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.

The cloud–ionosphere discharge: A newly observed thunderstorm phenomenon

This paper deals with a luminous electric discharge that forms in the mesospheric region between thundercloud tops and the ionosphere at 90-km altitude. These cloud–ionosphere discharges (CIs), following visual reports dating back to the 19th century, were finally imaged by a low-light TV camera as part of the “SKYFLASH” program at the University of Minnesota in 1989. Many observations were made by various groups in the period 1993–1996. The characteristics of CIs are that they have a wide range of sizes from a few kilometers up to 50 km horizontally; they extend from 40 km to nearly 90 km vertically, with an intense region near 60–70 km and streamers extending down toward cloud tops; the CIs are partly or entirely composed of vertical luminous filaments of kilometer size. The predominate color is red. The TV images show that the CIs usually have a duration less than one TV field (16.7 ms), but higher-speed photometric measurements show that they last about 3 ms, and are delayed 3 ms after an initiating cloud–ground lightning stroke; 95% of these initiating strokes are found to be “positive”—i.e., carry positive charges from clouds to ground. The preference for positive initiating strokes is not understood. Theories of the formation of CIs are briefly reviewed.

Operative GABAergic inhibition in hippocampal CA1 pyramidal neurons in experimental epilepsy

Patch–clamp recordings of CA1 interneurons and pyramidal cells were performed in hippocampal slices from kainate- or pilocarpine-treated rat models of temporal lobe epilepsy. We report that γ-aminobutyric acid (GABA)ergic inhibition in pyramidal neurons is still functional in temporal lobe epilepsy because: (i) the frequency of spontaneous GABAergic currents is similar to that of control and (ii) focal electrical stimulation of interneurons evokes a hyperpolarization that prevents the generation of action potentials. In paired recordings of interneurons and pyramidal cells, synchronous interictal activities were recorded. Furthermore, large network-driven GABAergic inhibitory postsynaptic currents were present in pyramidal cells during interictal discharges. The duration of these interictal discharges was increased by the GABA type A antagonist bicuculline. We conclude that GABAergic inhibition is still present and functional in these experimental models and that the principal defect of inhibition does not lie in a complete disconnection of GABAergic interneurons from their glutamatergic inputs.

Continuous and lurching traveling pulses in neuronal networks with delay and spatially decaying connectivity

Propagation of discharges in cortical and thalamic systems, which is used as a probe for examining network circuitry, is studied by constructing a one-dimensional model of integrate-and-fire neurons that are coupled by excitatory synapses with delay. Each neuron fires only one spike. The velocity and stability of propagating continuous pulses are calculated analytically. Above a certain critical value of the constant delay, these pulses lose stability. Instead, lurching pulses propagate with discontinuous and periodic spatio-temporal characteristics. The parameter regime for which lurching occurs is strongly affected by the footprint (connectivity) shape; bistability may occur with a square footprint shape but not with an exponential footprint shape. For strong synaptic coupling, the velocity of both continuous and lurching pulses increases logarithmically with the synaptic coupling strength gsyn for an exponential footprint shape, and it is bounded for a step footprint shape. We conclude that the differences in velocity and shape between the front of thalamic spindle waves in vitro and cortical paroxysmal discharges stem from their different effective delay; in thalamic networks, large effective delay between inhibitory neurons arises from their effective interaction via the excitatory cells which display postinhibitory rebound.

Synchronization of oscillatory responses in visual cortex correlates with perception in interocular rivalry

In subjects suffering from early onset strabismus, signals conveyed by the two eyes are not perceived simultaneously but in alternation. We exploited this phenomenon of interocular suppression to investigate the neuronal correlate of binocular rivalry in primary visual cortex of awake strabismic cats. Monocularly presented stimuli that were readily perceived by the animal evoked synchronized discharges with an oscillatory patterning in the γ-frequency range. Upon dichoptic stimulation, neurons responding to the stimulus that continued to be perceived increased the synchronicity and the regularity of their oscillatory patterning while the reverse was true for neurons responding to the stimulus that was no longer perceived. These differential changes were not associated with modifications of discharge rate, suggesting that at early stages of visual processing the degree of synchronicity rather than the amplitude of responses determines which signals are perceived and control behavioral responses.

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 α 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.

Overexpression of a Shaker-type potassium channel in mammalian central nervous system dysregulates native potassium channel gene expression

The nervous system maintains a delicate balance between excitation and inhibition, partly through the complex interplay between voltage-gated sodium and potassium ion channels. Because K+ channel blockade or gene deletion causes hyperexcitability, it is generally assumed that increases in K+ channel gene expression should reduce neuronal network excitability. We have tested this hypothesis by creating a transgenic mouse that expresses a Shaker-type K+ channel gene. Paradoxically, we find that addition of the extra K+ channel gene results in a hyperexcitable rather than a hypoexcitable phenotype. The presence of the transgene leads to a complex deregulation of endogenous Shaker genes in the adult central nervous system as well as an increase in network excitability that includes spontaneous cortical spike and wave discharges and a lower threshold for epileptiform bursting in isolated hippocampal slices. These data suggest that an increase in K+ channel gene dosage leads to dysregulation of normal K+ channel gene expression, and it may underlie a mechanism contributing to the pathogenesis of human aneuploidies such as Down syndrome.

A role for Src kinase in spontaneous epileptiform activity in the CA3 region of the hippocampus

Members of the Src family of nonreceptor protein tyrosine kinases (PTKs) have been implicated in the regulation of cellular excitability and synaptic plasticity. We have investigated the role of these PTKs in in vitro models of epileptiform activity. Spontaneous epileptiform discharges were induced in vitro in the CA3 region of rat hippocampal slices by superfusion with the potassium channel blocker 4-aminopyridine in Mg2+-free medium. In hippocampal slices treated in this fashion, Src kinase activity was increased and the frequency of epileptiform discharges could be greatly reduced by inhibitor of the Src family of PTKs, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), but not by the inactive structural analog 4-amino-7-phenylpyrazol[3,4-d]pyrimidine (PP3). 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine also reduced epileptiform activity induced by either 4-aminopyridine or Mg2+-free medium alone. These observations demonstrate a role for Src family PTKs in the pathophysiology of epilepsy and suggest potential therapeutic targets for antiepileptic therapy.

Negative pH, efflorescent mineralogy, and consequences for environmental restoration at the Iron Mountain Superfund site, California

The Richmond Mine of the Iron Mountain copper deposit contains some of the most acid mine waters ever reported. Values of pH have been measured as low as −3.6, combined metal concentrations as high as 200 g/liter, and sulfate concentrations as high as 760 g/liter. Copious quantities of soluble metal sulfate salts such as melanterite, chalcanthite, coquimbite, rhomboclase, voltaite, copiapite, and halotrichite have been identified, and some of these are forming from negative-pH mine waters. Geochemical calculations show that, under a mine-plugging remediation scenario, these salts would dissolve and the resultant 600,000-m3 mine pool would have a pH of 1 or less and contain several grams of dissolved metals per liter, much like the current portal effluent water. In the absence of plugging or other at-source control, current weathering rates indicate that the portal effluent will continue for approximately 3,000 years. Other remedial actions have greatly reduced metal loads into downstream drainages and the Sacramento River, primarily by capturing the major acidic discharges and routing them to a lime neutralization plant. Incorporation of geochemical modeling and mineralogical expertise into the decision-making process for remediation can save time, save money, and reduce the likelihood of deleterious consequences.

Grafts of adenosine-releasing cells suppress seizures in kindling epilepsy

Adenosine is an inhibitor of neuronal activity in the brain. The local release of adenosine from grafted cells was evaluated as an ex vivo gene therapy approach to suppress synchronous discharges and epileptic seizures. Fibroblasts were engineered to release adenosine by inactivating the adenosine-metabolizing enzymes adenosine kinase and adenosine deaminase. After encapsulation into semipermeable polymers, the cells were grafted into the brain ventricles of electrically kindled rats, a model of partial epilepsy. Grafted rats provided a nearly complete protection from behavioral seizures and a near-complete suppression of afterdischarges in electroencephalogram recordings, whereas the full tonic–clonic convulsions in control rats remained unaltered. Thus, the local release of adenosine resulting in adenosine concentrations <25 nM at the site of action is sufficient to suppress seizure activity and, therefore, provides a potential therapeutic principle for the treatment of drug-resistant partial epilepsies.

Long-term plasticity in interneurons of the dentate gyrus

Single interneurons influence thousands of postsynaptic principal cells, and the control of interneuronal excitability is an important regulator of the computational properties of the hippocampus. However, the mechanisms underlying long-term alterations in the input–output functions of interneurons are not fully understood. We report a mechanism of interneuronal plasticity that leads to the functional enhancement of the gain of glutamatergic inputs in the absence of long-term potentiation of the excitatory synaptic currents. Interneurons in the dentate gyrus exhibit a characteristic, limited (≈8 mV) depolarization of their resting membrane potential after high-frequency stimulation of the perforant path. The depolarization can be observed with either whole-cell or perforated patch electrodes, and it lasts in excess of 3 h. The long-term depolarization is specific to interneurons, because granule cells do not show it. The depolarization requires the activation of Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and the rise of intracellular Ca2+, but not N-methyl-d-aspartate (NMDA) receptor activation. Data on the maintenance of the depolarization point to a major role for a long-term change in the rate of electrogenic Na+/K+-ATPase pump function in interneurons. As a result of the depolarization, interneurons after the tetanus respond with action potential discharges to previously subthreshold excitatory postsynaptic potentials (EPSPs), even though the EPSPs are not potentiated. These results demonstrate that the plastic nature of the interneuronal resting membrane potential underlies a unique form of long-term regulation of the gain of excitatory inputs to γ-aminobutyric acid (GABA)ergic neurons.

G protein activation kinetics and spillover of gamma-aminobutyric acid may account for differences between inhibitory responses in the hippocampus and thalamus.

We have developed a model of gamma-aminobutyric acid (GABA)ergic synaptic transmission mediated by GABAA and GABAB receptors, including cooperativity in the guanine nucleotide binding protein (G protein) cascade mediating the activation of K+ channels by GABAB receptors. If the binding of several G proteins is needed to activate the K+ channels, then only a prolonged activation of GABAB receptors evoked detectable currents. This could occur if strong stimuli evoked release in adjacent terminals and the spillover resulted in prolonged activation of the receptors, leading to inhibitory responses similar to those observed in hippocampal slices. The same model also reproduced thalamic GABAB responses to high-frequency bursts of stimuli. In this case, prolonged activation of the receptors was due to high-frequency release conditions. This model provides insights into the function of GABAB receptors in normal and epileptic discharges.

Electrical and synaptic properties of embryonic luteinizing hormone-releasing hormone neurons in explant cultures.

Voltage- and ligand-activated channels in embryonic neurons containing luteinizing hormone-releasing hormone (LHRH) were studied by patch-pipette, whole-cell current and voltage clamp techniques. LHRH neurons were maintained in explant cultures derived from olfactory pit regions of embryonic mice. Cells were marked intracellularly with Lucifer yellow following recording. Sixty-two cells were unequivocally identified as LHRH neurons by Lucifer yellow and LHRH immunocytochemistry. The cultured LHRH neurons had resting potentials around -50 mV, exhibited spontaneous discharges generated by intrinsic and/or synaptic activities and contained a time-dependent inward rectifier (Iir). Voltage clamp analysis of ionic currents in the LHRH neuron soma revealed a tetrodotoxin-sensitive Na+ current (INa) and two major types of K+ currents, a transient current (IA), a delayed rectifier current (IK) and low- and high-voltage-activated Ca2+ currents. Spontaneous depolarizing synaptic potentials and depolarizations induced by direct application of gamma-aminobutyrate were both inhibited by picrotoxin or bicuculline, demonstrating the presence of functional gamma-aminobutyrate type A synapses on these neurons. Responses to glutamate were found in LHRH neurons in older cultures. Thus, embryonic LHRH neurons not yet positioned in their postnatal environment in the forebrain contained a highly differentiated repertoire of voltage- and ligand-gated channels.