Cocaine-responsive gene expression changes in rat hippocampus.

Chronic cocaine use is known to elicit changes in the pattern of gene expression within the brain. The hippocampus plays a critical role in learning and memory and may also play a role in mediating behaviors associated with cocaine abuse. To profile the gene expression response of the hippocampus to chronic cocaine treatment, cDNA hybridization arrays were used to illuminate cocaine-regulated genes in rats treated non-contingently with a binge model of cocaine (45 mg/kg/day, i.p.) for 14 days. Validation of mRNA changes illuminated by hybridization array analysis was accomplished by measuring immunoreactive protein (via specific immunoblots). The induction of protein kinase Calpha, potassium channel 1.1, and metabotropic glutamate receptor 5 seen by hybridization arrays was confirmed at the level of protein. Immunoblot screening of previously described cocaine-responsive genes demonstrated increased levels of protein tyrosine kinase 2, beta-catenin, and protein kinase Cepsilon. While some of these changes exist in previously described cocaine-responsive models, others are novel to any model of cocaine use. The inductions of potassium channel 1.1, protein tyrosine kinase 2 and metabotropic glutamate receptor 5 are novel findings to hippocampal cocaine-responsive gene expression. These proteins have been shown to subserve learning and memory and/or long-term potentiation functions within the hippocampus. Additionally, these genes are known to interact with one another, forming a more complex pattern of gene expression changes.The findings suggest altered expression of genes with a number of different functions in the rat hippocampus after a 'binge' style of non-contingent cocaine administration. These changes in gene expression may play roles in neuronal plasticity and the behavioral phenomena associated with cocaine abuse.     

脂代谢相关三基因突变小鼠肝组织基因表达差异研究

目的研究3个脂代谢相关基因联合突变小鼠与野生型小鼠肝脏基因表达差异及其与血脂代谢紊乱和动脉粥样硬化病变的关系。方法应用BiostarM-40S型小鼠cDNA表达谱芯片检测不同基因型小鼠肝脏基因表达的差异，并观察不同年龄小鼠血浆总胆固醇和甘油三脂的浓度以及主动脉内膜形态变化。结果在被测的4000条基因中，与野生型小鼠比较，5周龄三基因突变小鼠肝脏基因表达上调92条，下调105条，脂代谢相关基因中与胆固醇合成相关的基因表达下调，与甘油三脂代谢相关的肝脂肪酶基因表达水平上调。糖代谢、细胞骨架蛋白和免疫等相关的基因表达也有明显差异。5周龄的三基因突变小鼠血浆总胆固醇和甘油三脂水平明显高于野生型小鼠，伴有主动脉内膜损伤，并随年龄增长而加重。结论三基因突变导致肝脏中与脂类、糖类以及免疫等相关的多种基因表达改变，可能共同参与了血脂代谢紊乱和动脉粥样硬化的发生发展。     

Traumatic brain injury induces adipokine gene expression in rat brain

Traumatic brain injury (TBI) induces cachexia and neuroinflammation which profoundly impact patient recovery. Adipokine genes such as leptin (ob), resistin (rstn) and fasting-induced adipose factor (fiaf) are implicated in energy metabolism and body weight control and are also associated with chronic low grade inflammation. Since central rstn and fiaf expression was increased following hypoxic/ischemic brain injury, we hypothesized that these genes would also be induced in the rat brain following TBI. Realtime RT-PCR detected a 2-2.5-fold increase in ob mRNA in the ipsilateral cortex and thalamus 12h following lateral fluid percussion (FP)-induced brain injury. Fiaf mRNA was elevated 5-7.5-fold in cortex, hippocampus and thalamus, and modest increases were also detectable in the contralateral brain. Remarkably, rstn mRNA was elevated in ipsilateral (150-fold) and in contralateral (50-fold) hippocampus. To test whether these changes were part of an inflammatory response to TBI we also examined the effects of an intracerebral injection of lipopolysaccharide (LPS). We determined that central injection of LPS produced some, but not all, of the changes seen after TBI. For example, in contrast to the stimulatory influence of TBI, LPS had no effect on ob expression in any brain region, though fiaf and rstn mRNA levels were significantly elevated in both ipsi- and contralateral cortex. In conclusion: (a) brain-derived adipokines could be involved in the acute pathology of traumatic brain injury partly through modulation of central inflammatory responses, but also via leptin-mediated neuroprotective effects and (b) TBI-induced brain adipokines may induce the metabolic changes observed following neurotrauma.     

Up-regulation of murine double minute clone 2 (MDM2) gene expression in rat brain after morphine, heroin, and cocaine administrations

Repeated administration of addictive drugs induces neuronal apoptosis and the underlying mechanisms are not clear. Our present study investigated the effects of treatments with different addictive drugs on gene expression of murine double minute clone 2 (MDM2), a key negative regulator of p53 and an important mediator in cell apoptosis. The level of MDM2 gene expression in rat brain was assessed using in situ hybridization histochemistry. In normal adult rat brain, MDM2 expression was at a very low level but MDM2 mRNA-positive cells were detected in various regions including cortex, hippocampus, thalamus, amygdala, periaqueductal gray and locus ceruleus. After a single morphine injection, MDM2 gene expression increased significantly in hippocampus, amygdala and cortex; however, such up-regulation of MDM2 gene expression was significantly reduced after repeated morphine administration. Moreover, 24 h after cessation of chronic morphine exposure, MDM2 mRNA increased again to a level comparable to that of the acute morphine group. Acute heroin or cocaine administration also significantly increased MDM2 gene expression in hippocampus, but not in cortex. In thalamus, no change was detected after acute or chronic treatment with morphine, heroin, or cocaine. Thus we demonstrated for the first time that the administration of addictive drugs regulate MDM2 gene expression in distinct rat brain regions and these data suggest that MDM2 may play an important role in the development of drug addiction.     

Chronic morphine treatment and withdrawal induce up-regulation of c-Jun N-terminal kinase 3 gene expression in rat brain

Chronic opiate applications produce long-term impacts on many functions of the brain and induce tolerance, dependence, and addiction. It has been demonstrated that opioid drugs are capable to induce apoptosis of neuronal cells, but the mechanism is not clear. c-Jun N-terminal kinase 3 (JNK3), specifically expressed in brain, has been proved to mediate neuronal apoptosis and is involved in opiate-induced cell apoptosis in vitro. The present study investigated the effect of opioid administration on expression of JNK3, an important mediator involved in apoptosis of neurons, in rat brain. Our results showed that single or chronic injection of morphine resulted in a 45-50% increase in the level of JNK3 mRNA in frontal cortex, while no significant change was detected in other brain regions such as thalamus, hippocampus and locus coeruleus. Similar to what was observed after the acute or chronic morphine administration, no significant change in JNK3 expression was detected in locus coeruleus following cessation of the chronic morphine administration. However, interestingly, sustained elevation of JNK3 expression peaked on day 14 after cessation of morphine treatment was observed in the brain regions such as hippocampus and thalamus, where acute or chronic morphine treatment did not cause any significant change in JNK3 gene expression. The increased JNK3 mRNA in these brain areas returned to the control levels in 28 days following cessation of chronic morphine treatment. Taken together, these results demonstrated for the first time that the expression of JNK3 gene is regulated by opioids and that chronic opioid administration and withdrawal could induce sustained elevation of JNK3 mRNA in many important brain areas. The changes in JNK3 gene expression in brain induced by chronic opioid treatment may play a role in opioid-induced apoptosis and neurotoxicity.     

Region specific gene expression profile in mouse brain after chronic corticotropin releasing factor receptor 1 activation: the novel role for diazepam binding inhibitor in contextual fear conditioning

We have previously reported that repeated central administration of sub-anxiogenic doses of the corticotropin releasing factor 1 (CRF₁) agonist Cortagine, termed “priming,” elicits a phenotype of increased anxiety-like behaviors in the elevated plus maze (EPM) and open-field test, and enhanced retention of contextual conditioned fear in C57BL/6J mice. Observed behavioral changes were functionally coupled to CRF₁-mediated elevated central cholecystokinin (CCK) tone in discrete brain regions. However, the changes in gene expression that mediated “priming”-induced behavioral and concurrent molecular changes in specific brain regions remained unknown. In the present study, a complementary DNA microarray analysis was used to investigate gene expression profiles in the hippocampus and prefrontal cortex (PFC) of C57BL/6J mice following the “priming” procedure. Here, we report that chronic stimulation of CRF₁, by i.c.v. administration of 10 ng Cortagine for five days, brought about alterations in the expression of a wide range of hippocampal (31 genes) and PFC (18 genes) genes, implicated in anxiety and aversive memory formation. These expression changes involved genes associated with signal transduction, neurotransmitter secretion, synaptic transmission, myelination, and others involved in the transport, biosynthesis, and binding of proteins. In particular, several genes of the protein kinase A (PKA) and protein kinase C (PKC) signaling cascades, known to be involved in synaptic plasticity, such as neurogranin, calmodulin 3, and the PKA regulatory subunit 1 b were found to be upregulated in the PFC and hippocampus of CRF₁ agonist “primed” mice. Moreover, we show pharmacologically that one of the newly implicated memory regulatory elements, diazepam-binding inhibitor (DBI) is functionally involved in hippocampus-dependent enhancement of contextual fear, a cardinal phenotypic feature of the “primed” mice. Finally, an interaction network mapping of the altered genes and their known interacting partners identified additional molecular candidates responsible for CRF₁-mediated hypersensitive fear circuitry.     

Activity-regulated cytoskeleton-associated protein in rodent brain is down-regulated by high fat diet in vivo and by 27-hydroxycholesterol in vitro

Growing evidence strongly suggests that high fat diet (HFD) has an important role in some neurodegenerative disorders, including Alzheimer's disease (AD). To identify new cellular pathways linking hypercholesterolemia and neurodegeneration, we analyzed the effects of HFD on gene expression in mouse brain. Using cDNA microarrays and real time RT-PCR, we found that HFD has a mild, but significant effect on the expression of several genes. The altered genes include molecules linked to AD pathology and others of potential interest for neurodegeneration. We further investigated the effect of HFD on the activity-regulated cytoskeleton-associated protein (Arc). Expression of Arc was decreased in cerebral cortex and hippocampus of HFD-fed animals. From the known regulatory mechanisms of Arc expression, HFD reduced N-methyl-D-aspartate receptor (NMDAR) activity, as seen by decreases in tyrosine phosphorylation of NMDAR2A and levels of NMDAR1. Additionally, we demonstrated that 27-hydroxycholesterol, a cholesterol metabolite that enters the brain from the blood, decreases Arc levels as well as NMDAR and Src kinase activities in rat primary hippocampal neurons. Finally, we showed that Arc levels are decreased in the cortex of AD brains. We propose that one of the mechanisms, by which hypercholesterolemia contributes to neurodegenerative diseases, could be through Arc down-regulation caused by 27-hydroxycholesterol.     

甲基苯丙胺对相关脑区的神经毒性作用

目的:研究甲基苯丙胺(MA)对大鼠纹状体、海马、额叶皮质等脑区神经细胞的毒性作用以及对大鼠行为的影响。方法:H-E染色、Glees银浸染观察神经元和轴突的变化;高效液相色谱检测上述脑区多巴胺(DA)及其代谢产物含量;免疫组化胶质纤维酸性蛋白(GFAP)检测胶质细胞增生情况。结果:MA对上述脑区神经细胞和轴突有损伤作用,表现为神经细胞变圆,极性消失;胶质细胞增生,噬神经细胞现象、胶质小结形成;神经轴索扭曲,节段性增粗,轴索间隙增宽;GFAP阳性星形细胞增多;纹状体DA及其代谢产物含量显著降低,海马、额叶皮质DA含量明显降低;大鼠行为改变明显。结论:MA对大鼠中枢神经系统多脑区神经元有明显的毒性作用,可导致上述脑区DA含量下降和大鼠行为改变。     

基因表达谱芯片筛选局灶性脑缺血大鼠脑相关基因的研究

目的:应用基因芯片技术研究局灶性脑缺血大鼠脑组织与假手术组大鼠脑组织基因表达谱的差异,探索局灶性脑缺血的发病机制。方法:将4096种大鼠基因PCR产物用CartesianPixsys7500点样仪按微矩阵排列制成基因芯片;按一步法抽提脑局灶性缺血大鼠脑组织与假手术组大鼠脑组织的总RNA;将等量的脑局灶性缺血大鼠脑组织与假手术组大鼠脑组织RNA分别逆转录合成以Cy5和Cy3标记的cDNA一链做探针,混合后与上述基因芯片杂交。用AxonGenepix4000B扫描仪扫描芯片荧光信号图像,用GenepixPro4.0软件进行数字化处理和分析后比较两种组织基因表达谱的差异。结果:局灶性脑缺血大鼠与假手术组大鼠脑组织基因表达谱分析,发现211个差异表达基因,其中12个基因低表达,199个基因高表达。结论:本研究从脑局灶性缺血大鼠脑组织中筛选出大量的差异表达基因,说明这些基因可能参与局灶性脑缺血后脑损伤的发生及发展过程,从而为局灶性脑缺血的诊治提供新思路。     

Expression of the glucocorticoid-induced receptor mRNA in rat brain

The glucocorticoid-induced receptor (GIR) is an orphan G-protein-coupled receptor awaiting pharmacological characterization. GIR was originally identified in murine thymoma cells, and shows a widespread, yet not completely complementary distribution in mouse and human brain. Expression of the mouse GIR gene is modulated by dexamethasone in the brain and periphery, suggesting that GIR function is directly responsive to glucocorticoid signals. The rat GIR was cloned from rat prefrontal cortex by our group and was shown to be up-regulated following chronic amphetamine. The physiological role of GIR in the rat is not known at present. In order to gain a clearer understanding of the potential functions of GIR in the rat, we performed a detailed mapping of GIR mRNA expression in the rat brain. GIR mRNA showed widespread distribution in forebrain limbic and thalamic structures, and a more restricted distribution in hindbrain areas such as the spinal trigeminal nucleus and the median raphe nucleus. Areas with moderate to high levels of GIR include olfactory regions such as the nucleus of olfactory tract, hippocampus, various thalamic nuclei, cortical layers, and some hypothalamic nuclei. In comparison with previous studies, significant regional differences exist in GIR distribution in mouse and rat brain, particularly in the thalamus, striatum and in hippocampus at a cellular level. Overall, the expression of GIR in rat brain more closely approaches that seen previously in human than mouse, suggesting that rat models may be more informative for understanding the role of GIR in glucocorticoid physiology and glucocorticoid-related disease states. GIR mRNA distribution in the rat indicates a potential role of this receptor in the control of feeding and ingestive behavior, regulation of stress and emotional behavior, learning and memory, and, drug reinforcement and reward.     

Antidepressant drug treatment induces Arc gene expression in the rat brain

The mechanism underlying the therapeutic effect of antidepressants is not known but neuroadaptive processes akin to long-term potentiation have been postulated. Arc (Activity-regulated, cytoskeletal-associated protein) is an effector immediate early gene implicated in LTP and other forms of neuroplasticity. Recent data show that Arc expression is regulated by brain 5-hydroxytryptamine neurones, a target of many antidepressants. Here in situ hybridisation and immunohistochemistry were used to examine whether Arc expression in rat brain is altered by antidepressant drug treatment. Repeated administration of the monoamine reuptake inhibitors paroxetine, venlafaxine or desipramine induced region-specific increases in Arc mRNA. These increases were greatest in regions of the cortex (frontal and parietal cortex) and hippocampus (CA1 layer) and absent in the caudate putamen. Repeated treatment with the monoamine oxidase inhibitor, tranylcypromine, increased Arc mRNA in a similar fashion to the monoamine reuptake inhibitors. The antidepressant drugs also increased the number of Arc-immunoreactive cells in the parietal cortex. Acute antidepressant injection, and repeated administration of the antipsychotic drug chlorpromazine, produced either limited or no changes in Arc mRNA. The data suggest that chronic treatment with antidepressant drugs induces Arc gene expression in specific regions across the rat forebrain. Up-regulation of Arc expression may be part of the process by which antidepressant drugs achieve long-term changes in synaptic function in the brain.     

Differential alterations of GABAA receptor (α1, β2, γ2 subunit) expression and increased seizure susceptibility in rat offspring from morphine-addicted mothers: Beneficial effect of dextromethorphan

Although prenatal morphine exposure experimentally induces seizures in rat offspring, underlying mechanisms remain unclear. This study addresses whether prenatal morphine exposure altered subunit compositions of γ-aminobutyric acid receptor subtype A (GABA_AR) in the hippocampal CA1 area and temporal cortex and increased seizure susceptibility of young rat offspring, at a representative age (postneonatal days 14; P14). Therapeutic efficacy of dextromethorphan (a noncompetitive antagonist of N-methyl-d-aspartate receptors (NMDARs)), in such offspring was also evaluated. From P7 to 14, Sprague–Dawley rat offspring were intraperitoneally (ip) injected a representative dose of dextromethorphan (3 mg/kg) twice a day. At P14, some offspring were ip injected pentylenetetrazol to estimate seizure susceptibility, while the others were studied for GABA_AR subunit (α1, β2, γ2) expression. Prenatal morphine exposure caused the up-regulated α1 subunit and down-regulated β2/γ2 subunit expression of GABA_AR within hippocampus and temporal cortex in rat offspring associated to increase seizure susceptibility. The magnitudes of upregulated α1 subunit and downregulated β2 subunit expression in the hippocampus were greater than which in the temporal cortex. The use of dextromethorphan markedly reversed the prenatal morphine-induced alterations, indicating the possible therapeutic actions of dextromethorphan. These results suggest that the altered subunit compositions (α1, β2, γ2) of GABA_AR in the hippocampal CA1 area and temporal cortex may contribute, at least in part, to the increased seizure susceptibility of rat offspring subjected to prenatal morphine exposure. More importantly, dextromethorphan may be a promising clinical agent acting against these alterations.     

c－fos基因在听源性惊厥易感大鼠脑的表达──原位杂交组织化学法研究

运用原位杂交技术，以遗传性听源性惊厥易感大鼠Ｐ７７ＰＭＣ为对象，发现听源性惊厥可诱导大鼠脑内ｃ－ｆｏｓ基因快速、大量、短暂性表达。ｃ－ｆｏｓｍＲＮＡ分布于大脑皮层、梨状皮层、杏仁复合体、海马齿状回、上丘脑、背侧丘脑、下丘脑部分核团、下丘、蜗神经核、蓝斑及小脑等处。惊厥后皮层下结构中ｃ－ｆｏｓ基因表达变化程度超过皮层的变化，尤其是下丘、蜗神经核与惊厥时程有明显关系。推测皮层下结构对听源性惊厥的发生有重要意义。Ｐ＜０．０１讨论本文结果说明听源性惊厥同其它因素诱导的惊厥一样［３］，可诱导大鼠脑内ｃ－ｆｏｓ基因的表达，表达涉及到大脑皮层、海马齿状回、丘脑、下丘、蜗神经核等结构，其中以皮层下结构如丘脑、下丘、蜗神经核表达变化最显著。原位杂交显示的ｃ－ｆｏｓ基因表达特征类似于Ｎｏｒｔｈｅｒｎ杂交结果即快速、大量和短暂性。由于不同部位在惊厥活动中的作用差别，因此用原位杂交可以显示每一结构内ｃ－ｆｏｓ基因表达特点。如在惊厥后３０ｍｉｎ，海马齿状回中７０％以上的神经元单位胞质面积上银粒计数超过２０个，而梨状皮层及运动皮层仅占５～１３．８％。有报告指出海马齿状回为钙离子通道和ＮＭＤＡ受体高密度区域［４］，推测Ｃａ２＋和ＮＭＤＡ?     

Microarray analysis of VEGF-responsive genes in myometrial endothelial cells

There is evidence that the vasculature of different organs display different functional characteristics in response to cytokines and growth factors. The aim of this study was to use cDNA gene expression microarray to analyse changes in gene expression following stimulation of myometrial microvascular endothelial cells (MMECs) with vascular endothelial growth factor (VEGF). Primary isolates of MMECs were obtained from fresh hysterectomy specimens and purified with magnetic beads. Cells were stimulated with 15 ng/ml VEGF for 3, 6 and 12 h, and two unstimulated experiments served as controls. A total of six arrays was performed over these time-points. A total of 110 genes were identified as up-regulated by VEGF, 19% of which (21 genes) have previously been reported as up-regulated by VEGF or by angiogenesis. Among the novel genes to be up-regulated by VEGF were brain-derived growth factor, oxytocin receptor and estrogen sulphotransferase. The significance of the genes identified in the physiological and pathological functioning of the myometrial vasculature is discussed.     

Gene expression profiles of transcripts in amyloid precursor protein transgenic mice: up-regulation of mitochondrial metabolism and apoptotic genes is an early cellular change in Alzheimer's disease

Alzheimer's disease (AD) is a progressive neurodegenerativedisease characterized by the impairment of cognitive functionsand by beta amyloid (Aß) plaques in the cerebral cortexand the hippocampus. Our objective was to determine genes thatare critical for cellular changes in AD progression, with particularemphasis on changes early in disease progression. We investigatedan established amyloid precursor protein (APP) transgenic mousemodel (the Tg2576 mouse model) for gene expression profilesat three stages of disease progression: long before (2 monthsof age), immediately before (5 months) and after (18 months)the appearance of Aß plaques. Using cDNA microarraytechniques, we measured mRNA levels in 11 283 cDNA clonesfrom the cerebral cortex of Tg2576 mice and age-matched wild-type(WT) mice at each of the three time points. This gene expressionanalysis revealed that the genes related to mitochondrial energymetabolism and apoptosis were up-regulated in 2-month-old Tg2576mice and that the same genes were up-regulated at 5 and 18 monthsof age. These microarray results were confirmed using northernblot analysis. Results from in situ hybridization of mitochondrialgenes—ATPase-6, heat-shock protein 86 and programmed celldeath gene 8—suggest that the granule cells of the hippocampaldentate gyrus and the pyramidal neurons in the hippocampus andthe cerebral cortex are up-regulated in Tg2576 mice comparedwith WT mice. Results from double-labeling in situ hybridizationsuggest that in Tg2576 mice only selective, over-expressed neuronswith the mitochondrial gene ATPase-6 undergo oxidative damage.These results, therefore, suggest that mitochondrial energymetabolism is impaired by the expression of mutant APP and/orAß, and that the up-regulation of mitochondrial genesis a compensatory response. These findings have important implicationsfor understanding the mechanism of Aß toxicity inAD and for developing therapeutic strategies for AD. ^* To whom correspondence should be addressed at: Neurogenetics Laboratory, Neurological Sciences Institute, Oregon Health and Science University, 505 NW 185th Avenue, Beaverton, OR 97006, USA. Tel: +1 5034182625; Fax: +1 5034182501; Email: reddyh{at}ohsu.edu     

Nonketotic hyperglycinemia: Biochemical, molecular, and neurological aspects

Summary Nonketotic hyperglycinemia (NKH) is a metabolic disorder with autosomal recessive inheritance, causing severe, frequently lethal, neurological symptoms in the neonatal period. The metabolic lesion of NKH is in the glycine cleavage system (GCS), a complex enzyme system with four enzyme components; P-, T-, H-, and L-protein. The enzymatic analysis revealed that 86% of the patients with NKH are deficient of P-protein activity. The cDNA clones encoding all four components were isolated and their primary structures were determined. Several mutations have been identified in P- and T-protein genes: One missense mutation, S564I, in P-protein gene accounts for 70% of the mutant alleles in Finland where the incidence of NKH is unusually high. The immunochemical andin situ hybridization analyses revealed that the strong GCS expression was observed in rat hippocampus, olfactory bulbus, and cerebellum. The distribution resembled that ofN-methyl-d-aspartic acid (NMDA) receptor which has binding site for glycine. It is, therefore, suggested that the neurological disturbance in NKH may be caused by excitoneurotoxicity through the NMDA receptor allosterically activated by high concentration of glycine. Based on the hypothesis the NMDA antagonists such as ketamine and dextromethorphan were administered to the patients. We treated three neonatal case with dextromethorphan and it ameliorated their findings on electroencephalogram and behavior in two out of three patients. Thus the GCS is suggested to play a role in regulation of glycine level around the NMDA receptor.