Identification of a novel gene with RING-H2 finger motif induced after chronic antidepressant treatment in rat brain

Previously, we have identified 200 cDNA fragments as antidepressant related genes/ESTs. In this study, using these cDNAs, we developed our original cDNA microarray for rapid secondary screening of candidate genes as the novel therapeutic targets. With this microarray, we found that the expression of a novel gene, ADRG34, was significantly increased in rat hippocampus which had been chronically treated with a selective serotonin reuptake inhibitor antidepressant, sertraline. RT-PCR analysis also demonstrated the induction of ADRG34 at mRNA levels in rat hippocampus and the frontal cortex. This cDNA encoded 685 amino acid residues containing a RING-H2 finger motif at the carboxy-terminal. Sequence analysis of ADRG34 with the EMBL/GenBank database showed significant homology to mouse and human kf-1 gene. Our data suggest that ADRG34, a possible rat homologue of kf-1, may be one of the common functional molecules induced after chronic antidepressant treatment.     

GABA and affective disorders

Recently sufficient evidence has accumulated to propose that a central GABAergic dysfunction may be primarily related to the pathology of affective disorders and that antidepressant mechanisms (pharmacological or electroconvulsive therapy, ECT) have an intrinsic GABAergic component. In depressed patients GABA levels are reported to be low in the CSF and plasma, and GABA synthesis is decreased in some brain areas, including the frontal cortex. GABAmimetics such as progabide and fengabine exert a therapeutic effect in depression. In behavioural laboratory models GABAmimetics exhibit antidepressant-like actions in the olfactory bulbectomized rat and in rats submitted to an inescapable shock (learned helplessness). Furthermore, antidepressant GABAmimetics decrease paradoxical sleep. In the olfactory bulbectomized rat, GABAB receptors are downregulated in the frontal cortex and in the learned helplessness model, GABA release is diminished in the hippocampus. These decreases are reversed by antidepressants in parallel with their behavioural activities. An intrinsic activity of widely varied antidepressants and ECT is the upregulation of GABAB receptors in the frontal cortex. This, together with the downregulation of beta-adrenergic receptors induced by these compounds, and the GABAB modulation of the beta-adrenergic second messenger system, strongly suggest that both GABAergic and beta-adrenergic responses are inherent to an antidepressant effect.     

KF-1 Ubiquitin Ligase: Anxiety Suppressor Model

Anxiety disorders are the most popular psychiatric disease in any human societies irrespective of nation, culture, religion, economics or politics. Anxiety expression mediated by the amygdala may be suppressed by signals transmitted from the prefrontal cortex and hippocampus. KF-1 is an endoplasmic reticulum (ER)-based E3-ubiquitin (Ub) ligase with a RING-H2 finger motif at the C-terminus. The kf-1 gene expression is up-regulated in the frontal cortex and hippocampus in rats after anti-depressant treatments. The kf-1 null mice show no apparent abnormalities, but exhibit selectively pronounced anxiety-like behaviors or increased timidity-like responses. The kf-1 orthologous genes had been generated after the Poriferan emergence, and are found widely in all animals except insects, arachnids and threadworms such as Drosophila, Ixodes and Caenorhabditis, respectively. This suggests that the kf-1 gene may be relevant to some biological functions characteristic to animals. Based on these observations, the Anxiety Suppressor Model has been proposed, which assumes that KF-1 Ub ligase may suppress the amygdala-mediated anxiety by degrading some anxiety promoting protein(s), such as a neurotransmitter receptor, through the ER-associated degradation pathway in the frontal cortex and hippocampus. According to this model, the emotional sensitivity to environmental stresses may be regulated by the cellular protein level of KF-1 relative to that of the putative anxiety promoter. The kf-1 null mice should be useful in elucidating the molecular mechanisms of the anxiety regulation and for screening novel anxiolytic compounds, which may block the putative anxiety promoter.     

Electroconvulsive Therapy Induces Neurogenesis in Frontal Rat Brain Areas

Electroconvulsive therapy (ECT) is an effective therapy for several psychiatric disorders, including severe major depression, mania and certain forms of schizophrenia. It had been proposed that ECT acts by modulating local plasticity via the stimulation of neurogenesis. In fact, among antidepressant therapies, ECT is the most robust enhancer of neurogenesis in the hippocampus of rodents and non-human primates. The existence of ECT-triggered neurogenesis in other brain areas, particularly in those adjacent to the other main locus of neurogenesis, the subventricular zone (SVZ), had so far remained unknown. Here we show that ECT also strongly enhances neurogenesis in frontal brain areas, especially in the rostro-medial striatum, generating specific, small-size calretinin-positive interneurons. We provide here the first evidence that ECT stimulates neurogenesis in areas outside the hippocampus. Our data may open research possibilities that focus on the plastic changes induced by ECT in frontal limbic circuitry.     

Evidence for increased expression of the vesicular glutamate transporter, VGLUT1, by a course of antidepressant treatment

The therapeutic effect of a course of antidepressant treatment is believed to involve a cascade of neuroadaptive changes in gene expression leading to increased neural plasticity. Because glutamate is linked to mechanisms of neural plasticity, this transmitter may play a role in these changes. This study investigated the effect of antidepressant treatment on expression of the vesicular glutamate transporters, VGLUT1-3 in brain regions of the rat. Repeated treatment with fluoxetine, paroxetine or desipramine increased VGLUT1 mRNA abundance in frontal, orbital, cingulate and parietal cortices, and regions of the hippocampus. Immunoautoradiography analysis showed that repeated antidepressant drug treatment increased VGLUT1 protein expression. Repeated electroconvulsive shock (ECS) also increased VGLUT1 mRNA abundance in regions of the cortex and hippocampus compared to sham controls. The antidepressant drugs and ECS did not alter VGLUT1 mRNA abundance after acute administration, and no change was detected after repeated treatment with the antipsychotic agents, haloperidol and chlorpromazine. In contrast to VGLUT1, the different antidepressant treatments did not commonly increase the expression of VGLUT2 or VGLUT3 mRNA. These data suggest that a course of antidepressant drug or ECS treatment increases expression of VGLUT1, a key gene involved in the regulation of glutamate secretion.     

Identification of a novel splice variant of heat shock cognate protein 70 after chronic antidepressant treatment in rat frontal cortex.

In this study, we identified a novel splice variant of 70-kDa heat shock cognate protein (HSC70), while screening differentially expressed molecules in rat brain after chronic antidepressant treatment. This clone, named HSC49, lacked 470 bp of nucleotides of rat HSC70. HSC49 encoded 442 amino acid residues with a calculated molecular mass of 48.6 kDa. DNA sequence analysis revealed that HSC49 lacked the entire Exon 7 and Exon 8 of the HSC70 gene. Chronic treatment with antidepressant, imipramine or sertraline, induced a 38.5 or 22.5% increase in mRNA levels in rat frontal cortex, respectively, when compared to controls. Western blot analysis also revealed that the protein expression of HSC49 was increased after antidepressant treatment. Our data suggest that HSC49 may be one of the common molecules induced after chronic antidepressant treatment.     

Effect of Electroconvulsive Shock on Mitochondrial Respiratory Chain in Rat Brain

It is well described that impairment of energy production has been implicated in the pathogenesis of a number of diseases. Although several advances have occurred over the past 20 years concerning the use and administration of electroconvulsive therapy (ECT) to minimize its side effects, little progress has been made in understanding its mechanism of action. In this work, our aim was to measure the activities of mitochondrial respiratory chain complexes II and IV and succinate dehydrogenase from rat brain after acute and chronic electroconvulsive shock (ECS). Our results showed that mitochondrial respiratory chain enzymes activities were increased after acute ECS in hippocampus, striatum and cortex of rats. Besides, we also demonstrated that complex II activity was increased after chronic ECS in cortex, while hippocampus and striatum were not affected. Succinate dehydrogenase, however, was inhibited after chronic ECS in striatum, activated in cortex and not affected in hippocampus. Finally, complex IV was not affected by chronic ECS in hippocampus, striatum and cortex. Our findings demonstrated that brain metabolism is altered by ECS.     

Neurochemical studies on Indian Hypericum perforatum L

The effect of acute administration of 50% standardised ethanolic extract of Indian Hypericum perforatum (IHp) was studied on the rat brain concentrations of monoamines and their metabolites in five different brain regions, viz. hypothalamus, hippocampus, striatum, pons-medulla and frontal cortex by a HPLC technique. IHp extract was administered at the doses of 50 and 200 mg/kg, p.o. and the brain monoamines were assayed after 30 min of the treatment. IHp treatment significantly decreased the levels of serotonin (5-HT) and its metabolite 5-hydroxy indole acetic acid (5-HIAA) and 5-HT turnover in all the brain regions assayed. On the other hand, IHp treatment significantly augmented the levels of norepinephrine (NE) and its metabolite methylhydroxy phenyl glycol (MHPG) and NE turnover in all the brain regions studied. Similarly, the levels of dopamine (DA) were also significantly augmented in the hypothalamus, striatum and frontal cortex. Likewise, the levels of dihydroxyphenyl acetic acid (DOPAC), the major metabolite of DA, were also increased in these brain areas. Pharmacological studies with IHp extract have shown two major behavioural actions, namely, anxiolytic and antidepressant effects. The present findings tend to rationalise these observations, reduced 5-HT activity being consonant with anxiolytic and increased NA and DA activity being consonant with antidepressant action.     

Expression of BDNF and TrkB Phosphorylation in the Rat Frontal Cortex During Morphine Withdrawal are NO Dependent

Nitric oxide (NO) mediates pharmacological effects of opiates including dependence and abstinence. Modulation of NO synthesis during the induction phase of morphine dependence affects manifestations of morphine withdrawal syndrome, though little is known about mechanisms underlying this phenomenon. Neurotrophic and growth factors are involved in neuronal adaptation during opiate dependence. NO-dependent modulation of morphine dependence may be mediated by changes in expression and activity of neurotrophic and/or growth factors in the brain. Here, we studied the effects of NO synthesis inhibition during the induction phase of morphine dependence on the expression of brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), nerve growth factor (NGF), and insulin-like growth factor 1 (IGF1) as well as their receptors in rat brain regions after spontaneous morphine withdrawal in dependent animals. Morphine dependence in rats was induced within 6 days by 12 injections of morphine in increasing doses (10–100 mg/kg), and NO synthase inhibitor L-N^G-nitroarginine methyl ester (L-NAME) (10 mg/kg) was given 1 h before each morphine injection. The expression of the BDNF, GDNF, NGF, IGF1, and their receptors in the frontal cortex, striatum, hippocampus, and midbrain was assessed 40 h after morphine withdrawal. L-NAME treatment during morphine intoxication resulted in an aggravation of the spontaneous morphine withdrawal severity. Morphine withdrawal was accompanied by upregulation of BDNF, IGF1, and their receptors TrkB and IGF1R, respectively, on the mRNA level in the frontal cortex, and only BDNF in hippocampus and midbrain. L-NAME administration during morphine intoxication decreased abstinence-induced upregulation of these mRNAs in the frontal cortex, hippocampus and midbrain. L-NAME prevented from abstinence-induced elevation of mature but not pro-form of BDNF polypeptide in the frontal cortex. While morphine abstinence did not affect TrkB protein levels as well as its phosphorylation status, inhibition of NO synthesis decreased levels of phosphorylated TrkB after withdrawal. Thus, NO signaling during induction of dependence may be involved in the mechanisms of BDNF expression and processing at abstinence, thereby affecting signaling through TrkB in the frontal cortex.     

反复苯丙胺刺激对大鼠脑部蛋白羰基化的影响

目的:为了进一步研究苯丙胺神经毒性作用机制,我们对大鼠进行不同时长的反复苯丙胺刺激,检测大鼠部分脑区中蛋白羰基化的变化情况,我们的研究为苯丙胺的成瘾及治疗提供了新的理论依据。方法:分别对大鼠进行1d、3d、7d、10d及14d的苯丙胺反复刺激,进行旷场测试检测其活动量变化后,采用DNPH法检查的大鼠大脑前皮层、海马区、杏仁核三大脑区总蛋白的蛋白羰基化水平变化,探讨反复苯丙胺刺激对大鼠脑部蛋白羰基化的影响。结果:苯丙胺刺激7d及14d时,大鼠活动量出现了显著性增加,同时大鼠前皮层总蛋白的蛋白羰基化也出现了显著性增加,而海马区及杏仁核区域总蛋白的蛋白羰基化没有明显变化。结论:反复苯丙胺刺激能够增加大鼠活动量及大脑前皮层总蛋白蛋白羰基化水平。     

Effects of Electroconvulsive Shock on Tetrahydrobiopterin and GTP-Cyclohydrolase Activity in the Brain and Adrenal Gland of the Rat

The effects of a single and repeated electroconvulsive shock (ECS) (300 mA, 0.2 s) on tetrahydrobiopterin (BH4) levels and GTP-cyclohydrolase activity in the brain and adrenal glands of rats were examined. Twenty-four hours after the last ECS treatment (one/day for 7 days), biopterin levels were significantly elevated in the locus coeruleus, hippocampus, frontal cortex, hypothalamus, ventral tegmental area, and adrenal gland. There were no changes in biopterin levels after a single application of ECS. GTP-cyclohydrolase activity was significantly increased in the locus coeruleus, frontal cortex, hippocampus, hypothalamus, and adrenal gland 24 h after repeated ECS and remained elevated in certain tissues up to 8 days after the last treatment. Kinetic analysis of adrenal and locus coeruleus GTP-cyclohydrolase 1 day after 7 days of ECS showed significant changes in both Km and Vmax values. These data suggest that the long-term increases in BH4 levels and GTP-cyclohydrolase activity after repeated ECS may play a part in the mediation of the antidepressant effects of ECS.     

Characterization of N-[3H]Methylcarbamylcholine Binding Sites and Effect of N-Methylcarbamylcholine on Acetylcholine Release in Rat Brain

The present experiments show that N-[3H]-methylcarbamylcholine ([3H]MCC) binds specifically and with high affinity to rat hippocampus, frontal cortex, and striatum. The highest maximal density of binding sites was apparent in frontal cortex and the lowest in hippocampus. [3H]MCC binding was potently inhibited by nicotinic, but not muscarinic, agonists and by the nicotinic antagonist dihydro-beta-erythroidine in all three brain regions studied. The effect of unlabeled MCC on acetylcholine (ACh) release from slices of rat brain was tested. The drug significantly enhanced spontaneous ACh release from slices of hippocampus and frontal cortex, but not from striatal slices. This effect of MCC to increase ACh release from rat hippocampus and frontal cortex was antagonized by the nicotinic antagonists dihydro-beta-erythroidine and d-tubocurarine, but not by alpha-bungarotoxin or by the muscarinic antagonist atropine. The MCC-induced increase in spontaneous ACh release from hippocampal and frontal cortical slices was not affected by tetrodotoxin. The results suggest that MCC might alter cholinergic transmission in rat brain by a direct activation of presynaptic nicotinic receptors on the cholinergic terminals. That this alteration of ACh release is apparent in hippocampus and frontal cortex, but not in striatum, suggests that there may be a regional specificity in the regulation of ACh by nicotinic receptors in rat brain.     

Effect of semax on the temporary dynamics of brain-derived neurotrophic factor and nerve growth factor gene expression in the rat hippocampus and frontal cortex

Semax is a synthetic peptide, which consists of the N-terminal adrenocorticotropic hormone fragment (4-7) (ACTH4-7) and C-terminal Pro-Gly-Pro peptide. Semax promotes neuron survival in hypoxia, increases selective attention and memory storage. It was shown that this synthetic peptide exerted a number of gene expressions, especially brain derived neurotrophic factor gene (Bdnf) and nerve growth factor gene (Ngf). Temporary dynamics of Bdnf and Ngf ex- pression in rat hippocampus and frontal cortex under Semax action (50 mg/kg, single intranasal administration) was studied in this work. It was shown that the studied gene expression levels changed significantly both in the hippocampus and the frontal cortex tissues 20 minutes after the peptide preparation application. The expression levels decreased in the hippocampus and increased in the frontal cortex. Forty minutes after Semax administration both gene expression levels returned to the level typical of control tissues. After that they increased significantly by 90 minutes after experiment start. Bdnf and Ngf expression levels decreased up to the control levels by 8 hours after medicine applying maximum gene expression levels were attained. Thus, Semax administration results in rapid, long-term, and specific activation of Bdnf and Ngf expression changes in different rat brain departments.     

Repeated treatment with mirtazepine induces brain-derived neurotrophic factor gene expression in rats.

Recent studies indicate a role of the brain-derived neurotrophic factor (BDNF) in the pathophysiology of depression, as well as in the mechanism of action of antidepressant drugs (ADs). It has been shown that serum BDNF levels are decreased in depressed patients. Moreover, antidepressant treatment increases serum BDNF levels and it is positively correlated with medication response. In addition, repeated administration of ADs induces an increase in rat hippocampal or cortical BDNF gene expression. Since the most potent effect of ADs on BDNF gene expression was found after prolonged treatment, in the present study we investigated the influence of repeated treatment (twice daily for 14 days) of the new AD mirtazapine (5 or 10 mg/kg) on BDNF mRNA level (the Northern blot) in rat hippocampus and cerebral cortex. Imipramine was used as a reference compound. The experiment was carried out on male Wistar rats. The tissue for biochemical assays was collected 24 h after the last doses of mirtazapine and imipramine. We also studied the effect of repeated mirtazapine on the action of the 5-HT2A receptor agonist (+/-)DOI in the behavioral test (head twitches induced by (+/-)DOI) in rats. The obtained results showed that, like imipramine (10 mg/kg), mirtazapine (10 mg/kg) increased BDNF gene expression in both the examined brain regions: in the hippocampus by 24.0 and 26.5%, in the cerebral cortex by 29.9 and 41.5%, respectively, compared with the vehicle-treated control. Neither mirtazapine nor imipramine administered repeatedly at a lower dose (5 mg/kg) significantly changed BDNF mRNA levels in the hippocampus and cerebral cortex. Repeated treatment with mirtazapine (10, but not 5 mg/kg) inhibited the behavioral syndrome induced by (+/-)DOI. This study provides first conclusive evidence that repeated mirtazapine administration increases BDNF mRNA levels; moreover, it indicates that the enhancement of BDNF gene expression may be essential for the clinical effect of mirtazapine.     

G(olf) protein levels in rat striatum are increased by chronic antidepressant administration and decreased by olfactory bulbectomy

There are many studies of the mechanisms of antidepressants; however, most of these studies were conducted on the hippocampus or frontal cortex. In the present study, we hypothesized that the nucleus accumbens and caudate/putamen might be major targets for antidepressant effects. Thus, we focused on G(olf) protein, a stimulant alpha-subunit of G protein that is coupled with the dopamine D1 receptor and specifically expressed in the striatum (nucleus accumbens, caudate/putamen and olfactory tubercle) in the rat brain. We examined the effects of chronic administration of imipramine, fluvoxamine, maprotiline and, as a negative control, cocaine on the level of G(olf) protein in the rat striatum. We also examined the effect of olfactory bulbectomy. Chronic imipramine treatment (10 mg/kg for 2 or 4 weeks) significantly increased the level of G(olf) in the striatum (by 17% or 18%, respectively), although this increase was not apparent after only 1 week of treatment. The time course of these changes corresponded well to that of the clinical efficacy of imipramine. Chronic fluvoxamine and maprotiline treatment (20 mg/kg for 2 weeks) also significantly increased the level of G(olf) (by 9% and 25%, respectively), but cocaine did not alter it significantly. Bulbectomy decreased the G(olf) protein level by 9%. The increases in G(olf) protein after chronic administration of these three different classes of antidepressants and the decrease after bulbectomy suggest that G(olf) protein may play an important role in the antidepressant effect.     

Brain lipid changes after repetitive transcranial magnetic stimulation: potential links to therapeutic effects?

Repetitive transcranial magnetic stimulation (rTMS) is increasingly used in the management of neurologic disorders such as depression and chronic pain, but little is known about how it could affect brain lipids, which play important roles in membrane structure and cellular functions. The present study was carried out to examine the effects of rTMS on brain lipids at the individual molecular species level using the novel technique of lipidomics. Rats were subjected to high frequency (15 Hz) stimulation of the left hemisphere with different intensities and pulses of rTMS. The prefrontal cortex, hippocampus and striatum were harvested 1 week after rTMS and lipid profiles analyzed by tandem mass spectrometry. rTMS resulted in changes mainly in the prefrontal cortex. There were significant alterations in plasmalogen phosphatidylethanolamines, phosphatidylcholines, and increases in sulfated galactosylceramides or sulfatides. Plasmalogen species with long chain polyunsaturated fatty acids (PUFAs) showed decrease in abundance together with corresponding increase in lysophospholipid species suggesting endogenous release of long chain fatty acids such as docosahexaenoic acid (DHA) in brain tissue. The hippocampus showed no significant changes, whilst changes in the striatum were often opposite to that of the prefrontal cortex. It is postulated that changes in brain lipids may underlie some of the clinical effects of rTMS.     

经颅磁刺激对癫痫病灶脑电相关维数的影响

利用脑功能指标——大鼠病灶区脑电的相关维数,研究低频经颅磁刺激对慢性颞叶癫痫大鼠脑功能改善的作用。对一组颞叶癫痫大鼠施予频率为0．5Hz、强度为0．4T、20次／日、连续一周的低频重复性经颅磁刺激(rTMS)．在rTMS前后,分别测取颞叶癫痫大鼠责任病灶区皮层和海马区的脑电,重构时间延迟吸引子,用G-P算法估算反映对应脑区功能状态的相关维数。研究结果显示：施予适量的rTMS(0.4T、20次／日、连续一周),使颞叶癫痫大鼠海马和相应皮层脑电的相关维数比刺激前明显升高。研究表明适量的rTMS有抑制癫痫的作用。     

Intranasal Administration of Nerve Growth Factor Produces Antidepressant-Like Effects in Animals

Many works showed that nerve growth factor (NGF) injected into the brain of animal model emerges potential antidepressant effects. However, this route of administration significantly restricts the application of NGF clinically. Here, we reported that intranasal NGF could provide an alternative to intraventricular injection. The behavioral analysis showed that intranasal administration of NGF reduced the immobility time in forced swimming test (FST) and tail suspension test (TST) in mice. Likewise, intranasal NGF increased the sucrose intake and the locomotor activity in rats after unpredictable chronic mild stress (UCMS). Furthermore, intranasal NGF increased the levels of monoamine neurotransmitters (norepinephrine, dopamine) in the frontal cortex and hippocampus and affected the number of 5-bromodeoxyuridine (BrdU), c-fos and caspase-3 positive neurons in dentate gyrus of hippocampus in rats after UCMS. In summary, intranasal NGF had significant antidepressant effects on animal models of depression and this route of administration may provide a promising way to deliver NGF to brain in a therapeutic perspective.     

丙戊酸钠活化大鼠海马和额叶ERK-1/2信号传导通路

为探讨慢性服用丙戊酸钠对中枢神经系统细胞外调控激酶 (ERK) 1/ 2信号传导通路活性的影响 ,阐明丙戊酸钠治疗躁狂抑郁症作用的可能分子机制 ,将 4 0只雄性Wistar大鼠随机分为实验组和对照组 ,每组各 2 0只 .实验组大鼠用含丙戊酸钠的饲料喂养 ,对照组大鼠用常规饲料喂养 ,4周后取大鼠海马和额叶组织制备蛋白质样本 ,蛋白质印迹方法分析海马和额叶组织丝裂原活化的蛋白激酶激酶 (MEK)、ERK 1/ 2、MAPK活化的蛋白激酶 1(RSK1)、cAMP效应元件结合因子 (CREB)的磷酸化水平以及Bcl 2的表达水平 ,电泳迁移率变动分析(EMSA)方法分析海马和额叶组织激活蛋白 1(AP 1)的DNA结合活性 .与对照组比较 ,丙戊酸钠显著增强海马和额叶MEK、ERK 1/ 2、RSK1、CREB和AP1的活性 ,上调海马和额叶Bcl 2的表达 .结果表明 :慢性服用丙戊酸钠激活中枢神经系统ERK 1/ 2信号传导通路、上调中枢神经系统Bcl 2蛋白表达 ,这些作用可能与丙戊酸钠治疗躁狂抑郁症的作用有关