The distribution of the orphan bombesin receptor subtype-3 in the rat CNS.

Bombesin receptor subtype 3 (BRS-3) is an orphan G-protein coupled receptor that shares between 47 and 51% homology with other known bombesin receptors. The natural ligand for BRS-3 is currently unknown and little is known about the mechanisms regulating BRS-3 gene expression. Unlike other mammalian bombesin receptors that have been shown to be predominantly expressed in the CNS and gastrointestinal tract, expression of the BRS-3 receptor in the rat brain has previously not been observed. To gain further understanding of the biology of BRS-3, we have studied the distribution of BRS-3 mRNA and protein in the rat CNS. The mRNA expression pattern was studied using reverse transcription followed by quantitative polymerase chain reaction. Using immunohistological techniques, the distribution of BRS-3 protein in the rat brain was investigated using a rabbit affinity-purified polyclonal antiserum raised against an N-terminal peptide. The BRS-3 receptor was found to be widely expressed in the rat brain at both mRNA and protein levels. Particularly strong immunosignals were observed in the cerebral cortex, hippocampal formation, hypothalamus and thalamus. Other regions of the brain such as the basal ganglia, midbrain and reticular formation were also immunopositive for BRS-3. In conclusion, our neuroanatomical data provide evidence that BRS-3 is as widely expressed in the rat brain as other bombesin-like peptide receptors and suggest that this receptor may also have important roles in the CNS, mediating the functions of a so far unidentified ligand.

Growth hormone secretagogue receptors in rat and human gastrointestinal tract and the effects of ghrelin.

The peptide hormone ghrelin is known to be present within stomach and, to a lesser extent, elsewhere in gut. Although reports suggest that gastric function may be modulated by ghrelin acting via the vagus nerve, the gastrointestinal distribution and functions of its receptor, the growth hormone secretagogue receptor (GHS-R), are not clear and may show signs of species-dependency. This study sought to determine the cellular localisation and distribution of GHS-R-immunoreactivity (-Ir) using immunofluorescent histochemistry and explore the function of ghrelin in both human and rat isolated gastric and/or colonic circular muscle preparations in which nerve-mediated responses were evoked by electrical field stimulation. The expression of GHS-R-Ir differed to a greater extent between species than between gut regions of the same species. Both the human and rat gastric and colonic preparations (n=3 each) expressed GHS-R-Ir within neuronal cell bodies and fibres, cells associated with gastric glands and putative entero-endocrine and/or mast cells. Smooth muscle cells and epithelia were devoid of GHS-R-Ir and only rat preparations expressed GHS-R-Ir on nerve fibres associated with the muscle layers. GHS-R-Ir was fully competed in all cases in pre-adsorption studies and antiserum specificity was confirmed using a cell line transiently expressing the rat GHS-R. In rat isolated forestomach circular muscle, ghrelin 0.1-10 microM had no effect on smooth muscle tension but concentration-dependently facilitated the amplitude of contractions evoked by excitatory nerve stimulation (n=4-7; P<0.05 for each concentration versus vehicle; n=18). When examined under similar conditions, in both rat distal colon (n=4-6, P>0.05 each) and human ascending (n=3) and sigmoid (n=1) colon, these concentrations of ghrelin were without effect (P>0.05 each). The data suggest that ghrelin has the potential to profoundly affect gastrointestinal functions in both species and at least one of these functions is to exert a gastric prokinetic activity. Moreover, we suggest that this activity of ghrelin is mediated via the enteric nervous system, in addition to known vagus nerve-dependent mechanisms.

Protein distribution of the orexin-2 receptor in the rat central nervous system.

Orexin-A and -B are neuropeptides mainly expressed in the lateral hypothalamic area (LHA). A role for orexins was first demonstrated in the regulation of feeding behaviour. Subsequently, the peptides have been implicated in the control of arousal. To date, two receptors for orexins have been characterised: orexin-1 and -2 receptors (OX-R1 and OX-R2). Both receptor genes are widely expressed within the rat brain. Particularly high expression of both receptor genes in certain hypothalamic and pons nuclei could be responsible for the orexigenic and arousal properties of the peptides. It is, however, presently unclear if one given receptor subtype or both subtypes may mediate a specific biological effect of orexins such as an increase in food intake. We have recently reported the distribution of the OX-R1 protein in the rat nervous system. In this study, we report the distribution of the OX-R2 protein in the rat brain and spinal cord using specific anti-peptide antisera raised against the OX-R2 protein. We also assess the expression profile of the OX-R2 gene in different brain regions. Immunolabelling for the OX-R2 protein was observed in brain regions that exhibited OX-R1-like immunoreactivity (cerebral neocortex, basal ganglia, hippocampal formation, and many other regions in the hypothalamus, thalamus, midbrain and reticular formation). Differences in the OX-R1 and OX-R2 distribution were, however, noticed in the hippocampus, hypothalamus and dorso-lateral pons.

Distribution and expression of TREK-1, a two-pore-domain potassium channel, in the adult rat CNS.

TREK-1 is a member of the two-pore-domain potassium channel family which is expressed predominantly in the CNS. Using an anti-peptide polyclonal antiserum, we have determined the distribution of TREK-1 in the brain and spinal cord of adult rats. Specificity of the antiserum was tested using a TREK-1-transfected cell line and confirmed with c-myc-tagged TREK-1. In thin tissue sections, immunoreactivity was widespread throughout the rat brain and spinal cord. TREK-1-like signals were observed in the cerebral cortex, basal ganglia, hippocampus, and various other subcortical nuclei in the hypothalamus, thalamus, mesencephalon and rhombencephalon. TREK-1 labelling appeared to be over the entire cell membrane, including the cell body and processes. Cells that morphologically resembled projection neurones and interneurones but not glial cells were labelled. As interneurones and known GABAergic projection neurones were the predominant population labelled, we investigated the possibility that TREK-1 is expressed in GABA-containing neurones using a specific anti-GABA antiserum. Expression of TREK-1 in GABA-containing neurones was observed in a number of areas, including the isocortex, hippocampus and thalamus. Thus, TREK-1 expression defines a unique and specific subset of interneurones and principal cells. These studies indicate a widespread distribution of TREK-1 potassium channels throughout the rat brain and spinal cord, with expression in a number of areas being demonstrated to be present on GABA-containing neurones.

Gene expression and protein distribution of the orexin-1 receptor in the rat brain and spinal cord.

Orexins-A and -B are neuropeptides derived from a single precursor prepro-orexin. The mature peptides are mainly expressed in the lateral hypothalamic and perifornical areas. The orexins have been implicated in the control of arousal and appear to be important messengers in the regulation of food intake. Two receptors for orexins have been characterised so far: orexin-1 and -2 receptors. To gain a further understanding of the biology of orexins, we studied the distribution of the orexin-1 receptor messenger RNA and protein in the rat nervous system. We first assessed the expression profile of the orexin-1 receptor gene (ox-r1) in different regions by using quantitative reverse transcription followed by polymerase chain reaction. Using immunohistochemical techniques, we investigated the distribution of orexin-1 receptor protein in the rat brain using a rabbit affinity-purified polyclonal antiserum raised against an N-terminal peptide. The orexin-1 receptor was widely and strongly expressed in the brain. Thus, immunosignals were observed in the cerebral cortex, basal ganglia, hippocampal formation, and various other subcortical nuclei in the hypothalamus, thalamus, midbrain and reticular formation. In particular, robust immunosignals were present in many hypothalamic and thalamic nuclei, as well as in the locus coeruleus. The distribution of the receptor protein was generally in agreement with the distribution of the receptor messenger RNA in the brain as reported previously by others and confirmed in the present study. In addition, we present in situ hybridisation and immunohistochemical data showing the presence of orexin-1 receptor messenger RNA and protein in the spinal cord and the dorsal root ganglia. Finally, due to the shared anatomical and functional similarities between orexins and melanin-concentrating hormone, we present a comparison between the neuroanatomical distribution of the orexin-1 receptor and melanin-concentrating hormone receptor protein-like immunoreactivities in the rat central nervous system, and discuss some functional implications. In conclusion, our neuroanatomical data are consistent with the biological effects of orexins on food intake and regulation of arousal. In addition, the data suggest other physiological roles for orexins mediated through the orexin-1 receptor.

The distribution of the mRNA and protein products of the melanin-concentrating hormone (MCH) receptor gene, slc-1, in the central nervous system of the rat.

Melanin-concentrating hormone (MCH), a 19 amino acid cyclic peptide, is largely expressed in the hypothalamus. It is implicated in the control of general arousal and goal-orientated behaviours in mammals, and appears to be a key messenger in the regulation of food intake. An understanding of the biological actions of MCH has been so far hampered by the lack of information about its receptor(s) and their location in the brain. We recently identified the orphan G-protein-coupled receptor SLC-1 as a receptor for the neuropeptide MCH. We used in situ hybridization histochemistry and immunohistochemistry to determine the distribution of SLC-1 mRNA and its protein product in the rat brain and spinal cord. SLC-1 mRNA and protein were found to be widely and strongly expressed throughout the brain. Immunoreactivity was observed in areas that largely overlapped with regions mapping positive for mRNA. SLC-1 signals were observed in the cerebral cortex, caudate-putamen, hippocampal formation, amygdala, hypothalamus and thalamus, as well as in various nuclei of the mesencephalon and rhombencephalon. The distribution of the receptor mRNA and immunolabelling was in good general agreement with the previously reported distribution of MCH itself. Our data are consistent with the known biological effects of MCH in the brain, e.g. modulation of the stress response, sexual behaviour, anxiety, learning, seizure production, grooming and sensory gating, and with a role for SLC-1 in mediating these physiological actions.

Distribution of somatostatin receptor subtypes in rat lumbar spinal cord examined with gold-labelled somatostatin and anti-receptor antibodies.

Using gold-labelled somatostatin, somatostatin binding sites were predominantly found in laminae I-III, X and on motorneurones of the rat lumbar spinal cord. A comparison with immunohistochemical staining using antisera against somatostatin receptor sequences revealed that the marked binding in laminae I-III coincided with the presence of somatostatin receptor-like immunoreactivity for the receptor subtypes 1, 2 and 3. Binding sites on motorneurones were only paralleled by an immunoreaction for subtype 3. In lamina X, however, the lack of a positive immunoreaction indicates that in this part other subtypes may be present.