Effects of phenelzine and imipramine on the steady-state levels of mRNAs that encode glutamic acid decarboxylase (GAD67 and GAD65), the GABA transporter GAT-1 and GABA transaminase in rat cortex

There is an increasing body of evidence suggesting that GABA plays an important role in the therapeutic effects of antidepressant/antipanic drugs. Phenelzine and imipramine are efficacious in the treatment of depression and panic disorder and phenelzine has been reported to elevate GABA levels while imipramine enhances GABA release in rat brains. In the present study, using a multiprobe quantitative solution hybridization assay, we measured the steady-state levels of mRNAs that encode glutamic acid decarboxylase (GAD₆₇ and GAD₆₅), the GABA transporter GAT-1 and GABA transaminase (GABA-T) in rat cortex after treatment with constant infusion (via osmotic minipumps) of phenelzine or imipramine for a short-term (3 days) or long-term (21 days) period. We found that none of the treatments gave rise to significant changes in the steady-state levels of mRNAs encoding GAD₆₇, GAD₆₅ or GABA-T at any time point. The steady-state levels of GAT-1 mRNA were increased significantly (23%) after long-term, but not by short-term, treatment with phenelzine. Imipramine treatment, short- or long-term, did not alter the steady-state levels of GAT-1 mRNA. These results suggest that the GABA enhancing effects of phenelzine or imipramine in rat cortex do not affect the steady-state levels of mRNAs that encode GAD₆₇, GAD₆₅ and GABA-T. Further, the previously observed increases in GABA levels or GABA release induced by these drugs are probably not a consequence of changes in the expression of these genes. Received: 20 June 1997 / Accepted: 23 September 1997     

羟丁酸钠对大鼠局灶性脑缺血再灌注损伤的保护作用及与GABA的关系

目的　研究羟丁酸钠对大鼠局灶性脑缺血再灌注损伤的保护作用及该作用与γ 氨基丁酸 (GABA)的关系。方法　采用线栓法建立大鼠局灶性脑缺血再灌注损伤模型 ,观察损伤大鼠行为功能缺失并称量脑梗死区域组织重量。用高效液相 -荧光法测定大鼠脑脊液中氨基酸的含量并计算GABA/Glu的比值。结果　羟丁酸钠可明显降低缺血再灌注后大鼠行为学评分分值 ,减小脑梗死组织重量 ,增加GA BA/Glu的比值。结论　羟丁酸钠对大鼠局灶性脑缺血再灌注损伤有明显的保护作用 ,该作用与其提高抑制性氨基酸GABA的含量 ,维持脑内兴奋性氨基酸和抑制性氨基酸的动态平衡 ,产生抑制性保护作用有关     

The effect of low concentrations of an organic lead compound on the transport and release of putative neurotransmitters

Tri-n-butyl lead acetate at concentrations of 5.10^?5-10^?7 M was effective in inhibiting high affinity uptake and in stimulating release of accumulated radioactive putative neurotransmitters by mouse brain homogenates. Mercurycontaining compounds and lead acetate were much less potent in inhibiting such transport and stimulating release. It is suggested that the neurotoxicity of organic lead compounds is, in part, related to effects on neurotransmission and that dopaminergic synapses are especially susceptible to such toxicity.     

Pharmacological characterisation of a cell line expressing GABA B1b and GABA B2 receptor subunits

The gamma-aminobutyric acid (GABA(B)) receptor has been shown to be a heterodimer consisting of two receptor subunits, GABA(B1) and GABA(B2). We have stably co-expressed these two subunits in a CHO cell line, characterised its pharmacology and compared it to the native receptor in rat brain membranes. Radioligand binding using [3H]CGP54626A demonstrated a similar rank order of potency between recombinant and native receptors: CGP62349>CGP54626A>SCH 50911>3-aminopropylphosphinicacid(3-APPA)>GABA>baclofen>saclofen>phaclofen. However, differences were observed in the affinity of agonists, which were higher at the native receptor, suggesting that in the recombinant system a large number of the receptors were in the low agonist affinity state. In contrast, [35S]GTPgammaS binding studies did not show any differences between recombinant and native receptors with the full agonists GABA and 3-APPA. Measurement of cAMP accumulation in the cells revealed a degree of endogenous coupling of the receptors to G-proteins. This is most likely to be due to the high expression levels of receptors (B(max)=22.5+/-2.5pmol/mg protein) in this experimental system. There was no evidence of GABA(B2) receptors, when expressed alone, binding [3H]CGP54626A, [3H]GABA, [3H]3-APPA nor of GABA having any effect on basal [35S]GTPgammaS binding or cAMP levels.     

SNP、GLU及GABA对纹状体神经元活动的影响

目的　探讨一氧化氮(NO)在纹状体(STR)信息传递中的作用,对帕金森综合征(PD)的发病机制有更深入的了解,对PD的临床诊断及治疗有更深远的帮助。方法　本实验采用多管玻璃微电极微电泳方法观察了微电泳硝普钠(SNP)、谷氨酸(GLU)及γ- 氨基丁酸(GABA)对大鼠STR神经元自发放电的影响。同时观察了NO对GLU及GABA神经传递的影响。结果　实验观察到微电泳SNP可使77%(51 /66)受试神经元放电频率加快,其兴奋作用可被单硝基L -精氨酸甲酯(L NAME)所拮抗。在微电泳GLU或GABA过程中,给予SNP可增强GLU的兴奋作用,拮抗GABA的抑制作用。给予NOS抑制剂L- NAME可拮抗GLU的兴奋作用。结论　实验结果表明NO、GABA、GLU等神经递质活动在STR的同一神经元有会聚作用,NO、GLU对STR神经元有兴奋作用,而GABA对STR神经元有紧张性抑制作用。     

黄芩苷对γ-氨基丁酸受体和抗焦虑症作用研究

目的观察黄芩苷与γ-氨基丁酸（GABA）受体的关系及其所具有的抗焦虑作用。方法采用同位素标记GABA受体结合实验观察黄芩苷与GABA受体的关系;采用高台十字迷宫实验和四孔箱实验观察动物服用黄芩苷后行为的改变。结果 GABA受体竞争结合试验显示,黄芩苷可竞争性抑制50%GABA受体苯二氮类结合浓度（IC50）为（117.62±8.50）μmol·L-1,抑制结合常数（Ki）为（77.10±4.79）μmol·L-1。黄芩苷可增加实验动物进入十字迷宫开放侧次数,百分率和滞留时间,减少在四孔箱中探头和竖身的次数。这些变化与黄芩苷剂量有明显的关系,可被GABA受体阻断剂Ro 15-1788阻断。结论黄芩苷是GABA结合位点的配体,具有一定的抗焦虑作用。     

Chronic difluoromethylornithine treatment impairs spatial learning and memory in rats

Recent evidence suggests that polyamines putrescine, spermidine and spermine are essential in maintaining normal cellular function. The present study investigated the effects of chronic treatment of difluoromethylornithine (DFMO, 3% in drinking water), a potent inhibitor of putrescine synthesis, for 54 consecutive days on animals'behavior and neurochemical levels in the CA1, CA2/3 and dentate gyrus sub-regions of the hippocampus and the prefrontal cortex. The DFMO group showed performance impairments in the place navigation and the probe test conducted 24 h after the training in the reference memory version of the water maze task, but not in the elevated plus maze, open field, object recognition, cued navigation and the working memory version of the water maze task when compared to the control group (drinking water only). DFMO treatment resulted in approximately 80-90% and 20% of reductions in the putrescine and spermidine levels, respectively, in the four brain regions examined, and a small reduction in agmatine level in the CA2/3, with no effects on spermine, glutamate and γ-aminobutyrate. The DFMO group showed decreased body weight relative to the control one. However, there were no significant differences between groups in the normalized brain, kidney and liver weights. The present study demonstrates that chronic treatment of DFMO depletes putrescine and decreases spermidine levels in the brain, inhibits growth, and impairs spatial learning and memory in the reference memory version of the water maze specifically. These findings merit further investigation to fully understand the functional role of endogenous polyamines in learning and memory.     

谷氨酸和γ-氨基丁酸对原代培养星形胶质细胞神经甾体合成释放的影响

目的研究氨基酸类神经递质对原代培养大鼠大脑皮质星形胶质细胞神经甾体合成释放的影响。方法采用原代培养的大鼠大脑皮质星形胶质细胞,分别加入不同浓度的谷氨酸和γ-氨基丁酸处理48h;采用固相萃取结合高效液相色谱质-谱联用分析方法提取分离和测定细胞培养液中游离型(脱氢表雄酮,DHEA;孕烯醇酮,PREG;别孕烯醇酮,AP)及结合型神经甾体(脱氢表雄酮硫酸酯,DHEAS;孕烯醇酮硫酸酯,PREGS)。结果与生理盐水对照组比较,谷氨酸处理使PREG和PREGS水平明显下降,DHEAS水平明显升高;γ-氨基丁酸处理使PREG水平明显降低,AP水平增加。结论谷氨酸和γ-氨基丁酸两种神经递质对原代培养的星形胶质细胞PREG合成释放均呈抑制作用;谷氨酸对DHE-AS、γ-氨基丁酸对AP的合成释放分别呈现明显促进作用;高剂量的谷氨酸还可以抑制PRGES的合成和释放。     

Opiate antagonists, morphine and spatial memory in rats

To assess the possible role of endogenous opioids on spatial memory, rats were administered morphine (1-15 mg/kg), naloxone (1-10 mg/kg), or naltrexone (0.1-10 mg/kg) at varying times after or prior to completing the first 4 choices in an 8 arm radial maze. None of these agents consistently affected retention, suggesting that endogenous opioid systems do not play a major role in modulating neural mechanisms that maintain accurate spatial memory.     

The effect of the convulsant allylglycine (2-amino-4-pentenoic acid) on the activity of glutamic acid decarboxylase and the concentration of GABA in different regions of guinea pig brain

Intraperitoneal administration of allylglycine to guinea pigs resulted in convulsions approximately 3 hr later. The concentration of GABA and the activity of GAD were significantly reduced in three brain areas, namely the cochlear nucleus, inferior colliculus and cerebral cortex, with the smallest changes being observed in the cortex. There were large in vitro regional variations in the extent of the allylglycine inhibition in brain areas from guinea pig, cat and rat, with those areas rich in GAD activity being least affected. Endogenous GAD activities in the brain regions were found to be inversely correlated with the percentage allylglycine inhibition (P < 0.005). Other inhibitors of GAD activity i.e. NaCl, Zn²⁺ and thiosemicarbazide showed no such regional variation of inhibition. The results suggest that the regional differences in allylglycine inhibition reflect anomalies of the metabolism of the drug per se, and probably do not indicate regional differences in GABA turnover and metabolism.     

Studies on GABA and cardiovascular function using in vivo and in vitro technics

Recent in vivo and in vitro studies regarding the involvement of GABAergic systems in cardiovascular function have been reviewed and analyzed. It seems evident, from studies conducted with various GABA agonists and GABA antagonists, that activation of central GABAergic systems reduces sympathetic outflow from the hindbrain area of mammals, thereby producing bradycardia and hypotension. As GABA agonists can relax cerebral arteries, blood-borne GABA might also play a role in cerebrovascular regulation. Further research concerning the mechanisms involved in GABA agonist-induced cardiovascular responses could lead to the development of agents that might be effective in treating certain cardiovascular disorders of man, such as hypertension, cerebral atherosclerosis, migraine, and stroke.     

蛇床子素对脑缺血/再灌注大鼠海马LTP及氨基酸含量的影响

目的研究蛇床子素对脑缺血/再灌注大鼠海马齿状回突触传递活动及海马内氨基酸含量的影响。方法 SD大鼠随机分为正常组、假手术组、模型组、蛇床子素组(25.0、12.5 mg.kg-1)和尼莫地平组(1.0 mg.kg-1),腔内线栓法制作右侧大脑中动脉栓塞(middle cerebral artery occlusion,MCAO)模型,缺血后2 h再灌。采用2,3,5-氯化三苯基四氮唑(TTC)染色法记录大鼠脑梗死体积、Morris水迷宫法观察大鼠空间学习记忆能力、电生理学方法记录中枢突触传递长时程增强(long-term potentiation,LTP)现象、高效液相色谱法记录大鼠海马内氨基酸含量的变化。结果蛇床子素(25.0、12.5 mg.kg-1,ip)可减少大鼠脑梗死体积,剂量依赖性地缩短大鼠寻找站台的时间,增强大鼠海马齿状回高频刺激(high frequency stimulation,HFS)诱导的LTP,降低再灌后72 h内海马内谷氨酸含量,蛇床子素(25.0 mg.kg-1)对于γ-氨基丁酸产生先降低后升高的调节作用,与尼莫地平组作用结果一致。结论蛇床子素改善学习记忆障碍、增强脑缺血/再灌注大鼠海马齿状回HFS诱导的LTP的作用,与调节海马内谷氨酸、γ-氨基丁酸水平有关。     

Effect of antidepressants on GABA(B) receptor function and subunit expression in rat hippocampus

Laboratory and clinical studies suggest that depression is associated with changes in the hippocampus and that this brain region is a major target for antidepressant drugs. Given the data suggesting that GABA(B) receptor antagonists display antidepressant properties, the present study was undertaken to assess the effect of antidepressant administration on GABA(B) receptors in the rat hippocampus to determine whether changes in this regional receptor system may play a role in the response to these agents. Rats were administered (i.p.) the monoamine oxidase inhibitors tranylcypromine (10mg/kg) or phenelzine (10mg/kg), the tricyclic antidepressant desipramine (15 mg/kg), or fluoxetine (5mg/kg), a selective serotonin re-uptake inhibitor, once daily for seven consecutive days. Two hours following the last drug treatment the hippocampal tissue was prepared for defining the distribution and quantity of GABA(B) receptor subunits using in situ hybridization and for assessing GABA(B) receptor function by quantifying baclofen-stimulated [(35)S]-GTPgammaS binding. All of these antidepressants selectively increased the expression of the GABA(B(1a)) subunit in hippocampus, having no consistent effect on the expression of GABA(B(1b)) or GABA(B(2)). Moreover, except for fluoxetine, these treatments increased GABA(B) receptor function in this brain region. The results indicate that an enhancement in the production of hippocampal GABA(B(1a)) subunits may be a component of the response to antidepressants, supporting a possible role for this receptor in the symptoms of depression and the treatment of this condition.     

Effects of cholinergic and monoaminergic antagonists and tranquilizers upon spatial memory in rats

To explore the pharmacological mechanisms of the spatial memory, performance on the radial arm maze was tested in rats under the following drugs, using a within-subject design; scopolamine (0.25 and 0.5 mg/kg), methylscopolamine (0.5 and 1 mg/kg), phentolamine (5 and 10 mg/kg), propranolol (10 and 20 mg/kg), chlorpromazine (1 and 2 mg/kg), and chlordiazepoxide (5 and 10 mg/kg). The number of correct choices was significantly decreased by scopolamine, while the other drugs, including methylscopolamine, showed no effects on the correct choices. Almost all drugs affected the running time. These findings indicate that the brain cholinergic system is involved in the spatial memory.     

夏天无总生物碱对痴呆大鼠学习记忆障碍及中枢胆碱能神经系统功能的影响

目的　观察夏天无总生物碱 (Xiatianwutotalalkaloids,XA)对喹啉酸损毁海马所致大鼠学习记忆障碍和中枢胆碱能神经系统功能的影响。方法　采用双侧海马CA1区微注射喹啉酸造成大鼠学习记忆障碍模型 ,在造模前 1wk至造模后 3wk内 ,大鼠每天igXA 0 2 5 ,0 5 ,1mg·kg-1。采用三等分Y迷宫测定大鼠学习记忆能力 ,并采用比色法检测海马乙酰胆碱酯酶 (acetylcholinesterase ,AChE)活性和大脑皮层乙酰胆碱 (acetylcholine ,ACh)含量的变化。结果　大鼠双侧海马内微注射喹啉酸可引起大鼠学习记忆功能障碍 ,海马中AChE活性下降 ,大脑皮层ACh含量减少 ;XA可显著改善喹啉酸诱导的痴呆大鼠学习记忆功能障碍 ,同时明显抑制模型大鼠海马的AChE活性 ,提高大脑皮层ACh含量。结论　XA可改善喹啉酸损毁海马造成的痴呆大鼠学习记忆功能障碍 ,而其机制可能与抑制脑AChE活性 ,从而提高ACh含量 ,增强中枢胆碱能系统的功能有关。     

Alteration of GABA metabolism in mammalian brain by l-alpha-amino-beta-chloropropionic acid hydroxamide and related compounds.

The administration of L-alpha-amino-beta-chloropropionic acid hydroxamide (L-ACPH) to mice brought about an inhibition in GABA-T activity in the brain of the animals, a significant inhibition occurring with dosage levels as low as 0.25 mmol/kg. Minimum levels of GABA-T activity were reached 3 h after administration of the drug. Brain glutamic acid decarboxylase, DOPA decarboxylase and aspartate aminotransferase activities were not altered by the L-ACPH but alanine aminotransferase activity was totally inhibited. Slight changes in structure caused great changes in the potency of the drugs. For example, the elongation of the L-ACPH structure by one carbon, or a change in the configuration of the amino group from L- to D-, caused a significant decrease in GABA inhibition. The chloro and hydroxamide groups were necessary for inhibitory activity. The administration of L-ACPH to mice delayed the onset of drug induced seizures but had a less noticeable effect against maximal electroshock. The addition of L-ACPH to crude extracts from brain, or to preparations of semipurified GABA-T, also inhibited GABA-T activity. Again the development of the inhibition was time-dependent. Possible mechanisms of action with respect to L-ACPH induced inhibition of GABA-T activity are discussed in the light of the data presented.     

Effects of methylmercuric chloride on high affinity uptake of certain putative neurotransmitters in neuroblastoma and glioma cell cultures

The effect of methylmercuric chloride (CH₃HgCl) on high affinity uptake of certain putative neurotransmitters on mouse neuroblastoma (NBE^?) and rat glioma (C-6) cells in culture 3 days after treatment was studied. The energydependent accumulation of radioactive choline was decreased in NB cells afte treatment with CH₃HgCl (1 μm), whereas the accumulation of radioactive glycine or glutamate was increased. The uptake of radioactive glycine or glutamate in glioma cells was decreased after treatment with CH₃HgCl (0.3 μM). High affinity uptake of choline was not demonstrable in glioma cells. Low concentrations of CH₃HgCl affect the accumulation of glycine and glutamate in glioma and NB cells in an opposite manner.     

Role of GABA(A) receptors in the ethanol-mediated inhibition of extracellular signal-regulated kinase

In the present study, we demonstrate the involvement of GABA(A) receptors in the ethanol-mediated modulation of extracellular signal-regulated kinases (ERK). Intraperitoneal (i.p.) administration of ethanol (3.5 g), flurazepam (75 mg) or (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cycloheptane-5,10-iminemaleate (MK-801) (0.4 mg/kg body weight) decreased, while picrotoxin (10 mg/kg body weight) increased, the phosphorylation of ERK following 10 min of their injection. However, the picrotoxin-induced phosphorylation of ERK was inhibited by ethanol, but was not affected by MK-801. These results indicate that ethanol's inhibitory effect on ERK phosphorylation may involve the modulation of GABA(A) receptor function.     

Electrophysiological actions of gamma-aminobutyric acid and clomethiazole on recombinant GABA(A) receptors

Clomethiazole is a gamma-aminobutyric acid (GABA)-mimetic agent with anticonvulsant, sedative and neuroprotective properties. The pharmacological actions of clomethiazole that underlie its functional profile have not been fully explored, but are known to result from an interaction with the GABA(A) receptor. Here, we present a quantitative electrophysiological study of clomethiazole action at human recombinant GABA(A) receptors. Whole-cell currents were recorded from murine L(tk-) cells stably transfected with either alpha1, beta1 and gamma 2 or alpha1, beta2 and gamma 2 GABA(A) receptor subunits. Clomethiazole directly activated GABA(A) currents in alpha1/beta1/gamma 2- and alpha1/beta2/gamma 2-containing cells, with EC(50) values of 0.3 and 1.5 mM, respectively. A low concentration of clomethiazole (30 micro M) also potentiated the action of GABA in both cell types, equivalent to a 3-fold increase in potency and up to 1.8-fold increase in maximal current. Both direct activation and gamma-aminobutyric acid potentiation are likely to contribute to the in vivo profile of clomethiazole.     

Biphasic effect of citral, a flavoring and scenting agent, on spatial learning and memory in rats

Although some central effects of citral have been reported, cognitive effects on spatial memory have not been investigated. The evidence showed that citral can regulate the synthesis of retinoic acid (RA), which exerts a vital function in the development and maintenance of spatial memory. In this study, we applied Morris water maze to test the effect of citral on animals' spatial learning and memory. To elucidate the mechanism of this effect, we also measured the retinoic acid concentration in rats' hippocampus by high performance liquid chromatography (HPLC). Our data implied biphasic effects of citral. The low dose (0.1 mg/kg) of citral improved the spatial learning capability, and enhanced the spatial reference memory of rats, whereas the high dose (1.0 mg/kg) was like to produce the opposite effects. Meanwhile, the low dose of citral increased the hippocampal retinoic acid concentration, while the high dose decreased it. Due to the quick elimination and non-bioaccumulation in the body, effects of citral on spatial memory in this study seemed to be indirect actions. The change in hippocampal retinoic acid concentration induced by different doses of citral might be responsible for the biphasic effect of citral on spatial learning and memory.