Postnatal maturation of GABA-immunoreactive neurons of rat medial prefrontal cortex.

A light microscopic immunocytochemical approach has been used to examine the distribution and maturation of gamma-aminobutyric acid- (GABA) containing cells in rat medial prefrontal cortex (mPFC) at progressive postnatal stages. Between P1 and P5, labeled cells in the cortical plate show less differentiated morphological characteristics when compared to cells in the deeper laminae. By P10, however, most labeled cells in superficial laminae show more differentiated characteristics with some having a distinctive multipolar appearance. Between P1 and P5, there is a significant increase (50%) in the density of GABA-containing cells in the superficial laminae, while concurrently there is an overall decreases in the subjacent deeper laminae. As the cortex continues to expand, there is a corresponding decrease in the density of GABA-immunoreactive cells in the outer two-thirds of the cortical mantle until approximately P15, stabilizing at 20-25 cells/100,000 microns2 for all laminae. Between P1 and P15, there is also a significant increase (133%) in the average size of labeled cells, followed by a gradual decrease of 30% between P15 and P41. During P1-7, there is a marked increase in the density of labeled axosomatic terminals in both the superficial (200%) and deep laminae (116%). In the superficial layers, however, the density of labeled terminals again increases by 86% between P12 and P18. In general, the present findings are consistent with the idea that there is a progressive maturation of the intrinsic GABAergic system in rat mPFC in a classic "inside-out" pattern, and this involves extensive postnatal changes occurring during the first 3 postnatal weeks.

A subtype of nicotinic cholinergic receptor in rat brain is composed of alpha 4 and beta 2 subunits and is up-regulated by chronic nicotine treatment.

The subunit composition and pharmacological regulation of rat neuronal nicotinic cholinergic receptors were assessed. Specific immunoprecipitation was determined in solubilized rat brain homogenates using [3H]cytisine, a high affinity agonist at nicotinic receptors, in conjunction with polyclonal antisera generated against nonhomologous domains of the various subunits comprising this receptor class. In all brain regions tested, only antisera generated against the alpha 4 and beta 2 subunits were able to immunoprecipitate specifically receptors labeled by [3H]cytisine. Thus, these sera were further characterized in order to validate and optimize their use in the immunoprecipitation protocol. Preincubation of solubilized receptors from rat forebrain with antisera generated against the alpha 2, alpha 3, alpha 5, beta 3, or beta 4 subunits did not decrease the amount of precipitable alpha 4 or beta 2 subunit. On the other hand, when either anti-alpha 4 or anti-beta 2 serum was used to immunoprecipitate solubilized receptors from rat forebrain, the supernatants contained little if any remaining receptors that could be specifically precipitated by either antibody. Because these antisera do not cross-react, the data indicate that alpha 4 and beta 2 subunits are associated with each other in at least one neuronal nicotinic receptor subtype that has high affinity for agonists. Moreover, these results imply that all alpha 4 subunits that are labeled by [3H]cystisine are coupled to beta 2 subunits. We also present evidence that the alpha 4/beta 2 subtype characterized in this report is significantly increased in the cortex of rats chronically treated with nicotine.

Electroconvulsive shock increases alpha 1b- but not alpha 1a-adrenoceptor binding sites in rat cerebral cortex.

Repeated administration of electroconvulsive shock (ECS) increases [3H]prazosin binding to alpha 1-adrenoceptors in rat cerebral cortex. In contrast, [3H]WB4101 binding in cortex has been reported to be unchanged after ECS. [3H]Prazosin labels two alpha 1-adrenoceptor subtypes, termed alpha 1a and alpha 1b, whereas [3H]WB4101 labels the alpha 1a subtype preferentially. The purpose of this study was to determine whether ECS increases one or both alpha 1-adrenoceptor subtypes in rat cerebral cortex. We found that treatment of rats with ECS once daily for 10-12 days increased [3H]prazosin binding in cortex by about 25% but did not significantly alter [3H]WB4101 binding to alpha 1-adrenoceptors. Measurement of alpha 1a and alpha 1b receptors by competition analysis of the selective alpha 1a antagonist 5-methylurapidil against [3H]prazosin and measurement of [3H]prazosin binding in homogenates preincubated with chlorethylclonidine, which alkylates alpha 1b binding sites, also indicated that the ECS-induced increase in alpha 1-adrenoceptors is confined to the alpha 1b subtype. In contrast to its effect on [3H]prazosin binding, ECS did not increase phosphoinositide hydrolysis as measured by [3H]inositol 1-phosphate accumulation in slices of rat cerebral cortex stimulated by either norepinephrine or phenylephrine. The failure of ECS to increase [3H]inositol 1-phosphate accumulation stimulated by phenylephrine, which is a partial agonist for this response, suggests that spare receptors do not account for the apparent absence of effect of ECS on alpha 1-adrenoceptor-mediated phosphoinositide hydrolysis.

[3H]cytisine binding to nicotinic cholinergic receptors in brain.

Cytisine, a ganglionic agonist, competes with high affinity for brain nicotinic cholinergic receptors labeled by any of several nicotinic 3H-agonist ligands. Here we have examined the binding of [3H]cytisine in rat brain homogenates. [3H]Cytisine binds with high affinity (Kd less than 1 nM), and specific binding represented 60-90% of total binding at all concentrations examined up to 15 nM. The nicotinic cholinergic agonists nicotine, acetylcholine, and carbachol compete with high affinity for [3H]cytisine binding sites, whereas among nicotinic receptor antagonists only dihydro-beta-erythroidine competes with high affinity (in the nanomolar range). Comparison of binding in several brain regions showed that [3H]cytisine binding is higher in the thalamus, striatum, and cortex than in the hippocampus, cerebellum, or hypothalamus. The pharmacology and brain regional distribution of [3H]cytisine binding sites are those predicted for neuronal nicotinic receptor agonist recognition sites. The high affinity and low nonspecific binding of [3H]cytisine should make it a very useful ligand for studying neuronal nicotinic receptors.