Cerebral Cortical Cultures Research Articles

Antiserum to activity-dependent neurotrophic factor produces neuronal cell death in CNS cultures: immunological and biological specificity

Activity-dependent neurotrophic factor (ADNF) is a glia-derived protein that is neuroprotective at femtomolar concentrations. ADNF is released from astroglia after treatment with 0.1 nM vasoactive intestinal peptide (VIP). To further assess the biological role of ADNF, antiserum was produced following sequential injections of purified ADNF into mice. Anti-ADNF ascites fluid (1:10,000) decreased neuronal survival by 45–55% in comparison to untreated cultures or those treated with control ascites. The neuronal death after anti-ADNF treatment was observed in cultures derived from the spinal cord, hippocampus or cerebral cortex at similar IC 50's. Using a terminal deoxynucleotidyl transferase in situ assay to estimate apoptosis in cerebral cortical cultures, anti-ADNF was shown to produce a 70% increase in the number of labeled cells in comparison to controls. In spinal cord cultures, anti-ADNF treatment produced a 20% decrease in choline acetyltransferase activity in comparison to controls. Neuronal cell death produced by the antiserum to ADNF was prevented in cultures co-treated with purified ADNF or ADNF-15, an active peptide derived from the parent ADNF. In vitro binding between the anti-ADNF and ADNF-15 was demonstrated with size exclusion chromatography. Comparative studies with other growth factors (insulin-like growth factor-1, platelet-derived growth factor, nerve growth factor, epidermal growth factor, ciliary neurotrophic growth factor, and neurotrophin-3) demonstrated that only ADNF prevented neuronal cell death associated with electrical blockade. These investigations indicated that an ADNF-like substance was present in cultures derived from multiple locations in the central nervous system and that ADNF-15 exhibited both neuroprotection and immunogenicity. ADNF appears to be both a regulator of activity-dependent neuronal survival and a neuroprotectant. © 1997 Elsevier Science B.V. All rights reserved.

Extracellular ATP induces formation of AP-1 complexes in astrocytes via P2 purinoceptors.

The transcription activator protein-1 (AP-1) complex is a heterodimer consisting of Fos and Jun family members. We found that extracellular ATP stimulated AP-1 DNA binding activity in cerebral cortical astrocyte cultures. This activity was maximal at 1 h and persisted for at least 3 h post-treatment. Shift-Western blotting indicated the presence of c-Fos in the AP-1 complexes. Stimulation of AP-1 binding by ATP was due to activation of P2 rather than P1 purinoceptors. The protein kinase C (PKC) inhibitor Ro 31-8220 markedly reduced P2 purinoceptor-mediated AP-1 induction. The induction of AP-1 complexes by ATP may contribute to changes in gene expression which underlie the trophic effects of extracellular ATP on astrocytes.

ω-Agatoxin IVA and excitotoxicity in cortical neuronal cultures

Because the P- and Q-type Ca 2+ channel antagonist w-agatoxin IVA inhibits glutamate release, agatoxin-related drugs could have a role in the treatment of excitotoxic neurologic disorders such as stroke. We examined the effect of w-agatoxin IVA on excitotoxicity (lactate dehydrogenase release) induced by depolarization with veratridine or ouabain, or by N-methyl- d-aspartate in neuron-enriched cerebral cortical cultures. ω-Agatoxin IVA (≤5300 nM) failed to reduce excitotoxicity, as did the L-type Ca 2+ channel antagonist nimodipine and the N-type antagonist w-conotoxin GVIA. Drugs that inhibit glutamate release may not necessarily be neuroprotective.

Neuroprotective strategy for Alzheimer disease: intranasal administration of a fatty neuropeptide.

Neurodegenerative diseases, in which neuronal cell disintegrate, bring about deteriorations in cognitive functions as is evidenced in millions of Alzheimer patients. A major neuropeptide, vasoactive intestinal peptide (VIP), has been shown to be neuroprotective and to play an important role in the acquisition of learning and memory. A potent lipophilic analogue to VIP now has been synthesized, [stearyl-norleucine17]VIP ([St-Nle17]VIP), that exhibited neuroprotection in model systems related to Alzheimer disease. The beta-amyloid peptide is a major component of the cerebral amyloid plaque in Alzheimer disease and has been shown to be neurotoxic. We have found a 70% loss in the number of neurons in rat cerebral cortical cultures treated with the beta-amyloid peptide (amino acids 25-35) in comparison to controls. This cell death was completely prevented by cotreatment with 0.1 pM [St-Nle17]VIP. Furthermore, characteristic deficiencies in Alzheimer disease result from death of cholinergic neurons. Rats treated with a cholinergic blocker (ethylcholine aziridium) have been used as a model for cholinergic deficits. St-Nle-VIP injected intracerebroventricularly or delivered intranasally prevented impairments in spatial learning and memory associated with cholinergic blockade. These studies suggest both an unusual therapeutic strategy for treatment of Alzheimer deficiencies and a means for noninvasive peptide administration to the brain.

Mechanisms of estrogen action during neural development: Mediation by interactions with the neurotrophins and their receptors?

Estrogen enhances the growth and differentiation of neurites within the developing forebrain. A critical issue is whether these developmental actions of estrogen are mediated directly or indirectly by means of autocrine responses or local paracrine mechanisms, through interactions with growth factors, such as the neurotrophins, and their receptors. Support for the latter hypothesis comes from our recent observations of co-expression of estrogen receptor mRNA with the mRNAs for the neurotrophins and their receptors; differential and reciprocal up-regulation of estrogen and NGF receptor mRNA and protein expression by estrogen in adult female rat sensory neurons, PC12 cells; and cerebral cortical cultures; and putative estrogen response elements in the NGF, BDNF, trkA and p75 genes. Estrogen and the neurotrophins may influence each other's actions by regulating receptor and ligand availability or by reciprocal regulation at the level of signal transduction or gene transcription. The neurotrophins may serve as regulatory “switches” for the apparent developmentally-regulated, differential pattern of estrogen receptor regulation by its ligand, whereby their ability to increase estrogen receptor levels significantly may be sufficient to override the intrinsic suppressive action of estrogen on its receptor. Estrogen and the neurotrophins, acting in concert and reciprocally, may stimulate the synthesis of proteins required for neuronal differentiation, survival and maintenance of function.

Molecular neurobiological approach to the pathogenesis of epilepsy: a preliminary study

We studied the effects and the interactions of some candidate genes related to the pathogenesis of epilepsy using a domestic audiogenic epilepsy-prone rat, matched with the epilepsy-resistant Wistar rat, and primary fetal cerebral cortical neuronal cell cultures of both. The preliminary results showed that there was a possible abnormality of the CCK gene at the post-translational stage in the early postnatal period in P77PMC rat brain; the later rapidly increased rate of CCK-8 synthesis in the hippocampus and subcortical region may represent a compensatory response to the neuronal pathways involved in audiogenic seizures. CCK-8 can decrease the NMDA (1 microM)-induced free intracellular Ca2+ concentration, so it seems to be an inhibitory neuromodulator. In neuronal cell cultures, the NMDA (0.1 microM)-induced c-fos mRNA expression on culture day 18 in vitro was higher in P77PMC than Wistar rats (P < 0.05). Interleukin-1 (20 nM) can induce endogenous opioid mRNA expression and peptide release in neuronal cell cultures, which can be abolished by the addition of antisense oligos of c-fos/c-jun to cell cultures treated with interleukin-1. Both interleukin-1 and opioids can increase the free intracellular Ca2+ concentration, and their specific antagonists can reverse their effects, so they both seem to be excitatory neuromodulators in the CNS.

Kynurenine pathway and cerebral ischemia

Accumulating evidence suggests functional interaction between brain-derived neurotrophic factor (BDNF) and metabotropic glutamate receptor (mGluR) signaling pathways in the central nervous system (CNS). To date, eight subtypes of mGluRs, mGluR1–8, have been identified, and a previous study suggested that BDNF leads to down-regulation of GluR2 mRNA in rat cerebral cortical cultures. However, precise transcriptomic effects of BDNF on other mGluRs and their cellular significance on the BDNF signaling pathway remain largely unknown. In this study, we assessed the transcriptomic effects of BDNF on mGluR1–8 in primary cultures of rat cerebral cortical neurons, and transcriptomic impacts of mGluR(s) whose expression is regulated by BDNF, on BDNF target genes. Real-time quantitative PCR (RT-qPCR) revealed that stimulation of the cultures with 100 ng/mL BDNF led to marked reductions not only in the gene expression levels of mGluR2, but also in those of mGluR3, both of which belong to group II mGluRs (mGluR II). There were, on the other hand, no changes in the amounts of mGluR I (mGluR1 and 5) and III (mGluR4, 6, 7, and 8) mRNA. Further, 10 ng/mL of BDNF, which mainly activates the high-affinity BDNF receptor, TrkB, but not the low-affinity receptor, p75NTR, was able to induce down-regulation of mGluR II mRNA. The BDNF-induced suppression of mGluR II was not significantly attenuated in the presence of tetrodotoxin (TTX), a blocker for voltage-gated sodium channels. In addition, on stimulation with BDNF (100 ng/mL), no significant down-regulation of mGluR II mRNA was seen in cultured astrocytes, which only express the truncated form of TrkB. Finally, we assessed the transcriptomic effect of mGluR II on the expressions of BDNF target genes, BDNF and activity-regulated cytoskeleton-associated protein (Arc). LY404039, an mGluR II agonist, enhanced the BDNF-induced up-regulation of BDNF, but not Arc. On the other hand, LY341495, an mGluR II antagonist, down-regulated BDNF mRNA levels. Collectively, these observations demonstrated the detailed functional interaction between BDNF and mGluR II: Activation of mGluR II positively regulates self-induced BDNF expression, and, in turn, BDNF negatively regulates the gene expression of mGluR II in a neuronal activity-independent manner, in cortical neurons, but not in astrocytes.

FGF-2 and IL-1β regulate S-100β mRNA levels in rat cerebral cortical astrocyte cultures : D.A. Hinkle ∗, J.P. Harney ∗∗, P.J. Yarowsky #, D.C. Hilt §, and P.M. Wise ∗∗. Depts. of ∗Physiology and #Pharm., University of Maryland School of Medicine, Balt., MD 21201, §AMGEN, Thousand Oaks, CA 91320, and Dept. of ∗∗Physiology, Univ. of Kentucky College of Medicine, Lexington,

FGF-2 and IL-1β regulate S-100β mRNA levels in rat cerebral cortical astrocyte cultures : D.A. Hinkle ∗, J.P. Harney ∗∗, P.J. Yarowsky #, D.C. Hilt §, and P.M. Wise ∗∗. Depts. of ∗Physiology and #Pharm., University of Maryland School of Medicine, Balt., MD 21201, §AMGEN, Thousand Oaks, CA 91320, and Dept. of ∗∗Physiology, Univ. of Kentucky College of Medicine, Lexington,

Vulnerability of oligodendroglia to glutamate: pharmacology, mechanisms, and prevention

Periventricular white matter injury, the principal variety of brain injury of the human premature infant, involves differentiating oligodendroglia. Nothing is known of the biochemical mechanism of oligodendroglial death in this disorder. Because an early event in periventricular white matter injury is ischemia-induced axonal disruption and because such axonal destruction could lead to a marked increase in local concentrations of glutamate, we evaluated the vulnerability of differentiating oligodendroglia to glutamate in a culture model. Oligodendroglia were isolated from mixed-glial primary cultures by a selective detachment technique and grown in a primary culture under conditions that lead to differentiation. These oligodendroglia were found to be highly vulnerable to glutamate-induced cell death. The EC50 for glutamate for a 24 hr exposure was approximately 200 microM, comparable to the value reported for neurons in conventional cerebral cortical cultures. Astrocytes, in contrast, were shown to be resistant to as much as 5 mM glutamate. Study of glutamate receptor antagonists and glutamate transport substrates showed that the glutamate-induced oligodendroglial death was not related to a receptor mechanism, as operates in neurons, but rather was secondary to glutamate uptake by the oligodendroglia. Glutamate transport by high-affinity, sodium-dependent and by sodium-independent systems was shown. The central importance of glutamate uptake for the toxic effect of glutamate was shown by total prevention of the oligodendroglial toxicity by the simultaneous inhibition of glutamate uptake by the specific inhibitor D,L-threo-beta-hydroxyaspartate. Subsequent observations showed that the toxicity of glutamate was mediated by free radical attack, the consequence of glutathione depletion, apparently caused by the action of a glutamate-cystine exchange mechanism that results in cystine and thereby glutathione depletion. Thus, addition of cystine or cysteine totally prevented the glutamate toxicity to oligodendroglia. Second, glutamate exposure led to cystine efflux. Third, glutathione levels decreased markedly in cells exposed to glutamate, and this marked decrease preceded the loss of cell viability. Fourth, glutamate toxicity could be prevented totally by exposure to different free radical scavengers, vitamin E and idebenone. The data thus show that glutamate is highly toxic to oligodendroglia. Moreover, the findings raise the possibilities that such glutamate toxicity is operative in the oligodendroglial cell death associated with ischemic processes that disrupt axons, such as periventricular white matter injury of the premature infant, and that novel therapies directed against glutamate transport, glutathione depletion, and free radical attack might be beneficial in prevention of that injury.

Chapter 9 NMDA receptors and AMPA/kainate receptors mediate parallel injury in cerebral cortical cultures subjected to oxygen-glucose deprivation

This chapter discusses two distinct hypotheses to explain a greater value of N -methyl- D -aspartate (NMDA) antagonists as neuroprotective agents in focal ischemia compared with global ischemia—including, (1) the toxic potential of NMDA receptor overactivation is small, and is only a critical factor in neuronal death under certain special conditions, such as in the partial energy depletion associated with the focal ischemic penumbra, (2) The toxic potential of NMDA receptor overactivation is large, but limited by endogenous mechanisms especially active in global ischemia. Only when NMDA receptor-mediated toxicity is suppressed does a more slowly triggered toxicity of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)/kainate receptors become apparent. The critical difference between these two hypotheses is the rank order of NMDA versus AMPA/kainate receptors, with respect to excitotoxic potential. In both hypotheses, the occurrence of calcium overload is suggested to trigger neurodegeneration. Because AMPA/kainate receptor families mediate most fast excitatory synaptic transmission throughout the CNS, they may contribute heavily to sodium overload during and after an ischemic insult, leading subsequently to calcium overload.

Widespread Distribution of Synaptophysin, a Synaptic Vesicle Glycoprotein, in Growing Neurites and Growth Cones.

Synaptophysin, a 38-kD glycoprotein, is one of the most abundant of the integral membrane proteins of small synaptic vesicles. The protein is widely distributed at synapses throughout the nervous system, where it is believed to be involved in the exocytosis of stored neurotransmitter. We show here that synaptophysin is also widely expressed in growing neurites and growth cones both in vitro and in vivo. In dissociated rat cerebral cortical cultures anti-synaptophysin antiserum (G-95) stains growth cones punctately as soon as they emerge from the cell body. In early cultures all neurites are immunoreactive. Later, synaptophysin is redistributed to become concentrated in axonal varicosities. In developing rat embryos, synaptophysin is expressed in the growing axons of, for instance, the spinal commissural interneurons and the parallel fibres of the cerebellar granule cells long before these neurons have established synaptic connections. These observations suggest that synaptic vesicle proteins like synaptophysin are functionally important in neuronal development.

Programmed cell death: its possible contribution to neurotoxicity mediated by calcium channel antagonists

Organic calcium channel antagonists attenuate ischemic or excitotoxic neuronal injury, probably by limiting Ca 2+ influx through the voltage-gated calcium channels. However, the possibility that calcium channel antagonists may compromise neuronal survival with long-term exposure has not been systematically examined. In the present study, we report that cerebral cortical cultures exposed for 2 days to either nifedipine, verapamil, diltiazem, or flunarizine, undergo selective neuronal degradation in a concentration-dependent fashion. This degeneration could be attenuated by protein synthesis inhibitors cycloheximide and actinomycin-D. Cortical cultures incubated for 2 days in low calcium media also exhibit widespread neuronal damage, which is similarly blocked by cycloheximide. Although we cannot exclude other possibilities, these findings suggest that a decrease in intraneuronal calcium levels may trigger synthesis of proteins mediating neuronal cell death. Regardless of the exact toxic mechanisms involved, additional studies on neurotoxicity of calcium channel antagonists seem warranted since some of these compounds are currently being clinically used.

Comparative neurotoxic potential of glutamate, endothelins, and plateletactivating factor in cerebral cortical cultures

The excitatory amino acid glutamate, the peptides endothelin-1 and -3, and the phospholipid platelet-activating factor have been implicated in ischemic injury to the nervous system. To determine if, like glutamate, endothelins and platelet-activating factor are directly toxic to neurons, we examined their effects on lactate dehydrogenase release and trypan blue dye exclusion in rat cerebral cortical cultures. Glutamate (1 mM) increased lactate dehydrogenase release by ∼ 75% and reduced the number of cells excluding trypan blue dye by ∼ 50%. In contrast, endothelins (0.5 and 100 nM) and platelet-activating factor (0.1 and 10 μM) had no effect on these indices of cell injury. Endothelins and platelet-activating factor appear more likely to act on blood vessels than on neurons or glia as mediators of ischemic injury.

Cyclic GMP modulators and excitotoxic injury in cerebral cortical cultures

N-Methyl- d-aspartate (NMDA) receptor activation generates nitric oxide (NO) and cyclic GMP (cGMP) and produces ‘excitotoxic’ neuronal injury. To examine the possible role of cGMP in excitotoxicity, we evaluated the effects of agents that stimulate or inhibit cGMP activity on the release of lactate dehydrogenase from neuron-enriched cortical cultures. cGMP analogs exhibited no toxicity, and inhibitors of guanylate cyclase or of cGMP-dependent enzymes failed to protect cultures from the toxic effects of NMDA or the NO donor sodium nitroprusside. These findings argue against a role for cGMP in the pathogenesis of excitotoxic neuronal injury.

Neuroprotective effects of graded reoxygenation following chronic hypoxia in neuronal cell cultures

The present study was undertaken to investigate the comparative effects of rapid vs graded correction of chronic hypoxia in vitro. Cerebral cortical cell cultures obtained from fetal mice were exposed to 5% O 2 for 24 h and returned immediately to room air for the following 24 h (Group I); comparable cultures were exposed to 5% O 2 for 24 h followed by 10% O 2 for an additional 24 h before return to room air (Group II). At the conclusion of the experimental protocol (time 0), partial pressure of oxygen in the bathing medium of Group I cultures was significantly higher than that of Group II and non-hypoxic controls (151 mmHg vs 124 and 132 mmHg, respectively; P < 0.05). Throughout the recovery period, Group II cultures evidenced improved neuronal survival (e.g. 35,800 vs 17,700 neurons/culture well at time 0, P < 0.01), decreased lactate dehydrogenase efflux into the bathing medium, relative preservation of neuronal morphology, as well as higher specific and clonazepam-displaceable benzodiazepine binding and GABA uptake. Glutamate binding was not differentially affected and glutamine synthetase activity, a predominantly glial marker, was only modestly increased after graded reoxygenation. These results demonstrate that gradual reoxygenation after prolonged hypoxia in vitro (i) improves neuronal survival compared to rapid reoxygenation and (ii) delays the manifestations of metabolic dysfunction even though the length of hypoxic exposure is increased. The findings are also consistent with the concept that a period of relative hyperoxia may contribute to hypoxia-induced neuronal injury.

Degenerative and axon outgrowth-altering effects of phencyclidine in human fetal cerebral cortical cells

Babies born to mothers abusing the psychotomimetic drug phencyclidine (PCP), often show profound deficits in CNS function. It is now reported that PCP can cause progressive degeneration and death in human fetal cerebral cortical neurons in culture and sublethal levels of PCP can inhibit axon outgrowth. In cerebral cortical cell cultures established from 14 week fetuses, exposure to 500 μM PCP resulted in a progressive degeneration of neurons over a 2–8 day period, characterized by early reversible vacuolation of the soma and later irreversible neurite fragmentation and cell death. A sublethal concentration of PCP (100 μM) suppressed axon outgrowth. The adverse effects of PCP on neurons were apparently not due to actions on N- methyl- d-aspartate (NMDA) receptors because: the neurons were not responsive to NMDA during the first 3 weeks in culture; concentrations of PCP that should block NMDA receptors maximally (1–50 μM) did not affect axon outgrowth or cell survival; and another NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV) did not cause neurodegeneration or affect axon outgrowth. Exposure of cultures to the sigma receptor ligand, pentazocine (10 μM), did not significantly affect the survival of neurons and haloperidol did not reduce PCP-induced neurodegeneration, indicating that the effects of PCP were not mediated by sigma receptors. Degenerative effects, similar to those elicited by PCP, were observed in cortical neurons exposed to the K + channel blockers, 4-aminopyridine and tetraethylammonium, a finding consistent with the possibility that the degenerative actions of PCP were mediated by its known inhibitory effects on K + channels. In support of the latter possibility, it was found that PCP caused a progressive elevation in intracellular levels of calcium during several days of exposure. In addition to affecting neurons, PCP and K + channel blockers caused vacuolation and degeneration of astrocytes. Taken together with previous data, indicating that PCP can be concentrated and retained in the fetal CNS, the present data suggest that high levels of PCP can disrupt the normal development of neural circuitry in the human fetus, which would be expected to result in profound functional impairments.

Ethanol inhibits excitotoxicity in cerebral cortical cultures

Excitatory amino acid neurotransmitters have been implicated in the pathogenesis of cerebral ischemia and related forms of acute neuronal injury. Because ethanol inhibits cellular signaling mechanisms activated by excitatory amino acids, we examined its effect on excitatory amino acid-induced toxicity in neuron-enriched cultures prepared from rat cerebral cortex. Both glutamate and N- methyl- d-aspartate (NMDA) were toxic to cultured cortical cells, as demonstrated by a reduction in their ability to exclude trypan blue dye, and this toxicity was reversed by the NMDA antagonist MK-801. Ethanol (100 mM) provided partial protection from the excitotoxic effect of NMDA. Thus ethanol, like conventional excitatory amino acid antagonists, can attenuate excitotoxic neuronal injury in vitro.

Beta-Amyloid peptides destabilize calcium homeostasis and render human cortical neurons vulnerable to excitotoxicity

In Alzheimer's disease (AD), abnormal accumulations of beta-amyloid are present in the brain and degenerating neurons exhibit cytoskeletal aberrations (neurofibrillary tangles). Roles for beta-amyloid in the neuronal degeneration of AD have been suggested based on recent data obtained in rodent studies demonstrating neurotoxic actions of beta-amyloid. However, the cellular mechanism of action of beta-amyloid is unknown, and there is no direct information concerning the biological activity of beta-amyloid in human neurons. We now report on experiments in human cerebral cortical cell cultures that tested the hypothesis that beta-amyloid can destabilize neuronal calcium regulation and render neurons more vulnerable to environmental stimuli that elevate intracellular calcium levels. Synthetic beta-amyloid peptides (beta APs) corresponding to amino acids 1-38 or 25-35 of the beta-amyloid protein enhanced glutamate neurotoxicity in cortical cultures, while a peptide with a scrambled sequence was without effect. beta APs alone had no effect on neuronal survival during a 4 d exposure period. beta APs enhanced both kainate and NMDA neurotoxicity, indicating that the effect was not specific for a particular subtype of glutamate receptor. The effects of beta APs on excitatory amino acid (EAA)-induced neuronal degeneration were concentration dependent and required prolonged (days) exposures. The beta APs also rendered neurons more vulnerable to calcium ionophore neurotoxicity, indicating that beta APs compromised the ability of the neurons to reduce intracellular calcium levels to normal limits. Direct measurements of intracellular calcium levels demonstrated that beta APs elevated rest levels of calcium and enhanced calcium responses to EAAs and calcium ionophore. The neurotoxicity caused by EAAs and potentiated by beta APs was dependent upon calcium influx since it did not occur in calcium-deficient culture medium. Finally, the beta APs made neurons more vulnerable to neurofibrillary tangle-like antigenic changes induced by EAAs or calcium ionophore (i.e., increased staining with tau and ubiquitin antibodies). Taken together, these data suggest that beta-amyloid destabilizes neuronal calcium homeostasis and thereby renders neurons more vulnerable to environmental insults.

Regulation of Cholecystokinin Octapeptide Secretion by Rat Cerebral Cortical Cells in Primary Culture*

The regulation of cholecystokinin octapeptide (CCK-8) secretion was studied using a fetal cerebral cortical cell culture system. After 2-3 weeks in culture, the cells were utilized for short-term release experiments. CCK-8 was measured by RIA and its identity was confirmed by HPLC. Depolarization of the cells with K+ (6 x 10(-2) M) evoked CCK-8 release and this response was blocked by the Ca++ channel blocker verapamil (2 x 10(-5) M) and by Ca++ free medium. The Na+ channel opener veratridine (10(-4) M) stimulated CCK-8 release and was blocked by the Na+ channel blocker tetrodotoxin (10(-6) M) and by Ca++ free medium. The adenylate cyclase activator forskolin (10(-5) M) markedly increased CCK-8 secretion. No changes in CCK-8 release were induced by epinephrine, norepinephrine, dopamine, acetylcholine, or 5-hydroxytryptamine at 10(-5) and 10(-4) M, but gamma-aminobutyric acid (GABA) at 10(-4) M inhibited CCK-8 release. GABA inhibition was reversed by the GABA antagonist picrotoxin (10(-4) M). Both picrotoxin (10(-4) M) and bicuculline (10(-3) M), another GABA receptor antagonist, alone stimulated CCK-8 secretion. These data show that CCK-8 secretion by cerebral cortical cells 1) is stimulated by cell membrane depolarization in a calcium-dependent fashion, 2) is regulated by cAMP, 3) is unaffected by the neurotransmitters characteristic of corticopetal systems, 4) is tonically inhibited by GABA.

NGF and bFGF protect rat hippocampal and human cortical neurons against hypoglycemic damage by stabilizing calcium homeostasis

NGF and bFGF have recently been shown to have biological activity in central neurons, but their normal functions and mechanisms of action are unknown. Since central neurons are particularly vulnerable to hypoglycemia that occurs with ischemia or insulin overdose, we tested the hypothesis that growth factors can protect neurons against hypoglycemic damage. NGF and bFGF each prevented glucose deprivation-induced neuronal damage in human cerebral cortical and rat hippocampal cell cultures (EGF was ineffective). Protection was afforded when the growth factors were administered before (NGF and bFGF) or up to 12 hr following (NGF) the onset of hypoglycemia. Direct measurements of intracellular calcium levels and manipulations of calcium influx demonstrated that sustained elevations in intracellular calcium levels mediated the hypoglycemic damage. NGF and bFGF each prevented the hypoglycemia-induced elevations of intracellular calcium. These findings indicate that growth factors can stabilize neuronal calcium homeostasis in central neurons and thereby protect them against environmental insults.