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The central nervous system of Lophius piscatorius has a cluster of large neurons located at the boundary between the medulla oblongata and spinal cord. The DNA content of these neurons was evaluated by microfluorimetric methods. Results demonstrated a DNA content ranging from a minimum of 8C in the smaller to over 1000C in the larger neurons of L. piscatorius cluster. These data are exceptional for the nervous system of Vertebrates, and known only for the giant neurons of Mollusca.

Brain aminopeptidase activity has been suggested as a candidate for the regulation and biotransformation of several neuropeptides. In this paper, changes in Lys- and Leu-aminopeptidase activities in rat brain hemispheres, cerebellum and medulla were examined in 1-, 3-, 5- and 7-days postnatal subjects. Aminopeptidase activities were studied by measuring the rate of hydrolysis of the artificial chromogenic substrates Lys- and Leu-2-naphthylamides (fluorimetrically detected in triplicate). Both enzyme activities show a decrease on the 3rd day of life followed by increases on the 5th and 7th day postbirth. It is suggested that these activities could play a part in the neurochemical changes that take place during axonal and dendritic growth in the rat brain.

Neurons in the rostral ventromedial medulla (RVM) are important in the opioid modulation of dorsal horn nociceptive transmission. Systemically administered morphine inhibits one class of RVM cells, the on-cells; excites a second class of RVM cells, the off-cells; and has no effect on a third class, neutral cells. In contrast, iontophoretic application of morphine inhibits on-cells but does not alter the activity of either off- or neutral cells. The present study addresses whether the differential sensitivity to exogenous opioids is correlated with a differential termination pattern onto the three classes of RVM neurons by afferents containing endogenous opioids. Intracellular recordings were made from RVM neurons in rats under light halothane anesthesia. Physiologically characterized neurons were injected with Neurobiotin and then subsequently visualized with a Texas red fluorophore. Thick (50 microns) sections containing labeled RVM cells were processed for enkephalin immunoreactivity (ENK-IR) using an FITC fluorophore and then optically sectioned at 1.5 micron intervals using a dual-channel confocal laser scanning microscope. ENK-IR appositions were found on the somata and dendrites of all on-cells. Although ENK-IR varicosities were also apparently apposed to off- and neutral cells, the density of such appositions was significantly less than the density of ENK-IR appositions onto on-cells. The greater overall density of ENK-IR appositions onto on-cells was apparently due to a concentration of appositions on the soma and proximal dendrites of these neurons. These results support a model of RVM function in which endogenous opioid peptides produce an antinociceptive action by a direct inhibitory action on on-cells that facilitate nociceptive transmission. This on-cell inhibition may produce an additional antinociceptive effect by removing a possible on-cell inhibition of off-cells, which are thought to inhibit nociceptive transmission.

gamma-Aminobutyric acid immunoreactive perikarya and boutons in the nucleus tractus solitarius of the cat were examined at both the light and electron microscopic level. Immunoreactive neurones were found predominantly in the parvocellular subdivision of the nucleus tractus solitarius and to a lesser degree in all the other subdivisions of the nucleus tractus solitarius and the dorsal vagal motonucleus. All the immunoreactive perikarya observed were similar in size and morphology. gamma-Aminobutyric acid immunoreactive boutons were observed throughout the nucleus tractus solitarius. However, in contrast to its high content of immunoreactive perikarya the parvocellular subdivision contained the lowest density of immunoreactive boutons. Ultrastructural examination of immunoreactive boutons in the different regions of the nucleus tractus solitarius revealed that they formed synaptic specializations, predominantly with dendritic shafts, all of which were of the symmetric type. This pattern of innervation was observed throughout the medial, commissural, ventrolateral and parvocellular subdivisions of the nucleus tractus solitarius.

The alpha 2 subunit of the glycine receptor is expressed transiently in the rat brain during early development suggesting that this subunit may be replaced by the alpha 1 subunit in the adult brain. The expression of glycine receptor alpha 2 subunit mRNA was investigated in the 7-day-old rat brain by in situ hybridization histochemistry using oligonucleotide probes specific for this subunit. Neurons expressing alpha 2 subunit mRNA were found to be widely and abundantly distributed throughout brain. We compared the distribution of neurons expressing alpha 2 subunit mRNA with that of neurons expressing alpha 1 or beta subunit mRNA. In the lower brainstem, the location of the neurons expressing alpha 2 subunit mRNA was very similar to that of the neurons with alpha 1 or beta subunit mRNA. Neurons expressing beta subunit mRNA were widespread and numerous in the forebrain, where neurons with alpha 1 subunit mRNA were uncommon. The locations of the neurons labeled by the alpha 2 probe were very similar to those of the cells labeled by the beta probe. These findings suggest that the alpha 2 subunit is not only expressed by immature neurons containing the alpha 1 subunit, but is also common to most immature neurons having the glycine receptor. However, it should be noted that several neurons contained beta and/or alpha 1 subunit mRNA but lacked alpha 2 subunit mRNA, suggesting that the glycine receptor is heterogeneous in its composition during brain development.

Neuropeptide FF (NFF) is an amidated octapeptide of bovine origin. It has some antiopioid-like effects and it elevates blood pressure when injected intravenously in rats. NFF-immunoreactive nerve cells and terminals are localized in large numbers in the dorsomedial caudal brainstem which is a region involved in central regulation of blood pressure. We compared the localization of NFF-immunoreactive neurons with medullary catecholamine-synthesizing neurons by using immunohistochemical double-labeling and light microscopic mirror methods. NFF and tyrosine hydroxylase coexisted in a minor portion of the NFF neurons in the nucleus of the solitary tract and occasional cell bodies were stained with both NFF and PNMT (phenylethanolamine N-methyltransferase) antisera. The results have anatomical correlation with previous pharmacological reports, suggesting that NFF is present in neuronal systems involved in cardiovascular reflex arcs.

The distribution of thyrotropin-releasing hormone (TRH)-like immunoreactivity (LI) has been studied in the grey monkey (Macaca fascicularis) spinal cord and medulla oblongata by the use of indirect immunofluorescence and the peroxidase-antiperoxidase (PAP) technique. Furthermore, double-labeling experiments were performed in order to study colocalization of 5-hydroxytryptamine (5-HT)- and substance P-LI. A dense innervation of TRH-immunoreactive (IR) varicose fibers was found in the ventral horn motor nuclei, in the region surrounding the central canal, in the intermediolateral cell column, and in the dorsal horn laminae II and III. In addition, cell bodies harboring TRH-LI were found in the dorsal horn laminae II-IV. In the ventral horn, many of the large cell bodies and their proximal dendrites were totally encapsulated by TRH-IR fibers. From double-labeled sections a high degree of coexistence could be established between TRH-/5-HT-LI, TRH-/substance P-LI, and 5-HT-/substance P-LI in fibers in the motor nuclei; as a consequence, a large proportion of these fibers should harbor TRH-/5-HT-/substance P-LI. A coexistence between TRH-/5-HT-LI could also be demonstrated in the intermediolateral cell column. However, no unequivocal coexistence could be found between TRH-/substance P-LI and 5-HT-/substance P-LI in this region. In the dorsal horn, no clear coexistence could be encountered for any of the above indicated combinations. Electron microscopic analysis of material from the lumbar lateral motor nucleus demonstrated TRH-IR terminals making synapses with large cell bodies and dendrites. In addition, contacts lacking synaptic specializations could also be verified. In the medulla oblongata, with the use of the PAP technique, a large number of cell bodies containing TRH-LI were encountered in the midline raphe nuclei and in nucleus reticularis lateralis. A similar distribution pattern could be found for 5-HT-LI, but no cell bodies containing substance P-LI could be seen in these regions. Chemical analysis of specimens from cervical, thoracic, and lumbar spinal cord revealed higher concentrations of TRH- and 5-HT-LI in the ventral quadrants, whereas substance P-LI dominated in the dorsal quadrants. Thus, the concentrations of TRH-, 5-HT-, and substance P-LI was in accordance with the observed regional variation in density of IR-fibers and varicosities found in the spinal cord. We have shown that TRH-LI has a distribution in the monkey spinal cord and medulla oblongata similar to that previously demonstrated in other species.(ABSTRACT TRUNCATED AT 400 WORDS)

The distribution of neurokinin B (NKB) was determined by immunocytochemistry with antisera directed toward its amino terminus. Immunoreactive perikarya were detected in the main and accessory olfactory bulbs, cortical regions, the olfactory tubercle, the bed nucleus of the stria terminalis, the diagonal band of Broca, the nucleus accumbens, the septum, the neostriatum, several hypothalamic nuclei, the superior colliculus, the central gray, the substantia nigra, the medullary reticular formation, and the external cuneate nucleus. The distribution of NKB-containing perikarya revealed by immunocytochemistry was similar to the distribution of protachykinin B-containing cells previously visualized by in situ hybridization. Immunoreactive nerve fibers and terminals were detected in all major subdivisions of the brain. The levels of NKB measured by radioimmunoassay were highest in the hypothalamus. The distribution of NKB in the rat brain was similar to the distribution of substance P; however, there were several regions where the two distributions were clearly different.

The immunohistochemical detection of the protein, Fos, has been used as an anatomical marker of activated neurons. Three conscious rats were hemorrhaged (4 ml, 20-25% of blood volume) and the distribution of Fos-stained neurons was compared to that in 4 rats which did not have blood removed. In hemorrhaged rats, a higher concentration of Fos-stained neurons was present in the lamina terminalis, particularly the subfornical organ and organum vasculosum of the lamina terminalis, and in the supraoptic and paraventricular nuclei of the hypothalamus. In the medulla, Fos-stained neurons were restricted to the nucleus of the tractus solitarius, area postrema and the ventrolateral medulla. We hypothesize that those neurons are involved in mediating the physiological responses to hemorrhage.

Morphometric techniques have been developed to quantitatively characterize groups of transmitter-identified neuronal profiles, such as cell groups, dendrite and nerve terminal fields. These morphometric techniques will be illustrated by introducing some general tools for image analysis which can be considered as a background for the present specific applications. The following methods have been included: (1) methods to identify and quantitatively characterize, from both numerical and geometrical standpoints, groups of profiles in a two- and three-dimensional frame; (2) methods to evaluate the evenness of a certain distribution of profiles in the plane; (3) methods to identify subgroups of profiles based on their different spatial or optical density; and (4) methods to compare the distributions of two or more groups of profiles. The applications of these general tools to some neuroanatomical problems, such as cell group definition and description, have been illustrated. Practical examples performed on immunocytochemical preparations of neuronal profile populations are also given. Finally, the potentiality of numerical classification to classify and compare morphometric data has been shown. As an example, numerical classification methods have been applied to the morphometric and microdensitometric analysis of adrenaline/neuropeptide Y costoring neuronal systems of the brainstem in adult and aged rats.

Using a specific antiserum raised against synthetic neuropeptide Y, we examined the localization of immunoreactivity in the brain and hypophysis of the cloudy dogfish, Scyliorhinus torazame, by the peroxidase-antiperoxidase method. Immunoreactive perikarya were demonstrated in the ganglion of the nervus terminalis, the dorsocaudal portions of the pallium dorsale, the basal telencephalon, and the nucleus lateralis tuberis and the nucleus lobi lateralis in the hypothalamus. Labeled perikarya were also found in the tegmentum mesencephali, the corpus cerebelli, and the medulla oblongata. Some of the immunoreactive neurons in the hypothalamus were of the CSF-contacting type. The bulk of the labeled fibers in the nervus terminalis ran toward the basal telencephalon, showing radial projections and ramifications. Large numbers of these fibers coursed into the nucleus septi caudoventralis and the nucleus interstitialis commissurae anterioris, where they became varicose and occasionally formed fine networks or invested immunonegative perikarya. In the diencephalon, immunoreactive fibers were observed throughout the hypothalamus, e.g., in the pars neurointermedia of the hypophysis, the subependymal layer of the lobus inferior hypothalami, and in the neuropil of the posterior (mammillary) recess organ. Labeled fibers were scattered throughout the rest of the brain stem and were also seen in the granular layer of the cerebellum. These results suggest that, in the dogfish brain, neuropeptide Y or a related substance is involved in a variety of physiological processes in the brain, including the neuroendocrine control of the hypophysis.

We have previously shown that the serotonergic regulation of renin secretion from the kidneys is mediated by a renin-releasing factor (RRF) that is present in both plasma and hypothalamus. The present studies were designed to determine the distribution of RRF in the brain and peripheral tissues and to test whether RRF release could be stimulated in vitro from hypothalamo-hypophyseal explants. RRF levels were determined in vitro by measuring renin release from kidney cortical slices. Addition of hypothalamic extract to rat kidney slices produced a dose-dependent increase in renin release. RRF was measurable in most brain areas with the highest renin-releasing activity in the hypothalamus, cerebral cortex, medulla oblongata and cerebellum. To determine which brain regions contain RRF cell bodies, rats received an intracerebroventricular injection of colchicine to inhibit axonal transport and concentrate RRF in the perikarya. After colchicine treatment, RRF activity in the cerebral cortex, medulla oblongata and cerebellum decreased. In contrast, the hypothalamus had increased RRF activity suggesting that RRF cell bodies are localized in the hypothalamus. Superfusion of hypothalamo-hypophyseal explants with a high potassium Krebs-Ringer solution stimulated RRF release, suggesting that depolarization of hypothalamic neurons can stimulate RRF secretion. Nephrectomy produced a significant increase in RRF concentration in the hypothalamus, suggesting that RRF neurons respond to decreased renin activity or other kidney-related substances in the circulation. The determination of RRF in peripheral tissue revealed minimal renin-releasing activity in the liver, spleen and skeletal muscle extracts. High performance chromatography of hypothalamic extract on a GPC-100 column revealed RRF activity in fractions that were estimated to have a molecular weight of 5,000. These studies suggest that RRF-containing cell bodies in the hypothalamus respond to depolarization by releasing RRF into the circulation. In addition, the hypothalamic content of RRF is regulated by the kidney. Altogether, these data suggest that RRF neurons are part of a neuroendocrine system that regulates renin secretion from the kidneys.

Further immunocytochemical analysis of the neuroblasts with SRIF-like immunoreactivity (ir) was carried out on the chick embryo medulla and pons. 5 or 100 microns rombencephalon sections were obtained from 60 White Leghorn chick embryos at stages (E = Embryonic days) ranging from E4 1/2 to E18 and incubated with rabbit polyclonal antibodies against synthetic cyclic Somatostatin-14, according to PAP-DAB technique. In the medulla and pons the ir appeared as from E12. From E12 to E13 1/2-E14 the ir distribution gradually changed. From E14 to E18 numbers and spatial arrangement of the positive neuroblast groups did not show substantial changes; in these respects the ir distributional pattern proved to be markedly different from the one observed by the Authors in adult animals. Moreover, from E13 to E15 the positive neuroblast density appeared to be higher than that of positive neurons in adults. These results are consistent with a possible SRIF local regulative role.

The effects of the local application of drugs acting on 5-HT2 receptors in the nucleus tractus solitarius (NTS) on the heart rate and blood pressure were investigated in normal and nodose ganglionectomized anaesthetized rats. The unilateral micro-injection of an agonist such as 2,5-dimethoxy-3-bromo-amphetamine (DOB) (0.1-0.5 pmol) or 2,5-dimethoxy-3-nitroamphetamine (DON) (0.1-0.5 pmol) produced a dose-dependent hypotension and bradycardia in both intact and ganglionectomized animals. These cardiovascular effects were similar to those observed after the unilateral micro-injection of low doses (pmol) of 5-HT, and could be prevented by the prior micro-injections of the 5-HT2 antagonists ketanserin, ritanserin and piremperone. These findings support the hypothesis that 5-HT2 receptors within the NTS play a role in the reflex regulation of blood pressure. In addition, it was also observed that the micro-injection of subthreshold doses of 5-HT or DOB significantly enhanced the hypotension and bradycardia produced by the unilateral micro-injection of N-methyl-D-aspartate (NMDA). The potentiation of NMDA depressor effects by 5-HT or DOB could be totally prevented by ketanserin or piremperone, suggesting that 5-HT acting upon 5-HT2 receptors in the NTS may intervene in the reflex control of blood pressure by modulating the glutamatergic transmission.

The central cardiovascular and dipsogenic effects of angiotensin II involve interactions with norepinephrine, dopamine, and serotonin. Our findings that angiotensin II receptors and substance P immunoreactivity show a parallel distribution in the dorsal medulla and that angiotensin II releases substance P from perfused rat medulla slices revealed the potential for a functional relation between these peptidergic systems as well. Additional evidence suggests that the heptapeptide angiotensin-(1-7) exerts its biological activities via selective angiotensin receptor subtypes. Thus, we compared the effects of these two peptides on release of substance P and monoamines in perfused slices of medulla and hypothalamus from 77 male Sprague-Dawley rats. Transmitter levels were determined in 6-minute collections of perfusate before (basal), during (experimental), and after (recovery) perfusion with either angiotensin-(1-7), angiotensin II, or Krebs' solution alone (control). Substance P was measured by radioimmunoassay and monoamines and their metabolites by high-performance liquid chromatography with electrochemical detection. In the medulla, 2 microM angiotensin II but not angiotensin-(1-7) significantly increased efflux of substance P (221 +/- 87% of basal) and norepinephrine (130 +/- 17% of basal) during the experimental period. The effect of angiotensin II on substance P was sustained into the recovery period. Dopamine and its metabolite 3,4-dihydroxyphenylacetic acid were not detected in this brain region. In the hypothalamus, both angiotensin-(1-7) and angiotensin II increased substance P (169 +/- 30% and 141 +/- 35% of basal, respectively); the effect of angiotensin II was sustained throughout the recovery period.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutamate immunoreactivity was found in 19% and 21% of the neurons of the central autonomic nuclei projecting into the subretrofacial nucleus (SRF) in the rostral ventrolateral medulla of Wistar-Kyoto rat (WKY) and spontaneously hypertensive rat (SHR), respectively, using a double-labeling technique in combination with glutamate immunocytochemistry. Double-labeled neurons were distributed in 22 nuclei or subnuclei in the limbic system, hypothalamus, midbrain, pons and medulla. The average number of glutamate-immunoreactive neurons per thousand in SHR was significantly higher in the ipsilateral lateral parabrachial nucleus (P less than 0.05) and Koelliker-Fuse nucleus (P less than 0.01) than in WKY, while it was significantly lower in the ipsilateral medial subnucleus (P less than 0.05) and the commissure subnucleus (P less than 0.05) of the nucleus tractus solitarii in SHR than in WKY. The results indicate that: (1) glutamate-immunoreactive neurons (possibly glutamatergic) in many central autonomic nuclei project into the sympathetic vasomotor control neurons in the SRF; (2) the large population of glutamate-immunoreactive neurons in the lateral parabrachial nucleus and the Koelliker-Fuse nucleus of SHR is likely to increase excitatory inputs to the SRF vasomotor control neurons, while the smaller population of glutamate-immunoreactive neurons in the medial and commissure subnuclei of the nucleus tractus solitarii is likely to decrease excitatory inputs to the GABAergic neurons intrinsic to the SRF.

Immunoradiometrical determinations of beta-endorphin (beta-EP) levels in 29 discrete brain regions from a series of victims of "Sudden Infant Death Syndrome" yielded a uniformly low levels profile in various areas of telencephalon, thalamus, pons, cerebellum and medulla oblongata. This low levels profile was interrupted by intermediate and high beta-EP levels in the midbrain and in two diencephalic zones. This study provides, for the first time, a comprehensive, neurochemically determined regional profile of beta-EP levels in the brain of the human infant.

An important step in the posttranslational modification of many bioactive neuropeptides, the carboxy-terminal amidation of glycine-extended peptides, is catalyzed by peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3). The expression of the gene encoding this enzyme was examined in adult rat brain by in situ hybridization histochemistry and immunocytochemistry. PAM mRNA transcripts and PAM-like immunoreactivity were detected in all major brain areas with the exception of the cerebellum. Very high levels of PAM mRNAs were found in the hypothalamic magnocellular neurons, the hippocampal formation, and olfactory cortex. These areas also showed strong PAM-like immunoreactivity. Regions known to contain high levels of amidated neuropeptides also expressed high levels of PAM mRNA. The observed heterogeneous PAM mRNA levels may reflect differences in the peptidergic activity of different neuronal systems. Interestingly, all pyramidal neurons of the hippocampus expressed very high levels of PAM mRNA, although no identified amidated peptide matches this distribution completely. Furthermore, PAM was not expressed exclusively in neuronal tissue but was also present in non-neuronal tissue. PAM transcripts could be localized in certain ventricular ependymal cells, with the highest expression in the lateral ventricle. Localization of PAM to non-neuronal cells and neurons not known to produce alpha-amidated peptides suggests that these cells may be producing as yet unidentified amidated neuropeptides.

The effects of whole body microwave exposure on the central nervous system (CNS) of the rat were investigated. Rats weighing from 250 to 320 g were exposed for 1 h to whole body microwave with a frequency of 2450 MHz at power densities of 5 and 10 mW.cm-2 at an ambient temperature of 21-23 degrees C. The rectal temperatures of the rats were measured just before and after microwave exposure and mono-amines and their metabolites in various discrete brain regions were determined after microwave exposure. Microwave exposure at power densities of 5 and 10 mW.cm-2 increased the mean rectal temperature by 2.3 degrees C and 3.4 degrees C, respectively. The noradrenaline content in the hypothalamus was significantly reduced after microwave exposure at a power density of 10 mW.cm-2. There were no differences in the dopamine (DA) content of any region of the brain between microwave exposed rats and control rats. The dihydroxyphenyl acetic acid (DOPAC) content, the main metabolite of DA, was significantly increased in the pons plus medulla oblongata only at a power density of 10 mW.cm-2. The DA turnover rates, the DOPAC:DA ratio, in the striatum and cerebral cortex were significantly increased only at a power density of 10 mW.cm-2. The serotonin (5-hydroxytryptamine, 5-HT) content in all regions of the brain of microwave exposed rats was not different from that of the control rats. The 5-hydroxyindoleacetic acid (5-HIAA) content in the cerebral cortex of microwave exposed rats was significantly increased at power densities of 5 and 10 mW.cm-2.(ABSTRACT TRUNCATED AT 250 WORDS)

Beta-endorphin (BE) and neurotensin (NT) are two neuropeptides which induce apneas. In infants who died of Sudden Infant Death Syndrome (SIDS) we measured, in brainstem and CSF, BE and NT by IRMA and RIA respectively. BE and NT levels are compared to same aged infant and adult controls. CSF BE level was significantly higher in SIDS than in the two control groups (86 +/- 14 vs 33 +/- 13 and 16 +/- 5 pmol/l). In 6 SIDS victims NT and BE were assayed in 5 brainstem sections, each of them divided in median, intermediate and lateral parts. We found high levels of BE in every fragment (3-11 pmol/mg protein) while NT elevated values were restricted to the mesencephalic regions (1.4-12 pmol/mg), the medial pons (6 pmol/mg) and the intermediate parts of the medulla (including the olive: 1.3-1.6 pmol/mg). These results support the hypothesis that NT and/or BE could induce or participate to the fetal issue of SIDS.

The localization of acidic fibroblast growth factor (aFGF) in the male mouse brain was studied with biochemical and immunocytochemical techniques. Using two peptide-based aFGF antisera directed against independent epitopes, Western gel analysis of dissected brain demonstrated significant levels of aFGF immunoreactivity in the pons-medulla, hypothalamus and cerebellum. The cortex contained much less immunoreactivity. Consistent with the biochemical data, immunocytochemical analysis with the same two antisera demonstrated that aFGF immunoreactivity is localized in neuronal cell bodies in these regions. Numerous immunoreactive neurons were observed in the reticular formation of the pons and medulla, as well as in several other brainstem nuclei and areas. Immunoreactive neurons were also present in the lateral and medial hypothalamus, and some thalamic, subthalamic and epithalamic nuclei. In the basal ganglia, immunoreactive neurons were present in the amygdala and septum. Few intensely stained immunoreactive neurons were observed in the striatum, pallidum and neocortex. Limbic cortices contained more numerous immunoreactive neurons than neocortex. These results support the concept that aFGF is present in the brain, where it is heterogeneously distributed in neuronal cell bodies in regions involved in sensory, extrapyramidal motor, limbic and autonomic functions. The results are consistent with various neurotrophic, mitogenic, and neuromodulatory functions associated with aFGF in the mammalian central nervous system.

The detailed anatomical distribution of iron in the post-mortem human brain has been studied using Perl's and Turnbull's methods with the diaminobenzidine intensification procedure for the demonstration of non-haem Fe3+ and Fe2+, respectively. Attention to methodological procedures has revealed that even brief immersion of tissue in routinely used fixatives causes a reduction of staining intensity in areas of high iron content and, often, loss of staining in areas of low iron content. Optimal staining is obtained using frozen section briefly fixed for 5 min in 4% formalin and Perl's stain (Fe3+) with diaminobenzidine intensification. Highest levels of stainable iron were found in the extrapyramidal system with the globus pallidus, substantia nigra zona reticulata, red nucleus and myelinated fibres of the putamen showing highest staining reactivity. Moderate staining intensity with Perl's technique was found in the majority of forebrain, midbrain and cerebellar structures with the striatum, thalamus, cortex and deep white matter, substantia nigra zona compacta, and cerebellar cortex showing consistent staining patterns with intensification of Perl's stain. The brain-stem and spinal cord generally only showed staining with the intensification procedure and even this was of low intensity. Microscopically the non-heam iron appears to be found predominantly in glial cells as fine cytoplasmic granules which in heavily stained areas coalesce to fill the entire cell. Iron-positive granules appear to be free in the neuropil and also around blood vessels in the globus pallidus, striatum and substantia nigra. The neuropil shows a fibrous impregnation when stained for iron which is, in part, derived from glial processes, myelinated fibres and fibre bundles. Neurones, in general, show only very low reactivity for iron, and this is difficult to discern due, often, to the higher reactivity of the surrounding neuropil. In the globus pallidus and substantia nigra zona reticulata, neurones with highly stainable iron content are found with granular cytoplasmic iron reactivity similar to that seen in the local glial cells. Our results are comparable with those of early workers, but with the use of intensification extend the distribution of non-haem iron to areas previously reported as negative. No apparent correlation of iron staining with known neurotransmitter systems is seen and the predilection for the extrapyramidal system is not easily explained, though the non-haem iron in the brain appears to be as a storage form in the iron storage protein ferritin. The localization of iron in the brain provides a foundation for the study of iron in certain neurodegenerative diseases such as Parkinson's disease, where iron has been implicated in the pathogenesis.

GABA-synthesizing neurons were identified in the medulla of the rat by peroxidase-antiperoxidase (PAP) immunohistochemistry for glutamic acid decarboxylase (GAD). Using diaminobenzidine (DAB) either alone or intensified with silver, a relatively large number of GAD-immunoreactive neurons were evident within the reticular formation, raphe nuclei and vestibular nuclei. In all these areas, profuse GAD-immunoreactive varicosities appeared to contact the soma and dendrites of both non-GABA and GABA neurons. These observations suggest that GABA neurons may act as interneurons or local projection neurons within the medulla and accordingly exert a potent inhibitory and/or disinhibitory control on bulbar projection neurons. Within the ventral reticular formation (pars alpha and ventralis of the gigantocellular reticular field) and raphe magnus, large numbers of prominent GAD-immunoreactive neurons resembled in size and morphology and overlapped in distribution the serotonin-immunoreactive neurons of the same regions. However, by sequential double immunostaining utilizing DAB as a chromogen for serotonin (5-HT) and benzidine dihydrochloride (BDHC) for GAD, it was found that GAD-containing neurons were distinct from 5-HT-containing neurons. Following injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the upper cervical spinal cord and combined processing for WGA-HRP (using tetramethylbenzidine [TMB] with cobalt) and immunohistochemistry (with DAB), a contingent of spinally projecting neurons were found to contain GAD. The GAD-immunoreactive reticulo- and raphe-spinal neurons were most frequent within the pars alpha and ventralis of the gigantocellular reticular fields and the raphe magnus, where they were approximately equal in number to the coexistent, but distinct 5-HT spinally projecting neurons. GABA neurons of the medulla may thus contribute directly to the bulbar inhibitory influence upon spinal sensory and motor systems.

Somatostatin-14-like immunoreactivity (S14LI) and somatostatin-28(1-12)-like immunoreactivity (S28(1-12)LI) in the brain of LEC (Long Evans Cinnamon) rats with hepatic encephalopathy were measured. Significant reduction of both S14LI and S28(1-12)LI was observed in the hypothalamus, medulla oblongata, striatum and spinal cord. Both of the immunoreactivities in the hypothalamus of these rats were approx. 50% of those in LEC rats without hepatic encephalopathy. The amounts of reduction of S14LI significantly correlated with those of reduction of S28(1-12)LI. No significant difference in gel chromatographic profiles of S14LI and S28(1-12)LI was observed between LEC rats with and without hepatic encephalopathy. These results suggest that the reduction of somatostatin-like immunoreactivity in LEC rats with hepatic encephalopathy may be caused by a decrease in production of prosomatostatin rather than altered degradation.

Thyrotropin-releasing hormone-immunoreactive nerve terminals heavily innervate the dorsal motor nucleus and nucleus of the solitary tract, whereas cell bodies containing thyrotropin-releasing hormone residue most densely in the hypothalamus and raphe nuclei. By using double-labeling techniques accomplished by retrograde transport of Fluoro-Gold following microinjection into the dorsal motor nucleus/nucleus of the solitary tract combined with immunohistochemistry for thyrotropin-releasing hormone, it was demonstrated that thyrotropin-releasing hormone-immunoreactive neurons projecting to the dorsal motor nucleus/nucleus of the solitary tract reside in the nucleus raphe pallidus, nucleus raphe obscurus, and the parapyramidal region of the ventral medulla, but not in the paraventricular nucleus of the hypothalamus. The parapyramidal region includes an area along the ventral surface of the caudal medulla, lateral to the pyramidal tract and inferior olivary nucleus and ventromedial to the lateral reticular nucleus. Varying the position of the Fluoro-Gold injection site revealed a rostral to caudal topographic organization of these raphe and parapyramidal projections.

The nucleus tractus solitarii (NTS) has long been considered as the first central relay for gustatory and visceral afferent informations only. However, data obtained during the past ten years, with neuroanatomical, biochemical and electrophysiological techniques, clearly demonstrate that the NTS is a structure with a high degree of complexity, which plays, at the medullary level, a key role in several integrative processes. The NTS, located in the dorsomedial medulla, is a structure of small size containing a limited number of neurons scattered in a more or less dense fibrillar plexus. The distribution and the organization of both the cells and the fibrillar network are not homogeneous within the nucleus and the NTS has been divided cytoarchitectonically into various subnuclei, which are partly correlated with the areas of projection of peripheral afferent endings. At the ultrastructural level, the NTS shows several complex synaptic arrangements in form of glomeruli. These arrangements provide morphological substrates for complex mechanisms of intercellular communication within the NTS. The NTS is not only the site of vagal and glossopharyngeal afferent projections, it receives also endings from facial and trigeminal nerves as well as from some renal afferents. Gustatory and somatic afferents from the oropharyngeal region project with a crude somatotopy within the rostral part of the NTS and visceral afferents from cardiovascular, digestive, respiratory and renal systems terminate viscero-topically within its caudal part. Moreover the NTS is extensively connected with several central structures. It projects directly to multiple brain regions by means of short connections to bulbo-ponto-mesencephalic structures (parabrachial nucleus, motor nuclei of several cranial nerves, ventro-lateral reticular formation, raphe nuclei...) and long connections to the spinal cord and diencephalic and telencephalic structures, in particular the hypothalamus and some limbic structures. The NTS is also the recipient of several central afferent inputs. It is worth to note that most of the structures that receive a direct projection from the NTS project back to the nucleus. Direct projections from the cerebral cortex to the NTS have also been identified. These extensive connections indicate that the NTS is a key structure for autonomic and neuroendocrine functions as well as for integration of somatic and autonomic responses in certain behaviors. The NTS contains a great diversity of neuroactive substances. Indeed, most of the substances identified within the central nervous system have also been detected in the NTS and may act, at this level, as classical transmitters and/or neuromodulators.(ABSTRACT TRUNCATED AT 400 WORDS)

The cellular distribution of parvalbumin-like immunoreactivity (PA-LI) in the human brain was investigated by peroxidase-antiperoxidase methods using antiserum to rat skeletal muscle parvalbumin. PA-LI was present in non-pyramidal neurons of the cerebral cortices, stellate cells, basket cells and Purkinje cells in cerebellar cortices, and neurons of some nuclei in human brain stem; the distribution of PA-LI in human brain was very similar to that in rat brain. These results indicate that PA-LI is widely distributed in a specific subpopulation of neurons of the human brain.

Amino acid sequence of human C-type natriuretic peptide (CNP) has recently been deduced to be identical to those of porcine and rat CNPs in the bioactive unit of C-terminal 22 residues (CNP-22) (1). Thus, tissue concentrations and molecular forms of immunoreactive (ir-) CNP in human brain and heart were determined or characterized using a radioimmunoassay established for porcine CNP. In human brain (hypothalamus and medullapons), ir-CNP was detected at a concentration of 1.04 pmol/g, being about 25 times or 70 times higher than ir-atrial (A-type) natriuretic peptide (ANP) or ir-brain (B-type) natriuretic peptide (BNP). CNP was present mainly as CNP-53, with CNP-22 as well as 13K CNP (presumed to be pro-CNP) as minor components. In heart, 1 approximately 5 pmol/g of ir-CNP was detected in both atrium and ventricle, but this ir-CNP was shown to be derived from crossreactivity of ANP. These results demonstrated that human CNP functions exclusively in the central nervous system in contrast to ANP and BNP which mainly function in the circulation system.

gamma-Aminobutyric acid (GABA) and glycine are major inhibitory neurotransmitters that are released from nerve terminals by exocytosis via synaptic vesicles. Here we report that synaptic vesicles immunoisolated from rat cerebral cortex contain high amounts of GABA in addition to glutamate. Synaptic vesicles from the rat medulla oblongata also contain glycine and exhibit a higher GABA and a lower glutamate concentration than cortical vesicles. No other amino acids were detected. In addition, the uptake activities of synaptic vesicles for GABA and glycine were compared. Both were very similar with respect to substrate affinity and specificity, bioenergetic properties, and regional distribution. We conclude that GABA, glycine, and glutamate are the only major amino acid neurotransmitters stored in synaptic vesicles and that GABA and glycine are transported by similar, if not identical, transporters.

Our study investigates the distribution of neurophysins (Nph), proteins that are part of the precursors for vasopressin and oxytocin, and calcitonin gene-related peptide (CGRP) in the human brainstem by immunohistochemistry. Both peptides were found in discrete regions of the human hindbrain. Nph could be demonstrated exclusively in fibers and punctate perineural varicosities that were travelling within the mesencephalic central gray, substantia nigra, as well as locus coeruleus, medial longitudinal fascicle, raphe, nucleus of the solitary tract, lateral reticular nucleus and area postrema. A few varicosities were seen in the substantia gelatinosa of the spinal trigeminal tract and its continuation into the dorsal horn of the cervical spinal cord. In contrast to these observations. CGRP-immunoreactive fibers were found to be densest in the spinal tract of the trigeminal nerve and the dorsal horn of the spinal cord. In addition, fibers and varicosities could be demonstrated in numerous distinct brain regions, such as locus coeruleus and subcoeruleus, solitary tract, cuneate nucleus, raphe and periaqueductal gray. CGRP-immunoreactivity was also present in perikarya in the ventral horn of the spinal cord, as well as motor nuclei of cranial nerves, i.e., hypoglossal nucleus, ambiguous nucleus. Our results suggest that Nph-immunoreactivity in the human brainstem may be present predominantly within long fiber projections from hypothalamic neurosecretory nuclei, in analogy to data obtained from rodents, whereas CGRP may play a role in the branchiomotor system as well as in intrinsic or extrinsic projections involved in autonomic regulation and integration of sensory information.

The immunohistochemical localization of P400/inositol 1,4,5-trisphosphate (InsP3) receptor protein was studied in developing and adult mouse brain by using monoclonal antibodies. The developmental expression pattern of P400/InsP3 receptor protein differed among different classes of neurons. It was first detected in the somata of immature Purkinje cells at embryonic day 17, in the ventrolateral region of the posterior vermis in the cerebellum. Axonal immunoreactivity within the cerebellar nuclei was first present at postnatal day 3. Neurons in the retrosplenial cortex, the anterior olfactory nucleus, and the CA1 region of the hippocampus expressed immunoreactivity earlier than other regions of the brain. In the adult brain, not only the Purkinje cell but also many other types of cells in many areas of the brain expressed P400/InsP3 receptor, though to a lesser extent. These included the neurons in the striatum, globus pallidus, nucleus accumbens septi, anterior olfactory nucleus, olfactory tubercle, precommissural hippocampus, hippocampus, substantia nigra, cerebral cortex, pons, and certain hypothalamic nuclei. Forebrain cortical regions that receive afferents from the olfactory bulb, such as the anterior olfactory nucleus, olfactory tubercle, prepiriform cortex, entorhinal cortex, and amygdala, exhibited distinct immunoreactivity, while olfactory bulb was almost devoid of staining. Immunoreactivity in the axonal pathways was also found in the limbic-hypothalamic pathways, strionigral projection, and part of the corpus callosum. Results of Western blot analysis and 3H-InsP3 binding assay were consistent with the qualitative regional differences of immunoreactivity demonstrated by immunohistochemical study. The location of InsP3 receptor in the brain correlates well with the InsP3 binding sites demonstrated by an autoradiographic study.

The organization of the neuroanatomical substrate which provides the supraspinal catecholaminergic innervation of the upper thoracic spinal cord in the rat was studied by means of retrograde labelling of neurons with primuline and other dyes, combined with simultaneous catecholamine fluorescence (FAGLU-method). It was shown that spinally projecting catecholaminergic neurons lie predominantly in the A6 and A11 groups, while a minority of these neurons are also located in the A1/C1 and A5 groups. The number of catecholamine-containing neurons in the A11 group in the rat was estimated to be 173 +/- 4. Most of them (up to 86%) were retrogradely labelled with primuline. The possible functional role of dopaminergic diencephalospinal, noradrenergic pontospinal, and adrenergic bulbospinal neuronal systems in cardiovascular control is discussed.

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

Heterogeneity of rat brain angiotensin II receptors was revealed by quantitative autoradiography after incubation with 125I-Sar1-angiotensin II and displacement with the angiotensin II antagonists CGP 42112 A and DuP-753 and by receptor sensitivity to dithiothreitol. Receptors in areas involved in cardiovascular and fluid control--the subfornical organ, nucleus of the solitary tract, paraventricular nucleus, and area postrema--are displaced by DuP-753 with an IC50 of 1 x 10(-7) M, are sensitive to 5 mM dithiothreitol, and thus are angiotensin II type-1. Receptors in the inferior olive are displaced by CGP 42112 A (IC50, 1 x 10(-9) M) but not by DuP-753 in concentrations up to 10(-4) M, are insensitive to 5 mM dithiothreitol, and thus are angiotensin II type-2.

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

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

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

The fact that GABA receptor agonists and antagonists influence nociceptive thresholds when microinjected into the rostroventral medulla or in the spinal cord may reflect the involvement of GABAergic neuronal elements in endogenous antinociceptive pathways. In the present study we used immunocytochemistry and retrograde tract tracing to investigate the contribution of GABAergic projection neurons to the antinociceptive network linking the midbrain periaqueductal gray matter (PAG), the nucleus raphe magnus (NRM), and the spinal cord dorsal horn. The tracer, WGAapoHRP-Au was injected into either the NRM or the spinal cord and the distribution of labeled neurons in sections of the PAG and medulla, respectively, was studied. The same sections were immunostained to demonstrate GABA-immunoreactive neurons. Although GABA-immunoreactive neurons were abundant in the PAG, only 1.5% were retrogradely labeled from the NRM. Similarly, very few GABA-immunoreactive neurons within the cytoarchitectural boundaries of the NRM were retrogradely labeled from the spinal cord. A much higher proportion of GABA-immunoreactive neurons in the region lateral to the NRM, however, were retrogradely labeled from the spinal cord. Eighteen percent of GABA-immunoreactive neurons were retrogradely labeled in the nucleus reticularis paragigantocellularis; conversely, 15% of the retrogradely labeled neurons in this region were GABA-immunoreactive. These results indicate that GABAergic projections constitute a very minor component of the PAG-NRM-spinal cord pathway; however, there is a significant contribution of GABAergic neurons to the spinal projections that originate lateral to the NRM. The majority of GABAergic neurons in the PAG and NRM are presumed to be inhibitory interneurons that directly or indirectly regulate activity in efferent pathways from these regions.

We studied the ultrastructure and the synaptic arrangement of glutamate-immunoreactive terminals in rats, in the superficial laminae of the spinal cord, the brainstem cuneate nucleus, and the thalamic ventroposterolateral nucleus, where a role for glutamate as neurotransmitter has been suggested by biochemical, physiological and pharmacological approaches. The antiserum employed was raised against glutaramate conjugated to keyhole limpet hemocyanin with glutaraldehyde, and was used for pre-embedding staining with an avidin-biotin-peroxidase method and for post-embedding staining with an immunogold procedure. Both methods yielded similar results, consisting of labeling of selected terminals in all the areas examined. Double immunogold labeling on the same thin section using antisera against gamma-amino-butyric acid (GABA) or substance P (SP), in combination with the anti-glutamate serum, showed that staining for glutamate and GABA was present in different terminals in all the regions examined; glutamate and SP were co-localized in a few terminals only in the superficial laminae of the spinal cord. By performing immunogold staining in combination with anterograde tracing, glutamate immunoreactivity could be localized in identified primary afferents to the dorsal spinal cord and cuneate nucleus, and in lemniscal afferents to the thalamus.

By the use of the indirect immunofluorescence and in situ hybridization techniques, the distribution of calcitonin gene-related peptide (CGRP)-like immunoreactivity (LI) and CGRP mRNA was studied in the spinal cord as well as in the midline raphe nuclei and the hypoglossal nucleus in the medulla oblongata of the monkey (Macaca fascicularis). In the spinal cord only a few large neurons in the motor nucleus contained CGRP-LI, while a majority of the neurons in the hypoglossal nucleus contained CGRP-LI. A relatively dense innervation by CGRP-immunoreactive (IR) fibers was also seen close to cell bodies and proximal dendrites of large neurons in the motor nucleus, especially in its ventral part. 5-Hydroxytryptamine (5-HT)-, substance P- and thyrotropin-releasing hormone (TRH)-IR varicosities were also observed in a similar position around large neurons in the motor nucleus. Double labeling disclosed that the majority of CGRP-IR axon terminals also contained 5-HT-LI. Expression of CGRP mRNA was found in neurons in the medullary midline raphe nuclei and in large neurons in the motor nucleus at the cervical spinal cord level. In adjacent sections of the medulla oblongata, CGRP-labeled neurons in the midline raphe nuclei also expressed preprotachykinin mRNA. The present results show that CGRP- and 5-HT-LI coexist in fibers within the motor nucleus of the monkey spinal cord and that this coexistence is probably due to the presence of CGRP in the descending bulbospinal, serotonergic pathway.

The diagnosis of feline alpha-mannosidosis is made by demonstrating deficient activity of the enzyme alpha-mannosidase or an elevation of its undergraded substrate in body fluids or tissue. In this study the storage of specific sugar residues in the brain and spinal cord in a case of feline alpha-mannosidosis was examined by means of nine different biotinylated lectins and the avidin-biotin-peroxidase method on formalin-fixed, paraffin-embedded tissue sections. The lectin staining pattern strongly correlated with the known biochemical findings of the stored oligosaccharides in feline alpha-mannosidosis and was different from the lectin reactivity of normal cat tissues. This confirms that lectin histochemistry is a simple, relatively inexpensive and reliable method for diagnosing alpha-mannosidosis in cats.

We have developed sensitive and specific radioimmunoassays (RIA) for salmon gonadotropin-releasing hormone (sGnRH) and chicken GnRH-II (cGnRH-II). Synthetic sGnRH and cGnRH-II(2-10) were conjugated to bovine serum albumin and injected into rabbits to raise specific antisera. The antiserum against sGnRH showed cross-reactivities of 1.58 and 0.08% for cGnRH-II and lamprey GnRH, respectively. The antiserum against cGnRH-II showed cross-reactivities of 0.05 and 0.01% for sGnRH and lamprey GnRH, respectively. Both antisera were observed not to cross-react with mammalian GnRH and cGnRH-I or other peptide hormones. Synthetic sGnRH and cGnRH-II were iodinated using the chloramine-T method. The iodinated GnRH was purified by HPLC using a reverse-phase C18 column. The RIA system was developed as a double antibody method. Brain extracts of rainbow trout showed displacement curves which were parallel to the sGnRH and cGnRH-II standards in each RIA. HPLC analysis followed by RIA has revealed that rainbow trout brain contains two types of GnRH: sGnRH and cGnRH-II. Total sGnRH content in the brain was about three-fold higher than that of cGnRH-II. In the olfactory bulbs, telencephalon, optic tectum-thalamus, hypothalamus, and pituitary, sGnRH content (per region) was higher than cGnRH-II content, whereas cerebellum and medulla oblongata contained much more cGnRH-II than sGnRH. sGnRH content in the optic tectum-thalamus and pituitary was the highest in 1-year-old immature fish and 3-year-old mature fish, respectively. Medulla oblongata showed the highest cGnRH-II content in all groups. sGnRH concentrations (per milligram of protein) were high in the pituitary and intermediate in the olfactory bulbs, hypothalamus, and telencephalon. In all groups, the cGnRH-II concentration was high in the medulla oblongata, whereas the concentration in the olfactory bulbs and pituitary gland was below the detectable limit in most individuals.

Distribution of dynorphin A (DyA) immunoreactivity in the nucleus of the solitary tract (NTS) was examined in rats after various surgical transections by light and electron microscopic immunohistochemistry. In colchicine-treated animals DyA immunostained perikarya were seen in each subdivision of the NTS. In intact rats, dense network of immunopositive nerve fibers was localized light microscopically, and synaptic contacts were found between DyA immunopositive structures (axo-axonic, axo-dendritic synapses), electron microscopically. Surgical transections medial, caudal or rostral to the nucleus did not alter the distribution pattern of DyA in the NTS. Lesion immediately lateral to the nucleus resulted in an ipsilateral appearance of immunostained cell bodies. Vagal and glossopharyngeal afferents (including baroreceptor fibers) terminate in the medial and commissural subnucleus of the NTS. Two days after extracranial vagotomy, synaptic contacts between degenerated presynaptic boutons and DyA immunopositive postsynaptic elements were observed in both medial and commissural part of the NTS. These observations provide morphological evidence suggesting that (1) axons of dynorphin A-containing cell bodies form an intrinsic network inside the nucleus; (2) these DyA cells receive direct peripheral inputs through the vagus nerve, and (3) projecting DyA neurons may exist in the NTS, they may innervate medullary, rather than forebrain, higher brainstem or spinal cord neurons.

The effect of methionine on cerebral content of dopamine, serotonin and major acidic metabolites was studied in distinct CDF-1 mouse brain regions. Methionine was administered 30 mg/kg for five trials over 24 h and mice were sacrificed 30 min post the terminal treatment. Methionine exerted differential effects on regional brain concentrations of 3,4-dihydroxyphenylacetic acid and serotonin. The results suggest methionine-mediated decreases of dopamine turnover in the hippocampus and medulla compared to increases of serotonin turnover in the cerebral cortex and midbrain regions. The results indicate a central action for methionine as related to endogenous neurotransmitters measured which may precede the abnormal O- or N-dimethylation ascribed to methionine and the insuing adverse effects postulated in schizophrenia.

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

An immunohistochemical study of MBP distribution in the brainstem of neonate till 16 d old rats based on the peroxidase-antiperoxidase method is described. Axons already invested with immunoreactive sheaths were found in neonate rats in the ventral funiculus of the cervical spinal cord and in the medial longitudinal fascicle of the medulla oblongata. Fibres commencing with myelination showed a closely spaced array of varicosities in longitudinal sections which diminished gradually. A caudo-rostral decrease in density of myelinated fibres in the brainstem was found in the medial and dorsal longitudinal fascicles. In contrast to other pathways, myelination in the fibres of the corticospinal tract in the brainstem occurred in a strictly synchronized pattern. The same temporal pattern of myelination was also observed in the cervical corticospinal tract, except that a few myelinated fibres had been visible much earlier within the area of the tract. At the exit of cranial nerves, the transitorial zone from central to peripheral myelin was outlined by a decrease in immunostaining.