J. Victor Nadler

Kainic acid as a tool for the study of temporal lobe epilepsy

Abstract Temporal lobe epilepsy (limbic epilepsy, complex partial epilepsy, psychomotor epilepsy) is the most devastating form of epilepsy commonly encountered in the adult population. The attacks involve loss of consciousness, thus limiting performance of normal functions and exposing the individual to bodily injury. Moreover, long-standing or pharmacologically intractable temporal lobe epilepsy is frequently associated with the loss of neurons from the hippocampus and other brain regions (Ammons horn sclerosis (AHS)). Unfortunately, pharmacologically intractable cases are rather common, owing to the relatively low efficacy against this condition of the available anticonvulsants. Progress in the understanding and treatment of temporal lobe epilepsy would be greatly facilitated by the availability of an animal model which reproduced the behavioral, electrographic and pathological features of this condition. Here I review evidence which indicates that the kainic acid (KA)-treated rat possesses many of the features required of such a model.

Selective reinnervation of hippocampal area CA1 and the fascia dentata after destruction of CA3-CA4 afferents with kainic acid

Intraventricular injections of kainic acid were used to destroy the hippocampal CA3-CA4 cells, thus denervating the inner third of the molecular layer of the fascia dentata and stratum radiatum and stratum oriens of area CA1. The responses of intact afferents to such lesions were then examined histologically. The hippocampal mossy fibers densely reinnervated the inner portion of the dentate molecular layer after bilateral destruction of CA4 neurons and to a lesser extent after unilateral destruction. Septohippocampal fibers replaced CA4-derived fibers in the dentate molecular layer only after particularly extensive bilateral CA4 lesions. Medial perforant path fibers showed no anatomical response to any of these lesions. Neither septohippocampal, temporoammonic nor mossy fibers proliferated in or grew into the denervated laminae of area CA1. These results show a preferential ordering in the reinnervation of dentate granule cells which is not readily explained by proximity to the degenerating fibers and also that removal of CA3-CA4-derived innervation more readily elicits translaminar growth in the fascia dentata than in area CA1. These results may be relevant to clinical situations in which neurons of the hippocampal end-blade are lost.

Kainic acid neurotoxicity toward hippocampal formation: dependence on specific excitatory pathways.

Abstract Rat hippocampal neurons are extremely sensitive to the neurotoxic action of the potent convulsant, kainic acid. It has been suggested that kainic acid destroys neurons through a specific interaction with excitatory glutamatergic afferent fibers. We have tested this hypothesis by making selective lesions of putatively glutamatergic or non-glutamatergic hippocampal afferent fibers three days prior to injecting kainic acid either intraventricularly or directly into the hippocampal formation. Both destruction of hippocampal mossy fibers with colchicine and transection of these fibers markedly reduced the subsequent toxicity of intraventricular kainic acid toward CA3 neurons, but degeneration of the mossy fibers conferred no protection against kainic acid injected locally. Conversely, removal of projections from the entorhinal cortex or medial septum protected dentate granule cells and all but the most medial CA1 pyramidal cells from destruction by locally injected kainic acid, but did not alter the hippocampal toxicity of intraventricular kainic acid. A commissurotomy little affected the hippocampal lesion made by either route of administration. Both intraventricularly and locally injected kainic acid destroyed neurons in extrahippocampal limbic regions. The pattern of damage could not be accounted for merely by diffusion of the toxin from the site of injection. All four types of hippocampal deafferentation markedly attenuated this extrahippocampal toxicity. These results emphasize the dependence of kainic acid neurotoxicity on specific excitatory circuitry. The identity of the critical circuit depends on the route by which kainic acid is administered, but not on the use of glutamate as a transmitter. We suggest that kainic acid destroys neurons indirectly, by initiating a prolonged status epilepticus that is lethal to seizure-sensitive neurons, such as the hippocampal pyramidal cells.

The gene encoding proline dehydrogenase modulates sensorimotor gating in mice

Hemizygous cryptic deletions of the q11 band of human chromosome 22 have been associated with a number of psychiatric and behavioural phenotypes, including schizophrenia. Here we report the isolation and characterization of PRODH, a human homologue of Drosophila melanogaster sluggish-A (slgA), which encodes proline dehydrogenase responsible for the behavioural phenotype of the slgA mutant. PRODH is localized at chromosome 22q11 in a region deleted in some psychiatric patients. We also isolated the mouse homologue of slgA (Prodh), identified a mutation in this gene in the Pro/Re hyperprolinaemic mouse strain and found that these mice have a deficit in sensorimotor gating accompanied by regional neurochemical alterations in the brain. Sensorimotor gating is a neural filtering process that allows attention to be focused on a given stimulus, and is affected in patients with neuropsychiatric disorders. Furthermore, several lines of evidence suggest that proline may serve as a modulator of synaptic transmission in the mammalian brain. Our observations, in conjunction with the chromosomal location of PRODH, suggest a potential involvement of this gene in the 22q11-associated psychiatric and behavioural phenotypes.

The neuroprotective agent riluzole inhibits release of glutamate and aspartate from slices of hippocampal area CA1.

Riluzole is believed to exert its anticonvulsant and neuroprotective actions by reducing glutamate release. This study demonstrated that 10-30 microM riluzole reduces the K(+)-evoked release of glutamate and aspartate from slices of hippocampal area CA1. Only higher concentrations reduced gamma-aminobutyrate (GABA) release. These actions of riluzole were not occluded by tetrodotoxin. Riluzole did not diminish the ability of glutamate analogues to depolarize CA1 pyramidal cells, as determined from grease-gap recordings. Therefore the anticonvulsant and neuroprotective actions of riluzole in the hippocampus may be at least partly explained by its ability to inhibit glutamate/aspartate release from synaptic terminals.

The Recurrent Mossy Fiber Pathway of the Epileptic Brain

The dentate gyrus is believed to play a key role in the pathogenesis of temporal lobe epilepsy. In normal brain the dentate granule cells serve as a high-resistance gate or filter, inhibiting the propagation of seizures from the entorhinal cortex to the hippocampus. The filtering function of the dentate gyrus depends in part on the near absence of monosynaptic connections among granule cells. In humans with temporal lobe epilepsy and in animal models of temporal lobe epilepsy, dentate granule cells form an interconnected synaptic network associated with loss of hilar interneurons. This recurrent mossy fiber pathway mediates reverberating excitation that can reduce the threshold for granule cell synchronization. Factors that augment activity in this pathway include modest increases in [K+]o; loss of GABA inhibition; short-term, frequency-dependent facilitation (frequencies of 1–2 Hz); feedback activation of kainate autoreceptors; and release of zinc from recurrent mossy fiber boutons. Factors that diminish activity include short-term, frequency-dependent depression (frequencies <1 Hz); feedback activation of type II metabotropic glutamate receptors; and the potential release of GABA, neuropeptide Y, adenosine, and dynorphin from recurrent mossy fiber boutons. The axon sprouting and reactive synaptogenesis that follow seizure-related brain damage can also create or strengthen recurrent excitation in other brain regions. These changes are expected to facilitate participation of these regions in seizures. Thus, reactive processes that are often considered important for recovery of function after most brain injuries probably contribute to neurological dysfunction in epilepsy.

Status epilepticus-induced hilar basal dendrites on rodent granule cells contribute to recurrent excitatory circuitry

Mossy fiber sprouting into the inner molecular layer of the dentate gyrus is an important neuroplastic change found in animal models of temporal lobe epilepsy and in humans with this type of epilepsy. Recently, we reported in the perforant path stimulation model another neuroplastic change for dentate granule cells following seizures: hilar basal dendrites (HBDs). The present study determined whether status epilepticus‐induced HBDs on dentate granule cells occur in the pilocarpine model of temporal lobe epilepsy and whether these dendrites are targeted by mossy fibers. Retrograde transport of biocytin following its ejection into stratum lucidum of CA3 was used to label granule cells for both light and electron microscopy. Granule cells with a heterogeneous morphology, including recurrent basal dendrites, and locations outside the granule cell layer were observed in control preparations. Preparations from both pilocarpine and kainate models of temporal lobe epilepsy also showed granule cells with HBDs. These dendrites branched and extended into the hilus of the dentate gyrus and were shown to be present on 5% of the granule cells in pilocarpine‐treated rats with status epilepticus, whereas control rats had virtually none. Electron microscopy was used to determine whether HBDs were postsynaptic to axon terminals in the hilus, a site where mossy fiber collaterals are prevalent. Labeled granule cell axon terminals were found to form asymmetric synapses with labeled HBDs. Also, unlabeled, large mossy fiber boutons were presynaptic to HBDs of granule cells. These results indicate that HBDs are present in the pilocarpine model of temporal lobe epilepsy, confirm the presence of HBDs in the kainate model, and show that HBDs are postsynaptic to mossy fibers. These new mossy fiber synapses with HBDs may contribute to additional recurrent excitatory circuitry for granule cells. J. Comp. Neurol. 428:240–253, 2000.

Development of cholinergic innervation in the hippocampal formation of the rat: I. Histochemical demonstration of acetylcholinesterase activity

Abstract The postnatal development of acetylcholinesterase (AChE) activity in the hippocampal formation of the developing rat brain, as demonstrated histochemically by the copper-thiocholine technique, serves as a marker for the ingrowing cholinergic afferent fibers. The discrete laminar pattern of staining characteristic of the adult hippocampal formation develops entirely after birth. Stain deposit is observable earliest (about 4 days after birth) at the septal end of the hippocampus. During the following week, AChE activity can be demonstrated in successively temporal segments until, about 11 days after birth, all parts of the hippocampal formation exhibit activity. Within each segment, the pattern of developing activity suggests association with three distinct fiber projections emanating from the fimbria, each with its own characteristic time of appearance and rate of growth: (1) a projection through stratum oriens of hippocampus regio inferior to stratum oriens of regio superior; (2) fibers which cross straum pyramidale of regio inferior, run in the suprapyramidal zone of that region and continue into the supra- and infragranular zones in the external leaf of the dentate gyrus; (3) a projection through stratum oriens of regio inferior which continues into the supra- and infragranular zones in the internal leaf of the dentate gyrus.

Regulation of glutamate and aspartate release from slices of the hippocampal CA1 area: effects of adenosine and baclofen

Abstract: Glutamate and/or aspartate is the probable transmitter released from synaptic terminals of the CA3‐derived Schaffer collateral, commissural, and ipsilateral associational fibers in area CA1 of the rat hippocampal formation. Slices of the CA 1 area were employed to test the effects of adenosine‐and 7‐aminobutyrate (GABA)‐related compounds on the release of glutamate and aspartate from this projection. Under the conditions of these experiments, the release of glutamate and aspartate evoked by 50 mM K+ was more than 90% Ca2+‐dependent and originated predominantly from the CA3‐derived pathways. Adenosine reduced the K+‐evoked release of glutamate and aspartate by a maximum of about 60%, but did not affect the release of GABA. This action was reversed by 1 μM 8‐phenyltheophylline. The order of potency for adenosine analogues was as follows: l‐N6‐phenylisopropyl‐adenosine > N6‐cyclohexyladenosine > d‐N6‐phenylisopro‐pyladenosine ≅ 2‐chloroadenosine > adenosine ≫5′‐N‐ethylcarboxamidoadenosine. 8‐Phenyltheophylline (10 μM) by itself enhanced glutamate/aspartate release, whereas di‐pyridamole alone depressed release. These results support the view that adenosine inhibits transmission at Schaffer col‐lateral‐commissural‐ipsilateral associational synapses mainly by reducing transmitter release and that these effects involve the activation of an A1 receptor. Neither adenosine, l‐N6‐phenylisopropyladenosine, nor 8‐phenyltheophylline affected the release of glutamate or aspartate evoked by 10 μM ve‐ratridine. The differing effects of adenosine compounds on release evoked by K+ and veratridine suggest that A1 receptor activation either inhibits Ca2+ influx through the voltage‐sensitive channels or interferes with a step subsequent to Ca2+ entry that is coupled to the voltage‐sensitive Ca2+ channels in an obligatory fashion. Neither baclofen nor any other agent active at GABAB or GABAA receptors affected glutamate or aspartate release evoked by elevated K+ or veratridine. Therefore, either baclofen does not inhibit transmission at these synapses by depressing transmitter release or else it does so in a way that cannot be detected when a chemical depolarizing agent is employed.

Loss and reacquisition of hippocampal synapses after selective destruction of CA3-CA4 afferents with kainic acid.

Intraventricular injections of kainic acid were used to destroy the hippocampal CA3-CA4 cells bilaterally in rats, thus denervating the inner third of the molecular layer of the fascia dentata and stratum radiatum of area CA1. Electron microscopic studies showed that this lesion reduced the synaptic density of the CA1 stratum radiatum by an average of 86%. The synaptic density of the inner third of the dorsal dentate molecular layer declined by two-thirds and the corresponding zone of the ventral dentate molecular layer by about half. Within 6-8 weeks the synaptic density of these laminae had been restored to the control value or nearly so. In the CA1 stratum radiatum about 72% of the synaptic contacts destroyed by the lesion were replaced, the inner third of the ventral dentate molecular layer recovered 75% of its lost synapses and the inner third of the dorsal dentate molecular layer apparently recovered virtually all of them. The newly formed synapses did not differ noticeably from those normally present. A kainic acid lesion reduced the synaptic density of the outer two-thirds of the dentate molecular layer by 30% within 3-5 days, despite a virtual absence of presynaptic degeneration in that zone. This result implies a substantial disconnection of perforant path synapses. It did not appear to depend on the extent of denervation of the inner zone. The loss of perforant path synapses was completely reversible. We suggest that the dentate granule cells shed a portion of their synapses in response to a substantial loss of neurons to which they project and regained them when their axons had formed new synaptic connections.