Astrocytes repress the neuronal expression of GLAST and GLT glutamate transporters in cultured hippocampal neurons from embryonic rats

Glutamate extracellular levels are regulated by specific transporters. Five subtypes have been identified. The two major ones, GLAST and GLT (glutamate transporters 1 and 2, respectively), are localized in astroglia in normal mature brain. However, in neuron-enriched hippocampal cultures, these proteins are expressed in neurons during the early in vitro development (Plachez et al., 2000). Here, we show that, in these cultures, GLAST and GLT neuronal expression is transient and no longer observed after 7 days in vitro, a stage at which the few astrocytes present in the culture are maturing. Moreover, we demonstrate that these few astrocytes are responsible for the repression of this neuronal expression. Indeed, addition of conditioned medium prepared from primary cultures of hippocampal astrocytes, to cultured hippocampal neurons, rapidly leads to the suppression of neuronal GLAST expression, without affecting neuronal GLT expression. However, when neurons are seeded and co-cultured on a layer of hippocampal astrocytes, they do not develop any immunoreactivity towards GLAST or GLT antibodies. Altogether, these results indicate that glia modulate the expression of GLAST and GLT glutamate transporters in neurons, via at least two distinct mechanisms. Neuronal GLAST expression is likely repressed via the release or the uptake of soluble factors by glia. The repression of neuronal GLT expression probably results from glia-neuron interactions. This further reinforces the fundamental role of direct or indirect neuron-glia interactions in the development of the central nervous system.     

Early calpain-mediated proteolysis following AMPA receptor activation compromises neuronal survival in cultured hippocampal neurons

In this work, we investigated the involvement of calpains in the neurotoxicity induced by short-term exposure to kainate (KA) in non-desensitizing conditions of AMPA receptor activation (cyclothiazide present, CTZ), in cultured rat hippocampal neurons. The calpain inhibitor MDL28170 had a protective effect in cultures treated with KA plus CTZ (p < 0.01), preventing the decrease in MTT reduction caused by exposure to KA (p < 0.001). Caspase inhibition by ZVAD-fmk was not neuroprotective against the toxic effect of KA. At 1 h after treatment, we could already observe significantly increased calpain activity, which was prevented by MDL 28170 and NBQX. Western blot analysis of calpain substrates, GluR1, neuronal nitric oxide synthase (nNOS) and nonerythroid spectrin (fodrin), showed a time-dependent and MDL 28170-sensitive proteolysis of these proteins. This effect was due to calpains, but not caspases, since ZVAD-fmk was ineffective in preventing proteolytic events. Breakdown products of fodrin (BDPs) were detected as early as 15 min after exposure to KA. Overall, these results show early activation of calpains following activation of AMPA receptors as well as compromise of neuronal survival, likely due to proteolytic events that affect proteins involved in neuronal signaling.     

Long-term potentiation in cultured hippocampal neurons

Studies performed on low-density primary neuronal cultures have enabled dissection of molecular and cellular changes during N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP). Various electrophysiological and chemical induction protocols were developed for the persistent enhancement of excitatory synaptic transmission in hippocampal neuronal cultures. The characterisation of these plasticity models confirmed that they share many key properties with the LTP of CA1 neurons, extensively studied in hippocampal slices using electrophysiological techniques. For example, LTP in dissociated hippocampal neuronal cultures is also dependent on Ca(2+) influx through post-synaptic NMDA receptors, subsequent activation and autophosphorylation of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and an increase in alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor insertion at the post-synaptic membrane. The availability of models of LTP in cultured hippocampal neurons significantly facilitated the monitoring of changes in endogenous postsynaptic receptor proteins and the investigation of the associated signalling mechanisms that underlie LTP. A central feature of LTP of excitatory synapses is the recruitment of AMPA receptors at the postsynaptic site. Results from the use of cell culture-based models started to establish the mechanism by which synaptic input controls a neuron's ability to modify its synapses in LTP. This review focuses on key features of various LTP induction protocols in dissociated hippocampal neuronal cultures and the applications of these plasticity models for the investigation of activity-induced changes in native AMPA receptors.     

Comparative study of endoplasmic reticulum stress-induced neuronal death in rat cultured hippocampal and cerebellar granule neurons

In this study, experiments were performed to characterize further the pathways responsible for neuronal death induced by endoplasmic reticulum (ER) stress in cultured hippocampal neurons (HPN) and cerebellar granule neurons (CGN) using tunicamycin (TM) and amyloid beta-peptide (Abeta). Exposure of HPN to Abeta or TM resulted in a time-dependent increase in the expression of 78-kDa glucose-regulated protein (GRP78) and caspase-12, an ER-resident caspase. In contrast, in CGN, although a drastic increase in the expression of GRP78 was found as was the case in HPN, no up-regulation of caspase-12 was detected. These results were consistent with immunohistochemical results that there were far lower number of caspase-12-positive cells in the cerebellum than in the cerebral cortex and hippocampus, and that caspase-12-positive cells were not identified in the external granule cell layer of the cerebellum of P7 rats. In CGN, a significant increase in the expression of C/EBP homologous protein (CHOP) protein was detected after exposure to Abeta or TM, whereas no such an increase in the protein expression was observed in HPN. In addition, S-allyl-L-cysteine (SAC), an organosulfur compound purified from aged garlic extract, protected neurons against TM-induced neurotoxicity in HPN but not in CGN, as in the case of Abeta-induced neurotoxicity. These results suggest that the pathway responsible for neuronal death induced by Abeta and TM in HPN differs from that in CGN, and that a caspase-12-dependent pathway is involved in HPN while a CHOP-dependent pathway is involved in CGN in ER stress-induced neuronal death.     

Fast inactivating potassium current in cultured hippocampal neurons

Using a voltage-clamp whole-cell technique, we studied transmembrane currents in hippocampal neurons after their long-lasting cultivation. Based on the activational characteristics and data on pharmacological sensitivity, we isolated and described an A-type voltage-activated fast inactivating potassium current (FIPC). This transient FIPC was activated at −50… −40 mV and was rather sensitive to 4-aminopyridine (4-AP). Extracellular application of 5 mM 4-AP decreased the FIPC amplitude by 75%, while application of 10 mM tetraethylammonium evoked no significant changes in it. Kinetics of FIPC activation could be described by one exponent in the fourth degree. With variations of the membrane potential from −40 to 30 mV, the activation time constant varied from 2.8 to 1.5 msec. Inactivation kinetics was described by one exponent with the time constant varying from 37 msec at −45 mV to 18 msec at 40 mV. Stationary activation and inactivation curves could be described by Boltzmann's equation; a half value of the level of stationary inactivation was reached at −80 mV, while stationary activation was attained at −25 mV. Kinetics of deinactivation (from stationary inactivation) was monoexponential with the time constant of 41 msec. It is supposed that FIPC through the membrane of hippocampal neurons is provided by the type Kv4.2 potassium channels.     

Brain-derived neurotrophic factor protects cultured rat hippocampal neurons from aluminum maltolate neurotoxicity

Aluminum is environmentally abundant but not an essential trace element. Although there is increasing evidence suggesting the implication of aluminum in the pathogenesis of Alzheimer's disease, it is still controversial. We found and report here that aluminum maltolate, a stable and hydrophilic aluminum complex, causes death of primary cultured rat hippocampal neurons in a time- and dose-dependent manner. Degenerated neurons were TUNEL-positive. Immunohistochemical detection of synapsin I and microtubule associated protein 2 revealed the synapse loss between neurons intoxicated by aluminum maltolate. To explore the mechanism underlying its neurotoxicity, we administered various pharmacological compounds prior to the application of aluminum maltolate, and found that brain-derived neurotrophic factor (BDNF) markedly attenuated the neurotoxicity. Furthermore, aluminum maltolate inhibited the elevation of intracellular calcium levels caused by BDNF. Our results suggest the involvement of BDNF in the molecular mechanism underlying neurotoxicity induced by aluminum maltolate.     

Over-expression of antioxidant enzymes protects cultured hippocampal and cortical neurons from necrotic insults

There is now considerable knowledge concerning neuron death following necrotic insults, and it is believed that the generation of reactive oxygen species (ROS) and oxidative damage play a pivotal role in the neuron death. Prompted by this, we have generated herpes simplex virus-1 amplicon vectors over-expressing the genes for the antioxidant enzymes catalase (CAT) or glutathione peroxidase (GPX), both of which catalyze the degradation of hydrogen peroxide. Over-expression of each of these genes in primary hippocampal or cortical cultures resulted in increased enzymatic activity of the cognate protein. Moreover, each enzyme potently decreased the neurotoxicity induced by kainic acid, glutamate, sodium cyanide and oxygen/glucose deprivation. Finally, these protective effects were accompanied by parallel decreases in hydrogen peroxide accumulation and the extent of lipid peroxidation. These studies not only underline the key role played by ROS in the neurotoxicity of necrotic insults, but also suggest potential gene therapy approaches.     

Fractal analysis of a voltage-dependent potassium channel from cultured mouse hippocampal neurons.

The kinetics of ion channels have been widely modeled as a Markov process. In these models it is assumed that the channel protein has a small number of discrete conformational states and the kinetic rate constants connecting these states are constant. In the alternative fractal model the spontaneous fluctuations of the channel protein at many different time scales are represented by a kinetic rate constant k = At1-D, where A is the kinetic setpoint and D the fractal dimension. Single-channel currents were recorded at 146 mM external K+ from an inwardly rectifying, 120 pS, K+ selective, voltage-sensitive channel in cultured mouse hippocampal neurons. The kinetics of these channels were found to be statistically self-similar at different time scales as predicted by the fractal model. The fractal dimensions were approximately 2 for the closed times and approximately 1 for the open times and did not depend on voltage. For both the open and closed times the logarithm of the kinetic setpoint was found to be proportional to the applied voltage, which indicates that the gating of this channel involves the net inward movement of approximately one negative charge when this channel opens. Thus, the open and closed times and the voltage dependence of the gating of this channel are well described by the fractal model.     

Gamma-interferon promotes differentiation of cultured cortical and hippocampal neurons

Clinical and experimental evidence suggests that the development of the brain may be modulated by soluble growth factors traditionally associated with cells of the immune system. As part of an investigation into agents modulating early neural differentiation, we examined the effects of the lymphokine gamma-interferon (IFN-gamma) on the development of cultured cortical and hippocampal neurons from embryonic rats and mice. We report here that recombinant IFN-gamma, at concentrations of 0.2-10 U/ml (50-2500 pg/ml, 3-150 pM), affects the differentiation of embryonic central neurons. IFN-gamma increased the number of cells expressing neurofilament (NF) protein, the growth of primary and secondary neurites on NF-expressing somas, and the extent of cell aggregation observed in culture. IFN-gamma-induced increases in the numbers of NF-positive cells were seen in the virtual absence of differentiated astrocytes, and in mixed neuron-glia cultures. Our results thus indicate that at physiologically relevant concentrations IFN-gamma acts, either directly on neurons and their precursor cells and/or indirectly via nonneuronal cell stimulation, to promote the differentiation of immature neurons.     

缺糖缺氧对体外培养海马神经元的影响

目的：观察缺糖缺氧诱导的培养海马神经元损伤。方法：取培养12d的海马神经元,在缺糖缺氧条件下分别培养0．5～4h后取出,换原神经元培养液在常氧条件下继续培养24h。用0．4％台盼蓝染色,检测神经元坏死,并用TUNEL法检测神经元凋亡,计算存活、坏死和凋亡神经元所占百分率。同时用图像分析仪测定存活、坏死和凋亡神经元的胞体面积、周长和等园直径。结果：培养的海马神经元急性缺糖缺氧后0．5～4h,随缺糖缺氧时间的延长,坏死神经元逐渐增多,缺糖缺氧后0．5～2h再恢复糖和氧供应后24h,凋亡神经元明显增多。图像分析的结果表明,坏死神经元的胞体面积、周长和等园直径均明显大于凋亡神经元。结论：缺糖缺氧可引起海马神经元严重损伤,在急性缺糖缺氧后0．5～4h引起的神经元死亡以坏死为多见,但在缺糖缺氧后0．5～2h再恢复糖和氧供应后24,神经元死亡则以凋亡为多见。     

Distribution of single-channel conductances in cultured rat hippocampal neurons

1. The nonhomogeneous spatial distribution of ionic channels in neurons has been implied from intracellular recordings at somatic and dendritic locations. These reports indicate that Na- and Ca-dependent regenerative currents are distributed differently throughout the neuron. Although a variety of K conductances and a noninactivating Na conductance have been described in intracellular studies, little is known about the spatial distribution of inward and outward currents throughout different regions of the neuron. 2. We recorded from cell-attached patches from cultured hippocampal cells from 1-day-old rats. The cells were cultured for 3-21 days. The spatial distribution of a variety of ionic channels was determined by comparing the conductances from somatic and dendritic membranes. Single-channel currents obtained from cell-attached patches were identified by the time course of ensemble (averaged) responses, voltage dependence, and the effect of channel blocking agents. 3. We consistently observed that only the rapidly inactivating inward current was localized to the soma. The other channel types that we studied, including an inward noninactivating, delayed rectifier and transient A-type currents, were observed in both the somatic and dendritic regions. 4. We suggest that the distribution of ionic conductances that we have observed may be functional in limiting excitability during development of neurons.     

Experimental observations on the development of polarity by hippocampal neurons in culture

In culture, hippocampal neurons develop a polarized form, with a single axon and several dendrites. Transecting the axons of hippocampal neurons early in development can cause an alteration of polarity; a process that would have become a dendrite instead becomes the axon (Dotti, C. G., and G. A. Banker. 1987. Nature (Lond.). 330:254-256). To investigate this phenomenon more systematically, we transected axons at varying lengths. The greater the distance of the transection from the soma, the greater the probability for regrowth of the original axon. However, it was not the absolute length of the axonal stump that determined the response to transection, but rather its length relative to the lengths of the cell's other processes. If one process was greater than 10 microns longer than the others, it invariably became the axon regardless of its identity before transection. Conversely, when a cell's processes were nearly equal in length, it was impossible to predict which would become the axon. In these cases, axonal outgrowth began only after a long latency. During this interval, the processes appeared to be in dynamic equilibrium, some growing for short distances while others retracted. When one process exceeded the others by a critical length, it rapidly elongated to become the axon. The establishment of neuronal polarity during normal development may similarly involve an interaction among processes whose identities have not yet been determined. When, by chance, one exceeds the others by a critical length, it becomes specified as the axon.     

Early presynaptic changes during plasticity in cultured hippocampal neurons

Long-lasting increase in synaptic strength is thought to underlie learning. An explosion of data has characterized changes in postsynaptic (pstS) AMPA receptor cycling during potentiation. However, changes occurring within the presynaptic (prS) terminal remain largely unknown. We show that appearance of new release sites during potentiation between cultured hippocampal neurons is due to (a) conversion of nonrecycling sites to recycling sites, (b) formation of new releasing sites from areas containing diffuse staining for the prS marker Vesicle-Associated Membrane Protein-2 and (c) budding of new recycling sites from previously existing recycling sites. In addition, potentiation is accompanied by a release probability increase in pre-existing boutons depending upon their individual probability. These prS changes precede and regulate fluorescence increase for pstS GFP-tagged-AMPA-receptor subunit GluR1. These results suggest that potentiation involves early changes in the prS terminal including remodeling and release probability increase of pre-existing synapses.     

肾上腺髓质素降低培养海马神经元胞内游离钙离子浓度

经荧光探针Fluo 3-AM标记细胞内游离钙后,用激光共聚焦显微镜检测肾上腺髓质素(adrenomedullin,ADM)对原代培养大鼠海马神经元内游离钙浓度([Ca^2 ]1)的影响。实验结果如下：(1)ADM(0．01-1．0μmol／L)浓度依赖性地降低细胞内钙浓度。(2)降钙素基因相关肽受体阻断剂(calcitonin gene-related peptide,CGRP8-37)预处理可部分抑制ADM的效应。(3)ADM可显著抑制高钾引起的[Ca^2 ]1增加。(4)ADM可显著抑制三磷酸肌醇(inositol 1,4,5-trisphosphate,IP3)引起的内钙释放,而对兰尼定(ryanodine)引起的内钙释放无显著影响。以上结果提示,ADM降低培养海马神经元内游离钙浓度,此作用与其抑制IP,引起的内钙释放有关,ADM对静息状态下的Ca^2 内流无影响,但可显著抑制高钾引起的Ca^2 内流,CGRP受体介导了ADM的上述效应。     

Nicotine induces dendritic spine remodeling in cultured hippocampal neurons

Cholinergic neurons in the CNS are involved in synaptic plasticity and cognition. Both muscarinic and nicotinic acetylcholine receptors (nAChRs) influence plasticity and cognitive function. The mechanism underlying nAChR‐induced plasticity, however, has remained elusive. Here, we demonstrate morphological changes in dendritic spines following activation of α4β2* nAChRs, which are expressed on glutamatergic pre‐synaptic termini of cultured hippocampal neurons. Exposure of the neurons to nicotine resulted in a lateral enlargement of spine heads. This was abolished by dihydro‐β‐erythroidine, an antagonist of α4β2* nAChRs, but not by α‐bungarotoxin, an antagonist of α7 nAChRs. Tetanus toxin or a mixture of 2‐amino‐5‐phosphonovaleric acid and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, antagonists of NMDA‐ and AMPA‐type glutamate receptors, blocked the nicotine‐induced spine remodeling. In addition, nicotine exerted full spine‐enlarging response in the post‐synaptic neuron whose β2 nAChR expression was knocked down. Finally, pre‐treatment with nicotine enhanced the Ca²⁺‐response of the neurons to glutamate. These data suggest that nicotine influences the activity of glutamatergic neurotransmission through the activation of pre‐synaptic α4β2 nAChRs, resulting in the modulation of spinal architecture and responsiveness. The present findings may represent one of the cellular mechanisms underlying cholinergic tuning of brain function.

     

S-adenosyl-L-methionine protects the hippocampal CA1 neurons from the ischemic neuronal death in rat

We investigated the effect of S-adenosyl-L-methionine (SAMe) on the prevention of the delayed neuronal death in rats subjected to transient and brief forebrain ischemia. As the results, SAMe dose-dependently protected the hippocampal CA1 neurons from degeneration and necrosis, whose effect was suppressed by simultaneous administration of S-adenosyl-L-homocysteine, a potent inhibitor in transmethylation. No protective effect was observed in CDP-choline, phosphatidylcholine and L-methionine. Therefore, it is necessary for the prevention of the delayed neuronal death to enhance cerebral SAMe level and to activate transmethylation using SAMe as a methyl donor in postischemic brain.     

Regulation of neuronal polarity

The distinctive polarized morphology of neuronal cells is essential for the proper wiring of the nervous system. The rodent hippocampal neuron culture established about three decades ago has provided an amenable in vitro system to uncover the molecular mechanisms underlying neuronal polarization, a process relying on highly regulated cytoskeletal dynamics, membrane traffic and localized protein degradation. More recent research in vivo has highlighted the importance of the extracellular environment and cell–cell interactions in neuronal polarity. Here, I will review some key signaling pathways regulating neuronal polarization and provide some insights on the complexity of this process gained from in vivo studies.