The influence of calcium transients on intracellular pH in cortical neurons in primary culture

The objective of this study was to assess the influence of Ca²⁺ influx on intracellular pH (pH_i) of neocortical neurons in primary culture. Neurons were exposed to glutamate (100–500 μM) or KCl (50 mM), and pH_i was recorded with microspectroflurometric techniques. Additional experiments were carried out in which calcium influx was triggered by ionomycin (2 μM) or the calcium ionophore 4-Br-A23187 (2 μM). Glutamate exposure either caused no, or only a small decrease in pH_i (ΔpH ≈ 0.06 units). When a decrease was observed, a rebound rise in pH_i above control was observed upon termination of glutamate exposure. In about 20% of the cells, the acidification was more pronounced (ΔpH ≈ 0.20 units), but all these cells had high control pH_i values, and showed gradual acidification. Exposure of cells to 50 mM KCl consistently increased pH_i. Since this increase was similar in the presence and nominal absence of HCO₃⁻, it probably did not reflect influx of HCO₃⁻ via a Na⁺-HCO₃⁻ symporter. Furthermore, since it occurred in the absence of external Ca²⁺ (or a measurable rise in Ca_i²⁺) it seemed independent of Ca²⁺ influx. It is tentatively concluded that the rise in pH_i was due to reduced passive influx of H⁺ along the electrochemical gradient, which is reduced by depolarization. In Ca²⁺-containing solutions, depolarization led to a rebound increase in pH_i above control. This, and the rebound found after glutamate transients, may reflect Ca²⁺-triggered phosphorylation and upregulation of the Na⁺/H⁺ antiporter which extrudes H⁺ from the cell. Ionomycin and 4-Br-A23187 gave rise to a large rise in Ca_i²⁺ and to alkalinization of the cell (ΔpH ≈ 0.5). Since amiloride or removal of Na⁺ from the external solution did not alter the rise in pH_i, it was probably not due to accelerated H⁺ extrusion. However, removal of Ca²⁺ from extracellular fluid prevented the rise, suggesting that it was secondary to Ca²⁺/2H⁺ exchange across plasma membranes.     

Role of calcium in sigma-mediated neuroprotection in rat primary cortical neurons

Since unique calcium dynamics have been reported for toxic (40–80 M) and non-toxic (5–10 μM) concentrations of glutamate, we evaluated the effect of neuroprotective sigma ligands on glutamate and potassium chloride (KCI)-stimulated changes in [Ca²⁺]; using 12–15 day old primary rat neuronal cortical cultures. In approximately 80% of the neurons tested, 80 μM glutamate caused a sustained calcium flux previously shown to be associated with neurotoxicity. The majority of sigma ligands that were evaluated altered glutamate-induced calcium flux. For example, the primary effect of maximally neuroprotective concentrations of the sigma ligands dextromethorphan, (+)-pentazocine, (+)-cyclazocine, (+)-SKF 10047, carbetapentane and haloperidol was a shift from a sustained, to either a biphasic or a monophasic transient calcium response indicative of neuroprotection. (+)-3-PPP, previously shown not to be neuroprotective in this model system, failed to alter glutamate-induced calcium flux. In contrast to glutamate, KCl (50 mM) produced changes in [Ca²⁺]_i which were not neurotoxic to the neurons as measured by LDH release. The primary response observed in 59% of the neurons treated with 50 mM KCl alone was an initial spike in [Ca²⁺]_i which abruptly declined then plateaued above basal levels throughout the 12 min of analysis (modified sustained response). The highly selective sigma ligands produced a shift from the modified sustained response to a monophasic transient calcium response. Again, (+)-3-PPP had no effect on KCl-induced calcium dynamics. Of the PCP-related sigma ligands only (+)-SKF-10047 consistently attenuated the KCl-induced calcium flux. Collectively, these results indicate that modulation of [Ca²⁺]_i through receptor and voltage-gated calcium channels contributes significantly to sigma mediated neuroprotection.     

头孢曲松钠对谷氨酸作用后神经细胞的保护作用

目的研究头孢曲松钠对谷氨酸作用后神经细胞活性、谷氨酸摄取能力以及对细胞内钙超载反应的影响。方法将体外培养的大鼠脑皮层神经细胞分为对照组和头孢曲松钠组,比较谷氨酸作用后两组神经细胞形态、细胞活性和谷氨酸重摄取能力的改变;运用激光共聚焦显微镜(CLSM)测定静息状态、谷氨酸短暂作用后以及谷氨酸作用后加入谷氨酸转运体1(GLT1)抑制剂二氢红藻氨酸(DHK)条件下,两组细胞内Ca2+荧光强度的改变;酶联免疫法检测培养上清液中脑源性神经营养因子(BDNF)、白细胞介素-6(IL-6)水平。结果两组神经细胞形态无差别,头孢曲松钠组较对照组神经细胞的细胞活性以及谷氨酸摄取率增加;静息状态下两组细胞内Ca2+荧光强度的差别无统计学意义,谷氨酸短暂作用后头孢曲松钠组较对照组细胞内Ca2+荧光强度上升缓慢但恢复迅速,加入DHK后,两组细胞内Ca2+荧光强度变化基本相同,差异消失;头孢曲松钠组较对照组细胞培养上清液中BDNF含量明显增加,而两组IL-6含量无差别。结论头孢曲松钠可以增强谷氨酸作用后的神经细胞活性,提高神经细胞的谷氨酸摄取能力,拮抗谷氨酸内钙超载作用,增加神经营养因子的表达,对神经细胞具有保护作用。     

AMPA receptor contribution to methylmercury-mediated alteration of intracellular Ca2+ concentration in human induced pluripotent stem cell motor neurons

α motor neurons (MNs) are a target of the environmental neurotoxicant methylmercury (MeHg), accumulating MeHg and subsequently degenerating. In mouse spinal cord MN cultures, MeHg increased intracellular Ca²⁺ [Ca²⁺]_i; the AMPA receptor (AMPAR) antagonist CNQX delayed the increase in [Ca²⁺]_i, implicating the role of AMPARs in this response. Here we used human induced pluripotent stem cell-derived MNs (hiPSC-MNs), to characterize the role of MN AMPARs in MeHg neurotoxicity. Acute exposure to MeHg (0.1, 0.2, 0.5, 1 and 1.5 μM), fura-2 microfluorimetry, and a standard cytotoxicity assay, were used to examine MN regulation of [Ca²⁺]_i, and cytotoxicity, respectively. Contribution of Ca²⁺-permeable and impermeable AMPARs was compared using either CNQX, or the Ca²⁺-permeable AMPAR antagonist N-acetyl spermine (NAS). MeHg-induced cytotoxicity was evaluated following a 24 h delay subsequent to 1 h exposure of hiPSC-MNs. MeHg caused a characteristic biphasic increase in [Ca²⁺]_i, the onset of which was concentration-dependent; higher MeHg concentrations hastened onset of both phases. CNQX significantly delayed MeHg’s effect on onset time of both phases. In contrast, NAS significantly delayed only the 2nd phase increase in fura-2 fluorescence. Exposure to MeHg for 1 h followed by a 24 h recovery period caused a concentration-dependent incidence of cell death. These results demonstrate for the first time that hiPSC-derived MNs are highly sensitive to effects of MeHg on [Ca²⁺]_i, and cytotoxicity, and that both Ca²⁺-permeable and impermeable AMPARs contribute the elevations in [Ca²⁺]_i.     

Reduced mitochondrial manganese-superoxide dismutase activity exacerbates glutamate toxicity in cultured mouse cortical neurons

Studies of neuronal injury and death after cerebral ischemia and various neurodegenerative diseases have increasingly focused on the interactions between mitochondrial function, reactive oxygen species (ROS) production and glutamate neurotoxicity. Recent findings suggest that increased mitochondrial ROS production precedes neuronal death after glutamate treatment. It is hypothesized that under pathological conditions when mitochondrial function is compromised, extracellular glutamate may exacerbate neuronal injury. In the present study, we focus on the relationship between mitochondrial superoxide production and glutamate neurotoxicity in cultured cortical neurons with normal or reduced levels of manganese-superoxide dismutase (MnSOD) activity. Our results demonstrate that neurons with reduced MnSOD activity are significantly more sensitive to transient exposure to extracellular glutamate. The increased sensitivity of cultured cortical neurons with reduced MnSOD activity is characteristically subject only to treatment by glutamate but not to other glutamate receptor agonists, such as N-methyl- -aspartate, kainate and quisqualate. We suggest that the reduced MnSOD activity in neurons may exacerbate glutamate neurotoxicity via a mechanism independent of receptor activation.     

Potassium-induced enhancement of persistent inward current in hippocampal neurons in isolation and in tissue slices

Previous work suggested a role for the voltage-dependent persistent sodium current, I(Na,P), in the generation of seizures and spreading depression (SD). Ordinarily, I(Na,P) is small in hippocampal neurons. We investigated the effect of raising external K(+) concentration, [K(+)](o), on whole-cell persistent inward current in freshly isolated hippocampal CA1 pyramidal neurons. I(Na,P) was identified by TTX-sensitivity and dependence on external Na(+) concentration. When none of the ion channels were blocked, I(Na,P) was not usually detectable, probably because competing K(+) current masked it, but after raising [K(+)](o) I(Na,P) appeared, while K(+) currents diminished. With K(+) channels blocked, I(Na,P) could usually be evoked in control solution and raising [K(+)](o) caused its reversible increase in most cells. The increase did not depend on external calcium [Ca(2+)](o). In CA1 pyramidal neurons in hippocampal slices a TTX-sensitive persistent inward current was always recorded and when [K(+)](o) was raised, it was reversibly enhanced. Strong depolarization evoked irregular current fluctuations, which were also augmented in high [K(+)](o). The findings support a role of potassium-mediated positive feedback in the generation of seizures and spreading depression.     

Neuroprotective effects of the glutamate release inhibitor 619C89 in temporary middle cerebral artery occlusion

619C89 is a use-dependent Na⁺ channel antagonist that decreases the release of glutamate during ischemia. The efficacy of this drug in reducing infarction volume 72 h after occlusion of the middle cerebral artery (MCA) for 2 h in rats (n = 93) was determined by analysis of TTC-stained coronal section of the brain. Doses of 10, 20, 30 and 50 mg/kg of study drug given i.v. prior to MCA occlusion significantly (P < 0.05−0.01) reduced infarction volume in cortex compared to vehicle controls. Only the 50 mg/kg dose reduced infarction volume in the striatum (P < 0.05). Administration of 50 mg/kg of 619C89 30 and 60 min after the onset of ischemia reduced cortical infarction volume (P < 0.05), but there was no effect when the drug was given 5 min after reperfusion. No post-treatment regimen reduced striatal infarction volume. These results confirm the neuroprotective effects of 619C89 in temporary focal ischemia.     

Differential neurotoxicity induced by -DOPA and dopamine in cultured striatal neurons

The neurotoxicity of -DOPA and dopamine (DA) on striatal neurons was examined by using primary cultures of rat striatum. Exposure to -DOPA and DA at concentrations of 30–300 μM induced dose-dependent cell death in both younger cultures (3 days in culture, 3 DIC) and elder cultures (10 days in culture, 10 DIC). The cytotoxicity of -DOPA and DA was also dependent on the exposure time (6–24 h). Ascorbic acid (200 μM) inhibited both -DOPA- and DA-induced cytotoxicity in 3 DIC cultures, whereas it provided significant protection against DA- but not -DOPA-induced cytotoxicity in 10 DIC cultures. The -DOPA cytotoxicity in 10 DIC cultures was prevented by a non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and by an NMDA receptor antagonist, MK-801. Neither antagonist prevented DA cytotoxicity. -DOPA did not affect the viability of 10 DIC cultures, though it elicited marked toxicity in 3 DIC cultures. These results suggest that there are two components in the mechanisms that mediate the -DOPA neurotoxicity on striatal neurons: one is autoxidation-relevant and the other is autoxidation-irrelevant. With respect to the latter, glutamate receptor stimulation may be involved. In contrast, autoxidation plays an important role in DA neurotoxicity.     

Protective effect of nerve growth factor against glutamate-induced neurotoxicity in cultured cortical neurons

The effect of recombinant human nerve growth factor (hNGF) and mouse NGF on cultured rat cortical neurons was examined. The DNA fragment coding the human NGF gene was isolated and inserted downstream from the SV40 promoter in a plasmid containing the dihydrofolate reductase cDNA, and this plasmid was introduced into Chinese hamster ovary (CHO) cells to establish cells producing recombinant hNGF. The recombinant hNGF protein secreted by CHO cells was confirmed to be biologically active in an assay using PC12 cells. Brief exposure of cortical cells to glutamate followed by incubation with glutamate-free medium reduced cell viability by 60–70% when compared with the control culture. Simultaneous addition of recombinant hNGF or mouse NGF to rat cortical cultures with glutamate did not affect this reduction of cell viability. However, 24 h pretreatment of rat cortical cultures with recombinant hNGF or mouse NGF resulted in a significant reduction of glutamate-induced neuronal damage. Mouse NGF also protected cortical neurons against N-methyl-d-aspartate (NMDA)- and kainate-induced neuronal damage. These findings suggest that NGF can protect cortical neurons against glutamate-induced neurotoxicity.     

Distribution of ω-Conotoxin GVIA binding sites in teleost cerebellar and electrosensory neurons

The distribution of ω-Conotoxin GVIA (CgTx) binding sites was used to localize putative N-type Ca²⁺ channels in an electrosensory cerebellar lobule, the eminentia granularis pars posterior, and in the electrosensory lateral line lobe of a gymnotiform teleost (Apteronotus leptorhynchus). The binding sites for CgTx revealed by an anti-CgTx antibody had a consistent distribution on somatic and dendritic membranes of specific cell types in both structures. The distribution of CgTx binding was unaffected by co-incubation with nifedipine or Aga Toxin IVA, blocking agents for L- and P-type Ca²⁺ channels, respectively. Incubation with CgTx in the presence of varying levels of extracellular Ca²⁺ altered the number but not the cell types exhibiting immunolabel. A punctate immunolabel was detected on somatic membranes of granule and stellate cell interneurons in both the eminentia granularis pars posterior and the electrosensory lateral line lobe. Punctate CgTx binding sites were also present on spherical cell somata and on the large presynaptic terminals of primary afferents that terminate on spherical cells in the electrosensory lateral line lobe. No label was detected in association with distal dendritic membranes of any cell class, or with parallel fibers in the respective molecular layers. Binding sites for CgTx in the eminentia granularis are consistent with the established role for N-type Ca²⁺ channels in cell migrations, an activity which is known to persist in this layer in adult Apteronotus. The distribution of labeled stellate cells with respect to topographic maps in the electrosensory lateral line lobe further suggest that N-type Ca²⁺ channels are expressed in relation to functional activity across these sensory maps. © 1996 Wiley-Liss, Inc.     

Agmatine induces glutamate release and cell death in cultured rat cerebellar granule neurons

We investigated the effect of agmatine on cell viability of rat cerebellar granule neurons in a high-K+ (27.5 mM) medium. Exposure of cultured rat cerebellar granule neurons to agmatine (200-800 microM) resulted in a significant decrease in cell viability. Agmatine-induced neuronal death began to occur 6-12 h after addition, and gradually progressed. The agmatine neurotoxicity was attenuated by N-methyl-D-aspartate (NMDA) receptor antagonists and by enzymatic degradation of L-glutamate with glutamic pyruvic transaminase. Furthermore, a significant increase in extracellular L-glutamate concentration was detected before cell death occurred. In addition, agmatine-induced glutamate release and cell death were both blocked by pretreatment with botulinum toxin C, which is known to specifically inhibit the exocytosis. The agmatine neurotoxicity was not observed when extracellular K+ concentration was lower (10 mM). These results suggest that agmatine induces glutamate release through the exocytosis and thereby causes NMDA receptor-mediated neuronal death in conditions in which extracellular K+ concentrations are elevated.     

σ receptor-mediated neuroprotection against glutamate toxicity in primary rat neuronal cultures

The role of the putative σ receptor in mediating neuroprotection against glutamate-induced neuronal injury was examined in mature cultured rat cortical neurons. With the exception of the selective σ₁ ligand (+)-3-PPP, all of the σ ligands tested were neuroprotective, preventing glutamate-induced morphological changes and increases in LDH release. Their rank order of neuroprotective potency (and EC₅₀ values) was as follows: (+)-SKF 10,047 (0.81 μM) > (+)- cyclazocine (2.3 μM) > dextromethorphan (3.1 μM) = haloperidol (3.7 μM) > (+)-pentazocine (8.5 μM) > DTG (42.7 μM) = carbetapentane (46.3 μM). When corrected for relative σ versus PCP binding affinity, it appears that a positive correlation exists between neuroprotective potency and σ₁ site affinity. However, there does not appear to be a significant correlation between neuroprotective potency and the σ₂ site. Critically, none of the σ ligands were neurotoxic when tested alone at concentrations at least 5–30 times their respective neuroprotective EC₅₀ values. Results from preliminary experiments with the selective σ₁ ligand (+)-pentazocine indicated that σ-mediated neuroprotection may involve the buffering of glutamate-induced calcium flux. Collectively, the results of these in vitro experiments demonstrate that σ ligands are neuroprotective and therefore deserve further exploration as potential therapeutic agents in in vivo models of CNS injury and neurodegenerative disorders.     

Acute ethanol alters calcium signals elicited by glutamate receptor agonists and K depolarization in cultured cerebellar Purkinje neurons

The effect of acute ethanol on Ca²⁺ signals evoked by ionotropic (iGluR) and metabotropic (mGluR) glutamate receptor (GluR) activation and K⁺ depolarization was examined in cultured rat cerebellar Purkinje neurons to assess the ethanol sensitivity of these Ca²⁺ signaling pathways. Mature Purkinje neurons 3 weeks in vitro were studied. iGluRs were activated by (RS)-α-amino-3-hydroxyl-5 methyl-4-isoxazolepropionic acid (AMPA; 1 and 5 μM) and domoate (5 μM). mGluRs were activated by (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD; 300 μM) and (R,S)-3,5-dihydroxyphenylglycine (DHPG; 200 μM). These agents and K⁺ (150 mM) were applied from micropipettes by brief (1 s) microperfusion pulses. Ca²⁺ levels were monitored at 2–3 s intervals during pre- and post-stimulus periods using microscopic digital imaging and the Ca²⁺ sensitive dye fura-2. iGluR and mGluR agonists and K⁺ produced abrupt increases in intracellular Ca²⁺ that slowly recovered to baseline resting levels. Acute exposure to ethanol at 33 mM (150 mg%) and 66 mM (300 mg%) significantly reduced the amplitude of the Ca²⁺ signals to iGluR agonists and K⁺ with little or no effect on Ca²⁺ signals to mGluR agonists. In contrast, acute ethanol at 10 mM (45 mg%) had no effect on the Ca²⁺ signals to the iGluR agonist AMPA but significantly enhanced the Ca²⁺ signals to the mGluR agonist DHPG. These results show that ethanol modulates Ca²⁺ signaling linked to GluR activation in a receptor subtype specific manner, and suggest that Ca²⁺ signaling pathways linked to GluR activation and membrane depolarization may be important mechanisms by which ethanol alters the transduction of excitatory synaptic signals at glutamatergic synapses and thereby affects intercellular and intracellular communication in the CNS.     

Cholecystokinin-induced protection of cultured cortical neurons against glutamate neurotoxicity

The effects of cholecystokinin (CCK) on glutamate-induced neurotoxicity were examined using cultured rat cortical neurons. Brief exposure of glutamate followed by an incubation with normal solution for more than 60 min reduced cell viability by 60–70%, compared with control values. Glutamate-induced neurotoxicity was significantly inhibited by MK-801 and ketamine, which are non-competitive blockers of N-methyl-d-aspartate (NMDA) receptors. Octapeptide CCK-8S and CCK-related decapeptide ceruletide at concentrations of 10⁻⁹−10⁻⁷ M dose-dependently reduced glutamate-induced neurotoxicity. A desulfated analog CCK-8NS, which acts selectively as an antagonist of CCK_B receptors, also reduced glutamate neurotoxicity. The neuroprotective effects of CCK were antagonized by L-365260, a CCK_B receptor antagonist, but not by L-364718, a CCK_A receptor antagonist. These results suggest that CCK protects cortical neurons against NMDA receptor-mediated glutamate neurotoxicity via CCK_B receptors.     

Mechanisms of cholecystokinin-induced protection of cultured cortical neurons against receptor-mediated glutamate cytotoxicity

The protective effects of cholecystokinin (CCK) against glutamate-induced cytotoxicity were examined using cultured neurons obtained from the rat cerebral cortex. Cell viabiilty was significantly reduced when the cultures were briefly exposed to glutamate or (NMDA) and then incubated with normal medium for 60 min. A 60-min exposure to kainate also reduced cell viability. CCK protected cortical neurons against glutamate-, NMDA- and kainate-induced cytotoxicity. Glutamate- and NMDA-induced cytotoxicity was also reduced by , a nitric oxide (NO) synthase inhibitor. However, CCK did not prevent the cytotoxic effects of sodium nitroprusside (SNP) which spontaneously releases NO. Moreover, CCK did not affect NMDA-induced CA²⁺ influx measured with rhod-2, a fluorescent Ca²⁺ indicator. Therefore, release of a NO-like factor from the cerebral cortex was assayed using the thoracic artery in vitro. When the artery was incubated with minced cerebral tissues, glutamate elicited marked relaxation. SNP also elicited relaxation of the smooth muscle. CCK inhibited glutamate-induced relaxation but did not affect that induced by SNP. These results indicate that CCK prevents NMDA receptor-mediated cytotoxicity without reducing the Ca²⁺ influx. It is suggested that CCK inhibits NO-formation triggered by NMDA receptor activation.     

HIV-1 envelope protein evokes intracellular calcium oscillations in rat hippocampal neurons

Treatment of single rat hippocampal neurons with 200 pM recombinant HIV-1 envelope glycoprotein, gp120, resulted in large increases in the intracellular free calcium concentration ([Ca2+]i) as measured with indo-1-based microfluorimetry. Three patterns of [Ca2+]i increases were observed: in one pattern the [Ca2+]i rose rapidly and transiently as a single peak, in a second pattern gp120 induced [Ca2+]i oscillations that subsided when the protein was removed, and in a third pattern the oscillations continued long after washout of gp120. Both single peak and oscillatory [Ca2+]i increases were completely blocked by the Ca2+ channel blocker nitrendipine (1 microM). The sustained oscillatory responses were also blocked completely and reversibly by the N-methyl-D-aspartate (NMDA) receptor antagonist CGS19755 (10 microM) and the Na+ channel blocker tetrodotoxin (1 microM). Complete block by antagonists of Ca2+, Na+, and NMDA-gated ion channels suggests that at least two cells are required to maintain the [Ca2+]i oscillations. We hypothesize that gp120 acts as an excitotoxin by increasing synaptic activity in the network of neurons established in primary culture.     

Hyperosmolar sodium chloride is toxic to cultured neurons and causes reduction of glucose metabolism and ATP levels,an increase in glutamate uptake,and a reduction in cytosolic calcium

Elevation of serum sodium, hypernatremia, which may occur during dehydration or treatment with sodium chloride, may cause brain dysfunction and damage, but toxic mechanisms are poorly understood. We found that exposure to excess NaCl, 10–100 mmol/L, for 20 h caused cell death in cultured cerebellar granule cells (neurons). Toxicity was due to Na⁺, since substituting excess Na⁺ with choline reduced cell death to control levels, whereas gluconate instead of excess Cl⁻ did not. Prior to cell death from hyperosmolar NaCl, glucose consumption and lactate formation were reduced, and intracellular aspartate levels were elevated, consistent with reduced glycolysis or glucose uptake. Concomitantly, the level of ATP became reduced. Pyruvate, 10 mmol/L, reduced NaCl-induced cell death. The extracellular levels of glutamate, taurine, and GABA were concentration-dependently reduced by excess NaCl; high-affinity glutamate uptake increased. High extracellular [Na⁺] caused reduction in intracellular free [Ca²⁺], but a similar effect was seen with mannitol, which was not neurotoxic. We suggest that inhibition of glucose metabolism with ensuing loss of ATP is a neurotoxic mechanism of hyperosmolar sodium, whereas increased uptake of extracellular neuroactive amino acids and reduced intracellular [Ca²⁺] may, if they occur in vivo, contribute to the cerebral dysfunction and delirium described in hypernatremia.     

Swelling-activated calcium signalling in cultured mouse primary sensory neurons

The effects of hypo-osmotic membrane stretch on intracellular calcium concentration ([Ca(2+)](i)), cell volume and cellular excitability were investigated in cultured mouse primary sensory trigeminal neurons. Hypotonic solutions (15--45%) led to rapid cell swelling in all neurons. Swelling was accompanied by dose-dependent elevations in [Ca(2+)](i) in a large fraction of neurons. Responses could be classified into three categories. (i) In 57% of the neurons [Ca(2+)](i) responses had a slow rise time and were generally of small amplitude. (ii) In 21% of the neurons, responses had a faster rise and were larger in amplitude. (iii) The remaining cells (22%) did not show [Ca(2+)](i) responses to hypo-osmotic stretch. Slow and fast [Ca(2+)](i) changes were observed in trigeminal neurons of different sizes with variable responses to capsaicin (0.5 microM). The swelling-induced [Ca(2+)](i) responses were not abolished after depletion of intracellular Ca2+ stores with cyclopiazonic acid or preincubation in thapsigargin, but were suppressed in the absence of external Ca(2+). They were strongly attenuated by extracellular nickel and gadolinium. Hypotonic stimulation led to a decrease in input resistance and to membrane potential depolarization. Under voltage-clamp, the [Ca(2+)](i) elevation produced by hypotonic stimulation was accompanied by the development of an inward current and a conductance increase. The time course and amplitude of the [Ca(2+)](i) response to hypo-osmotic stimulation showed a close correlation with electrophysiological properties of the neurons. Fast [Ca(2+)](i) responses were characteristic of trigeminal neurons with short duration action potentials and marked inward rectification. These findings suggest that hypo-osmotic stimulation activates several Ca(2+)-influx pathways, including Gd(3+)-sensitive stretch-activated ion channels, in a large fraction of trigeminal ganglion neurons. Opening of voltage-gated Ca(2+) channels also contributes to the response. The pattern and rate of Ca(2+) influx may be correlated with functional subtypes of sensory neurons.     

Astrocyte-conditioned medium attenuates glutamate-induced apoptotic cell death in primary cultured spinal cord neurons of rats

Abstract

Objectives:

To better understand the neuroprotective role of astrocytes in spinal cord injury (SCI), we investigated whether astrocyte-conditioned medium (ACM) can attenuate glutamate-induced apoptotic cell death in primary cultured spinal cord neurons.

Methods:

Spinal cord neurons were pretreated with ACM for 24?hours. Subsequently, they were exposed to glutamate (125?μM) for 1?hour. The neurons were then incubated for 24?hours. Following that, measurements assessing cell viability and lactate dehydrogenase (LDH) release were performed. Apoptosis was confirmed through cell morphology using Hoechst 33342 staining and terminal deoxynucleotidyl transferase dUTP-mediated nicked end labeling (TUNEL) assay. Assessment for expression of apoptotic enzymes, including Caspase-3, Bcl-2 and Bax, was performed using Western Blot Analysis.

Results:

Astrocyte-conditioned medium pretreatment of neurons showed both an increase in spinal cord neuron viability and a decrease in LDH release in a dose-dependent pattern. Moreover, pretreatment seems to attenuate glutamate-induced apoptotic cell death, antagonise glutamate-induced up-regulation of Caspase-3 expression and downregulate Bcl-2/Bax protein expression ratio.

Conclusions:

By attenuating glutamate-induced apoptotic cell death in primary cultured spinal cord neurons of rats, ACM seems to provide a neuroprotective effect by regulating apoptosis-related protein expression. Our results provide an experimental basis for clinical applications and potential therapeutic use of ACM in SCI.     

Basic fibroblast growth factor modulates synaptic transmission in cultured rat hippocampal neurons

Basic fibroblast growth factor (bFGF) is one of the effective growth factors that protect neurons against excitotoxic/ischemic injury and promote neuronal survival. In the present study, we examined the acute modulative effect of bFGF on synaptic transmission by monitoring spontaneous intracellular Ca²⁺ ([Ca²⁺]i]) oscillation, the amplitudes of which reflect excitatory and inhibitory inputs. The hippocampal cells from embryonic day 18 rats were cultured for 11–14 days, and changes in [Ca²⁺]i of single neurons were measured by a microfluometrical technique with fura-2. The amplitude of spontaneous oscillation was decreased by 10 ng/ml bFGF, but not by nerve growth factor (10–1000 ng/ml). Acidic FGF (1000 ng/ml) had a weaker depressant effect. The effect of bFGF was counteracted by suramin. bFGF did not affect the increase in [Ca²⁺]i evoked by glutamate agonists, NMDA or kainate, indicating that glutamate receptors are not involved in the mechanism. This is supported by similar results that kainate-evoked current was not affected by bFGF. On the other hand, bicuculline masked the effect of bFGF on the Ca²⁺ oscillation. But GABA-evoked current was slightly decreased by bFGF. These results suggest the possible role of bFGF in modulating GABAergic rather than glutamatergic neurotransmission.