Charles F. Zorumski

The functional anatomy and pathology of lithium-pilocarpine and high-dose pilocarpine seizures

Subcutaneous treatment of rats with low doses of lithium and pilocarpine or a high dose of pilocarpine results in a severe seizure--brain damage syndrome. Rats thus treated were studied with multiple-depth electrodes, quantitative [14C]2-deoxyglucose autoradiography, and light and electron microscopy. Rats receiving lithium-pilocarpine did not differ from high-dose pilocarpine rats in behavioral, electrographic, metabolic or histopathological findings, but lithium-pilocarpine reproduced the syndrome more reliably and with a lower acute mortality rate. Organized electrographic seizure activity developed just prior to the onset of behavioral forelimb clonus and appeared to originate from ventral forebrain in the vicinity of the ventral pallidum and/or nucleus accumbens. From these sites activity spread rapidly to involve other regions. Once initiated, electrographic seizures persisted for hours. Increased glucose utilization was found in most brain regions during the period of continuous seizure activity. The greatest increases were found in the ventral pallidum, globus pallidus, hippocampus, entorhinal cortex, amygdala, lateral septum, substantia nigra, ventrobasal and mediodorsal thalamus and frontal motor cortex. Animals sustaining seizures displayed a disseminated pattern of neural degeneration not involving globus pallidus or ventral pallidum but otherwise coinciding with the above pattern of enhanced glucose utilization. No consistent correlation was observed between the pattern of brain damage and known regions of high muscarinic cholinergic receptor density. Ultrastructurally, the cytopathological changes, like those associated with various other sustained seizure syndromes, resemble the excitotoxic type of damage glutamate is known to cause. This seizure-brain damage syndrome and that induced by systemic kainic acid appear to be similar in behavioral but not in electrophysiological or metabolic manifestations. During kainic acid seizures, electrographic changes are first recorded in the hippocampus while they are first detected in the ventral forebrain region in pilocarpine seizures. Pilocarpine also induced metabolic activation of ventral forebrain sites not activated by kainic acid. The cytopathology associated with the two syndromes is identical in type but not in pattern, the cholinergic model being characterized by much greater neocortical and slightly less hippocampal damage. Further study of these cholinergic models may provide new insights into the roles of the major excitatory neurotransmitter systems (cholinergic and glutamergic) in limbic epilepsy.

Ketamine, phencyclidine, and Mk-801 protect against kainic acid-induced seizure-related brain damage

Summary: Recent evidence implicates the endogenous excitotoxin, glutamate (Glu), in several neurologic disorders, including seizure‐related brain damage. Ketamine, phencyclidine, and MK‐801, which are noncompetitive antagonists of the N‐methyl‐D‐aspartate (NMDA) subtype of Glu receptor (but do not antagonize kainic acid receptors) were tested in the present study for their effects on behavioral and/or electrographic seizures and seizure‐related brain damage induced by kainic acid. Behavioral seizure activity was reduced by these agents, as was spread of electrographic seizures to neocortex, but seizures recorded from deep brain regions such as hippocampus, piriform cortex, and amygdala were not significantly diminished. All three agents prevented seizure‐related brain damage in the amygdala, piriform cortex, thalamus, and CA1 region of the hippocampus but conferred little or no protection in the lateral septum and CA3 region of the hippocampus. The regional selectivity of the neuroprotective effect suggests that NMDA receptors may play a more dominant role in seizure‐related brain damage in some brain regions than in others. The ability of NMDA antagonists to prevent seizure‐related damage in several brain regions without suppressing seizure activity suggests that in these brain regions persistent seizure activity can be maintained by other transmitter systems, with or without NMDA receptor participation, but that seizure‐related brain damage is critically dependent on NMDA receptor participation.

Enhancement of hippocampal excitatory synaptic transmission by platelet-activating factor

The biologically active lipid platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine; PAF) is a mediator of inflammatory and immune responses, and it accumulates in the brain during convulsions or ischemia. We have examined whether PAF may play a second messenger role in the central nervous system by studying effects on synaptic transmission in cultured hippocampal neurons. Carbamyl-PAF, a nonhydrolyzable PAF analog with a similar pharmacologic profile, augmented glutamate-mediated, evoked excitatory synaptic transmission and increased the frequency of spontaneous miniature excitatory synaptic events without increasing their amplitude or altering their time course. This compound had no significant effect on gamma-aminobutyric acid-mediated inhibitory synaptic responses. Lyso-PAF, the biologically inactive metabolic intermediate, had no effect on synaptic transmission. Moreover, the enhancement of excitatory synaptic transmission by carbamyl-PAF was blocked by a PAF receptor antagonist. These results indicate a specific presynaptic effect of PAF in enhancing excitatory synaptic transmission in cultured rat hippocampal neurons.

Functional network dysfunction in anxiety and anxiety disorders.

A recent paradigm shift in systems neuroscience is the division of the human brain into functional networks. Functional networks are collections of brain regions with strongly correlated activity both at rest and during cognitive tasks, and each network is believed to implement a different aspect of cognition. We propose here that anxiety disorders and high trait anxiety are associated with a particular pattern of functional network dysfunction: increased functioning of the cingulo-opercular and ventral attention networks as well as decreased functioning of the fronto-parietal and default mode networks. This functional network model can be used to differentiate the pathology of anxiety disorders from other psychiatric illnesses such as major depression and provides targets for novel treatment strategies.

A benzodiazepine recognition site associated with the non-NMDA glutamate receptor

GYKI 52466 is a benzodiazepine molecule that has muscle relaxant and anticonvulsant properties not attributable to a gamma-aminobutyric acid receptor-mediated mechanism. Here it is shown that GYKI 52466 exerts no blocking action at N-methyl-D-aspartate (NMDA) glutamate receptors, but acts noncompetitively to block ion currents and associated excitotoxicity, including ischemic neuronal degeneration, mediated through non-NMDA glutamate receptors. The inhibition of non-NMDA responses by GYKI 52466 is antagonized by cyclothiazide, hydrochlorothiazide, and diazoxide, benzothiadiazide drugs that inhibit non-NMDA receptor desensitization. These results suggest that non-NMDA receptor-ion channel complexes may contain a novel benzodiazepine recognition site where receptor desensitization is regulated; this postulated site represents a promising new target for rational development of drugs to treat neurological disorders.

Nitric oxide inhibitors attenuate N-methyl-d-aspartate excitotoxicity in rat hippocampal slices

To investigate whether nitric oxide (NO) plays a role in the neurotoxicity produced by N-methyl-D-aspartate (NMDA) we have examined the effects of NO inhibitors on NMDA-mediated neurodegeneration in the CA1 region of rat hippocampal slices. L-NG-Monomethylarginine, L-NG-nitroarginine and hemoglobin markedly diminished the toxicity produced by activation of NMDA receptors without interfering with NMDA receptor-mediated ion currents or synaptic responses. The neuroprotective effects are reversed by coapplication of L-arginine with the NO synthase inhibitors. These results suggest that activation of the NO system is an important component of the biochemical cascade leading to neurodegeneration produced by NMDA receptors.

Paired-pulse modulation of fast excitatory synaptic currents in microcultures of rat hippocampal neurons.

1. Paired‐pulse modulation of excitatory non‐N‐methyl‐D‐aspartate (non‐NMDA) receptor‐mediated autaptic currents and conventional monosynaptic (interneuronal) excitatory postsynaptic currents (EPSCs) was investigated in microcultures of rat hippocampal neurons, where polysynaptic influences are eliminated. 2. Most autaptic currents and EPSCs exhibited paired‐pulse depression in response to paired stimuli. Depression was sensitive to the level of transmitter release, which was varied by manipulating extracellular Ca2+ and Mg2+ concentrations. Paired‐pulse facilitation emerged in many cells at low levels of transmitter release. 3. Paired‐pulse depression and facilitation could be differentially expressed at two distinct postsynaptic targets of a single presynaptic cell, and the form of modulation was not dependent upon the transmitter phenotype of the postsynaptic cell. 4. Paired‐pulse depression recovered exponentially with a time constant of approximately 5 s, although in most neurons a much faster component of recovery was detected. Recovery from paired‐pulse facilitation was well described by a single exponential of 380 +/‐ 57 ms. 5. Under conditions of robust paired‐pulse depression of evoked responses, spontaneous autaptic and postsynaptic currents (sEPSCs, presumed miniature EPSCs) occurred at an enhanced frequency immediately following evoked responses. The decay of the frequency increase mirrored the time course of recovery from paired‐pulse facilitation of evoked responses examined under conditions of reduced transmitter release. 6. Several lines of evidence suggested a large presynaptic component to paired‐pulse depression. In eight out of nine cells no depression in sEPSC amplitudes was detected following conditioning stimulation. Simultaneously recorded glial glutamate uptake currents showed depression similar to neuronal evoked EPSCs. Finally, NMDA receptor‐mediated EPSC paired‐pulse depression at positive potentials was similar to non‐NMDA EPSC depression. 7. Neither adenosine nor glutamate feedback onto presynaptic receptors is likely to mediate paired‐pulse depression, because neither competitive nor non‐competitive inhibitors of the actions of these agents diminished paired‐pulse depression.

Kindling with rapidly recurring hippocampal seizures

Bipolar electrodes, stereotactically implanted in the hippocampus of adult rats, were used to deliver 10 s trains of suprathreshold tetanic electrical stimuli every few minutes. As indices of seizure intensity, durations of the afterdischarges triggered by these stimuli were measured, and the accompanying behaviors were scored on a 5-point scale. After 2-3 h, prolonged afterdischarges appeared in conjunction with severe limbic seizures, separated by periods of approximately 60 min. After 3-9 h, the stimulation was withheld until the following day. Upon reinstitution of the stimuli, intense seizures were seen at the onset, and the cycle time between them was shortened. Enhanced responsiveness to a fixed stimulus persisted for several months, the longest period tested. In addition, the enhanced epileptogenicity showed transference and was not stimulus-specific. These studies, using stimuli with low intertrain frequency and short interstimulus intervals, establish a robust and rapidly-developing model of epileptogenesis in the hippocampus that is comparable to traditional kindling.

Domoic acid: A dementia-inducing excitotoxic food poison with kainic acid receptor specificity

Domoic acid (Dom), a rigid analog of the excitotoxic amino acids, glutamate and kainic acid, is believed to be the mussel neurotoxin responsible for a recent food poisoning incident in Canada that killed some people and left others with memory impairment. Since the literature contains very little information pertaining to Dom excitotoxicity, we have systematically evaluated the neuroexcitatory properties of Dom in vitro (cultured hippocampal neurons) and its neurotoxic properties both in vitro (chick embryo retina) and in vivo (adult rat). In the in vitro experiments, the properties of Dom were compared with those of kainic acid, N-methyl-D-aspartate (NMDA) and quisqualate, each of which is a prototypic agonist at a different subtype of glutamate receptor. Currents induced in hippocampal neurons by Dom and kainic acid were identical and displayed a linear current/voltage relationship (in contrast to NMDA currents) and were nondesensitizing (in contrast to quisqualate currents). Dom currents were not blocked by NMDA antagonists but were blocked by CNQX, an antagonist of non-NMDA receptors. In the chick embryo retina, Dom induced a lesion pattern having the same distinctive characteristics as a kainic acid lesion which differs from that induced by either NMDA or quisqualate, and the Dom lesion was blocked by CNQX but not by NMDA antagonists. Subcutaneous administration of Dom (2.5-3 mg/kg) to adult rats resulted in an acute seizure-brain damage syndrome almost identical to that induced in rats by KA (12 mg/kg) and having important features analogous to the neurotoxic syndrome observed in the human food poison victims.

Neurosteroid Access to the GABAA Receptor

GABAA receptors are a pivotal inhibitory influence in the nervous system, and modulators of the GABAA receptor are important anesthetics, sedatives, anticonvulsants, and anxiolytics. Current views of receptor modulation suggest that many exogenous drugs access and bind to an extracellular receptor domain. Using novel synthetic steroid analogs, we examined the access route for neuroactive steroids, potent GABAA receptor modulators also produced endogenously. Tight-seal recordings, in which direct aqueous drug access to receptor was prevented, demonstrated that steroids can reach the receptor either through plasma membrane lateral diffusion or through intracellular routes. A fluorescent neuroactive steroid accumulated intracellularly, but recordings from excised patches indicated that the intracellular reservoir is not necessary for receptor modulation, although it can apparently equilibrate with the plasma membrane within seconds. A membrane impermeant neuroactive steroid modulated receptor activity only when applied to the inner membrane leaflet, demonstrating that the steroid does not access an extracellular modulatory site. Thus, neuroactive steroids do not require direct aqueous access to the receptor, and membrane accumulation is required for receptor modulation.