Detailed localization of regulator of G protein signaling 2 messenger ribonucleic acid and protein in the rat brain.

Regulator of G protein signaling (RGS) proteins are a recently identified family of proteins which dampen G protein-coupled receptor-mediated signaling by accelerating the intrinsic GTPase activity of Galpha subunits of heterotrimeric G proteins. More than 20 different RGSs have been identified and at least 10 are expressed in the CNS. The present study describes in detail the localization in the rat brain of one member of this family, RGS2. The distribution of RGS2 mRNA and protein has been studied in parallel by performing in situ hybridization and immunoautoradiography on adjacent rat brain sections. Our localization study reveals that RGS2 mRNA and protein are widely expressed in the brain. Protein and mRNA are mostly colocalized such as in neocortex, piriform cortex, caudate-putamen, septum, hippocampus, locus coeruleus. Some mismatches were also observed such as presence of mRNA but not protein in the paraventricular nucleus, the substantia nigra pars compacta and the red nucleus, suggesting that RGS2 protein is present in neuronal projections. Previous reports describing an induction of RGS2 mRNA in the rat striatum after psychostimulants (amphetamine, cocaine) led us to focus on the distribution of RGS2 in the basal ganglia circuitry. The absence of RGS2 mRNA and protein in the globus pallidus suggests that RGS2 would play its regulatory role more in the direct (striatonigral) than in the indirect (striatopallidal) striatal output pathway. In addition, to delineate the implication of RGS2 in pre- and/or postsynaptic functions in the basal ganglia, we performed lesions of the nigrostriatal pathway by 6-hydroxydopamine (6-OHDA) and striatal quinolinic acid lesions. The 6-OHDA lesion did not modify RGS2 mRNA or protein levels in the caudate-putamen whereas the intrastriatal quinolinic acid infusion caused a marked reduction of RGS2 mRNA and protein in the lesioned zone. These data indicate that RGS2 is predominantly expressed in intrinsic striatal neurons. Moreover, the absence of detectable change in RGS2 expression after injections of 6-OHDA suggests also that RGS2 is not primarily involved in the hypersensitization of postsynaptic dopamine receptors observed after lesion of the nigrostriatal pathway.

Use of the beta-imager for rapid ex vivo autoradiography exemplified with central nervous system penetrating neurokinin 3 antagonists.

The neurokinin 3 (NK3) receptor antagonists represent a novel class of pharmacological agents, which are currently under evaluation for the treatment of psychiatric disorders. An efficient brain penetration is one of the main prerequisites to further evaluate compounds displaying high potency to bind the NK3 receptor. The present report describes a method for determining the in vivo occupancy of central NK3 receptors after peripheral administration of drugs. An ex vivo measurement of NK3 receptor occupancy by quantitative autoradiography employing [3H]senktide as the radioligand has been developed. The speed of the method, which is usually considered low due to the time dedicated to film exposure (from weeks to months), has been considerably increased by the use of the beta-imager. The high sensitivity of this new radioimager was used to visualize and quantitatively analyze the [3H]senktide binding sites in brain sections within hours. Using this method, we have demonstrated that the reference NK3 antagonist SR142801 dose dependently occupied the NK3 receptors in the gerbil brain after subcutaneous administration with an ED50 of 0.85 mg/kg. The less active enantiomer SR142806 occupied the NK3 receptors only by 25% at the highest used dose of 10 mg/kg. These values are in accordance with the reported behavioral effects of the compounds. Our results indicate that ex vivo receptor occupancy measurements can be dependently used to predict the central activity of NK3 antagonists. More generally, the combination of ex vivo receptor autoradiography with the beta-imager detection constitutes a new and fast method to evaluate the brain penetration of drug candidates.

Decreased hypothalamic serotonin levels in adult rats treated neonatally with clomipramine.

Early postnatal treatment with the antidepressant drug clomipramine has repeatedly been shown to lead to behavioural and physiological changes in adult rats. To provide some neurochemical correlates to these studies we have measured a number of monoaminergic parameters in the brains of adult (one year old) rats that were treated twice daily with 15 mg/kg clomipramine from postnatal day 2-14. The most consistent finding was that the hypothalamic levels of serotonin (5-HT) were decreased and those of the dopamine metabolite dihydroxyphenylacetic acid (DOPAC) were increased in rats irrespectively whether they went through a range of behavioural and physiological tests or not. The numbers of beta-adrenoceptors in the frontal cortex and of alpha 2-adrenoceptors in the amygdala/piriform cortex were not changed. The decrease in hypothalamic 5-HT concentrations appears to be up to now the most consistent neurochemical alteration in adult rats that were neonatally treated with antidepressant drugs. It is, however, not clear what the relation is with the functional changes in these rats, that are proposed by some authors as an animal model for depression.

Development and isoproterenol-induced regulation of adrenoceptor binding in cultured rat neocortical explants is seen only with the beta-1, not with the beta-2 subtype.

The presence and time-course of beta-adrenoceptor density in cultured explants of neocortex obtained from 6-day-old rat pups were investigated using a [125I]ICYP binding assay. A delayed, but more pronounced, increase in the receptor expression was observed as compared to the situation previously described in vivo. These changes only occurred for the beta 1-subtype of the receptor, whereas the beta 2-subtype binding remained constant up to 3 weeks in vitro. The delay of beta 1-adrenoceptor expression may be due to the incomplete presence of the proper maturational input, and the late enhancement of receptor expression to upregulation related to the absence in vitro of noradrenergic input. Decreased beta-adrenoceptor levels could be induced by chronic treatment of the beta-agonist isoproterenol (1 microM) introduced either for 3 or 13 days. Again, changes in density were found only for the beta 1-adrenoceptor binding sites. There is no reduction of receptor density following return to control conditions for 10 days after a 3-day treatment with isoproterenol, demonstrating the ability of this model to attain its final receptor density notwithstanding the developmental insult.

Early postnatal appearance of enhanced noradrenaline content in the brain of vasopressin-deficient Brattleboro rat; normal adrenoceptor densities and aberrant influences of vasopressin treatment.

The course of postnatal development of noradrenaline (NA) and its unconjugated free metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG), as well as the influence on early chronic vasopressin treatment, were investigated in various brain regions of the hereditary vasopressin-deficient (homozygous di/di) Brattleboro rat. In addition, the densities of the adrenergic receptor subtypes were measured in adult brain. Brain NA levels of di/di pups appeared enhanced already at 7 days of age when compared with data of heterozygous (+/di) controls. This was also seen in areas not known to receive a vasopressinergic input, e.g. the frontal cortex. Levels of MHPG also differed between genotypes, but changes were slight and either a decrease or increase, depending on age and region tested. Saturation analyses of alpha 1-, alpha 2-, and beta-adrenoceptor binding on crude membrane preparations of some brain regions revealed no differences in adulthood. Chronic treatment with vasopressin between 6 and 13 days of age reduced the enhanced NA brain levels throughout the brain of the di/di Brattleboro pups. The known vasopressin-mediated enhancement of NA turnover in adult brain was also measurable in +/di pups of this neonatal period (MHPG/NA ratios), indicating the early maturation of the interaction of vasopressinergic and NAergic systems. However, the dose-response in the di/di Brattleboro rat was biphasic with a decrease at a low dose of vasopressin. Since changes were found throughout the brain, it was concluded that vasopressin deficiency had altered the maturation of NA neurons of the locus coeruleus which may be due to the absence of a presumed inhibitory control of vasopressin on synthesis and storage mechanisms at the perikaryal level.

Increased loss of brain DNA in the neonatal vasopressin-deficient Brattleboro rat, but not in normal rat treated with vasopressin antagonist.

In order to establish whether vasopressin (VP) influences brain cell survival, [3H]thymidine was injected in 10-day-old vasopressin-deficient Brattleboro rat pups, as well as in Wistar pups treated, neonatally, with the VP antagonist dP[Tyr(Me)2]VP followed by subsequent measurement of [3H]DNA in olfactory bulbs and cerebellum days and weeks thereafter. Results show, first of all, that the incorporation of [3H]thymidine into DNA was enhanced in the homozygous (HOM) Brattleboro, when compared with the heterozygous (HET; non-vasopressin-deficient) controls. The difference is due to the greater and prolonged tissue availability of [3H]thymidine, possibly pointing to an altered thymidine uptake and/or metabolism. Between postnatal days 25 and 39 no differences were seen in [3H]DNA content of the brain parts of the HET and Wistar control rats. For the HOM rats, however, a loss of [3H]DNA was seen (up to 8%), indicating that increased postnatal brain cell death might occur in the mutant. The antagonist treatment in Wistar rat up to 21 days of age failed to show a similar effect. It is proposed that general growth impairments, rather than VP receptor-mediated effects, lead to the brain cell loss.