Robert S. Sloviter

“Epileptic” brain damage in rats induced by sustained electrical stimulation of the perforant path. I. Acute electrophysiological and light microscopic studies

Sustained electrical stimulation of the perforant path in urethane-anesthetized rats evoked hippocampal granule cell population spikes and epileptiform discharges. After stimulation, recurrent inhibition in the granule cell layer was abolished. Light microscopic analysis revealed a highly reproducible pattern of hippocampal damage to dentate pyramidal basket cells, hilar cells in general and CA3 and CA1 pyramidal cells. CA2 pyramidal cells and dentate granule cells were relatively unaffected. When perforant path stimulation on one side of the brain evoked bilateral granule cell discharges, damage was bilateral. Unilateral hippocampal seizures were associated with unilateral hippocampal damage. Rapid Golgi-stained hippocampi exhibited spherical dendritic swellings at the sites of termination of excitatory entorhinal afferents to the hippocampus and in the mossy fiber region. Electrical stimulation of a single excitatory afferent to the hippocampus appears to reproduce the epileptic pattern of hippocampal damage without using convulsant drugs and without causing motor convulsions. It is suggested that seizure-associated brain damage is caused by excessive pre-synaptic release of excitatory transmitter that induces intracellular post-synaptic changes that lead to dendritic swelling and cell death.

Epileptic brain damage is replicated qualitatively in the rat hippocampus by central injection of glutamate or aspartate but not by GABA or acetylcholine.

Repeated intraventricular injection of the excitatory amino acids glutamate and aspartate for one hour produced morphologic changes in the hippocampus that were qualitatively identical to the acute and chronic changes seen in the brains of human epileptics and in experimental animals in which hippocampal seizure activity was induced by kainic acid or electrical stimulation of the perforant path. Light and electron microscopy revealed acute effects of glutamate and aspartate consisting of glial and dendritic swelling and neuronal soma necrosis (dark cell degeneration). Electron microscopy showed the focal dendritic swelling induced by glutamate or aspartate to be of the axon-sparing type with presynaptic terminals relatively unaffected. Four weeks after injection, irreversible neuron loss and reactive gliosis had occurred. The inhibitory amino acid gamma-aminobutyric acid caused acute glial swelling similar to that caused by glutamate and aspartate but did not produce neurotoxic effects, indicating that glial swelling may not be causally related to neuronal death but may be the result of amino acid uptake. The excitatory non-amino acid acetylcholine produced no direct, periventricular hippocampal damage or glial swelling but did produce dendritic swelling in the CA3 region innervated by the perforant path, presumably as a result of acetylcholine-induced seizure activity in this pathway. Glutamate and aspartate also caused glial and neuronal changes in other periventricular structures, e.g., septum, hypothalamus, caudate and habenula, as well as in the most dorsal portion of the cerebellum. Dendritic swelling induced by glutamate and aspartate in the cerebellar molecular layer was accompanied by acute necrosis of Purkinje cell somata. These results suggest that seizure-associated brain damage is initiated by excessive endogenous excitatory amino acid receptor activation.

A simplified timm stain procedure compatible with formaldehyde fixation and routine paraffin embedding of rat brain

A simplified sulphide/silver technique for visualizing transitional metals in rat brain is described. It consists of: (1) sulphide perfusion before formaldehyde perfusion, (2) routine paraffin embedding and sectioning, and (3) silver staining with physical developer. This method is compatible with formaldehyde fixation, eliminates the need for post-fixation in Carnoys solution before embedding and does not require low paraffin temperatures during embedding. Once embedded, brains can be stored for months before sectioning without loss of stain quality.

“Epileptic” brain damage in rats induced by sustained electrical stimulation of the perforant path. II. Ultrastructural analysis of acute hippocampal pathology

Sustained electrical stimulation of the perforant path evokes granule cell population spikes and epileptiform discharges, abolishes recurrent inhibition in the granule cell layer and induces a reproducible pattern of hippocampal damage (see preceding paper, this volume, for electrophysiological and light microscopic findings). Electron microscopic findings described here reveal that the hippocampal damage is identical in pattern and cytopathological detail to that associated with sustained limbic seizures induced by chemical convulsants such as kainic acid, folic acid and dipiperidinoethane. Acutely swollen dendritic segments distributed in a laminar pattern corresponding closely with the termination of putative glutamate or aspartate-containing fibers, including those of the perforant path, were a conspicuous finding. Cell bodies of CA1 and CA3 pyramidal neurons and various interneurons in the hilus and elsewhere displayed degenerative changes ranging from mild to severe. Both the dendritic and somal degenerative changes closely resemble the excitotoxic type of damage that the putative transmitters glutamate and aspartate are known to cause. It is proposed, therefore, that sustained electrical stimulation of the perforant path results in excessive synaptic release and accumulation of glutamate (or aspartate) at numerous dendrosomal receptors in the hippocampus with consequent degeneration of the dendrosomal structures housing these receptors. Early excitotoxic effects on interneurons that mediate recurrent inhibition may play an important role in the observed loss of recurrent inhibition and in the evolution of subsequent excitotoxic degeneration in the hippocampus.

Sustained electrical stimulation of the perforant path duplicates kainate-induced electrophysiological effects and hippocampal damage in rats

Electrical stimulation of the perforant path of urethane-anesthetized rats for 24 h evoked hippocampal granule cell population spikes, epileptiform discharges, and abolished the recurrent inhibition of granule cells (as determined with the twin pulse technique). After 24 h of stimulation, hilar interneurons of area dentata and the regions of the CA3 pyramidal cells which receive granule cell input were damaged; CA2 pyramidal cells and dentate granule cells were relatively unaffected. Pericellular spaces possibly indicative of acute gliotoxicity were observed in the CA1 pyramidal cell body region. These results establish a causative relationship between excessive presynaptic neuronal activity and postsynaptic neuronal damage.

On the relationship between kainic acid-induced epileptiform activity and hippocampal neuronal damage

The electrophysiological effects of kainic acid on evoked hippocampal granule cell activity and recurrent inhibition (as measured with the twin-pulse technique) were examined in urethaneanesthetized rats. In the 3-hr period after injection, kainic acid (10 mg/kg i.v.) caused repetitive cycles of decreased recurrent inhibition, granule cell epileptiform discharges and depression of evoked activity. These electrophysiological effects were accompanied by twitching of the vibrissae, mild hyperpyrexia and “wet-dog” shakes which, despite anesthesia, often occurred at the end of an epileptiform granule cell discharge. Four hours after the injection of kainic acid, recurrent inhibition was abolished and granule cell spike amplitudes in response to perforant path stimuli were significantly greater than saline controlor pre-drug values. Concomitantly, kainic acid caused acute damage to dentate hilar interneurons and to the cell body and proximal apical dendritic regions of CA3 pyramidal cells. These results indicate that an early effect of kainic acid is impairment of recurrent inhibition, possibly as a result of damage to inhibitory interneurons. The presumably resultant epileptiform activity of dentate granule cells and acute damage to the region of the pyramidal cells which receive dense excitatory input from granule cell axon terminals supports the view that epileptiform activity plays a significant role in the neurotoxic effects of kainic acid.

Serotonergic properties of β-phenethylamine in rats

Current research interest in β-phenethylamine (PEA) derives, in part, from a possible role for this endogenous compound in paranoid schizophrenia. Systemic administration of PEA caused a behavioral syndrome in rats (simultaneous forepaw padding, side-to-side headweaving or head tremor and splayed hindlimbs) which reflects activation of serotonin (5-HT) receptors in the central nervous system. This syndrome in rats was also produced by large doses of amphetamine, a drug structurally related to PEA, which can induce a behavioral state in man similar to paranoid schizophrenia. Reduction of endogenous 5-HT concentrations by p-chlorophenylalanine or 5,7-dihydroxytryptamine prevented the 5-HT behavioral syndrome caused by amphetamine, but not by PEA. The presumed 5-HT receptor antagonists, methysergide and mianserine, blocked these behavioral responses to both amphetamine and PEA. Therefore, it seems most likely that PEA, in addition to its catecholaminergic actions, produces serotonergic effects in rats by a direct 5-HT agonist action. This is in contrast to amphetamine which apparently causes the same serotonergic behavioral effects by releasing endogenous 5-HT.

Effect of Morphine on ‘Wet-Dog’ Shakes Caused by Cerebroventricular Injection of Serotonin

Intraventricular administration of serotonin to rats causes wet-dog shakes, a sign of morphine withdrawal. The frequency of shakes is dose-dependent. Shaking is potentiated by pretreatment with an inhibitor of monoamine oxidase or with 5,7-dihydroxytryptamine, and is depressed by morphine or serotonin receptor blockers. Depression of serotonin-induced shaking by morphine is reversed rapidly by naloxone. However, naloxone did not reverse the inhibition of wet-dog shakes caused by serotonin receptor blockers.

Serotonin agonist actions of p-chlorophenylalanine.

Abstract p -Chlorophenylalanine (pCPA), in combination with a monoamine oxidase inhibitor, caused a behavioural syndrome shown previously to be due to serotonin receptor activation. p -Chlorophenyl-ethylamine (pCPEA), a decarboxylation product of p CPA, also caused the behavioural syndrome. Decarboxylase inhibition prevented the syndrome after p CPA administration, but not after p CPEA. Prior depletion of brain serotonin to 18% of control prevented the syndrome caused by p CPA (plus monoamine oxidase inhibitor), or by pPCEA alone. Subsequent replacement of serotonin in depleted rats by 5-hydroxytryptophan restored the behavioural effects of p CPA and p CPEA. The results show that p CPA (in combination with an inhibitor of monoamine oxidase) caused serotonin receptor activation. The behavioural syndrome is probably elicited by displacement of presynaptic serotonin by p CPEA. As p CPA apparently has a long biological half-life, continued synthesis of p CPEA may cause anomalous behavioural effects not correlated with serotonin depletion. This possibility should be considered in behavioural studies with p CPA, especially during early time periods when serotonin concentrations are not yet reduced.

Methionine enkephalin-induced shaking behavior in rats: dissociation from brain serotonin mechanisms.

Abstract Intracerebroventricular injection of methionine enkephalin (met-enkephalin) caused dosedependent shakes in rats. The role of endogenous 5-hydroxytryptamine (5-HT) in met-enkephalin-induced shakes was investigated since shaking behavior has been associated with activation of 5-HT receptors. Met-enkephalin-induced shakes were not affected significantly by pretreatment with p-chlorophenylalanine, 5,7-dihydroxytryptamine, or methysergide. Naloxone inhibited the shakes caused by met-enkephalin. The results suggest that met-enkephalin elicits shaking behavior by a mechanism independent of central 5-HT.