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.     

Attenuation of N-methyl-D-aspartate-mediated cytoplasmic vacuolization in primary rat hippocampal neurons by mood stabilizers

Recent post-mortem and brain imaging studies suggest that decreased neuronal and glial densities may account for cell loss in vulnerable brain regions such as the hippocampus and the frontal cortex in patients with bipolar disorder. Investigations into the mechanisms of action of mood stabilizers suggest that these drugs may regulate the expression of neuroprotective genes and protect against excitotoxicity. In this study, we characterized the ultrastructural appearance of rat hippocampal neurons pretreated with mood stabilizers and then exposed to the glutamate receptor agonist N-methyl-D-aspartate. Using transmission electron microscopy we found that rat hippocampal neurons exposed to 0.5 mM N-methyl-D-aspartate for 10 min produced more cytoplasmic vacuolization than in control neurons. Chronic treatment with mood stabilizers, lithium, valproate or carbamazepine for 7 days at therapeutically relevant concentrations fully attenuated N-methyl-D-aspartate-mediated cytoplasmic vacuolization. These results suggest that inhibition of neurotoxicity may be involved in the action of mood stabilizers.     

D1/D5 dopamine receptors stimulate intracellular calcium release in primary cultures of neocortical and hippocampal neurons

D1/D5 dopamine receptors in basal ganglia, hippocampus, and cerebral cortex modulate motor, reward, and cognitive behavior. Previous work with recombinant proteins revealed that in cells primed with heterologous G(q/11)-coupled G-protein-coupled receptor (GPCR) agonists, the typically G(s)-linked D1/D5 receptors can stimulate robust release of calcium from internal stores when coexpressed with calcyon. To learn more about the intracellular signaling mechanisms underlying these D1/D5 receptor regulated behaviors, we explored the possibility that endogenous receptors stimulate internal release of calcium in neurons. We have identified a population of neurons in primary cultures of hippocampus and neocortex that respond to D1/D5 dopamine receptor agonists with a marked increase in intracellular calcium (Ca) levels. The D1/D5 receptor stimulated responses occurred in the absence of extracellular Ca(2+) indicating the rises in Ca involve release from internal stores. In addition, the responses were blocked by D1/D5 receptor antagonists. Further, the D1/D5 agonist-evoked responses were state dependent, requiring priming with agonists of G(q/11)-coupled glutamate, serotonin, muscarinic, and adrenergic receptors or with high external K(+) solution. In contrast, D1/D5 receptor agonist-evoked Ca(2+) responses were not detected in neurons derived from striatum. However, D1/D5 agonists elevated cAMP levels in striatal cultures as effectively as in neocortical and hippocampal cultures. Further, neither forskolin nor 8-Br-cAMP stimulation following priming was able to mimic the D1/D5 agonist-evoked Ca(2+) response in neocortical neurons indicating that increased cAMP levels are not sufficient to stimulate Ca release. Our data suggest that D1-like dopamine receptors likely modulate neocortical and hippocampal neuronal excitability and synaptic function via Ca(2+) as well as cAMP-dependent signaling.     

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.     

Regulation of apolipoprotein E secretion in rat primary hippocampal astrocyte cultures

Apolipoprotein E isoforms may have differential effects on a number of pathological processes underlying Alzheimer's disease. Recent studies suggest that the amount, rather than the type, of apolipoprotein E may also be an important determinant for Alzheimer's disease. Therefore, understanding the regulated synthesis of apolipoprotein E is important for determining its role in Alzheimer's disease.We show here that in rat primary hippocampal astrocyte cultures, dibutyryl-cAMP increased apolipoprotein E secretion with time in a dose-dependent manner (to 177% at 48 h) and that retinoic acid potentiated this effect (to 298% at 48 h). Dibutyryl-cAMP also gave a rapid, albeit transient, increase of apolipoprotein E mRNA expression (to 200% at 1 h). In contrast, the protein kinase C activator phorbol 12-myristate 13-acetate decreased both apolipoprotein E secretion (to 59% at 48 h) and mRNA expression (to 22% at 1 h). Phorbol 12-myristate 13-acetate also reversed the effects of dibutyryl-cAMP. Apolipoprotein E secretion was also modulated by receptor agonists for the adenylyl cyclase/cAMP pathway. Isoproterenol (50 nM, a beta-adrenoceptor agonist) enhanced, while clonidine (250 nM, an alpha2-adrenoceptor agonist) decreased, secreted apolipoprotein E. We also analysed the effects of agonists for the phospholipase C/protein kinase C pathway. Arterenol (1 microM, an alpha1-adrenoceptor agonist) and serotonin (2.5 microM) enhanced, whereas carbachol (10 microM, an acetylcholine muscarinic receptor agonist) decreased secreted apolipoprotein E. The effects of these non-selective receptor agonists were modest, probably due to effects on different signalling pathways. Arterenol also potentiated the isoproterenol-mediated increase. We also show that phorbol 12-myristate 13-acetate and dibutyryl-cAMP have opposite effects on nerve growth factor, as compared to apolipoprotein E, secretion, suggesting that the results obtained were unlikely to be due to a general effect on protein synthesis.We conclude that astrocyte apolipoprotein E production can be regulated by factors that affect cAMP intracellular concentration or activate protein kinase C. Alterations in these signalling pathways in Alzheimer's disease brain may have consequences for apolipoprotein E secretion in this disorder.     

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。     

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.     

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.     

Identification of somatostatin-containing neurons in primary cultures of rat cerebral cortex

The expression of somatostatin-like immunoreactivity was investigated in primary cultures of dissociated cerebral cortical tissue from the rat. A subpopulation of neurons in the cortical cultures exhibited intense staining for somatostatin. These somatostatin-immunoreactive neurons corresponded to 1.25% of the total neuronal population. Stained neurons were typically small with a soma size of 10-20 micron. The majority of somatostatin-containing cells had stellate and bipolar morphology, with the bipolar class predominating.     

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.     

Mechanisms involved in tetanus-induced potentiation of fast IPSCs in rat hippocampal CA1 neurons

In the present study, possible mechanisms involved in the tetanus-induced potentiation of gamma-aminobutyric acid-A (GABA-A) receptor-mediated inhibitory postsynaptic currents (IPSCs) were investigated using the whole cell voltage-clamp technique on CA1 neurons in rat hippocampal slices. Stimulations (100 Hz) of the stratum radiatum, while voltage-clamping the membrane potential of neurons, induces a long-term potentiation (LTP) of evoked fast IPSCs while increasing the number but not the amplitude of spontaneous IPSCs (sIPSCs). The potentiation of fast IPSCs was input specific. During the period of IPSC potentiation, postsynaptic responses produced by 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride and baclofen, GABA-A and GABA-B agonists respectively, were not significantly different from control. CGP 36742, a GABA-B antagonist, blocked the induction of tetanus-induced potentiation of evoked and spontaneous IPSCs, while GTPgammaS, an activator of G proteins, substitution for GTP in the postsynaptic recording electrode did not occlude potentiation. Since GABA-B receptors work through G proteins, our results suggest that pre- but not postsynaptic GABA-B receptors are involved in the potentiation of fast IPSCs. A tetanus delivered when GABA-A responses were completely blocked by bicuculline suggests that GABA-A receptor activation during tetanus is not essential for the induction of potentiation. Rp-cAMPs, an antagonist of protein kinase A (PKA) activation, blocks the induction of potentiation of fast IPSCs. Forskolin, an activator of PKA, increases baseline evoked IPSCs as well as the number of sIPSCs, and a tetanic stimulation during this enhancement uncovers a long-term depression of the evoked IPSC. Sulfhydryl alkylating agents, N-ethylmaleimide and p-chloromercuribenzoic acid, which have been found to presynaptically increase GABA release and have been suggested to have effects on proteins involved in transmitter release processes occurring in nerve terminals, occlude tetanus-induced potentiation of evoked and spontaneous IPSCs. Taken together our results suggest that LTP of IPSCs originates from a presynaptic site and that GABA-B receptor activation, cyclic AMP/PKA activation and sulfhydryl-alkylation are involved. Plasticity of IPSCs as observed in this study would have significant implications for network behavior in the hippocampus.     

Whole-cell patch-clamp analysis of voltage-dependent calcium conductances in cultured embryonic rat hippocampal neurons

1. Voltage-dependent calcium currents in embryonic (E18) hippocampal neurons cultured for 1-14 days were investigated using the whole-cell patch-clamp technique. 2. Calcium currents were isolated by removing K+ from both the internal and external solutions. In most recordings the external solution contained tetrodotoxin, tetraethylammonium ions, and low concentrations of Na+, whereas the internal solution contained the large cations and anions, N-methyl-D-glucamine and methanesulphonate, and an adenosine 5'-triphosphate (ATP) regenerating system (Forscher and Oxford, 1985) to retard "run-down" of Ca currents. 3. Under these conditions, the sustained inward current triggered during depolarizing steps was enhanced when extracellular [Ca2+] ([Ca2+]0) was raised from 2 to 10 mM and abolished when [Ca2+]0 was lowered to 0.1 mM or by addition of Co2+ ions. These results indicate that the inward current was carried primarily by Ca2+ ions and was designated ICa. This current may be comparable to the "high-voltage-activated" Ca current described in other preparations. 4. In cells cultured for 1-3 days, ICa was small or absent (less than 20 pA for cells 1 day in culture and less than 80 pA for cells 3 days in culture). Although ICa decayed considerably during depolarizing steps, there was little evidence of the transient calcium current (T current) that was recorded in approximately 40% of cells cultured longer than 6 days. Maximal (i.e., the largest) ICa increased from 20 to 80 pA in 1- to 3-day cells to 150-450 pA in cells cultured for longer than 6 days. 5. The decay of ICa elicited by depolarizations from holding potentials of -60 mV or more negative was usually greatest for the maximal ICa. Replacement of extracellular Ca2+ (4 mM) with Ba2+ (2 mM) resulted in a substantial decrease in the extent of decay of ICa and a shift of the I-V relation in the hyperpolarizing direction. 6. Qualitative data obtained from experiments in which different levels of internal Ca2+ buffering were employed demonstrated that, on average, the decay of ICa was reduced as the capacity and/or rate of buffering was increased. The mean decay of ICa in cells buffered with 5 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was 9 +/- 7 (SD) %, (n = 12) and 25 +/- 12%, (n = 12) for cells buffered with the same concentration of ethyleneglycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).(ABSTRACT TRUNCATED AT 400 WORDS)     

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.