Douglas W. Bonhaus

Anticonvulsant and antiepileptogenic actions of MK-801 in the kindling and electroshock models

The actions of MK-801, a noncompetitive antagonist at the N-methyl-d-aspartate subtype of excitatory amino acid receptor, were investigated on the development of kindling and on seizures in the electroshock and kindling models. The drug MK-801 potently and effectively suppressed the tonic hindlimb extension component of electroshock-induced seizures; it also suppressed both the electrophysiological and behavioral manifestations of the development of kindling. In contrast to its effects on electroshock-induced seizures and the development of kindling, MK-801 only partly reduced the duration of seizures in fully kindled animals and did not elevate the threshold for afterdischarge despite the use of a large dose, associated with profound untoward behavioral effects. Together with previous findings, these results support the idea that noncompetitive blockade of NMDA receptors markedly inhibits the development of kindling. The diminished effectiveness of MK-801 against kindled seizures suggests that MK-801 will not be a clinically-useful anticonvulsant against complex partial seizures.

Decreased density, but not number, N-methyl-D-aspartate, glycine and phencyclidine binding sites in hippocampus of senescent rats

Aging is associated with reduced rates of kindling and spatial learning. Blockade of N-methyl-D-aspartate (NMDA) receptors in young animals produces effects similar to those of aging. These findings raise the possibility that age-dependent reductions in neuronal plasticity are the consequence of decreased NMDA receptor-mediated neurotransmission. Conceivably this reduction could be due to an alteration in the NMDA receptor itself. To test this idea we quantified ligand binding to 3 distinct sites on the NMDA receptor/channel complex in hippocampal membranes prepared from 3- and 24-month-old Fischer-344 rats. The binding parameters of the NMDA, glycine and non-competitive antagonist (A.K.A. phencyclidine) sites on the NMDA receptor/channel complex were examined using [3H]3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid ([3H]CPP), [3H]glycine and [3H]N-(1-[2-thienyl]cyclohexyl)3,4-piperidine ([3H]TCP), respectively. Aging produced no change in the affinity or stoichiometry of the binding sites. Aging was associated with a 30% reduction in the density of each of the 3 binding sites (when expressed as sites/mg membrane protein). However, this reduction in receptor density was the consequence of increased protein content in the hippocampus of aged animals, not a reduction in the number of binding sites. These findings suggest that a selective alteration in the NMDA receptor/channel complex itself does not account for the age-dependent reductions in neuronal plasticity.

The role of substantia nigra in the development of kindling: pharmacologic and lesion studies

The role of substantia nigra (SN) in the development of kindling was investigated. Microinjection of gamma-vinyl gamma-aminobutyric acid (GVG), a gamma-aminobutyric acid (GABA) transaminase inhibitor, into the SN bilaterally retarded kindling development by 77%. GVG injected dorsal to the SN did not alter the kindling rate. By contrast, lesions of the SN, whether by thermocoagulation or by microinjected neurotoxin, N-methyl-D,L-aspartate, facilitated kindling development by 27-44%. Thermocoagulative lesions dorsal to the SN did not affect the rate of kindling development. Thus these two manipulations, each presumed to suppress the activity of the SN, resulted in opposite effects on kindling development. We interpret the pharmacologic findings to indicate that the intact SN can powerfully facilitate kindling development. However, the SN is not vital for kindling development, since kindling can be established after destruction of a considerable portion of SN. Whether the increased rate of kindling development following SN lesions is due solely to the absence of SN remains unclear.

Intranigral dynorphin-1-13 suppresses kindled seizures by a naloxone-insensitive mechanism

Numerous lines of evidence indicate that the substantia nigra (SN) facilitates the propagation of seizures in kindling and in other seizure models. Intranigral injection of dynorphin-1-13 exerted a potent seizure suppressant action in kindled rats. This seizure suppressant action was dose dependent, spatially specific for the area of the SN and was not blocked by naloxone (2 mg/kg i.p.). This finding extends previous work indicating that treatments which reduce SN output exert an anticonvulsant action and further suggests that opioid peptides endogenous to the SN may regulate seizure susceptibility in the kindling model.

Anticonvulsant action of intranigral γ-vinyl-GABA: role of noradrenergic neurotransmission☆

Intranigral γ-vinyl-GABA (GVG) suppresses electroshock seizures (ES). This anticonvulsant action was blocked by systemic treatment with the α2-antagonist idazoxan. Consequently, we tested the idea that intranigral GABA mimetics suppress ES by increasing noradrenergic (NE) neuronal activity. Contrary to our hypothesis. GVG decreased NE turnover. This result indicates that while the seizure-suppressant effect of intranigral GVG requires α2-mediated NE neurotransmission, the mechanism of this anticonvulsant action is not by increasing NE neuronal activity.

NMDA Receptor Plasticity in the Kindling Model

The N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptor serves a critical role in the development and stabilization of synapses in the developing nervous system (Cline et al., 1987) and in plasticity of the adult nervous system, particularly with respect to formation of some forms of learning and memory (Morris et al., 1986; Mondadori et al., 1989) Its role in these processes almost certainly derives from two unique features of this ionotropic neurotransmitter receptor: 1) its regulation by magnesium which results in its sensitivity to membrane voltage, thereby endowing it with associative properties (MacDonald et al., 1982; Flatman et al., 1983; Nowak et al., 1984; Mayer et al., 1984); and 2) its permeability to calcium (MacDermott et al., 1986), a second messenger capable of controlling a host of calcium sensitive enzymes.

TCP binding: A tool for studying NMDA receptor-mediated neurotransmission in kindling

Findings from numerous pharmacological and electrophysiological studies have uniquely implicated the N-methyl-D-aspartate (NMDA) receptor in kindling. Recent findings indicate that this receptor is regulated by ligands acting at both amino acid (NMDA and glycine) and ion (Zn++ and Mg++) binding sites. To examine the role of the NMDA receptor in kindling it will be necessary to understand how ligands for these different binding sites interact to control activation of the NMDA receptor. To this end we examined a biochemical tool for measuring opening of the NMDA receptor-gated ion channel (NMDA channel). [3H]N-(1-[thienyl] cyclohexyl)piperidine (TCP) binding to brain membranes is stimulated by NMDA and glycine receptor agonists. We have shown that NMDA and glycine increase TCP binding by increasing the access of TCP to its site. Moreover, the pharmacology of the NMDA and glycine binding sites regulating TCP binding is identical to that of the sites regulating NMDA evoked currents. These findings strongly suggest that glycine and NMDA regulate TCP binding by increasing the opening of the NMDA channel. That is NMDA and glycine increase the overall time that the channel is open thereby increasing the time available for TCP to diffuse to its binding site. These findings support the use of TCP binding (association rate) as a marker of channel opening and thereby permit measurement of NMDA receptor activation and ligand binding under identical conditions. This will allow direct testing the hypothesis that an alteration in the NMDA receptor/channel complex itself underlies the increased seizure response of kindled animals.

N-Methyl-D-Aspartate (NMDA) Receptors and the Kindling Model

This chapter will consider recent pharmacologic, electrophysiologic, and biochemical studies examining the role of a subtype of excitatory amino receptor, the NMDA receptor, in the development of kindling and expression of kindled seizures.

A radiohistochemical measure of [3H]TCP binding to the activated NMDA-receptor-gated ion channel in rat brain

The N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptor is linked to an ion channel that is blocked by the phencyclidine analog N-(1-[thienyl]cyclohexyl)piperidine (TCP). Previous studies have shown that NMDA and glycine act together to increase the access of [3H]TCP to its binding site, presumably by increasing channel opening; NMDA/glycine-enhanced [3H]TCP binding performed under non-equilibrium conditions thereby serves as a dynamic molecular marker of channel activation. In this study we tested whether NMDA and glycine regulate [3H]TCP binding in slide-mounted brain sections. Striking activation of the NMDA ion channel was observed in neo- and allocortex; dentate gyrus and strata radiatum and oriens of CA1 and CA3 of hippocampal formation; and certain amygdaloid nuclei (basomedial, basolateral, and cortical). Other nuclei of the amygdala (central, posterolateral, and medial), basal ganglia, and numerous regions within the diencephalon, brainstem and cerebellum showed relatively little activation of the NMDA ion channel. Our demonstration of enriched NMDA/glycine-stimulated [3H]TCP binding in stratum radiatum of hippocampal region CA1 but not in cerebellar granule cell layer correlates with electrophysiologic studies that showed NMDA channel ion flux in CA1 but not in cerebellar granule cell layer in adult rats. These data demonstrate that NMDA and glycine regulation of [3H]TCP binding can be quantified with a radiohistochemical method to provide a regional measure of the activated NMDA-receptor-gated ion channel. This technique is a powerful tool for functional analysis of the NMDA receptor/channel complex in both physiologic and pathologic states.

Activity of locus coeruleus neurons in amygdala kindled rats: role in the suppression of afterdischarge

Kindling is a model of epilepsy. The mechanisms of kindling development are unknown but may involve attenuation of noradrenergic neurotransmission. Single unit recordings, pharmacologic and lesion techniques were used to test the hypothesis that the increased seizure duration of kindled rats is the consequence of an inactivation of noradrenergic neurons in the locus coeruleus (LC). No difference was found between unkindled (naive) and kindled rats in the firing rates of recorded LC neurons either between or during seizures in the paralyzed, ventilated condition. Moreover, in naive rats, frank destruction of the LC did not lengthen seizure duration. We conclude that the lengthened seizure duration of kindled rats, in the paralyzed, ventilated conditions, is not the consequence of inactivation of the LC since the firing rate of recorded LC neurons in kindled rats was not decreased and since destruction of the LC in naive rats did not lengthen seizure duration. If attenuation of noradrenergic neurotransmission does contribute to the kindling phenomenon, then the LC is not likely to be the site at which this attenuation occurs.