Neurotoxicity Induced by Glutamate in Glucose-Deprived Rat Hippocampal Slices is Prevented by GMP

Guanosine-5-monophosphate (GMP) was evaluated as a neuroprotective agent against the damage induced by glutamate in rat hippocampal slices submitted to glucose deprivation. In slices maintained under physiological conditions, glutamate (0.01 to 10 mM), Kainate, alpha-amino-3-hydroxi-5-methylisoxazole-propionic acid (AMPA), N-methyl-D-aspartate (NMDA), 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD), or L-2-amino-4-phosphonobutanoic acid (L-AP4) (100 M) did not alter cell membrane permeability, as evaluated by lactate dehydrogenase (LDH) release assay. In slices submitted to glucose deprivation, GMP (from 0.5 mM) prevented LDH leakage and the loss of cell viability induced by 10 mM glutamate. LDH leakage induced by Kainate, AMPA, NMDA or 1S,3R-ACPD was fully prevented by 1 mM GMP. However, glutamate uptake was not altered in slices submitted to glucose deprivation and glutamate analogues. Glucose deprivation induced a significant decrease in ATP levels which was unchanged by addition of glutamate or GMP. Our results show that glucose deprivation decreases the energetic charge of cells, making hippocampal slices more susceptible to excitotoxicity and point to GMP as a neuroprotective agent acting as a glutamatergic antagonist.     

Suppression of protein kinase Cepsilon mediates 17beta-estradiol-induced neuroprotection in an immortalized hippocampal cell line

Although estrogens are neuroprotective in a variety of neuroprotection models, the precise underlying mechanisms are currently not well understood. Here, we examined the role of protein kinase C (PKC) in mediating estrogen-induced neuroprotection in the HT-22 immortalized hippocampal cell line. The neuroprotection model utilized calcein fluorescence to quantitate cell viability following glutamate insults. 17beta-Estradiol (betaE2) protected HT-22 cells when treatment was initiated before or after the glutamate insult. The inhibition of PKC by bis-indolylmaleimide mimicked and enhanced betaE2-induced neuroprotection. In contrast, the inhibition of specific PKC isozymes (alpha and beta) by Go6976, inhibition of 1-phosphatidylinositol 3 kinase by wortmannin, or inhibition of protein kinase A by H-89, did not alter cell viability, suggesting a specific involvement of PKC in an isozyme-dependent manner. We further examined whether estrogen interacts with PKC in a PKC isozyme-specific manner. Protein levels and activity of PKC isozymes (alpha, delta, epsilon, and zeta) were assessed by western blot analysis and radiolabeled phosphorylation assays respectively. Among the isozymes tested, betaE2 altered only PKCepsilon; it reduced the activity and membrane translocation of PKCepsilon in a manner that correlated with its protection against glutamate toxicity. Furthermore, betaE2 reversed the increased activity of membrane PKCepsilon induced by glutamate. These data suggest that the neuroprotective effects of estrogens are mediated in part by inhibition of PKCepsilon activity and membrane translocation.     

Disruption of postsynaptic GABA receptor clusters leads to decreased GABAergic innervation of pyramidal neurons

We have used RNA interference (RNAi) to knock down the expression of the gamma2 subunit of the GABA(A) receptors (GABA(A)Rs) in pyramidal neurons in culture and in the intact brain. Two hairpin small interference RNAs (shRNAs) for the gamma2 subunit, one targeting the coding region and the other one the 3'-untranslated region (UTR) of the gamma2 mRNA, when introduced into cultured rat hippocampal pyramidal neurons, efficiently inhibited the synthesis of the GABA(A) receptor gamma2 subunit and the clustering of other GABA(A)R subunits and gephyrin in these cells. More significantly, this effect was accompanied by a reduction of the GABAergic innervation that these neurons received. In contrast, the gamma2 shRNAs had no effect on the clustering of postsynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, postsynaptic density protein 95 (PSD-95) or presynaptic glutamatergic innervation. A gamma2-enhanced green fluorescent protein (EGFP) subunit construct, whose mRNA did not contain the 3'-UTR targeted by gamma2 RNAi, rescued both the postsynaptic clustering of GABA(A)Rs and the GABAergic innervation. Decreased GABA(A)R clustering and GABAergic innervation of pyramidal neurons in the post-natal rat cerebral cortex was also observed after in utero transfection of these neurons with the gamma2 shRNAs. The results indicate that the postsynaptic clustering of GABA(A)Rs in pyramidal neurons is involved in the stabilization of the presynaptic GABAergic contacts.     

TGF-beta2 neutralization inhibits proliferation and activates apoptosis of cerebellar granule cell precurors in the developing cerebellum

Transforming growth factor beta 2 (TGF-beta2) plays a critical role in growth, differentiation and cell death, but its function in the developing cerebellum is still uncertain. In this study we analyzed the effects of TGF-beta2 on ex vivo developing cerebellar slice cultures. Proliferation of granule cell precursors peaked ex vivo in the same developmental window as in vivo (P8-P14). Addition of recombinant TGF-beta2 could extent the proliferation of granule cell precursors and induced a second late proliferation wave. In contrast, antibody neutralization of TGF-beta2 strongly reduced proliferation and induced neurodegeneration. TGF-beta2 neutralization resulted in apoptotic cells, which showed caspase 3 activation. Taken together our results demonstrate that TGF-beta2 is a novel growth and survival factor for granule cells precursors in the developing cerebellum.     

Detection of hypoxia-evoked ATP release from chemoreceptor cells of the rat carotid body

The carotid body (CB) is a chemosensory organ that detects changes in chemical composition of arterial blood and maintains homeostasis via reflex control of ventilation. Thus, in response to a fall in arterial PO(2) (hypoxia), CB chemoreceptors (type I cells) depolarize, and release neurotransmitters onto afferent sensory nerve endings. Recent studies implicate ATP as a key excitatory neurotransmitter released during CB chemoexcitation, but direct evidence is lacking. Here we use the luciferin-luciferase bioluminescence assay to detect ATP, released from rat chemoreceptors in CB cultures, fresh tissue slices, and whole CB. Hypoxia evoked an increase in extracellular ATP, that was inhibited by L-type Ca(2+)channel blockers and reduced by the nucleoside hydrolase, apyrase. Additionally, iberiotoxin (IbTX; 100 nM), a blocker of O(2)-sensitive Ca(2+)-dependent K(+) (BK) channels, stimulated ATP release and largely occluded the effect of hypoxia. These data strongly support a neurotransmitter role for ATP in carotid body function.     

印度胶树茎、叶制片新尝试

对于印度胶榕这种特殊类的材料制片,在取材、抽气和染色等过程中,常出现抽气不净和染色不均匀及乳汁脱不掉等问题,影响观察效果。从操作程序上进行了改进,取得了明显的效果,对于特殊植物材料制片有一定的参考价值。     

不同训练方式建立大鼠空间记忆后海马结构NMDA受体表达的变化

短期强化训练能否建立可靠的空间长时记忆？用不同训练方式建立空间记忆后,大鼠海马结构NMDA受体的表达发生怎样的变化？目前尚未见明确报道。本研究应用Morris水迷宫方法分别采用以下模式对大鼠进行训练：空间长时记忆训练模式（LT组）、空间短时记忆训练模式（ST组）以及短期强化训练模式（SRT组）,对不同训练模式建立的空间记忆进行了比较,应用免疫荧光组织化学方法检测各组大鼠海马结构NMDA／NR1受体表达的变化。结果表明,Morris水迷宫训练过程中,LT和SRT组大鼠寻找站台的半均潜伏期和策略均无显著性差异：记忆检测发现,除LT组大鼠在站台所在象限的停留时间明显长于SRT组大鼠外,两组大鼠寻找站台的潜伏期和策略以及穿越站台的次数均无显著性差异。ST组大鼠海马结构NMDA／NR1的免疫反应强度与对照组相比,无显著差异。但是,LT和SRT组大鼠海马CA1区锥体细胞联及齿状回的颗粒细胞层NMDA／NR1免疫荧光反应都明显增强,两组之间比较无显著差异,但是两组分别与对照组和ST组相比均有显著性差异。上述结果提示,短期强化训练可建立与长期训练基本相同的空间长时记忆。大鼠海马结构CA1区和齿状回NMDA受体表达的增加,可能是空间长时记忆形成的机制之一。     

Implication of apoE isoforms in cholesterol metabolism by primary rat hippocampal neurons and astrocytes

Apolipoprotein E (apoE) has been genetically linked to late-onset Alzheimer's disease. From the three common alleles (epsilon2, epsilon3 and epsilon4), epsilon4 has been suggested to promote amyloid beta (Ass) plaque fibrillation, one hallmark of Alzheimer's disease. It has been demonstrated that altered lipid content of hippocampal plasma membrane coincides with the disease. In this study, we show for the first time that the apoE dependent cholesterol metabolism in hippocampal neurons is higher than that of hippocampal astrocytes. Further, apoE-bound cholesterol is highly incorporated in membranous compartments in hippocampal neurons, whereas hippocampal astrocytes show higher intracellular distribution. This is an effect that coincides with cell-type dependent difference of low density lipoprotein receptor (LDLR) family member expression. Hippocampal neurons express high levels of the LDLR related protein (LRP), whereas hippocampal astrocytes are highly positive for LDLR. We could also demonstrate an apoE isoform (apoE2, apoE3 and apoE4) dependent cholesterol uptake in both cells types. In hippocampal neurons, we could find a decreased apoE4-bound cholesterol uptake. In contrast, hippocampal astrocytes show decreased internalization of apoE2-bound cholesterol. In addition, lipidated apoE4 is little associated with neurites in hippocampal neurons in comparison to the other two isoforms. In contrary, hippocampal astrocytes show faint apoE2 immunocytostaining intensity. Data presented indicate that the role of apoE4 in cholesterol homeostasis and apolipoprotein cell association is more pronounced in hippocampal neurons, showing significant alterations compared to the other two isoforms, suggesting that hippocampal neurons are affected by apoE4 associated altered cholesterol metabolism compared to hippocampal astrocytes.     

Dietary supplementation with acetyl-l-carnitine in seizure treatment of pentylenetetrazole kindled mice

In spite of the availability of new antiepileptic drugs a considerable number of epilepsy patients still have pharmacoresistant seizures, and thus there is a need for novel approaches. Acetyl-l-carnitine (ALCAR), which delivers acetyl units to mitochondria for acetyl-CoA production, has been shown to improve brain energy homeostasis and protects against various neurotoxic insults. To our knowledge, this is the first study of ALCAR's effect on metabolism in pentylenetetrazole (PTZ) kindled mice. ALCAR or the commonly used antiepileptic drug valproate, was added to the drinking water of mice for 25days, and animals were injected with PTZ or saline three times a week during the last 21days. In order to investigate ALCAR's effects on glucose metabolism, mice were injected with [1-(13)C]glucose 15min prior to microwave fixation. Brain extracts from cortex and the hippocampal formation (HF) were studied using (1)H and (13)C NMR spectroscopy and HPLC. PTZ kindling caused glucose hypometabolism, evidenced by a reduction in both glycolysis and TCA cycle turnover in both brain regions investigated. Glutamatergic and GABAergic neurons were affected in cortex and HF, but the amount of glutamate was only reduced in HF. Slight astrocytic involvement could be detected in the cortex. Interestingly, the dopamine content was increased in the HF. ALCAR attenuated the PTZ induced reduction in [3-(13)C]alanine and the increase in dopamine in the HF. However, TCA cycle metabolism was not different from that seen in PTZ kindled animals. In conclusion, even though ALCAR did not delay the kindling process, it did show some promising ameliorative effects, worthy of further investigation.