Estrogen and phytoestrogen regulate the mRNA expression of adrenomedullin and adrenomedullin receptor components in the rat uterus

The serum estrogen surge in the uterus triggers precisely-timed physiological and biochemical responses required establishing and maintaining pregnancy. Previous reports have shown that consumption of phytoestrogen-containing plants may disrupt the precise control of pregnancy. To evaluate the effects of phytoestrogens in the uterus, we screened for estradiol (E2)-inducible genes in immature rat uteri. We identified the gene for receptor-activity-modifying protein 2 (Ramp2), known to be a component of the adrenomedullin (ADM) receptor, as responsive to both E2 and the phytoestrogen coumestrol (Cou). We further examined the expression of ADM and ADM signaling components Ramp2, Ramp3, and CRLR in the immature rat uterus and found that both E2 and Cou regulated these genes expression. In addition, treatment with ADM increased uterine weight and edema similar to that observed after Cou treatment. Our findings indicated that the phytoestrogen caused the abnormal induction of vasoactive factors in the uterus.     

Neonatal ovariectomy and pituitary-adrenal responsiveness in the adult rat.

Litters of female rat pups were handled daily from birth to weaning. When 12 days old the pups were ovariectomized or received sham surgery. At 70 days sham and ovariectomized animals were decapitated immediately or placed within a novel environment for 10, 20, or 40 min before decapitation. Basal plasma levels of androstenedione in ovariectomized animals were approximately half that of intact sham controls, and stress caused only a small rise in androstenedione in this group. Androstenedione levels in ovariectomized animals were consistently at or below the lower sensitivity of the radioimmunoassay, indicating that circulating levels of androstenedione in the female rat are primarily of ovarian origin. Basal ACTH and corticosterone levels did not differ significantly between the two groups. However, stress-induced levels of ACTH and corticosterone were significantly depressed in ovariectomized subjects compared to controls following exposure to the novel environment. Furthermore, while ACTH levels started to return to baseline by 40 min in controls, this effect was not observed in ovariectomized animals. These findings demonstrate that prepubertal removal of ovarian humoral factors can have a long-term impact on the responsiveness of the hypothalamic-pituitary-adrenal axis to stress.     

Opiates inhibit neurogenesis in the adult rat hippocampus

Recent work implicates regulation of neurogenesis as a form of plasticity in the adult rat hippocampus. Given the known effects of opiates such as morphine and heroin on hippocampal function, we examined opiate regulation of neurogenesis in this brain region. Chronic administration of morphine decreased neurogenesis by 42% in the adult rat hippocampal granule cell layer. A similar effect was seen in rats after chronic self-administration of heroin. Opiate regulation of neurogenesis was not mediated by changes in circulating levels of glucocorticoids, because similar effects were seen in rats that received adrenalectomy and corticosterone replacement. These findings suggest that opiate regulation of neurogenesis in the adult rat hippocampus may be one mechanism by which drug exposure influences hippocampal function.     

Chronic restraint stress up-regulates GLT-1 mRNA and protein expression in the rat hippocampus: reversal by tianeptine

Excitatory amino acids play a key role in stress-induced remodeling of dendrites in the hippocampus as well as in suppression of neurogenesis in the dentate gyrus. The regulation of extracellular glutamate levels has been suggested as a potential mechanism through which repeated stress causes dendritic remodeling of CA3 pyramidal neurons. Accordingly, the current study examined the distribution and regulation of the glia glutamate transporter GLT-1 and the recently identified GLT isoform, GLT-1b, in the hippocampus of rats subjected to chronic restraint stress (CRS). We also examined the ability of the antidepressant tianeptine, which blocks CRS-induced dendritic remodeling, to modulate CRS-mediated changes in GLT-1 and GLT-1b expression. CRS increased GLT-1 mRNA expression in the dentate gyrus and CA3 region of Ammon's horn, increases that were inhibited by tianeptine. CRS more selectively increased GLT-1 protein levels in the subregion where dendritic remodeling is most prominent, namely the CA3 region, increases that were also inhibited by tianeptine administration. In contrast, GLT-1b mRNA expression was not modulated in the hippocampus in any of these groups, but CRS increased GLT-1b protein levels in all hippocampal subfields examined, increases that were unaffected by tianeptine treatment. These results point to the importance of understanding the mechanism for the differential and subregional regulation of GLT-1 isoforms in neuronal and glial compartments in the hippocampus as a basis for understanding the effects of chronic stress on structural plasticity as well as the neuroprotective properties of agents such as tianeptine.     

Neurotransmitter modulation of glucocorticoid receptor mRNA levels in the rat hippocampus.

Glucocorticoids in the hippocampus mediate adaptive responses elicited by stressful stimuli. In this study we investigated glucocorticoid receptor gene expression in the rat hippocampus following acute stress. A significant decrease in glucocorticoid receptor mRNA levels was observed in the hippocampus less than 1 h after the onset of stress. This decrease was inhibited by administering either MK-801, diazepam or propranolol prior to exposure to stress. The effect of diazepam on the stress-induced decrease in hippocampal glucocorticoid receptor mRNA was reversed by Ro-15-1788, suggesting that it is mediated by central benzodiazepine receptors, i.e. GABA-A. These results indicate that NMDA, GABA-A and beta-adrenergic receptors are involved in the mechanism of the stress-induced decrease in glucocorticoid receptor mRNA levels in the rat hippocampus.     

Effects of ovariectomy on GnRH mRNA, proGnRH and GnRH levels in the preoptic hypothalamus of the female rat

Experiments were carried out to investigate the effects of ovariectomy on gonadotropin-releasing hormone (GnRH) messenger RNA (mRNA), proGnRH and GnRH peptide levels in the hypothalamus of female rats. Intact proestrous female rats and female rats, which had been ovariectomized for 2 weeks, were sacrificed at 9.00 h and the preoptic area (POA) and basal hypothalamus (BH) were dissected out and frozen on dry ice. One group of tissues from proestrous control and ovariectomized females were extracted in acetic acid, centrifuged at 13,000 g and the supernatant purified on a C18 column. The purified extract was then radioimmunoassayed for proGnRH, using a specific antiserum to rat proGnRH (ARK-2), and for GnRH using the E1-14 antiserum. Total cellular RNA was isolated from another group of tissues and prepared as Northern blots. Hybridization with 32P-labeled GnRH cRNA was used to detect GnRH mRNA. A third group of proestrous and ovariectomized female rats were perfused, and 50 microns vibratome sections were cut. These were immunostained with proGnRH or GnRH antiserum, followed by in situ hybridization with 35S-labeled GnRH cRNA to detect GnRH mRNA. Based on the histochemical staining, mRNA was colocalized to the cell soma of neurons containing proGnRH and GnRH throughout the POA and BH. Based on the radioimmunoassay, proGnRH levels were 2 times higher in the POA versus the BH, but GnRH levels were 6-7 times higher in the BH. Ovariectomy significantly decreased proGnRH levels in both the POA and BH, while GnRH decreased in the BH. In contrast, quantitative Northern blot analysis demonstrated that ovariectomy had no effect on mRNA levels in the POA and BH. These data indicate that the effects of ovariectomy on proGnRH and GnRH levels are a result of altered translation, posttranslational processing and/or secretion of GnRH.     

大鼠海马CA3区谷氨酸能、γ-氨基丁酸能神经元的增龄性变化

目的 探讨大鼠海马CA3区谷氨酸能、γ-氨基丁酸(GABA)能神经元增龄性变化的规律.方法 SD大鼠分为1～2,4～5,11～12和≥24月龄4组,常规石蜡海马连续冠状切片,HE染色和谷氨酸、GABA免疫组织化学染色,图像分析仪测量谷氨酸、GABA免疫反应阳性产物的光密度及其阳性神经元的多种形态学参数.结果 大鼠海马CA3区谷氨酸、GABA免疫反应阳性产物的光密度及其阳性神经元多种形态学参数随增龄而减小.结论 海马CA3区谷氨酸、GABA能神经元随年龄增长而逐渐出现衰老性变化,为海马衰老病变的研究提供形态学依据.     

大鼠降结肠上皮γ-氨基丁酸及谷氨酸脱羧酶的表达特征

目的:检测γ-氨基丁酸(gamma-aminobutyricacid,GABA)和谷氨酸脱羧酶(glutamicaciddecarboxylase,GAD)在大鼠降结肠上皮的表达及分布特征,并探讨GABA与上皮细胞分化增殖的关系.方法:用免疫荧光及激光共聚焦显微扫描技术,检测GABA、GAD65及GAD67在大鼠降结肠上皮中的表达,并以麦芽凝聚素组织化学染色与免疫荧光结合的双重染色显示GABA和GAD65表达细胞的分布特征.同时,用RT-PCR和原位分子杂交方法检测GADmRNA的表达.此外,用3H-胸腺嘧啶放射自显影法显示降结肠上皮的增殖带.结果:RT-PCR显示降结肠黏膜中GAD65及GAD67mRNA均阳性,原位杂交显示阳性杂交信号主要分布在上皮细胞的隐窝和腔面,且GAD65信号较GAD67强.GABA及GAD65免疫反应阳性细胞主要分布在降结肠的腔面和隐窝的上1/3上皮细胞的胞质,而GAD67阳性细胞仅分布腔面,此外,GABA及GAD65阳性染色也见于黏膜固有层.双重染色显示杯状细胞中GABA及GAD65均阴性.3H-胸腺嘧啶标记阳性细胞主要在隐窝的中下段.结论:GABA及GAD65分布在大鼠降结肠上皮的成熟带及功能带,GABA系统可能参与上皮细胞的分化与增殖的调节.     

Antidepressants increase glucocorticoid and mineralocorticoid receptor mRNA expression in rat hippocampus in vivo.

Adrenal corticosteroids bind to hippocampal glucocorticoid (GR) and mineralocorticoid receptors (MR), thereby affecting neurochemical transmission leading to altered mood, behaviour and neuroendocrine control. Serotoninergic (5-HT) and noradrenergic projections innervate the hippocampus, interacting with corticosteroid-sensitive cells. We have previously demonstrated that lesions of 5-HT neurons reduce hippocampal GR and MR mRNA levels and now examine whether acute or chronic treatment with antidepressant drugs, which potentiate endogenous monoamines by inhibiting their reuptake, affect hippocampal GR and MR mRNA expression in vivo. Rats were treated with amitriptyline (20 mg/kg.day-1), desipramine (10 mg/kg.day-1) or citalopram (20 mg/kg.day-1). After 2 or 14 days animals were killed, RNA extracted and GR and MR mRNA expression quantified by slot blot hybridization. Amitriptyline for 2 days led to a significant increase in MR (by 23 +/- 6%, compared with saline-treated controls), but not GR, mRNA expression. After 14 days amitriptyline, expression of both MR (87 +/- 27% rise) and GR mRNA (56 +/- 18% rise) had increased significantly in hippocampus, but not in parietal cortex. Desipramine for 14 days also increased MR (60 +/- 9%) and GR (42 +/- 9%) mRNA expression, though 14 days of citalopram altered only MR mRNA expression (17 +/- 5%). Thus, antidepressant drug administration elevates MR and GR mRNA expression in hippocampus, but not parietal cortex, in a time-dependent manner.     

Chronic ethanol-induced subtype- and subregion-specific decrease in the mRNA expression of metabotropic glutamate receptors in rat hippocampus

BACKGROUND: Chronic ethanol consumption is known to induce adaptive changes in the hippocampal glutamatergic transmission and alter NMDA receptor binding and subunit expression. Metabotropic glutamate (mGlu) receptors have been shown to function as modulators of neuronal excitability and can fine tune glutamatergic transmission. This study was aimed to determine whether chronic ethanol treatment could change the messenger RNA (mRNA) expression of mGlu receptors in the hippocampus. METHODS: Male Sprague Dawley rats were fed a Lieber-DeCarli liquid diet with 5% (w/v) ethanol or isocaloric amount of maltose for 2 months. Quantitative in situ hybridization was carried out using coronal brain sections through the hippocampus. RESULTS: The results revealed decreases in mRNA expression of several mGlu receptors in different subregions of the hippocampus. In the dentate gyrus, mGlu3 and mGlu5 receptor mRNA levels were significantly lower in the ethanol-treated rats than in the control rats. In the CA3 region, the mRNA expression of mGlu1, mGlu5, and mGlu7 receptors showed substantial decreases after ethanol exposure. The mGlu7 receptor mRNA levels were also declined in the CA1 region and the polymorph layer of the dentate gyrus. No changes were found in mRNA expression of mGlu2, mGlu4, and mGlu8 receptors. CONCLUSIONS: Considering the involvement of hippocampal mGlu receptors in learning and memory processes as well as in neurotoxicity and seizure production, the reduced expression of these receptors might contribute to ethanol withdrawal-induced seizures and also may play a role in cognitive deficits and brain damage caused by long-term ethanol consumption.     

Fetal alcohol exposure alters GAP-43 phosphorylation and protein kinase C responses to contextual fear conditioning in the hippocampus of adult rat offspring

Abstract : Background: The growth‐ and plasticity‐associated protein GAP‐43 plays a significant role in the establishment and remodeling of neuronal connections. We have previously shown that GAP‐43 levels, protein kinase C (PKC) activity, and GAP‐43 phosphorylation increase during contextual fear conditioning and that fetal alcohol exposure (FAE) decreases PKC activity and GAP‐43 phosphorylation in the hippocampus of adult offspring. Drawing on these observations, we hypothesized that FAE manifests its cognitive impairment by disrupting PKC activation and membrane translocation, thereby decreasing GAP‐43 phosphorylation and function. Methods: Three groups of pregnant rat dams (FAE and two control diet groups) were placed on different diet regimens. Offspring from each of these groups were placed into each of four test groups, a contextual fear conditioned (CFC) group, a naïve unhandled group, and two nonlearning stress control groups. Hippocampi were dissected, homogenized, and used to prepare a cytosolic and a membrane fraction. These fractions were probed for total GAP‐43, PKC‐phosphorylated GAP‐43, and several PKC subtypes. PKC activity also was measured in total homogenates. Results: Compared with both control diet groups, FAE animals showed a deficit in the activation of PKC in the hippocampus at 24 hr but not at 1.5 hr after CFC. Likewise, we found that the amount of GAP‐43 and its phosphorylation were decreased 24 hr after CFC in FAE rats but not at early times after training. Analysis of the translocation of various PKC isoforms revealed that FAE animals had decreased levels of membrane‐bound PKC β₂ and PKC ε 24 hr after CFC. Conclusions: Considering the role of PKC activation and GAP‐43 phosphorylation in synaptic plasticity, our results suggest that deficient translocation of PKC β₂ and PKC ε in the hippocampus may mediate the electrophysiological and behavioral deficits observed in fetal alcohol exposed animals.