Regional distribution of regulators of G-protein signaling (RGS) 1, 2, 13, 14, 16, and GAIP messenger ribonucleic acids by in situ hybridization in rat brain.

Regulators of G-protein signaling (RGS) proteins are a novel family of GTPase-activating proteins that interact with Galpha subunits of the Gi/o, Gz, Gq and G(12/13) subfamilies to dampen G-protein-coupled receptor (GPCR)-mediated signaling by accelerating intrinsic Galpha-GTPase activity. In the present study, we report on messenger ribonucleic acid (mRNA) localization in rat brain of six RGS genes by in situ hybridization. The distribution patterns of RGS2, RGS13, RGS14 and GAIP (Galpha interacting protein) overlapped in most brain regions examined. Highest regional expression was observed for RGS2 in the cerebral cortical layers, striatum, hippocampal formation, several thalamic and hypothalamic nuclei and hindbrain regions such as the pontine, interpeduncular and dorsal raphe nuclei. Levels of RGS14 mRNA closely paralleled those of RGS2 expression levels throughout most brain regions. RGS13 mRNA was enriched in the hippocampal formation, amygdala, mammillary nuclei as well as the pontine and interpeduncular nuclei. GAIP expression levels were highest in the hippocampal formation with moderate to low levels present in all other regions studied. Of the six RGS genes probed, RGS16 mRNA displayed a discrete localization predominantly in the thalamic midline/intralaminar and principal relay nuclei, and the hypothalamic suprachiasmatic nucleus. RGS1 mRNA signal was not detected in brain. In conclusion, the in situ hybridization studies for RGS2, RGS13, RGS14, RGS16 and GAIP mRNAs extend our knowledge of the distribution of RGS genes expressed in the rat central nervous system, and indicate overlapping RGS-enriched regions that may be indicative of functional diversification in GPCR signaling pathway modulation.

Pindolol-insensitive [3H]-5-hydroxytryptamine binding in the rat hypothalamus; identity with 5-hydroxytryptamine7 receptors.

Pindolol-insensitive [3H]-5-hydroxytryptamine ([3H]-5-HT) binding to rat hypothalamic membranes was pharmacologically and functionally characterized to resolve whether this procedure selectively labels 5-HT7 receptors. Consistent with a previous report, 3 microM and not 100 nM pindolol was required to occupy fully 5-HT1A and 5-HT1B receptors. Remaining [3H]-5-HT binding was saturable (KD, 1.59+/-0.21 nM; Bmax, 53.8+/-3.1 fmol x mg protein(-1)). Displacement of [3H]-5-HT with metergoline and 5-CT revealed shallow Hill slopes (<0.5) but seven other compounds had slopes >0.8 and pKi values and the rank order of affinity were significantly correlated (r = 0.81 and 0.93, respectively) with published [3H]-5-HT binding to rat recombinant 5-HT7 receptors. In the presence of pindolol, 5-HT-enhanced accumulation of [32P]-cyclic AMP was unaffected by the 5-HT4 antagonist RS39604 (0.1 microM) or the 5-ht6 antagonist Ro 04-6790 (1 microM) but significantly attenuated by mesulergine (250 nM), ritanserin (450 nM) or methiothepin (200 nM) which have high affinity for the 5-HT7 receptor. Intracerebroventricular pretreatment with the serotonergic neurotoxin 5,7-dihydroxytryptamine, 5,7-DHT, elevated the [3H]-5-HT Bmax 2 fold, indicating that the hypothalamic 5-HT7 receptor is post-synaptic to 5-HT nerve terminals and regulated by synaptic 5-HT levels. These results suggest that, in the presence of 3 microM pindolol, [3H]-5-HT selectively labels hypothalamic binding sites consistent with functional 5-HT7 receptors.

Antisense oligonucleotide-induced reduction in 5-hydroxytryptamine7 receptors in the rat hypothalamus without alteration in exploratory behaviour or neuroendocrine function.

The effect of a 5-hydroxytryptamine7 (5-HT7) receptor-directed antisense oligonucleotide on rat behaviour and neuroendocrine function was investigated. Six days of intracerebroventricular 5-HT7 antisense oligonucleotide treatment significantly reduced [3H]5-HT binding to hypothalamic 5-HT7 receptors, whereas cortical 5-HT2C density remained unchanged. In rats on a food-restricted diet, both antisense and mismatch oligonucleotides reduced food intake and body weight compared with that in vehicle-treated controls by day 4 of administration. 5-HT7 antisense oligonucleotide administration did not affect exploratory or locomotor activity in photocell activity monitors on day 4 or elevated plus-maze behaviour on day 6 of intracerebroventricular treatment. 5-HT7 antisense oligonucleotide did not affect plasma corticosterone or prolactin levels or 5-HT turnover in either 5-HT cell body or terminal areas. These data demonstrate that intracerebroventricular 5-HT7 antisense oligonucleotide administration selectively reduced rat hypothalamic 5-HT7 receptor density without affecting any of the biochemical or behavioural measures. The results suggest that this antisense protocol could be a valuable tool to investigate central 5-HT7 receptor functions, and that this receptor is not critical for the control of neuroendocrine function or food intake.

Distribution of heterotrimeric G-protein beta and gamma subunits in the rat brain.

Heterotrimeric G-proteins, comprising alpha, beta and gamma subunits, have been shown to play a central role in coupling multiple receptors to a variety of enzymes and ion channels. In vitro studies have demonstrated the existence of selective interactions between various alpha, beta and gamma subunits, as well as between specific heterotrimers and target receptor and effector proteins. However, little is known of the physiological relevance of such associations, and the determinants of specificity in G-protein signaling within the brain remain largely unidentified. To investigate the possibility that specific heterotrimeric interactions result from discrete localizations of the G-protein subunits within the brain, we have used the technique of in situ hybridization to map the distribution of G-protein beta and gamma subunits in the rat brain. Beta1, beta2, beta3 and beta5 subunits were found to be widely expressed throughout the rat brain, whilst beta4 and the G-protein gamma subunit messenger RNAs generally showed more discrete expression patterns. The expression patterns for these subunits suggest that individual beta and gamma subunits may be co-expressed in certain cell types and brain regions; a particularly intriguing and striking co-localization was observed in the case of beta4 and gamma2 subunit messenger RNAs in layer VI of the occipital cortex. The localizations of the G-protein beta and gamma subunits, and their potential coupling to various receptor/effector systems, are discussed.