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The spinal cord contains noradrenergic (NA) pathways which descend from cell bodies in the medulla oblongata and pons to terminate at all levels in the spinal gray matter. The present studies sought to determine the patterns of postnatal development of pre- and postsynaptic elements of NA transmission in the spinal cord. Significant presynaptic development is evident at birth as reflected by substantial high-affinity uptake of norepinephrine (NE) into synaptosomes (0.65-0.90 pmol/mg protein). There is a subsequent increase in uptake on postnatal day (PND) 5, followed by a decrease in 5-10 days to essentially adult levels, starting on PND 20 (0.30-0.35 pmol/mg protein). This decrease in NE uptake occurs coincident with increases in the density of postsynaptic alpha 1 and beta adrenergic receptors and also NE-stimulated accumulation of 3',5'-cyclic adenosine monophosphate (cAMP). Peaks in the development of alpha 1 receptors (PND 10) and beta receptors (PND 20) and NE-stimulated cAMP accumulation (PND 15) were also followed by decreases to adult levels. The neurotoxin 6-hydroxydopamine (6-OHDA) was administered at birth to determine the effects of denervation on the development of the spinal NA systems. At each day following 6-OHDA, synaptosomal uptake of [3H]NE was reduced by two-thirds compared with control values. alpha 1 and beta adrenergic receptor binding are uniformly increased along with a parallel increase in NE-stimulated accumulation of cAMP. While uniformly increased over control, the pattern of postnatal increases and decreases in receptors and cAMP accumulation is maintained.(ABSTRACT TRUNCATED AT 250 WORDS)

We sought to characterize regional heterogeneity of astrocytes using adrenergic receptor sites as cellular markers. Primary cultures made from 6 regions of neonatal rat brain consisted almost exclusively of astrocytes. Membranes from astrocytes cultured 1-3 weeks were prepared for radioligand binding assays of beta- and alpha 2-adrenergic sites using the ligands [3H]dihydroalprenolol and [3H]p-aminoclonidine, respectively. Receptor expression was not affected by time in culture. Astrocytes from different brain regions varied up to 3-fold with respect to number but not affinity for both classes of adrenergic binding site with a rank order of cerebral cortex = superior colliculus greater than hippocampus = ventral midbrain greater than or equal to caudate nucleus greater than or equal to hypothalamus. Binding to beta- and alpha 2-adrenergic receptors was positively correlated across brain regions. Astrocytic receptor binding in each region did not correspond to total receptor levels assessed by quantitative autoradiography. We conclude that: (a) astrocytes are markedly heterogeneous between major brain regions with respect to expression of adrenergic binding sites; (b) regional variations in the density of adrenergic binding sites in brain reflect, in part, local specialization of astrocytes; and (c) a substantial proportion of the adrenergic binding sites in some brain regions may be on astrocytes.

The agonist specificity pattern of the beta-adrenergic adenylate cyclase in glial primary cultures was not typical of either beta 1- or beta 2-adrenergic receptors. The dose-response curves for adrenaline did not correspond to simple mass action kinetics and their computer analysis suggests the presence of both beta 1- and beta 2-adrenergic-sensitive adenylate cyclase (58 plus or minus 17% and 42 plus or minus 17% respectively). Similar properties of beta 1- and beta 2-adrenergic-sensitive adenylate cyclases were found by computer analysis of the dose-response curves for isoprenaline in the presence of a constant concentration of practolol (a selective beta 1 antagonist) (55 plus or minus 10% and 45 plus or minus 10% of beta 1- and beta 2-sensitive adenylate cyclase respectively). The curves for displacement of [3H]dihydroalprenolol by practolol confirm these results. For purpose of comparison, the beta-adrenergic receptors of meningeal cells in cultures were subjected to similar analysis. The results clearly showed that these cells exclusively contained beta 2-adrenergic receptors.

Forced immobilization is a severe stress in rats which diminishes levels of epinephrine in specific nuclei in the hypothalamus and brain stem, suggesting that release of epinephrine is stimulated to a rate which exceeds the rate of its replacement. In the pineal gland, frog erythrocytes, C6 astrocytoma cells and rat brain, beta-adrenoceptor agonists appear to regulate the number of their receptors. Exposure to high concentrations of an agonist leads to apparent decrease in receptors reflected by a decrease in maximal specific binding of antagonists. The apparent decreases in receptors have been shown to be attended by decreases in physiologic responsiveness. In C6 astrocytoma cells, beta-agonists stimulate methylation of phosphatidylethanolamine to increase formation of membrane phosphatidylcholine which in turn appears to enhance activation of adenyl cyclase. Interference with the metabolism of phospholipids by exposure to phospholipase A2 inhibitor, mepacrine (quinacrine), prevents agonist-induced desensitization of beta-adrenoceptors in astrocytoma cells. In the present study repeated immobilization stress has been found to decrease significantly the number of beta-adrenoceptors in hypothalamus and brain stem while increasing the number of alpha 2-adrenoceptors. The desensitization of beta-adrenoceptors was prevented by treatment with mepacrine.

Two weeks after noradrenergic denervation, beta-adrenergic receptor binding increased in 2 of 3 crude fractions of rat cortical homogenates, while the relative distribution of binding was unchanged on continuous sucrose density gradients of the crude synaptosomal fraction. When rat glioma cells were fractionated by the same methods, a band of membranes with beta-adrenergic receptor binding sedimented at the same sucrose density as synaptosomes. Thus membrane receptors remained associated with synaptosomes even in the absence of noradrenergic nerve terminals suggesting that a portion of these receptors are either glial or associated with non-noradrenergic nerve terminals.

The effects of 3 cannabinoids, delta 9-tetrahydrocannabinol (delta 9-THC), 11-OH-delta 9-tetrahydrocannabinol (11-OH-delta 9-THC) and cannabidiol (CBD) on the binding of [3H]dihydroalprenolol [( 3H]DHA) to mouse brain beta-adrenergic receptors were determined. In vitro, delta 9-THC and 11-OH-delta 9-THC increased the specific binding of [3H]DHA. The increased specific binding of [3H]DHA was due to an increase in receptor affinity as indicated by a decrease in the dissociation constant (Kd). CBD had no effect on binding. Chronic administration of delta 9-THC in vivo caused a decrease in the number of [3H]DHA binding sites with no change in Kd.

The effect of thyroid status on beta-adrenergic receptor binding and 5'-nucleotidase activity was studied in the forebrain and the cerebellum of the rat during the first 5 postnatal weeks. The developmental increase in beta-adrenergic receptor binding was significantly depressed in thyroid deficiency in both the forebrain and the cerebellum. The effect was more pronounced in the cerebellum, where at day 35 the concentration and the total number of beta-adrenergic receptor sites were reduced by 35% and 50% respectively. In contrast, hyperthyroidism had no significant effect on the development of beta-adrenergic receptors in the brain. On the other hand, hyperthyroidism led to a sustained increase in the forebrain in the activity of 5'-nucleotidase, an enzyme which is also associated with plasma membranes and has been proposed to play some role in neurotransmission. In thyroid deficiency the enzyme activity was markedly depressed. The effect was significant from day 12 in the cerebellum and from day 21 in the forebrain, the maximal depression, at day 21, being 55% and 45% respectively. In comparison with these plasma membrane markers, the accretion of membranous proteins was less affected: although this was retarded in hypothyroidism and advanced in hyperthyroidism there was no residual effect at 35 days except those attributable to changes in organ size. The results indicated, therefore, that the biochemical specialization of cells, as reflected in certain plasma membrane constituents, are chatacteristically influenced in the developing brain by thyroid disorders.

The effects of acute and chronic treatment with psychomotor stimulants on specific binding of [3H]dihydroalprenolol to beta-adrenoceptors in rat brain were examined. At a dose of 10 mg/kg both acute and chronic treatment with cocaine and chronic treatment with D-amphetamine (10 mg/kg) caused increased binding of [3H]dihydroalprenolol. The molecular mechanism for this enhanced binding appears to be augmentation of the density of beta-adrenoceptors in rat brain. At a lower dose (5 mg/kg), however, chronic administration of D-amphetamine caused a decrease in the density of beta-adrenoceptors in rat brain. Chronic treatment with either D-amphetamine (10 mg/kg) or cocaine induced a marked increase in the magnitude of cyclic AMP accumulation in rat brain slices elicited by norepinephrine. Acute as well as chronic administration of D-amphetamine in vivo inhibited the temperature-dependent uptake of [3H]norepinephrine in rat brain synaptosomal homogenates, but no such inhibition was observed after chronic or acute treatment with cocaine. The results suggest that psychomotor stimulants induce beta-adrenoceptor supersensitivity which may be involved in the phenomenon of reverse tolerance and possibly psychosis in humans. The development of beta-adrenoceptor supersensitivity does not appear to be mediated through alterations in norepinephrine transport at the presynaptic sites.

In these studies we have characterized differences between spontaneously hypertensive rats (SHR) and normotensive Wistar/Kyoto (WKY) rats with respect to rates of neuronal uptake of norepinephrine (NE) and dopamine (DA), and beta-adrenergic receptor (dihydroalprenolol; [3H]DHA) binding in the central nervous system. We find that SHR have greater rates of NE uptake in the frontal cortex, cerebellum, hypothalamus and pons-medulla during early development, and that these changes are accounted for, at least in the cerebral cortex, by an increased Vmax of the NE uptake mechanism. In addition, we find a decrease in the Bmax for [3H]DHA binding, suggestive of down-regulation of beta-adrenergic receptors of this region. In contrast to the results for NE uptake, we have measured significant decreases in DA uptake in the frontal cortex of the SHR at several postnatal ages. Decreases in DA uptake were also observed in the striatum of SHR although these changes were found only in animals approximately 6 weeks of age. From these results we have suggested that NE neurons projecting to a number of brain regions have elevated functional activity, while more regionally selective decreases in dopaminergic functional activity are characteristic of the SHR. We have further proposed that these changes in catecholamine neurons of the central nervous system may play an important role in the development of both the hypertension and behavioral hyperactivity exhibited by these animals.

beta-Adrenergic receptors and the activities of adenylate cyclase, phosphodiesterase and protein kinase were examined in two human glioma cell lines, U 251 and LM, as well as in rat C6 glioma. [3H]Dihydroalprenolol binding to beta-adrenergic receptors was specific, saturable and of high affinity in each cell line. The dissociation constant (Kd) and maximal binding (Bmax) extrapolated from Scatchard curves were Kd = 17.4 +/- 3.2 nM and Bmax = 1110 +/- 197 fmol/mg protein for the U-251 cells; Kd = 14.4 +/- 2.2 nM and Bmax = 655 +/- 105 fmol/mg protein for the LM cells; and Kd = 5.6 +/- 1.1 nM and Bmax = 454 +/- 80 fmol/mg protein for the C6 glioma cells. L-Isoproterenol stimulated cyclic AMP formation in all 3 cell lines. beta-Adrenergic agonists also increased calcium-dependent and calcium non-dependent phosphodiesterase activity in these tumor cells. Cytosolic protein kinase in the 3 cell lines phosphorylated exogenous histone as a substrate. The phosphorylation was enhanced by cyclic AMP. Cytosolic protein kinase also phosphorylated endogenous cytosolic macromolecules. The phosphorylated proteins had molecular weights of 30,000, 51,000 and 90,000 in the two human glioma cell lines. The present results indicate that human glioma cell lines have functional beta-adrenergic receptors linked to adenylate cyclase. These beta-receptors can also regulate phosphodiesterase activity and cyclic AMP in human glioma cells can activate protein kinase and induce the phosphorylation of specific proteins.

Bacterial endotoxin (E. coli O111:B4) induces, 4 days after intraperitoneal injection, a 30% reduction of guinea pig lung beta-adrenoceptor number (Bmax). No change in affinity (Kd) for the receptors occurred. Bilateral electrolytic lesions centered in the anterior hypothalamic nucleus prevent this reduction in Bmax and even reverse the reduction into a small increase in beta-adrenoceptor number. Since it is known from the literature data that anterior hypothalamic lesions as well as beta-adrenoceptor stimulants have an inhibitory influence on the immune system, the mechanism by which these lesions inhibit the reduction of beta-adrenoceptor sites after bacterial endotoxin and influence immune functions, may be related.

We have determined maximum binding capacities for beta-adrenergic binding sites in developing cat visual cortex. These increase quickly from birth to 4 weeks, after which time a slower increase reaches maximal levels at 12 weeks. Dark-rearing and monocular eyelid suture have no influence on this developmental profile.

In order to define the adrenergic receptors in small pial blood vessels, we studied [3H]dihydroalprenolol (DHA) binding, [3H]dihydroergocryptine (DHE) binding, and catecholamine-stimulated adenylate cyclase in membranes prepared from the hog pia. [3H]dha bound to a saturable number of sites (0.09 pmol/mg protein) with a high affinity to (10 mM). The ability of a small series of adrenergic agents to compete for [3H]DHA binding was as expected for binding to beta-adrenergic membrane receptors. The receptor binding appeared to be composed largely of beta 2-adrenergic sites but some sites with beta 1 properties were detected. The beta-adrenergic agonist, isoproterenol, stimulated adenylate cyclase activity by 20% in pial membranes in the presence of GTP. The potency of catecholamines in stimulating adenylate cyclase correlated well with theif sites (0.39 pmol/mg) on these membranes. alpha-Adrenergic agents were potent competitors for [3H]DHE binding but dopamine, serotonin and histamine had effects only at high concentrations. These data demonstrate the presence of alpha- and beta-adrenergic membrane receptors and beta-adrenergically stimulated adenylate cyclase activity in small pial blood vessels from the hog.

Alpha 1, alpha 2, beta 1 and beta 2 adrenergic receptors were measured in the pre-frontal cortex of parkinsonian patients post-mortem. The number of beta 2 receptors was the same in control and parkinsonian subjects. alpha 1 and beta 1 receptors increased in number, particularly in demented parkinsonian patients, while alpha 2 receptors decreased. The affinity constants were unchanged. The modifications seem to be related to lesion of the noradrenergic pathway from the locus coeruleus to the cortex. The relationship between this lesion and the symptoms of dementia and depression in parkinsonian patients is discussed.

To clarify the functional development of the descending monoaminergic input to the chick spinal cord we have studied the ontogeny of beta-adrenergic receptors by measuring the specific binding the tritiated dihydroalprenolol (DHA). In addition, we examined the ability of isoproterenol to stimulate the accumulation of cyclic AMP in slices of developing chick spinal cord. Results show that the chick spinal cord contains a high density of beta-adrenergic receptors that are apparently linked to adenylate cyclase. During development, both the density of beta-receptors, as determined by the specific binding of DHA, and the response of tissue slices to isoproterenol underwent marked changes. beta-Adrenergic receptors (approximately 4 fmol/mg tissue) were first detected on the fourteenth day in ovo. Receptor density increased to approximately 20 fmol/mg by day 20. Between day 20 and the time of hatching, a sharp increase in receptor density, to approximately 50 fmol/mg, was seen. The density of receptors remained high until the second day after hatching, fell off to approximately 30 fmol/mg by the fourth day, and remained relatively unchanged through day 30. The response of spinal cord slices to isoproterenol showed a similar pattern of development with the peak response (7-fold increase in levels of cyclic AMP) occurring at or near the time of hatching. During the period between day 18 in ovo and the time of hatching, when both the response of tissue slices to isoproterenol and the density of beta-receptors increased markedly, the activity of phosphodiesterase did not change. Therefore, the pronounced changes in adrenergic responsiveness that occurred near the time of hatching appear to be related primarily to changes in the density of beta-adrenergic receptors coupled to adenylate cyclase. Such developmental changes in the density of beta-adrenergic receptors and adrenergic responsiveness may play an important role in determining the functional state of the descending monoaminergic systems in the chick spinal cord.

The relationship of alpha- and beta-adrenergic receptors to the noradrenergic innervation of the rat hippocampal formation was studied with histofluorescent, biochemical, and radioligand binding methods. The dentate gyrus received a major portion of the innervation and contained twice the norepinephrine content of the hippocampal gyrus. However, the density of beta-adrenergic receptors, determined by [3H]dihydroalprenolol (DHA) binding, was approximately equal in both gyri. By contrast, a presumed alpha-adrenergic binding site was relatively concentrated in the dentate gyrus, roughly correlating with the distribution of the noradrenergic innervation. Although a significant concentration of beta-adrenergic receptors in the pyramidal cell layer had been suggested in previous studies with fluorescent analogs of propranolol, direct microchemical measurements of [3H]DHA binding in stratum pyramidalis and stratum radiatum showed that beta-adrenergic receptors were uniformly distributed in the hippocampal gyrus. These data, demonstrating a ubiquitous distribution of beta-adrenergic binding in a brain region with a well-defined noradrenergic input, suggest that a portion, perhaps a majority, of beta-adrenergic receptors are not associated with noradrenergic nerve terminals.

Specific bindings of [3H]clonidine and [3H]dihydroalprenolol [( 3H]DHA) to crude synaptic membranes were measured after cerebral cortical slices or synaptic membranes were pre-incubated with isoproterenol, 200 microM at 37 degrees C for 40 min, in 7- and 70-day-old rats. Isoproterenol caused a significant increase of the Bmax value of [3H]clonidine binding sites at both days 7 and 70, without changing the Kd. Scatchard analysis of [3H]clonidine binding to adult synaptic membranes which was examined by using a wide range of [3H]clonidine concentration (0.05-15 nM), showed that the Bmax in only high-affinity binding sites was 4-fold increased by pretreatment of synaptic membranes with isoproterenol 200 microM. In contrast, the Bmax of [3H]DHA binding sites was significantly reduced by isoproterenol at 7 and 70 days. These results suggest that the possible interrelationship between beta- and alpha 2-adrenoceptors which exists in synaptic membranes, matures by day 7 in the cerebral cortex of rats.

Intraperitoneal administration of 0.8 mg/kg N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) to rats significantly reduced alpha-adrenoceptor binding in cerebral cortical membranes without affecting beta-adrenoceptor sites. A selective reduction in alpha 1-adrenoceptors was achieved by injecting yohimbine prior to EEDQ, whereas prazosin pretreatment yielded a selective reduction in alpha 2-adrenoceptor binding. Administration of EEDQ decreased norepinephrine-stimulated inositol phosphate and cyclic adenosine monophosphate (cAMP) accumulation in cerebral cortical tissue as well as the cAMP response to isoproterenol in combination with 6-fluoronorepinephrine without modifying the second messenger response to isoproterenol alone. The results suggest that, under the proper conditions, EEDQ administration can selectively diminish rat brain alpha-adrenoceptor number and function, yielding a procedure that may be useful for defining the behavioral and physiological properties of these sites.

The tritiated adrenergic antagonists [3H]dihydroalprenolol ([3H]DHA; beta-receptors), [3H]prazosin ([3H]PRZ; alpha 1-receptors), and [3H]idazoxan ([3H]IDA; alpha 2-receptors) were used to determine the distribution of these sites in 5 defined areas of the adult rat cerebral cortex. The highest density of [3H]PRZ binding was found in the prefrontal cortex, with a lower and homogeneous distribution for the frontal, parietal, occipital and temporal areas. The [3H]IDA binding sites were fairly uniform for all areas, except for the temporal cortex where it was very dense. In contrast, beta-adrenoceptors labelled by [3H]DHA were very homogeneous for all the regions examined. The functional significance of the distribution of alpha 1, alpha 2 and beta-adrenoceptors is discussed in relation to the catecholamine innervation and monoamine contents measured by high performance liquid chromatography.

Sertraline, a potent and selective inhibitor of serotonin uptake, was chronically administered to laboratory rats. Using in vitro receptor autoradiographic techniques, we found that the binding of [3H]dihydroalprenolol ([3H]DHA) was reduced in cortex layers IV-VI. Results of a saturation experiment indicated that the reduction in cortex layer IV was due to a change in number but not affinity of beta-adrenergic receptors.

Brain tissues from aged rats have an impaired ability to increase beta-adrenergic receptors in response to reduced noradrenergic input, but can down-regulate these receptors in response to repeated administration of desmethylimipramine (DMI). In this study we compared the ability of brain tissues from young (3-month) and aged (20- to 26-month) rats to restore their density of beta-adrenergic receptors following desmethylimipramine (DMI)-induced receptor subsensitivity. Either DMI or saline was administered i.p. twice daily for 7 days to groups of young and aged rats. At various times after drug administration [3H]dihydroalprenolol (DHA) binding was determined in homogenates of pineal gland and cerebral cortex. Four hours after the last dose of DMI there was a decrease in DHA binding in both brain areas of young and aged rats. In young rats DHA binding in these tissues returned to control levels by 2 days after DMI administration. In contrast, in aged rats it took 8 and 16 days for DHA binding to recover in cerebrum and pineal, respectively. The concentration and half-life for the disappearance of DMI from serum and cerebrum were significantly greater in aged rats than in young rats, but the differences do not entirely explain the delayed recovery of beta-receptors in the aged rats. The results suggest that beta-adrenergic receptors of brain tissues from aged rats cannot recover from beta-receptor subsensitivity as readily as those from young rats. If this recovery process requires the synthesis of new receptors, then this synthetic mechanism may be impaired with age.(ABSTRACT TRUNCATED AT 250 WORDS)

The response of pre- and postsynaptic beta-adrenoreceptors to depletion of brain norepinephrine (NE) with reserpine in the rat was characterized by studying the anterograde and retrograde axonal transport of presynaptic receptors and the receptor binding changes induced in postsynaptic frontal cortex cells. Anterograde transport was shown to occur by the linear accumulation of [3H]dihydroalprenolol ([3H]DHA) binding sites (by in vitro binding assay) proximal to a 6-hydroxydopamine (6-OHDA) lesion placed in the ascending pathway of the locus coeruleus and was blocked by more proximal lesions in the pathway. Retrograde transport was demonstrated by the accumulation of [125I]iodocyanopindolol binding distal to similar lesions. Autoradiograms from sections of 6-OHDA injected brains were produced with [3H]DHA binding in the presence of the beta 2-agonist, zinterol, and suggested that the anterograde accumulation of binding sites was primarily of the beta 1-subtype. A single injection of reserpine (5 mg/kg, i.p.) produced a long lasting (6-8 weeks), biphasic decrease in cortical NE levels with nadirs and 4 and 28 days (10% and 45% of control, respectively). Frontal cortex binding of [3H]DHA increased to a maximum at 7-14 days and again at 28 days post-reserpine (230% and 167% of control, respectively). These increases were not prevented by the destruction of presynaptic noradrenergic nerve terminals with intraventricular administration of 6-OHDA 1 day prior to sacrifice and therefore appeared to take place solely in postsynaptic cells. Presynaptic, anterograde axonal transport of beta-receptors was completely blocked from 4-14 days post-reserpine, increased to 323% of control at 21 days, was blocked again at 6 weeks and returned to control by 8 weeks. Retrograde transport of beta-receptors followed a similar pattern suggesting that the presynaptic alterations in beta-receptors in noradrenergic neurons of the locus coeruleus take place independently from those in postsynaptic cortical beta-receptors as a response to NE depletion by reserpine.

Circadian adrenoceptor rhythms were measured in the cortex, hypothalamus, cerebellum and pons-medulla of WKY and SHR rats. The 24-h mean binding to alpha 1-, and beta-adrenoceptors was higher in SHR than WKY in all regions:greatest differences were found at the end of the dark phase. The most consistent and large amplitude circadian rhythms in adrenoceptor binding occurred in the hypothalamus; strain differences in wave form of the rhythm were the most noticeable phenomenon. In the pons-medulla SHR rats showed decreased rhythm amplitudes. In the cerebellum, beta-adrenoceptor binding was higher in SHR rats at all time points, with an identical rhythm to that in WKY rats. Thus regional differences between SHR and WKY rats were expressed in changes in many circadian rhythm parameters. There were no differences between SHR and WKY in the ratios of pre-synaptic: post-synaptic receptors. High affinity binding to alpha-adrenoceptors showed similar circadian rhythms to total specific binding, indicating that the short-term fluctuations cannot be attributed to change in affinity state. Whether these modifications in circadian rhythm characteristics are related to the development or maintenance of hypertension in the SHR strain remains to be investigated.

Selective neuronal lesions have been utilized in efforts to localize binding sites in rat brain for beta-adrenergic, gamma-aminobutyric acid (GABA), histamine H1 and benzodiazepine receptors. The various receptors respond differentially to lesions both in extent of change and in time course. After kainate lesions in the corpus striatum, benzodiazepine receptors are depleted up to 45% at 45--78 days but are unaffected after 7 days. By contrast striatal GABA receptors are increased at 7 days but depleted at later times. Thus both striatal benzodiazepine and GABA receptors appear to be associated at least in part with intrinsic neurons. In the cerebellum both benzodiazepine and GABA receptors are reduced in kainate treated rats and in Nervous mice, mutants which lack Purkinje cells. The most pronounced dissimilarity between benzodiazepine and GABA receptors occurs in Weaver mice, which selectively lack granule cells and display a 60% reduction in GABA receptors but a 40% augmentation in benzodiazepine receptors. A major portion of cerebellar GABA receptors, therefore, appear to be localized to granule cells. Striatal beta-adrenergic receptors are reduced following intrastriatal kainate injections but are unaffected by cerebral cortex ablation, suggesting an association with intrinsic neurons but not with axon terminals of the corticostriate pathway. While intraventricular injections of 6-hydroxydopamine enhance [3H]dihydroalprenolol binding to beta-adrenergic receptors in the cerebral cortex and hippocampus, such binding is not augmented in the corpus striatum, brain stem, midbrain or thalamus-hypothalamus by this treatment. Moreover, medial forebrain bundle lesions, which destroy ascending adrenergic neurons, fail to alter cerebral cortical or striatal beta-adrenergic receptors. Thus denervation-elicited increases in beta-adrenergic receptors vary with brain region and the type of denervating lesion. Histamine H1-receptors are the most resistant of all to neuronal lesions. In the corpus striatum [3H]mepyramine binding is unaffected by cerebral cortex ablation, nigral injections of 6-hydroxydopamine or brain stem hemisection. In the hippocampus, medial forebrain bundle lesions, intrahippocampal kainate injection, and fimbria and fornix transection largely fail to alter [3H]mepyramine binding. Accordingly, a major portion of these receptors may be associated with nonneuronal elements such as glia or blood vessels.

The effects of chronic morphine treatment and morphine withdrawal on beta-adrenergic receptor density and electrophysiological responsiveness in rat hippocampus were examined. Chronic treatment of rats with morphine for 14 days resulted in a 19% increase in the number of beta-adrenergic receptors in hippocampus, as measured by the binding of the specific antagonist [3H]dihydroalprenolol (DHA). In comparison, the number of specific binding sites for [3H]DHA was decreased 27% in hippocampus in morphine-withdrawn animals, compared to saline-treated controls. These alterations in beta-adrenergic receptor density were not accompanied by a significant change in the dissociation constant (Kd) for [3H]DHA or in the inhibitory constants (Ki) for the displacement of the [3H]-antagonist by either norepinephrine or isoproterenol. Electrophysiological experiments in the in vitro hippocampal slice preparation revealed that responses to threshold as well as maximal concentrations of isoproterenol were significantly enhanced in morphine-dependent animals, compared to controls, whereas electrophysiological responsiveness to maximal concentrations of isoproterenol was decreased in slices from morphine-withdrawn rats. The results of this study indicate that beta-adrenergic receptors in hippocampus are up-regulated during the development of morphine dependence and down-regulated during opiate withdrawal. These changes in hippocampal beta-adrenergic receptor density are likely to be of functional relevance since they are manifested in a corresponding increase and decrease, respectively, in electrophysiological responsiveness to an exogenously administered beta-adrenergic receptor agonist.

Previous work has identified an effect of circulating estrogens on the number of central adrenergic binding sites. We have further characterized this effect by performing the experiments in vitro and have taken advantage of a well-described hypothalamic preparation in which diethylstilbestrol (DES), is known to elevate cAMP levels through a pathway which involves adrenoreceptors. We show that DES induces a reciprocal change in the numbers of alpha- and beta-adrenergic binding sites in incubated hypothalami obtained from intact immature female rats as well as from ovariectomized adult rats. The alpha-adrenergic binding sites are reduced by 25-30% whereas the beta-adrenergic sites are increased by 60-100%. The effect is maximal at 3 h in vitro (20 microM DES) and largely reversible following a 2 h wash in the absence of DES. Using the change in beta-adrenergic binding sites as a probe, we were further able to show that estradiol (100 microM) and 2-hydroxyestradiol (50 microM) had no effect. Further, the effect of DES was not blocked by the anti-estrogens clomiphene or tamoxifen. Since DES is able to elevate beta-adrenergic binding sites in hypothalamus and amygdala (brain areas known to contain high levels of estrogen receptors) but has no effect in cerebellum, we conclude that we have observed an effect of DES not shared by estradiol but which may be confined to estrogen target areas of the brain.

The stimulation of adenylate cyclase by dopamine and various beta-adrenergic agonists has been investigated in homogenates from 3 areas of cat brain: the cerebral cortex, cerebellum and hippocampus. The purpose of the study was to determine whether the beta-arenergic receptors coupled to adenylate cyclase could be classified as either beta 1 and beta 2 subtypes in the different regions studied. The stimulation of adenylate cyclase by the beta-adrenergic agonist, (-)isoproterenol (5 X 10(-6) M), was completely blocked by the specific beta-adrenergic antagonist, (p)alprenolol (1-(-5) M), but not by the dopaminergic antagonist, fluphenazine (10(-5) M), whereas the stimulation of adenylate cyclase by (-)epinephrine (10(-4) M) was blocked to varying extents by these two drugs in each of the 3 regions studied. The (-)epinephrine effect was always blocked in the combined presence of (p)alprenolol and fluphenazine. The adenylate cyclase stimulation by (p)epinephrine which is not blocked by (p)alprenolol was due to interaction of (p)epinephrine with a dopaminergic-sensitive adenylate cyclase which has been characterized in cerebral cortex, hippocampus and cerebellum. Regional differences in the affinity of beta-adrenergic-sensitive adenylate cyclase for various agonists were investigated in the presence of fluphenazine (10(-5) M). In the cerebellum the potency order was (+/-)protokylol greater than (+/-)hydroxybenzylisoproterenol greater than (+/-)isoproterenol greater than (-)epinephrine greater than (+/-)salbutamol greater than (-)norepinephrine, indicating the presence of a beta 2-adrenergic receptor. In the cerebral cortex the potency order was (-)isoproterenol greater than +/-)protokylol greater than (+/-)hydroxybenzylisoproterenol greater than (-)epinephrine = (-)norepinephrine ((+/-)salbutamol being inactive). A similar pattern was found in the hippocampus indicating the presence of a beta 1-adrenergic receptor in these two regions. (+/-)Salbutamol was a partial agonist in the cerebellum and a competitive antagonist in the cerebral cortex. The ratio of the antagonist potencies of (+/-)practolol and (+/-)butoxamine preferential beta 1- and beta 2-adrenergic antagonists respectively, to block the stimulation of adenylate cyclase was 25 in the cerebellum, compared to 0.5 in the cerebral cortex and 1.6 in the hippocampus. These results confirm the presence of a beta 2 subtype of receptor coupled to adenylate cyclase in the former and beta 1 subtypes in the latter two regions. The comparison between the affinities of a series of beta-adrenergic agonists and antagonists for the beta-adrenergic receptors coupled with an adenylate cyclase in cerebral cortex and cerebellum with their affinities for well characterized beta 2-adrenergic receptors in lung and beta 1-adrenergic receptor in heart substantiated this conclusion.

The effect of acute and chronic electric shocks on beta-adrenergic receptor binding in the rat's brain was investigated. Beta-adrenergic receptor subsensitivity in the cortex was induced by chronic shocks, but not by acute shocks. This reduction appears to be due to a decreased number of receptors. It seems that stress, by increasing intrasynaptic norepinephrine levels resulting from an accelerated turnover rate, causes beta-adrenergic receptor subsensitivity.

The guanyl nucleotides GTP and GDP are endogenous factors in the rabbit cerebellum which contribute to the in vitro coupling of the beta-adrenergic receptor to adenylate cyclase. Repeated washing of the particulate material removes these endoactivity to beta-adrenergic agonists. The endogenous GTP and GDP, isolated by high pressure liquid chromatography, mimic the ability of the supernatant both to restore the coupling of receptor and enzyme and to increase basal enzyme activity. The effectiveness of GDP reflects its conversion to GTP during the assay. GTP does not affect either the number of [3H]dihydroalprenolol binding sites or the affinity of these sites for L-isoproterenol; furthermore, GTP does not cause a shift in activation affinity of enzyme activity for isoproterenol. Thus, the guanyl nucleotides are endogenous constituents of the mammalian brain which are essential for the functional 'coupling' of beta-adrenergic receptors to adenylate cyclase.

Neurotransmitter receptor binding of 5 ligands was examined in the striatum, substantia nigra (SN) and frontal cortex of rats which had received either unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal pathway (NSP) or unilateral kainic acid lesions of the striatum. 6-OHDA lesions of the NSP significantly reduced [3H]dihydroalprenolol ([3H]DHA) and [3H]naloxone ([3H]Nal) binding by 31% and 28% respectively, in the denervated striatum compared to the contralateral side. Scatchard analysis revealed that the alteration in [3H]DHA binding was not due to a change in the affinity of the beta-adrenergic receptor for [3H]DHA. In marked contrast to these changes in the striatum, destruction of the NSP resulted in a significant increase in [3H]DHA and [3H]Nal binding by 44% and 26%, respectively, in the frontal cortex of the lesioned compared to the control side. 6-OHDA lesions in the NSP did not alter striatal receptor binding for [3H]quinuclidinyl benzilate ([3H]QNB), [3H]muscimol ([3H]Mus) or [3H]flunitrazepam ([3H]Flu). Similarily, intrastriatal kainic acid injections did not alter striatal receptor binding for [3H]Nal, [3H]Flu or [3H]Mus. Of the various receptor densities measured in the SN after the above lesions the only alteration observed was a 43% increase in [3H]Flu binding following 6-OHDA lesions of the NSP. Scatchard analysis indicated no change in the affinity of the benzodiazepine receptor for [3H]Flu. 6-OHDA lesions of the NSP did not alter [13H]QNB or [3H]Nal binding in the SN. Striatal kainic acid lesions did not alter nigral [3H]QNB or [3H]Flu binding. The results are discussed in terms of neurotransmitter localization and plasticity within the striatum, SN and frontal cortex.

The pretreatment of rat cerebral cortical membranes with 10 or 100 microM isoproterenol at 37 degrees C for 40 min caused a significant elevation of the Bmax value of [3H]clonidine binding but pretreatment at 4 degrees C did not affect the value. The isoproterenol-induced increase in the Bmax value of the binding was higher in 50 mM Tris-HCl buffer (pH 7.7) than in Krebs-Ringer solution. In 50 mM Tris-HCl buffer (pH 7.7), treatment with isoproterenol reduced the Bmax value of [3H]dihydroalprenolol binding but neither Kd nor Bmax of [3H]WB 4101 binding was affected by this treatment. Fifty microM propranolol or 100 microM GTP produced a significant reduction in isoproterenol-induced elevation of the Bmax value of [3H]clonidine binding. In contrast, 100 microM cyclic AMP did not affect the control binding and 0.1 or 1 mM theophylline did not affect the isoproterenol-induced elevation of the binding. The only Bmax value in high affinity binding of [3H]clonidine was increased by isoproterenol. It is suggested that isoproterenol increases the density of alpha 2-adrenoceptors in a temperature-dependent manner. The direct interaction between beta- and alpha 2-receptor molecules and/or their indirect interaction, mediated by GTP regulatory proteins, would exist in the cerebral cortical membranes of rats.

Rats were subjected to REM sleep deprivation by the flower pot method for 7 days. Another group of rats received either desipramine (10 mg/kg, i.p.), a tricyclic antidepressant, or saline (i.p.) once daily for 7 days. beta-Adrenergic receptor sensitivity in rat brain cortices was assessed by determining the maximum number of [3H]dihydroalprenolol binding sites by Scatchard analysis. Administration of desipramine caused a 26% reduction in beta-adrenergic receptor density (P less than 0.01) while REM sleep deprivation reduced beta-adrenergic receptor density by 13%, which was not statistically significant.

The development of calmodulin, calmodulin-dependent adenylate cyclase and beta-adrenergic receptors was studied in the rat brain. Membrane-bound calmodulin detected was approximately 40-50% of the total calmodulin throughout the postnatal development of either in the cerebrum or cerebellum. No significant difference was found between the quantitative patterns of the membrane-bound and cytosolic calmodulin during the entire period of postnatal development in either of these tissues. Both the cytosolic and membrane-bound calmodulin were present in low concentrations in the immature brain after birth. Their contents rapidly increased during the second postnatal week. Subsequently, the cytosolic calmodulin content remained constant, but showed a considerable decrease in the particulate fraction after day 14. Basal adenylate cyclase activity in the rat cerebrum slowly increased up to the second postnatal week and decreased after day 14. The responsiveness to calmodulin of this enzyme remained unaltered during postnatal development, whereas fluoride and guanine nucleotide sensitivities increased in the same period. The maximum number of (-)-[3H]dihydroalprenolol binding site sharply increased during day 9-14 in the rat cerebrum, although the dissociation constant Kd of the binding site was not affected by age. The results in the latter study suggest that calmodulin-dependent adenylate cyclase may be already present in the earlier postnatal ages of the rat brain, while the beta-adrenergic receptor and guanine nucleotide regulatory unit, both of which are required for a hormone-sensitive adenylate cyclase, may sharply increase in the second postnatal week.

Methylazoxymethanol acetate (MAM)-induced cortical hypoplasia resulted in a 2-fold increase in the concentration of norepinephrine and its metabolite, 3-methoxyl-4-hydroxyphenylglycol sulfate, and a 26% decrease in the Bmax for the beta-receptor ligand, [3H]dihydroalprenolol (DHA) in the rat cortex. Chronic treatment with desmethylimipramine did not further decrease DHA-labeled sites although prior lesion of the noradrenergic terminals with 6-hydroxydopamine markedly increased the number in the MAM-lesioned cortex.

Binding assays of [3H]dihydroalprenolol ([3H]DHA), [3H]prazosin and [3H]clonidine have been performed on whole brain (minus cerebellum) homogenates of the convulsive mutant mice quaking (qk) and the controls of the same strain (C57BL/6J:B6). In 70-day-old mutants (which fully exhibit the qk convulsive phenotype), the binding of [3H]DHA to beta-adrenoceptor binding sites was not different from the controls, whereas the binding capacities of [3H]prazosin and [3H]clonidine to alpha 1-and alpha 2-adrenoceptor sites, respectively, were greatly enhanced. The biphasic ontogenic pattern of alpha 2-adrenoceptors had a greater amplitude in the brain of 30- to 90-day-old mutants than in the corresponding B6 controls. In mutants younger than 30 days or older than 90 days, the number of alpha 2-adrenoceptor sites was not modified. The number of alpha 1-adrenoceptor binding sites was increased in the brain of the mutants, only in animals older than 70 days. In younger mice, the postnatal modulation of alpha 1-adrenoceptor sites was identical to the controls. Regional studies were performed in 70-day-old mice. [3H]clonidine binding was increased in the brainstem of the mutants, and to a lesser extent in the cerebral cortex, while it was slightly diminished in the hypothalamic area. [3H]prazosin binding was also increased in the brainstem of the mutants, and decreased in the olfactory bulbs. Our results suggest that the convulsions of the qk mutants are selectively associated with modifications of alpha- and not beta-adrenoceptor binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

The concentration and location of adrenergic receptors in cat visual cortex have been determined by radioligand binding techniques using [3H]prazosin (alpha 1-adrenergic receptors), [3H]yohimbine (alpha 2-adrenergic receptors) and [3H]dihydroalprenolol (beta-adrenergic receptors). Saturable high affinity binding sites for all of these ligands were found. The beta-adrenergic receptor population was resolved into beta 1- and beta 2-sites that were present in the ratio 35:65. The laminar distributions of the alpha 1-, alpha 2- and beta-adrenergic receptors were different. The alpha 1- and beta-adrenergic receptors were very similarly localized, being seen in upper layers (I, II and III) and lower layers (layers V and VI). The labelling in upper layers was greater than that in lower layers, more so for alpha 1-adrenergic receptors than beta-adrenergic receptors. alpha 2-Adrenergic receptors were seen in a single band that occupied layer II and III but did extend to the pial surface. These results indicate that the effect of norepinephrine on neuronal activity in cat visual cortex will depend upon the layer in which it is released. Our results provide a basis for further physiological studies of the role of norepinephrine in the processing of visual information.

Exposure of rats to 25 min anoxia within 24 h following birth caused behavioural as well as biochemical changes during their development and maturity. Following postnatal anoxia, a significant increase in the concentration of the cholinergic muscarinic receptors in the hippocampus was noted at the early stages of development, between 6 and 20 days of age, but reached normal values at 40 days of age. However, at this age, significant increase in the concentration of beta-adrenergic receptors in the hippocampus was found, which remained significantly high during maturity and adulthood, as compared to controls. Rats submitted postnatally to anoxia exhibited hyperactivity in the open field which was maximal at 20-25 days of age and declined towards normal values at 40 days of age. At maturity, between 60 and 80 days of age, these rats showed poor performance in a complex 6-choice discrimination learning but not in simple differential conditioning. Possible correlations between the behavioural and biochemical findings are discussed.

The relationship between brain beta-adrenergic receptors and adaptation to stress was studied in rats subjected to repeated restraint stress. The stress was found to produce a reduction in the density of these receptors in the hypothalamus, cerebral cortex and brain stem. This change first appeared after 4-7 days and persisted for the duration of the two-week stress. Adaptation, as judged by resistance to the anorexic and gastric lesion-inducing effects of the stress, occurred progressively over the full two-week period. The loss of beta-receptors was found to correlate positively with the degree of adaptation. The relationship was strongest for the hypothalamus but was also apparent in the cortex and brain stem. These findings support the hypothesis that a reduction in the number of brain adrenergic receptors is one of the biochemical factors underlying adaptation to stress.

Bilateral lesion of the ascending noradrenergic fibers in the dorsal bundle of adult Wistar rats with 4 micrograms 6-hydroxydopamine caused extensive depletion of norepinephrine in all forebrain areas, but led to a 54% increase in norepinephrine levels in the cerebellum. beta-Adrenergic receptor binding of [3H]dihydroalprenolol was significantly increased in all forebrain areas depleted of norepinephrine except hypothalamus. The increase in [3H]dihydroalprenolol binding was due to 62% and 34% increases in the number of beta-receptor sites in the frontal cerebral cortex and hippocampus respectively. Binding of [3H]WB-4101 to alpha 1-adrenergic receptors after dorsal bundle lesion was augmented generally to a lesser extent than beta-receptor binding, with significantly increased numbers of sites only in the frontal cortex (74%), thalamus (20%) and septum. Both alpha 1- and beta-receptor binding sites were reduced in number by 25-28% in the cerebellum of dorsal bundle-lesioned rats, whereas intraventricular administration of 6-hydroxydopamine to adult rats, which depletes norepinephrine in the cerebellum by 96%, increased cerebellar alpha 1- and beta-receptor binding by 33-40%. Binding of [3H]clonidine to forebrain alpha 2-adrenergic receptors was significantly elevated in the frontal cortex, but reduced in the amygdala and septum, after dorsal bundle lesion.