Maxine M. Okazaki

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.

Ultrastructural features and synaptic connections of hilar ectopic granule cells in the rat dentate gyrus are different from those of granule cells in the granule cell layer.

Several investigators have shown the existence of dentate granule cells in ectopic locations within the hilus and molecular layer using both Golgi and retrograde tracing studies but the ultrastructural features and synaptic connections of ectopic granule cells were not previously examined. In the present study, the biocytin retrograde tracing technique was used to label ectopic granule cells following injections into stratum lucidum of CA3b of hippocampal slices obtained from epileptic rats. Electron microscopy was used to study hilar ectopic granule cells that were located 20-40 microm from the granule cell layer (GCL). They had ultrastructural features similar to those of granule cells in the GCL but showed differences, including nuclei that often displayed infoldings and thicker apical dendrites. At their origin, these dendrites were 6 microm in diameter and they tapered down to 2 microm at the border with the GCL. Both biocytin-labeled and unlabeled axon terminals formed exclusively asymmetric synapses with the somata and proximal dendrites of hilar ectopic granule cells. The mean number of axosomatic synapses for these cells was three times that for granule cells in the GCL. Together, these data indicate that hilar ectopic granule cells are postsynaptic to mossy fibers and have less inhibitory input on their somata and proximal dendrites than granule cells in the GCL. This finding is consistent with recent physiological results showing that hilar ectopic granule cells from epileptic rats are more hyperexcitable than granule cells in the GCL.

Protective effects of mossy fiber lesions against kainic acid-induced seizures and neuronal degeneration.

The effects of a hippocampal mossy fiber lesion have been determined on neuronal degeneration and limbic seizures provoked by the subsequent intracerebroventricular administration of kainic acid to unanesthetized rats. Mossy fiber lesions were made either by transecting this pathway unilaterally or by destroying the dentate granule cells unilaterally or bilaterally with colchicine. All control rats eventually developed status epilepticus and each temporally discrete seizure that preceded status epilepticus was recorded from the hippocampus ipsilateral to the kainic acid infusion before the contralateral hippocampus. A mossy fiber lesion of the ipsilateral hippocampus prevented the development of status epilepticus in 26% of subjects and in 52% of subjects seizures were recorded from the contralateral hippocampus before the ipsilateral hippocampus. Unlike electrographic records from other treatment groups, those from rats which had received a bilateral colchicine lesion exhibited no consistent pattern indicative of seizure propagation from one limbic region to another. A bilateral, but not a unilateral, mossy fiber lesion also dramatically attenuated the behavioral expression of the seizures. Regardless of its effects on kainic acid-induced electrographic and behavioral seizures, a mossy fiber lesion always substantially reduced or completely prevented the degeneration of ipsilateral hippocampal CA3-CA4 neurons. This protective effect was specific for those hippocampal neurons deprived of mossy fiber innervation. Neurons in other regions of the brain were protected from degeneration only when the mossy fiber lesion also prevented the development of electrographic status epilepticus. These results suggest that the hippocampal mossy fibers constitute an important, though probably not an obligatory, link in the circuit responsible for the spread of kainic acid seizures. Degeneration of CA3-CA4 neurons appears to depend upon (1) the duration of hippocampal seizure activity and (2) an as yet undefined influence of or interaction with the mossy fiber projection which enhances the neurodegenerative effect of the seizures.

N-methyl-D-aspartate receptor autoradiography in rat brain after angular bundle kindling.

The specific binding of L-[3H]glutamate to N-methyl-D-aspartate (NMDA) receptors in brain regions of kindled rats was visualized autoradiographically and quantitated. When assayed 28 days after the last evoked seizure, NMDA receptor binding had declined by 7-11% in stratum radiatum of the dorsal hippocampal area CA1, in both deep and superficial layers of the motor cortex and in layers I-IV of the somatosensory cortex. No significant changes were detected in any other brain region nor in any region examined 1 day after the last evoked seizure. These findings suggest that the enhanced activation of NMDA receptors in kindled rats cannot be explained by an increased expression of these receptors. Rather, kindling leads to a regionally-selective down regulation of NMDA receptor binding.

Glutamate receptor involvement in dentate granule cell epileptiform activity evoked by mossy fiber stimulation.

In many persons with temporal lobe epilepsy, dentate granule cells form an interconnected synaptic network. This recurrent mossy fiber circuit mediates reverberating excitation that may facilitate seizure propagation by synchronizing granule cell discharge. The involvement of specific glutamate receptors in granule cell epileptiform activity evoked by stimulating the mossy fibers was investigated with use of rat hippocampal slices superfused with bicuculline, with or without increasing [K+](o) to 6 mM. The occurrence of short-latency mossy fiber-evoked granule cell epileptiform activity in slices from pilocarpine-treated rats correlated with the presence and extent of recurrent mossy fiber growth. Blockade of AMPA receptors nearly abolished the orthodromic component of the response; subsequent antagonism of kainate receptors as well appeared to have no further action. Antagonism of NMDA receptors reduced the duration of epileptiform discharge, but increased the amplitude of population spikes within the evoked burst. Thus AMPA and NMDA, but perhaps not kainate, receptors play an important role in this type of epileptiform activity. Activation of type II metabotropic glutamate receptors, which inhibits the release of glutamate from mossy fiber boutons, reduced the magnitude of epileptiform discharge. This action was reversed by a partial agonist of these receptors. However, neither an agonist nor an antagonist of type III metabotropic glutamate receptors significantly altered the response. Considering the importance of synchronous granule cell discharge for seizure propagation from the entorhinal cortex to the hippocampus, agonists of type II metabotropic glutamate receptors may be useful in suppressing such discharge both experimentally and clinically.

Kainate and quisqualate receptor autoradiography in rat brain after angular bundle kindling

The kainate and quisqualate types of excitatory amino acid receptor were visualized autoradiographically in brain sections from rats kindled by stimulating the angular bundle. Kainate receptors were labeled with [3H]kainate and quisqualate receptors with L-[3H]glutamate. When assayed one day after the last evoked seizure, kainate receptor binding had declined by 24-29% in stratum lucidum of hippocampal area CA3 and by 12-14% in the inner third of the dentate molecular layer, but was unchanged in the neocortex and basolateral amygdala. Saturation binding curves revealed that, under the conditions of these experiments, [3H]kainate labeled a single class of binding sites with a KD of 33-36 nM. In stratum lucidum of area CA3, kindling reduced the density of kainate receptors without altering their affinity for kainate. At the same time, quisqualate receptor binding had declined by 20-35% in many layers of the hippocampal formation and neocortex, but remained unchanged in the basolateral amygdala. Repeated stimulation or repeated seizures were required to produce these effects, since both kainate and quisqualate receptor binding were unchanged one day after a single afterdischarge. These receptor changes largely or completely reversed during a 28-day period without further stimulation. Thus maintenance of the kindled state probably cannot be explained by a long-lasting change in the expression of kainate or quisqualate receptors. The transient, regionally-selective down-regulation of these receptors may represent a compensatory response of forebrain neurons to repeated stimulation or seizures.

Kainic Acid Seizures and Neuronal Cell Death: Insights from Studies of Selective Lesions and Drugs

Hippocampal pathology (Ammon’s horn sclerosis (AHS)) is a well-recognized finding in the brains of temporal lobe epileptics (Meldrum and Corsellis, 1984). Classical AHS involves extensive loss of neurons from hippocampal area CA1 (h1, Sommer sector), a less extensive neuronal deficit in the CA3-CA4 area (h3-h5, endblade, endfolium), and relative sparing of neurons in the h2 area (‘resistant zone’; area CA2 and the adjacent portion of area CA3a which contains the mossy fiber endbulb) and in the fascia dentata. Most commonly, some neuronal loss in other brain regions, particularly the amygdala, thalamus and cerebral neocortex, accompanies the hippocampal lesion. Although the near-total loss of neurons from area CA1 is the most striking feature of AHS in many patients, there is reason to believe that the most vulnerable neurons are the CA3 hippocampal pyramidal cells and the morphologically diverse neurons of area CA4 (Margerison and Corsellis, 1966).

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.

Mossy fiber lesion reduces the probability that kainic acid will provoke CA3 hippocampal pyramidal cell bursting.

Hippocampal slices prepared from rats which had received a mossy fiber lesion differed in their response to 50 nM kainic acid. Those slices in which the mossy fiber projection had been substantially destroyed were significantly less likely to develop epileptiform bursting in area CA3 than slices in which the mossy fiber projection was only modestly damaged. Similarly, mossy fiber lesions prevent the development of electrographic status epilepticus after intracerebroventricular administration of kainic acid in 26% of rats. Therefore mossy fiber lesions probably act, both in vivo and in vitro, by reducing the sensitivity of CA3 hippocampal pyramidal cells to the epileptogenic action of kainic acid.

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