Utility Of Cns Cell Cultures As A Model To Help Understand The Pathophysiology Of Epilepsy And The Mechanisms Of Action Of Antiepileptic Drugs

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Neuronal and Synaptic Physiology and Pharmacology

Ionic Currents Underlying Action Potentials in Cortical Neurons

Our understanding of the physiologic basis of the action potential (AP) was derived from pioneering studies of the squid giant axon and other similar preparations. In these cells and axons, APs are generated by the opening of voltage-dependent Na channels followed by a rapid voltage-sensitive inactivation. In addition, voltage-dependent K channels open to facilitate repolarization in many neurons. In a few invertebrate neurons, investigators also demonstrated that voltage-dependent Ca currents could sustain APs. Early studies in cell culture demonstrated that some vertebrate, including mammalian, neurons had Ca-dependent APs as well. These were first demonstrated in chick DRG neurons (Dichter and Fischbach, 1977), a preparation that then became very useful for studying mechanisms by which neuromodulators were able to affect Ca currents (Dunlap and Fischbach, 1978). Voltage-dependent Ca APs were also demonstrated in mammalian cortical neurons, both in slice preparations (Schwartzkroin and Slawsky, 1977) and in culture. Under baseline conditions, these currents were not apparent because of the rapid depolarization-repolarization sequence initiated by Na and K currents, but when Na currents were inhibited, the cortical neurons developed slower, powerful Ca-dependent APs (Dichter and Zona, 1989; Dichter et al., 1983). Once the Ca APs were identified, it was noted that some groups of neurons in cortex, hippocampus, and thalamus had quite prominent Ca APs and tended to fire bursts of action potentials when depolarized. These neurons appeared capable of acting as "pacemaker" neurons for seizure-like discharges (Wong and Prince, 1978), and hypotheses about the origin of rhythmic discharges in cortex, hippocampus, and thalamocortical circuits were developed based on these findings. Similar observations were made on various K currents that were characterized in cortical and hippocampal neurons, some of which contributed to a neuron's having a propensity for endogenous bursting.

Physiology and Pharmacology of Inhibitory Synapses

Earlyin vivo studies demonstrated that inhibitory postsy-naptic potentials (IPSPs) were generated by opening Cl channels and were associated with an increase in membrane conductance. GABA had been tentatively identified as the likely inhibitory neurotransmitter in mammalian forebrain, but glycine was also thought to be significant as a neuro-transmitter. Studies in mammalian CNS cell culture demonstrated that GABA was the main, if not exclusive, neurotransmitter responsible for postsynaptic inhibition (Dichter, 1980; White et al., 1980) and that glycine likely played no, or very little, role in forebrain inhibition. These studies also showed that GABAergic inhibition was very labile at most synapses and declined rapidly with repetitive stimulation, mostly as a result of presynaptic factors (Wilcox and Dichter, 1994) (i.e., not due to receptor desensitization or changes in Cl gradient). These discoveries in cell culture led to extensive examinations of the pharmacology of GABAer-gic synapses, including the elucidation of the mechanisms of action of some of the most important AEDs available.

From both biochemical (e.g., receptor binding) studies on processed brain tissue and from studies at the cellular level in cell culture, the GABAa receptor was recognized as a GABA receptor complex (GRC). Studies utilizing cell cultures first described the allosteric modulation of the GRC by benzodiazepines (Choi et al., 1977), a group of drugs used extensively to treat status epilepticus and other forms of seizures but whose mechanism of action had been unknown. Similar studies demonstrated that barbiturates, another class of antiepileptic drug, could allosterically modulate the GRC at a different site (Macdonald and Barker, 1977, 1978; Macdonald and McLean, 1982). Further, unlike the benzo-diazepines, barbiturates at high concentrations could directly activate the GRC. Studies of membrane patches removed from cultured neurons also demonstrated how these drugs worked at the single channel level (Macdonald et al., 1988). Other work focused on a number of new AEDs that were developed based on animal models and whose mechanisms of action are unknown. At least one of these drugs, topira-mate, appears to work partially by potentiating GABA-mediated inhibition as demonstrated in hippocampal cultures (White et al., 1997, 2000).

GABA also activates other classes of receptors, most notably GABAB receptors which are found on both somas and axon terminals, and GABAC receptors which are most prominent in retina. Much of what we know of the physiology of GABAB receptors is derived from studies in cell culture. These receptors are G-protein coupled to Ca channels in axon terminals, where they block Ca entry and thereby downregulate neurotransmitter release. They are also G-protein coupled to K channels on somas and den-drites, where they mediate slow and prolonged inhibition (Bowery et al., 1983, 1984, 1989; Bowery et al., 1984). Both these sets of events have important implications for epilep-tiform mechanisms. For example, it is thought that activation of GABAB-mediated inhibition plays a role in the termination of interictal discharges and possibly in the termination of seizures (Sperk et al., 2004). In addition, increased activation of GABAB receptors is thought to contribute to enhanced bursting in thalamocortical relay neurons and the generation of spike-wave discharges in primary generalized epilepsy (Hosford et al., 1992, 1995). Inhibition of GABA release by activation of GABAb receptors at GABAergic terminals has also been implicated in the reduction of inhibition seen with repetitive activation of inhibitory synapses (Deisz and Prince, 1989). In addition, activation of GABAB receptors is likely to be responsible for the complex effects of two new AEDs that were designed to increase GABA levels at inhibitory synapses: tiagabine (by blocking GABA uptake) and vigabatrin (by blocking GABA metabolism). Both agents are antiseizure drugs effective against partial seizures. However, both can also enhance primary generalized absence seizures and possibly status epilepticus, presumably by allowing released GABA to act on presynaptic GABA terminals and paradoxically decreasing inhibition (Oh and Dichter, 1994) or by enhancing hyperpolarization on thalamocortical neurons and thereby enhancing synchronized spike-wave activity.

Physiology and Pharmacology of Excitatory Synapses

The physiology of excitatory synapses was much less well understood than even inhibitory synapses before the introduction of neuronal cell cultures. Unlike IPSPs, excitatory PSPs (EPSPs) were often not associated with measurable conductance changes and had unusual current-voltage (I-V) characteristics. The main hypothesis to explain these observations was that EPSPs occurred far out on dendrites and the characteristics that one could measure in the soma were distorted by the cable properties of the neurons. In addition, although glutamate and aspartate were hypothesized to be the excitatory neurotransmitters in the brain, their identities had not been confirmed and the cellular mechanisms by which these acidic amino acids depolarized neurons were not understood. This picture changed relatively rapidly once these properties could be analyzed in vitro. The response of neurons to applied glutamate was characterized (Ransom et al., 1977); the odd I-V curve was attributed to a voltage-dependent block by Mg of a subpopulation of glutamate receptors (Ascher and Nowak, 1988; Ascher et al., 1988); different glutamate receptor subtypes (e.g., AMPA, NMDA, kainate) were identified and characterized (Dingledine et al., 1990; McLennan, 1983; Westbrook, 1994), and the physiology of excitatory synapses was much better understood. Moreover, a virtual "industry" of glutamate pharmacology was born.

Each of the advances in our understanding of the mechanisms of excitatory transmission had direct impact on epilepsy research. The nature of the large depolarizing events seen both during interictal discharges and ictal events was characterized within the framework of different glutamate receptor events. The role of NMDA receptors especially was emphasized as an important component of epileptiform activity, and the autoregenerative nature of NMDA-mediated synaptic events (with depolarization reducing the Mg block of NMDA receptors, which in turn promoted further depolarization by glutamate) became a critical part of the explanation for the all-or-nothing development of some forms of epileptic activity.

The characterization of excitatory synaptic transmission was also determined at the single-channel level. AMPA receptors were demonstrated to have very rapid and unusual desensitization properties such that they opened once in response to agonist and then desensitized until agonist was removed (Tang et al., 1989; Trussell et al., 1988). NMDA receptor-coupled channels, however, continued to open and close for as long as glutamate remained present. In addition, it was demonstrated that NMDA receptors were very sensitive to changes in extracellular pH, becoming inhibited at acidic values and enhanced by alkalosis, within pH ranges that were found in the brain during physiologic or pathological stimuli (Tang et al., 1990). Investigators also demonstrated that most AMPA receptors were permeable to only monovalent cations, whereas NMDA receptors were permeable to Ca as well. As the molecular biology of the receptors was further understood, AMPA receptors that lacked a properly edited version of the GluR2 subunit were found to be permeable to Ca. These channels occurred naturally on some inhibitory interneurons (Hollmann et al., 1991) and were proposed as a contributing component of some disease states (Bennett et al., 1996; Pellegrini-Giampietro et al., 1997). Each of these discoveries had great implications for our understanding of epileptic processes and for current theories of epileptogenesis. For example, the use of hyperventilation to stimulate epileptic changes in the electroencephalogram (EEG) may be due to the brief metabolic alkalosis induced by this procedure. In addition, the vulnerability of subsets of inhibitory interneurons to hypoxia may be related to their Ca-permeable AMPA receptors.

In addition to the fast depolarization mediated by ionotrophic glutamate receptors, it was also noted that glutamate could produce a slow and long-lasting change in neuronal properties as a result of activation of "metabotropic" glutamate receptors. The discovery and characterization of these receptors also added significantly to our understanding of how epileptiform events could become established and propagate throughout the brain (Ghauri et al., 1996; Lee et al., 2002).

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