创伤后应激障碍大鼠杏仁核PDE-4和CREB的表达

目的探讨PDE-4与CREB在创伤后应激障碍(post-traumatic stress disorder,PTSD)大鼠杏仁核内的表达变化,为进一步阐明PTSD增强恐惧记忆机制研究提供基础。方法成年雄性大鼠40只,随机分为PTSD模型1d、7d、14d组及正常对照组,采用单一连续刺激(single-prolonged stress,SPS)方法建立PTSD模型,应用免疫组化及Real-time PCR方法检测大鼠杏仁核PDE-4及CREB蛋白表达及mRNA表达变化。结果 PTSD造模后1d PDE-4蛋白表达显著降低,于PTSD 7d时降至最低,第14d时回升,但仍显著低于正常,PDE-4mRNA表达与蛋白表达同步变化;CREB蛋白在PTSD造模后1d表达显著增加,在PTSD 7d时升至最高,第14d时下降,但仍显著高于正常,其m PNA表达与蛋白表达同步变化。结论创伤应激刺激能诱发大鼠杏仁核PDE-4和CREB的表达变化,且PDE-4和CREB表达呈反向变化。     

大鼠局灶性脑缺血后缺血边缘区NG_2细胞的表达

目的观察局灶性脑缺血后缺血边缘区海马和皮层NG2细胞的动态表达,探讨其在脑缺血神经损伤与修复过程中所起的作用。方法将大鼠随机分为假手术组(sham)和脑缺血再灌注组,脑缺血再灌注组采用线栓法制备大鼠大脑中动脉阻塞再灌流模型(MCAO),假手术组不插入线栓,采用免疫荧光组织化学法结合共聚焦显微镜成像观察sham组及脑缺血后3d,7d,30d不同时间点缺血边缘区的海马CA1区和皮层区NG2的动态表达情况。结果脑缺血再灌注后缺血边缘区海马和皮层NG2胶质细胞表达增加,缺血后7d最明显。结论脑缺血后缺血边缘区存在NG2细胞的增生和形态变化可能与脑缺血后损伤修复密切相关。     

杏仁核点燃模型癫痫样放电传播途径研究

目的 :探讨杏仁核点燃模型癫痫样放电的传播途径。方法 :选择健康Wistar大鼠 3 0只以电刺激杏仁核的方式制作杏仁核点燃癫痫模型 ,于右侧杏仁核、左侧海马及右侧额叶皮质埋植电极记录脑电活动 ,观察电刺激杏仁核时在杏仁核、海马及额叶皮质出现癫痫样放电的潜伏期、最低刺激强度及癫痫样放电的持续时间。结果 :杏仁核出现癫痫样放电时 ,海马及皮质均未记录到癫痫样放电。而当杏仁核、海马及皮质三处出现癫痫样放电时的最低刺激强度依次增大 ,潜伏期依次延长 ,海马处癫痫样放电的持续时间最长。结论 :杏仁核点燃模型癫痫样放电可能由杏仁核经海马传至皮层 ,海马可能为癫痫样放电传播的重要结构     

大鼠脑缺血再灌注后神经元和星形胶质细胞凋亡的比较研究

目的比较研究大鼠局灶性脑缺血再灌注后神经元和星形胶质细胞的凋亡规律。方法建立大鼠大脑中动脉阻塞(middle cerebral artery occlusion,MCAO)再灌注模型,在缺血再灌注后1、3、7、14d断头取脑,应用流式细胞分选技术和原位末端标记法分别检测各组MCAO后不同时期神经元和星形胶质细胞凋亡情况。结果局灶性脑缺血再灌注后,海马区星形胶质细胞凋亡数量超过神经元,其凋亡以再灌注3d最为显著,而神经元则以7d最为显著;而皮层区神经元凋亡数量超过星形胶质细胞,两种细胞凋亡均在再灌注后7d达高峰。结论脑缺血再灌注后,皮层和海马区的神经元及星形胶质细胞均可发生凋亡,海马区星形胶质细胞比皮层区更易凋亡,而皮层区神经元比海马区更易凋亡。     

亚慢性地卓西平诱导的精神分裂样小鼠前额叶和海马脑区MIF蛋白表达的变化

目的:探讨亚慢性地卓西平(MK-801)诱导的精神分裂样小鼠模型中前额叶和海马脑区巨噬细胞迁移抑制因子(Macrophage migration inhibitory factor,MIF)蛋白表达的变化。方法:将24只7周龄小鼠随机分为对照组、MK-801组和MK-801+奥氮平(olanzapine,olz)组(n=8),三组小鼠分别接受0.9%生理盐水、MK-801(0.6 mg/kg)和MK-801(0.6 mg/kg)+奥氮平(2.5 mg/kg)给药,持续4周。小鼠行为学通过旷场试验、社交实验进行评价,免疫印迹法检测小鼠前额叶和海马组织中MIF蛋白的表达。结果:MK-801处理后,小鼠活动量增加,社交功能受损,且都能被抗精神分裂症药物奥氮平显著改善。MK-801组小鼠前额叶皮层中MIF蛋白表达与对照组比较无明显统计学差异(P0.05),而海马脑区中MIF蛋白表达较对照组明显升高(P0.05);MK-801+奥氮平组小鼠前额叶皮层中MIF蛋白表达较MK-801组无显著变化,而海马脑区中MIF蛋白表达较MK-801组明显降低(P0.05)。结论:亚慢性给予MK-801诱导的精神分裂样小鼠海马脑区中MIF蛋白水平升高,提示MIF蛋白可能参与MK-801诱导的精神分裂样行为。     

PTSD样行为异常大鼠杏仁核Ca^2＋/CaM的改变

目的观察创伤后应激障碍（PTSD）样行为异常大鼠杏仁核细胞内Ca^2＋信号及Ca M（钙调蛋白）表达变化,有望揭示PTSD的部分发病机制。方法成年健康雄性Wistar大鼠60只,随机分为连续单一刺激（single prolonged stress,SPS）模型的12h、1d、4d、7d、14d组及正常对照组,采用荧光探针标记法、免疫组化、Western blott等方法,检测PTSD样行为异常大鼠杏仁核神经元游离Ca^2＋含量和钙调蛋白（Ca M）的表达变化。结果SPS刺激后大鼠杏仁核神经元游离Ca^2＋浓度（nmol/L）于12h内升高,24h增至顶峰,4d开始下降,14d恢复正常。CaM的表达于SPS刺激后4d表达最多,之后渐趋下降。结论杏仁核Ca^2＋信号调控与Ca M表达变化,可能与PTSD样大鼠恐惧增强的发病机制相关。     

焦虑性抑郁模型大鼠脑区单胺递质含量与神经因子表达的变化

目的研究焦虑性抑郁模型大鼠海马、杏仁核、前额叶皮质内单胺递质的含量变化及脑内神经营养因子的表达趋势,探讨其可能的发病机制。方法 60只SD大鼠随机分为正常对照组、溶媒对照组、焦虑模型组、抑郁模型组、焦虑性抑郁模型组,每组12只。采用慢性束缚应激联合皮质酮注射的方法建立焦虑性抑郁大鼠模型,造模时间为21 d,造模结束后采用高架十字迷宫测试,旷场实验,强迫游泳实验评价大鼠的焦虑和抑郁样行为,HPLC-ECD法检测大鼠海马、杏仁核、前额叶皮质的单胺递质5-HT、NE、DA含量,蛋白印迹法检测大鼠各脑区神经营养因子BDNF、NT-3的含量。结果焦虑性抑郁模型组大鼠在进入开臂的时间、次数、旷场中自主活动次数均与焦虑组相当,与对照组及抑郁组比较差异有显著性(P0.01或P0.05),在强迫游泳中的不动时间显著增加,与对照组及焦虑组对比差异有显著性(P0.01);同时,与对照组比较,焦虑性抑郁模型组大鼠海马5-HT、杏仁核及前额叶皮质区的5-HT和NE含量均显著下降(P0.01或P0.05);此外,与对照组比较,焦虑性抑郁模型组大鼠各脑区BDNF、NT-3含量显著下降(P0.01或P0.05),同时与焦虑组比较,BDNF含量显著下降(P0.05)。结论焦虑性抑郁模型组大鼠具有显著的焦虑及抑郁样行为,其发病机制可能与脑内海马、杏仁核、前额叶皮质区域的单胺递质含量降低及神经营养因子BDNF、NT-3表达下调有关。     

PTSD样情感行为异常大鼠杏仁核Caspase 9的改变

目的观察创伤后应激障碍(PTSD)样行为异常大鼠杏仁核神经元Caspase 9表达变化,有望揭示PTSD的部分发病机制。方法采用国际认定的SPS方法刺激建立大鼠PTSD模型,取成年健康雄性Wistar大鼠60只,随机分为SPS模型的1d、4d、7d组及正常对照组,采用免疫荧光法、免疫印迹和逆转录-聚合酶链式反应检测大鼠杏仁核Caspase 9的表达变化。结果SPS刺激后大鼠杏仁核神经元细胞内Caspase 9于1d开始逐渐升高,7d时表达最多;Caspase 9 mRNA的变化与之相一致。结论海马Caspase 9的表达变化,可能是PTSD大鼠情感行为异常的重要病理生理基础之一。     

AMPA受体和相关蛋白在束缚应激大鼠相关脑区的表达变化及逍遥散对其影响

目的：观察海马及杏仁核α-氨基羟甲基恶唑丙酸（AMPA）受体亚基和相关调节蛋白在束缚应激状态下蛋白表达变化及逍遥散的调节作用。方法：使用每天捆绑3 h的方法制作慢性束缚应激动物模型,并用逍遥散进行干预,分别于7 d后和21 d后用Western blot方法检测各组大鼠海马CA1区、CA3区、齿状回（DG）和杏仁核的AMPA受体亚基GluR2/3及N-乙基顺丁烯二酰亚胺敏感性的融合蛋白（NSF）、PKC作用蛋白1（PICK1）蛋白表达的情况。结果：7 d应激可使DG和杏仁核的GluR2/3、NSF表达显著降低（P均〈0.05）,使PICK1在CA1区的表达量显著增多（P〈0.05）,逍遥散对PICK1变化显示出一定调节作用。21 d应激可使CA1区的GluR2/3、NSF表达升高,其中GluR2/3有显著性差异（P〈0.01）,而在杏仁核表达有降低趋势,逍遥散对其均有显著调节作用（均为P〈0.05）,21 d应激使杏仁核PICK1表达量出现升高趋势,逍遥散可显著降低其表达（P〈0.05）。结论：AMPA受体在短期重复应激和慢性应激状态下反应不同,海马和杏仁核反应相反,逍遥散对慢性应激状态下AMPA受体表达的调节作用较短期重复应激强。     

创伤后应激障碍大鼠杏仁核海马复合体Ca^2＋-CaM—CaMKⅡa变化的研究

探讨创伤后应激障碍（PTSD）大鼠杏仁核海马复合体Ca^2＋-CaM-CaMKⅡa信号通路的变化,为揭示PTSD发病机制提供实验依据。海马的部分功能由杏仁核调节实现。采用目前国际公认的连续单-刺激（single prolonged stress,SPS）模型刺激方法制作PTSD模型,成年雄性Wistar大鼠60只,随机分为SPS模型组的12h、1d、4d、7d及正常组。     

Differential Response of Central Dopaminergic System in Acute and Chronic Unpredictable Stress Models in Rats

We aimed to evaluate the response of dopaminergic system in acute stress (AS) and chronic unpredictable stress (CUS) by measuring dopamine (DA) levels, its receptor densities in the frontal cortex, striatum, hippocampus, amygdala and orbito-frontal cortex regions of rat brain, and investigated the corresponding behavioral locomotor changes. Involvement of D₁ receptor was also examined during AS and CUS using A 68930, a D₁ selective agonist. Rats were exposed to AS (single immobilization for 150 min) and CUS (two different stressors for 7 days). AS significantly decreased the DA levels in the striatum and hippocampus, and A 68930 pretreatment significantly reverted these changes. However, in the frontal cortex significantly increased DA levels were remain unchanged following A 68930. CUS led to a decrease of DA levels in the frontal cortex, striatum and hippocampus, which were normalized by A 68930. Saturation radioligand binding assays revealed a significant decrease in the number of D₁-like receptors in the frontal cortex during CUS, which were further decreased by A 68930 pretreatment. However, in the striatum and hippocampus, A 68930 pretreatment reduced the CUS induced increase in the number of D₁-like receptors. No significant changes were observed in the amygdala and orbito-frontal cortex during AS and CUS, while D₂-like receptors were unchanged in all the brain regions studied. Locomotor activity was significantly decreased in both the stress models, A 68930 pretreatment significantly increased stereotypic counts and horizontal activity. Thus, present investigation provide insights into the differential regional response of dopaminergic system during AS and CUS. Further, neurochemical and behavioral effects of D₁ agonist pretreatment suggest specific modulatory role of D₁ receptor under such stressful episodes.     

Postnatal Development of Cholecystokinin-Like Immunoreactivity and Its mRNA Level in Rat Brain Regions

Developmental changes of preprocholecystokinin mRNA (CCK mRNA) and cholecystokinin-like immunoreactivity (CCK-LI) were examined in rat brain regions (frontal cortex, colliculi, hippocampus, striatum, and cerebellum) using RNA dot blot assays with cholecystokinin (CCK) cDNA and radioimmunoassay, respectively. The CCK-LI levels in all regions examined were very low at birth. Excluding the cerebellum, the levels in these regions increased postnatally and reached adult values at 28 days of age. In contrast to CCK-LI, CCK mRNA levels changed dramatically during development. A considerable amount of CCK mRNA was detected in the frontal cortex and hippocampus at birth. The changes in the level of CCK mRNA in the frontal cortex and colliculi paralleled those of CCK-LI, including a rapid increase from 7 to 14 days of age. The synthesis of CCK mRNA preceded the appearance of CCK-LI. CCK mRNA levels in the hippocampus and striatum exhibited a transient increase, with a peak at 14 days of age. In the adult brain, the CCK mRNA levels were high in the frontal cortex, moderate in the hippocampus and colliculi, and low in the striatum. The cerebellum contained only a negligible amount of CCK mRNA during development. The relatively high level of CCK-LI compared with the low level of CCK mRNA in the striatum supports the idea that most of the striatal CCK-LI is supplied from extrastriatal regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative immunochemistry on neuronal loss, reactive gliosis and BBB damage in cortex/striatum and hippocampus/amygdala after systemic kainic acid administration

Cell specific markers were quantified in the hippocampus, the amygdala/pyriform cortex, the frontal cerebral cortex and the striatum of the rat brain after systemic administration of kainic acid. Neuron specific enolase (NSE) reflects loss of neurons, glial fibrillary acidic protein (GFAP) reflects reactive gliosis, and brain levels of serum proteins measures blood-brain-barrier permeability. While the concentration of NSE remained unaffected in the frontal cerebral cortex and the striatum, their GFAP content increased during the first three days. In the hippocampus and amygdala, NSE levels decreased significantly. GFAP levels in the hippocampus were unaffected after one day and decreased in the amygdala/pyriform cortex. After that, GFAP increased strikingly until day 9 or, in the case of amygdala/pyriform cortex, even longer. This biphasic time course for GFAP was accompanied by a decrease of S-100 during days 1-9 followed by a significant increase at day 27 above the initial level. The regional differences in GFAP and S-100 could result from the degree of neuronal degeneration, the astrocytic receptor set-up and/or effects on the blood-brain barrier.     

Changes in Antioxidant Defense Enzymes after d-amphetamine Exposure: Implications as an Animal Model of Mania

Studies have demonstrated that oxidative stress is associated with amphetamine-induced neurotoxicity, but little is known about the adaptations of antioxidant enzymes in the brain after amphetamine exposure. We studied the effects of acute and chronic amphetamine administration on superoxide dismutase (SOD) and catalase (CAT) activity, in a rodent model of mania. Male Wistar rats received either a single IP injection of d-amphetamine (1 mg/kg, 2 mg/kg, or 4 mg/kg) or vehicle (acute treatment). In the chronic treatment rats received a daily IP injection of either d-amphetamine (1 mg/kg, 2 mg/kg, or 4 mg/kg) or vehicle for 7 days. Locomotor behavior was assessed using the open field test. SOD and CAT activities were measured in the prefrontal cortex, hippocampus, and striatum. Acute and to a greater extent chronic amphetamine treatment increased locomotor behavior and affected SOD and CAT activities in the prefrontal cortex, hippocampus and striatum. Our findings suggest that amphetamine exposure is associated with an imbalance between SOD and CAT activity in the prefrontal cortex, hippocampus and striatum.     

Regional distribution and relative amounts of glutamate decarboxylase isoforms in rat and mouse brain.

The levels of the two isoforms of glutamate decarboxylase (GAD) were measured in 12 regions of adult rat brain and three regions of mouse brain by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting with an antiserum that recognizes the identical C-terminal sequence in both isoforms from both species. In rat brain the amount of smaller isoform, GAD65, was greater than that of the larger isoform, GAD67, in all twelve regions. GAD65 ranged from 77-89% of total GAD in frontal cortex, hippocampus, hypothalamus, midbrain, olfactory bulb, periaqueductal gray matter, substantia nigra, striatum, thalamus and the ventral tegmental area. The proportion of GAD65 was lower in amygdala and cerebellum but still greater than half of the total. There was a strong correlation between total GAD protein and GAD activity. In the three mouse brain regions analysed (cerebellum, cerebral cortex and hippocampus) the proportion of GAD65 (35,47, and 51% of total GAD) was significantly lower than in the corresponding rat-brain regions. The amount of GAD67 was greater than the amount of GAD65 in mouse cerebellum and was approximately equal to the amount of GAD65 in mouse cerebral cortex and hippocampus.     

Effect of repeated convulsive seizures on brain GABA levels

Repeated audiogenic seizures (4 times a day for 14 days), in genetically selected sensitive mice, induce a significant decrease in GABA level in the following brain areas: nucleus caudatus, posterior colliculus, occipital and frontal cortex, cerebellum, substantia nigra, hippocampus, amygdala, and temporal cortex. No variations were observed in olfactory bulbs, pons medulla, hypothalamus, thalamus, or cochlear area.     

Brain creatine kinase activity in an animal model of mania

There is evidence pointing to dysfunction at the mitochondrial level as an important target for the understanding of the pathophysiology of bipolar disorder (BD). We assessed creatine kinase (CK) activity in rats submitted to an animal model of mania which included the use of lithium and valproate. In the acute treatment, amphetamine (AMPH) or saline was administered to rats for 14 days, and between day 8 and 14, rats were treated with either lithium, valproate or saline. In the maintenance treatment, rats were pretreated with lithium, valproate or saline, and between day 8 and 14, AMPH or saline were administered. In both experiments, locomotor activity was assessed by open-field test and CK activity was evaluated in hippocampus, striatum, cerebellum, whole cortex and prefrontal cortex. Our results showed that mood stabilizers reversed AMPH-induced behavioral effects. Moreover, AMPH (acute treatment) inhibited CK activity in hippocampus, striatum and cortex, but not in cerebellum and prefrontal cortex, and administration of lithium or valproate did not reverse the enzyme inhibition. In the maintenance treatment, AMPH decreased CK activity in saline-pretreated rats in hippocampus, striatum and cortex, but not in cerebellum and prefrontal cortex. AMPH administration in lithium- or valproate-pretreated animals decreased CK activity in hippocampus, striatum and cortex. Our results showed that AMPH inhibited CK activity and that mood stabilizers were not able to reverse and/or prevent the enzyme inhibition. These findings reinforce the hypothesis that mitochondrial dysfunction plays an important role in the pathophysiology of BD.     

Stimulation of adenylyl cyclase and induction of brain-derived neurotrophic factor and TrkB mRNA by NKH477, a novel and potent forskolin derivative

The present study was undertaken to examine whether NKH477, a novel and potent water-soluble forskolin derivative, stimulates adenylyl cyclase and regulates brain-derived neurotrophic factor (BDNF) and TrkB expression in the rat brain. Administration of NKH477 at a dose of 1.0 mg/kg, but not 0.1 mg/kg, increased levels of cyclic AMP (cAMP) in a time-dependent manner in frontal cortex and hippocampus. Repeated administration of NKH477 (1.0 mg/kg) for 7 or 14 days also increased levels of cAMP in these two brain regions, indicating that the response does not desensitize with chronic treatment. In addition, administration of NKH477 at the 1 mg/kg dose increased the expression of BDNF and TrkB mRNA in frontal cortex and hippocampus. This effect was observed after single, as well as repeated (7 or 14 days), administration of NKH477. These results demonstrate that NKH477 administration rapidly increases cAMP levels in brain and provides evidence that stimulation of this second messenger system increases the expression of BDNF and TrkB mRNA.     

Stimulated D(1) dopamine receptors couple to multiple Galpha proteins in different brain regions

Previous studies have revealed that activation of rat striatal D(1) dopamine receptors stimulates both adenylyl cyclase and phospholipase C via G(s) and G(q), respectively. The differential distribution of these systems in brain supports the existence of distinct receptor systems. The present communication extends the study by examining other brain regions: hippocampus, amygdala, and frontal cortex. In membrane preparations of these brain regions, selective stimulation of D(1) dopamine receptors increases the hydrolysis of phosphatidylinositol/phosphatidylinositol 4,5-biphosphate. In these brain regions, D(1) dopamine receptors couple differentially to multiple Galpha protein subunits. Antisera against Galpha(q) blocks dopamine-stimulated PIP(2) hydrolysis in hippocampal and in striatal membranes. The binding of [(35)S]GTPgammaS or [alpha-(32)P]GTP to Galpha(i) was enhanced in all brain regions. Dopamine also increased the binding of [(35)S]GTPgammaS or [alpha-(32)P]GTP to Galpha(q) in these brain regions: hippocampus = amygdala > frontal cortex. However, dopamine-stimulated binding of [(35)S]GTPgammaS to Galphas only in the frontal cortex and striatum. This differential coupling profile in the brain regions was not related to a differential regional distribution of the Galpha proteins. Dopamine induced increases in GTPgammaS binding to Galpha(s) and Galpha(q) was blocked by the D(1) antagonist SCH23390 but not by D(2) receptor antagonist l-sulpiride, suggesting that D(1) dopamine receptors couple to both Galpha(s) and Galpha(q) proteins. Co-immunoprecipitation of Galpha proteins with receptor-binding sites indicate that in the frontal cortex, D(1) dopamine-binding sites are associated with both Galpha(s) and Galpha(q) and, in hippocampus or amygdala, D(1) dopamine receptors couple solely to Galpha(q). The results indicate that in addition to the D(1)/G(s)/adenylyl cyclase system, brain D(1)-like dopamine receptor sites activate phospholipase C through Galpha(q) protein.     

Decrease of Glial Fibrillary Acidic Protein in Rat Frontal Cortex Following Aluminum Treatment

Aluminum lactate was injected either intraperitoneally or stereotactically into the lateral cerebral ventricles of rats. Rats were killed at various times after treatment, and frontal cortex, hippocampus, and striatum were dissected out. Microtiter plate-based sandwich ELISA and immunohistochemistry were used to measure the glial fibrillary acidic protein (GFAP) concentration. GFAP levels were significantly decreased in frontal cortex 7 days after a single lateral ventricular injection of aluminum lactate and 14 days following systemic treatment. In contrast, neither hippocampus nor striatum exhibited any significant changes in the content of this astrocytic intermediate filament protein after aluminum treatment. Levels of a predominantly astroglial enzyme, glutamine synthetase, were also selectively reduced in the frontal cortex following intraventricular injection of aluminum. This depression exhibited a regional and temporal specificity similar to that found for GFAP. These results suggest a selective and progressive diminution of astrocytic responsivity in frontal cortex following either systemic or intraventricular aluminum dosing. The depression of GFAP levels reported here, which was found in the rat cerebral cortex 7-14 days after aluminum treatment in a species that does not form neurofilamentous aggregates, may reflect extended impairment of astrocytic function and suggests that these cells may be the primary targets of aluminum neurotoxicity.