Bruce Nock

Effects of 5, 7-dihydroxytryptamine on serotonin1 and serotonin2 receptors throughout the rat central nervous system using quantitative autoradiography

The effects of the serotonin neurotoxin 5,7-dihydroxytryptamine (5,7-DHT), on serotonin1 (5-HT1) and 5-HT2 receptors were investigated using the high degree of resolution provided by quantitative autoradiography in an effort to determine the synaptic location of these receptors. 5,7-DHT treatment resulted in a decrease in 5-HT1 binding in the dentate gyrus and CA3c/4 of the anterior hippocampus and in the dorsal raphe nucleus, whereas no changes were observed in the posterior hippocampus nor in many other brain structures. 5-HT2 receptors exhibited no changes in any brain area examined in response to 5,7-DHT treatment, despite over 90% serotonin depletion in most of the forebrain nuclei examined. The results indicate that at least some of the 5-HT1 sites labelled by [3H]5-HT in the hippocampus and dorsal raphe nucleus are presynaptic, whereas 5-HT2 receptors are probably postsynaptic. In addition, the distribution profiles of 5-HT1 and 5-HT2 binding sites were compared in the rat central nervous system at various anatomical levels. 5-HT1 binding sites were identified using [3H]5-HT, while 5-HT2 binding sites were labelled with [3H]ketanserin. Both receptor subtypes displayed distinctly different localization patterns, which, in most cases was the inverse of the other pattern. In the brainstem it is significant that 5-HT2 receptors are concentrated in the facial nucleus and the motor nucleus of the trigeminal nerve, areas known to influence head and facial movement. The serotonin-mediated head-shake response occurs when 5-HT2 receptors are activated. In contrast, 5-HT1 receptors are distributed throughout the brainstem and in specific portions of the spinal cord. These areas are thought to control the serotonin behavioral syndrome and this behavior is 5-HT1A-mediated. All raphe nuclei were devoid of 5-HT2 receptors; only 5-HT1 receptor were found in these nuclei. Correlations with serotonin terminal distribution patterns are discussed. The pattern of 5-HT2 receptor distribution was also compared with the pattern of alpha 1 receptors, using [3H]prazosin in order to determine whether [3H]ketanserin significantly labels alpha 1 receptors. Although some similarities exist, overlap of binding did not occur in other nuclei, indicating that alpha 1 contamination of this system is probably negligible.

Nitric oxide control of steroidogenesis: Endocrine effects of NG-nitro-L-arginine and comparisons to alcohol

Recent studies suggest that nitric oxide (NO) may regulate hormone biosynthesis and secretion. This was tested by treating male rats with NG-nitro-L-arginine methyl ester (NAME), a NO synthase inhibitor, and measuring serum and testicular interstitial fluid testosterone and serum corticosterone, luteinizing hormone (LH), and prolactin (PRL). The effect of NG-nitro-L-arginine (NA), a less-soluble form of the same NO synthase inhibitor, on the reproductive suppressant actions of alcohol was also examined. NAME increased testosterone and corticosterone secretion dose-dependently without affecting LH and PRL secretion. The alcohol-induced suppression of testosterone or LH secretion was not altered by treatment with NA. Although effects of NAME and NA on other systems may be involved, these results indicate that testicular and adrenal steroidogenesis are negatively regulated by endogenous NO and that NO does not regulate LH and PRL secretion or inhibit the testicular steroidogenic pathway in the same way as alcohol.

Regional Specificity of Gamma-Aminobutyric Acid Receptor Regulation by Estradiol

In vitro quantitative autoradiography and the microdissection technique of Palkovitz were used to examine the effects of estradiol-17 beta on GABAA receptors and on glutamic acid decarboxylase in discrete areas of rat brain. Under the conditions examined, estradiol did not affect glutamic acid decarboxylase activity. However, treatment with estradiol decreased GABAA receptor binding in a majority of areas that contain high levels of intracellular estradiol receptors and in a number of areas that contain few or no estradiol receptors. Within one brain area, the ventromedial nucleus of the hypothalamus, the estradiol effect was mapped and found to occur within the estradiol-sensitive ventrolateral portion and the surrounding dendritic plexus. Time- and dose-response relationships were region specific suggesting that estradiol might influence GABAA-receptor binding through multiple mechanisms. Estradiol does not appear to interact directly with GABAA receptors since addition of estradiol to the assay system did not affect binding. Our findings suggest that one way estradiol might affect neuroendocrine and other centrally mediated processes is through effects on GABAA-receptor binding.

Autoradiography of [3H]U-69593 binding sites in rat brain: evidence for κ opioid receptor subtypes

In vitro quantiative autoradiography was used to determine the distribution of [3H]U-69593 binding sites in rat brain. The highest density of binding was found in areas of the tel- and diencephalon while certain areas of the mes- and metencephalon were moderately labeled. The distribution of [3H]U-69593 binding sites corresponds closely (but not precisely) to the distribution of sites labeled by [3H]EKC and [3H]bremazocine at room temperature but differs substantially from the distribution of sites labeled by [3H]EKC at 4°C (as described by others). These anatomical findings support previous biochemical evidence indicating that [3H]U-69593 selectively labels a κ opioid receptor subtype with characteristics that differ from the κ receptors labeled by [3H]EKC at 4°C.

Neurotransmitter modulation of steroid action in target cells that mediate reproduction and reproductive behavior

Two major functional interactions between steroid hormones and neurotransmitters are generally recognized. First, steroids affect neurotransmission, and second, through effects on hypothalamic peptides that regulate anterior pituitary function neurotransmitters affect steroid secretion. In recent years, evidence has accumulated which indicates that neurotransmitters can also affect steroid action within postsynaptic steroid target cells. We review evidence for this relationship in pineal, uterus and hypothalamus and propose that the modulation of target cell responsiveness to steroids is an important mechanism by which neurotransmitters affect steroid-dependent processes. The operation of such a mechanism provides a means for environmental, behavioral and emotional events to rapidly and selectively alter steroid effects on behavior and physiology.

Changes in noradrenergic transmission alter the concentration of cytoplasmic progestin receptors in hypothalamus

Summary These experiments were designed to determine whether drug-induced changes in NA transmission affect lordosis behavior of female guinea pigs by altering steroid action within hypothalamic target cells. In the first experiment, we examined the effects of the dopamine-β-hydroxylase inhibitor, U-14,624, on cytoplasmic progestin receptors (measured using a one-point [3H]R5020 binding assay) in hypothalamus (HYPO), preoptic area (POA), cerebral cortex (CORT) and midbrain (MB) of estradiol benzoate (EB)-primed females. At 12 h after U-14,624 administration, specific binding of [3H]R5020 was 36% less in cytosol from HYPO (binding in POA, CORT, and MB was not affected by U-14,624) in drug-treated than in non-drug-treated controls. To determine whether this reduction in [3H]R5020 binding was due to competition of U-14,624 with [3H]R5020 for progestin receptors, we examined the effects of U-14,624 on [3H]R5020 binding in vitro. U-14,624 had no effects on [3H]R5020 binding under these conditions. By using a range of [3H]R5020 concentrations to assay cytoplasmic progestin receptors, we found that the reduction in [3H]R5020 binding on hypothalamic cytosol after U-14,624 treatment was due to a lower concentration of progestin receptors rather than to a lower progestin receptor affinity for [3H]R5020. Several lines of evidence indicate that the lower (compared to non-drug-treated females) concentration of these receptors was attributable to a reduction in NA transmission. First, U-14,624 caused a significant reduction in regional brain NE content. Second, activation of α-adrenergic receptors with clonidine completely reversed the effects of U-14,624 on cytoplasmic progestin receptors, although clonidine had no effect on progestin receptors when administered alone. Third, blockade of α-adrenergic receptors by i.p. injection of phenoxybenzamine (Pb) resulted in a relative reduction of specific [3H]R5020 binding in hypothalamic (but not POA, CORT or MB) cytosol of EB-primed females. There was a 3–4 h delay between the blockade of α-receptors by Pb (determined using a [3H]WB4101 binding assay) and a significant effect on the concentration of progestin receptors in cytoplasm of HYPO. The effects of Pb on hypothalamic progestin receptors did not appear to be due to a peripheral action of the drug. When administered intraventricularly, Pb caused a relative reduction in specific [3H]R5020 binding in hypothalamic (but not POA, CORT or MB) cytosol of EB-primed females. The lower concentration of progestin receptors in HYPO after drug treatment also did not appear to be attributable to the release of adrenal progesterone. The concentrations of progesterone in plasma at 0.5, 2, 4, and 12 h after Pb and at 12 h after U-14,624 administration were not different from control values. Furthermore, the concentration of cytoplasmic progestin receptors was lower only in HYPO after Pb and U-14,624 treatment, whereas injection of 100 μg progesterone caused a reduction in the concentration of these receptors in all brain areas examined. The lower concentration of cytoplasmic progestin receptors in HYPO after drug treatment might be attributable to a drug-induced interference with the EB-induced increase in the concentration of these receptors. This hypothesis is supported by the finding that Pb has no effect on hypothalamic progestin receptors in the absence of EB priming. Thus, changes in NA transmission might alter EB action in HYPO in addition to altering target cell sensitivity (through effects on progestin receptor concentration) to progestins. On the basis of these results we propose that the modulation of target tissue responsiveness to steroids is an important mechanism by which neurotransmitters affect steroid-dependent processes. The operation of such a mechanism would provide a rapid means by which environmental, behavioral and emotional events could modulate steroid-dependent behaviors and anterior pituitary function.

Noradrenergic transmission and female sexual behavior of guinea pigs.

Treatment with the dopamine beta-hydroxylase (DBH) inhibitor U-14,624 (50, 100, or 150 mg/kg) blocked the induction of lordosis behavior be estradiol benzoate (EB) and progesterone (P) in ovariectomized guinea pigs. After treatment with U-14,624 (100 mg/kg), norepinephrine (NE) content of medial basal hypothalamus, preoptic area and cortex was reduced (by 55%) and dopamine (DA) content of medial basal hypothalamus was increased (by 155%) during the period when females treated with EB and P normally display lordosis. Treatment with the NE receptor stimulator clonidine (1.0 mg/kg) restored lordosis behavior in females treated with EB, P, and U-14,624 (100 mg/kg), but the putative DA and serotonin (5-HT) receptor blockers pimozide (1.0 mg/kg) and methysergide (20.0 mg/kg) were ineffective in this respect. Thus, inhibition of lordosis after treatment with U-14,624 appeared to be attributable primarily to a reduction in NE neurotransmission, rather than to increase in DA or 5-HT activity. Because clonidine induced lordosis in females treated with EB, P, and U-14,624, it seemed unlikely that the facilitatory effects of clonidine on lordosis were mediated by activation of presynaptic alpha-adrenergic receptors (i.e. inhibitory NE autoreceptors) rather than by postsynaptic alpha-receptors. In addition, pretreatment with the postsynaptic alpha-adrenergic antagonist phenoxybenzamine (20.0 mg/kg) blocked the facilitation of lordosis by clonidine (1.0 mg/kg) in females primed with EB alone and with EB plus P. Thus, the facilitatory effects of clonidine on lordosis appear to be mediated by activation of postsynaptic alpha-adrenergic (i.e. NE) receptors. The results of this study provide further evidence that NE neurotransmission facilitates the expression of female sexual behavior in guinea pigs.

Distribution ofα2-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.

Gender-linked differences in the expression of physical dependence in the rat.

In earlier studies, it was shown that there were gender differences in several aspects of the pharmacological profile of morphine, including its antinociceptive activity, discriminative stimulus properties and its reinforcing effects. The purpose of the present studies was to examine whether there might also be gender-related differences in the development of physical dependence, as reflected in the expression of an opiate withdrawal syndrome upon cessation of chronic morphine administration. We found that a more severe spontaneous withdrawal syndrome was produced by chronic morphine injections or morphine pellet implantation in male rats than in females. The duration of the withdrawal syndrome was also longer. In contrast to our observations with spontaneous withdrawal, we found no gender differences in the naloxone-precipitated withdrawal syndrome induced by chronic morphine administration. These observations suggest that there are gender differences only in the expression of the spontaneous withdrawal syndrome, but not in the neuro-adaptive changes associated with the generation of physical dependence as reflected by naloxone-precipitated withdrawal.

Chronic corticosterone treatment impairs spontaneous alternation behavior in rats.

The present study used behavioral and morphological measures to assess hippocampal integrity in adult male rats after 8 weeks of daily corticosterone (10 mg/kg) injections. Behavioral testing during the final week of treatment revealed that spontaneous alternation behavior, a behavioral marker of hippocampal damage, was reduced in experimental animals without influencing exploration. Physiological assessment indicated that steroid exposure produced functional changes characteristic of prolonged exposure to stress or elevated plasma corticosterone, i.e., lower body weight and thymic involution. However, hippocampal cell loss was not observed in experimental rats. The data suggest that prolonged elevation of plasma corticosterone may significantly disrupt a hippocampal-sensitive behavior without producing gross morphological changes.