Effects of ovarian hormones on levels of luteinizing hormone in plasma and on serotonin concentrations in discrete brain nuclei

Levels of serotonin were measured in microdissected, individual brain nuclei in ovariectomized rats after treatment with ovarian hormones. Regions sampled included nuclei in the forebrain, rostral and medial hypothalamus, and midbrain tegmentum. Estradiol benzoate decreased levels of luteinizing hormone in plasma but did not affect serotonin levels in any region. Progesterone alone elevated serotonin content in the nucleus tractus diagonal's and ventral tegmental area. The combined estrogen plus progesterone regime produced a surge in plasma luteinizing hormone and also markedly elevated serotonin in the median eminence. These results may be of significance for ovarian hormonal regulation of gonadotropin secretion and reproductive behavior.     

Effects of tramadol on alpha2-adrenergic receptors in the rat brain

In recent years, it has been postulated that tramadol, used mainly for the treatment of moderate to severe pain, might display a potential as an antidepressant drug. The present study investigated the effects of acute and repeated tramadol administration on the binding of [³H]RX 821002, a selective α₂-adrenergic receptor ligand, in the rat brain. Male Wistar rats were used. Tramadol (20 mg/kg, i.p.) administered acutely (single dose), at 24 h after dosing, induced a significant decrease in the α₂-adrenergic receptors in all brain regions studied. The most pronounced effects were observed in all subregions of the olfactory system, nucleus accumbens and septum, thalamus, hypothalamus, amygdala, and cerebral cortex. Repeated treatment with tramadol (20 mg/kg, i.p., once daily for 21 days) also induced statistically significant downregulation of [³H]RX 821002 binding sites in the rat brain. However, the effect—although statistically significant—was less pronounced than in the group treated acutely with the drug. Since drugs such as mianserin and mirtazapine are potent antagonists of central α₂-adrenergic receptors and are effective antidepressants, it is tempting to suggest that, in addition to other alterations induced by tramadol, downregulation of these receptors may represent a potential antidepressant efficacy. On the other hand, one should be careful to avoid the treatment of chronic pain with tramadol in patients already receiving antidepressant drugs. Tramadol-induced downregulation of α₂-adrenergic receptors—when combined with ongoing antidepressant therapy with drugs, which themselves inhibit serotonin reuptake or are antagonists of α₂-adrenergic receptors—might cause threatening complications.     

Effects of the noradrenergic neurotoxin DSP4 on spatial memory in the rat

Summary Patients with attention-deficit/hyperactivity disorder (ADHD) show various cognitive impairments such as deficits in attention or working memory. Most symptoms of ADHD are thought to be associated with a dysbalance between the neurotransmitters noradrenaline and dopamine in the brain. In order to investigate the role of noradrenaline in this context we have produced a central depletion of noradrenaline in rats by administering different doses (10, 20 or 50 mg/kg body weight) of the neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) and assessing the behavioral consequences with a modified hole board task. The administration of DSP4 affected the working memory error, while reference memory and motor functions were not affected. The use of different doses of DSP4 to influence prefrontal functions and to understand the dysbalance of dopamine and noradrenaline in ADHD appears to be a promising approach. Correspondence: Klaus W. Lange, Department of Experimental Psychology, University of Regensburg, 93040 Regensburg, Germany     

Effect of noradrenergic denervation by neonatal DSP-4 on peptide neurotransmitter systems in the rat brain.

The specific effects of a neonatal treatment of rats with the noradrenergic neurotoxin DSP-4 were investigated in the adult rat by measuring monoamine and peptide transmitter levels in eight brain regions. When applied concomitantly with a 5-hydroxytryptamine uptake blocker, neonatal DSP-4 induced a selective depletion of noradrenaline (NA) in cortex, hippocampus and amygdala. An increase of NA was observed in the medulla and substantia nigra/ventral tegmental area and a decrease in dopamine was observed in the thalamus. The vasoactive intestinal polypeptide levels were markedly elevated in the DSP-4 treated rats in almost all CNS regions whereas those of three other neuropeptides remained unchanged. It is concluded that certain adaptational changes can be observed in the peptide systems of the CNS upon disruption of the development of the noradrenergic innervation.     

New studies and perspectives on the noradrenergic receptor system in depression: effects of the alpha 2-adrenergic agonist clonidine

In an attempt to understand the dynamics of noradrenergic function in depression, we evaluated neuroendocrine, biochemical, cardiovascular, and behavioral responses to the acute intravenous administration of the alpha 2-adrenergic agonist, clonidine, in depressed patients and normal controls. Significantly more variance was observed in the depressed patients than the controls for most indices of basal noradrenergic output including plasma norepinephrine (NE) and 3-methoxy-4-hydroxyphenylglycol (MHPG). Growth hormone, plasma MHPG, and heart rate responses to clonidine were reduced in the depressed patients compared to the controls, all suggesting reduced responsiveness of alpha 2-adrenergic receptors in depression. Baseline levels of cortisol were elevated in the depressed patients compared to the controls. Clonidine decreased cortisol to normal levels in the depressed patients but had little effect in the controls. Thus the depressed patients manifested a significantly increased cortisol response to clonidine. These data raise the possibility that the hypercortisolemia of depression may be related to noradrenergic dysfunction. Clonidine also significantly reduced anxiety in the depressed patients, particularly those with elevated basal plasma MHPG, but not in controls. These results suggest that diminished alpha 2-adrenergic responsiveness as documented by decreased endocrine, biochemical, and physiological responses to clonidine may be related to the depressive and anxiety symptoms as well as the neuroendocrine disturbances characteristic of many depressed patients.     

Effects of drugs which influence noradrenergic activity on metenkephalin levels in specific brain areas

The effects of pretreatment with clonidine, an alpha 2 adrenoceptor agonist, and isobutylmethylxanthine, a phosphodiesterase inhibitor, were measured on the metenkephalin content of the locus coeruleus, the amygdala and the nucleus periventricularis thalamis of rat brain. No significant effect of either drug on metenkephalin content could be detected.     

Rapid changes in brain aromatase activity in the female quail brain following expression of sexual behaviour

In male quail, oestrogens produced in the brain (neuro‐oestrogens) exert a dual action on male sexual behaviour: they increase sexual motivation within minutes via mechanisms activated at the membrane but facilitate sexual performance by slower, presumably nuclear‐initiated, mechanisms. Recent work indicates that neuro‐oestrogens are also implicated in the control of female sexual motivation despite the presence of high circulating concentrations of oestrogens of ovarian origin. Interestingly, aromatase activity (AA) in the male brain is regulated in time domains corresponding to the slow “genomic” and faster “nongenomic” modes of action of oestrogens. Furthermore, rapid changes in brain AA are observed in males after sexual interactions with a female. In the present study, we investigated whether similar rapid changes in brain AA are observed in females allowed to interact sexually with males. A significant decrease in AA was observed in the medial preoptic nucleus after interactions that lasted 2, 5 or 10 minutes, although this decrease was no longer significant after 15 minutes of interaction. In the bed nucleus of the stria terminalis, a progressive decline of average AA was observed between 2 and 15 minutes, although it never reached statistical significance. AA in this nucleus was, however, negatively correlated with the sexual receptivity of the female. These data indicate that sexual interactions affect brain AA in females as in males in an anatomically specific manner and suggest that rapid changes in brain oestrogens production could also modulate female sexual behaviour.     

Effects of suckling on serum prolactin levels and catecholamine concentrations and turnover in discrete brain regions

The effects of suckling on serum prolactin levels and catecholamine concentrations and turnover were examined in several discrete brain regions. Turnover rates were assessed by using the synthesis inhibitor alpha-methyltyrosine (alpha-MT) in combination with microdissection techniques for the removal of individual brain regions and sensitive radioenzymatic assays for norepinephrine (NE) and dopaime (DA). Prolactin secretion was induced by mothers experiencing 6 h of pup removal with subsequent pup replacement. Suckling or the administration of alpha-MT to mothers resulted in a marked increase in circulating titers of prolactin. A decrease in steady-state NE concentrations in the anterior hypothalamus and a decrease in steady-state DA concentrations in the ventromedial nucleus were noted in suckled mothers. The comparison of relative rates of NE depletion after alpha-MT treatment revealed a suckling-induced increase in turnover in the ventromedial nucleus and a suckling-induced decrease in turnover in the anterior hypothalamus. Neither suckling nor alpha-MT treatment produced any changes in NE or DA turnover rates in the arcuate nucleus or median eminence. These findings demonstrate that suckling-induced activation of prolactin results in changes in noradrenergic processes in the ventromedial and anterior hypothalamic nuclei. This suggests an involvement of noradrenergic systems in suckling-induced prolactin release.     

Stress and β-adrenergic receptor binding in the rat's brain

The effect of acute and chronic electric shocks on β-adrenergic receptor binding in the rat's brain was investigated. β-adrenergic receptor subsensitivity in the corter 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 β-adrenergic receptor subsensitivity.     

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.     

Distribution of aromatase in the brain of the Japanese quail, ring dove, and zebra finch: an immunocytochemical study

An immunocytochemical peroxidase-antiperoxidase procedure using a purified polyclonal antibody raised against human placental aromatase was used to localize aromatase-containing cells in the brain of three avian species: the Japanese quail, the ring dove, and the zebra finch. In quail and dove, immunoreactive cells were found only in the preoptic area and hypothalamus, with a high density of positive cells being present in the medial preoptic area, in the septal area above the anterior commissure, in the ventromedial nucleus of the hypothalamus, and in rostral part of the infundibulum. Immunoreactivity was weaker in zebra finches, and no signal could therefore be detected in the ventromedial and tuberal hypothalamus. The positive material was localized in the perikarya and in adjacent cytoplasmic processes, including the full length of axons always leaving a clear unstained cell nucleus. These features could be observed in more detail on sections cut from perfused brains and stained with an alkaline phosphatase procedure. The distribution of aromatase immunoreactivity was similar in the three species although minor differences were observed in the preoptic area. The localization of labelled neurons coincided with the distribution of aromatase activity as studied by in vitro radioenzyme assays on brain nuclei dissected by the Palkovits punch method. There was one striking exception to this rule: no immunoreactivity was detected in the zebra finch telencephalon, while assays had shown the presence of an active enzyme in several nuclei such as the robustus archistriatalis, the hyperstriatum ventrale pars caudale, and the hippocampus and area parahippocampalis. The origins of this discrepancy and the functional role of the aromatase observed in the axons are discussed.     

Neurotoxic effects of DSP-4 on the central noradrenergic system in male zebra finches

When administered systemically, the noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) appears to target the noradrenergic innervation originating in the locus coeruleus causing long-term decrements in noradrenergic function. In songbirds, DSP-4-treatment decreased female-directed singing by males and copulation solicitation responses of females to male songs. However, DSP-4 treatment in songbirds did not lower measures of NE function in the brain to the same extent as it does in mammals. The current study had two goals: determining if two DSP-4 treatments 10 days apart would cause significant decrements in noradrenergic function in male zebra finches and determining if, as in other species, the noradrenergic innervation of midbrain and cortical areas would be profoundly affected while hypothalamic areas were spared. Dopamine-beta-hydroxylase immunoreactivity (DBH-ir) was quantified in thirteen brain regions (five vocal control nuclei, one auditory nucleus, two hypothalamic nuclei, and five additional areas that demonstrated high DBH labeling in controls). Within 20 days, DSP-4 treatment profoundly reduced the number of DBH-ir cells in both the locus coeruleus and ventral subcoeruleus. Unlike a previous study, DBH labeling delineated four out of five vocal control nuclei and an auditory nucleus. As expected, DSP-4 treatment significantly decreased DBH labeling in all areas examined in the mesencephalon and telencephalon without significantly affecting DBH-ir in hypothalamic areas. This double treatment regime appears to be much more effective in decreasing noradrenergic function in songbirds than the single treatment typically used.     

Effects of morphine on luteinizing hormone secretion and catecholamine turnover in the hypothalamus of estrogen-treated rats

This study examined the effects of morphine sulfate and naloxone alone or in combination on phasic luteinizing hormone (LH) secretion in estrogen-treated ovariectomized rats. Thereafter, the effects of morphine on initial concentrations, rate constants and rates of turnovers of norepinephrine and dopamine were evaluated in untreated or morphine-injected, estrogen-primed rats. Morphine, when given at 12.30 h, completely suppressed the spontaneous LH surges which occur in steroid-treated rats. The opiate antagonist, naloxone, (12.15 h) markedly amplified and advanced the time of LH release and a combination of morphine and naloxone produced peak afternoon LH values which were intermediate between those obtained in controls and in rats receiving only naloxone. Norepinephrine (NE) and dopamine (DA) turnover were calculated from data obtained in groups of rats sacrificed 0,45 or 90 min after administering 300 mg/kg b. wt. i.p. ofα-methyl-p-tyrosine (α-MPT) at 10.00 or 15.00 h. In these experiments, the medial preoptic nucleus (MPN) and the median eminence (ME) were microdissected and analyzed for changes in NE and DA concentrations by a radioenzymatic procedure. In estrogen-treated rats, NE rate constants and turnover significantly increased at 15.00 vs 10.00 h in MPN and ME concomitant with increases in serum LH. Morphine blocked both increases in rate constants and turnover in the MPN and ME and also significantly reduced initial concentrations of NE in the MPN. None of the DA parameters measured in MPN or ME changed in estrogen-treated controls between morning and afternoon. Further, while morphine did not affect DA turnover in the MPN, DA turnover declined in the ME. These data add to accumulating evidence which demonstrates an important modulatory role for hypothalamic opiate neurons in regulating catecholamine activity and gonadotropin secretion.     

Effects of testosterone on alpha2A-adrenergic receptor expression in the rat brain

Androgens are involved in regulation of behaviour through intracellular mechanisms owing to their receptors. Involvement of intercellular messengers such as brain norepinephrine and adrenergic receptors (ARs) is seemed to be necessary to realise hormone-dependent behavioural effects. Castration of adult male rats, which decreases copulatory activity in the animals, was accompanied by a significant increase in 3H-clonidine (alpha2-AR agonist) binding site density in the frontal cortex. The levels of mRNA for the alpha2A-ARs (measured by RT-PCR) were increased in the brainstem of castrated males in parallel to the changes in cortical ARs densities. Testosterone treatment, that activates copulatory behaviour in castrates, down regulated alpha2A-AR mRNA levels in the brainstem and 3H-clonidine binding sites densities in the cortex, where terminals of the brain stem neurones are situated. Unlike in the brainstem, castration caused a decrease in alpha2A-AR mRNA in the cortex and testosterone up-regulated this mRNA in the cortical region. The data suggested that down-regulation of alpha2-ARs densities in the cortex that is induced by testosterone can be preferentially related to alpha2-ARs subpopulation which is expressed by the brainstem neurones and imported into the cortex by axons of these neurones.     

Transient elevation of locus coeruleus alpha 2-adrenergic receptor binding during the early stages of amygdala kindling

Enhancement of noradrenergic neurotransmission retards, but does not prevent, the development of kindling. The firing activity of noradrenergic locus coeruleus (LC) neurons is partially regulated by axon collateral recurrent inhibition mediated via alpha 2-adrenergic receptors. We tested the hypothesis that LC autoinhibitory alpha 2-adrenergic receptors may change during the kindling process thereby altering LC excitability. Specific binding of the alpha 2-adrenergic receptor antagonist [3H]RX781094 (idazoxan) was measured in the LC of rats at 3 different stages of kindling development using in vitro neurotransmitter receptor autoradiography techniques. Specific [3H]RX781094 binding was elevated significantly in rats kindled to two Class 1 kindled motor seizures. No differences in binding were observed in animals kindled to Class 3 or Class 5 kindled motor seizures. Saturation of binding experiments indicated that the increase in binding following two Class 1 kindled motor seizures was due to an increase in the total number of alpha 2-receptors without a change in the affinity of the binding site for [3H]RX781094. The transient increase in number of LC alpha 2-adrenergic receptors is consistent with the idea that noradrenergic neurotransmission inhibits the early progress of kindling development, but then subsequently becomes ineffective in maintaining the inhibition during later stages of kindling development.     

Effects of prenatal malnutrition on GABAA receptor alpha1, alpha3 and beta2 mRNA levels

Exposure of pregnant rats to protein malnutrition throughout pregnancy alters the developing hippocampus, leading to increased inhibition and selective changes in hippocampal-mediated behaviors. Given that GABA mediates most inhibitory neurotransmission, we asked whether selective changes in the levels of GABA receptor subunit mRNAs might result. Quantitative RNase protection profiling of 12 GABAA and GABAB receptor subunit mRNAs show that alpha1 and beta2 decrease in the adult (P90) hippocampal formation of prenatally malnourished rats, while the levels of alpha3 are increased. Moreover, the distribution of alpha1, alpha3 and beta2 mRNAs remains unchanged in CA1 and CA3 hippocampal subfields relative to dentate gyrus. The data suggest that prenatal malnutrition produces global changes of certain GABAA, but not GABAB, receptor mRNAs in the hippocampal formation.     

Effects of thyrotropin-releasing hormone on catecholamine concentration in various structures of the rat brain

In an attempt to identify the site of TRH-action in the central nervous system (CNS), we have measured catecholamine levels in discrete brain regions of rats after an intravenous administration of TRH or a long-term intraventricular administration of TRH using Alzet's osmotic minipump. An intravenous administration of 50 micrograms TRH caused a rapid increase in dopamine (DA) concentration in the septal nucleus and then returned to the basal level at 30 min after injection. The concentration of DA in the septal nucleus after the long-term TRH intraventricular administration was also significantly higher than that in the controls. These results indicate that a long-term intraventricular administration of TRH as well as a bolus intravenous injection of TRH may affect the release or metabolism of DA in the septal nucleus.     

Serum protein leakage in aged human brain and inhibition of ligand binding at alpha 2-adrenergic and cholinergic binding sites

Serum proteins are known to extravasate into the brain parenchyma in senile and presenile dementia (Glenner: Hum. Pathol. 16:433-435, 1986; Wisniewski and Kozlowski: Ann. NY Acad. Sci. 396:119-129, 1982). We have recently demonstrated that human serum Cohn fraction IV (alpha-globulin enriched) inhibits ligand binding at putative dopamine and serotonin2 receptors labeled by [3H]spiroperidol in human brain (Andorn, Pappolla, Fox, Klemens, and Martello: Proc. Natl. Acad. Sci. USA 83:4572-4575, 1986). We now demonstrate that serum proteins can be identified in the neuropil and in neuronal cell bodies in normal aged brain, that alpha-globulin-enriched fractions inhibit ligand binding at alpha 2-adrenergic and muscarinic binding sites in human brain as well, and that serum proteins can be identified within neuronal cytoplasm and axons.     

Distribution of alpha 2-adrenergic receptor mRNAs in the rat CNS.

alpha 2-Adrenergic receptors (ARs) are involved in central nervous system (CNS) control of blood pressure. It is now known that there are three human genes that encode subtypes of alpha 2-ARs, but little is known regarding the distribution of these subtypes throughout the CNS. The availability of receptor clones allows the mapping of mRNAs encoding the individual alpha 2-AR subtypes in the CNS. In this communication, we report that there are three, closely related rat alpha 2-AR genes. We have developed subtype-specific hybridization probes from each of these genes and have used these reagents to measure alpha 2-AR subtype mRNA accumulation in extracts of discrete regions of the rat CNS. We found that mRNAs encoding the alpha 2A-AR and alpha 2C-AR subtypes are distributed widely, but unevenly, throughout the rat CNS. The A subtype is prominent in the midbrain, brainstem, spinal cord, pituitary and diencephalon while the C subtype predominates in basal ganglia and cerebellum. The cortex, olfactory bulb and hippocampus contain roughly equal amounts of the alpha 2A- and alpha 2C-AR mRNAs. A third subtype's (alpha 2B-AR) mRNA is far less abundant in brain tissues, and is only found in the diencephalon.     

Analysis of platelet alpha2-adrenergic receptor activity in stable coronary artery disease patients on dual antiplatelet therapy

Combined antiplatelet therapy reduces recurrent atherothrombotic events in stable coronary disease patients; however, high residual platelet reactivity measured ex vivo still raises concerns as a condition related to treatment failure. Alpha-2 adrenoceptor enhances platelet reactivity and might contribute to this phenomenon. For the present study, 121 stable angina patients on standard dual antiplatelet therapy (75 mg clopidogrel and 100 mg acetylsalicylic acid) were recruited. Born aggregometry was performed with adenosine diphosphate (ADP), collagen and epinephrine. To verify platelet adrenergic activity, potentiation by low-dose epinephrine and inhibition by selective alpha-2 receptor blocker atipamezole were determined. To assess the P2Y(12)-specific residual activity, cangrelor was used. Plasma norepinephrine, soluble CD40-ligand, high-sensitivity-C-reactive protein (hsCRP) - and in 24 subjects platelet P-selectin positivity were measured. Epinephrine - at very low concentration (10(-9)g/ml) - significantly potentiates (1.25 microM ADP: 26.5% vs. 43%; 5 microM ADP: 53% vs. 64.5%; collagen: 17% vs 42%, p < 0.001) while atipamezole inhibits ADP- and collagen-induced platelet aggregations (1.25 microM ADP: 26.5% vs. 23%; 5 microM ADP: 53% vs. 47%; collagen: 17% vs. 11%, p < 0.001). Patients with high adrenergic activity have significantly increased baseline ADP- and collagen-induced platelet aggregation. Based on cangrelor's efficacy, these patients have significantly more residual P2Y(12) activity as well. HsCRP and soluble CD40-ligand levels were similar. In conclusion, stable coronary heart disease patients with prominent adrenoceptor activity in vitro have significantly increased platelet aggregability and more functional P2Y(12) receptor, indicating poor inhibitory response to thienopyridines. Therefore, platelet adrenergic receptor represents a considerable, dynamic factor of high residual platelet reactivity and might contribute to cardiovascular events indicating failure of antiplatelet therapy.