Regulation of γ-aminobutyric acidB (GABAB) receptors in cerebral cortex during the estrous cycle

We examined binding of the GABA_B receptor agonist baclofen to brain synaptic membranes as a function of the natural variations in gonadal steroids that occur during the estrous cycle of the adult rat. We found that the binding of baclofen to neocortical membranes varied systematically as a function of the estrous cycle, with the lowest binding occuring during the estrus stage. Binding to archicortical (hippocampal) and hypothalamic preparations also varied with the estrous cycle, except that the lowest level of binding in these latter cases occurred during the diestrus stage. The variation of [³H]baclofen binding during the estrous cycle was different with respect to the binding of [³H]muscimol, an agonist for GABA_A receptors, and [³H]8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), an agonist for serotonin 5-HT_1A receptors that shares similar G proteins and effectors with GABA_B receptors. Saturation binding studies of cortical GABA_B receptors showed that apparent receptor density (B_max) rather than affinity (K_d)best accountd for the change in binding during the estrous cycle in that B_max), like total specific binding, was at a minimum during the estrus stage. The robust regulation of GABA_B receptors in neocortex was unexpected and its functional significance is at present unknown. However, the correlation of the menstrual cycle with mood and other behavioral changes, and the correlations of the estrous and menstrual cycles with seizure susceptibility, may somehow depend upon hormonal regulation of transmitter systems such as the one we have observed here.     

Changes in progesterone receptor isoforms content in the rat brain during the oestrous cycle and after oestradiol and progesterone treatments

We studied the effects of oestradiol and progesterone on progesterone receptor (PR) isoform content in the brain of ovariectomized rats and in intact rats during the oestrous cycle by Western blot analysis. In the hypothalamus and the preoptic area of ovariectomized rats, PR-A and PR-B content was increased by oestradiol, whereas progesterone significantly diminished the content of both PR isoforms after 3 h of treatment in the hypothalamus, but not in the preoptic area. In the hippocampus, only PR-A content was significantly increased by oestradiol while progesterone significantly diminished it after 12 h of treatment. In the frontal cortex, no treatment significantly modified PR isoform content. During the oestrous cycle, the lowest content of PR isoforms in the hypothalamus was observed on diestrus day and, by contrast, in the preoptic area, the highest content of both PR isoforms was observed on diestrus day. We observed no changes in PR isoform content in the hippocampus during the oestrous cycle. These results indicate that the expression of PR isoforms is differentially regulated by sex steroid hormones in a regionally specific manner.     

Regionally specific modulation of brain apolipoprotein E in the mouse during the estrous cycle and by exogenous 17beta estradiol

Studies have suggested that 17beta estradiol (E2) can modify apolipoprotein E (apoE) expression. The current study determined if apoE protein varied in different regions of the mouse brain as a function of the estrous cycle and if E2 could increase apoE protein expression. In this study apoE concentration was lowest on estrus in the hippocampus, cingulate cortex and frontal cortex. In contrast, apoE concentration was highest on estrus in the olfactory bulb and cerebellum. There were no differences in the striatal apoE expression throughout the estrous cycle. Exogenous E2 significantly raised tissue levels of apoE in the olfactory bulb and cerebellum at 5 days after treatment. There was a slight, but nonsignificant increase in cortical expression of apoE and no change in striatum. Immunocytochemical localization studies found estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) in cortical neurons and glia. In the cerebellum and olfactory bulb, ERbeta was seen primarily in glia. ERalpha was not observed in the cerebellum and was rare in the olfactory bulb. Neither ERalpha nor ERbeta was seen in the striatum. Our data show regional differences in the production of apoE throughout the estrous cycle. In addition, exogenous E2 has regionally specific effects on apoE expression. Regional variability in apoE production appears to vary as a function of the estrogen receptor subtype.     

Expression of intracellular progesterone receptors in rat brain during different reproductive states, and involvement in maternal behavior

Progesterone is one of a complex of hormones which influences the occurrence of maternal behavior in rats. The present study provides information on progesterone's mechanism and possible neural site(s) of action with respect to maternal responsiveness. Progesterone can exert cellular effects by acting on membrane receptors or by acting on intracellular receptors. In the first experiment we show that RU 486 can antagonize progesterone's inhibitory effect on maternal behavior. Since RU 486 acts as an antagonist to progesterone's action at its intracellular receptor, these results support the involvement of that receptor in maternal behavior control. The second experiment employs immunocytochemical techniques to detect the number of cells in various forebrain regions which contain intracellular progesterone receptors during different reproductive states. The number of cells which contained progesterone receptors was higher toward the end of pregnancy (progesterone is presumably exerting its effects on maternal behavior at this time) when compared to either early pregnancy or lactation in the following forebrain regions: anteroventral periventricular nucleus of the preoptic area; medial preoptic area; ventral part of the bed nucleus of stria terminalis; ventrolateral division of the ventromedial nucleus; arcuate nucleus; anterior paraventricular nucleus of the hypothalamus; and medial amygdala. The possible involvement of these regions as a site or sites where progesterone might exert its effects on maternal behavior is discussed.     

缺血再灌注对大鼠海马CA1及DG神经元内19S蛋白酶体影响的激光扫描共聚焦显微镜研究

目的探讨缺血再灌注对大鼠海马CA1及海马齿状核(DG)神经元内19S蛋白酶体的影响。方法采用20min全脑缺血的大鼠模型,20只大鼠分为5组,分别为假手术组及按照再灌注时间分为30min组,4h组,24h组,72h组,每组4只。采用含有4%多聚甲醛的PBS液体进行灌注,取出脑组织,放于多聚甲醛中固定24h后行冠状切片,应用免疫组织化学法标记抗19S蛋白酶体抗体,应用激光共聚焦显微镜对组织切片进行观察。结果大鼠海马区CA1神经元内19S蛋白酶体在缺血再灌注30min后开始减少,4h略增高,然后逐渐减少,直至72h细胞大部份死亡;DG神经元内的19S蛋白酶体也于再灌注30min后减少,4h略增高,然后逐渐减少,至24h程度最重,72h则有所恢复。结论全脑缺血再灌注后,海马CA1及DG神经元内19S蛋白酶体的变化影响了神经元内蛋白的降解,是导致缺血后神经元死亡的一个因素。     

Changes in the content of estrogen alpha and progesterone receptors during differentiation of mouse embryonic stem cells to dopamine neurons

Embryonic stem cells (ESC) can differentiate to derivatives of the three embryonic germ layers. Dopamine neurons have been produced from mouse and human ESC. This in vitro induction mimics the developmental program followed by dopaminergic cells in vivo. Production of dopamine neurons might have clinical applications for Parkinson's disease, which has a higher incidence in men than in women, suggesting a protective role for sex hormones, particularly progesterone and estradiol. These hormones exert many of their effects through the interaction with their nuclear receptors. In this study, we used a described 5-stage protocol for dopamine neuron differentiation of ESC, allowing neuronal commitment as evidenced by specific markers and by behavioural recovery of hemiparkinsonian rats after grafting. We studied the expression of steroid hormone receptors by immunoblot during this procedure and found an increase in the content of both A and B isoforms of progesterone receptor (PR) and a decrease in estrogen receptor alpha (ER-alpha) when cells were at the neural/neuronal stages, when compared with the amount found in initial pluripotent conditions. We also found the same pattern of PR and ER-alpha expression by immunocytochemistry. Ninety-two percent of dopamine neurons expressed progesterone receptors and only 19% of these neurons co-expressed tyrosine hydroxylase and ER-alpha. These results show a differential expression pattern of ER-alpha and PR isoforms during neuronal differentiation of ESC.     

The expression and binding of kainate receptors is modified in different brain regions by glutamate neurotoxicity during postnatal rat development

Kainic acid receptor (KA-R) subunits are differentially expressed during brain development, and they modulate both neural growth and survival. High concentrations of glutamate in the brain can induce neuronal injury through these receptors, altering normal development. However, it is unclear whether KAR subunit expression itself is also modified by neonatal exposure to high glutamate. To analyze this, monosodium glutamate (4 mg/g of body weight) was subcutaneously administered on postnatal days 1, 3, 5 and 7, and the expression of GluR5, GluR6, KA1 and KA2, as well as [³H]-kainic acid (KA-R) binding, was evaluated on postnatal days 14, 21, 30 and 60 in different regions of rat brain. As a result, high levels of GluR5 expression associated with strong [³H]-kainic acid binding were observed on postnatal days 30 and 60 in the cerebral cortex of rats exposed to glutamate. Similarly, the changes induced by glutamate administration in the expression of the KA1 and KA2 subunits were paralleled by those of [³H]-kainic acid binding in the striatum at postnatal days 21 and 30. In contrast, while KAR subunits were over expressed in the hippocampus, no changes were observed in [³H]-kainic acid binding in adult rats that had been exposed to glutamate. Therefore, glutamate modifies both the expression of kainic acid receptor subunits and kainic acid binding in a determined spatial and temporal manner, which may be indicative of a regional susceptibility to glutamate neurotoxicity.     

Light and electron microscopic immunocytochemical visualization of 5-HT1B receptors in the rat brain

Specific antipeptide antibodies were used for the immunohistochemical visualization of 5-HT_1B receptors in the rat brain. A dense, specific 5-HT_1B receptor-like immunoreactivity was found in the globus pallidus, the dorsal subiculum and the substantia nigra. At the light microscope level, immunostaining was diffuse within the neuropil but absent from cell bodies. Observations at the electron microscope level in the substantia nigra showed immunoperoxidase staining in fine unmyelinated axons and nerve terminals.     

Facilitating antidepressant-like actions of estrogens are mediated by 5-HT1A and estrogen receptors in the rat forced swimming test

Previous studies have shown that 17beta-estradiol (E2) induces antidepressant-like actions per se and potentiates those produced by fluoxetine (FLX) in the forced swimming test (FST). The aim of the present work was to explore the participation of serotonin 1A receptors (5-HT1A) and estrogen receptors (ERs) in the antidepressant-like actions of E2, FLX or their combination in the FST. Although all antidepressants reduce behavioral immobility, antidepressants that modulate serotonergic neurotransmission increase swimming behavior whereas those that modulate the catecholaminergic neurotransmission increase climbing behavior. Thus, using this animal model, it is possible to infer which neurotransmitter system is modulating the action of an antidepressant compound. Ovariectomized female Wistar rats were used in all experiments. In the first experiment, an effective dose of E2 (10 microg/rat, -48 h) was combined with several doses (0.5, 1.0 and 2 mg/kg) of RU 58668 (a pure ER antagonist) 48 h previous to the FST. The second experiment evaluated the action of (1 mg/kg, -48 h or -23, -5 and -1 h) WAY 100635 (5-HT1A receptor antagonist) on the antidepressant-like action of FLX (10 mg/kg, -23, -5 and -1 h). In the third experiment, the effect of RU 58668 (2 mg/kg, -48) or WAY 100635 (1 mg/kg, -48 h) on the antidepressant-like action of the combination of a sub-optimal dose of E2 (2.5 microg/rat, -48 h) plus a non-effective dose of FLX (2.5 mg/kg, -23,-5 and -1 h) was evaluated. The results showed that RU 58668, the antagonist to the ER, canceled the antidepressant-like action of E2 in a dose-dependent manner. The antagonist to the 5-HT1A receptor blocked the antidepressant action of FLX only when administered simultaneously with FLX, i.e. -23, -5 and -1 h before the FST. Finally, the administration of both RU 58668, and WAY100635 canceled the antidepressant-like action of the combination of E2/FLX. These results imply that both 5-HT1A receptors and ERs participate in the facilitating actions of E2 on the antidepressant-like action of FLX in the FST.     

Dopaminergic neurotransmission in somatodendritic and terminal areas of the rat brain: susceptibility to modulation by D 1 and D2 receptors and to axotomy

Summary We have investigated the influence of D1 and D2 dopamine receptor active drugs on dopamine (DA) release in substantia nigra (SN), striatum and limbic forebrain in intact and in hemisected rats in vivo. DA release was indirectly assessed as 3-methoxytyramine (3-MT) accumulation following monoamine oxidase inhibition by pargyline. Hemisection per se had no effect on the 3-MT accumulation in the SN. Neither, had SCH 23390, SK & F28393, or cisflupentixol any effect in the SN in intact animals or in the lesioned side in hemisected animals. SCH 23390 slightly increased the 3-MT accumulation both in the striatum and limbic forebrain, indicating a stimulatory action on DA release, but SK & F38393 had no effect in these brain regions. A difference between the striatum and the limbic forebrain was that the effects of SCH 23390, and cis-FPX were almost abolished following hemisection in the limbic fore-brain, but only partially reduced in the striatum. In summary, our data give further support for the concept that neither D 1 nor D 2 dopamine receptors have any pronounced influence on the DA release in the SN. The data also indicate operational differences in the feedback regulation of limbic versus striatal dopaminergic transmission.     

The influence of gonadal hormones on neuronal excitability, seizures, and epilepsy in the female

It is clear from both clinical observations of women, and research in laboratory animals, that gonadal hormones exert a profound influence on neuronal excitability, seizures, and epilepsy. These studies have led to a focus on two of the primary ovarian steroid hormones, estrogen and progesterone, to clarify how gonadal hormones influence seizures in women with epilepsy. The prevailing view is that estrogen is proconvulsant, whereas progesterone is anticonvulsant. However, estrogen and progesterone may not be the only reproductive hormones to consider in evaluating excitability, seizures, or epilepsy in the female. It seems unlikely that estrogen and progesterone would exert single, uniform actions given our current understanding of their complex pharmacological and physiological relationships. Their modulatory effects are likely to depend on endocrine state, relative concentration, metabolism, and many other factors. Despite the challenges these issues raise to future research, some recent advances have helped clarify past confusion in the literature. In addition, testable hypotheses have developed for complex clinical problems such as "catamenial epilepsy." Clinical and animal research, designed with the relevant endocrinological and neurobiological issues in mind, will help advance this field in the future.     

Effects of chronic administration of caffeine on adenosine A1 and A2 receptors in rat brain

Chronic administration of caffeine (75 mg/kg/day) to rats for 12 days increased [3H]R-PIA binding in the cerebral cortex and cerebellum and [3H]NECA binding to high affinity receptor sites in the striatum. The results indicate that both adenosine A1 and A2 receptor subtypes possess mechanisms of adaptation to chronic caffeine treatment. In addition, adenosine A1 receptor binding shows heterogenous neuroanatomical pattern indicating that the A1 response to caffeine treatment presents regional variation in the rat brain.     

Expression and distribution of kinin B1 receptor in the rat brain and alterations induced by diabetes in the model of streptozotocin

A role for kinin B1 receptors was suggested in the spinal cord and peripheral organs of streptozotocin (STZ)-diabetic rats. The present study aims at determining whether B1 receptors are also induced and over-expressed in the brain of STZ-rats at 2, 7, and 21 days post-treatment. This was addressed by in situ hybridization using the [35S]-UTPalphaS-labeled riboprobe and by in vitro autoradiography with the radioligand [125I]-HPP-des-Arg10-Hoe 140. In control rats, B1 receptor mRNA was found widely distributed in many brain regions. Low mRNA levels were found in thalamus and hypothalamus (7-12 nCi/g) while high mRNA signals were detected in cortical regions and hippocampus (18-29 nCi/g). In diabetic rats, B1 receptor mRNA was markedly increased in hippocampus, temporal/parietal cortices and amygdala at 2 and 7 days (+88 to +150%). Low densities of B1 receptor binding sites were detected in all analyzed regions in control rats (0.18-0.37 fmol/mg tissue). In diabetic rats, B1 receptor binding sites were significantly increased in hippocampus, amygdala, temporal/parietal, and perhinal/piriform cortices (+ 55 to + 165 %) at 7 days only. Results highlight an early but transient and reversible up-regulation of B1 receptors in specific brain regions of STZ-diabetic rats. This may offer the advantage of reducing putative central side effects with B1 receptor antagonists if used for the treatment of diabetic complications in the periphery.     

Changes in the immunoreactivities of an opioid peptide leumorphin in the hypothalamus and anterior pituitary during the estrous cycle of the rat and their relation to sexual behavior

Leumorphin, an opioid peptide whose functions are unknown, is found in mammalian brain and pituitary and stimulates lordosis behavior in estrogen-treated female rats. To elucidate the role of leumorphin in the physiological control of female sexual behavior, the levels of immunoreactive (ir) leumorphin as well as ir dynorphin (dynorphin A) were measured in the rat brain and pituitary during the estrous cycle. There was a clear variation of ir leumorphin in the hypothalamus and anterior pituitary during the estrous cycle. The levels of ir leumorphin in the hypothalamus and anterior pituitary on the afternoon of proestrus were significantly higher (P less than 0.01) than those on the afternoons of estrus and metestrus. The rise in the hypothalamic levels of ir leumorphin on the afternoon of proestrus was correlated with the receptivity of lordosis during the estrous cycle. Furthermore, there was a close correlation with ir dynorphin levels. These findings are in agreement with studies demonstrating a common precursor for leumorphin and dynorphin. Ir leumorphin in the hippocampus and neurointermediate pituitary did not change significantly during the estrous cycle. Because the leumorphin antiserum used recognizes rimorphin (dynorphin B) 1.78 times more than porcine leumorphin on a molar basis, high performance-gel permeation chromatography was done on pooled extracts of hypothalamus taken at proestrus and estrus. The peak in the leumorphin-like substance in the activation of sexual behavior is discussed.     

Autoregulatory properties of dorsal raphe 5-HT neurons: Possible role of electrotonic coupling and 5-HT1D receptors in the rat brain

In the present study, the hypothesis that somatodendritic availability of 5-hydroxytryptamine (5-HT) could be regulated independently of the firing activity of dorsal raphe 5-HT neurons was tested. The 5-HT pathway was electrically stimulated at the level of the ventromedial tegmentum and the ensuing action potentials, recorded in the dorsal raphe, met all criteria for antidromic invasion of 5-HT neurons. The latency of antidromic spikes was current-dependent and the changes in latency were of quantal nature. This observation suggests an electrotonic coupling between 5-HT neurons. Stimulation of the ventromedial tegmentum also induced a decrease in the probability of firing of 5-HT neurons. This reduction in 5-HT neuron firing activity is a 5-HT-mediated response, due to an increased bioavailability of the neurotransmitter in the biophase of somatodendritic 5-HT_1A autoreceptors. The intravenous administration of the 5-HT₁ agonists TFMPP and RU 24969 reduced the duration of suppression of firing induced by the 5-HT-pathway stimulation, without altering the spontaneous firing rate of 5-HT neurons. The effect of TFMPP and RU 24969 on duration of suppression was blocked by (±)mianserin, a drug with high affinity for the rat 5-HT_1D, but not 5-HT_1B, receptors. On the other hand, (−)propranolol, a mixed 5-HT antagonist also blocked the effect of TFMPP. However, the selective 5-HT_1A antagonist (+)WAY 100135 did not alter the effect of TFMPP. These results, in keeping with previous anatomical studies, suggest the existence of electrotonic coupling of 5-HT neurons and indicate that 5-HT release in the rat dorsal raphe nucleus may be controlled independently of firing-regulating 5-HT_1A autoreceptors. They also suggest that 5-HT_1D receptors may play a role in this regulatory function of 5-HT neurons. © 1996 Wiley-Liss, Inc.     

Seizures and neuronal damage induced in the rat by activation of group I metabotropic glutamate receptors with their selective agonist 3,5-dihydroxyphenylglycine

While it is well documented that the overactivation of ionotropic glutamate receptors leads to seizures and excitotoxic injury, little is known about the role of metabotropic glutamate receptors (mGluRs) in epileptogenesis and neuronal injury. Intracerebroventricular (i.c.v.) infusion of the group I mGluR specific agonist (R,S)-3,5-dihydroxyphenylglycine (3,5-DHPG) (1.5 μmol) to conscious rats produced severe and delayed seizures (onset at 4 hr) in 70% of the animals. The i.c.v. infusion of the group I mGluR non-selective agonist 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) (2 μmol) produced a similar rate of severe seizures, but with an early onset (0.6 hr). The analysis of motor activity showed that 3,5-DHPG elicited higher central stimulatory action than did 1S,3R-ACPD. Histopathological analysis of the hippocampus showed that 3,5-DHPG produced severe neuronal damage mainly in the CA1 pyramidal neurons and, to a lesser extent, in the CA3. Although 1S,3R-ACPD infusion also induced a slight injury of the CA1 and CA3 pyramidal neurons, damage was greater in the CA4 and dentate gyrus cells. In conclusion, the in vivo activation of group I mGluRs with the selective agonist 3,5-DHPG produces hyperexcitatory effects that lead to seizures and neuronal damage, these effects being more severe than those observed after infusion of the non-selective agonist 1S,3R-ACPD. J. Neurosci. Res. 51:339–348, 1998. © 1998 Wiley-Liss, Inc.     

Immunohistochemical detection of A1 adenosine receptors in rat brain with emphasis on localization in the hippocampal formation, cerebral cortex, cerebellum, and basal ganglia

Polyclonal antisera were generated against two identical regions of rat and human A₁ adenosine receptors using synthetic multiple-antigenic-peptides as immunogens. Western blotting showed that the antisera recognized a single protein in brain of the expected size for A₁ receptors. Immunohistochemistry of CHO cells transfected with the rat or human A₁ adenosine receptor cDNAs showed robust labeling of the cell surface. In contrast, labeling was not apparent over non-transfected CHO cells, nor over CHO cells expressing A_2a receptors. The pattern of immunoreactivity in rat brain was similar to that expected for A₁ adenosine receptors. In contrast to receptor autoradiography or in situ hybridization methods, immunohistochemistry allowed identification of individually labeled cells and processes. Heavy labeling was apparent in many brain regions. In the hippocampal formation, strong labeling was present on granule cell bodies and dendrites, mossy fibers, and pyramidal neurons. In cerebellum, basket cells were the most heavily labeled cell type. Less intense staining was present over granule cells. In cerebral cortex, pyramidal cells were the most heavily labeled cell type, and some interneurons were also labeled. In the basal ganglia, 43% of neurons in the globus pallidus were labeled. In the caudate-putamen region, 38% of neurons were labeled. Heavy labeling was present in most thalamic nuclei, and moderate to heavy labeling was seen in many brainstem nuclei. These data identify specific cellular sites of A₁ receptor expression and support the concept of cellular specificity of A₁ adenosine receptor action.     

Modulation of feeding behaviour by blocking purinergic receptors in the rat nucleus accumbens: a combined microdialysis, electroencephalographic and behavioural study

The nonspecific P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), the nonspecific P1 receptor antagonist 8-(p-sulphophenyl)-theophylline (8-SPT) and the combination of both were applied by retrograde microdialysis into the nucleus accumbens (NAc) before and during feeding of 18-h food-deprived rats. In addition to the registration of behavioural parameters, such as the amount and duration of food intake, the feeding-induced changes in dopamine (DA) concentration and the concomitant changes of neuronal activity in the NAc and the ventral tegmental area (VTA) were simultaneously determined. The perfusion with PPADS (20 microm) diminished the amount of food intake and the duration of feeding. Furthermore, the P2 receptor antagonist blocked the feeding-induced DA release and prevented the feeding-elicited changes of the electroencephalography (EEG) power distribution which was characterised by an increase in the power of the 8.0-13.0-Hz frequency band in the NAc and the VTA. The effects of PPADS could be completely prevented by the concomitantly perfused adenosine receptor antagonist 8-SPT (100 microm). When given alone, 8-SPT increased the amount of food ingested, the duration of feeding and the EEG power of the higher frequency range, particularly between 19.0 and 30.0 Hz, in both the NAc and the VTA. The feeding-elicited DA release was supplemented to the enhanced DA level caused by the perfusion with 8-SPT in an additive manner. The P2 and P1 receptor antagonists interact antagonistically in the modulation of feeding behaviour and the feeding-induced changes of EEG activity suggesting that both endogenous extracellular ATP and adenosine are involved in the regulation of the feeding-associated mesolimbic neuronal activity in a functionally antagonistic manner.