Calcium influx constitutes the ionic basis for the maintenance of glutamate-induced extended neuronal depolarization associated with hippocampal neuronal death.

Excessive activation of neuronal glutamate receptors has been implicated in the pathophysiology of stroke, epilepsy, and traumatic brain injury. Previously, it has been demonstrated that excitotoxic glutamate exposure results in the induction of an extended neuronal depolarization (END), as well as protracted elevations in free intracellular calcium ([Ca(2+)](i)). Both END and the prolonged [Ca(2+)](i) elevations were shown to correlate with subsequent neuronal death. In the current study, we used whole-cell current-clamp electrophysiology and fura-ff Ca(2+) imaging to determine the electrophysiological basis of END. We found that removal of extracellular Ca(2+) but not Na(+) in the post-glutamate period resulted in complete reversal of END, allowing neurons to rapidly repolarize to their initial resting membrane potential (RMP). In addition, removal of extracellular Ca(2+) was sufficient to eliminate the protracted [Ca(2+)](i) elevations induced by excitotoxic glutamate exposure. To investigate the mechanism through which extracellular Ca(2+) was effecting these changes, pharmacological antagonists of well-characterized routes of Ca(2+) entry were tested for their effects on END. Antagonists of glutamate receptors and voltage-gated Ca(2+) channels (VGCCs) had no significant effect on the membrane potential of neurons in END. Likewise, inhibitors of the Na(+)/Ca(2+) exchange (NCX) were ineffective. In contrast, addition of 500 microM ZnCl(2) or 100 microM GdCl(3) to control extracellular medium (containing normal levels of extracellular Ca(2+)) in the post-glutamate period resulted in rapid and complete reversal of END. Addition of 1mM CdCl(2) to control medium had only modest effects on END. These data provide the first direct evidence that END induced by excitotoxic glutamate exposure is caused by an influx of extracellular Ca(2+) and demonstrate that the previously irreversible condition of END can be reversed by removing extracellular Ca(2+). In addition, understanding the electrophysiological basis of this novel Ca(2+)-induced extended depolarization may provide an insight into the pathophysiology of stroke, traumatic brain injury, and other forms of neuronal injury.

Anticonvulsant activities of ethanolic extract and aqueous fraction isolated from Delphinium denudatum.

Dried roots of Delphinium denudatum Wall. are a popular folk remedy for the treatment of epilepsy in the traditional Unani system of medicine in the sub-continent. We carried out anticonvulsant screening of the ethanolic extract (EE) and aqueous fraction (AF) of this plant utilising the maximal electroshock (MEST) and subcutaneous pentylenetetrazole (scPTZ), bicuculline (scBIC), picrotoxin (scPTX) and strychnine (scSTN) tests for anticonvulsant activity. EE had weak dose-dependent anticonvulsant effects on seizures induced by PTZ and BIC. AF exhibited dose-dependent activity against hind limb tonic extension phase (HLTE) of MEST and comparatively stronger anticonvulsant activity against seizures induced by PTZ and BIC. The results suggest the presence of potent anticonvulsant compounds in AF of D. denudatum and deserve further investigation for isolation of active compounds and elucidation of the mechanism of anticonvulsant action.

Glutamate injury-induced epileptogenesis in hippocampal neurons: an in vitro model of stroke-induced "epilepsy".

BACKGROUND AND PURPOSE: Stroke is the major cause of acquired epilepsy. The mechanisms of ischemia-induced epileptogenesis are not understood, but glutamate is associated with both ischemia-induced injury and epileptogenesis in several models. The objective of this study was to develop an in vitro model of epileptogenesis induced by glutamate injury in hippocampal neurons as observed during stroke. METHODS: Primary hippocampal cultures were exposed to 5 micromol/L glutamate for various durations. Whole-cell current clamp electrophysiology was used to monitor the acute effects of glutamate on neurons and chronic alterations in neuronal excitability up to 8 days after glutamate exposure. RESULTS: A single, 30-minute, 5-micromol/L glutamate exposure produced a subset of neurons that died and a larger population of injured neurons that survived. Neuronal injury was characterized by prolonged reversible membrane depolarization, loss of synaptic activity, and neuronal swelling. Surviving neurons manifested spontaneous, recurrent, epileptiform discharges in neural networks characterized by paroxysmal depolarizing shifts and high-frequency spike firing that persisted for the life of the culture. CONCLUSIONS: This study demonstrates that glutamate injury produced a permanent epileptiform phenotype expressed as spontaneous, recurrent epileptiform discharges for the life of the hippocampal neuronal culture. These results suggest a novel in vitro model of glutamate injury-induced epileptogenesis that may help elucidate some of the mechanisms that underlie stroke-induced epilepsy.

Anticonvulsant activities of the FS-1 subfraction isolated from roots of Delphinium denudatum.

Delphinium denudatum Wall. (Ranunculaceae) is a medicinal herb used for the treatment of epilepsy in the subcontinent. The present study reports the anticonvulsant activities in the maximal electroshock test (MEST) and subcutaneous pentylenetetrazole (PTZ), bicuculline (BIC), picrotoxin (PIC)-induced seizures of the FS-1 subfraction (FS-1) that was obtained by purification of an aqueous fraction isolated from the roots of D. denudatum. In CF 1 mice, FS-1 (600 mg/kg i.p.) exhibited very potent anticonvulsant activity that was comparable to the effects of the well-known antiepileptic drug phenytoin (20 mg/kg) in MEST and protected 100% animals from hind limb tonic extension phase of this model. FS-1 also suppressed PTZ-induced threshold seizure and the loss of the righting reflex with tonic fore and hind limb extension by 100%, similar to the antiepileptic drug valproic acid (350 mg/kg). BIC-induced seizures were suppressed in 80% of the animals. FS-1 exhibited weak anticonvulsant effect on PIC-induced seizures, however, it significantly reduced mortality and delayed the onset of seizures. FS-1 had no effect on strychnine (STN)-induced extensor seizures. The results demonstrate the broad and potent anticonvulsant activity of the compounds in FS-1 of D. denudatum.

Cellular actions of topiramate: blockade of kainate-evoked inward currents in cultured hippocampal neurons.

PURPOSE: This study was undertaken to evaluate the effects of topiramate (TPM) on excitatory amino acid-evoked currents. METHODS: Kainate and N-methyl-D-aspartate (NMDA) were applied to cultured rat hippocampal neurons by using a concentration-clamp apparatus to selectively activate the AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid)/kainate and NMDA receptor subtypes, respectively. The evoked membrane currents were recorded by using perforated-patch whole-cell voltage-clamp techniques. RESULTS: TPM partially blocked kainate-evoked currents with an early-onset reversible phase (phase I) and a late-onset phase (phase II) that occurred after a 10- to 20-min delay and did not reverse during a 2-h washout period. Application of dibutyryl cyclic adenosine monophosphate (cAMP; 2 mM) during washout after phase II block enhanced reversal, with the kainate current amplitude being restored by approximately 50%. Phase II but not phase I block was prevented by prior application of okadaic acid (1 microM), a broad-spectrum phosphatase inhibitor, suggesting that phase II block may be mediated through interactions with intracellular intermediaries that alter the phosphorylation state of kainate-activated channels. Topiramate at 100 microM blocked kainate-evoked currents by 90% during phase II, but had no effect on NMDA-evoked currents. The median inhibitory concentration (IC50) values for phase I and II block of kainate currents were 1.6 and 4.8 microM, respectively, which are within the range of free serum levels of TPM in patients. CONCLUSIONS: The specific blockade of the kainate-induced excitatory conductance is consistent with the ability of TPM to reduce neuronal excitability and could contribute to the anticonvulsant efficacy of this drug.

Effects of topiramate on sustained repetitive firing and spontaneous recurrent seizure discharges in cultured hippocampal neurons.

PURPOSE: In this study, we examined the effects of topiramate (TPM) on the electrophysiologic properties of cultured rat hippocampal pyramidal neurons. METHODS: Whole-cell current-clamp recording techniques were used to determine the effects of TPM on sustained repetitive firing (SRF), spontaneous epileptiform-burst firing, and spontaneous recurrent seizures (SRS). RESULTS: Topiramate at therapeutic concentrations (10-100 microM) significantly decreased or abolished SRF in a dose-dependent and partially reversible manner. When transiently exposed to a medium in which Mg2+ is omitted, hippocampal neurons in culture develop SRS ("epilepsy") and epileptiform discharges. Application of TPM at concentrations ranging from 10 to 100 microM to cells displaying seizure activity caused a concentration-dependent decrease in the number of action potentials within a burst and in the average duration of epileptiform activity. Both effects were partially reversed during a 5- to 30-min drug washout period. CONCLUSIONS: These effects on the electrophysiologic properties of cultured neurons are consistent with the concept that TPM exerts modulatory effects on voltage-dependent Na+ and/or Ca2+ conductances responsible for the generation and propagation of action potentials. Topiramate also may inhibit synaptic conductances responsible for transmission of epileptiform discharges.

Inhibition of calcium/calmodulin kinase II alpha subunit expression results in epileptiform activity in cultured hippocampal neurons.

Several models that develop epileptiform discharges and epilepsy have been associated with a decrease in the activity of calmodulin-dependent kinase II. However, none of these studies has demonstrated a causal relationship between a decrease in calcium/calmodulin kinase II activity and the development of seizure activity. The present study was conducted to determine the effect of directly reducing calcium/calmodulin-dependent kinase activity on the development of epileptiform discharges in hippocampal neurons in culture. Complimentary oligonucleotides specific for the alpha subunit of the calcium/calmodulin kinase were used to decrease the expression of the enzyme. Reduction in kinase expression was confirmed by Western analysis, immunocytochemistry, and exogenous substrate phosphorylation. Increased neuronal excitability and frank epileptiform discharges were observed after a significant reduction in calmodulin kinase II expression. The epileptiform activity was a synchronous event and was not caused by random neuronal firing. Furthermore, the magnitude of decreased kinase expression correlated with the increased neuronal excitability. The data suggest that decreased calmodulin kinase II activity may play a role in epileptogenesis and the long-term plasticity changes associated with the development of pathological seizure activity and epilepsy.

In vitro status epilepticus causes sustained elevation of intracellular calcium levels in hippocampal neurons.

Calcium ions and calcium-dependent systems have been implicated in the pathophysiology of status epilepticus (SE). However, the dynamics of intracellular calcium ([Ca2+]i) levels during SE has not yet been studied. We have employed the hippocampal neuronal culture (HNC) model of in vitro SE that produces continuous epileptiform discharges to study spatial and dynamic changes in [Ca2+]i levels utilizing confocal laser scanning microscopy and the calcium binding dye, indo-1. During SE, the average [Ca2+]i levels increased from control levels of 150-200 nM to levels of 450-600 nM. This increased [Ca2+]i was maintained for the duration of SE. Following SE, [Ca2+]i levels gradually returned to basal values. The duration of SE was shown to affect the ability of the neuron to restore resting [Ca2+]i levels. Both N-methyl-D-aspartate (NMDA) receptor-gated and voltage-gated Ca2+ channels (VGCCs) contributed to the increased calcium entry during SE. Moreover, this elevation in [Ca2+]i occurred in both the nucleus and cytosol. These results provide the first dynamic measurement of [Ca2+]i during prolonged electrographic seizure discharges in an in vitro SE model and suggest that prolonged epileptiform discharges give rise to abnormal sustained increases in [Ca2+]i levels that may play a role in the neuronal cell damage and long-term plasticity changes associated with SE.

Long-lasting decrease in neuronal Ca2+/calmodulin-dependent protein kinase II activity in a hippocampal neuronal culture model of spontaneous recurrent seizures.

Ca2+/calmodulin-dependent protein kinase II (CaM Kinase II) activity was evaluated in a well-characterized in vitro model of epileptiform activity. Long-lasting spontaneous recurrent seizure (SRS) activity was induced in hippocampal neuronal cultures by exposure to low Mg2+ media for 3 h. Analysis of endogenous Ca2+/calmodulin-dependent phosphorylation revealed a significant long-lasting decrease in 32P incorporation into the alpha (50 kDa) and beta (60 kDa) subunits of CaM kinase II in association with the induction of SRS activity in this preparation. Ca2+/calmodulin-dependent substrate phosphorylation of the synthetic peptides, Autocamtide-2 and Syntide II, was also significantly reduced following the induction of SRSs and persisted for the life of the neurons in culture. The decrement in CaM kinase II activity associated with low Mg2+ treatment remained significantly decreased when values were corrected for changes in levels of alpha subunit immunoreactivity and neuronal cell loss. Addition of the protein phosphatase inhibitors, okadaic acid and cyclosporin A, to the phosphorylation reaction did not block the SRS-associated decrease in substrate phosphorylation, indicating that enhanced phosphatase activity was not a contributing factor to the observed decrease in phosphate incorporation. The findings of this study demonstrate that CaM kinase II activity is decreased in association with epileptogenesis observed in these hippocampal cultures and may contribute to the production and maintenance of SRSs in this model.

Prolonged activation of the N-methyl-D-aspartate receptor-Ca2+ transduction pathway causes spontaneous recurrent epileptiform discharges in hippocampal neurons in culture.

The molecular basis for developing symptomatic epilepsy (epileptogenesis) remains ill defined. We show here in a well characterized hippocampal culture model of epilepsy that the induction of epileptogenesis is Ca2+-dependent. The concentration of intracellular free Ca2+ ([Ca2+]i) was monitored during the induction of epileptogenesis by prolonged electrographic seizure activity induced through low-Mg2+ treatment by confocal laser-scanning fluorescent microscopy to directly correlate changes in [Ca2+]i with alterations in membrane excitability measured by intracellular recording using whole-cell current-clamp techniques. The induction of long-lasting spontaneous recurrent epileptiform discharges, but not the Mg2+-induced spike discharges, was prevented in low-Ca2+ solutions and was dependent on activation of the N-methyl-D-aspartate (NMDA) receptor. The results provide direct evidence that prolonged activation of the NMDA-Ca2+ transduction pathway causes a long-lasting plasticity change in hippocampal neurons causing increased excitability leading to the occurrence of spontaneous, recurrent epileptiform discharges.

Physiological and pharmacological alterations in postsynaptic GABA(A) receptor function in a hippocampal culture model of chronic spontaneous seizures.

Cultured rat hippocampal neurons previously exposed to a media containing no added Mg2+ for 3 h begin to spontaneously trigger recurrent epileptiform discharges following return to normal medium, and this altered population epileptiform activity persisted for the life of the neurons in culture (> 2 wk). Neurons in "epileptic" cultures appeared similar in somatic and dendritic morphology and cellular density to control, untreated cultures. In patch-clamp recordings from hippocampal pyramidal cells from "epileptic," low Mg2+ pretreated hippocampal cultures, a rapid (within 2 h of treatment), permanent (lasting > or = 8 days) and statistically significant 50-65% reduction in the current density of functional gamma-aminobutyric acid-A (GABA(A)) receptors was evident when the GABA responses of these cells were compared with control neurons. Functional GABA receptor current density was calculated by determining the maximal response of a cell to GABA 1 mM application and normalizing this response to cellular capacitance. Despite the marked GABA efficacy differences noted above, the potency of GABA in activating chloride currents was not significantly different when the responses to control and "epileptic" pyramidal cells to multiple concentrations of GABA were compared. The EC50 for GABA was 4.5 +/- 0.2 (mean +/- SE) for control neurons and 3.5 +/- 0.4 microM, 5.2 +/- 0.5 microM, 3.7 +/- 0.3 microM, and 4.6 +/- 0.3 microM for epileptic neurons 2 h, 2 days, 3 days, and 8 days after low Mg2+ pretreatment, respectively. Modulation of GABA responses by the benzodiazepine, clonazepam, was significantly reduced in epileptic neurons compared with controls. The kinetically determined clonazepam 100 nM GABA augmentation efficacy decreased from 44.1% in control neurons to 9.3% augmentation in neurons recorded from cultures 10 days posttreatment. The kinetics of GABA current block by the noncompetitive antagonist picrotoxin were determined in hippocampal cultured neurons, and an IC50 of 14 microM determined. Bath application of picrotoxin at half of the IC50 concentration (7 microM) induced epileptiform activity in control cultures and this activity appeared very similar to the epileptiform activity induced by prior low Mg2+ treatment. This concentration of picrotoxin was determined experimentally to block 30% of the GABA(A)-mediated receptor responses in these cultures, and this level of block was sufficient to trigger spontaneous epileptiform activity. The 50% reduction of GABA responses induced as a permanent consequence of low Mg2+ treatment therefore was determined to be sufficient in and of itself to induce the spontaneous epileptiform activity, which was also a consequence of this treatment.

Inability to restore resting intracellular calcium levels as an early indicator of delayed neuronal cell death.

The hippocampus is especially vulnerable to excitotoxicity and delayed neuronal cell death. Chronic elevations in free intracellular calcium concentration ([Ca2+]i) following glutamate-induced excitotoxicity have been implicated in contributing to delayed neuronal cell death. However, no direct correlation between delayed cell death and prolonged increases in [Ca2+]i has been determined in mature hippocampal neurons in culture. This investigation was initiated to determine the statistical relationship between delayed neuronal cell death and prolonged increases in [Ca2+]i in mature hippocampal neurons in culture. Using indo-1 confocal fluorescence microscopy, we observed that glutamate induced a rapid increase in [Ca2+]i that persisted after the removal of glutamate. Following excitotoxic glutamate exposure, neurons exhibited prolonged increases in [Ca2+]i, and significant delayed neuronal cell death was observed. The N-methyl-D-aspartate (NMDA) channel antagonist MK-801 blocked the prolonged increases in [Ca2+]i and cell death. Depolarization of neurons with potassium chloride (KCl) resulted in increases in [Ca2+]i, but these increases were buffered immediately upon removal of the KCl, and no cell death occurred. Linear regression analysis revealed a strong correlation (R = 0.973) between glutamate-induced prolonged increases in [Ca2+]i and delayed cell death. These data suggest that excitotoxic glutamate exposure results in an NMDA-induced inability to restore resting [Ca2+]i (IRRC) that is a statistically significant indicator of delayed neuronal cell death.

Recurrent spontaneous seizure activity in hippocampal neuronal networks in culture.

1. Experiments were carried out using intracellular recording techniques on hippocampal neurons maintained in culture to determine if populations of hippocampal neurons could be induced to develop spontaneously recurring epileptiform discharges. This study demonstrates the conversion of normal hippocampal neurons in culture by a brief Mg(2+)-free treatment into a preparation of cells that permanently manifested recurrent, spontaneous seizure discharges. These electrographic seizure discharges illustrated the same electrographic properties seen in human epilepsy and were observed for the life of the culture. 2. The epileptic activity was shown to occur synchronously in populations of neurons and to be controlled by clinically useful anticonvulsant drugs. 3. This new cell culture model of epileptic activity provides a powerful tool to investigate the molecular mechanisms underlying the induction, maintenance, and termination of this "epileptic condition" in vitro and demonstrates that neuronal networks in culture can be transformed to manifest permanently spontaneous recurrent electrographic seizures.

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.

Excitotoxicity affects membrane potential and calmodulin kinase II activity in cultured rat cortical neurons.

BACKGROUND AND PURPOSE: Glutamate-induced excitotoxicity has been implicated as a causative factor for selective neuronal loss in ischemia and hypoxia. Toxic exposure of neurons to glutamate results in an extended neuronal depolarization that precedes delayed neuronal death. Because both delayed neuronal death and extended neuronal depolarization are dependent on calcium, we examined the effect of glutamate exposure on extended neuronal depolarization and calcium/calmodulin-dependent protein kinase II (CaM kinase II) activity. METHODS: Three-week-old cortical cell cultures from embryonic rats were exposed to 500 microM glutamate and 10 microM glycine or to control medium for 10 minutes. Cells were examined for neuronal toxicity, electrophysiology, and biochemical alterations. In one set of experiments, whole-cell current clamp recording was performed throughout the experiment. In a parallel experiment, cortical cultures were allowed to recover from glutamate exposure for 1 hour, at which time the cells were homogenized and CaM kinase II activity was assayed using standard techniques. RESULTS: Excitotoxic exposure to glutamate resulted in extended neuronal depolarization, which remained after removal of the glutamate. Glutamate exposure also resulted in delayed neuronal death, which was preceded by significant inhibition of CaM kinase II activity. The excitotoxic inhibition of CaM kinase II correlated with neuronal loss, was N-methyl-D-aspartate receptor-mediated, and was not due to autophosphorylation of the enzyme. CONCLUSIONS: Glutamate-induced delayed neuronal toxicity correlates with extended neuronal depolarization and inhibition of CaM kinase II activity. Because inhibition of CaM kinase II activity significantly preceded the histological loss of neurons, the data suggest that modulation of CaM kinase II activity may be involved in the cascade of events resulting in loss of calcium homeostasis and delayed neuronal death.

Electrophysiology of glutamate neurotoxicity in vitro: induction of a calcium-dependent extended neuronal depolarization.

1. Physiological responses of hippocampal pyramidal neurons in primary culture to prolonged glutamate (GLU) exposure (500 microM in all experiments) were studied with the use of patch electrodes and whole-cell current-clamp recording techniques. In some experiments, perforated patch recordings were employed with electrodes containing the pore-forming antibiotic nystatin. 2. After washout of GLU after a 10-min exposure, pyramidal neurons remained depolarized by greater than or equal to 20 mV from rest for the duration of the recording (30 min to less than 4 h). This depolarization was accompanied by a 57.8% increase in membrane conductance and was termed an extended neuronal depolarization (END). The percentage of neurons in which END was induced varied with the duration of GLU exposure, with a 4-, 6-, 8-, 10-, and 20-min GLU exposure eliciting END in 12.5, 41.7, 81.8, 100, and 100% of neurons. END induction appeared to be an all-or-none phenomenon, because END levels did not differ when compared across GLU exposure times. 3. During the END, cells retained both the ability to fire action potentials and the ability to respond to GLU, appeared viable when examined anatomically, and still excluded vital dyes. This supports the conclusion that END is not a nonspecific consequence of cell death. Rather, END is a discrete physiological process triggered by prolonged GLU exposure. The results raise questions concerning the reversibility of END induction, i.e., can neurons be "rescued" once END is induced, or will these cells inevitably go on to die? 4. END induction was dependent on a rise in intracellular free calcium ([Ca]i). END was prevented by strong buffering of [Ca]i or by substitution of external Ba2+ for Ca2+. However, substitution of Mn2+ for Ca2+ still permitted END induction. In cells recorded with the perforated-patch technique, maintaining normal [Ca]i levels, END could be induced, but less readily than under unbuffered [Ca]i conditions. 5. END could not be induced by a 10- to 20-min current-clamp depolarization to 0 mV, nor by 10-min GLU application while the membrane potential was voltage clamped at rest in a solution containing 1 mM Mg2+. In addition, END induction by GLU could be blocked by application of MK-801 (10-30 microM) but not 6-cyano-7-nitroquinoxaline-2,3-dione [CNQX (100-200 microM)]. 6. The dependence of both delayed neuronal cell death and END induction on GLU exposure duration were similar.(ABSTRACT TRUNCATED AT 400 WORDS)

Excitatory amino acid receptor activation produces a selective and long-lasting modulation of gene expression in hippocampal neurons.

Activation of excitatory amino acid (EAA) receptors in cultured hippocampal neurons causes down-regulation of the protein ligatin, a receptor for phosphoglycoproteins and a marker protein for membrane-vesicle transport systems. This reduction occurs at both physiologic and excitotoxic levels of glutamate stimulation and is accompanied by a significant decrease in steady state levels of ligatin mRNA. Reduction in ligatin mRNA occurs within 60 min and persists 24 h later. Steady state levels of mRNAs encoding cyclophilin, an ubiquitous cytosolic protein, and neuron specific-enolase (N-SE) are not diminished by glutamate receptor activation, demonstrating that down-regulation of ligatin mRNA was not a result of general catabolism. Further, this reduction in ligatin mRNA occurred without induction of HSP 70. Pharmacological studies using selective antagonists and agonists indicate that this down-regulation of ligatin gene expression is predominantly mediated by the N-methyl-D-aspartate (NMDA) subclass of EAA receptors and that Ca2+ is required. This is the first report that EAA receptor activation in hippocampal neurons can pretranslationally down-regulate gene expression in a rapid and long-lasting manner under physiologic, as well as cytotoxic conditions. The data support the hypothesis that modulation of neuronal gene expression may represent a molecular mechanism mediating some of the long-lasting functional and pathophysiological effects of EAA on cell function.

Neurotoxic activation of glutamate receptors induces an extended neuronal depolarization in cultured hippocampal neurons.

Intracellular recording revealed that cytotoxic activation of excitatory amino acid receptors by glutamate or N-methyl-D-aspartate (NMDA) elicited an extended neuronal depolarization (END) of at least 5 h duration following washout of glutamate in hippocampal neurons in culture. During END, cells were still responsive to glutamate, and still able to fire sodium spikes. END induction could be blocked by concurrent application of D-2-amino-5-phosphonovalerate (APV) or MK-801, but not 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), during the glutamate exposure. The induction of END by excitotoxic glutamate receptor activation may play a role in the pathophysiology of glutamate toxicity.

Membrane currents in identified lactotrophs of rat anterior pituitary.

Qualitative features of the primary inward and outward current components of identified lactotrophs of the rat anterior pituitary were examined. Identification of lactotrophs in heterogeneous dissociated anterior pituitary cultures was accomplished by application of the reverse hemolytic plaque assay. Currents in lactotrophs were subsequently examined using whole-cell or patch recording techniques. Two components of inward calcium current were observed: a transient component and a sustained component. The transient component activated at voltages as negative as -50 mV and was the major contributor to total lactotroph calcium current. The sustained component activated at voltages above about -10 mV. The 2 currents could be qualitatively separated by differences in inactivation properties and in sensitivity to cadmium. At least 3 components of outward current were distinguished. Either 30 mM TEA or 0 calcium eliminated a major portion of sustained outward current. This is likely to represent primarily calcium- and voltage-activated potassium current. The remaining current could be further differentiated into a transient current component that could be inactivated with conditioning potentials above -60 mV. A slowly activating and deactivating potassium current remained following inactivation of the transient current. Although the time course of the transient current is reminiscent of "A" current, activation of this current required potentials above -30 mV. Candidates for the single-channel currents that underlie the whole-cell outward currents were observed in cell-attached recordings. When combined with patch-clamp electrophysiological methods, the reverse hemolytic plaque assay promises to be a powerful technique for the electrophysiological characterization of specific cell subtypes in heterogeneous dissociated cell populations.

Central nervous sensitization and dishabituation of reflex action in an insect by the neuromodulator octopamine.

Habituation of excitatory synaptic inputs onto identified motor neurons of the locust metathoracic ganglion, driven electrically and by natural stimuli, was examined using intracellular recording. Rapid progressive reduction in amplitude of EPSPs from a variety of inputs onto fast-type motor neurons occurred. The habituated EPSPs were quickly dishabituated by iontophoretic release of octopamine from a microelectrode into the neuropilar region of presumed synaptic action. The zone within which release was effective for a given neuron was narrowly-defined. With larger amounts of octopamine applied at a sensitive site the EPSP became larger than normal, and in many instances action potentials were initiated by the sensitized response. Very small EPSPs onto a motor neuron, which were associated with proprioceptive feedback, and which were originally too small to be detected above the noise, were potentiated to a level of several mV by the iontophoresed octopamine. A DUM neuron (presumed to be octopaminergic) was found, whose direct stimulation was followed by a strong dishabituating and sensitizing action leading to spikes, of inputs to an identified flexor tibiae motor neuron. The action and its time course were closely similar to those evoked by octopamine iontophoresed into the neuropil in the region of synaptic inputs to the motor neuron. It is concluded that DUM (octopaminergic) neurons exert large potentiating actions on central neuronal excitatory synaptic transmission in locusts.