Regulation of [3H]mazindol binding to subhypothalamic areas: involvement in glucoprivic feeding

The distribution of low-affinity sodium-sensitive binding sites of [3H]mazindol were studied in rat hypothalamic nuclei. Using microdissection methods, it was demonstrated that the highest level of [3H]mazindol binding is localized to the paraventricular nucleus (PVN) and the lowest binding is observed in the lateral hypothalamus. Following food deprivation, a significant decrease in [3H]mazindol binding in the PVN and ventromedial hypothalamus (VHM) were observed. Refeeding food-deprived rats resulted in restoration of the level of binding in the PVN, and this was correlated with changes in blood glucose levels. Thus, changes in the binding of [3H]mazindol in the PVN may reflect local changes in glucose levels. In related studies, the involvement of the PVN in the regulation of food deprivation or 2-deoxyglucose (2-DG)-induced food intake was studied. Application of amphetamine (20 micrograms) into the PVN had no effect on food deprivation induced feeding, but significantly inhibited 2-DG induced (glucoprivic) feeding. The PVN may play an important role in the glucostatic regulation of feeding and in mediating the anorectic action of amphetamine and related anorectic drugs on glucoprivic feeding.     

Distribution of α-adrenergic, β-adrenergic and dopaminergic receptors in discrete hypothalamic areas of rat

Catecholamine receptor binding sites were measured in discrete hypothalamic nuclei or regions as well as in certain extrahypothalamic areas of the adult male rat. For each assay, discrete areas were microdissected from frozen tissue sections and pooled from several animals. Specific high affinity binding sites were assessed at fixed ligand concentrations for [³H]p-aminoclonidine (PAC) and [³H](2-C 2′,6′-(CH₃O)₂ phenoxyethylamino)-methylbenzodioxan (WB-4101) for α-adrenergic receptor sites, for [³H]dihydroalprenolol (DHA) for β-adrenergic receptor sites, and for [³H]2-amino-6, 7-dihydroxy-1,2,3,4-tetrahydronaphtalene (ADTN) and [³H]spiroperidol in the presence of cinanserin for dopaminergic receptor sites.Regional variations in [³H]WB-4101 binding were relatively small in magnitude, with most hypothalamic and extrahypothalamic areas possessing between 60 and 90% of the binding in frontal cortex. [³H]PAC binding showed a wider range of binding density across brain areas than did [³H]WB-4101, but, in general, variations in [³H]PAC binding paralleled those in [³H]WB-4101 binding. In hypothalamus, binding was characterized as being predominantly to α₁-receptors in the of [³H]WB-4101 and to α₂-receptors in the case of [³H]PAC. The medial hypothalamic areas exhibited a somewhat higher density of these α-adrenergic sites than did the lateral hypothalamus (perifornical hypothalamus and medial forebrain bundle). Also, the ratio of [³H]PAC to [³H]WB-4101 binding differed in different hypothalamic areas, ranging from 1.5:1 to 4:1. The median eminence was exceptional in that it contained appreciable [³H]PAC but no significant [³H]WB-4101 binding sites at the radioligand concentrations tested. Binding of [³H]DHA to β-adrenergic receptors varied over approximately a 3-fold range in the different hypothalamic areas, with binding highest in the medial forebrain bundle and the medial preoptic area, and lowest in the periventricular, dorsomedial and posterior hypothalamic nuclei, the median eminence and the zona incerta. The ratio of β-adrenergic to α-adrenergic binding sites was generally lower in the medial than in the lateral hypothalamic areas and higher in the extrahypothalamic areas examined than in the hypothalamus. With regard to [³H]spiroperidol and [³H]ADTN binding to dopaminergic sites, the striatum, nucleus accumbens and olfactory tubercle showed a greater density of [³H]spiroperidol than of [³H]ADTN sites, in contrast to the hypothalamus where [³H]ADTN binding was more predominant. Within the hypothalamus, [³H]ADTN binding was relatively uniform, while [³H]spiroperidol binding was quite high in four hypothalamic areas (lateral perifornical area, medial forebrain bundle, paraventricular and dorsomedial nuclei), intermediate in the median eminence and arcuate nucleus, and low or not detectable in all other hypothalamic areas.     

Down-regulation of alpha 2-adrenoceptors involved in growth hormone control in the hypothalamus of infant rats receiving short-term clonidine administration

In infant rats short-term administration of the alpha 2-adrenoceptor agonist, clonidine (CLO), induces refractoriness to the growth hormone (GH)-releasing effect of an acute CLO challenge. CLO reportedly stimulates GH release via increased release of GH-releasing hormone (GHRH) from the hypothalamus. Based on these premises, in this study we investigated the possibility that repeated CLO administration may induce down-regulation of hypothalamic alpha 2-adrenoceptors, involved in GH control, thus prohibiting the GH-releasing effect of the drug. alpha 2-Adrenoceptor binding was determined in different brain regions of 10-day-old rats pretreated for 5 days with CLO (150 micrograms/kg, b.i.d.) and killed 14 h after last CLO administration. [3H]p-Aminoclonidine [( 3H]PAC) was used as the specific ligand of alpha 2-adrenoceptors. Treatment with CLO decreased by about 30% the maximum number of binding sites (Bmax) in areas of the mediobasal hypothalamus (MBH) involved in the stimulatory control of GH secretion, i.e. nucleus periventricularis arcuatus, nucleus ventromedialis hypothalami and nucleus lateralis hypothalami. Reduction of Bmax for [3H]PAC binding was observed also in the nucleus periventricularis hypothalami, an area involved in the inhibitory control of GH secretion and, among extrahypothalamic areas, only in the cortex piriformis. In no brain areas was the affinity constant (Kd) for [3H]PAC binding significantly changed after CLO pretreatment. Binding studies performed with a specific ligand of alpha 1-adrenoceptors, [3H]prazosin, showed that the effect of CLO was specific since no changes in the Bmax or Kd were present in either hypothalamic or extrahypothalamic regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tritium-sensitive film autoradiography of guinea pig brain alpha 2-noradrenergic receptors

Tritium-sensitive film autoradiography was used to determine the distribution of alpha2-noradrenergic receptors (i.e. [3H]p-aminoclonidine binding sites) in guinea pig forebrain. Alpha 2-Receptors are heterogeneously distributed throughout the forebrain. Many limbic system structures, such as bed nucleus of stria terminalis, medial preoptic area, medial amygdaloid nucleus and lacunosum molecular layer in hippocampus were heavily labeled. We did not quantify receptor density in areas containing principally white matter but the optical density in those areas was similar to film background suggesting a very low receptor density. Low receptor concentrations were also found in areas that do not contain a high percentage of white matter, such as lateral septum and ventromedial hypothalamic nucleus.     

Galanin/alpha2-adrenoceptor interactions in telencephalic and diencephalic regions of the rat

The aim of this study was to evaluate whether galanin could affect central alpha2-adrenoceptors in telencephalic and diencephalic regions in the rat using quantitative receptor autoradiography with the alpha2 agonist radioligand [3H]p-aminoclonidine. Galanin 1 nM significantly and substantially increased the Kd value of the [3H]p-aminoclonidine binding sites in the medial hypothalamus and amygdala by 86% (p < 0.01) and 73% (p < 0.05) respectively. The Bmax value was only significantly increased with 3 nM galanin in the amygdala and the medial hypothalamus (both p < 0.05). The antagonist M35 counteracted the increase of the Kd values of the alpha2-adrenoceptor agonist binding sites produced by galanin 1 nM in the amygdala and the medial hypothalamus (both p < 0.001). These findings suggest the existence of an antagonistic galanin/alpha2 adrenoceptor interaction in the medial hypothalamus and amygdala that may be of relevance for alpha2-adrenoceptor-regulated neuroendocrine functions and food intake.     

Hypothalamic paraventricular nucleus: interaction between alpha 2-noradrenergic system and circulating hormones and nutrients in relation to energy balance

Extensive evidence suggests that norepinephrine (NE) in the brain is active in the control of eating behavior. Central injection studies demonstrate a stimulatory effect of NE on food intake, a response which is mediated by alpha 2-noradrenergic receptors located in the medial hypothalamus, in particular the paraventricular nucleus (PVN). Activation of these PVN receptors stimulates ingestion specifically of carbohydrate-rich foods, and this response is believed to reflect the role of endogenous NE in controlling natural appetite for this macronutrient. This alpha 2-noradrenergic system in the PVN appears to be physiologically activated at the onset of the animals' active cycle, when there is a natural peak in preference for carbohydrate. At this time, the adrenal hormone corticosterone, which is known to play a major role in carbohydrate metabolism, is found to interact positively with NE in the potentiation of carbohydrate ingestion. Circulating glucose also influences the activity of PVN alpha 2-noradrenergic receptors at this time, and, moreover, alpha-noradrenergic stimulation of the PVN produces an increase in circulating levels of both corticosterone and glucose. This and other evidence has led to the hypothesis that NE in the PVN, through the activation of glucocorticoid- and glucose-sensitive alpha 2-receptor sites, is physiologically active in energy homeostasis, most particularly at the onset of the animal's active cycle. Specifically, this neurotransmitter in the PVN evokes a state of energy conservation. This state involves adjustments in carbohydrate ingestion as well as metabolism, that allow animals to maintain energy reserves by anticipating or responding to a depletion.     

Glucose regulates [H](+)-amphetamine binding and NaK ATPase activity in the hypothalamus: A proposed mechanism for the glucostatic control of feeding and satiety

Binding sites for [3H](+)-amphetamine in the hypothalamus may mediate the anorectic actions of amphetamine and related phenylethylamines. To investigate further the role of these sites in the central control of appetite, the binding of [3H](+)-amphetamine to the hypothalamus and brainstem was measured following food deprivation and refeeding, the onset of genetic obesity, or the administration of 2-deoxy-D-glucose. Food deprivation for 24 to 72 hours reduced the Bmax for [3H](+)-amphetamine binding in the hypothalamus and brainstem but not in other brain areas or peripheral tissues. The decrease in hypothalamic and brainstem [3H](+)-amphetamine binding observed following food deprivation was time-dependent and rapidly reversed by brief refeeding with either rat chow or a 10% glucose solution. Moreover the changes in [3H](+)-amphetamine binding were highly correlated to corresponding alterations in blood glucose concentration. Furthermore, D-glucose, but not L-glucose increases the number of hypothalamic [3H](+)-amphetamine binding sites when administered in vivo or when added to hypothalamic slices in vitro. These data suggest that the [3H](+)-amphetamine binding site in the hypothalamus and (or) brainstem may be coupled to a central "glucostat."     

The alpha 1-noradrenergic antagonist prazosin decreases the concentration of estrogen receptors in female rat hypothalamus

A series of experiments was performed to determine the effects of the alpha 1-noradrenergic antagonist, prazosin, on the concentration of estrogen receptors in female rat brain and pituitary gland. Prazosin caused a dose-dependent decrease in the concentration of cytosol estrogen receptors in mediobasal hypothalamus when injected 10 and 16 h prior to assay. The drug was without effect on the concentration of nuclear estrogen receptors in the absence of estradiol, indicating that the decreased concentration of cytosol estrogen receptors is not due to nuclear estrogen receptor accumulation. Scatchard analysis confirmed that prazosin treatment decreases the concentration of cytosol estrogen receptors without influencing the apparent affinity of the receptors for [3H]estradiol. The prazosin-induced decrease in the concentration of cytosol estrogen receptors in the mediobasal hypothalamus was transient with maximal effects occurring between 8 and 12 h after a single injection. Competition analysis confirmed that prazosin is not an effective competitor for binding to the estrogen receptor in vitro. The effects of prazosin on the estrogen receptor system could not be attributed to modulation of the levels of norepinephrine or dopamine. Assay of the levels of norepinephrine and dopamine in hypothalamus and preoptic area after prazosin injection revealed no effects of prazosin on the level of either of these catecholamines. An estradiol injection resulted in the predicted decrease in the concentration of estrogen receptors accumulating in hypothalamic cell nuclei, suggesting that the cytosol estrogen receptors that decrease in concentration are functional receptors. Prazosin treatment did not result in a decrease in the effectiveness of estradiol in the induction of cytosol progestin receptors in the mediobasal hypothalamus, suggesting that the cells are regulated by the alpha 1-noradrenergic system may not be those cells in which progestin receptors are also induced. These experiments provide further evidence that the noradrenergic system modulates the concentration of estrogen receptors, and perhaps sensitivity to estradiol, in some cells within the rat hypothalamus.     

Interaction between alpha 2- and beta-adrenergic receptors in rat cerebral cortical membranes: clonidine-induced reduction in agonist and antagonist affinity for beta-adrenergic receptors.

The interaction between alpha 2- and beta-adrenergic receptors was investigated in rat cerebral cortical membranes. Clonidine inhibition of [3H]dihydroalprenolol ([3H]DHA) binding resulted in biphasic competition curves with a mean Hill coefficient of 0.45. The addition of 1 microM yohimbine caused a rightward shift of the first portion of the clonidine inhibition curve. In the presence of 1 microM clonidine, the maximum concentration which did not inhibit [3H]DHA binding, inhibition curves of [3H]DHA binding by isoproterenol shifted to the right. A mean Hill coefficient increased from a control value of 0.63 to 0.76. Computer modeling analysis revealed that 1 microM clonidine decreased a beta-adrenergic high-affinity state from 28% to 13%. However, the addition of 1 microM yohimbine completely prevented the clonidine-induced reduction in the beta-adrenergic high-affinity state. In the presence of 200 microM GTP, the effect of clonidine was not observed. In addition, Kd and Bmax values for [3H]p-aminoclonidine ([3H]PAC) binding were not significantly changed by the addition of 100 nM isoproterenol, the maximum concentration which did not inhibit [3H]PAC binding. Moreover, isoproterenol inhibition of [3H]PAC binding resulted in steep competition curves with a mean Hill coefficient of 0.97. The addition of 1 microM alprenolol did not affect the isoproterenol inhibition curve. These data demonstrated that clonidine caused a decrease in agonist and antagonist affinity for beta-adrenergic receptors, while isoproterenol did not modulate the binding characteristics of alpha 2-adrenergic receptors. Furthermore, these results suggest that regulation between alpha 2- and beta-adrenergic receptors is not bidirectional, but is instead unidirectional from alpha 2-adrenergic receptors to beta-adrenergic receptors.     

Autoradiographic study of alpha1- and alpha2-noradrenergic and serotonin1A receptors in the spinal cord of normal and chronically transected cats

Serotoninergic and noradrenergic drugs have been shown to initiate and/or modulate locomotion in cats after spinal cord transection and in patients suffering from spinal cord injuries. To establish a firmer basis for locomotor pharmacotherapy, the distribution of alpha1- and alpha2-noradrenergic and serotonin1A (5-HT1A) receptors was examined in the spinal cord of control cats and of from animals with spinal cord transection at T13 some weeks or months previously. In control cats, the highest levels of alpha1-noradrenergic receptors, labeled with [3H]prazosin, were found in laminae II, IX, and X. The alpha2-noradrenergic receptors, labeled with [3H]idazoxan, were found mainly in laminae II, III, and X, with moderate densities in lamina IX. After spinal transection, both receptors did not change in segments above the lesion. At 15 and 30 days after spinal transection, binding significantly increased in laminae II, III, IV, and X for alpha2 and in laminae I, II, III, and IX for alpha1 receptors in lumbar segments. For longer survival times, binding densities returned to near control values. The 5-HT1A receptors, labeled with [3H] 8-hydroxy-dipropylaminotetralin, were found mainly in laminae I-IV and X. After spinal transection, binding significantly increased only in laminae II, III, and X of lumbar segments at 15 and 30 days. Thereafter, binding returned to control values. The pronounced upregulation of different monoaminergic receptors observed in the lumbar region in the first month after spinal transection suggests that these receptors may be important during the period when cats normally recover functions such as locomotion of the hindlimbs.     

Autoradiographic localization of alpha5 subunit-containing GABAA receptors in rat brain

Multiple subtypes of GABAA receptors are expressed in the rat central nervous system (CNS). To determine the distribution and proportion of alpha5 subunit containing receptors, quantitative autoradiographic analyses were performed with both [3H]L-655,708 and [3H]Ro15-1788, an alpha5 selective and a non selective benzodiazepine binding site ligand, respectively. High densities of [3H]L-655,708 binding sites were observed in hippocampus and olfactory bulb, where alpha5 receptors accounted for 20-35% of total [3H]Ro15-1788 binding sites. Low levels of [3H]L-655,708 sites were associated with the cortex as well as amygdala, thalamic, hypothalamic and midbrain nuclei. These observations indicate that although [3H]L-655,708 binding sites have an overall low expression in rat CNS, they may contribute significantly to GABAergic inhibition in specific brain regions.     

Further pharmacological characterization of [3H]idazoxan binding sites in rat brain: evidence for predominant labeling of alpha 2-adrenergic receptors.

In addition to binding to alpha 2-adrenergic receptors, the antagonist [3H]idazoxan has been reported to bind to non-adrenergic sites in a number of tissues and species. In the present study, the pharmacological nature of [3H]idazoxan binding sites in rat brain slices has been examined using radioligand binding and autoradiographic techniques. In Na2KHPO4 buffer, four drugs with high affinity for alpha 2-adrenergic binding sites were potent inhibitors of [3H]idazoxan binding, with the rank order of potency being RX821002 greater than phentolamine greater than yohimbine greater than (-)epinephrine. Non-linear regression analysis resolved all competition curves into two components, with the high affinity site representing the majority of total [3H]idazoxan binding. In autoradiographic studies performed in Na2KHPO4 buffer, all alpha 2-selective ligands displaced greater than or equal to 75% of total [3H]idazoxan binding to most brain regions. These findings indicate that the major component of [3H]idazoxan binding was to sites that are alpha 2-adrenergic in nature. [3H]Idazoxan binding was also examined in glycylglycine buffer. In contrast to binding in Na2KHPO4 buffer, the proportion of low affinity sites was significantly increased in glycylglycine buffer. Autoradiographic studies confirmed these findings. These pharmacological data are consistent with our previously reported conclusions that, under appropriate assay conditions, [3H]idazoxan predominantly labels alpha 2-adrenergic binding sites in rat brain. These sites are widely distributed and have pharmacological characteristics consistent with those previously reported for alpha 2A-adrenergic receptors.     

Distribution of alpha 2-adrenergic receptors in the brain of the Japanese quail as determined by quantitative autoradiography: implications for the control of sexually dimorphic reproductive processes

With the use of [3H]p-aminoclonidine (PAC), alpha 2-adrenergic binding sites were mapped in the brain of the Japanese quail (Coturnix coturnix japonica). The sites were labeled with the use of in vitro quantitative autoradiography. Special attention was given to areas implicated in the control of sexually dimorphic reproductive processes including sexual behavior. Preliminary competition experiments found that [3H]PAC binding on tissue sections exhibited a pharmacology appropriate to the alpha 2 receptor. Binding sites were found to be heterogeneously distributed throughout the brain. Some of the highest levels of specific binding were found in several areas regulating reproductive function such as the preoptic area, the supraoptic nucleus, the infundibulum, and the medial mammillary nucleus of the infundibulum. [3H]PAC labeled precisely the morphologically dimorphic preoptic medial nucleus but no sexual dimorphism in density of receptor binding was identified. However, dimorphism in density of receptor binding was identified in two areas: the medial mammillary nucleus and the mesencephalic intercollicular nucleus. The former area appears to be involved in the regulation of gonadotrophin secretion and the latter area has been implicated in the control of vocal behavior. These neurochemical dimorphisms may contribute to the regulation of two sexually dimorphic reproductive processes, gonadotropin secretion and courtship vocalizations.     

Central alpha 1 adrenoceptor subtypes in narcolepsy-cataplexy: a disorder of REM sleep

The present study suggests the specific involvement within the central nervous system of an alpha 1 adrenoceptor subtype in a behavior, the control of cataplexy, a pathological analogue of rapid eye movement (REM) sleep atonia. Experiments have shown that prazosin, an alpha 1 antagonist, dramatically aggravates canine narcolepsy-cataplexy through a central mechanism, and that [3H]prazosin binding sites are increased in the amygdala of narcoleptic dogs. However, the corresponding Scatchard plots were curvilinear and best fit was obtained with a two-site model, suggesting the existence of two [3H]prazosin binding sites. These two sites (high and low affinity [3H]prazosin binding sites) met the criteria for authentic receptors and were respectively very similar to the alpha 1a and alpha 1b (high and low affinity for WB4101, respectively) subtypes recently described in the rat and rabbit. Our results of in vivo pharmacology and in vitro [3H]prazosin binding in canine narcolepsy now clearly implicate the low affinity [3H]prazosin binding site (alpha 1b) in canine narcolepsy: (1) Prazosin, an alpha 1 antagonist with similar affinity for both subtypes, was much more potent in increasing cataplexy than WB4101, a compound with more affinity for the alpha 1a receptor. (2) Chlorethylclonidine and phenoxybenzamine, two irreversible blockers of the alpha 1 receptors with more affinity for the alpha 1b receptors, aggravate cataplexy for up to two weeks. (3) The alpha 1 receptor upregulation previously reported by our group in the amygdala of narcoleptic dogs was due to a selective increase in the low affinity [3H]prazosin binding sites. A role for noradrenaline in REM sleep regulation has been suspected for many years, but has never been clearly elucidated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative anatomy of alpha(2) and beta adrenoceptors in the adult and developing brain of the marine teleost the red porgy (Pagrus pagrus, Sparidae): [(3)H]clonidine and [(3)H]dihydroalprenolol quantitative autoradiography and receptor subtypes immunohistochemistry

The present study aimed to determine the anatomic distribution and developmental profile of alpha(2) and beta adrenoceptors (AR) in marine teleost brain. Alpha 2 and beta adrenoceptors were studied at different developmental stages by using [(3)H]clonidine and [(3)H]dihydroalprenolol, respectively, by means of in vitro quantitative autoradiography. Furthermore, immunohistochemical localization of the receptor subtypes was performed to determine their cellular distribution. Saturation studies determined a high-affinity component of [(3)H]clonidine and [(3)H]dihydroalprenolol binding sites. High levels of both receptors were found in preglomerular complex, ventral hypothalamus, and lateral torus. Dorsal hypothalamus and isthmus included high levels of alpha(2) AR, whereas pretectum and molecular and proliferative zone of cerebellum were specifically characterized by high densities of beta AR. From the first year of life, adult levels of both AR were found in most medial telencephalic, hypothalamic, and posterior tegmental areas. Decreases in both receptors densities with age were prominent in ventral and posterior telencephalic, pretectal, ventral thalamic, hypothalamic, and tegmental brain regions. Immunohistochemical data were well correlated with autoradiography and demonstrated the presence of alpha(2A), alpha(2C), beta(1), and beta(2) AR subtype-like immunoreactivity. Both the neuronal (perikaryal or dendritic) and the glial localization of receptors was revealed. The localization and age-dependent alterations in alpha(2) and beta AR were parallel to plasticity mechanisms, such as cell proliferation in periventricular thalamus, hypothalamus, and cerebellum. In addition, the biochemical characteristics, distribution pattern, and neuronal or glial specificity of the receptors in teleost brain support a similar profile of noradrenergic transmission in vertebrate brain evolution.     

Effects of diet and obesity on brain α1- and α2-noradrenergic receptors in the rat

The chronic feeding of a sweetened condensed milk/corn oil diet (CM diet) to adult male rats produced significant increases in body weight and levels of plasma insulin in 34% of the rats fed this diet with respect to chow-fed controls. Levels of alpha 1-noradrenergic receptor binding were lower (32%) in the hypothalamic ventromedial nucleus (VMN) of only those rats which became obese (DIO rats) with respect to both chow-fed controls and those rats which resisted the development of obesity on the CM diet (DR rats). Also, alpha 1-noradrenergic binding was inversely proportional to body weight gain in the VMN (r = -0.831). alpha 2-Noradrenergic receptors were 30-37% lower in both the DIO and DR rats in the dorsomedial nucleus and dorsal area of the hypothalamus, and the medial dorsal area and nucleus reuniens of the thalamus. The similar decreases in alpha 2-noradrenergic receptors in both the DIO and DR rats in these areas suggested that dietary factors alone were responsible for these changes. There were no significant differences from chow-fed rats for hypothalamic dopamine (D2) or beta-noradrenergic (beta 1- and beta 2-) receptors in either DR or DIO rats. These results indicate that VMN alpha 1-noradrenergic receptors co-vary with body weight and implicate a role for alpha 1-receptors in the VMN in the central neuronal regulation of body weight.     

Specific bindings of prostaglandin D2, E2 and F2 alpha in postmortem human brain

Binding activities specific for each of [3H]prostaglandin (PG) D2, E2 and F2 alpha were detected in the P2 fraction of the human brain homogenates. The bindings were time-dependent, saturable and of high affinity; Kd values were 30 nM for all the PG bindings. Regional distribution of these binding activities was determined by measuring specific bindings with 10 nM [3H]PG-D2, [3H]PG-E2 and [3H]PG-F2 alpha in the P2 fractions from 17 brain regions. The PG-D2 binding activity was high in the hypothalamus, amygdala and hippocampus followed by cerebellar nuclei, thalamus, nucleus accumbens and cerebral cortex. The PG-E2 binding sites were similarly concentrated in the hypothalamus and the limbic system, but, unlike the PG-D2 binding, no significant binding of [3H]PG-E2 was observed in cerebellar nuclei, cerebellar cortex and putamen. Compared with these two PG bindings, PG-F2 alpha binding activity was low in many areas, but significant binding was detected in the amygdala, cingulate cortex, cerebellar medulla, hippocampus, nucleus accumbens, midbrain and hypothalamus. These results suggest the presence and specific distribution of three distinct types of PG binding activities, i.e. specific binding of PG-D2, PG-E2 and PG-F2 alpha, in the human brain.     

Binding of [3H]p-aminoclonidine to two sites, alpha 2-adrenoceptors and imidazoline binding sites: distribution of imidazoline binding sites in rat brain

Binding sites labeled by [3H]p-aminoclonidine [( 3H]PAC) were investigated by the competitive analysis with imidazoline and non-imidazoline derivatives. Phenylethylamine derivatives displaced only the part of specific sites for [3H]PAC, which was considered as alpha 2-adrenoceptor, whereas imidazoline derivatives, such as clonidine and tolazoline, competed for a further specific binding of [3H]PAC to the non-adrenergic sites, in addition to the alpha 2-adrenoceptor. Because the non-adrenergic sites were specific for the imidazoline structure, they were termed imidazoline sites. The imidazoline sites were not distributed uniformly among rat brain regions. In striatum, hippocampus and medulla oblongata, they occupied 39.6, 33.0 and 36.5% of the specific binding of [3H]PAC, respectively. Saturation isotherms revealed that Kd and Bmax of imidazoline sites for [3H]PAC were 3.09 +/- 0.59 nM, 27.4 +/- 1.7 fmol/mg protein and 2.23 +/- 0.29 nM, 21.0 +/- 1.5 fmol/mg protein in striatum and hippocampus, respectively. Because imidazoline binding sites also displayed weak affinities for imidazole compounds, such as histamine and cimetidine, the imidazoline site may be a subtype of histamine H2-receptor.     

Further evidence of noradrenergic regulation of rat hypothalamic estrogen receptor concentration: possible non-functional increase and functional decrease

The regulation of estrogen receptors by the alpha 2-noradrenergic system was studied. A single injection of the alpha 2-noradrenergic antagonist, yohimbine, caused a biphasic effect on the concentration of cytosol estrogen receptors in the mediobasal hypothalamus and anterior pituitary gland. A short-latency increase was seen at 1.5-3 h, followed by a longer-lasting decrease at 8-16 h. Scatchard analysis revealed that the apparent, short-latency increase is in the concentration of binding sites, not in the affinity of the receptor for [3H]estradiol. The increase in the concentration of cytosol estrogen receptors is not blocked by pretreatment with the alpha 2-noradrenergic agonist, clonidine. In addition, no increase is detected in the concentration of cell nuclear estrogen receptors accumulating in response to a saturating dose of estradiol. Therefore, the apparent increase in the concentration of cytosol estrogen receptors may not represent a functional increase in receptors. The decrease in the concentration of estrogen receptors, which occurs 8-16 h after yohimbine treatment, is also seen after injection of the alpha 2-adrenergic antagonist, idazoxan, and is not due to a change in the in vitro rate of association of the receptors with [3H]estradiol. Furthermore, the decrease seems to be a functional decrease in the concentration of receptors capable of cell nuclear accumulation in response to estradiol injection, as indicated by the results of experiments in which the concentration of cell nuclear estrogen receptors was assayed after estradiol injection. These experiments provide further support for the hypothesis that the alpha-noradrenergic system, and perhaps specifically the alpha 2-subtype, is involved in decreasing the concentration of estrogen receptors in parts of the brain and pituitary gland. This interaction provides a mechanism by which the environment could regulate the sensitivity of certain neurons to estradiol. However, the finding that the initial increase in the concentration of cytosol estrogen receptors after yohimbine treatment is not followed by the predicted increase in cell nuclear estrogen receptors after estradiol injection raises questions about the physiological relevance of the apparent increase under some conditions.     

The pharmaco-ontogeny of the paraventricular alpha 2-noradrenergic receptor system mediating norepinephrine-induced feeding in the rat.

The present study was undertaken to assess the functional ontogeny of alpha 2-noradrenergic receptors in the hypothalamic paraventricular nucleus (PVN) that mediate noradrenergic stimulation of feeding in the rat. Rat pups, ranging in age from 2 to 15 days, were removed from their mothers and implanted with a brain cannula directed unilaterally at the PVN or third ventricle. On the following day, each pup was implanted with an intra-oral cannula for oral infusion of milk or water that could be swallowed or rejected. Following a 1-h period of satiation, each pup received an intracerebral injection of saline, or a single dose of norepinephrine (NE, 0.01-100.0 nmol) or the alpha 2-noradrenergic receptor agonist clonidine (0.01-1.0 nmol). Milk or water intake was then assessed following a 1-h period of infusion. Injection of NE into the PVN and third ventricle significantly enhanced milk intake at 2 days of age. NE was 10-fold more potent in the PVN than in the ventricle. While paraventricular injections of NE stimulated milk and water intake equally at 2 days of age, NE produced a greater stimulation of milk than water intake at 15 days of age. Like NE, clonidine significantly enhanced milk intake at 2 days of age following injection into the PVN. Collectively, these findings suggest that alpha 2-noradrenergic receptors, mediating noradrenergic stimulation of feeding, are functionally mature very early in the postnatal development of the rat. Moreover, consistent with evidence in the adult rat, these findings indicate that alpha 2-noradrenergic receptors relevant to feeding are located in the vicinity of the PVN.