Excision-activated chloride channels in cultured postnatal rat hippocampal neurons

The non-enveloped picornaviruses, which are particularly resistant to physicochemical inactivation, include the aetiological agents of poliomyelitis, hepatitis A and E and infectious common cold (rhinovirus). In this work we used human rhinovirus type 5 (RV-5) cultivated in VERO cells to study the photoinactivating effects of several phthalocyanines and naphthobenzoporphyrazines. Free RV-5 was photoinactivated by aluminium trisulphonated naphthobenzoporphyrazine at 5 x 10(-8) M concentration. This photosensitizer was also active on replicating virus when the infected VERO cells were treated with 5 x 10(-6) M concentration followed by a very short illumination period. On the other hand, the ZnPc(3-MeO-Py)4 phthalocyanine, which possesses four positive charges, does not photoinactivate free rhinovirus, but this molecule protects VERO cells against RV-5 infection when added to the cultures before virus inoculation, in the presence or absence of subsequent illumination, and may therefore be considered as an antiviral agent in itself.     

Involvement of GABAA receptors in the outgrowth of cultured hippocampal neurons

Whereas GABA is a major inhibitory neurotransmitter in the adult central nervous system, recent experiments performed in our laboratory have shown that the activation of GABAA receptors in the hippocampus leads to excitatory effects during the early post-natal period. The possible consequence of a depolarizing effect of GABA was assessed on the neuritic outgrowth of embryonic hippocampal neurons in culture. No morphological alterations were observed when hippocampal neurons were cultured for three days in the presence of muscimol, a GABAA receptor agonist. In contrast, the neuritic outgrowth of cultured hippocampal neurons was profoundly affected by the presence of bicuculline in the culture medium. In the presence of this GABAA receptor antagonist neurons displayed a reduction in the number of primary neurites and branching points, resulting in a concomitant decrease of the total neuritic length. Thus, this study suggests that GABA, acting on GABAA subtype of receptors, is able to affect the development of the hippocampus.     

Effects of ethanol on glucose transporter expression in cultured hippocampal neurons

Glucose transport was studied in primary hippocampal neuron cultures exposed to ethanol. Immunofluorescent staining with antibodies against neuron-specific enolase and glial fibrillary acidic protein identified approximately 95% of the cultured cells as neurons. Western blot analysis was conducted with polyclonal antisera to glucose transporter isoforms GLUT1 and GLUT3. As previously seen in astrocytes, GLUT1 protein was regulated by the culture medium glucose content. Exposure to 50 and 100 mM of ethanol for 5 hr induced dose-dependent reductions in GLUT1 and GLUT3 protein. In contrast, GLUT1 mRNA abundance was increased relative to controls under the same conditions. Glucose uptake, measured with the nonmetabolized analog, 2-deoxy-D-glucose, was reduced by 50 and 100 mM of ethanol in four experiments. These results indicate a direct effect of ethanol on neuronal glucose transporter expression, which may play a role in the neurotoxic effects of alcohol.     

Calcium-induced activation of the mitochondrial permeability transition in hippocampal neurons

The mitochondrial permeability transition (mPT) has been implicated in both excitotoxic and apoptotic neuronal cell death, despite the fact that it has not been previously identified in neurons. To study the mPT in hippocampal neurons, cultures were loaded with the mitochondrial dye JC-1 and observed with confocal and conventional microscopy. After pretreatment with 4Br-A23187 and subsequent calcium addition, the initially rodlike mitochondria increased in diameter until mitochondria became rounded in appearance. Morphological changes reversed when calcium was removed by EGTA. When neurons were loaded with both fura-2-AM and rhodamine 123, calcium loading produced an increase in cytosolic calcium, mitochondrial depolarization, and similar alterations in mitochondrial morphology. Smaller calcium challenges produced calcium cycling, delaying morphological changes until after secondary depolarization and calcium release to the cytosol. In neurons exposed to glutamate, confocal observation of JC-1 fluorescence revealed comparable changes in mitochondrial morphology that were prevented when barium was substituted for calcium, or following pretreatment with the mPT inhibitor, cyclosporin A. These experiments establish conditions in which the mPT could be observed in situ in neurons in response to calcium loading. In addition, the timing of changes suggested that induction of the permeability transition in situ represents a sequence of multiple events that may reflect the multiple open conformations of the mPT pore.     

Glutamate-dependent astrocyte modulation of synaptic transmission between cultured hippocampal neurons

The idea that astrocytes merely provide structural and trophic support for neurons has been challenged by the demonstration that astrocytes can regulate neuronal calcium levels. However, the physiological consequences of astrocyte-neuron signalling are unknown. Using mixed cultures of rat hippocampal astrocytes and neurons we have determined functional consequences of elevating astrocyte calcium levels on co-cultured neurons. Electrical or mechanical stimulation of astrocytes to increase their calcium level caused a glutamate-dependent slow inward current (SIC) in associated neurons. Microinjection of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) into astrocytes to prevent the stimulus-dependent increase in astrocyte calcium level, blocks the appearance of the neuronal SIC. Pharmacological manipulations indicate that this astrocyte-dependent SIC is mediated by extracellular glutamate acting on N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors. Additionally, stimulation of astrocytes reduced the magnitude of action potential-evoked excitatory and inhibitory postsynaptic currents through the activation of metabotropic glutamate receptors. The demonstration that astrocytes modulate neuronal currents and synaptic transmission raises the possibility that astrocytes play a neuromodulatory role by controlling the extracellular level of glutamate.     

Cannabinoid receptor agonists protect cultured rat hippocampal neurons from excitotoxicity

Cannabinoid receptor agonists act presynaptically to inhibit the release of glutamate. Because other drugs with this action are known to reduce excitotoxicity, we tested several cannabimimetics in a model of synaptically mediated neuronal death. Reduction of the extracellular Mg2+ concentration to 0.1 mM evoked a repetitive pattern of intracellular Ca2+ concentration ([Ca2+]i) spiking that, when maintained for 24 hr, resulted in significant neuronal death. The [Ca2+]i spiking and cell death in this model result from excessive activation of N-methyl-D-aspartate receptors, as indicated by the inhibition of both [Ca2+]i spiking and neuronal death by the N-methyl-D-aspartate receptor antagonist CGS19755 (10 microM). The cannabimimetic drug Win55212-2 (100 nM) completely blocked [Ca2+]i spiking and prevented neuronal death induced by low extracellular Mg2+ concentrations. These effects on [Ca2+]i spiking and viability were stereoselective and were prevented by the CB1 receptor antagonist SR141716 (100 nM). The partial agonist CP55940 (100 nM) also afforded significant protection from excitotoxicity. Cannabimimetic drugs did not protect cells from the direct application of glutamate (30 microM). These data suggest that cannabimimetic drugs may slow the progression of neurodegenerative diseases.     

Excitotoxic activation of the NMDA receptor results in inhibition of calcium/calmodulin kinase II activity in cultured hippocampal neurons

Neurotoxic effects of excitatory amino acids have been implicated in various neurological disorders, and have been utilized for excitotoxic models of delayed neuronal cell death. The excitotoxic glutamate-induced, delayed neuronal cell death also results in inhibition of calcium/calmodulin-dependent kinase II (CaM kinase II). In this report, we characterized the glutamate-induced inhibition of CaM kinase II in relation to loss of intracellular calcium regulation and delayed neuronal cell death. Glutamate (500 microM for 10 min), but not KCl (50 mM), exposure resulted in a significant inhibition of CaM kinase II activity. The inhibition of CaM kinase II activity was observed immediately following excitotoxic glutamate exposure and present at every time point measured. Glutamate-induced inhibition of kinase activity and delayed neuronal cell death was dependent upon both the activation of the NMDA glutamate receptor subtype and the presence of extracellular calcium. The relationship between inhibition of CaM kinase II activity and loss of intracellular calcium regulation was also examined. Experimental conditions which resulted in significant neuronal cell death and inhibition of CaM kinase II activity also resulted in a long-term loss of intracellular calcium regulation. Thus, inhibition of CaM kinase II activity occurred under experimental conditions which resulted in loss of neuronal viability and loss of neuronal calcium regulation. Since the glutamate-induced inhibition of CaM kinase II activity preceded neuronal cell death, the data support the hypothesis that inhibition of CaM kinase II activity may play a significant role in excitotoxicity-dependent, delayed neuronal cell death.     

Synaptic vesicle endocytosis mediates the entry of tetanus neurotoxin into hippocampal neurons

Tetanus neurotoxin causes the spastic paralysis of tetanus by blocking neurotransmitter release at inhibitory synapses of the spinal cord. This is due to the penetration of the toxin inside the neuronal cytosol where it cleaves specifically VAMP/synaptobrevin, an essential component of the neuroexocytosis apparatus. Here we show that tetanus neurotoxin is internalized inside the lumen of small synaptic vesicles following the process of vesicle reuptake. Vesicle acidification is essential for the toxin translocation in the cytosol, which results in the proteolytic cleavage of VAMP/ synaptobrevin and block of exocytosis.     

Modulation of neurite branching by protein phosphorylation in cultured rat hippocampal neurons

The control of branching of axons and dendrites is poorly understood. It has been hypothesized that branching may be produced by changes in the cytoskeleton [F.J. Diez-Guerra, J. Avila, MAP2 phosphorylation parallels dendrite arborization in hippocampal neurones in culture, NeuroReport 4 (1993) 412-419; P. Friedrich, A. Aszodi, MAP2: a sensitive cross-linker and adjustable spacer in dendritic architecture, FEBS Lett. 295 (1991) 5-9]. The assembly and stability of microtubules, which are prominent cytoskeletal elements in both axons and dendrites, are regulated by microtubule-associated proteins, including tau (predominantly found in axons) and MAP2 (predominantly found in dendrites). The phosphorylation state of tau and MAP2 modulates their interactions with microtubules. In their low-phosphorylation states, tau and MAP2 bind to microtubules and increase microtubule assembly and/or stability. Increased phosphorylation decreases these effects. Diez-Guerra and Avila [F.J. Diez-Guerra, J. Avila, MAP2 phosphorylation parallels dendrite arborization in hippocampal neurones in culture, NeuroReport 4 (1993) 412-419] found that protein phosphorylation correlates with neurite branching in cultured rat hippocampal neurons, and hypothesized that increased protein phosphorylation stimulates neurite branching. To test this hypothesis, we cultured rat hippocampal neurons in the presence of specific modulators of serine-threonine protein kinases and phosphatases. Inhibitors of several protein kinases, which would be expected to decrease protein phosphorylation, reduced branching. KT5720, an inhibitor of cyclic AMP-dependent protein kinase, and KN62, an inhibitor of Ca(2+)-calmodulin-dependent protein kinases, inhibited branching of both axons and dendrites. Calphostin C and chelerythrine, inhibitors of protein kinase C, inhibited branching of axons but not dendrites. Treatments that would be expected to increase protein phosphorylation, including inhibitors of protein phosphatases (okadaic acid, cyclosporin A and FK506) and stimulators of PKA (SP-cAMPS) or PKC (phorbol 12-myristate 13-acetate), increased dendrite branching. Only FK506 and phorbol 12-myristate 13-acetate stimulated axon branching. A subset of these agents was tested to confirm their effects on protein phosphorylation in this preparation. Okadaic acid, FK506 and SP-cAMPS all increased protein phosphorylation; KT5720 and KN62 decreased protein phosphorylation. On Western blots, the position of MAP2c extracted from cultures exposed to okadaic acid was slightly shifted toward higher molecular weight, suggesting greater phosphorylation, while the position of MAP2c from cultures exposed to KT5720 and KN62 was slightly shifted toward lower molecular weight, suggesting less phosphorylation. We conclude that protein phosphorylation modulates both dendrite branching and axon branching, but with differences in sensitivity to phosphorylation and/or dephosphorylation by specific kinases and phosphatases.     

Vasoactive intestinal polypeptide enhances the GABAergic synaptic transmission in cultured hippocampal neurons

The whole-cell mode of patch-clamp techniques was used to investigate the effect of vasoactive intestinal polypeptide (VIP) on spontaneous gamma-aminobutyric acid (GABA)-mediated inhibitory postsynaptic currents (IPSCs) of cultured hippocampal neurons. Application of VIP caused a significant increase in the frequency of spontaneous IPSCs with a reversible and dose-dependent manner. VIP had no effect on the mean amplitude and kinetic parameters of spontaneous IPSCs. In the presence of tetrodotoxin, VIP increased the frequency of miniature inhibitory postsynaptic currents (mIPSCs) without affecting their mean magnitude. Forskolin, but not its inactive analog 1,9-dideoxyforskolin, mimicked the stimulatory effect of VIP on spontaneous IPSCs and mIPSCs. VIP and forskolin failed to modulate GABAergic IPSCs in the presence of Rp-cAMPs, a cell permeable antagonist of cAMP-dependent protein kinase (PKA). Calcium channel blocker CdCl2 did not prevent VIP and forskolin from increasing the frequency of mIPSCs. These results suggest that the activation of presynaptic VIP receptor enhances the GABAergic synaptic transmission in cultured hippocampal neurons through the cAMP-PKA pathway and that VIP is likely to increase GABA release by directly stimulating the vesicular release apparatus.     

Ibudilast protects against neuronal damage induced by glutamate in cultured hippocampal neurons

1. The effect of ibudilast, a drug that has been clinically used for asthma and the improvement of cerebrovascular disorders, was examined on glutamate neurotoxicity in cultured neurons from rat hippocampus. 2. The extent of neuronal damage induced by exposure of the neurons to glutamate for 5 min was estimated by the activity of lactate dehydrogenase (LDH) released from degenerated neurons into the medium during a 24 h postexposure period. When ibudilast was added into all pre-incubation, exposure and postexposure media, the extent of neuronal damage decreased to approximately half that of control at an ibudilast concentration of 43 mumol/L. 3. The neuroprotective effects of ibudilast were dose-dependent. Sufficient protection was detected even when ibudilast was added only into the postexposure medium. 4. The extent of 45Ca2+ influx during glutamate exposure was slightly reduced by the addition of ibudilast. Intracellular cAMP, as measured by radioimmunoassay, was increased by neuronal exposure to glutamate and then decreased after the removal of glutamate; however in the presence of ibudilast, AMP was maintained at the high level. 5. These results suggest that protection against glutamate neurotoxicity by ibudilast is not only attributable to the inhibition of phenomena that occur during glutamate exposure, such as Ca2+ influx, but also to some beneficial metabolic changes that are induced by a sustained high level of intracellular cAMP.     

Rapid, activation-induced redistribution of ionotropic glutamate receptors in cultured hippocampal neurons

We have examined the membrane localization of an AMPA receptor subunit (GluR1) and an NMDA receptor subunit (NR1) endogenously expressed in primary cultures of rat hippocampal neurons. In unstimulated cultures, both GluR1 and NR1 subunits were concentrated in SV2-positive synaptic clusters associated with dendritic shafts and spines. Within 5 min after the addition of 100 microM glutamate to the culture medium, a rapid and selective redistribution of GluR1 subunits away from a subset of synaptic sites was observed. This redistribution of GluR1 subunits was also induced by AMPA, did not require NMDA receptor activation, did not result from ligand-induced neurotoxicity, and was reversible after the removal of agonist. The activation-induced redistribution of GluR1 subunits was associated with a pronounced (approximately 50%) decrease in the frequency of miniature EPSCs, consistent with a role of GluR1 subunit redistribution in mediating rapid regulation of synaptic efficacy. We conclude that ionotropic glutamate receptors are regulated in native neurons by rapid, subtype-specific membrane trafficking, which may modulate synaptic transmission in response to physiological or pathophysiological activation.     

Cable properties of cultured hippocampal neurons determined from sucrose-evoked miniature EPSCs

1. The passive cable properties of rat hippocampal neurons in dissociated culture were studied using focal application of hypertonic solution to locally elicit miniature excitatory postsynaptic currents (mEPSCs) on the soma and dendrites. Neurons were filled with Lucifer yellow and portions of their dendritic trees were measured. 2. The average mEPSC measured at the soma appeared smaller and slower as the site of sucrose application was made more distal. Normalizing to a 1-micron diam dendrite, the mean mEPSC peak amplitude and charge was reduced e-fold in 170 and 1,000 microns, respectively, and the mean mEPSC decay time constant was increased e-fold in 150 microns. However, for any particular sucrose site, individual mEPSCs varied widely in their amplitudes and time courses. Plots of individual peak amplitudes versus half-width or rise time showed much overlap for mEPSCs originating from sites as much as 100 microns apart. This suggests that use of such plots to estimate the electrotonic location of synaptic currents is highly prone to error. 3. Averaged mEPSCs recorded when applying sucrose at the soma were poorly fitted by an alpha function but were well-described by an equation of the form mxh, where m incorporates a rise-time constant tau 1 and h a decay time constant tau 2. Averaged fits to mean mEPSCs elicited at the somas of five cells gave (mean +/- SE): peak conductance = 832 +/- 126 pS, tau 1 = 0.29 +/- 0.06 ms, tau 2 = 3.03 +/- 0.24 ms, x = 4.7 +/- 0.7. 4. For three cells, the entire dendritic branch to which sucrose was applied was measured and used to construct a passive cable model. The specific membrane resistance (Rm) and intracellular resistivity (Ri) were varied systematically in the model (assuming membrane capacitance Cm = 1 microF/cm2) to search for the best agreement between the mean mEPSCs and the model. Optimal Rm was found to lie in the range 20-30 k omega cm2, Ri in the range 100-200 omega cm. 5. These results confirm those obtained by other methods and emphasize the considerable cable filtering of fast electrical events in cultured hippocampal neurons.     

Enhancement of neurotransmitter release induced by brain-derived neurotrophic factor in cultured hippocampal neurons

Brain-derived neurotrophic factor (BDNF), like other neurotrophins, has long-term effects on neuronal survival and differentiation; furthermore, recent work has shown that BDNF also can induce rapid changes in synaptic efficacy. We have investigated the mechanism(s) of these synaptic effects on cultured embryonic hippocampal neurons. In the presence of the GABAA receptor antagonist, picrotoxin, the application of BDNF (100 ng/ml) for 1-5 min increased the amplitude of evoked synaptic currents by 48 +/- 9% in 10 of 15 pairs of neurons and increased the frequency of EPSC bursts to 205 +/- 20% of the control levels. There was no detectable effect of BDNF on various measures of electrical excitability, including the resting membrane potential, input resistance, action potential threshold, and action potential amplitude. In addition, BDNF did not change the postsynaptic currents induced by the exogenous application of glutamate. BDNF did increase the frequency of miniature EPSCs (mEPSCs) (268.0 +/- 46.8% of control frequency), however, without affecting the mEPSC amplitude. The effect of BDNF on mEPSC frequency was blocked by the tyrosine kinase inhibitor K252a and also by the removal of extracellular calcium ([Ca2+]o). Fura-2 recordings showed that BDNF elicited an increase in intracellular calcium concentration ([Ca2+]c). This effect was dependent on [Ca2+]o; it was blocked by K252a and by thapsigargin, but not by caffeine. The results demonstrate that BDNF enhances glutamatergic synaptic transmission at a presynaptic locus and that this effect is accompanied by a rise in [Ca2+]c that requires the release of Ca2+ from IP3-gated stores.     

Very short-term plasticity in hippocampal synapses

Hippocampal pyramidal neurons often fire in bursts of action potentials with short interspike intervals (2-10 msec). These high-frequency bursts may play a critical role in the functional behavior of hippocampal neurons, but synaptic plasticity at such short times has not been carefully studied. To study synaptic modulation at very short time intervals, we applied pairs of stimuli with interpulse intervals ranging from 7 to 50 msec to CA1 synapses isolated by the method of minimal stimulation in hippocampal slices. We have identified three components of short-term paired-pulse modulation, including (i) a form of synaptic depression manifested after a prior exocytotic event, (ii) a form of synaptic depression that does not depend on a prior exocytotic event and that we postulate is based on inactivation of presynaptic N-type Ca2+ channels, and (iii) a dependence of paired-pulse facilitation on the exocytotic history of the synapse.     

Muscarinic receptors involved in hippocampal plasticity

The cholinergic septohippocampal system has been associated with learning and memory, as evidenced by the severe loss of these functions in lesioned animals as well as in senile demented patients. In an attempt to comprehend the physiological basis of the cholinergic innervation for hippocampal functions, numerous studies employed the in-vitro hippocampal slice preparation and analyzed the consequences of exposing the cells to cholinergic ligands. Many effects of activating a cholinergic receptor in the hippocampus were thus described, including blockade of several types of potassium conductances, yet few of these effects are intuitively related to the involvement of the cholinergic system in hippocampal plasticity. An alternative approach involves focusing on the possible effect of low concentration of cholinergic ligands on reactivity of the hippocampus to afferent stimulation. We found two new actions of acetylcholine (ACh); The first one is a fast onset, short lived increase in cellular responses to activation of the N-methyl-D-aspartate (NMDA) receptor, and the second one is a slow onset, long lasting increase in reactivity to afferent stimulation, resembling that produced by a tetanic stimulation, which we called muscarinic long term potentiation (LTPm). The latter effect is mediated by a postsynaptic M2 receptor, and it shares several properties with the more familiar tetanic LTP. In addition, LTPm involves a rise of intracellular calcium concentration and an activation of both a tyrosine kinase and a serine/threonine kinase. Intuitively, LTPm is better related to hippocampal plasticity than the other reported effects of ACh in the hippocampus. Indeed, aged rats, which are cognitively impaired, lack LTPm while they do express other muscarinic actions. It is proposed that LTPm is an important link between the cholinergic action and function in the hippocampus.     

Src activation in the induction of long-term potentiation in CA1 hippocampal neurons

Long-term potentiation (LTP) is an activity-dependent strengthening of synaptic efficacy that is considered to be a model of learning and memory. Protein tyrosine phosphorylation is necessary to induce LTP. Here, induction of LTP in CA1 pyramidal cells of rats was prevented by blocking the tyrosine kinase Src, and Src activity was increased by stimulation producing LTP. Directly activating Src in the postsynaptic neuron enhanced excitatory synaptic responses, occluding LTP. Src-induced enhancement of alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) receptor-mediated synaptic responses required raised intracellular Ca2+ and N-methyl-D-aspartate (NMDA) receptors. Thus, Src activation is necessary and sufficient for inducing LTP and may function by up-regulating NMDA receptors.     

NMDA-induced superoxide production and neurotoxicity in cultured rat hippocampal neurons: role of mitochondria

Excitotoxic mechanisms are believed to be involved in the death of neurons after trauma, epileptic seizures and cerebral ischaemia. We investigated the role of mitochondrial superoxide production in excitotoxic cell death of cultured rat hippocampal neurons. Brief exposure to the selective glutamate agonist N-methyl-D-aspartate (NMDA; 100-300 microM, 10 min) induced significant neuronal death, which was sensitive to cycloheximide (1 microM) and the caspase-1 inhibitor, acetyl-Tyr-Val-Ala-Asp-chloromethylketone (10 microM). Intracellular superoxide production was monitored semiquantitatively on sister cultures from the same platings using the oxidation-sensitive probe, hydroethidine. Brief exposures to toxic NMDA concentrations induced significant increases in superoxide production which correlated with the degree of neuronal injury. However, subtoxic NMDA exposures also produced moderate, yet statistically significant increases in superoxide production. Both NMDA-induced superoxide production and neurotoxicity were reduced by inhibition of mitochondrial electron transport using either sodium cyanide (1 mM), or a combination of rotenone (2 microM) and oligomycin (2 microM). The mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP, 1 microM) mimicked the effect of NMDA on mitochondrial superoxide production. Both NMDA-induced superoxide production and neurotoxicity were potentiated by FCCP (1 microM). Exposure to FCCP alone (1-10 microM, 10 min), however, failed to produce any toxicity. Our data suggest that mitochondrial superoxide production per se is not sufficient to trigger the degeneration of cultured hippocampal neurons, but that manipulation of mitochondrial activity alters NMDA-induced superoxide production and neurotoxicity.     

Nitric oxide acts as a retrograde messenger during long-term potentiation in cultured hippocampal neurons

We examined long-term potentiation (LTP) at synapses between hippocampal neurons in dissociated cell culture following presynaptic, postsynaptic, or extracellular application of a nitric oxide (NO) scavenger, an inhibitor of NO synthase, and a membrane-impermeant NO donor that releases NO only upon photolysis with UV light. Our results indicate that NO is produced in the postsynaptic neuron, travels through the extracellular space, and acts directly in the presynaptic neuron to produce long-term potentiation, supporting the hypothesis that NO acts as a retrograde messenger during LTP.