Protective effect of L-carnitine against bilirubin-induced neuronal cell death

There is growing evidence that glutamate receptor-mediated injury plays a crucial role in bilirubin neurotoxicity. L-carnitine (LC) has been shown to prevent glutamate-induced toxicity in neuronal cell culture. The purpose of this study is to assess whether LC is able to prevent bilirubin neurotoxicity. Unconjugated bilirubin at different concentrations was administered to cerebellar granular cell cultures prepared from 1-day-old Sprague-Dawley rats. The neuroprotective effect of LC was examined. LC at doses of 10(-6), 10(-5), 10(-4) and 10(-3) M was applied to culture flasks. LC at a dose of 10(-4) M significantly blocked bilirubin neurotoxicity. On the other hand, LC significantly increased bilirubin toxicity at a higher dose (10(-3) M). LC at the doses of 10(-5) and 10(-6) M was found to be ineffective. 10(-4) M LC decreased bilirubin-induced neuronal cell death from 47.72+/-3.68 to 27.23+/-5.14%, (P=0.003). The present study demonstrates, for the first time, that LC protects against bilirubin neurotoxicity in a dose-dependent manner in cerebellar granular cell culture of rats. Further research is needed to confirm our findings and to clarify the mechanisms responsible for the protective effect of LC.     

Estradiol enhances the neurotoxicity of glutamate in GT1-7 cells through an estrogen receptor-dependent mechanism

Glutamate plays an important role in neuroendocrine regulation of reproduction through acting on the N-methyl-D-asparate receptor (NMDAR) in the preoptic area (POA). However, a larger dose of glutamate is neurotoxic. Estradiol (E2) increases the responsiveness of neurons to glutamate through activation and/or expression of NMDAR. In order to investigate whether estradiol modulates the neurotoxic effect of glutamate on the neurons through estrogen receptor (ER), immortalized GT1-7 cells, which simultaneously express ER and NMDAR were used. Tamoxifen and ICI 182,780, ER antagonist, were used to investigate whether the ER is involved in the effect of estradiol on glutamate-induced neurotoxicity. MK-801, a NMDAR antagonist, was used to confirm the enhancement of NMDAR-mediated neurotoxicity by estradiol. Neurotoxicity was evaluated by cell viability and LDH efflux. Cell death was observed by flow cytometry and DNA fragmentation. The results showed that: (1) estradiol (10 nM, incubated for 3 days) significantly enhanced the glutamate-induced neuronal death; (2) the percentages of necrosis and apoptosis were elevated after glutamate treatment, and estradiol significantly enhanced the glutamate-induced cell death; (3) glutamate-induced DNA fragmentation was enhanced by E2-pretreatment; (4) the induction of cell death and increase of LDH efflux after glutamate treatment were also enhanced by E2-pretreatment; (5) both the tamoxifen and ICI 182,780 abolished the estradiol-enhanced NMDAR expression and neurotoxicity of glutamate; (6) higher dose of MK-801 (2 microM) was needed in E2-pretreated cells than in non-E2-pretreated group to block the glutamate-induced neurotoxicity. These results suggested that pretreatment of estradiol might enhance the expression of NMDAR and subsequent glutamate-induced neurotoxicity on the GT1-7 cells through an ER-dependent manner.     

p21(WAF1/Cip1) is not involved in kainic acid-induced apoptosis in murine cerebellar granule cells

Kainic acid (KA) treatment induced neuronal death and apoptosis in murine cerebellar granule cells (CGNs) cultures from both wild-type and knockout p21(-/-) mice. There was not statistically significant difference in the percentage of neuronal apoptosis among strains. KA-induced neurotoxicity was prevented in the presence of NBQX (20 microM) and GYKI 52446 (20 microM), but not by z-VAD-fmk, suggesting that caspases are not involved in the apoptotic process. Data suggest that p21(WAF/Cip) was unable to modulate KA-induced apoptosis in murine CGNs.     

Role of Transient Receptor Potential Channel 1 (TRPC1) in Glutamate-Induced Cell Death in the Hippocampal Cell Line HT22

Transient receptor potential channel 1 (TRPC1; a cation channel activated by store depletion and/or through an intracellular messenger) is expressed in a variety of tissues, including the brain. To study the physiological function of TRPC1, we investigated the role of endogenously expressed TRPC1 in glutamate-induced cell death, using the murine hippocampal cell line HT22. Knocking down TRPC1 mRNA using TRPC1-shRNA or blocking of TRPC channels using 2-APB (≥200 μM) robustly attenuated glutamate-induced cell death after 24 h of incubation with 5 mM glutamate. Glutamate toxicity in HT22 cells seems to involve metabotropic glutamate receptor mGluR5 since MPEP (2-methyl-6-(phenylethynyl)-pyridine), an mGluR5 antagonist (≥100 μM), abrogated glutamate toxicity. Furthermore, a direct activation of mGluR5 by CHPG [(RS)-chloro-5-hydroxyphenylglycine; 100 μM or 300 μM] promoted HT22 cell death. TRPC1 knock-down markedly reduced CHPG-induced cell death. These observations suggest that glutamate-induced cell death in HT22 cells activates mGluR5 receptors, which significantly increases Ca²⁺ influx through TRPC1 channels. TRPC1 knock-down prevented glutamate- and CHPG-induced cell death, suggesting that glutamate-induced toxicity in HT22 cells is mediated through TRPC1 channels and an mGluR5-dependent pathway. Together, this work provides evidence for a novel receptor activation pathway of TRPC1 in glutamate-induced toxicity.     

alpha-Amino-3-hydroxy-5-methyl-4-isoxazole propionate attenuates glutamate-induced caspase-3 cleavage via regulation of glycogen synthase kinase 3beta

Preconditioning of sublethal ischemia exhibits neuroprotection against subsequent ischemia-induced neuronal death. It has been indicated that glutamate, an excitatory amino acid, is involved in the pathogenesis of ischemia-induced neuronal death or neurodegeneration. To elucidate whether prestimulation of glutamate receptor could counter ischemia-induced neuronal death or neurodegeneration, we examined the effect of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), an ionotropic subtype of glutamate receptor, on excess glutamate-induced excitotoxicity using primary cortical neuronal cultures. We found that AMPA exerted a neuroprotective effect in a time- and concentration-dependent manner. A blocker of phosphatidylinositol-3 kinase (PI3K), LY294002 (10 microM), significantly attenuated AMPA-induced protection. In addition, Ser473 of Akt/PKB, a downstream target of PI3K, was phosphorylated by AMPA administration (10 microM). Glycogen synthase kinase 3beta (GSK3beta), which has been reported to be inactivated by Akt, was phosphorylated at Ser9 by AMPA. Ser9-phosphorylated GSK3beta or inactivated form would be a key molecule for neuroprotection, insofar as lithium chloride (100 microM) and SB216763 (10 microM), inhibitors of GSK3beta, also induced phosphorylation of GSK3beta at Ser9 and exerted neuroprotection, respectively. Glutamate (100 microM) increased cleaved caspase-3, an apoptosis-related cysteine protease, and caspase-3 inhibitor (Ac-DEVD-CHO; 1 microM) blocked glutamate-induced excitotoxicity in our culture. AMPA (10 microM, 24 hr) and SB216763 (10 microM) prominently decreased glutamate-induced caspase-3 cleavage. These findings suggest that AMPA activates PI3K-Akt and subsequently inhibits GSK3beta and that inactivated GSK3beta attenuates glutamate-induced caspase-3 cleavage and neurotoxicity.     

TNF alpha potentiates glutamate neurotoxicity by inhibiting glutamate uptake in organotypic brain slice cultures: neuroprotection by NF kappa B inhibition

Glutamate and the proinflammatory cytokine, tumor necrosis factor alpha (TNF alpha), have been suggested to contribute to neurodegenerative diseases. We investigated the interaction of TNF alpha and glutamate on neuronal cell death using fluorescence propidium iodide uptake in rat organotypic hippocampal-entorhinal cortex (HEC) brain slice culture that maintains the cytoarchitecture of the intact brain. Time course and concentration studies indicate that glutamate produced significant neuronal cell death in all four brain areas examined, for example, entorhinal cortex, hippocampal CA1 and CA3 fields, and dentate gyrus. TNF alpha alone at concentration of 20 ng/ml caused little or no detectable neuronal cell death, however, when combined with submaximal glutamate (3.3 mM), TNF alpha significantly increased and accelerated glutamate neurotoxicity. TNF alpha potentiation of glutamate neurotoxicity is blocked by NMDA receptor antagonists but not by AMPA antagonists CNQX and NBQX. Studies directly measuring [14C]-glutamate uptake in HEC slices indicate that TNF alpha dose-dependently inhibited glutamate uptake. Further, inhibitors of glial glutamate transporters potentiated glutamate neurotoxicity similar to TNF alpha. The antioxidant butylated hydroxytoluene (BHT) and the NF kappa B inhibitor PTD-p65 peptide inhibit NF kappa B activation and TNF alpha potentiation of glutamate neurotoxicity. BHT prevented the inhibition of TNFalpha on glutamate transport in HEC slices and also blocked nuclear translocation of NF kappa B subunit p65. These data indicate that TNF alpha and glutamate can act synergistically to induce neuronal cell death. TNF alpha potentiation of glutamate neurotoxicity through the blockade of glutamate transporter activity may represent an important mechanism of neurodegeneration associated with neuroinflammation.     

A single oral dose of geranylgeranylacetone attenuates kainic acid-induced seizures and neuronal cell death in rat hippocampus

The present study evaluated the potential effect of geranylgeranylacetone (GGA), which is known as an antiulcer agent, against kainic acid (KA)-induced neurotoxicity. Pretreatment with a single oral GGA dose (800 mg/kg, 2 days before KA) significantly attenuated KA-induced seizures and cell death in rat hippocampus. These effects of GGA were prevented by the coinjection of MK801, a noncompetitive N-methyl-D-aspartate glutamate receptor antagonist, which indicates that the protection was indeed mediated by glutamate receptor activation.     

Repeated intracerebroventricular infusion of nicotine prevents kainate-induced neurotoxicity by activating the alpha7 nicotinic acetylcholine receptor

We examined whether (-)-nicotine infusion can affect kainic acid (KA)-induced neurotoxicity in rats. Although treatment with a single nicotine infusion (0.5 or 1.0 microg/side, i.c.v.) failed to attenuate KA-induced neurotoxicity, repeated nicotine infusions (1.0 microg/side/day for 10 days) attenuated the seizures, the severe loss of cells in hippocampal regions CA1 and CA3, the increase in activator protein (AP)-1 DNA binding activity, and mortality after KA administration. alpha-Bungarotoxin and mecamylamine blocked the neuroprotective effects of nicotine. These results suggest that repeated nicotine treatment provides alpha7 nicotinic acetylcholine receptor-mediated neuroprotection against KA toxicity.     

Protective effect of topiramate on kainic acid–induced cell death in mice hippocampus

The protective effect of topiramate (TPM) on seizure-induced neuronal injury is well known; however, its molecular basis has yet to be elucidated. We investigated the effect and signaling mediators of TPM on seizure-induced hippocampal cell death in kainic acid (KA)-treated ICR mice. KA-induced hippocampal cell death was identified by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling. Immunoreactivity (IR) of p-Erk, p-Jnk, p-P38, and caspase-3, and caspase-3 activity were observed in the hippocampal region 3 h after KA (0.1 μg/5 μL, i.c.v.) administration, and/or TPM (100 mg/kg, i.p.) pretreatment. TPM attenuated seizure-induced neuronal cell death and reduced KA-induced p-Erk IR in the CA3 region of the hippocampus, but did not affect p-Jnk and p-P38. In addition, TPM reduced caspase-3 IR and activation by KA. KA-induced seizures were also suppressed by TPM pretreatment. TPM inhibits seizures, and decreases Erk phosphorylation and caspase-3 activation by KA, thereby contributing to protection from neuronal injury.     

Protection against kainate neurotoxicity by ginsenosides: attenuation of convulsive behavior, mitochondrial dysfunction, and oxidative stress

We previously demonstrated that kainic acid (KA)-mediated mitochondrial oxidative stress contributed to hippocampal degeneration and that ginsenosides attenuated KA-induced neurotoxicity and neuronal degeneration. Here, we examined whether ginsenosides affected KA-induced mitochondrial dysfunction and oxidative stress in the rat hippocampus. Treatment with ginsenosides attenuated KA-induced convulsive behavior dose-dependently. KA treatment increased lipid peroxidation and protein oxidation and decreased the reduced glutathione/oxidized glutathione (GSH/GSSG) ratio to a greater degree in the mitochondrial fraction than in the hippocampal homogenate. KA treatment resulted in decreased Mn-superoxide dismutase expression and diminished the mitochondrial membrane potential. Furthermore, KA treatment increased intramitochondrial Ca(2+) and promoted ultrastructural degeneration in hippocampal mitochondria. Treatment with ginsenosides dose-dependently attenuated convulsive behavior and the KA-induced mitochondrial effects. Protection appeared to be more evident in mitochondria than in tissue homogenates. Collectively, the results suggest that ginsenosides prevent KA-induced neurotoxicity by attenuating mitochondrial oxidative stress and mitochondrial dysfunction.     

Protective effect of topiramate on kainic acid-induced cell death in mice hippocampus.

The protective effect of topiramate (TPM) on seizure-induced neuronal injury is well known; however, its molecular basis has yet to be elucidated. We investigated the effect and signaling mediators of TPM on seizure-induced hippocampal cell death in kainic acid (KA)-treated ICR mice. KA-induced hippocampal cell death was identified by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Immunoreactivity (IR) of p-Erk, p-Jnk, p-P38, and caspase-3, and caspase-3 activity were observed in the hippocampal region 3 h after KA (0.1 microg/5 microL, i.c.v.) administration, and/or TPM (100 mg/kg, i.p.) pretreatment. TPM attenuated seizure-induced neuronal cell death and reduced KA-induced p-Erk IR in the CA3 region of the hippocampus, but did not affect p-Jnk and p-P38. In addition, TPM reduced caspase-3 IR and activation by KA. KA-induced seizures were also suppressed by TPM pretreatment. TPM inhibits seizures, and decreases Erk phosphorylation and caspase-3 activation by KA, thereby contributing to protection from neuronal injury.     

Protection in Glutamate-Induced Neurotoxicity by Imidazoline Receptor Agonist Moxonidine

In the present study we investigated the effects of mixed imidazoline-1 and α₂-adrenoceptor agonist, moxonidine, in glutamate-induced neurotoxicity in frontal cortical cell cultures of rat pups by dye exclusion test. Also, phosphorylated p38 mitogen activated protein kinases (p-p38 MAPK) levels were determined from rat frontal cortical tissue homogenates by two dimensional gel electrophoresis and semidry western blotting. Glutamate at a concentration of 10^?6 M was found neurotoxic when applied for 16 hr in cell cultures. Dead cell mean scores were 12.8 ± 0.5 for control and 52.3 ± 4.8 for glutamate (p < .001). On the other hand, p-p38 MAPK levels start to increase at a glutamate concentration of 10^?7 M for 20 min application. Moxonidine was found to have an U-shape neuroprotective effect in glutamate-induced neurotoxicity in neuronal cell culture experiments. Even though moxonidine did not induce neurotoxicity alone between the doses of 10^?8 to 10^?4 M concentrations in cell culture series, it caused the reduction of glutamate-induced dead cell population 23.07 ± 3.6% in 10^?6 M and 26.7 ± 2.1% in 10^?5 M concentrations (p <.001 for both, in respect to control values). The protective effect of moxonidine was confirmed in 10^?8 and 10^?7 M, but not in higher concentrations in glutamate neurotoxicity in gel electrophoresis and western blotting of p-p38 MAPK levels. In addition to other studies that revealed an antihypertensive feature of moxonidine, we demonstrated a possible partial neuroprotective role in lower doses for it in glutamate-mediated neurotoxicity model.     

Kainate-induced apoptosis correlates with c-Jun activation in cultured cerebellar granule cells

We have investigated the involvement of c-Jun in cell death induced by exposure of primary cultures of murine cerebellar granule cells to the glutamate receptor agonist kainate (KA) and evaluated its possible use as a marker for apoptosis. Using cerebellar granule cell neurones from postnatal day 7 mice, we found that 1 hr exposure to KA (1–1000 μM) induced a concentration-dependent neuronal cell death with characteristic apoptotic morphology, including cell shrinkage, neurite blebbing and DNA fragmentation. In addition KA-induced a concentration-dependent expression of c-Jun mRNA and protein as determined by in situ hybridization and immunocytochemistry respectively. DNA fragmentation was detected using terminal transferase-mediated nick-end (TUNEL) labelling and agarose gel electrophoresis. KA-induced cell death was significantly attenuated by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 μM), which shifted the concentration-response curve significantly rightward. The number of apoptotic cell bodies, determined by TUNEL, was also reduced by CNQX (50 μM), with only 15–20% of neurones staining positive after exposure to 1mM KA. In addition, the number of positively stained cells for c-Jun protein and mRNA was substantially reduced by CNQX (50 μM) as determined by random and representative cell counts. These results show for the first time that KA induced apoptotic neuronal death in cultured murine cerebellar granule cells involves the induction of c-Jun mRNA and protein, suggesting the involvement of this immediate early gene in excitotoxic receptor-mediated apoptosis and its potential use as a marker for apoptotic cell death. J. Neurosci. Res. 52:69–82, 1998. © 1998 Wiley-Liss, Inc.     

Intracellular glutathione levels determine cerebellar granule neuron sensitivity to excitotoxic injury by kainic acid

Glutathione (GSH) is a key component of the cellular defence cascade against injury caused by reactive oxygen species. Kainic acid (KA) is a potent central nervous system excitotoxin. KA-elicited neuronal death may result from the generation of ROS. The present study was undertaken to characterize the role of GSH in KA-induced neurotoxicity. Cultures of cerebellar granule neurons were prepared from 8-day-old rats, and used at 8, 14 and 20 days in vitro (DIV). Granule neurons displayed a developmental increase in their sensitivity to KA injury, as quantified by an ELISA-based assay with the tetrazolium salt MTT. At DIV 14 and 20, a 30-min challenge with KA (500 microM) reduced cell viability by 45% after 24 h, significantly greater (P<0.01) than the 22% cell loss with DIV 8 cultures. Moreover acute (30 min) KA exposure concentration-dependently reduced intracellular GSH and enhanced reactive oxygen species generation (evaluated by 2', 7'-dichlorofluorescein diacetate). In comparison to control, KA (500 microM) lowered GSH levels in DIV 8 granule neurons by 16% (P=0. 0388), and by 36% (P=0.0001) in both DIV 14 and DIV 20 neurons, after 30 min. Preincubation of granule neurons with the membrane permeant GSH delivery agent, GSH ethyl ester (5 mM), for 30 min significantly increased intracellular GSH content. Importantly, GSH ethyl ester reduced the toxic effects of KA, becoming significant at 1 mM (P=0.007 vs. KA-treated group), and was maximal at >/=2.5 mM (P<0.0001). GSH ethyl ester displayed a similar dose-dependence in its ability to counteract KA-induced depletion of cellular GSH. The data strengthen the notion that cellular GSH levels have a fundamental role in KA-induced neurotoxicity.     

Roles of adenosine receptors in the regulation of kainic acid-induced neurotoxic responses in mice

Kainic acid (KA) is a well-known excitatory and neurotoxic substance. In ICR mice, morphological damage of hippocampus induced by KA administered intracerebroventricularly (i.c.v.) was markedly concentrated on the hippocampal CA3 pyramidal neurons. In the present study, the possible role of adenosine receptors in hippocampal cell death induced by KA (0.1 microg) administered i.c.v. was examined. It has been shown that 3,7-dimethyl-1-propargylxanthine (DMPX; A2 adenosine receptors antagonist, 20 microg) reduced KA-induced CA3 pyramidal cell death. KA dramatically increased the phosphorylated extracellular signal-regulated kinase (p-ERK) immunoreactivities (IR) in dentate gyrus (DG) and mossy fibers. In addition, c-Jun, c-Fos, Fos-related antigen 1 (Fra-1) and Fos-related antigen 2 (Fra-2) protein levels were increased in hippocampal area in KA-injected mice. DMPX attenuated KA-induced p-ERK, c-Jun, Fra-1 and Fra-2 IR. However, 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine (PACPX; A1 adenosine receptor antagonist, 20 microg) did not affect KA-induced p-ERK, c-Jun, Fra-1 and Fra-2 IR. KA also increased the complement receptor type 3 (OX-42) IR in CA3 region of hippocampus. DMPX, but not PACPX, blocked KA-induced OX-42 IR. Our results suggest that p-ERK and c-Jun may function as important regulators responsible for the hippocampal cell death induced by KA administered i.c.v. in mice. Activated microglia, which was detected by OX-42 IR, may be related to phagocytosis of degenerated neuronal elements by KA excitotoxicity. Furthermore, it is implicated that A2, but not A1, adenosine receptors appear to be involved in hippocampal CA3 pyramidal cell death induced by KA administered i.c.v. in mice.     

Selective mGluR1 Antagonist EMQMCM Inhibits the Kainate-Induced Excitotoxicity in Primary Neuronal Cultures and in the Rat Hippocampus

Abundant evidence suggests that indirect inhibitory modulation of glutamatergic transmission, via metabotropic glutamatergic receptors (mGluR), may induce neuroprotection. The present study was designed to determine whether the selective antagonist of mGluR1 (3-ethyl-2-methyl-quinolin-6-yl)-(4-methoxy-cyclohexyl)-methanone methanesulfonate (EMQMCM), showed neuroprotection against the kainate (KA)-induced excitotoxicity in vitro and in vivo. In in vitro studies on mouse primary cortical and hippocampal neuronal cultures, incubation with KA (150 μM) induced strong degeneration [measured as lactate dehydrogenase (LDH) efflux] and apoptosis (measured as caspase-3 activity). EMQMCM (0.1-100 μM) added 30 min to 6 h after KA, significantly attenuated the KA-induced LDH release and prevented the increase in caspase-3 activity in the cultures. Those effects were dose- and time-dependent. In in vivo studies KA (2.5 nmol/1 μl) was unilaterally injected into the rat dorsal CA1 hippocampal region. Degeneration was calculated by counting surviving neurons in the CA pyramidal layer using stereological methods. It was found that EMQMCM (5-10 nmol/1 μl) injected into the dorsal hippocampus 30 min, 1 h, or 3 h (the higher dose only) after KA significantly prevented the KA-induced neuronal degeneration. In vivo microdialysis studies in rat hippocampus showed that EMQMCM (100 μM) significantly increased γ-aminobutyric acid (GABA) and decreased glutamate release. When perfused simultaneously with KA, EMQMCM substantially increased GABA release and prevented the KA-induced glutamate release. The obtained results indicate that the mGluR1 antagonist, EMQMCM, may exert neuroprotection against excitotoxicity after delayed treatment (30 min to 6 h). The role of enhanced GABAergic transmission in the neuroprotection is postulated.     

Inhibition of hexokinase leads to neuroprotection against excitotoxicity in organotypic hippocampal slice culture

During seizures, glucose concentrations are high in the hippocampus. Mitochondrial hexokinase (HK) catalyzes the first essential step of glucose metabolism and directly couples extramitochondrial glycolysis to intramitochondrial oxidative phosphorylation. The neuroprotective effects of an HK inhibitor, 3-bromopyruvate (3-BrPA), on kainic acid (KA)-induced excitotoxic injury were investigated. Hippocampal slices were prepared from hippocampi of 6-8-day-old rats using a tissue chopper and placed on a membrane insert. After a treatment with KA (5 μM) for 15 hr, neuronal death was quantified by propidium iodide (PI), cresol violet, and TUNEL staining. KA-induced cell death was significantly prevented by 30 μM 3-BrPA treatment. According to Western blots, the expression level of phospho-Akt increased after 3-BrPA treatment. The induction of long-term potentiation (LTP) at 48 hr after 3-BrPA treatment tended to increase in the CA1 area compared with the KA-only group, but the difference was not significant. Blocking the PI3 kinase/Akt pathway using LY294002 reversed the neuroprotective effect of 3-BrPA. These results suggest that inhibition of HK may play a protective role against neuronal death in KA-induced excitotoxic injury.     

Protective effects of vasoactive intestinal peptide against delayed glutamate neurotoxicity in cultured retina

The effects of vasoactive intestinal peptide (VIP) on glutamate-induced delayed death were examined using the primary cultures of rat retinal neurons. Effects of VIP on glutamate-induced neurotoxicity were evaluated by double staining with fluorescein diacetate and propidium iodide. Glutamate (1 mM) was applied to the culture for 10 min in the presence and absence of VIP, and visible cells enumerated 24 h after culture in normal medium. Effects of VIP on increase in the intracellular Ca²⁺ concentration and currents induced by glutamate in retinal neurons were investigated using the Ca²⁺ image analyzing system with fura-2 and whole-cell patch-clamp recording, respectively. The cAMP contents in retinal cultures were measured by radioimmunoassay. VIP (10 nM–1 μM) dose-dependently protected against glutamate-induced neurotoxicity in cultured retinal neurons. Protection by VIP (100 nM) against glutamate (1 mM)-induced neurotoxicity was antagonized by VIP6-28 (1 μM), a VIP antagonist, and H-89 (100 nM and 1 μM), a protein kinase A inhibitor. However, VIP had no effect on glutamate-induced inward currents nor glutamate-induced increase in the intracellular Ca²⁺ concentration. A 10-min exposure of VIP (100 nM) with glutamate (1 mM) resulted in an increase in the cAMP level to 446±58 from 22±1 pmol/mg protein. These findings suggest that VIP protects against the glutamate-induced neurotoxicity in retinal cultures by elevating the cAMP level via VIP receptors and thereby activating protein kinase A.     

Kainic acid-induced lipid peroxidation: protection with butylated hydroxytoluene and U78517F in primary cultures of cerebellar granule cells

The generation of free radicals in the progression of kainic acid (KA)-mediated neuronal death has been implicated in both in vitro and in vivo studies. In the present study, the association between KA-induced neurodegeneration and the appearance of lipid peroxidation products was investigated and compared to three well characterized free radical generating (FRG) systems: 200 μM ferrous ammonium sulfate (FAS), 20 μM opper (Cu²⁺), and 0.01 U/ml xanthine oxidase/ μM transferrin (XO). KA caused a dose-dependent increase in conjugated diene and lipid hydroperoxide formation as did the FRG systems. The antioxidant, butylated hydroxytoluene (BHT), decreased both FRG system- and KA-induced lipid peroxidation by approximately 60–70%. Unlike BHT, the potency of the lipid peroxidation inhibitor, U78517F, depended upon the system utilized to induce free radical generation. U78517F was most potent in attenuating FAS-induced lipid peroxidation (100 nM), followed by KA (1.5 μM), and then Cu²⁺ and XO (> 2 μM). Results were confirmed by measurement of cytolysis through the release of lactic dehydrogenase (LDH). These data provide further evidence that the generation of free radicals, subsequently leading to membrane disruption, is central to the mechanism of KA-elicited neuronal death in cultures of cerebellar granule cells.     

Localization of kainic acid-sensitive cells in mammalian retina

Short-term (15 minutes) in vitro exposure to kainic acid (KA), a rigid structural analog of L-glutamic acid (Glu), caused two morphologically distinct neuronal lesions in retinas of several species. In rabbit retina, one type of lesion was characterized by rapid swelling after exposure to low concentrations of KA (10^?4 M). This lesion was observed in elements of both plexiform layers and, more specifically, in cell bodies and neurites of horizontal cells that contact cones. A few cell bodies from the amacrine cell layer showed some limited swelling. The swelling was completely blocked when sodium was removed from the incubation medium. The second type of lesion was generally seen after longer exposures of after exposure to higher concentrations of KA and was evidenced by degeneration of neurons in the amacrine and ganglion cell layers. One exception was noted in that a few cells from the ganglion cell layer degenerated even under low exposure conditions. The second type of lesion was not blocked by removal of sodium ions. Photoreceptor cells appeared resistant to all effects of KA. The results suggest that a correlation may exist between certain KA-induced lesions of the retina and putative glutamoreceptive neurons. At the same time, the two types of retinal lesions produced by KA are morphologically and chemically differentiable and may be useful in elucidating the differences between specific, Glu-related toxicity and nonspecific toxicity of KA.