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The adrenergic phenotype was analysed in the rat's rostral dorsomedial medulla under normal conditions and 3 days after a single intraperitoneal injection of an eburnamine derivative, RU 24722, which increases tyrosine hydroxylase protein expression in the rostral portion of the nucleus tractus solitarius. This approach was investigated by a double immunofluorescence labelling of tyrosine hydroxylase and phenylethanolamine N-methyltransferase proteins. Under normal conditions, most adrenergic cell bodies are anatomically distributed in the dorsal and rostral medulla oblongata between the rostral part of the dorsal motor nucleus of the vagus nerve and the medial longitudinal fasciculus. Adrenergic neurons detected in this medullar region were distributed between both cell groups. Three days after the pharmacological RU 24722 treatment, an upregulation in tyrosine hydroxylase and phenylethanolamine N-methyltransferase protein expression was detected in both cell groups characterized by a highly increased number of tyrosine hydroxylase- and phenylethanolamine N-methyltransferase-containing cell bodies. The number of TH-mRNA containing neurons was also increased, indicating the transcriptional level of this regulation. These results demonstrated a particular neuronal plasticity of adrenergic phenotype in the medullary cell groups of adult rat.

We have performed a mapping of basic fibroblast growth factor (bFGF) immunoreactive (ir) glial and nerve cell populations in the male rat brain using a rabbit antibody raised against a synthetic peptide of bovine bFGF. Regional morphometric and microdensitometric analysis of the bFGF ir neuronal profiles in coronal brain sections was carried out by means of an automatic image analyser. The density and intensity of the bFGF ir glial profiles were subjectively evaluated. The bFGF immunoreactivity (IR) was detected within the cytoplasm of neurons, except within the pyramidal neurons of hippocampal CA2 region, the fasciola cinerea and the indusium griseum, where bFGF IR was present in the nucleus. In contrast, in glial cells bFGF IR was always found in the nucleus. Neuronal and glial IR was no longer observed after absorption of the bFGF antiserum with recombinant bFGF. Basic FGF IR was found in neuronal and glial cell populations throughout the brain as well as in the choroid plexus and in the ependymal cells lining the ventricles. Basic FGF ir nerve cells were found in all layers of both the neocortex and allocortex. Within the caudate putamen and the nucleus accumbens a low density of weak bFGF ir neuronal profiles was detected. The majority of the thalamic nuclei showed medium to high densities of moderate to strong bFGF ir neuronal profiles. All the hypothalamic nuclei, with the exception of the anterior and lateral hypothalamic area and of the ventral hypothalamic nucleus, contained a high density of bFGF ir profiles. The pons and the medulla oblongata were characterized by the presence of a large number of nuclei containing moderate to high densities of strong bFGF ir profiles. The Purkinje cell layer of the cerebellar cortex contained a high density of moderately bFGF ir profiles. A moderate density of strong bFGF ir nerve cell profiles was observed within all the laminae of the spinal cord, except within the II and III laminae where a high density of strongly ir profiles was found. Histogram analysis of total immunoreactivity showed that the distribution of bFGF ir profiles within the telencephalon and mesencephalon tend to be similar with regard to the central tendency and spread. Using Kendall's tau, a significant correlation between intensity and density values was obtained only in the diencephalon. The cytoplasmic bFGF IR found in distinct nerve cell populations all over the rat brain and spinal cord may represent forms of bFGF which can be released from the nerve cells via non-exocytotic mechanisms in view of the absence of an intracellular signal peptide in bFGF. The presence of nuclear bFGF IR within the glial cells all over the central nervous system (CNS) suggests an intracellular function of bFGF, such as the promotion of mitogenesis and/or participation in the transcriptional regulation of various genes.

Of the three major types of opioid receptors ( mu, delta, kappa) in the nervous system, mu-opioid receptor shows the highest affinity for morphine that exerts powerful effects on nociceptive, autonomic, and psychological functions. So far, at least two isoforms of mu-opioid receptors have been cloned from rat brain. The present study attempted to examine immunohistochemically the distribution of mu-opioid receptors in the rat central nervous system with two kinds of antibodies to recently cloned mu-opioid receptors (MOR1 and MOR1B). One antibody recognized a specific site for MOR1, and the other bound to a common site for MOR1 and MOR1B. Intense MOR1-like immunoreactivity (LI) was seen in the 'patch' areas and subcallosal streak in the striatum, medial habenular nucleus, medial terminal nucleus of the accessory optic tract, interpeduncular nucleus, median raphe nucleus, parabrachial nuclei, locus coeruleus, ambiguous nucleus, nucleus of the solitary tract, and laminae I and II of the medullary and spinal dorsal horns. Many other regions, including the cerebral cortex, amygdala, thalamus, and hypothalamus, also contained many neuronal elements with MOR1-LI. The distribution pattern of the immunoreactivity revealed with the antibody to the common site for MOR1 and MOR1B (MOR1/1B-LI) was almost the same as that of MOR1-LI. Both MOR1-LI and MOR1/1B-LI were primarily located in neuronal cell bodies and dendrites. However, the immunoreactivities were observed in the accessory optic tract, fasciculus retroflexus, solitary tract, and primary afferent fibers in the superficial layers of the medullary and spinal dorsal horns. The presynaptic location of MOR1-LI and MOR1/1B-LI was confirmed by lesion experiments: Enucleation, placing a lesion in the medial habenular nucleus, removal of the nodose ganglion, or dorsal rhizotomy resulted in a clear reduction of the immunoreactivities, respectively, in the nuclei of the accessory optic tract, some subnuclei of the interpeduncular nucleus, nucleus of the solitary tract, or laminae I and II of the spinal dorsal horn. The results indicate that the mu-opioid receptors are widely distributed in the brain and spinal cord, mainly postsynaptically and occasionally presynaptically. Opioids, including morphine, may inhibit the excitation of neurons via the postsynaptic mu-opioid receptors, and also suppress the release of neurotransmitters and/or neuromodulators from axon terminals through the presynaptic mu-opioid receptors.

The present study described an ultrastructural synaptic configurations between primary afferent terminals (PATs), cuneothalamic relay neurons (CTNs) and GABA-immunoreactive (GABA-IR) boutons in the cuneate nucleus of rats using cervicothoracic dorsal rhizotomies, retrograde transport of wheat germ agglutinin conjugated with horseradish peroxidase complex (WGA-HRP) and anti-GABA immunogold labelling methods. With this procedure, direct synaptic relationships between the PATs, CTNs and GABA-IR boutons have been demonstrated. The most remarkable feature was the observation whereby an immunogold-labelled GABA-IR bouton was presynaptic to a WGA-HRP labelled dendrite of CTN and a degenerating PAT; the same PAT was in turn presynaptic to the HRP-labelled dendrite. This was evident in ten out of a total of 133 synaptic configurations that were closely scrutinized. Results of this study support the concept that GABA-IR boutons are not only involved in presynaptic inhibition on the primary afferent input to the cuneothalamic relay neurons, but also exert a simultaneous postsynaptic inhibition on these cells.

The actin-based microfilament system is thought to play a critical role in neuronal development. We have determined specific changes in the composition of microfilaments accompanying neuronal morphogenesis. By using specific antibodies against the isoforms for tropomyosin (Tm) (Tm-5 and TmBr-1/-3) and actin (beta- and gamma-actin), we found that during early morphogenesis in vivo immature growing axons contain beta- and gamma-actin and Tm-5. In particular, Tm-5 is exclusively located in the immature axonal processes relative to the neuronal cell body. In contrast, beta-actin and Tm-5 are absent in mature, quiescent axons. This developmental loss from axons is associated with an approximately twofold downregulation of beta-actin and Tm-5 levels in the brain; gamma-actin levels do not change, and this molecule is widely distributed throughout neurons during development. The loss of beta-actin and Tm-5 from axons is accompanied by a progressive appearance of TmBr-1/-3. This apparent replacement of Tm-5 with TmBr-1/-3 occurs over a 2 d time period during rat embryonic hindbrain development and is conserved in evolution between birds and mammals. The loss of Tm-5 from axons involves a redistribution of this molecule to the cell soma and dendrites. These findings suggest that specialized microfilament domains are associated with the development and maintenance of neuronal polarity. We conclude that these Tm isoforms and beta-actin are subject to specific patterns of segregation associated with axonal development and neuronal differentiation. This provides a potential molecular basis for the temporal and spatial specificity of microfilament function during neuronal differentiation.

Acid sulfated glycosaminoglycans (sGAG) and chondroitin proteoglycans have been biochemically and immunohistochemically demonstrated in certain cortical areas of the CNS. Such molecules display an important role in cerebral function, modulating cell adhesion, migration and signal neuromediation. In the present study we have evidenced the presence of sGAG using the colloidal iron method and C0S, C4S- and C6S-proteoglycans by the immunohistochemical pre-embedding PAP method. Our results have demonstrated the presence of such molecules in several structures of nervous (axon terminals, neuronal bodies, dendrites) and non-nervous nature (glial processes, capillaries). This fact led us to suggest that the chondroitin localizations advocate for their different functions. In their various localization exist distinct proteoglycans constituted by chondroitins with diverse concentrations and other attached radicals.

The present study describes the distribution of alpha 2-adrenoceptors in the parabrachial and Kölliker-Fuse nucleus of the rat by employing the tritium-labeled alpha 2-receptor antagonist rauwolscine ([3H]-RAUW) as a ligand. The [3H]-RAUW binding was densitometrically quantified in five nuclei of the parabrachial (PB) complex in serial coronal sections. We found that cytoarchitectonically and anatomically distinct nuclei of the PB complex exhibit different numbers of [3H]-RAUW-binding sites. The largest number of binding sites was observed over the external lateral PB and caudally over the waist area of the PB. Lower numbers of binding sites were found in the remaining lateral PB nuclei, followed by the medial PB and the Kölliker-Fuse nucleus. In addition we disclosed that the internal lateral PB contains a very low number of binding sites while the external medial PB is marked by dense [3H]-RAUW binding. Also, the affinities of the binding sites differed between the PB areas. High affinities were observed in the external lateral PB, the remaining lateral PB nuclei and in the waist area of the PB, while the medial PB and the Kölliker-Fuse nucleus exhibited only low affinities for the ligand. Furthermore, saturation curves demonstrated non-linear profiles, indicating the presence of more than one population of binding sites in the PB nuclei for the radioligand. Our data demonstrate that the PB exhibits a distinct distribution of alpha 2-adrenergic binding sites. These correlate well with the cytoarchitectonically defined nuclei of the PB complex and with the pattern of ascending axons from the medial nucleus of the solitary tract and the area postrema terminating in the PB. Since a large number of these projection neurons utilize adrenaline or noradrenaline as their transmitters, we conclude that solitary-parabrachial neurotransmission to the forebrain is, at least in part, mediated via alpha 2-adrenoceptors.

In the present study, we assessed the extent of the thyrotropin-releasing hormone (TRH) input to motoneurons in the ambigual, facial, and hypoglossal nuclei of the rat using a combination of intracellular recording, dye filling, and immunohistochemistry. Twelve motoneurons in the rostral nucleus ambiguus were labelled by intracellular injection in vivo of Neurobiotin (Vector). Seven out of 12 ambigual motoneurons displayed rhythmic fluctuations of their membrane potential in phase with phrenic nerve discharge, whereas the other five had no modulations of any kind. Seven facial motoneurons and seven hypoglossal motoneurons were also filled with Neurobiotin. All three motor nuclei contained TRH-immunoreactive varicosities, with the largest numbers found in the nucleus ambiguus. Close appositions were seen between TRH-immunoreactive boutons and every labelled motoneuron. Respiratory-related motoneurons in the nucleus ambiguus received the largest number of TRH appositions with 74 +/- 38 appositions/neuron (mean +/- S.D.; n = 7). In contrast, nonrespiratory ambigual motoneurons received significantly fewer TRH appositions (11 +/- 5; n = 5; P < 0.05; Mann-Whitney U test). Facial motoneurons received about the same number of TRH appositions as nonrespiratory ambigual motoneurons, with 13 +/- 4 (n = 7). Hypoglossal motoneurons received the fewest appositions from TRH-containing boutons, with 8 +/- 2 (n = 7). There were no differences in the TRH inputs to respiratory and nonrespiratory motoneurons in the facial and hypoglossal nuclei. These results demonstrate that, among motoneurons in the medulla, respiratory motoneurons in the rostral nucleus ambiguus are preferentially innervated by the TRH-immunoreactive boutons.

The present study addresses the question whether metabolic dopamine can be immunocytochemically detected within non-dopaminergic catecholaminergic axonal fibers. For this purpose, confocal microscopy was used to analyze sections treated for the double fluorescence immunostaining of dopamine and either noradrenaline or phenylethanolamine-N- methyltransferase (the enzyme in adrenergic neurons that converts noradrenaline into adrenaline). Our data demonstrate that throughout the brain and spinal cord, the majority of the axonal fibers that reacted with the anti-phenylethanolamine-N-methyltransferase antibodies also exhibited faint to intense dopamine immunoreactivity. Similarly noradrenaline and dopamine immunoreactivities were frequently colocalized within axonal fibers innervating brain and spinal cord regions that receive a dense innervation from medullary noradrenergic neurons. On the contrary, dopamine was rarely detected within noradrenaline-immunoreactive fibers in those regions where the noradrenergic innervation essentially arises from noradrenergic neurons of the locus coeruleus. A similar differential dopamine immunostaining was observed in the corresponding neuronal perikarya of the medulla oblongata and the locus coeruleus. These data indicate that two types of non-dopaminergic catecholaminergic neurons can be distinguished according to their content in dopamine: (i) the noradrenergic and adrenergic neurons located in the medulla oblongata, whose cell bodies and axons contain high concentrations of metabolic dopamine and (ii) the noradrenergic neurons located in the mesencephalon, which contain low levels of metabolic dopamine.

Several lines of evidence suggest that pigment-dispersing hormone-immunoreactive neurons with ramifications in the accessory medulla are involved in the circadian system of insects. The present study provides a detailed analysis of the anatomical and neurochemical organization of the accessory medulla in the brain of the cockroach Leucophaea maderae. We show that the accessory medulla is compartmentalized into central dense nodular neuropil surrounded by a shell of coarse fibers. It is innervated by neurons immunoreactive to antisera against serotonin and the neuropeptides allatostatin 7, allatotropin, corazonin, gastrin/cholecystokinin, FMRFamide, leucokinin I, and pigment-dispersing hormone. Some of the immunostained neurons appear to be local neurons of the accessory medulla, whereas others connect this neuropil to various brain areas, including the lamina, the contralateral optic lobe, the posterior optic tubercles, and the superior protocerebrum. Double-label experiments show the colocalization of immunoreactivity against pigment-dispersing hormone with compounds related to FMRFamide, serotonin, and leucokinin I. The neuronal and neurochemical organization of the accessory medulla is consistent with the current hypothesis for a role of this brain area as a circadian pacemaking center in the insect brain.

The purposes of the present study were to: (1) characterize the GABAergic input to vasodepressor neurons in the caudal ventrolateral medulla of the cat, and (2) define more precisely the anatomical localization of these neurons in this species. This was done by microinjecting GABA receptor antagonists and agonists, and a negative allosteric modulator of the GABA receptor, namely, ethyl-beta-carboline-3-carboxylate, into the caudal ventrolateral medulla of alpha-chloralose-anesthetized animals while monitoring arterial blood pressure and heart rate. Localization studies where performed relating injection sites in the caudal ventrolateral medulla where cardiovascular responses were elicited, to neurons exhibiting immunoreactivity to tyrosine hydroxylase (TH) and phenethyl-N-methyl-transferase (PNMT). Microinjection of 1 and 10 ng of bicuculline into the caudal ventrolateral medulla produced decreases in mean blood pressure and heart rate of -34 +/- 6.4 and -49 +/- 9.2 mmHg, and -22 +/- 4.3 and -35 +/- 8.2 beats/min, respectively. Hypotension and bradycardia were also observed with picrotoxin microinjection (120 ng). Microinjection of muscimol (100-200 ng) and GABA (12 microgram) had no effect on mean blood pressure and heart rate. Microinjection of ethyl-beta-carboline-3-carboxylate also decreased mean blood pressure (-39 +/- 7.0 mmHg). The location of the micropipette tip after bicuculline microinjection in relation to TH and PNMT immunoreactive cells was as follows: (1) TH-immunoreactive cells of the A1 cell group were visible in the same relative location as the micropipette tip, and (2) no PNMT-positive cells were noted at the sites where bicuculline elicited hypotension. These results indicate that there is a tonic GABAergic input to neurons in the caudal ventrolateral medulla. The location of these neurons overlaps with the A1 cells.

The transient expression of estrogen receptor (ER) in the ventromedial subnucleus of the facial nucleus was previously detected in the newborn rat, and the expression of ER molecules was down-regulated by daily injections of estradiol. Here we examined possible involvement of aromatization in this process. ER molecules were measured by immunohistochemistry and in situ hybridization histochemistry after daily injections of testosterone propionate (TP; 100 micrograms/0.02 ml) and estradiol benzoate (EB; 10 micrograms/0.02 ml) in the male pups castrated within 24 h of birth. Daily injections of TP for 5 consecutive days did not suppress ER and ER mRNA in the facial nucleus, while they were both suppressed by daily injections of EB. Moreover, aromatase immunoreactivity was not detected in the facial nucleus of both castrated, TP injected and intact control males at 6 days of age. The present findings therefore suggest that ER molecules expressed transiently in the facial nucleus are not directly involved in masculine sexual differentiation of the brain in newborn rat.

The distribution of acidic fibroblast growth factor (aFGF) mRNA-expressing neurons was studied throughout the adult mouse central nervous system (CNS) with in situ hybridization histochemistry using a radiolabelled synthetic oligodeoxynucleotide probe complementary to the mRNA of human aFGF. We report here a widespread distribution of aFGF mRNA in several defined functional systems of the adult mouse brain, whereby the highest levels of aFGF mRNA were found in large somatomotor neurons in the nuclei of the oculomotor, trochlear, abducens, and hypoglossal nerves; in the motoneurons of the ventral spinal cord and the special visceromotor neurons in the motor nucleus of the trigeminal nerve; and in the facial and ambiguus nuclei. Labelled perikarya were also detected in all central structures of the auditory pathway including the level of the inferior colliculus, i.e., the lateral and medial superior nuclei; the trapezoid, cochlear, and lateral lemniscal nuclei; and parts of the anterior colliculus. Furthermore, many aFGF-positive cell bodies were found in the vestibular system and other structures projecting to the cerebellum, in the deep cerebellar nuclei, in somatosensory structures of the medulla (i.e., in the gracile, cuneate, and external cuneate nuclei), as well as in the spinal nucleus of the trigeminal nerve. The findings that aFGF mRNA is expressed in all components of several well-defined systems (i.e., in sensory structures) as well as in central neurons that process sensory information and, finally, in some efferent projections point towards a concept of aFGF expression primarily within certain neuronal circuitries.

Distribution of messenger RNA (mRNA) encoding the insulin-like growth factor (IGF) type 2 receptor (IGF2R) is investigated in the rat lower brainstem by in situ hybridization histochemistry. Cells with IGF2R mRNA are distributed widely in a region-specific manner. It is expressed in: (1) motor nuclei such as the oculomotor nucleus, trochlear nucleus, motor trigeminal nucleus, abducens nucleus, facial nucleus, ambiguus nucleus, dorsal motor nucleus of vagus and hypoglossal nucleus; (2) several sensory-related nuclei like the mesencephalic trigeminal nucleus, ventral nucleus of the lateral lemniscus, lateral and spinal vestibular nuclei, ventral and dorsal cochlear nuclei and nucleus of the trapezoid body; and (3) other regions such as the red nucleus, dorsal raphe nucleus, pontine nuclei, three cerebellar nuclei (medial, interposed and lateral), Purkinje cells, cells in the granular layer of the cerebellum, locus coeruleus, several areas of the reticular nucleus and area postrema.

Central expression of the protooncogene c-fos was used to examine areas receiving noxious sensory input from the rat temporomandibular joint (TMJ). Fos-like immunoreactivity (Fos-LI) in the caudal brainstem was visualized 2 hours after unilateral injection of the small-fiber-specific excitant/inflammatory irritant mustard oil into the TMJ region. Control animals received injection of either mustard oil into the subcutaneous fascia overlying the masseter muscle or mineral oil vehicle into the TMJ region. In all groups, Fos-LI was consistently observed ipsilaterally in the spinal trigeminal nucleus and cervical dorsal horn and, bilaterally, in the nucleus of the solitary tract and the ventrolateral medulla. The expression of Fos-LI ipsilaterally in the paratrigeminal nucleus was variable. Within the trigeminal sensory complex, Fos-LI was restricted to subnucleus caudalis and the caudal portions of subnucleus interpolaris near the level of the obex. Approximately 12% of Fos-LI cells in subnucleus caudalis and in the cervical dorsal horn were found in laminae III-VI. Compared to TMJ mustard oil injection, mineral oil injection produced less Fos-LI at all rostrocaudal levels, whereas subcutaneous mustard oil injection produced less Fos-LI in caudal subnucleus caudalis but similar amounts in the cervical dorsal horn. Neither of these injections yielded significant ipsilateral responses in subnucleus caudalis, indicating that Fos-LI in this region following TMJ mustard oil injection could be ascribed solely to small-fiber stimulation in the deep TMJ region. The wide rostrocaudal distribution of Fos-LI within the caudal brainstem reflects the distribution of TMJ-responsive nociceptive neurons that may underlie the spread and referral of pain from the TMJ region.

A converted form of the normal cellular prion protein (PrP) accumulates in the brains of sheep with scrapie. We describe an immunohistochemical method for identifying scrapie-associated PrP (PrPSc) in periodate-lysine-paraformaldehyde-fixed brain tissue, which provides adequate preservation of tissue morphology. After pretreatment of tissue sections with formic acid and hydrated autoclaving, we located PrPSc in the brains of 50 sheep with natural scrapie by use of antipeptide antisera raised against ovine PrP. No PrP was seen in 20 sheep without histopathologic signs of scrapie. PrPSc that did not stain for amyloid was present in the cytoplasm and at the cell membrane of both neurons and astrocytes. Large amounts of PrPSc were seen at the cell membrane of neurons in the medulla oblongata and pons, whereas PrPSc accumulated at the cell membrane of astrocytes of the glial limitans in all brain regions. PrPSc that stained for amyloid was located in the walls of blood vessels and perivascularly in the brains of 32 (64%) of 50 sheep, mainly in the thalamus and never in the pons or medulla oblongata. No apparent topographic relationship existed between PrPSc that stained for amyloid and PrPSc accumulation associated with neurons or astrocytes. In all scrapie-affected sheep, PrPSc was present in brain regions with vacuolation, but it could also be detected in regions with minimal or no vacuolation. We conclude that the immunohistochemical detection of PrP can be an important confirmative test in scrapie diagnosis.

Melanin-concentrating hormone (MCH) is a neuropeptide originally isolated from chum salmon pituitaries. To explore physiological roles of MCH in mammals, we studied the regional distribution of immunoreactive MCH in the rat tissues and the presence of immunoreactive MCH in human adrenal glands, adrenal tumors and plasma by radioimmunoassay, and the expression of MCH mRNA in rat tissues and human brain tissues by Northern blot analysis. Immunoreactive MCH was present in every region of rat brain and neurointermediate lobe of the pituitary gland, with the highest concentrations found in the hypothalamus (48.3 +/- 6.6 pmol/g wet weight, mean +/- SEM, n = 6). The immunoreactive MCH in rat hypothalamus, frontal lobe, and pons and medulla oblongata was eluted in the position of synthetic human/rat MCH in reverse-phase high-performance liquid chromatography. No immunoreactive MCH was detected in the rat peripheral tissues. Northern blot analysis showed that a single species of MCH mRNA (approximately 1 kb) was expressed specifically in the rat and human hypothalamus, but not detectable in other regions of brain or rat peripheral tissues. Immunoreactive MCH was not detected in human adrenal glands (< 0.5 pmol/g wet weight, n = 9) or adrenal tumors including pheochromocytomas. Immunoreactive MCH were not detected in plasma obtained from human healthy subjects (< 0.25 pmol/l) and rat (< 0.25 pmol/l).(ABSTRACT TRUNCATED AT 250 WORDS)

The objectives of the present study were 1) to utilize Fos immunohistochemistry as a marker for neuronal activity in order to examine the population of neurons in the medulla that is engaged by activation of nociceptive peripheral afferents and 2) to determine whether catecholamine-containing neurons in the medulla also express noxious heat-evoked Fos-like immunoreactivity. Noxious heating of the hindpaw evoked specific patterns of Fos-like immunoreactivity in the medulla in regions known to be involved in both nociceptive processing and cardiovascular regulation. Noxious heating of the hindpaw significantly increased the mean number of neurons expressing Fos-like immunoreactivity in the contralateral ventrolateral medulla. Increased numbers of Fos-positive neurons also were observed in both the ipsilateral and the contralateral A1 catecholamine cell groups. Similarly, in the contralateral medullary dorsal reticular fields, noxious heating of the hindpaw significantly increased the mean number of neurons expressing Fos-like immunoreactivity. In contrast, in the paramedian reticular nucleus, noxious heating of the hindpaw resulted in a significant decrease in the mean number of neurons expressing Fos-like immunoreactivity. No significant differences in the mean numbers of neurons expressing Fos-like immunoreactivity were noted in the A2, C1, or C2/C3 medullary catecholamine cell groups. These results suggest that noxious stimuli affect pools of neurons in the medulla with multiple physiological functions.

The distribution of nitric oxide producing neurones in the medulla oblongata of the cat was investigated using nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry, and nitric oxide synthase (NOS) immunohistochemistry. The pattern of staining obtained with both methods was found to be similar. Strongly diaphorase and NOS reactive neurones were present in the paramedian and lateral tegmental fields, including the regions occupied by the A1/C1 catecholamine cell groups, the nucleus ambiguus and lateral reticular nucleus, and in a number of sensory nuclei including the nucleus of the tractus solitarius and the dorsal column nuclei. The extent of co-localization of NADPH-diaphorase with a number of neuropeptides and neurotransmitters was investigated by combining NADPH-diaphorase histochemistry with immunocytochemistry for neuropeptide Y, somatostatin, glutamate, cholecystokinin and tyrosine hydroxylase. NADPH-diaphorase reaction product was observed in neurones immunoreactive for glutamate and somatostatin. These double-labelled cells were found in the paramedian region, lateral reticular field, the nucleus prepositus hypoglossi and in the rostral nucleus of the tractus solitarius. In the rostral ventrolateral medulla NADPH-diaphorase/somatostatin immunoreactive cells were found in the paragigantocellular nucleus. NADPH-diaphorase/glutamate immunoreactive cells overlapped the nucleus ambiguus, the lateral reticular nucleus and the A1/C1 catecholaminergic cell groups. In addition, a few NADPH-diaphorase/glutamate immunoreactive cells were found in the paraolivary area and gigantocellular tegmental field, in the external cuneate and infratrigeminal nuclei. The functional implications of the co-localization of nitric oxide with these neurotransmitters in areas of the medulla concerned with cardiovascular regulation is discussed.

Pre- and post-embedding immunocytochemistry were employed in this electron microscopic investigation of cuneate neurons that are enriched in GABA and in nitric oxide synthase, the enzyme responsible for the synthesis of nitric oxide. GABAergic neurons are local circuit interneurons; 10-20% of them also contain nitric oxide synthase. These are among the smallest GABA-positive perikarya. We describe a network of processes in the rat cuneate nucleus that are immunopositive for nitric oxide synthase. Axon terminals positive for nitric oxide synthase are small and make synapses mainly onto dendrites; they make only occasional axo-axonic contacts. Double-labelling immunocytochemistry verified that the large majority of terminals positive for nitric oxide synthase also contained GABA. However, most GABA-positive profiles were negative for nitric oxide synthase and GABA-positive terminals that are negative for nitric oxide synthase frequently made axo-axonic contacts. These results suggest that nitric oxide synthase is within a specialized subpopulation of interneurons in the cuneate nucleus.

This study attempts to determine if projections ascending from the guinea pig cochlear nucleus (CN) could be glutamatergic and/or aspartatergic. Multiple radio frequency lesions were made to ablate the right CN. The ablation was verified histologically. To identify the principal targets of CN efferents, silver impregnation methods were used to localize the preterminal degeneration of fibers in transverse sections of the brainstem 5 and 7 days after CN ablation. CN efferents projected heavily to the lateral superior olive (LSO) ipsilaterally, the medial superior olive (MSO) bilaterally, and contralaterally to the medial (MNTB) and ventral (VNTB) nuclei of the trapezoid body, the ventral (VNLL) and intermediate nuclei of the lateral lemniscus and the central nucleus of the inferior colliculus (ICc). There were smaller projections to the lateral nucleus of the trapezoid body ipsilaterally, the dorsal and dorsomedial periolivary nuclei bilaterally, and the dorsal nucleus of the lateral lemniscus contralaterally. There were sparse projections to the VNLL and ICc ipsilaterally and the CN contralaterally, and a very sparse projection to the contralateral LSO. To determine if CN efferents were glutamatergic and/or aspartatergic, the fresh brainstem was sectioned transversely and samples of the LSO, MSO, MNTB, VNLL, and ICc were taken to measure the electrically evoked release and the uptake of D-[3H]Asp and [14C]Gly or [14C]GABA 3-5 days after the CN ablation. The release studies suggest that only certain of the histologically identified projections ascending from the CN may be glutamatergic and/or aspartatergic. CN ablation depressed D-[3H]Asp release in the MSO bilaterally and in the contralateral MNTB and VNLL, suggesting that the CN efferents to these nuclei may use glutamate or aspartate as a transmitter. It was unclear whether a marginal depression of D-[3H]Asp release in the ipsilateral LSO reflected the presence of glutamatergic CN projections to this nucleus. D-[3H]Asp release in the ICc was unaffected, suggesting that CN efferents to this nucleus may not be glutamatergic. There were no deficits in D-[3H]Asp uptake. [14C]Gly release from the LSO and MSO was unchanged. [14C]Gly uptake was unchanged in the MSO and depressed only in the contralateral LSO, possibly reflecting subnormal uptake activity in endings contributed by contralateral MNTB cells that had lost their CN efferents.(ABSTRACT TRUNCATED AT 400 WORDS)

Quantitative microfluorometric evaluation indicated that, in Lophius piscatorius, the DNA content of large neurons of a cluster located at the boundary between the medulla oblongata and the spinal cord varied from 4C to more than 5000C. This finding must be considered exceptional for the nervous system of Vertebrates. DNA contents were correlated to both nuclear and animal size. Utilization of AT and GC specific fluorochromes showed that the increase in DNA content is due to differential genome amplification involving GC-rich DNA sequences.

In kernicterus bilirubin appears at higher concentrations in the basal ganglia, the cerebellum, and the medulla. The mechanism for this distribution is unknown, but might involve differences in bilirubin entry among brain regions. We infused [3H]bilirubin (30 mg/kg over 5 min) intravenously into young Sprague-Dawley rats (n = 21, weight 144 +/- 10 g, mean +/- SD). Blood was sampled from the tail, and groups of rats were killed at 15, 30, 45, 60, and 75 min by intravenous pentobarbital. After in situ flushing of the brain vasculature, the brain was dissected into seven regions, each of which was weighed, dissolved in Soluene-350, and subsequently scintillation counted. Blood samples were analyzed for bilirubin (including specific activity), albumin, and blood gases. Brain bilirubin concentrations in each of the regions were calculated by comparing the radioactivity in that region with the specific activity of bilirubin. Using a curve-fitting program (EXPOFIT), the concentrations of bilirubin in each brain region at 5 min (i.e. the end of bilirubin infusion) could be estimated, and were as follows (nmol/g +/- SD): cortex 6.4 +/- 4.4; hippocampus 6.2 +/- 3.3; striatum 6.3 +/- 4.5; midbrain 7.3 +/- 4.4; hypothalamus 6.3 +/- 4.6; cerebellum 6.7 +/- 4.1, and medulla 7.0 +/- 4.0. There were no significant differences in the bilirubin concentrations between brain regions (F6,140 0.205). The mechanism for preferential localization of bilirubin to the basal ganglia probably does not involve the acute entry of bilirubin into brain.

The distribution of the short (gamma 2S) and long (gamma 2L) subunits of the GABAA receptors in the rat brain has been revealed by light microscopy immunocytochemistry with novel subunit-specific antibodies (anti-gamma 2S and anti-gamma 2L). We have also used other subunit-specific antibodies including anti-gamma 2IL2 (which recognizes both gamma 2S and gamma 2L), anti-alpha 1COOH, and the monoclonal antibody 62-3G1 to beta 2/3 for comparing the regional and cellular distribution of the most abundant GABAA receptor subunits in the rat brain. The distributions of gamma 2S and gamma 2L immunoreactivities are similar throughout the brain although the relative intensity of both signals varies depending on the brain area and neuronal type. In the hippocampus, cerebral cortex, and olfactory bulb (particularly mitral, periglomerular, and tufted neurons), gamma 2S was more abundant than gamma 2L. In contrast, the inferior colliculus, medulla, and the cerebellar Purkinje cells displayed more gamma 2L than gamma 2S immunolabeling. An important difference in the distribution of the various subunits was found in cerebellum: gamma 2S and gamma 2L were predominantly localized in the molecular layer, whereas alpha 1 and beta 2/3 were more abundant in the granular layer. In the thalamus, gamma 2L and gamma 2S were less abundant than either alpha 1 or beta 2/3 subunits. The results showed that there is colocalization of gamma 2S and gamma 2L subunits in some brain areas and neuronal types, as well as areas of mismatch. Colocalization and mismatches were also found among alpha 1, beta 2/3, and gamma 2, probably resulting from the heterogeneity in the subunit composition of the GABAA receptors through the brain.

Although the nucleus tractus solitarii (NTS) has been established as the primary site of synaptic integration for the baroreceptor reflex, the higher-order pathways responsive to, and mediating, changes in vasomotor tone are not well characterized. We used immunohistochemistry to determine the distribution of cells expressing the Fos protein following pharmacologically induced, directionally specific changes in arterial pressure. The goal of this investigation was to determine if this immediate early gene product is differentially expressed in neurons of the rat brainstem following increased (pressor) versus decreased (depressor) arterial blood pressure (AP). Because brainstem catecholaminergic (CA) cell groups have been implicated in cardiovascular regulation, a double-labeling immunohistochemical procedure was used to examine the distribution of Fos in CA cells. Animals received continuous intravenous infusion of either a vasoconstrictor (l-phenylephrine hydrochloride), a vasodilator (sodium nitroprusside), or physiological saline. Extensive Fos-like immunoreactivity (FLI) was induced in both the pressor and depressor conditions in the NTS, caudal ventrolateral medulla (CVLM), rostral ventrolateral medulla (RVLM), A5, locus coeruleus (LC), Kolliker-Fuse, and parabrachial nucleus (PBN). These regions have all been implicated in central cardiovascular regulation. There were differences in the anatomical distribution of Fos-positive cells along the rostrocaudal axis of CVLM in the pressor and depressor conditions. Specifically, increased AP induced significantly more FLI cells within the rostral aspects of CVLM, whereas decreased AP resulted in a significantly greater number of FLI cells within the caudal CVLM. This result suggests that selective vasomotor responses differentially engaged discrete subsets of neurons within this brainstem region. Overall, approximately 50% of CA-immunoreactive cells were also FLI (CA-FLI) in the A1, A5, and A7 regions. Interestingly, increased AP produced significantly more CA-FLI double-labeled cells within the caudal than rostral A1 compared with depressor and control groups. Additionally, increased AP yielded significantly less CA-FLI double-labeled cells within the caudal A2 region. This suggests that CA barosensitive neurons in the CVLM/A1 and NTS/A2 regions are functionally segregated along the rostrocaudal axis of these structures. While twice as many PNMT-FLI double-labeled neurons were found in the C1-C3 regions following vasomotor changes versus saline control, there were no differences in the numbers or anatomical locations of labeled cells between pressor versus depressor groups. The results of this study indicate that (1) tonic changes in AP induce robust Fos expression in brainstem cardiovascular areas and (2) neurons responsive to specific directional changes in arterial pressure are segregated in some brainstem regions.

The distribution of VIP-immunoreactive neurons and fibers was detected in the extrahypothalamic areas of chicken brain by immunohistochemistry and light microscopy VIP-ir perikarya were found in the hippocampus and in the area parahippocampalis; in the area ventralis of Tsai, in the n. interpeduncularis, in the substantia nigra, in the substantia grisea centralis, in the locus coeruleus, in the n. subcoeruleus ventralis and in the n. pontis lateralis. VIP-ir fibers were seen in the lobus parolfactorius and throughout the brainstem mainly arranged in lateral and midsagittal position. This finding was discussed in relation to other studies performed on chicken and/or other avian brain. The distributional pattern of VIP-ergic system in the chicken brain suggests a possible involvement of VIP or VIP-like peptide in several neuroregulatory mechanisms.

Secretoneurin is a peptide of 33 amino acids generated in the brain by proteolytic processing of secretogranin II which is a member of the chromogranin/secretogranin family. The distribution of this newly characterized peptide was investigated by immunocytochemistry in the human brain stem. The staining pattern of secretoneurin-like immunoreactivity was compared with that of substance P in adjacent sections. Secretoneurin-like immunoreactivity appeared mainly in dot- and fiber-like structures with densities varying from low to very high. Only a low number of secretoneurin-immunoreactive perikarya was found. Pericellular staining of both secretoneurin-immunopositive and immunonegative cells was frequently observed in the area of the central gray, in the reticular formation and in the solitary nuclear complex. The medial part of the substantia nigra pars reticulata, the nucleus interpeduncularis, the area of the central gray, the raphe complex and the inferior olive displayed a high density of secretoneurin-like immunoreactivity. Furthermore, a very 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 secretoneurin-immunopositive. The staining patterns of secretoneurin- and substance P-like immunoreactivities were to a certain extent overlapping in several areas. The highest degree of coincidence was found in the substantia gelatinosa. This study demonstrated that secretoneurin is distinctly distributed in the human brain stem. Its distributional pattern indicates a role particularly in the modulation of afferent pain transmission and in the regulation of autonomic functions.

Vasopressin V1a receptor (V1aR) transcripts were localized in brain, pineal, and superficial brain vascular tissues of adult male rats using hybridization histochemistry and an [35S]riboprobe complementary to the messenger ribonucleic acid (mRNA) encoding the fifth to the midseventh transmembrane regions of the receptor. V1aR mRNA was extensively distributed throughout brain and was expressed in 1) superficial cells of the granule cell layers of the main olfactory bulb, hippocampal dentate gyrus, and cerebellum; 2) numerous anatomically distinct brain nuclei; 3) isolated cells dispersed throughout the central nervous system; 4) cells of the choroid plexus, occasional blood vessels in the olfactory bulb and interpeduncular nucleus, and extraparenchymal intracranial vasculature; and 5) some white matter structures. Numerous cells expressing V1aR transcripts were found in forebrain structures, including primary olfactory (piriform) cortex, the anterior and posterior olfactory nuclei; dorsal, intermediate, and ventral lateral septal nuclei; the septo-fimbrial nucleus and accumbens nucleus; and numerous hypothalamic regions with the most intense hypothalamic labeling in the arcuate, stigmoid, suprachiasmatic, and periventricular nuclei and the lateral hypothalamic area. Cells expressing V1aR transcripts were ubiquitous throughout the midbrain, pontine, and medullary regions. A lower intensity signal was found in cells of the parvocellular paraventricular and anteroventral nucleus of the thalamus, circumventricular organs including the pineal, and the subfornical organ. V1aR transcripts were not generally detected in parenchymal vasculature, but could be found over large blood vessels in the interpeduncular nucleus and medial olfactory bulb; transcripts were commonly detected in perivascular brain cells. V1aR mRNA was abundantly expressed by choroid plexus, endothelial cells of midline blood vessels between the main olfactory bulbs, and superficial vascular tissue on all brain surfaces. These data confirm the presence of the vascular/hepatic-type V1aR gene in brain tissue and document an extensive expression. The distribution of V1aR mRNA suggests that there are at least two types of vasopressin-responsive cells in brain: one type exemplified by lateral septal ara neurons innervated by classical axodendritic/somatic synaptic vasopressinergic terminals and a second, perivascular/vascular type that would facilitate humoral vasopressinergic signaling in the brain.

A population of depressor neurons in the caudal ventrolateral medulla that project to the rostral ventrolateral medulla may mediate the baroreceptor reflex. The aim of the present study was to determine the anatomical distribution of the population of neurons in the caudal ventrolateral medulla that mediate the baroreceptor reflex. Injection of the retrogradely transported tracer, rhodamine-labelled latex beads, into the pressor area of the rostral ventrolateral medulla of rats was used to identify neurons in the caudal ventrolateral medulla with projections to that area. Barosensitive neurons were identified by immunohistochemical detection of the protein Fos, a marker of neuronal activation, following infusion of the pressor agent phenylephrine (10 micrograms/kg/min, i.v. for 2 h n = 5). Isotonic saline was infused into control animals (n = 4). Neurons in the caudal ventrolateral medulla with projections to the rostral ventrolateral medulla were located at all rostrocaudal levels examined between 1 mm caudal and 0.4 mm rostral of the obex. Compared to saline infused rats, phenylephrine infusion induced a significant increase in the proportion of those neurons that expressed Fos (14% vs. 1% P < 0.000.1). These barosensitive neurons were found mainly at the level of the obex, between the lateral reticular nucleus and the nucleus ambiguus. In conclusion, this study is the first to show the distribution of the population of barosensitive neurons in the caudal ventrolateral medulla that project to the pressor region of the rostroventrolateral medulla. The results suggest there is a subpopulation of depressor neurons, confined to a small region of the rostral part of the caudal ventrolateral medulla, that are likely to be the interneurons that mediate the baroreceptor-reflex response.

The cuneate nucleus is a relay center for somatosensory information by receiving tactile and proprioceptive inputs from primary afferent fibers that ascend in the dorsal funiculus. The morphology, synaptic contacts, and neurochemical content of primary afferent terminals in the cuneate nucleus of rats were investigated by combining anterograde transport of horseradish peroxidase conjugated to wheat-germ agglutinin or to cholera toxin (injected in cervical dorsal root ganglia) with postembedding immunogold labeling for glutamate and GABA. Both tracers gave similar results. Two types of terminals were labeled: type I terminals were irregularly shaped, had a mean area of 4.0 microns 2, synapsed on several dendrites, and were contacted by other terminals, some of which were GABA positive. Type II terminals were dome-shaped, had a mean area of 2.18 microns 2, and made synaptic contact on a single dendrite. All the anterogradely labeled terminals (interpreted as endings of primary afferents) were enriched in glutamate but not in GABA. The finding that identified primary afferent terminals are enriched in glutamate with respect to other tissue profiles strongly suggests a neurotransmitter role for glutamate in this afferent pathway to the rat cuneate nucleus.

The distribution of cell bodies containing serotonin (5-HT)-, substance-P (SP)-, L-enkephalin (L-ENK)- and neuropeptide Y (NPY)-like immunoreactivity (IR) in the nucleus paragigantocellularis lateralis (PGCL) of the rat was studied immunocytochemically. Perikarya containing 5-HT-, SP- or L-ENK-IR were found throughout the rostro-caudal extent of the PGCL. Large, well-staining 5-HT cells densely clustered rather near the ventrolateral surface of the medulla. SP- and L-ENK-IR were distributed more extensively and located more lateraly. A few of perikary on containing NPY-IR were found primarily in the area from the medium to caudal region. The results provided morphological basis for further study of the neurochemical properties of PGCL neurons involved in the regulation of cardiovascular, respiratory and other functions in the rat.

A specific anti-imidazoline receptor (IR) protein antiserum was used to detect these putative receptors in brain and other tissues. In immunoblotting experiments, various brain areas and tissues expressed a double band of 29-30 kDa, and some of them bands of 47 and 66 kDa. The human medulla oblongata also showed bands of 44 and 50 kDa; and the rat adrenal medulla, bands of 58 and 86 kDa. Total immunoreactivity correlated with the density of IR binding sites in the various tissues. This is first evidence of heterogeneity of immunoreactive IRs in the mammalian brain.

Immunohistochemistry and retrograde tracing using cholera toxin B subunit colloidal gold (CTB-gold) has been used to identify neurons in the medulla that contain neuropeptide Y and project to the area of the intermediolateral cell column in either the upper (T2-T4) or the lower (T8-T9) thoracic spinal cord. The rostrocaudal distributions of neuropeptide Y neurons and neuropeptide Y/CTB-gold neurons have been compared with the distributions of adrenaline-synthesising, phenylethanolamine N-methyltransferase-containing neurons and phenylethanolamine N-methyltransferase/CTB-gold neurons visualised in adjacent sections. In particular areas of the rostral medulla similarities in the numbers and distributions of neuropeptide Y neurons and phenylethanolamine N-methyltransferase neurons suggested a coexistence of the peptide within the catecholamine neurons. However, at the most rostral levels of the rostral ventral medulla, the large numbers of phenylethanolamine N-methyltransferase neurons were not matched by similar numbers of neuropeptide Y neurons, so that the phenylethanolamine N-methyltransferase neurons in this area could not all contain neuropeptide Y. In the rostral ventral medulla fewer neuropeptide Y/CTB-gold neurons than phenylethanolamine N-methyltransferase/CTB-gold neurons were observed, so that these bulbospinal peptide neurons might define a subset of the phenylethanolamine N-methyltransferase/CTB-gold neurons, accounting for 25% of the total phenylethanolamine N-methyltransferase bulbospinal projection from the rostral ventral medulla. Other neuropeptide Y/CTB-gold neurons in the dorsal medulla are also likely to contain phenylethanolamine N-methyltransferase. Finally, a population of neuropeptide Y/CTB-gold neurons was identified in the caudal ventral medulla, these neurons appear not to contain catecholamine synthesising enzymes.

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

In this study we examined the distribution of calcitonin (CT) binding sites in the human medulla oblongata by in vitro autoradiography. In competition studies, the rank order of potency for calcitonins competing for 125I-salmon CT binding was salmon CT > porcine CT > human CT, which is consistent with physiologically relevant CT receptors in other systems. For the determination of CT binding in the human medulla, 20-micron cryostat sections were incubated with 125I-salmon CT in the presence or absence of 10(-6) M unlabelled salmon CT to map specific CT binding sites. Punctate binding was observed over discrete nuclei of the medulla. High binding densities were seen over subnuclei of the dorsal motor nucleus of the vagus, the nucleus of the solitary tract, the intermediate reticular zone, the gigantocellular and dorsal paragigantocellular nuclei, and the raphe obscurus nucleus. Moderate levels of binding were observed over the lateral paragigantocellular nucleus and the rostral extent of the epiolivary nucleus. The cuneate and gracile nuclei and the fiber tracts did not contain detectable binding, while the inferior olivary nucleus had moderate levels of nonspecific binding. The localization of calcitonin binding sites in the human presents similarities but also important differences to the distribution in rat and cat. The most notable difference is the extreme binding densities in the intermediate reticular zone of the human. The location of binding sites suggests involvement of calcitonin in regulation of autonomic function.

Immunocytochemical localization of tryptamine in rat brain was performed at light and electron microscope level. The immunoreactive cells were present in the mesencephalon, pons and medulla, around the interpendicular nucleus in the reticular substantia nigra and in the giganto cellularis ventralis nucleus. Immunoreactive fibres were present in the dorsal region of the brain stem, and innervated the hippocampus, the ependymal epithelium and the corpus striatum. An ultrastructural study revealed that tryptamine was present only in neuronal perikaryon and processes.

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

The coexistence of neurokinin A (NKA) with substance P (SP) and serotonin (5-HT) in ventral medullary neurons of the parapyramidal region and nucleus raphe pallidus of the rat was studied using multiple immunofluorescence labeling. Nearly all of the NKA-immunoreactive (IR) cells in the parapyramidal region and raphe pallidus were SP-IR nd 5-HT-IR, whereas about 70% of the SP-IR neurons and about 60% of the 5-HT-IR neurons contained NKA-IR. There were no apparent differences in the patterns of coexistence between parapyramidal and raphe pallidus neurons. NKA-IR neurons, which colocalized SP-IR and 5-HT-IR, were studied for projections to the lumbar and thoracic spinal cord by use of retrograde transport of fluorescent tracer. Whereas about 50% of the retrogradely labeled neurons of the parapyramidal region and raphe pallidus contained NKA-IR, nearly all of the NKA-IR neurons projected to the thoracic and lumbar spinal cord. In addition, some NKA-IR neurons in the ventral medulla were retrogradely labeled with tracer from localized injections into the thoracic intermediolateral cell column. In summary, this study demonstrated that NKA-IR is colocalized with SP-IR in bulbospinal serotonergic neurons of the parapyramidal region and raphe pallidus, which are known to regulate sensory, motor, and autonomic activities of the spinal cord.

The distribution of peptide and serotonin fibers in the nucleus of the solitary tract (NTS) and the dorsal motor nucleus of the vagus nerve (DMNX) in the pigeon (Columba livia) was investigated immunocytochemically. This information was correlated with the viscerotopic organization of the nuclei and with central NTS circuitry to suggest the role of the neurochemical containing fibers in the regulation of organ function. The distribution of fibers containing cholecystokinin (CCK), calcitonin gene-related peptide (CGRP), enkephalin (ENK), neuropeptide Y (NPY), neurotensin (NT), substance P (SP), somatostatin (SS), vasoactive intestinal peptide (VIP), and 5-hydroxytryptamine (5-HT) was determined. Each substance had a distinct distribution within the subnuclei of NTS-DMNX, but certain generalities can be deduced. In the DMNX, fibers immunoreactive for ENK, NT, and SP were found in greatest concentration, while CGRP and 5-HT immunoreactive fibers were the least dense. This suggests that ENK, NT, and SP may have a significant modulatory effect on gastrointestinal functions. In the NTS overall, ENK, NT, SP, and VIP fibers were found in high density, CCK, NPY, SS, and 5-HT fibers were found in moderate density, and CGRP fibers were found in low density. However, some individual NTS subnuclei were found to contain moderate to high concentrations of each of the substances, including CGRP. Fibers containing CCK, ENK, NT, SP, SS, and VIP in the medial dorsal NTS subnuclei may regulate gastroesophageal functions. The caudal part of subnucleus lateralis parasolitarius did not contain most of the substances, which suggests that pulmonary function is not modulated by these neurochemicals. The boundaries of a subnucleus could sometimes be demarcated by a change in density of immunoreactive fibers between adjacent subnuclei. This was particularly evident in NTS subnuclei medialis dorsalis anterior centralis and lateralis parasolitarius, and in DMNX subnucleus posterior dorsalis magnocellularis. The selective distribution of peptide and serotonin immunoreactive fibers in various subnuclei of NTS-DMNX suggests that these substances may be differentially involved in neural circuits that mediate cardiovascular and gastrointestinal functions.

1. [3H]-clonidine binding was investigated in membranes isolated from the ventral medulla oblongata of the rabbit, where clonidine produced a hypotensive effect which was not mediated by adrenoceptors. [3H]-clonidine specific binding, as defined by the difference between the binding of [3H]-clonidine in the presence and in the absence of 10 microM cirazoline, occurred at two sites: a high affinity site with a KD = 2.9 +/- 0.7 nM and a Bmax of 40 +/- 8 fmol mg-1 protein and a low affinity site with a KD = 18.2 +/- 0.4 nM and a Bmax of 66 +/- 14 fmol mg-1 protein. 2. The high affinity sites being catecholamine-sensitive were identified as alpha 2-adrenoceptors. The low affinity binding of [3H]-clonidine was insensitive to catecholamines, as well as to other alpha 2-adrenoceptor specific probes, and could be inhibited with high affinity only by compounds which lowered blood pressure when directly injected in the nucleus reticularis lateralis of the ventral brainstem, or by antagonists. 3. It was concluded that in the ventral medulla of the rabbit, [3H]-clonidine labelled alpha 2-adrenoceptors and imidazoline receptors (IRs). Only the latter were related to the hypotensive effects of clonidine and rilmenidine directly injected into the rostroventrolateral medulla oblongata (RVLM) of the rabbit. The methodological problems regarding the study of IRs with [3H]-clonidine are discussed.

The purpose of this study was to determine whether neuropeptide Y (NPY) terminals in the intermediolateral spinal cord originate from the rostral ventrolateral medulla (RVLM). Immunohistochemical staining of tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), phenylethanolamine-N-methyltransferase (PNMT), and NPY in the rat brainstem and spinal cord were performed in this study in order to examine consequences of lesions of the RVLM and of intracisternal injections of 6-hydroxydopamine (6-OHDA) on catecholamine and NPY immunoreactivity in the intermediolateral column (IML) of rats. In addition, ricin, a retrograde neurotoxin, was applied in the superior cervical ganglion (SCG) to determine its effect on catecholamine and NPY immunoreactivity in the IML. Computer-aided image analysis was used to quantify the immunohistochemical changes in the RVLM and spinal cord. The results demonstrated that many catecholamine- and NPY-containing neurons and/or fibers existed in the RVLM and their terminals were found in the IML. After administration of 6-OHDA intracisternally, the catecholamine and NPY immunoreactivities were decreased both in the brainstem and IML of the spinal cord. Following unilateral microinjection of 6-OHDA into the RVLM, the number of NPY- and catecholamine-containing neurons decreased and there was a reduction in neuron terminals on the ipsilateral side. After injection of ricin into the SCG, the catecholamine and NPY neurons of the medulla were not affected, whereas their terminals in the IML decreased ipsilaterally. These results indicate that most of the catecholamine- and NPY-immunoreactive terminals found in the IML originated in the RVLM. These terminals appear to project towards the superior cervical ganglia.

The present experiments investigated the possible relationship between portal hypertension and norepinephrine metabolism in the central nervous system (hypothalamus and medulla oblongata) and the portal vein in the rat. Group I (72), portal hypertensive, and group II (70) sham-operated animals, were sacrificed day 14, and endogenous norepinephrine content, uptake and release from hypothalamus, medulla oblongata, and portal vein were investigated. In group I our results showed increases in norepinephrine storage (69%; 8.3%) and release (19.7%; 43.8%) and a diminished uptake (42.3%; 27.5%) in the hypothalamus and medulla oblongata, respectively. Portal veins showed a decreased content and uptake (62.5% and 43.5%, respectively) and increased release (25%) compared to group II rats. These results suggest a close relationship between the central nervous system and rat portal hypertension, perhaps related to modifications of central sympathetic activity.

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

Recently, we proposed the hypothesis according to which the central hypotensive effect of clonidine and related substances could be related to an action upon specific receptors, requiring the imidazoline or imidazoline-like structures, rather than alpha 2-adrenoceptors. Since then, direct evidences have been accumulated to confirm the existence of a population of imidazoline specific binding sites in the brainstem of animals and man, more precisely in the Nucleus Reticularis Lateralis (NRL) region of the ventrolateral medulla (VLM), site of the antihypertensive action of clonidine. The purification of the putative endogenous ligand of the imidazoline receptors--named endazoline--is currently being attempted from human brain extracts. This new concept might at last lead to the expected dissociation of the pharmacological mechanisms involved, on the one hand, in the therapeutic antihypertensive effect, and on the other, in their major side-effect, which is sedation. In fact, it has been recently confirmed that hypotension is mediated by the activation of imidazoline preferring receptors (IPR) within the NRL region, while sedation is attributed to the inhibition of alpha 2-adrenergic mechanisms in the locus coeruleus, which is involved in the control of the sleep-waking cycle. The IPR may constitute an interesting target for new drugs in the treatment of arterial hypertension. Finally, dysfunctions of this modulatory system which could be involved in the pathophysiology of some forms of the hypertensive disease are under investigation.

We have determined the distribution of angiotensin II receptor subtypes in rabbit brain using in vitro autoradiography. AT1 receptors were found in very high concentrations in the forebrain circumventricular organs--the subfornical organ, organum vasculosum of the lamina terminalis, and the median eminence as observed in other mammals. However, there was very little labeling in the area postrema. In the paraventricular nucleus, median preoptic nucleus, supraoptic nucleus there were high levels of predominantly AT1 receptors. High densities of AT1 receptors were also found in the nucleus of the solitary tract and the rostral and caudal ventrolateral medulla. All of these regions have putative roles in the regulation of blood pressure and fluid and electrolyte balance. In the rabbit brain there is less AT2 receptor binding than the rat, with most AT2 binding found in the molecular layer of the cerebellum and in the septohypothalamic nucleus. In the subthalamic nucleus, the mediodorsal and ventroposterior nuclei of the thalamus, locus coeruleus and inferior olivary nuclei, areas containing mostly AT2 receptors in the rat, no binding was detected in the rabbit except in the locus coeruleus which contains moderate levels of AT1 receptors. Taken in conjunction with our previous results in the rat and human brains, these results reveal that AT1 receptors predominate in rostral forebrain, hypothalamus and autonomic control centers of the medulla oblongata in all three species. However, the distribution and density of AT2 bearing sites in regions such as the septum, thalamus subthalamic nuclei, locus coeruleus, cerebellum and inferior olivary nuclei show marked species differences.

A new approach to study angiotensin receptor distribution in the brain has been taken by developing antibodies to partial sequence of the angiotensin II (AII) type-1 receptor subtype (AT1) and demonstrating the presence of receptors with immunohistochemical staining. The antibody to a portion of the 3rd cytoplasmic loop of the AT1 receptor revealed distinctive punctate immunoreactive staining on cell bodies. The cell bodies were distributed in the forebrain in paraventricular and supraoptic nuclei, the organum vasculosum lamina terminalis, median preoptic area and subfornical organ. In the brainstem, the entire locus coeruleus was stained, together with the adjacent mesencephalic and motor nuclei of the trigeminal nerve. The auditory system including the cochlear nucleus and superior olivary nuclei were stained. In the medulla, all the structures involved in blood pressure control were stained including the nucleus of the solitary tract, the 12th nerve nuclei, the rostroventral lateral area and the nucleus ambiguous. Sites where AT2 receptors are located were not stained or staining was limited to specific area such as the medial accessory nucleus of the inferior olive. Immunocytochemical staining of AT1 receptors provides a new and more precise approach to the cellular localization of AII receptors.

It was shown that GSH concentration and glutathione peroxidase activity in hypothalamus (H) and sensomotor cortex (SC) were twice more than in ME. After single or double injection of diethylmaleate (DEM) two-phase change of the GSH level was observed in all the investigated structures. In the first 1-3 h phase, the pool of free GSH decreases by more than 50% as a result of conjugative effect of DEM. In the second phase which is about 3 days long the level of GSH is gradually restored mainly due to the activation of its biosynthesis. In various brain structures conjugation and reduction processes has their own peculiarities: conjugation processes predominate in H and SC while the reduction processes predominate in ME. These processes were almost two times intensified in all the investigated structures under double injection of DEM.

The origin of cardiac vagal preganglionic motoneurones in the rat is still controversial and knowledge of the chemistry of synaptic inputs onto these neurones is limited. In this investigation vagal preganglionic motoneurones innervating the heart were identified by the retrograde transport of cholera toxin conjugated to horseradish peroxidase (CT-HRP) combined with the immunocytochemical localization of 5-hydroxytryptamine. Injection of CT-HRP into the myocardium resulted in the retrograde labelling of neurones primarily in the ventral regions of the nucleus ambiguus (75.1%). Labelled neurones were also distributed in a narrow band through the reticular formation extending between the dorsal motor nucleus of the vagus nerve and the nucleus ambiguus (17.3%) as well as in the dorsal motor nucleus itself (7.6%). A combination of retrograde labelling with immunocytochemistry for 5-hydroxytryptamine revealed that the neuronal perikarya and the dendrites of cardiac vagal motoneurones in the nucleus ambiguus were often ensheathed in 5-hydroxytryptamine-immunoreactive axonal boutons. Electron microscopic examination of this material confirmed that there were synaptic specializations between these boutons and the cardiac vagal motoneurones. The identification of 5-hydroxytryptamine-containing synaptic inputs to this population of vagal motoneurones provides further detail towards the understanding of the regulation of heart rate by the parasympathetic nervous system.

The serotoninergic innervation of the dorsal column nuclei (DCN) was investigated in cats and owl monkeys (Aotus trivirgatus) with immunohistochemical methods. A dense network of serotonin-immunoreactive fibers was present in the reticular regions of DCN in cats, and in the pars triangularis of the cuneate nucleus and the peripheral and caudal regions of the gracile nucleus in owl monkeys. The cat's cluster regions and the monkey's rotund regions were more sparsely innervated. Electron microscopic examination showed that the labeled fibers were thin and unmyelinated. Vesicle-containing, terminal-like structures were small. They were in contact with dendrites, other terminals and cell bodies, but synapses were rare. The results demonstrate that the serotoninergic projection to the DCN in both cats and owl monkeys is heterogeneously distributed in a pattern that is faithfully related to the cytoarchitectonic subdivisions of the DCN. The densely innervated reticular regions in the DCN of cats and the corresponding regions in monkeys are predominantly involved in the processing of sensory information to the cerebellum, either directly, or indirectly through projections to the inferior olive, pontine gray, tectum, pretectum, red nucleus, or zona incerta. Thus, the present findings suggest that the serotoninergic innervation of the DCN is primarily related to the DCN's involvement in motor functions.

The present study, by the use of in vivo electrochemical detection of 5-hydroxyindole (peak '3') in the bulbo spinal serotonergic system at the medullary dorsal horn (MDH) level, investigated the effects of the new tricyclic antidepressant (TCA) tianeptine, which has been shown to be a specific serotonin (5-HT) uptake enhancer. It was found that acutely administered tianeptine (10 mg/kg, i.p.) induced a marked significant increase in peak 3 within the dorsal horn, an in vivo observation which is in accordance with the biochemical properties of tianeptine as studied in forebrain structures. In addition, the effect of tianeptine on the morphine-induced increase in 5-HT metabolism was investigated, by comparison with the previous data obtained with the specific 5-HT uptake inhibitor femoxetine in the MDH. It was shown that tianeptine can display additive effect with morphine (10 mg/kg, i.p.) on 5-HT metabolism at the MDH level. These results are discussed in relation to the effects of classical TCAs and the particular properties of tianeptine.