Temporal profiles of synaptic plasticity-related signals in adult mouse hippocampus with methotrexate treatment

Methotrexate, which is used to treat many malignancies and autoimmune diseases, affects brain functions including hippocampal-dependent memory function. However, the precise mechanisms underlying methotrexate-induced hippocampal dysfunction are poorly understood. To evaluate temporal changes in synaptic plasticity-related signals, the expression and activity of N-methyl-D-aspartic acid receptor 1, calcium/calmodulin-dependent protein kinase II, extracellular signal-regulated kinase 1/2, cAMP responsive element-binding protein, glutamate receptor 1, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor were examined in the hippocampi of adult C57BL/6 mice after methotrexate (40 mg/kg) intraperitoneal injection. Western blot analysis showed biphasic changes in synaptic plasticity-related signals in adult hippocampi following methotrexate treatment. N-methyl-D-aspartic acid receptor 1, calcium/calmodulin-dependent protein kinase II, and glutamate receptor 1 were acutely activated during the early phase (1 day post-injection), while extracellular signal-regulated kinase 1/2 and cAMP responsive element-binding protein activation showed biphasic increases during the early (1 day post-injection) and late phases (7-14 days post-injection). Brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor expression increased significantly during the late phase (7-14 days post-injection). Therefore, methotrexate treatment affects synaptic plasticity-related signals in the adult mouse hippocampus, suggesting that changes in synaptic plasticity-related signals may be associated with neuronal survival and plasticity-related cellular remodeling.     

Chronic inhibition of Ca(2+)/calmodulin kinase II activity in the pilocarpine model of epilepsy

The development of symptomatic epilepsy is a model of long-term plasticity changes in the central nervous system. The rat pilocarpine model of epilepsy was utilized to study persistent alterations in calcium/calmodulin-dependent kinase II (CaM kinase II) activity associated with epileptogenesis. CaM kinase II-dependent substrate phosphorylation and autophosphorylation were significantly inhibited for up to 6 weeks following epileptogenesis in both the cortex and hippocampus, but not in the cerebellum. The net decrease in CaM kinase II autophosphorylation and substrate phosphorylation was shown to be due to decreased kinase activity and not due to increased phosphatase activity. The inhibition in CaM kinase II activity and the development of epilepsy were blocked by pretreating seizure rats with MK-801 indicating that the long-lasting decrease in CaM kinase II activity was dependent on N-methyl-D-aspartate receptor activation. In addition, the inhibition of CaM kinase II activity was associated in time and regional localization with the development of spontaneous recurrent seizure activity. The decrease in enzyme activity was not attributed to a decrease in the alpha or beta kinase subunit protein expression level. Thus, the significant inhibition of the enzyme occurred without changes in kinase protein expression, suggesting a long-lasting, post-translational modification of the enzyme. This is the first published report of a persistent, post-translational alteration of CaM kinase II activity in a model of epilepsy characterized by spontaneous recurrent seizure activity.     

难治性颞叶癫痫患者脑组织脑源性神经营养因子表达明显高于正常对照者

脑源性神经营养因子在癫痫中的作用一直备受争议,现有的研究多为动物实验研究结果,本文采用光镜及电镜技术,观察24例难治性颞叶癫痫患者致痫灶组织病理及超微结构变化,结果显示颞叶癫痫患者致痫灶神经元变性,胶质细胞增生,细胞核空泡化,可见嗜神经细胞现象,免疫电镜及免疫组化显示难治性颞叶癫痫患者脑组织脑源性神经营养因子表达明显多于正常对照者,证实难治性癫痫患者致痫灶病理改变明显,且与脑源性神经营养因子表达变化有关。     

G蛋白偶联内向整流钾通道亚基2 mRNA在颞叶癫疒间大鼠海马内的表达

目的探讨G蛋白偶联内向整流钾通道亚基2(GIRK 2)在颞叶癫癎大鼠海马内的表达变化.方法应用腹腔注射海人酸致癎大鼠,采用原位杂交法检测大鼠海马GIRK 2 mRNA的表达.结果 GIRK 2 mRNA在癫癎大鼠海马齿状回表达增加,与正常对照组相比差异有显著性( P<0.01).结论癫癎大鼠海马内GIRK2增高是机体对神经元网络过度兴奋的代偿反应.     

Localization of CAM II Kinase-α, GAD, GluR2 and GABAA Receptor Subunit mRNAs in the Human Entorhinal Cortex

The human entorhinal cortex (ERC) is an important relay between neocortical association areas and the hippocampus. Pathology in this area, including disturbances in its unique cytoarchitecture and alterations in neurotransmitter receptor binding, has been implicated in several neuropsychiatric disorders but details of the patterns of gene expression for molecules involved in the major neurotransmitter systems in this cortex have been lacking. We used in situ hybridization histochemistry to localize the mRNAs for several proteins which are involved in excitatory and inhibitory neurotransmission in the human ERC. Labelling of mRNA for a glutamate receptor subunit (GluR2) and for a marker of glutamatergic cortical neurons (alpha type II calcium/calmodulin-dependent protein kinase) were distributed in a laminar manner which matched the cellular packing seen on the Nissl sections, with particularly high levels of labelling in the layer II (pre-á) cell clusters characteristic of this cortex. Cells labelled for the mRNA of 67 kDa glutamic acid decarboxylase, the synthesizing enzyme of GABA, were distributed diffusely throughout all layers, not concentrated in the cell clusters, and were present in higher numbers in layer Ill. The labelling of mRNAs for the α1, β2 and γ2 subunits of the GABA_A receptor, however, was distributed in a laminar pattern similar to that for GluR2 and CAM II kinase mRNAs, implying a high concentration of inhibitory synapses on the excitatory cells which express these mRNAs.     

Effect of propofol on brain-derived neurotrophic factor and tyrosine kinase receptor B in the hippocampus of aged rats with chronic cerebral ischemia

We intraperitoneally injected 10 and 50 mg/kg of propofol for 7 consecutive days to treat a rat model of chronic cerebral ischemia. A low-dose of propofol promoted the expression of brain-derived neurotrophic factor, tyrosine kinase receptor B, phosphorylated cAMP response element binding protein, and cAMP in the hippocampus of aged rats with chronic cerebral ischemia, but a high-dose of propofol inhibited their expression. Results indicated that the protective effect of propofol against cerebral ischemia in aged rats is related to changes in the expression of brain-derived neurotrophic factor and tyrosine kinase receptor B in the hippocampus, and that the cAMP-cAMP responsive element binding protein pathway is involved in the regulatory effect of propofol on brain-derived neurotrophic factor expression.     

颞叶癫痈大鼠海马内Rnd1 mRNA及蛋白表达变化

目的：动态观察小分子GTPase Rho家族的Rnd1 mRNA及其蛋白在氯化锂-毛果芸香碱（匹罗卡品）致痫大鼠模型海马中的表达变化，探讨其在颞叶癫痫发生发展中的作用。方法：在氯化锂-毛果芸香碱颞叶癫痫模型中应用逆转录聚合酶链反应（RT—PCR）检测癫痫持续发作（SE）后各时间点海马内Rnd1 mRNA的表达变化，并运用免疫组织化学染色法及Neo—Timm染色法分别检测齿状回门区、CA1区及CA3区中该蛋白在不同时间点的表达变化及苔藓纤维出芽（MFS）情况。结果：实验发现模型组于SE后8h内即出现Rnd1表达上调，SE后约1d达高峰，7d左右回复至对照组水平，此后其mRNA表达水平与对照组相似；而免疫组化染色发现Rnd1蛋白表达从SE后8h内即开始上调，约3d达高峰，至7d虽略有回落，但仍高于对照组水平，且这种情况可一直持续至慢性期。结论：急性期海马齿状回门区Rnd1表达上调可能通过促进MFS的发生参与了颞叶癫痫的发生。     

Mutant mice lacking ryanodine receptor type 3 exhibit deficits of contextual fear conditioning and activation of calcium/calmodulin-dependent protein kinase II in the hippocampus

As it is known that ryanodine receptor type 3 is expressed in the hippocampus, we examined the contribution of this receptor to contextual fear conditioning behavior and to the activation of Ca(2+)/calmodulin-dependent protein kinase II using mice lacking the receptor. Ryanodine receptor type 3-deficient mice exhibited impairments of performance in the contextual fear conditioning test, passive avoidance test, and Y-maze learning test. Both the activities of Ca(2+)/calmodulin-dependent protein kinase IIbeta and Ca(2+)/calmodulin-dependent protein kinase IIalpha were significantly increased in the experimental group compared to the control group in the hippocampus, but not in the cingulate cortex on the testing day 24 h after contextual fear training. However, the activities of Ca(2+)/calmodulin-dependent protein kinase IIbeta and alpha were almost the same in the experimental and control groups in the hippocampus on the training day. Ryanodine receptor type 3-deficient mice did not show the increment of Ca(2+)/calmodulin-dependent protein kinase IIbeta and alpha activities in the hippocampus on the testing day. In addition, these mutant mice showed the reduction of fear response in the elevated plus-maze test. The present results suggest that calcium-induced calcium release through the activation of ryanodine receptor type 3 in the hippocampus is important to the expression of the performance of contextual learning through the elevation of Ca(2+)/calmodulin-dependent protein kinase IIbeta and alpha activities.     

Long-lasting decreases of type II calmodulin kinase expression in kindled rat brains.

The kindling model of epilepsy is associated with long-lasting changes in type II calmodulin kinase (CaM kinase) activity and immunoreactivity. In order to determine the mechanism of these alterations, we measured gene expression of CaM kinase using in situ hybridization in septally kindled rat brains and paired controls using a 35S-labeled riboprobe for the beta subunit of the enzyme. We found CaM kinase mRNA concentrated in the hippocampus and other limbic structures. Kindling decreased hippocampal CaM kinase mRNA by 30% in CA1, 34% in CA2, 35% in CA3 41% in CA4, and 29% in the dentate gyrus. Hybridization was also decreased by 21% in the cerebral cortex but not in the lateral septum. These changes are similar in distribution and direction to those previously measured by immunohistochemistry. These data suggest that altered CaM kinase activity and immunoreactivity associated with kindling reflect long-lasting alterations in gene expression of this important synaptic protein, and provide further evidence for its possible importance in the kindling phenomenon.     

Effects of lateral ventricular transplantation of bone marrow-derived mesenchymal stem cells modified with brain-derived neurotrophic factor gene on cognition in a rat model of Alzheimer's disease

In the present study, transplantation of bone marrow-derived mesenchymal stem cells modified with brain-derived neurotrophic factor gene into the lateral ventricle of a rat model of Alzheimer’s disease, resulted in significant attenuation of nerve cell damage in the hippocampal CA1 region. Furthermore, brain-derived neurotrophic factor and tyrosine kinase B mRNA and protein levels were significantly increased, and learning and memory were significantly improved. Results indicate that transplantation of bone marrow-derived mesenchymal stem cells modified with brain-derived neurotrophic factor gene can significantly improve cognitive function in a rat model of Alzheimer’s disease, possibly by increasing the levels of brain-derived neurotrophic factor and tyrosine kinase B in the hippocampus.     

Gene expression profiling following chronic NMDA receptor blockade-induced learning deficits in rats

Acute treatments with MK-801, a noncompetitive antagonist of the NMDA glutamate receptor, induce spatial memory deficits in rodents. In the present study, we developed a low-dose chronic MK-801 treatment regimen that induced persistent learning deficits (determined by the Morris water maze task) after administration of the drug (0.2 mg/kg) every 12 h for 14 days. To determine the impact of such a treatment, changes in mRNA expression were investigated in the hippocampi and striata of treated animals using a cDNA membrane array followed by Western blots. Genes whose expression levels were found to be most altered included preprolactin (downregulated) and mitogen-activated protein kinase (MAP kinase 1; upregulated) in the hippocampus, and acyl-CoA synthetase (downregulated) and apolipoprotein D (upregulated) in the striatum. Furthermore, MAP kinase 1 and proteosome subunit beta precursor was found to meet selection criteria for upregulation in both the hippocampus and striatum. Among other genes found to be most changed in the hippocampus were protein kinase C beta I and II, protein tyrosine phosphatase 1beta, neuropilin I and II, adenosine receptor A1, and metabotropic glutamate receptor 2/3. The impact of some gene expression alterations on their corresponding protein levels was studied next. In the hippocampus, protein kinase C beta I and II, protein tyrosine phosphatase, neuropilin I and II, adenosine receptor A, metabotropic glutamate receptor 2/3, and in the striatum phosphatidyl inositol 4 kinase, mitogen-activated protein kinase 1, adenylyl cyclase II, dopamine receptors 1A and 2, and cytochrome C oxidase subunit Va gene and protein expression levels were found to be highly correlated. These results suggest the potential involvement of several genes and proteins in the neuropharmacological effects of MK-801 and possibly the persisting cognitive deficits induced by this repeated drug treatment.     

Hippocampal N-methyl-D-aspartate receptor subunit mRNA levels in temporal lobe epilepsy patients.

Changes in the subunit stoichiometry of the N-methyl-D-aspartate (NMDA) receptor (NMDAR) alters its channel properties, and may enhance or reduce neuronal excitability in temporal lobe epilepsy patients. This study determined whether hippocampal NMDA receptor subunit mRNA levels were increased or decreased in temporal lobe epilepsy patients compared with nonseizure autopsy cases. Hippocampal sclerosis (HS; n = 16), non-HS (n = 10), and autopsy hippocampi (n = 9) were studied for NMDAR1 (NR1) and NR2A-D mRNA levels by using semiquantitative in situ hybridization techniques, along with neuron densities. Compared with autopsy hippocampi, non-HS and HS patients showed increased NR2A and NR2B hybridization densities per dentate granule cell. Furthermore, non-HS hippocampi showed increased NR1 and NR2B mRNA levels per CA2/3 pyramidal neuron compared with autopsy cases. HS patients, by contrast, showed decreased NR2A hybridization densities per CA2/3 pyramidal neuron compared with non-HS and autopsy cases. These findings indicate that chronic temporal lobe seizures are associated with differential changes in hippocampal NR1 and NR2A-D hybridization densities that vary by subfield and clinical-pathological category. In temporal lobe epilepsy patients, these findings support the hypothesis that in dentate granule cells NMDA receptors are increased, and excitatory postsynaptic potentials should be strongly NMDA mediated compared with nonseizure autopsies. HS patients, by comparison, showed decreased pyramidal neuron NR2A mRNA levels, and this suggests that NMDA-mediated pyramidal neuron responses should be reduced in HS patients compared with non-HS cases.     

Antidepressant effect of electroacupuncture regulates signal targeting in the brain and increases brain-derived neurotrophic factor levels

Electroacupuncture improves depressive behavior faster and with fewer adverse effects than antidepressant medication. However, the antidepressant mechanism of electroacupuncture remains poorly understood. Here, we established a rat model of chronic unpredicted mild stress, and then treated these rats with electroacupuncture at Yintang(EX-HN3) and Baihui(DU20) with sparse waves at 2 Hz and 0.6 m A for 30 minutes, once a day. We found increased horizontal and vertical activity, and decreased immobility time, at 2 and 4 weeks after treatment. Moreover, levels of neurotransmitters(5-hydroxytryptamine, glutamate, and γ-aminobutyric acid) and protein levels of brain-derived neurotrophic factor and brain-derived neurotrophic factor-related proteins(Trk B, protein kinase A, and phosphorylation of cyclic adenosine monophosphate response element binding protein) were increased in the hippocampus. Similarly, protein kinase A and Trk B m RNA levels were increased, and calcium-calmodulin-dependent protein kinase II levels decreased. These findings suggest that electroacupuncture increases phosphorylation of cyclic adenosine monophosphate response element binding protein and brain-derived neurotrophic factor levels by regulating multiple targets in the cyclic adenosine monophosphate response element binding protein signaling pathway, thereby promoting nerve regeneration, and exerting an antidepressive effect.     

Neurotrophin expression in the hippocampus and cerebellum is affected by chronic placental insufficiency in the late gestational ovine fetus

Our aim was to determine the effects of chronic placental insufficiency (CPI) during late gestation on the expression of neurotrophic factors and their receptors in the hippocampus and cerebellum in the near-term fetus. Structural alterations were also assessed in these brain regions. CPI was induced in eight fetal sheep by umbilicoplacental embolization (UPE) from 120 to 140 days of gestation (term approximately 147d) such that fetal arterial O2 saturation (SaO2) was maintained at approximately 50% of pre-UPE values. Five non-UPE fetuses served as controls. UPE resulted in fetal hypoxemia, hypoglycaemia, and growth restriction. In hippocampi from UPE fetuses, there were reductions in the optical density (OD) of the immunoreactivity (IR) of brain-derived neurotrophic factor (BDNF) protein within the mossy fibre collaterals of the polymorphic layer and in stratum lucidum (p<0.05); there was no consistent effect on tyrosine-related kinase (Trk) B receptor or neurotrophin-3 (NT-3) expression. Within the cerebellum, there was an increase in BDNF-IR (p<0.05) in the molecular layer; however, Trk B-IR and NT-3-IR were unaltered. There were no significant alterations to the structural parameters measured in the hippocampus. We conclude that CPI in late gestation affects the expression of BDNF in the fetal hippocampus and cerebellum, but these changes do not have a well-defined relationship to structural outcome.     

Mu opioid receptor mRNA expression, binding, and functional coupling to G-proteins in human epileptic hippocampus

Mu opioid receptors (MOR) are known to be involved in seizure activity. The main goal of the present study was to characterize the MOR mRNA expression, binding, as well as G protein activation mediated by these receptors in epileptic hippocampus of patients with pharmacoresistant mesial temporal lobe epilepsy (TLE). In contrast with autopsy samples, hippocampus obtained from patients with mesial TLE demonstrated enhanced MOR mRNA expression (116%). Saturation binding experiments revealed significantly higher (60%) B(max) values for the mesial TLE group, whereas the K(d) values were not statistically different. Although mesial TLE group demonstrated high levels of basal binding for the G proteins (136%), DAMGO-stimulated [(35)S]GTPγS binding did not demonstrate significant alterations. In conclusion, our present data provide strong evidence that the epileptic hippocampus of patients with pharmacoresistant mesial TLE presents significant alterations in MOR. Such changes may represent adaptive mechanisms to compensate for other as yet unknown alterations.     

Decreased prefrontal CaMKII alpha mRNA in bipolar illness

Ca2+/calmodulin-dependent protein kinase II (CaMKII) plays critical roles in neurotransmission, synaptic plasticity, learning and memory. The aim of this study was to examine, by in situ hybridization, prefrontal cortical expression of CaMKII alpha mRNA in postmortem brains of unipolar, bipolar, schizophrenic, and control subjects. Compared to controls, bipolar patients had significantly lower levels of CaMKII alpha mRNA in laminae I-VI of Brodmann's area 9 and laminae I-III and VI of area 46. Unipolar patients also exhibited significantly lower levels of CaMKII alpha mRNA in laminae I-IV of area 9 than did controls. The significant decrease in CaMKII alpha mRNA in bipolar patients could be associated with some of the affective and cognitive alterations that have been linked to prefrontal cortical dysfunction in bipolar disorder, although this requires further direct examination.