Immunohistochemical localization of the cloned μ opioid receptor in the rat CNS

Three opioid receptor types have recently been cloned that correspond to the pharmacologically defined μ, δ and κ₁ receptors. In situ hybridization studies suggest that the opioid receptor mRNAs that encode these receptors have distinct distributions in the central nervous system that correlate well with their known functions. In the present study polyclonal antibodies were generated to the C terminal 63 amino acids of the cloned μ receptor (335–398) to examine the distribution of the μ receptor-like protein with immunohistochemical techniques. μ receptor-like immunoreactivity is widely distributed in the rat central nervous system with immunoreactive fibers and/or perikarya in such regions as the neocortex, the striatal patches and subcallosal streak, nucleus accumbens, lateral and medial septum, endopiriform nucleus, globus pallidus and ventral pallidum, amygdala, hippocampus, presubiculum, thalamic and hypothalamic nuclei, superior and inferior colliculi, central grey, substantia nigra, ventral tegmental area, interpeduncular nucleus, medial terminal nucleus of the accessory optic tract, raphe nuclei, nucleus of the solitary tract, spinal trigeminal nucleus, dorsal motor nucleus of vagus, the spinal cord and dorsal root ganglia. In addition, two major neuronal pathways, the fasciculus retroflexus and the stria terminalis, exhibit densely stained axonal fibers. While this distribution is in excellent agreement with the known μ receptor binding localization, a few regions, such as neocortex and cingulate cortex, basolateral amygdala, medial geniculate nucleus and the medial preoptic area fail to show a good correspondence. Several explanations are provided to interpret these results, and the anatomical and functional implications of these findings are discussed.     

Opposite alterations of NPFF1 and NPFF2 neuropeptide FF receptor density in the triple MOR/DOR/KOR-opioid receptor knockout mouse brains

Mice lacking the mu-delta-kappa-opioid receptor (MOR/DOR/KOR) genes and their corresponding wild-type littermates have been used to quantify NPFF(1) and NPFF(2) (neuropeptide FF) receptors by in vitro autoradiography in the central nervous tissues. Adjacent coronal sections were labelled with [125I]YVP ([125I]YVPNLPQRF-NH(2)) and [125I]EYF ([125I]EYWSLAAPQRF-NH(2)) as specific radioligands for NPFF(1) and NPFF(2) receptors, respectively. NPFF(2) receptors are predominantly expressed in both genotypes, but their density increases significantly in non cortical regions of mutant mice: 64% in the amygdaloid area, 89, 308, 1214 and 49% in the nucleus of the vertical limb of the diagonal band, substantia nigra, the vestibular nucleus and the spinal cord, respectively. In contrast, the density of the NPFF(1) subtype is lower than NPFF(2) in both genotypes and significantly decreased in some brain areas of mutant mice: -99, -90 and -90% in the nucleus of the vertical limb of the diagonal band, substantia nigra and the spinal cord, respectively. This study shows that mice lacking opioid receptors have brain region-dependent increases (NPFF(2)) and decreases (NPFF(1)) in NPFF receptors densities and suggests a different functional participation of each NPFF receptor subtype in the actions of opioids.     

Anti-opioid effects of neuropeptide FF receptors in the ventral tegmental area

The present study investigates the modulatory effects of neuropeptide FF (NPFF) receptors on the mesolimbic dopaminergic pathway controlled by opioid receptors. A stable NPFF₂ receptor agonist, dNPA, was injected into the ventral tegmental area (VTA) and the release of dopamine and serotonin within the nucleus accumbens (NAc), induced by intraperitoneal injection of morphine, was monitored using the brain microdialysis, in non-constrained rat. dNPA decreased systemic morphine-induced elevation of dopamine and serotonin metabolites within the NAc. Furthermore, co-injected with morphine into the VTA, NPFF inhibited morphine-induced stereotypy 60–120 min after the injection. This neurochemical and behavioural anti-opioid effect mediated by NPFF₂ receptors at the level of VTA suggests the involvement of NPFF in the rewarding effects of opiates on the mesolimbic dopamine system.     

Localization of glycine receptors in the rat central nervous system: an immunocytochemical analysis using monoclonal antibody

The localization of glycine receptors was immunocytochemically examined in the rat brain using a monoclonal antibody against the affinity-purified glycine receptor. Glycine receptors were concentrated in the lower brainstem, whereas no immunoreactivity was observed in the diencephalon and forebrain except in a few diencephalic nuclei. The highest density of receptors was found in the cranial motor nuclei, reticular formation, parabrachial area, dorsal and ventral cochlear nuclei, and dorsal and ventral tegmental nuclei. Differences were observed in the distribution of immunoreactive elements in the various brain regions. In the cerebellar cortex, the immunoreactivity was exclusively seen along the dendrites of the Purkinje cells. On the other hand, glycine receptors were detected on the cellular membrane of the soma of the cochlear nuclei, trigeminal motor nucleus, parabrachial area, lateral reticular nucleus, dorsal nucleus of the lateral lemniscus, cerebellar nuclei, trigeminal spinal nucleus, anterior horn and reticular formation. In other regions, the receptors were evenly distributed throughout the neuropil.     

Mu opioid receptors in rat ventral medulla: effects of endomorphin-1 on phrenic nerve activity

Anatomical and in vitro studies suggest that mu opioid receptors (MOR) on pre-B?tzinger complex neurons are responsible for opioid induced respiratory depression (Grey et al., Science 286 (1999) 1566). However, mu opioid agonists injected in vivo, in other regions of the ventral respiratory group (VRG), produce respiratory depression, suggesting that opioids are widely distributed in the VRG. We therefore re-examined the distribution of the MOR in the ventral medulla and found MOR-immunoreactive neurons and terminals in all subdivisions of the VRG. Furthermore, we determined, in rats, the effects of a MOR agonist (endomorphin-1, 10 mM, 60 nl, unilateral), microinjected into different subdivisions of the VRG, on phrenic nerve activity. Endomorphin-1 produced changes in phrenic nerve frequency and amplitude, throughout the VRG. Unexpectedly, endomorphin-1 microinjected into the B?tzinger and pre-B?tzinger complexes consistently increased phrenic nerve frequency. These results support the widespread distribution of MOR in the VRG and also indicate that endomorphin-1, a postulated endogenous ligand, may differentially regulate respiration.     

Distribution of SMI-32-immunoreactive neurons in the central auditory system of the rat

SMI-32 antibody recognizes a non-phosphorylated epitope of neurofilament proteins, which are thought to be necessary for the maintenance of large neurons with highly myelinated processes. We investigated the distribution and quantity of SMI-32-immunoreactive(-ir) neurons in individual parts of the rat auditory system. SMI-32-ir neurons were present in all auditory structures; however, in most regions they constituted only a minority of all neurons (10–30%). In the cochlear nuclei, a higher occurrence of SMI-32-ir neurons was found in the ventral cochlear nucleus. Within the superior olivary complex, SMI-32-ir cells were particularly abundant in the medial nucleus of the trapezoid body (MNTB), the only auditory region where SMI-32-ir neurons constituted an absolute majority of all neurons. In the inferior colliculus, a region with the highest total number of neurons among the rat auditory subcortical structures, the percentage of SMI-32-ir cells was, in contrast to the MNTB, very low. In the medial geniculate body, SMI-32-ir neurons were prevalent in the ventral division. At the cortical level, SMI-32-ir neurons were found mainly in layers III, V and VI. Within the auditory cortex, it was possible to distinguish the Te1, Te2 and Te3 areas on the basis of the variable numerical density and volumes of SMI-32-ir neurons, especially when the pyramidal cells of layer V were taken into account. SMI-32-ir neurons apparently form a representative subpopulation of neurons in all parts of the rat central auditory system and may belong to both the inhibitory and excitatory systems, depending on the particular brain region.     

Differential involvement of mu(1)-opioid receptors in endomorphin- and beta-endorphin-induced G-protein activation in the mouse pons/medulla

Several genetic mouse models of differential sensitivity to opioids have been used to investigate the mechanisms underlying individual variation in responses to opioids. The CXBK mice are inbred recombinant mice which have a lower level of mu(1)-opioid receptors than their parental strain. Endomorphin-1 and endomorphin-2 are endogenous opioid peptides that are highly selective for mu-opioid receptors, while beta-endorphin, which is also an endogenous opioid peptide, is non-selective for mu-, delta- and putative epsilon-opioid receptors. The present study was designed to investigate the effects of these endogenous opioid peptides on G-protein activation by monitoring guanosine-5'-o-(3-[35S]thio)triphosphate binding to pons/medulla membranes of CXBK mice and their parental strain C57BL/6 ByJ mice. Endomorphin-1 (0.1-10 microM), endomorphin-2 (0.1-10 microM) and beta-endorphin (0.1-10 microM) increased guanosine-5'-o-(3-[35S]thio)triphosphate binding to the pons/medulla membranes from C57BL/6 ByJ and CXBK mice in a concentration-dependent manner. However, the increases of guanosine-5'-o-(3-[35S]thio)triphosphate binding induced by either endomorphin-1 or endomorphin-2 in CXBK mice were significantly much lower than those in C57BL/6ByJ mice. However, no significant difference was found in the increases of the guanosine-5'-o-(3-[35S]thio)triphosphate binding induced by beta-endorphin in C57BL/6 ByJ and CXBK mice. Moreover, whereas the increase of guanosine-5'-o-(3-[35S]thio)triphosphate binding induced by 10 microM endomorphin-1 or endomorphin-2 were almost completely blocked by a mu-opioid receptor antagonist beta-funaltrexamine (10 microM) in both strains, the increase of guanosine-5'-o-(3-[35S]thio)triphosphate binding induced by 10 microM beta-endorphin was attenuated to approximately 70% of stimulation by co-incubation with 10 microM beta-funaltrexamine in both strains. The residual stimulation of [35S]guanosine-5'-o-(3-thio)triphosphate binding by 10 microM beta-endorphin in the presence of 10 microM beta-funaltrexamine was further attenuated by the addition of putative epsilon-opioid receptor partial agonist beta-endorphin (1-27) (1 microM) in both strains. Like the endomorphins, the synthetic mu-opioid receptor agonist [D-Ala(2),N-MePhe(4), Gly-ol(5)]enkephalin at 10 microM showed lower increases of guanosine-5'-o-(3-[35S]thio)triphosphate binding in CXBK mice than those in C57BL/6ByJ mice. However, there was no strain difference in the stimulation of guanosine-5'-o-(3-[35S]thio)triphosphate binding induced by 10 microM of the selective delta(1)-opioid receptor agonist [D-Pen(2,5)]enkephalin, delta(2)-opioid receptor agonist [D-Ala(2)]deltorphin II or kappa-opioid receptor agonist U50,488H. The results indicate that the G-protein activation by endomorphin-1 and endomorphin-2 in the mouse pons/medulla is mediated by both mu(1)- and mu(2)-opioid receptors. Moreover, beta-endorphin-induced G-protein activation in the mouse pons/medulla is, in part, mediated by mu(2)- and putative epsilon-, but not by mu(1)-opioid receptors.     

Differential involvement of μ1-opioid receptors in endomorphin- and β-endorphin-induced G-protein activation in the mouse pons/medulla

Several genetic mouse models of differential sensitivity to opioids have been used to investigate the mechanisms underlying individual variation in responses to opioids. The CXBK mice are inbred recombinant mice which have a lower level of μ₁-opioid receptors than their parental strain. Endomorphin-1 and endomorphin-2 are endogenous opioid peptides that are highly selective for μ-opioid receptors, while β-endorphin, which is also an endogenous opioid peptide, is non-selective for μ-, δ- and putative -opioid receptors. The present study was designed to investigate the effects of these endogenous opioid peptides on G-protein activation by monitoring guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding to pons/medulla membranes of CXBK mice and their parental strain C57BL/6ByJ mice. Endomorphin-1 (0.1–10 μM), endomorphin-2 (0.1–10 μM) and β-endorphin (0.1–10 μM) increased guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding to the pons/medulla membranes from C57BL/6ByJ and CXBK mice in a concentration-dependent manner. However, the increases of guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding induced by either endomorphin-1 or endomorphin-2 in CXBK mice were significantly much lower than those in C57BL/6ByJ mice. However, no significant difference was found in the increases of the guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding induced by β-endorphin in C57BL/6ByJ and CXBK mice. Moreover, whereas the increase of guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding induced by 10 μM endomorphin-1 or endomorphin-2 were almost completely blocked by a μ-opioid receptor antagonist β-funaltrexamine (10 μM) in both strains, the increase of guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding induced by 10 μM β-endorphin was attenuated to approximately 70% of stimulation by co-incubation with 10 μM β-funaltrexamine in both strains. The residual stimulation of [³⁵S]guanosine-5′-o-(3-thio)triphosphate binding by 10 μM β-endorphin in the presence of 10 μM β-funaltrexamine was further attenuated by the addition of putative -opioid receptor partial agonist β-endorphin (1–27) (1 μM) in both strains. Like the endomorphins, the synthetic μ-opioid receptor agonist [ -Ala²,N-MePhe⁴,Gly-ol⁵]enkephalin at 10 μM showed lower increases of guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding in CXBK mice than those in C57BL/6ByJ mice. However, there was no strain difference in the stimulation of guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding induced by 10 μM of the selective δ₁-opioid receptor agonist [ -Pen^2,5]enkephalin, δ₂-opioid receptor agonist [ -Ala²]deltorphin II or κ-opioid receptor agonist U50,488H.The results indicate that the G-protein activation by endomorphin-1 and endomorphin-2 in the mouse pons/medulla is mediated by both μ₁- and μ₂-opioid receptors. Moreover, β-endorphin-induced G-protein activation in the mouse pons/medulla is, in part, mediated by μ₂- and putative -, but not by μ₁-opioid receptors.     

Distribution of neuropeptide Y-like immunoreactivity in the rat central nervous system--II. Immunohistochemical analysis

The distribution of neuropeptide Y-like immunoreactivity in the rat brain and spinal cord was investigated by means of the peroxidase-antiperoxidase procedure of Sternberger using a rabbit anti-neuropeptide Y serum. A widespread distribution of immunostained cells and fibres was detected with moderate to large numbers of cells in the following regions: olfactory bulb, anterior olfactory nucleus, olfactory tubercle, striatum, nucleus accumbens, all parts of the neocortex and the corpus callosum, septum including the anterior hippocampal rudiment, ventral pallidum, horizontal limb of the diagonal band, amygdaloid complex. Ammon's horn, dentate gyrus, subiculum, pre- and parasubiculum, lateral thalamic nucleus (intergeniculate leaflet), bed nucleus of the stria terminalis, medial preoptic area, lateral hypothalamus, mediobasal hypothalamus, supramammillary nucleus, pericentral and external nuclei of the inferior colliculus, interpeduncular nucleus, periaqueductal central gray, locus coeruleus, dorsal tegmental nucleus of Gudden, lateral superior olive, lateral reticular nucleus, medial longitudinal fasciculus, prepositus hypoglossal nucleus, nucleus of the solitary tract and spinal nucleus of the trigeminal nerve. In the spinal cord cells were found in the substantia gelatinosa at all levels, the dorsolateral funiculus and dorsal gray commissure in lumbosacral cord. The pattern of staining was found to be similar to that observed with antisera to avian and bovine pancreatic polypeptide, but to differ in some respects from that observed with antisera to molluscan cardioexcitatory peptide. The presence of neuropeptide Y immunoreactive fibres in tracts such as the corpus callosum, anterior commissure, lateral olfactory tract, fimbria, medial corticohypothalamic tract, medial forebrain bundle, stria terminalis, dorsal periventricular bundle and other periventricular areas, indicated that in addition to the localisation of neuropeptide Y-like peptide(s) in interneurons in the forebrain, neuropeptide Y may be found in long neuronal pathways throughout the brain.     

Riboflavin-like inmunoreactive fibers in the monkey brain

Using an antiserum directed against the vitamin riboflavin, we studied the distribution of riboflavin-like immunoreactive structures in the monkey brain. In the mesencephalon, at the level of the mesencephalic-diencephalic junction, single riboflavin-like immunoreactive fibers were observed in its dorsal part, whereas a low density of immunoreactive fibers was found below the surface of the section and close to substantia nigra, and a high density was observed above the substantia nigra and close to the medial geniculate nucleus. In the thalamus, single riboflavin-like immunoreactive fibers were found in the ventral regions of the lateral posterior and the medial geniculate nuclei; a low density in the region located above the medial and lateral geniculate nuclei and a high density in the ventral part of the pulvinar nucleus and in the region extending from this latter to the caudate nucleus. Immunoreactive fibers were not observed in the medulla oblongata, pons, cerebellum, hypothalamus, basal ganglia and cerebral cortex. Moreover, no riboflavin-like immunoreactive cell bodies were observed in the monkey brain. The distribution of riboflavin-like immunoreactive fibers in the monkey suggests that this vitamin could be involved in several physiological mechanisms.     

A semi-quantitative atlas of 5-hydroxytryptamine-1 receptors in the primate brain

The regional distribution of 5-hydroxytryptamine-1 receptors in the primate brain was studied by semi-quantitative autoradiographic analysis of tritiated ligand binding. Areas showing the highest density of 5-hydroxytryptamine-1 receptors (greater than 200 fmol [3H]5-hydroxytryptamine bound per mg tissue), included the cerebral cortex (laminae I-II), claustrum, posterior cell group of the basal nucleus of Meynert, the infracommissural part of the globus pallidus, cortical amygdaloid nucleus, hippocampal formation (CA1-subiculum region, the anterior CA2, CA3 and CA4 regions and the molecular layer of the dentate gyrus), thalamic nuclei (parafascicular, parataenial, paraventricular and superior central lateral nuclei), substantia nigra pars reticulata, dorsal raphe nucleus and choroid plexus. The distribution of 5-hydroxytryptamine-1 receptors is compared to the distribution of both 5-hydroxytryptamine receptors and terminal fields of serotonergic projections as previously described in subprimates.     

Different motivational effects induced by the endogenous mu-opioid receptor ligands endomorphin-1 and -2 in the mouse

The present study was designed to investigate the motivational effects of the newly discovered endogenous mu-opioid receptor ligands, endomorphin-1 and endomorphin-2, using the conditioned place preference paradigm in mice. The binding properties of these peptides were first examined using an opioid binding assay. In membranes obtained from the mouse whole brain, the binding of [3H][D-Ala2, NMePhe4, Gly(ol)5]enkephalin (DAMGO; mu), but not of [3H][D-Phe2, D-Phe5]enkephalin (DPDPE; delta) or [3H]U69593 (kappa) selectively and concentration-dependently competed with that of endomorphin-1 and endomorphin-2, indicating that both endomorphin-1 and endomorphin-2 are specific ligands for mu-opioid receptors in the brain. Endomorphin-1 (1-30 nmol/mouse) given i.c.v. produced a dose-related place preference. This effect was abolished by pre-treatment with the mu-opioid receptor antagonist beta-funaltrexamine but not the delta-opioid receptor antagonist naltrindole or the kappa-opioid receptor antagonist nor-binaltorphimine. In contrast, endomorphin-2 (5.6 nmol/mouse) produced place aversion. This aversive effect was inhibited by nor-binaltorphimine as well as beta-funaltrexamine, but not by naltrindole. The place aversion produced by endomorphin-2 was also attenuated by pre-treatment with antiserum against the endogenous kappa-opioid receptor ligand dynorphin A (1-17). These findings indicate that endomorphin-1 may produce its rewarding effect via mu-opioid receptors. On the other hand, the aversive effect induced by endomorphin-2 may be associated with the stimulation of endomorphin-1-insensitive mu-opioid receptors and the activation of dynorphinergic systems in the mouse brain.     

C1 adrenergic neurons are contacted by presynaptic profiles containing DELTA-opioid receptor immunoreactivity

Ligands of the δ-opioid receptor tonically influence sympathetic outflow. Some of the actions of δ-opioid receptor agonists may be mediated through C1 adrenergic neurons in the rostral ventrolateral medulla. The goal of this study was to determine whether C1 adrenergic neurons or their afferents contain δ-opioid receptors. Single sections through the rostral ventrolateral medulla were labeled for δ-opioid receptor using the immunoperoxidase method and the epinephrine synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT) using the immunogold method, and examined at the light and electron microscopic level. Few (5% of 903) profiles dually labeled for PNMT and δ-opioid receptor were detected; most of these were dendrites with diameters <1.5 μm. δ-Opioid receptor immunoreactivity was affiliated with multivesicular bodies in dually labeled perikarya, whereas δ-opioid receptor immunoperoxidase labeling appeared as isolated clusters within both singly and dually labeled dendrites. The majority (83% of 338) of δ-opioid receptor-immunoreactive profiles were axons and axon terminals. δ-Opioid receptor-immunoreactive terminals averaged 0.75 μm in diameter, contained numerous large dense-core vesicles and usually formed appositions or asymmetric (excitatory-type) synapses with their targets. The majority (>50% of 250) of δ-opioid receptor-immunoreactive axons and axon terminals contacted PNMT-immunoreactive profiles. Most of the contacts formed by δ-opioid receptor-immunoreactive profiles (75% of 132) were on single-labeled PNMT-immunoreactive dendrites with diameters <1.5 μm.

The prominent localization of δ-opioid receptors to dense-core vesicle-rich presynaptic profiles suggests that δ-opioid receptor activation by endogenous or exogenous agonists may modulate neuropeptide release. Furthermore, the presence of δ-opioid receptors on axon terminals that form excitatory-type synapses with PNMT-immunoreactive dendrites suggests that δ-opioid receptor ligands may modulate afferent activity to C1 adrenergic neurons. The observation that some PNMT-immunoreactive neurons contain δ-opioid receptor immunoreactivity associated with multivesicular bodies and other intracellular organelles suggests that some C1 adrenergic neurons may present, endocytose and/or recycle δ-opioid receptors.     

Localization of Cells Containing Transforming Growth Factor- α Precursor Immunoreactivity in the Basal Ganglia of the Adult Rat Brain

Abstract

Transforming growth factor-α-like immunoreactivity (TGF-α-ir) was visualized in the adult rat forebrain using three antisera directed against carboxyterminal sequences in the TGF-α precursor. Using immunoperoxidase and immunofluorescence techniques with all three antisera, TGF-λ-ir was found to be present in a subpopulation of astrocytes in the forebrain. Striatal and pallidal regions of the basal ganglia were studied in detail. In the striatum, there was an uneven distribution of astrocytes containing TGF-α-ir, with the greatest number in the dorsal medial third of the caudate-putamen and the overlying corpus callosum/external capsule. In addition, the region of the caudate-putamen bordering the globus pallidus contained numerous clusters of TGF-α-ir astrocytes. The globus pallidus itself contained numerous and more evenly distributed TGF-α-ir astrocytes. Other pallidal structures-including the ventral pallidum, entopeduncular nucleus, and substantia nigra pars reticulata–contained moderate numbers of TGF-α-ir astrocytes. These results suggest that TGF-α precursor is present and, perhaps, synthesized in astrocytes. A related growth factor, epidermal growth factor (EGF), has also been reported to be present in pallidal regions of rat brain. Therefore, the TGF-α/EGF family of trophic factors may play an important role in the function of the central nervous system.     

Differential expression of ubiquitin within the rat brain: discretely localized increases following salt-loading

Ubiquitin is thought to be a universal component of all eukaryotic cells. The recent finding of ubiquitin as a component of abnormal neuronal filaments in various neurodegenerative diseases has prompted the need for knowledge of its distribution within the normal nervous system. To determine this distribution, the rat brain was examined immunocytochemically. Ubiquitin immunoreactivity was found within many regions of the rat brain, although to strikingly different degrees. Staining was most intense in the hypothalamic suprachiasmatic and supraoptic nuclei, as well as the anterodorsal nucleus of the thalamus, medial and lateral habenular nuclei and associated fibre tracts. Following salt-loading, ubiquitin immunoreactivity increased dramatically in axons of the hypothalamo-neurohypophysial tract and its site of termination, the posterior pituitary. In colchicine-treated rats the ubiquitin staining became more prominent in many areas of the brain including the supraoptic nucleus, the habenular nuclei, anterodorsal thalamic nucleus, the hypothalamic arcuate nucleus and cells within the ventral pallidum/substantia innominata region showing a distribution similar to nucleus basalis of Meynert. Partial anterior hypothalamic deafferentation indicated ubiquitin to be axonally-transported at a considerably slower rate than neuropeptides. The differential distribution of ubiquitin within the brain suggests an involvement in processes not heretofore considered.     

Tyrosine hydroxylase-immunoreactive cell groups in the brain of the teleost fishGnathonemus petersii

Different antibodies against tyrosine hydroxylase (TH) were used to obtain detailed information about the distribution, morphology and chemical differentiation of catecholaminergic neurons in the highly differentiated brain of the electric mormyrid fishGnathonemus petersii. The results show that the distribution of catecholaminergic neurons is much more widespread than was previously thought on the basis of dopamine and noradrenaline immunohistochemistry. Tyrosine hydroxylase-immunoreactive neurons were observed not only in clearly dopaminergic regions (the suprachiasmatic nucleus, the magnocellular hypothalamic nucleus and the area postrema) and noradrenergic cell groups (the locus coeruleus and inferior reticular cell group), but also in regions that do not, or only fragmentarily, display dopamine or noradrenaline immunoreactivity, including the ventral and intermediate telencephalon, the anterior and posterior preoptic cell group, the ventromedial thalamus, the pretectal region and the nucleus of the solitary tract, suggesting that they either represent depleted dopaminergic cell groups orl-dihydroxy phenlalanine- producing nuclei. Most TH-immunoreactive neurons are rather small (<10 μm) and have only a few slender processes, but neurons in the magnocellular hypothalamic nucleus and the inferior reticular formation are multipolar and larger (10–20 μm), while those of the locus coeruleus are even more than 20 μm in diameter. The hypothalamic paraventricular organ, which is strongly dopamine and noradrenaline immunoreactive, displays minimal TH immunoreactivity, suggesting that its cerebrospinal fluid-contacting neurons do not synthesize catecholamines, but acquire them from external sources.

Comparison with other teleosts shows that the catecholaminergic system in the brain of Gnathonemus is similarly organized as inCarassius, Gasterosteus, Anguilla andAperonotus, with some variations that may partly be due to technical reasons, and partly reflect true species differences. However, TH-immunoreactive neurons in the midbrain tegmentum were not observed, confirming previous conclusions that a major difference between teleosts and mammals concerns the absence of dopaminergic midbrain groups and correlated mesencephalo-telencephalic projections in teleosts.     

Endomorphin-2 inhibits GABAergic inputs to cardiac parasympathetic neurons in the nucleus ambiguus

The nucleus ambiguus is an area containing cardiac vagal neurons, from which originates most of the parasympathetic control regulating heart rate and cardiac function. GABAergic pathways to these neurons have recently been described, yet modulation of this GABAergic input and its impact upon cardiac vagal neurons is unknown. The nucleus ambiguus has been shown to contain mu-opioid receptors and endomorphin-1 and endomorphin-2, the endogenous peptide ligands for the mu-receptor, whilst microinjections of opioids in the ambiguus area evoke bradycardia. The present study therefore examined the effects of endomorphin-1, endomorphin-2 and DAMGO (a synthetic, mu-selective agonist) on spontaneous GABAergic IPSCs in cardiac parasympathetic neurons. Only endomorphin-2 (100 microM) produced a significant inhibition, of both the frequency (-22.8%) and the amplitude (-30.5%) of the spontaneous IPSCs in cardiac vagal neurons. The inhibitory effects of endomorphin-2 were blocked by naloxonazine (10 microM), a selective mu(1) receptor antagonist. Naloxonazine alone (10 microM) had a potentiating effect on the frequency of the GABAergic IPSCs (+161.43%) but not on the amplitude, indicating that GABA release to cardiac vagal neurons may be under tonic control of opioids acting at the mu(1) receptor. Endomorphin-2 did not reduce the responses evoked by exogenous application of GABA. These results indicate that endomorphin-2 acts on mu(1) receptors located on precedent neurons to decrease GABAergic input to cardiac vagal neurons located in the nucleus ambiguus. The subsequent increase in parasympathetic outflow to the heart may be one mechanism by which mu-selective opioids act to induce bradycardia.     

Regional localization of the mRNA coding for the neuropeptide cholecystokinin in the rat brain studied by in situ hybridization

The regional localization of mRNA coding for the neuropeptide cholecystokinin (CCK) has been studied in the rat brain by in situ hybridization using a 32P-labelled synthetic 32 mer oligonucleotide. Autoradiograms were quantified using computer-assisted microdensitometry. High levels of hybridization were observed in the neocortex, claustrum, endopiriform nucleus, cingular cortex, amygdala, olfactory bulb, hippocampus, ventral tegmental area, geniculate nucleus, several thalamic nuclei and substantia nigra compacta. Very weak signal was detected in the striatum, the cerebellum and the brainstem. The topographic distribution of CCK neurons observed overlaps in part with that previously described by immunohistochemical techniques. However, some discrepancies were also found, particularly in the thalamus. These results show that in situ hybridization with oligonucleotide probes together with a semiquantitative method described can be used to map the expression of the CCK mRNA in rat brain sections as well as its modification after pharmacological or physiological manipulations.     

Localization of chick retinal 24,000 dalton protein (visinin)-like immunoreactivity in the rat lower brain stem

The distribution of visinin, a 24,000 dalton peptide, in the lower brain stem of the rat was examined by means of an indirect immunofluorescent method. Visinin-immunoreactive structures were found to be unevenly distributed only in the neuronal elements. The following neuronal systems were strongly labeled by the antiserum; the Purkinje cell system, mammillotegmental system, habenulointerpeduncular system, the second layer of the superior colliculus, ventral tegmental area, substantia nigra pars lateralis, area medial to the medial geniculate body, parabrachial area, dorsal and ventral nuclei of the lateral lemniscus, pontine reticular formation just medial to the trigeminal principal nucleus, superior olivary nucleus, solitarii nucleus, external layer of the inferior colliculus and spinal trigeminal nucleus. The densities of the labeled fibers in these areas paralleled those of the labeled cells. In addition, highly dense visinin-immunoreactive fiber plexuses were seen in the zona compacta of the substantia nigra, lateral portion of the interpeduncular nucleus, ventral tegmental nucleus of Gudden and vestibular nucleus.     

Chemoarchitecture and afferent connections of the "olfactostriatum": a specialized vomeronasal structure within the basal ganglia of snakes

The olfactostriatum, a portion of the striatal complex of snakes, is the major tertiary vomeronasal structure in the ophidian brain, receiving substantial afferents from the nucleus sphericus, the primary target of accessory olfactory bulb efferents. In the present study, we have characterized the olfactostriatum of garter snakes (Thamnophis sirtalis) on the basis of chemoarchitecture (distribution of serotonin, neuropeptide Y and tyrosine hydroxylase) and hodology (afferent connections). The olfactostriatum is densely immunoreactive for serotonin and neuropeptide Y and shows moderate-to-weak immunoreactivity for tyrosine hydroxylase. In addition to afferents from the nucleus sphericus, the olfactostriatum receives inputs from the dorsal and lateral cortices, nucleus of the accessory olfactory tract, external and dorsolateral amygdalae, dorsomedial thalamic nucleus, ventral tegmental area and raphe nuclei. Double labeling experiments demonstrated that the distribution of serotonin and neuropeptide Y in this area almost completely overlaps the terminal field of projections from the nucleus sphericus. Also, serotonergic and dopaminergic innervation of the olfactostriatum likely arise, respectively, from the raphe nuclei and the ventral tegmental area, whereas local circuit neurons originate the neuropeptide Y immunoreactivity. These results indicate that the olfactostriatum of snakes could be a portion of the nucleus accumbens, with features characteristic of the accumbens shell, devoted to processing vomeronasal information. Comparative data suggest that a similar structure is present in the ventral striatum of amphibians and mammals.