Kinetic and pharmacological properties of high- and low-threshold calcium channels in primary cultures of rat hippocampal neurons

The kinetic, permeability and pharmacological properties of Ca currents were investigated in primary cultures of rat hippocampal neurons. The low-voltage-activated (LVA) Ca current turned on positive to –60mV and fully inactivated in a voltage-dependent way. This current was depressed by nickel (Ni, 40 M) and amiloride (500 M) and was insensitive to -conotoxin (-CgTx) (4 M) and to the Ca agonist Bay K 8644 (5 M). The high-voltage-activated (HVA) Ca current turned on positive to –40 mV and inactivated slowly and incompletely. This current was much less sensitive than the LVA current to Ni and amiloride but more sensitive to cadmium. CgTx blocked only partially this current (about 50%) in an irreversible way. Bay K 8644 had a clear agonistic action almost exclusively on the -CgTx-resistant HVA current component. The present results suggest that the HVA channels, quite homogeneous for their kinetic properties and sensitivity to holding potentials, can be pharmacologically separated in two classes: (i) -CgTx-sensitive and Bay-K-8644-insensitive (-S/BK-I) and (ii) -CgTx-insensitive and Bay-K-8644-sensitive (-I/BK-S), the latter displaying a stronger Cadependent inactivation.     

Agonist-induced fluctuations in cytoplasmic calcium in primary cultures of bovine endothelial cells.

Endothelial cells play an important role in the vascular responsiveness to many stimuli by releasing locally active agents. The intracellular signal which links the external stimulus to the release of the active compounds is almost certainly an elevation in cytoplasmic calcium (Cai2+). Thus a detailed knowledge of Cai2+ regulation is central to an understanding of the physiology and pharmacology of endothelial cells. The present experiments, on single bovine aortic endothelial cells, demonstrate that agonists stimulate complex changes in Cai2+. These include rapid and regular fluctuations in Cai2+ which are different from the oscillations reported in other endothelial cells and non-excitable cells. The fluctuations are completely abolished in media containing low calcium, 2 mM-cobalt or caffeine but are not affected if the cells are bathed in isotonic potassium solutions. The hypothesis is put forward that the fluctuations in Cai2+ are associated with localized influxes of calcium and are possibly involved with the recycling of calcium between the internal stores, the cytoplasm and the external medium.     

Pharmacological characterization of voltage-dependent calcium currents in rat hippocampal neurons.

The kinetics and pharmacology of voltage-dependent calcium (Ca) currents in primary cultures of hippocampal neurons were studied using the whole cell clamp technique. The low voltage-activated (LVA) Ca current was activated at -50 mV and completely inactivated within 100 ms. This current was insensitive to omega-conotoxin (omega-CgTx) and to the calcium agonist Bay K 8644. The high-voltage-activated (HVA) Ca current was activated at -20 mV and inactivated incompletely during pulses of 200 ms duration. The snail toxin omega-CgTx revealed two pharmacological components of the HVA Ca current, one irreversibly blocked and the other insensitive to the toxin. Bay K 8644 had a clear agonistic action mainly on the omega-CgTx insensitive component of the HVA Ca current.     

皮质酮快速升高大鼠脊髓背角神经元钙浓度

目的:观察皮质酮(CORT)对培养的脊髓背角神经元Ca2+浓度([Ca2+]i)的调节作用及机制。方法:培养新生SD大鼠脊髓背角神经元,激光共聚焦显微镜检测神经元[Ca2+]i的变化。结果:CORT可快速升高培养的脊髓背角神经元[Ca2+]i,且呈现剂量依赖性(P0.05);CORT诱导的神经元[Ca2+]i升高是以外钙内流为主(P0.01);百日咳毒素(G蛋白活化阻断剂)可阻断CORT所致的脊髓背角神经元[Ca2+]i升高(P0.01),而糖皮质激素受体拮抗剂RU38486对CORT的效应无抑制作用。结论:CORT通过非基因组途径快速增高培养的脊髓背角神经元[Ca2+]i。     

The glycine site modulates NMDA-mediated changes of intracellular free calcium in cultures of hippocampal neurons

Recent evidence indicates that the N-methyl-D-aspartate (NMDA) receptor-channel complex contains a glycine subunit whose activation may be necessary for channel operation. It has been previously shown that stimulation of the NMDA receptor leads to an increase in intracellular ionic Ca2+ [( Ca2+]i); therefore, we examined the role of the NMDA receptor-associated glycine site in modulating [Ca2+]i using the fluorescent dye Fura II in hippocampal neuron cultures. A 3-s pulse of 200 microM NMDA resulted in a mean [Ca2+]i increase of 363 nM above the average resting concentration of 122 nM. Perfusion of the glycine site antagonist 7-chlorokynurenate (Cl-Kyn) essentially eliminated the NMDA-induced alteration in [Ca2+]i. Either 40 microM glycine or 50 microM D-serine completely reversed the effect of Cl-Kyn, indicating that the drug was acting at the glycine site. The NMDA receptor antagonists 2-amino-5-phosphonovalerate (AP5) and ketamine, which bind to the glutamate recognition site and the ion channel, respectively, also blocked the NMDA-mediated [Ca2+]i response; however, glycine or D-serine did not reverse this effect. These data show that the glycine binding site coupled to the NMDA receptor modulates the NMDA-mediated increase in [Ca2+]i. Antagonists of the glycine site provide a new tool to investigate and possibly control neuroplasticity and neurotoxicity related to the NMDA receptor complex.     

Beta-amyloid-induced activation of caspase-3 in primary cultures of rat neurons

It is known that beta-amyloid peptide (Abeta) contributes to the neurodegeneration in Alzheimer's disease (AD) and operates through activation of an apoptotic pathway. Apoptotic signal is driven by a family of cysteine proteases called caspases. The beta-amyloid precursor protein (APP) is directly and efficiently cleaved by caspases during apoptosis, resulting in elevated beta-amyloid peptide formation. Cerebellar neurons from rat pups were treated with the aged Abeta(25-35) at 1 and 5 microM and fluorescence assays of caspase activity performed over 4 days. We observed an increase in caspase activity after 48 h treatment in both 1 and 5 microM treated cells, then (72-96 h) caspase activity decreased to control values. The data presented support the hypothesis that Abeta(25-35)-induced apoptosis is mediated by the activation of Caspase-3 and that this is a transient effect.     

Enrichment of unipolar brush cell-like neurons in primary rat cerebellar cultures

Unipolar brush cells are a distinct class of excitatory interneurons situated in the granular layer of the cerebellar cortex, where they form giant synapses with individual mossy fiber terminals. We have previously shown that primary cerebellar cell cultures from embryonic and postnatal rodents contain neurons displaying morphological and chemical phenotypes characteristic of unipolar brush cells in situ, including intense staining with calretinin antiserum. In cultures from both embryonic and postnatal rats, the large majority of calretinin-positive neurons are unipolar brush cells, while granule cells are usually calretinin-negative. A small percentage of putative Golgi/Lugaro cells also express calretinin. We demonstrate here that the developmental stage of the source tissue, the concentration of potassium in the medium, and treatment with glutamate after differentiation have substantial effects on the density of putative unipolar brush cells in the cultures. In dissociated cultures obtained from embryos at gestation day E18 and E20 and from pups at postnatal day P0, P2, P5, P8, and P10 grown in 25 mM KCl, the percentage calretinin-positive cells progressively decreases from 24% to 0.1% of total cells. In cultures from E20 embryos grown in physiological potassium (5 mM KCl), calretinin-positive cells are enriched to approximately 60% of total cells, while the majority of calretinin-negative cells die. In embryonic cultures exposed to high concentrations of glutamate after 12 days in vitro, calretinin-positive neurons have a survival advantage over calretinin-negative cells and represent up to 83% of total cells.     

Aging is associated with elevated intracellular calcium levels and altered calcium homeostatic mechanisms in hippocampal neurons

Aging is associated with increased vulnerability to neurodegenerative conditions such as Parkinson's and Alzheimer's disease and greater neuronal deficits after stroke and epilepsy. Emerging studies have implicated increased levels of intracellular calcium ([Ca²⁺]_i) for the neuronal loss associated with aging related disorders. Recent evidence demonstrates increased expression of voltage gated Ca²⁺ channel proteins and associated Ca²⁺ currents with aging. However, a direct comparison of [Ca²⁺]_i levels and Ca²⁺ homeostatic mechanisms in hippocampal neurons acutely isolated from young and mid-age adult animals has not been performed. In this study, Fura-2 was used to determine [Ca²⁺]_i levels in CA1 hippocampal neurons acutely isolated from young (4–5 months) and mid-age (12–16 months) Sprague–Dawley rats. Our data provide the first direct demonstration that mid-age neurons in comparison to young neurons manifest significant elevations in basal [Ca²⁺]_i levels. Upon glutamate stimulation and a subsequent [Ca²⁺]_i load, mid-age neurons took longer to remove the excess [Ca²⁺]_i in comparison to young neurons, providing direct evidence that altered Ca²⁺ homeostasis may be present in animals at significantly younger ages than those that are commonly considered aged (≥24 months). These alterations in Ca²⁺ dynamics may render aging neurons more vulnerable to neuronal death following stroke, seizures or head trauma. Elucidating the functionality of Ca²⁺ homeostatic mechanisms may offer an understanding of the increased neuronal loss that occurs with aging, and allow for the development of novel therapeutic agents targeted towards decreasing [Ca²⁺]_i levels thereby restoring the systems that maintain normal Ca²⁺ homeostasis in aged neurons.     

Characterization of GABAergic neurons in hippocampal cell cultures

Summary The morphological characteristics of GABAergic neurons and the distribution of GABAergic synaptic terminals were examined in cultures of hippocampal neurons from 4–35 daysin vitro. Neurons expressing GABA immunoreactivity represented about 6% of the total number of cultured neurons at all time points. Although the morphological characteristics of GABAergic cells suggested a heterogeneous population, GABAergic cells as a class were notably different from the non-GABAergic, presumably pyramidal cells. Most GABAergic cells had more fusiform or polygonal shaped somata, non-spiny and less tapering dendrites and appeared more phase-dense than nonGABAergic cells. Quantitative analysis revealed that GABAergic cells had fewer primary dendrites, more elongated dendritic arbors, and longer dendritic segments than non-GABAergic neurons-characteristics that are similar to GABAergic cellsin situ. Double immunostaining revealed that GAD65-positive varicosities were also immunopositive for synapsin I, suggesting that GAD65-positive varicosities that contacted somata and dendrites represented presynaptic specializations. Confocal microscopy revealed the proportion of the synaptic specializations on the cell soma that were GAD65-positive was greater than on the dendrites, suggesting that somata and dendrites differ in their ability to induce the formation of presynaptic specializations by GABAergic axons. These data indicate that the GABAergic cells that develop in culture exhibit distinctive morphological characteristics and participate in different synaptic interactions than nonGABA cells. Thus many of the features that distinguish GABAergic neurons in culture are reminiscent of the characteristics that distinguish GABAergic neuronsin situ.     

Thioredoxin-1 expression levels in rat hippocampal neurons in moderate hypobaric hypoxia

Previous studies have demonstrated that preconditioning (PC) with three sessions of moderate hypoxia significantly increases the expression of the antioxidant protein thioredoxin-1 (Trx-1) in the rat hippocampus by 3 h after subsequent acute severe hypoxia as compared with non-preconditioned animals. However, it remained unclear whether this increase in Trx-1 accumulation during PC is induced before severe hypoxia or is a modification of the response to severe hypoxia. This question was addressed in the present investigation using experiments on 12 adult male Wistar rats with studies of Trx-1 expression after PC without subsequent severe hypoxia. Immunocytochemical studies were performed 3 and 24 h after three episodes of moderate hypobaric hypoxia (three sessions of 2 h at 360 mmHg with 24-h intervals). Immunoreactivity to Trx-1 24 h after the last session was significantly decreased in neurons in all the areas of the hippocampus studied (CA1, CA2, CA3, and the dentate gyrus). Immunoreactivity in CA3 was also decreased 3 h after hypoxia. These results provide evidence that moderate preconditioning hypoxia itself not only does not increase, but even significantly decreases Trx-1 expression. Thus, increases in Trx-1 contents in the hippocampus of preconditioned animals after severe hypoxia are not associated with the accumulation of this protein during PC, but with a PC-induced modification of the reaction to severe hypoxia. Translated from Morfologiya, Vol. 133, No. 1, pp. 20–24, January–February, 2008.     

The APP670/671 mutation alters calcium signaling and response to hyperosmotic stress in rat primary hippocampal neurons

Altered calcium homeostasis is implicated in the pathogenesis of Alzheimer's disease and much effort has been put into understanding the association between the autosomal dominant gene mutations causative of this devastating disease and perturbed calcium signaling. We have focused our attention on the effect of the APP670/671 mutation on spontaneous calcium oscillations in embryonic hippocampal neurons derived from the tg6590 transgenic rat. Intracellular free calcium levels were imaged by confocal microscopy using the fluorescent dye fluo-3AM. Hyperosmotic shrinkage, which can occur in a variety of pathophysiological conditions, has been shown to induce multiple cellular responses, including activation of volume-regulatory ion transport, cytoskeletal reorganization, and cell death. When exposed to hyperosmotic stress (addition of 50 mM sucrose) the frequency of calcium oscillations was suppressed to an equal extent in both wild-type and transgenic cultures, but the transgenic neurons, in contrast to the wild-type neurons, responded with a significantly higher increase in the amplitude of oscillations. A decrease in cell viability was observed by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay in neurons exposed to the hyperosmotic medium for 30 h. Whereas this loss in cell viability was comparable in both sets of cultures, the amplitude of calcium oscillations in transgenic neurons exhibited a significantly greater decrease in the presence of the L-type calcium channel antagonist, nimodipine. These results suggest that APP670/671 transgenic neurons have impaired calcium homeostasis.     

Impaired calcium homeostasis in aged hippocampal neurons

Development of neurodegenerative diseases such as Alzheimer's and Parkinson's disease is strongly age-associated. The impairment of calcium homeostasis is considered to be a key pathological event leading to neuronal dysfunction and cell death. However, the exact impact of aging on calcium homeostasis in neurons remains largely unknown. In the present work we have investigated intracellular calcium levels in cultured primary hippocampal neurons from young (2 months) and aged (24 months) rat brains. Upon stimulation with glutamate or hydrogen peroxide aged neurons in comparison to young neurons demonstrated an increased vulnerability to these disease-related toxins. Measurement of calpain activity using Western blot analysis showed a significant increase in basal activity of calpains in aged neurons. The observed increase of calpain activity was correlated with elevated protein levels of μ-calpain. Ca²⁺-imaging experiments performed on living individual neurons using the dye calcium green demonstrated a twofold increase in intracellular calcium concentration in aged neurons as compared to young neurons. The observed changes of intracellular calcium in aged neurons might play a role in their increased vulnerability to neurodegeneration.     

Oestrogen effects on kainate-induced toxicity in primary cultures of rat cortical neurons

Oestrogens protect neurons against excitatory amino acid-induced toxicity; however data on their interaction with particular subtype of glutamate receptors are sparse. Therefore in the present study we investigated oestrogen effects on kainate neurotoxicity in primary cortical neurons. The data showed that both oestradiol-17 beta and oestrone (100 nM and 200 nM) reduced kainate toxicity by ca. 40%. Since tamoxifen only partly inhibited the above effects, we suggest that both genomic and non-genomic mechanisms are involved in the anti-kainate action of oestrogens.     

红藻氨酸对大鼠海马锥体细胞钙电流的影响

目的：应用膜片钳技术,观察红藻氨酸（KA）对大鼠海马锥体细胞ca2＋电流的影响,以研究癫痫的发病机制。方法：采用酶加机械分离法制备出生10～12d的大鼠海马锥体神经元标本,用全细胞膜片钳技术测定其生理学特性及观察KA对ca2＋电流的影响。结果：分离出的海马锥体细胞形态正常,有较长突起;用膜片钳技术证实,其保存了主要的离子通道活性。KA20μmol／L和100μmol／L的浓度均可使海马锥体细胞ca2＋电流峰值增大（n=8,P〈0．01）。结论：①大鼠海马锥体神经细胞具有明显的突起,细胞膜表面光洁、晕光好,适用于膜片钳实验研究;②KA使ca2＋内流增加,引起“Ca”超载”导致细胞毒性等一系列反应;③KA通过激活α-氨基羟甲基恶唑丙酸（AMPA）受体,诱发快速的兴奋性突触后电位（EPSP）,参与兴奋性突触传递。AMPA受体的激活可能是癫痫的发病机制之一。     

Endocannabinoid signalling triggered by NMDA receptor-mediated calcium entry into rat hippocampal neurons

Endocannabinoids are released from neurons in activity-dependent manners, act retrogradely on presynaptic CB₁ cannabinoid receptors, and induce short-term or long-term suppression of transmitter release. The endocannabinoid release is triggered by postsynaptic activation of voltage-gated Ca²⁺ channels and/or G_q-coupled receptors such as group I metabotropic glutamate receptors (I-mGluRs) and M₁/M₃ muscarinic receptors. However, the roles of NMDA receptors, which provide another pathway for Ca²⁺ entry into neurons, in endocannabinoid signalling have been poorly understood. In the present study, we investigated the possible contribution of NMDA receptors in endocannabinoid production by recording IPSCs in cultured hippocampal neurons. Under the conditions minimizing the activation of voltage-gated Ca²⁺ channels, local application of NMDA (200 μ m ) transiently suppressed cannabinoid-sensitive IPSCs, but not cannabinoid-insensitive IPSCs. This NMDA-induced suppression was abolished by blocking NMDA receptors, CB₁ receptors and diacylglycerol lipase, but not by inhibiting voltage-gated Ca²⁺ channels. When the postsynaptic neuron was dialysed with 30 m m BAPTA, the NMDA-induced suppression was reduced significantly. A lower dose of NMDA (20 μ m ) exerted little effect when applied alone, but markedly enhanced the cannabinoid-dependent suppression driven by muscarinic receptors or I-mGluRs. These data clearly indicate that the activation of NMDA receptors facilitates the endocannabinoid release either alone or in concert with the G_q-coupled receptors.     

Non-NMDA receptor mediates cytoplasmic Ca2+ elevation in cultured hippocampal neurones

Microscopic fluorometry of cultured rat hippocampal neurones revealed that the intracellular concentration of Ca2+ ([Ca2+]i) rises in response to the activation of excitatory amino acid receptor (EAA-R), which included not only N-methyl-D-aspartate subspecies but also kainate and quisqualate subspecies of EAA-R. Each EAA-R mediated [Ca2+]i rise consisted of the components dependent on and independent of the activity of the voltage-dependent Ca2+ channel. The receptor-mediated voltage-independent [Ca2+]i rise may be related to the modulation of synaptic transmission efficacy.     

Glutamate dependence of GABA levels in neurons of hypoxic and hypoglycemic rat hippocampal slices

Hypoxia may increase GABA levels in neurons by ATP depletion-induced activation of glutamate decarboxylase and by inhibiting GABA transaminase. Hypoglycemia, which also depletes ATP, reduces neuronal levels of GABA and its precursor glutamate. We examined whether differences in glutamate levels may contribute to these altered GABA levels in hippocampal slices. GABA levels were highly correlated with endogenous glutamate levels during both hypoxia and hypoglycemia (R=0.93 for combined data). Hypoxia maximally increased GABA levels (146+/-6.3% of control, S.E.M.) when glutamate remained above 90% of control levels and ATP was at 30% of control levels. Hypoglycemia with similar ATP levels and glutamate levels at 40% of control decreased GABA levels to 55% of control. Effects of inhibitors of glutamate decarboxylase and GABA transaminase suggested that increased synthesis and decreased catabolism may both contribute to increased hypoxic GABA levels. Immunocytochemical studies suggested that hypoxia increased GABA concentrations primarily in neurons and their processes, but not in glial cells. Severe hypoxic ATP depletion increased the release of both GABA and glutamate.Hypoxia increased GABA levels in neurons, while hypoglycemia with a similar severity of ATP depletion decreased GABA levels. Much of the difference may be related to lower levels of precursor glutamate during hypoglycemia. The twofold higher levels of neuroprotective GABA available for release during hypoxia may contribute to differences in the pathophysiology of these metabolic insults.