GABA[A] receptor level changes in female hamster forebrain following in vivo estrogen progesterone and benzodiazepine treatment: a quantitative autoradiography analysis

Summary The levels of gamma amino butyric acid (GABA_A) receptors (i.e. ³H-Muscimol binding sites) were determined by quantitative neurotransmitter receptors autoradiography in ovariectomized (OVX), OVX-estradiolo (E), OVX-progesterone (P), OVX-E+P, diazepam (DZ) and DZ + Ro15-1788 treated female hamsters.The various hormonal treatments altered ³H muscimol binding in many brain areas, whereas DZ and DZ + Ro15-1788 had little influence on GABA_A receptor levels at the onset of the blocked aggressive behavioral activity. For example, E, P and E+P all significantly increased ³H muscimol binding in medial preoptic area, ventromedial hypothalamic nuclei, and vertical diagonal bandmedial septal nucleus, whereas P treatment increased binding in the caudate-putamen and decreased it in reuniens nucleus of the thalamus. E and E+P treatments increased ³H muscimol binding in the corticomedial amygdala nucleus and hippocampus. Diazepam treatment decreased the GABA_A receptor binding in the caudate-putamen and basolateral amygdala, while having no effect in the other brain regions where hormone treatment was effective. In vitro incubation of brain sections with micromolar concentrations of E or P did not change muscimol binding. These results suggest that ovarian steroids, and benzodiazepines to a lesser extent, modulate ³H muscimol binding but do so in different brain regions. The hormone effects on ³H muscimol binding in the critical reproductive centres at a time period that coincides with the onset of its behavioral effect is consistent with a genomic controlled activity.     

Gonadal regulation of GABAA receptors in the different brain areas of the male Japanese quail

Summary Quantitative autoradiography was used to investigate the distribution and effects of gonadal hormones on [³H] muscimol (specific GABA_A receptor ligand) binding in the male Japanese quail brain. In gonadally intact Japanese quail brains, [³H] muscimol revealed a heterogeneous distribution with high GABA_A receptor levels in the cerebellum pars granularis (656 fmol/mg wet weight of tissue) and in the pars molecularis (405 fmol/mg wet weight of tissue). Low receptor levels were found in the nucleus preopticus anterior and the nucleus lateralis of the hypothalamic regions (<220 fmol/mg wet weight of tissue) as well as thalamic nuclei such as rotundus and pretectalis (220–261 fmol/ mg wet weight of tissue). Castration resulted in [³H] muscimol binding changes in both brain areas that contain steroid receptors and brain areas devoid of steroid receptors. In fact, castration led to high binding levels in the preopticus anterior nucleus and in the anterior neostriatum area, brain areas that are known to contain gonadal steroid receptors. Castration also elevated [³H] muscimol binding in the hyperstriatum ventrale and reduced binding levels in the paleostriatum augmentatum and the stratum griseum centrale area; all of these areas are known to be devoid of gonadal steroid receptors. At this point it was also important to know whether the gonadal steroid effect is due to alterations in the number of binding sites (B_max) and/or the affinity binding state (K_D). The saturation binding study, dealing with some of the areas described above in brains of male quails castrated or castrated and treated with testosterone or estradiol, demonstrated that the steroid replacement therapy was responsible for the changes of the B_max. Diminishing B_max values were displayed in the hypothalamic preoptic area and the hyperstriatum ventrale of the male quail treated with testosterone and estradiol while a reduced B_max was obtained in the anterior neostriatum of the quail treated with the former steroid. Our findings suggest that these steroids might control some centrally mediated behavior activities through effects on the maximum number of GABA_A binding sites in the male Japanese quail.     

Cyclothiazide binding to the GABAA receptor

In order to explore the molecular interaction between cyclothiazide (CTZ) and gamma-aminobutyric acidA (GABAA) receptors, possibly underlying inhibition of GABAA receptor currents, [3H]-CTZ was synthesized. Binding of [3H]-CTZ to rat brain synaptic membranes could be observed only in the presence of the GABAA receptor antagonist (-)[1S,9R]-bicuculline methiodide (BMI) (EC(50,BMI)=500+/-80microM). GABA decreased [(3)H]-CTZ binding induced by the presence 300microM and 3mM BMI with IC(50,GABA) values of 300+/-50microM and 5.0+/-0.7mM, respectively. Binding of CTZ to [3H]-CTZ labeled sites was characterized by IC(50,CTZ) values of 0.16+/-0.03muM ([BMI]=300microM) and 7.0+/-0.5microM ([BMI]=3mM). Binding of the diastereomeric fraction [3H]-(3R,1'S,4'S,5'R+3S,1'R,4'R,5'S)-CTZ induced by 3mM BMI was quantitatively the more significant in cerebrocortical and hippocampal membranes. It was characterized by IC(50,CTZ)=80+/-15nM and IC(50,GABA)=13+/-3mcapital EM, Cyrillic. In the absence of BMI, CTZ (1mM) significantly decreased GABA-induced enhancement of [3H]-flunitrazepam binding. Our findings suggest that functional inhibition may occur through binding of CTZ to an allosteric site of GABAA receptors. This allosteric site is possibly emerged in the receptor conformation, stabilized by BMI binding.     

Neurogenesis in the basal forebrain of the Chinese hamster (Cricetulus griseus). I. Time of neuron origin.

Summary The time of neuron origin has been determined in the basal ganglia and related basal forebrain structures of the Chinese hamster with the aid of ³H-thymidine autoradiography. Large-celled structures like the globus pallidus, nucleus of the horizontal limb of the diagonal band of Broca as well as large cells in the rostral part of the substantia innominata, in the caudate-putamen-complex and in the olfactory tubercle arise early (E₁₂–E₁₆), whereas medium-sized and small cells in the basal forebrain have a persistent origin over a much longer period. Neuron formation in the basal forebrain persists decrementally until P₄. A clear caudorostral spatiotemporal gradient as well as a distinct outside-in gradient have been observed in the caudate-putamen-complex. Medium-sized neurons in the neostriatum and in the nucleus accumbens, generated simultaneously, are usually arranged in scattered clusters. The present data on time of neuron origin strongly support other evidence which points to the conclusion that the nucleus accumbens can be considered as a ventromedial extension of the caudate-putamen-complex.     

The corticosterone receptive system in the brain of Tupaia belangeri visualized by in vivo autoradiography

Summary The present investigation deals with in vivo binding of ³H-corticosterone in the brains of tree shrews as visualized by autoradiography. Tree shrews were injected with ³H-corticosterone and brain sections were mounted on slides which were subsequently exposed on tritium sensitive film. The relative labeling of 20 different brain structures was determined densitometrically. The indusium griseum, which demonstrated the highest binding for corticosterone of all brain regions in the autoradiograms, was taken as reference and defined as 100% relative labeling (RL). As in other species, the hippocampal subdivisions of the tree shrew retained high amounts of the steroid (60 to 80% RL). In other parts of the limbic system, medium labeling intensities were observed with approximately 40% RL in the lateral septum. The amygdala was less intensely labeled revealing around 30% RL in the basal accessory, the cortical, central, and the lateral nuclei. Autoradiographic grey values in the ventral striatum and pallidum were comparable to those in the amygdala, but in the islands of Callejae they were approximately as high as in the lateral septum (44% RL). In contrast to previous reports dealing with other species, the tree shrew cerebellum also demonstrated a high binding capacity for corticosterone. The RL was nearly 60% in the cerebellar granular layer. This finding may indicate that the cerebellum also plays a role in mediating the effects of corticosterone in the central nervous system.Abbreviations Acb nucleus accumbens - ADX adrenalectomy - BAA nucleus basalis accessorius amygdalae - BLA nucleus basalis lateralis amygdalae - BMA nucleus basalis medialis amygdalae - bv blood vessel - CD nucleus caudatus - Ce nucleus centralis amygdalae - ChP plexus choroideus - Co nucleus corticalis amygdalae - DG fascia dentata; gran, granule cells - Hip hippocampus - I nucleus intercalatus amygdalae - ICj insulae Callejae - Ig indusium griseum - IO nucleus olivaris inferior - LA nucleus lateralis amygdalae - LS nucleus lateralis septi - OT optic tract - RL relative labeling - S subiculum - SFi septofimbrial nucleus - SI substantia innominata - SHy nucleus septohypothalamicus - Tu olfactory tubercle     

The age-dependent changes in the number of3H-ouabain binding sites in mammalian skeletal muscle

The influence of age on the binding of³H-ouabain in skeletal muscle has been characterized in rats, mice and guinea pigs. Measurements performed using biopsies and intact fibers obtained from different types of rat muscles showed that from birth to the 4th week of life, the number of³H-ouabain binding sites per unit weight increases up to 5-fold, followed by almost the same relative decrease to a plateau around 250 pmol/g wet wt at an age of 22 weeks. These changes were not associated with any major alterations in apparentK _D (1.7–3.1×10^–7M) dissociation rate or heterogeneity in binding characteristics. Measurements of 3-O-methylfluorescein phosphatase activity, an enzyme activity which is closely correlated to the Na–K-ATPase activity, confirmed the³H-ouabain binding data.In mice, the number of³H-ouabain binding sites showed similar, albeit less pronounced changes with age, a maximum being reached at the 4th week of life. In guinea pigs, the number of³H-ouabain binding sites per unit weight decreased by 60% from birth to maturity.The results indicate that the early development and differentiation of individual skeletal muscles is associated with a marked increase in the number of Na–K-pumps (when expressed as pmol/muscle), until at maturity a plateau is reached. However, when expressed as pmol/g wet wt the increase is followed by a decrease to a plateau. This may in part account for the relatively low digitalis sensitivity seen in infants as compared to newborn and mature individuals.     

Early developmental expression of leptin receptor gene and [125I]leptin binding in the rat forebrain

Leptin, via leptin receptors (Ob-R), regulates appetite and energy balance. Of the six isoforms of the receptor identified, so far, only the long form (Ob-Rb) can fully activate downstream signal transduction pathways. Although the expression and function of leptin receptors is well described in the adult brain, little is known about the ontogeny of leptin receptor system around the time of birth. In this study, the mRNA expression patterns of total leptin receptor, Ob-R, and the long signalling form of the receptor, Ob-Rb, were investigated in the brain of embryonic and newborn rats using in situ hybridisation and [125I]leptin binding. On embryonic day 18 (E18), Ob-R mRNA was detected in the choroid plexus and the ependymal layer of the third ventricle by in situ hybridisation. At E21, Ob-Rb mRNA was first observed in the arcuate and the ventral premammillary hypothalamic nuclei while at P3, receptor expression was also found in the dorsomedial nucleus. Other leptin target areas identified were the trigeminal ganglion, the thalamus and the hippocampus. Using quantitative receptor autoradiography specific [125I]leptin binding sites on the choroid plexus were found to increase with age in contrast to the ependymal layer of the third ventricle where levels decreased with age. Together these findings demonstrate that the leptin receptor system is differentially regulated during late gestation and early postnatal life in the rat.     

Status epilepticus causes a long-lasting redistribution of hippocampal cannabinoid type 1 receptor expression and function in the rat pilocarpine model of acquired epilepsy

Activation of the cannabinoid type 1 (CB1) receptor, a major G-protein-coupled receptor in brain, acts to regulate neuronal excitability and has been shown to mediate the anticonvulsant effects of cannabinoids in several animal models of seizure, including the rat pilocarpine model of acquired epilepsy. However, the long-term effects of status epilepticus on the expression and function of the CB1 receptor have not been described. Therefore, this study was initiated to evaluate the effect of status epilepticus on CB1 receptor expression, binding, and G-protein activation in the rat pilocarpine model of acquired epilepsy. Using immunohistochemistry, we demonstrated that status epilepticus causes a unique "redistribution" of hippocampal CB1 receptors, consisting of specific decreases in CB1 immunoreactivity in the dense pyramidal cell layer neuropil and dentate gyrus inner molecular layer, and increases in staining in the CA1-3 strata oriens and radiatum. In addition, this study demonstrates that the redistribution of CB1 receptor expression results in corresponding functional changes in CB1 receptor binding and G-protein activation using [3H] R+-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl](1-napthalen-yl)methanone mesylate (WIN55,212-2) and agonist-stimulated [35S]GTPgammaS autoradiography, respectively. The redistribution of CB1 receptor-mediated [35S]GTPgammaS binding was 1) attributed to an altered maximal effect (Emax) of WIN55,212-2 to stimulate [35S]GTPgammaS binding, 2) reversed by the CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A), 3) confirmed by the use of other CB1 receptor agonists, and 4) not reproduced in other G-protein-coupled receptor systems examined. These results demonstrate that status epilepticus causes a unique and selective reorganization of the CB1 receptor system that persists as a permanent hippocampal neuronal plasticity change associated with the development of acquired epilepsy.     

Rapid eye movement sleep deprivation induces changes in the high-affinity binding of [3H]-ouabain to the rat cortical membranes

Rapid eye movement sleep (REMS) suppresses seizures. On the other hand, REMS deprivation (REMSD) increases brain susceptibility to seizures. Sodium-potassium/ATPase is involved in the control of brain excitability. Ouabain, a cardiotonic glycoside, binds to a regulatory extracellular allosteric site in the sodium-potassium/ATPase inhibiting/stimulating its activity depending on its concentration. Endogenous ouabain-like substances exist in the brain; therefore, changes in the ouabain binding site may be involved in the increased brain excitability induced by REMSD. Adult, Wistar male rats were deprived of REMS for 96 hours by the flower-pot method (REMSD). A stress control group was kept in the same environment on a larger platform (LP). A third group of rats was kept in the same room in their home-cages (CONTROL). After REMSD all rats were sacrificed by decapitation and their cerebral cortex dissected. High-affinity [3H]-ouabain binding was carried out in cortical crude membrane preparation using 8 concentrations of [3H]-ouabain (1-24 nM). The results show a statistically significant increase of KD in the REMSD rats compared to both CONTROL and LP groups. There were no statistically significant differences in the Bmax among the experimental groups. There was also no change either in cortical activity of K+ stimulated p-nitrophenylphosphatase, the dephosphorylation reaction of phosphorylated sodium-potassium/ATPase or in Mg2+-stimulated p-nitrophenylphosphatase. An increase in the KD of [3H]-ouabain binding to the sodium-potassium/ATPase in REMSD rats indicates a lower affinity to the endogenous inhibitors/stimulators of the enzyme. Therefore, this decreased affinity of the endogenous ouabain-like substances may be involved in the increased excitability induced by REMSD.     

Distribution of norepinephrine transporters in the non-human primate brain

Noradrenergic terminals in the central nervous system are widespread; as such this system plays a role in varying functions such as stress responses, sympathetic regulation, attention, and memory processing, and its dysregulation has been linked to several pathologies. In particular, the norepinephrine transporter is a target in the brain of many therapeutic and abused drugs. We used the selective ligand [(3)H]nisoxetine, therefore, to describe autoradiographically the normal regional distribution of the norepinephrine transporter in the non-human primate central nervous system, thereby providing a baseline to which alterations due to pathological conditions can be compared. The norepinephrine transporter in the monkey brain was distributed heterogeneously, with highest levels occurring in the locus coeruleus complex and raphe nuclei, and moderate binding density in the hypothalamus, midline thalamic nuclei, bed nucleus of the stria terminalis, central nucleus of the amygdala, and brainstem nuclei such as the dorsal motor nucleus of the vagus and nucleus of the solitary tract. Low levels of binding to the norepinephrine transporter were measured in basolateral amygdala and cortical, hippocampal, and striatal regions. The distribution of the norepinephrine transporter in the non-human primate brain was comparable overall to that described in other species, however disparities exist between the rodent and the monkey in brain regions that play a role in such critical processes as memory and learning. The differences in such areas point to the possibility of important functional differences in noradrenergic information processing across species, and suggest the use of caution in applying findings made in the rodent to the human condition.     

Tritiated thymidine autoradiographic study on postnatal development of epididymal adipose tissue in the normal mouse

Summary Cell proliferation was studied in the developing epididymal adipose tissue of mice by means of³H-thymidine autoradiography. 4 mice in each age group were killed 30 min after a single injection of³H-thymidine given at the 6th, 10th, 16th, 21st, 28th, 35th, 42nd, 49th or 56th postnatal day. The labeling index of the adipose tissue was high for several postnatal days but it was highest on the 6th day. The labeling index decreased thereafter, and cell proliferation appeared to almost cease after 56 days. Another 16 mice were given a single injection of³H-thymidine at 6 days old; pairs of animals were killed at 30 min, 2, 4, 6, 8, 10, 14 and 18 days later. From the 2nd to 6th days of pulse labeling, the number of labeled fat cells increased significantly, whereas the number of labeled poorly differentiated mesenchymal cells decreased reciprocally. The labeling index of fat cells remained unchanged from the 6th to the 18th day. These results suggest that there is a population of fat cell-precursor cells in the mesenchymal tissue of the new born mouse, and that it takes 2–6 days for the precursor cells to become mature fat cells in the epidiymal fatty tissue.     

Roles of estrogen and progesterone in modulating renal nerve function in the rat kidney

The maintenance of extracellular Na⁺ and Cl^- concentrations in mammals depends, at least in part, on renal function. It has been shown that neural and endocrine mechanisms regulate extracellular fluid volume and transport of electrolytes along nephrons. Studies of sex hormones and renal nerves suggested that sex hormones modulate renal function, although this relationship is not well understood in the kidney. To better understand the role of these hormones on the effects that renal nerves have on Na⁺ and Cl^- reabsorption, we studied the effects of renal denervation and oophorectomy in female rats. Oophorectomized (OVX) rats received 17β-estradiol benzoate (OVE, 2.0 mg·kg^-1·day^-1, sc) and progesterone (OVP, 1.7 mg·kg^-1·day^-1, sc). We assessed Na⁺ and Cl^- fractional excretion (FE_Na⁺ and FE_Cl^-, respectively) and renal and plasma catecholamine release concentrations. FE_Na⁺, FE_Cl^-, water intake, urinary flow, and renal and plasma catecholamine release levels increased in OVX vs control rats. These effects were reversed by 17β-estradiol benzoate but not by progesterone. Renal denervation did not alter FE_Na⁺, FE_Cl^-, water intake, or urinary flow values vs controls. However, the renal catecholamine release level was decreased in the OVP (236.6±36.1 ng/g) and denervated rat groups (D: 102.1±15.7; ODE: 108.7±23.2; ODP: 101.1±22.1 ng/g). Furthermore, combining OVX + D (OD: 111.9±25.4) decreased renal catecholamine release levels compared to either treatment alone. OVE normalized and OVP reduced renal catecholamine release levels, and the effects on plasma catecholamine release levels were reversed by ODE and ODP replacement in OD. These data suggest that progesterone may influence catecholamine release levels by renal innervation and that there are complex interactions among renal nerves, estrogen, and progesterone in the modulation of renal function.     

Mitotic activity of the endothelium of mesenteric microvessels of various diameters in wistar rats

Laboratory of Microsurgery, Institute of Physiology, Siberian Branch, Academy of Medical Sciences of the USSR, Novosibirsk. (Presented by Academician of the Academy of Medical Sciences of the USSR Yu. I. Borodin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 110, No. 8, pp. 201–204, August, 1990.     

Muscarinic cholinergic receptor subtypes in cerebral cortex of Fisher 344 rats: a light microscope autoradiography study of age-related changes

The density and localization of muscarinic cholinergic M1-M5 receptor subtypes was investigated in frontal and occipital cortex of male Fisher 344 rats aged 6 months (young-adult), 15 months (mature) and 22 months (senescent) by combined kinetic and equilibrium binding and light microscope autoradiography. In 6-month-old rats, the rank order density of muscarinic cholinergic receptor subtypes was M1>M2>M4>M3>M5 both in frontal and occipital cortex. A not homogeneous distribution of different receptor subtypes throughout cerebrocortical layers of frontal or occipital cortex was found. In frontal cortex silver grains corresponding to the M1 and M2 receptor subtypes were decreased in 15- and 22-month-old groups. The M3 receptor density was remarkably and moderately decreased in layers II/III and V, respectively, of rats aged 15 and 22 months. A reduced M4 receptor density was observed in layer I and to a lesser extent in layer V of mature and senescent rats, whereas no age-related changes of M5 receptor were found. In occipital cortex a diminution of M1 receptor was observed in layers II/III and V of mature and senescent rats. The M2 receptor expression decreased in layer I of 15- and 22-month-old senescent rats, whereas M3-M5 receptors were unchanged with exception of a slight decrease of the M4 receptor in layer IV and of M5 receptor in layers II/III. These findings indicate a different sensitivity to aging of muscarinic receptor subtypes located in various cerebrocortical layers. This may account for the difficulty in obtaining relevant results in manipulating cholinoceptors to counter age-related impairment of cholinergic system.