (up to 10 days). In addition, expression of preproGAL mRNA was significantly decreased in the locus coeruleus (LC) of SHR following acute restraint (1 session) (P<0.05). These results provide the first evidence that both acute (LC) and chronic (CeA, SON) restraint stress is associated with alterations in preproGAL mRNA expression. As such, the present study provides further evidence linking neurons containing galanin with the central response to restraint stress., (Copyright 1999 Elsevier Science B.V.)

Recent studies have revealed brainstem-spinal pathways involved in the generation of receptive behavior in hamster and cat, and the enormous influence of estrogen on these pathways. The present study gives an overview of the location of estrogen receptor-alpha-immunoreactive neurons (ER-alpha-IR) in the brainstem of the female hamster. In the mesencephalon, ER-alpha-IR cells were found in the arcuate and peripeduncular nuclei as well as throughout the rostrocaudal extent of the periaqueductal gray (PAG), and the laterally adjoining tegmentum. In the caudal brainstem, groups of ER-alpha-IR cells were present in the ventrolateral parabrachial nucleus, the solitary nucleus, and in contrast to the cat, in the nucleus retroambiguus. No ER-alpha-IR cells were found in any other part of the brainstem. The functional implications of these findings are discussed.

Reticulospinal sympathoexcitatory neurons of rostral ventrolateral medulla (RVL) are selectively excited by hypoxia to elevate arterial pressure (AP) and cerebral blood flow (rCBF), that are elements of the oxygen-conserving (diving) reflex. We investigated whether KATP+-channels participate in this. Tolbutamide and glibenclamide, KATP+-channel blockers, microinjected into RVL in anesthetized rats, dose-dependently and site-specifically elevated AP and rCBF and potentiated responses to hypoxemia. KATP+-channels may mediate hypoxic excitation of oxygen-sensing RVL neurons., (Copyright 1999 Elsevier Science B.V.)

We investigated the role of glutamatergic projection from the parabrachial nucleus (PBN) complex to the rostral ventrolateral medulla (RVLM) in the PBN-induced suppression of reflex bradycardia in adult Sprague-Dawley rats that were maintained under pentobarbital anesthesia. Under stimulus conditions that did not appreciably alter the baseline systemic arterial pressure and heart rate, electrical (10-s train of 0.5-ms pulses, at 10-20 microA and 10-20 Hz) or chemical (L-glutamate, 1 nmol) stimulation of the ventrolateral regions and Köelliker-Fuse (KF) subnucleus of the PBN complex significantly suppressed the reflex bradycardia in response to transient hypertension evoked by phenylephrine (5 micrograms/kg iv). The PBN-induced suppression of reflex bradycardia was appreciably reversed by bilateral microinjection into the RVLM of the N-methyl-D-aspartate (NMDA)-receptor antagonist MK-801 (500 pmol) or the non-NMDA-receptor antagonist 6-cyano-7-nitroquinoxaline-2, 3-dione (50 pmol). Anatomically, most of the retrogradely labeled neurons in the ventrolateral regions and KF subnucleus of the ipsilateral PBN complex after microinjection of fast blue into the RVLM were also immunoreactive to anti-glutamate antiserum. These results suggest that a direct glutamatergic projection to the RVLM from topographically distinct regions of the PBN complex may participate in the suppression of reflex bradycardia via activation of both NMDA and non-NMDA receptors at the RVLM.

The neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) is expressed in various parts of the developing and adult rat brain, including the cerebellum. In situ hybridization was employed to localize the precise site of mRNA expression for PACAP and PACAP receptor I (PRI). During prenatal cerebellar development, PACAP mRNA was present in developing Purkinje cells and some deep cerebellar nuclei, whilst PRI mRNA was expressed by adjacent cells in the Purkinje cell layer (PCL). There was a shift in PRI mRNA expression to the external germinal cell layer around birth. PACAP decreased the number of neurons positive for the inhibitory neurotransmitter gamma-amino butyric acid (GABA) in cultures from embryonic cerebellum, but did not affect overall cell survival. In conclusion, our results show the pattern of PACAP mRNA expression in embryonic cerebellum and suggest a physiological role for PACAP on GABAergic cerebellar neurons.

Median preoptic nucleus (POMe) neurons are innervated by catecholaminergic and neuropeptide Y (NPY)-immunoreactive nerve terminals originating from the catecholamine area of the ventrolateral medulla (VLM). The possibility that such POMe neurons project to the VLM catecholamine area was investigated in the rat. Immunoelectron microscopy revealed synaptic contacts of tyrosine hydroxylase- and NPY-immunoreactive axon terminals onto POMe neurons retrogradely labeled from the VLM catecholamine area, suggesting the existence of bidirectional connections between these two regions., (Copyright 1999 Elsevier Science B.V.)

Autopsy samples from 17 Greenlanders and 12 Danes were analysed for total and organic mercury by atomic absorption spectrophotometry, and for cellular localisation by autometallography. The concentration of total mercury in the Greenlanders (median: 174 micrograms Hg/kg wet weight brain tissue, range 59-4782, highest in cerebellum: 492) was found to be significantly higher than in the Danish group (3.7 micrograms Hg/kg w.w., range 1.2-11.8). Furthermore, the total concentration of mercury was positively correlated to age (rho = 0.56, p < 0.05), and the fraction of methyl mercury was negatively correlated to age (rho = -0.66, p < 0.01) among the Greenlanders. This suggests an age dependent accumulation of total mercury and a slow transformation of methyl mercury to inorganic mercury in the brain. The autometallographically demonstrable mercury was primarily located in glia cells.

Activation of neurons in the ventromedial medulla by electrical stimulation or by microinjection of opioid or cholinergic agonists produces antinociception that is mediated in part by spinally-projecting noradrenergic neurons. Several lines of evidence indicate that these noradrenergic neurons are located in the pontine A7 catecholamine cell group. For example, anatomical studies have demonstrated that neurons in the ventromedial medulla project to the noradrenergic neurons in the A7 catecholamine cell group that provide the major noradrenergic innervation of the spinal cord dorsal horn. In addition, electrical and chemical stimulation of A7 neurons produces antinociception that can be reduced by intrathecal injection of alpha2-adrenoceptor antagonists. The present studies provide more direct evidence that activation of neurons in the ventromedial medulla produces antinociception by activating noradrenergic neurons in the A7 cell group. Neurons in the ventromedial medulla were stimulated by microinjecting the cholinergic agonist carbachol (5 microg) into sites in the nucleus raphe magnus or the nucleus gigantocellularis pars alpha of pentobarbital anesthetized Sprague-Dawley rats. In some experiments, the local anesthetic tetracaine (10 microg) was then microinjected near the A7 cell group to inactivate the spinally-projecting noradrenergic neurons. In other experiments, cobalt chloride (100 mM) was microinjected near the A7 cell group to block synaptic activation of spinally-projecting noradrenergic neurons. Microinjection of carbachol into sites in the ventromedial medulla produced antinociception, assessed using the tail flick test, that lasted more than 60 min. However, the effects of carbachol were attenuated by microinjection of either tetracaine or cobalt into sites near the A7 cell group neurons identified by tyrosine hydroxylase-immunoreactivity. Similar injections of tetracaine or cobalt more than 500 microm from the A7 neurons did not alter the antinociceptive effect of carbachol. These results support the conclusion that the antinociception produced by activating neurons in the ventromedial medulla is mediated in part by the subsequent activation of spinally-projecting noradrenergic neurons in the A7 cell group., (Copyright 1999 Elsevier Science B.V.)

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.

The influence of the dorsal reticular nucleus (DRt) on pain behaviour during the formalin test was studied in the rat by lesioning the nucleus through local application of electrical current or quinolinic acid. Animals in which the DRt was lesioned ipsilaterally to the paw injected with formalin spent less time in focused (licking, biting or scratching the injected paw) and total (focused pain behaviour plus protection of the injected paw during movements) pain behaviour, and showed paw-jerks less frequently than non-lesioned animals in both phases 1 and 2 of the test. Animals in which the DRt was lesioned contralaterally to the injected paw presented a decrease in total pain behaviour and number of paw-jerks only during phase 2. The number of superficial (laminae I-II) and deep (laminae III-VI) spinal dorsal horn cells expressing the c-fos proto-oncogene 2 h after subcutaneous injection of formalin was reduced by 34% and 50%, respectively, in animals with an ipsilateral DRt lesion as compared to non-lesioned rats. No differences in c-fos expression were observed after lesioning the DRt contralateral to the formalin injection. The results indicate that the DRt is involved in the facilitation of nociception during the formalin test by enhancing the response capacity of dorsal horn neurons to noxious stimulation. It is suggested that the pronociceptive action of the DRt is mediated by the reciprocal connections it establishes with the spinal dorsal horn.

Immunoreactivity for the substance P receptor (NK1 receptor) has been investigated by light and electron microscopy in the dorsal vagal complexes of adult rats and cats. The general pattern of NK1 immunoreactivity was similar for both rat and cat. Numerous NK1-immunoreactive neurons were present in the area postrema, the nucleus of the solitary tract, and the dorsal motor nucleus of the vagus nerve. The density of labelled neurons differed between the subnuclei of the nucleus of the solitary tract. Overall, the efferent neurons of the dorsal motor nucleus of the vagus nerve highly expressed NK1 when compared to neurons in the nucleus of the solitary tract. The results are discussed with reference to the viscerotopic organisation of the dorsal vagal complex. Ultrastructural analysis demonstrated that NK1 immunoreactivity was present only at the membrane surface of somatic and dendritic profiles of neurons. No labelling was found in axon terminals, axons, or glial processes. NK1 immunoreactivity, as revealed by a preembedding immunogold technique in serial ultrathin sections, was preferentially located at nonsynaptic sites. A semiquantitative study suggested that the density of NK1 receptors is statistically higher at membrane sites free of any contact (synaptic or not) with axon terminals. The subcellular localisation of NK1 immunoreactivity was similar for neurons of both rat and cat. These results suggest that in the dorsal vagal complex, substance P might act on NK1 receptors through a process of volume transmission.

The caudal ventrolateral medulla (CVLM) participates in the central control of airway caliber. For example, both electrical and chemical stimulation of the CVLM decrease total lung resistance by withdrawing cholinergic input to airway smooth muscle. Although cell bodies in the CVLM have been shown to play an important role in mediating the central control of airway caliber, the pharmacological mechanism in this brainstem region responsible for causing this airway dilation is unknown. We, therefore, examined the role played by ionotropic excitatory amino acid receptors in the CVLM in the control of airway caliber in chloralose-anesthetized dogs. We found that microinjection of 3.9 pmol of NMDA or AMPA or quisqualate into 12 sites in the CVLM decreased total lung resistance by 1.5 +/- 0.2 cm H2O l(-1) s(-1) (p < 0.05), and that microinjection of 3.9 pmol of kainic acid into 9 in the CVLM decreased total lung resistance by 0.5 +/- 0.1 cm H2O l(-1) s(-1) (p < 0.05). The decrease in total lung resistance evoked by either NMDA or AMPA or quisqualate was not different (p > 0.05) while that evoked by kainic acid was significantly smaller. Additionally, microinjection of NMDA or AMPA or quisqualate caused a small but significant decrease in mean arterial pressure and heart rate (p < 0.05). These experiments demonstrate that the airway dilation evoked by stimulation of excitatory amino acid receptors in the CVLM is mediated by both NMDA and non-NMDA receptors.

Gramicidin-perforated patch-clamp recording revealed phasic Cl(-)-mediated hyperpolarizations in respiratory neurons of the brainstem-spinal cord preparation from newborn rats. The in vitro respiratory rhythm persisted after block of gamma-aminobutyric acid (GABA), i.e. GABAA, receptor-mediated inhibitory postsynaptic potentials (IPSPs) with bicuculline and/or glycinergic IPSPs with strychnine. In one class of expiratory neurons, bicuculline unmasked inspiration-related excitatory postsynaptic potentials (EPSPs), leading to spike discharge. Bicuculline also blocked hyperpolarizations and respiratory arrest due to bath-applied muscimol, whereas strychnine antagonized similar responses to glycine. The reversal potential of respiration-related IPSPs and responses to GABA, muscimol or glycine was not affected by CO2/HCO3(-)-free solutions, but shifted from about -65 mV to values more positive than -20 mV upon dialysis of the cells with 144 instead of 4 mM Cl-. Impairment of GABA uptake with nipecotic acid or glycine uptake with sarcosine evoked a bicuculline- or strychnine-sensitive decrease of respiratory frequency which could lead to respiratory arrest. Also, the GABAB receptor agonist baclofen led to reversible suppression of respiratory rhythm. This in vitro apnoea was accompanied by a K+ channel-mediated hyperpolarization (reversal potential -88 mV) of tonic cells, whereas membrane potential of neighbouring respiratory neurons remained almost unaffected. Both baclofen-induced hyperpolarization and respiratory depression were antagonised by 2-OH-saclofen, which did not affect respiration-related IPSPs per se. The results show that synaptic inhibition is not essential for rhythmogenesis in the isolated neonatal respiratory network, although (endogenous) GABA and glycine have a strong modulatory action. Hyperpolarizing IPSPs mediated by GABAA and glycine receptors provide a characteristic pattern of membrane potential oscillations in respiratory neurons, whereas GABAB receptors rather appear to be a feature of non-respiratory neurons, possibly providing excitatory drive to the network.