Circuit mechanisms of seizures in the pilocarpine model of chronic epilepsy: cell loss and mossy fiber sprouting.

We used the pilocarpine model of chronic spontaneous recurrent seizures to evaluate the time course of supragranular dentate sprouting and to assess the relation between several changes that occur in epileptic tissue with different behavioral manifestations of this experimental model of temporal lobe epilepsy. Pilocarpine-induced status epilepticus (SE) invariably led to cell loss in the hilus of the dentate gyrus (DG) and to spontaneous recurrent seizures. Cell loss was often also noted in the DG and in hippocampal subfields CA1 and CA3. The seizures began to appear at a mean of 15 days after SE induction (silent period), recurred at variable frequencies for each animal, and lasted for as long as the animals were allowed to survive (325 days). The granule cell layer of the DG was dispersed in epileptic animals, and neo-Timm stains showed supra- and intragranular mossy fiber sprouting. Supragranular mossy fiber sprouting and dentate granule cell dispersion began to appear early after SE (as early as 4 and 9 days, respectively) and reached a plateau by 100 days. Animals with a greater degree of cell loss in hippocampal field CA3 showed later onset of chronic epilepsy (r = 0.83, p < 0.0005), suggesting that CA3 represents one of the routes for seizure spread. These results demonstrate that the pilocarpine model of chronic seizures replicates several of the features of human temporal lobe epilepsy (hippocampal cell loss, supra- and intragranular mossy fiber sprouting, dentate granule cell dispersion, spontaneous recurrent seizures) and that it may be a useful model for studying this human condition. The results also suggest that even though a certain amount of cell loss in specific areas may be essential for chronic seizures to occur, excessive cell loss may hinder epileptogenesis.

Synaptic reorganization by mossy fibers in human epileptic fascia dentata.

This study was designed to identify whether synaptic reorganizations occur in epileptic human hippocampus which might contribute to feedback excitation. In epileptic hippocampi, (n = 21) reactive synaptogenesis of mossy fibers into the inner molecular layer of the granule cell dendrites was demonstrated at the light microscopic and electron microscopic levels. There was no inner molecular layer staining for mossy fibers in autopsy controls (n = 4) or in controls with neocortex epilepsy having no hippocampal sclerosis (n = 2). Comparing epileptics to controls, there were statistically significant correlations between Timm stain density and hilar cell loss. Since hilar neurons are the origin of ipsilateral projections to the inner molecular layer, this suggests that hilar deafferentation of this dendritic zone precedes mossy fiber reafferentation. Quantitative Timm-stained electron microscopy revealed large, zinc-labelled vesicles in terminals with asymmetric synapses on dendrites in the inner molecular and granule cell layers. Terminals in the middle and outer molecular layers did not contain zinc, were smaller and had smaller vesicles. These histochemical and ultrastructural data suggest that in damaged human epileptic hippocampus, mossy fiber reactive synaptogenesis may result in monosynaptic recurrent excitation of granule cells that could contribute to focal seizure onsets.

Glutamate decarboxylase-immunoreactive neurons are preserved in human epileptic hippocampus.

The present study was designed to determine whether inhibitory neurons in human epileptic hippocampus are reduced in number, which could reduce inhibition on principal cells and thereby be a basis for seizure susceptibility. We studied the distribution of GABA neurons and puncta by using glutamate decarboxylase (GAD) immunocytochemistry (ICC) together with Nissl stains. Using quantitative comparisons of GAD-immunoreactive (GAD-IR) neurons and puncta in human epileptic hippocampus and in the normal monkey hippocampus, we found that GAD-IR neurons and puncta are relatively unaffected by the hippocampal sclerosis typical of hippocampal epilepsy where 50-90% of principal (non-GAD-IR) cells are lost. GAD-IR neurons and puncta were not significantly decreased compared with normal monkey. In 6 patients, prior in vivo electrophysiology demonstrated that the anterior hippocampus generated all seizures. The anterior and posterior hippocampus were processed simultaneously, and the counts of hippocampal GAD-IR neurons were numerically greater in anterior than in the posterior hippocampus, where no seizures were initiated. These results indicate that GABA neurons are intact in sclerotic and epileptogenic hippocampus. Computerized image analysis of puncta densities in fascia dentata, Ammon's horn, and subicular complex in epileptic hippocampi (n = 7) were not different from puncta densities in the same regions in normal monkey (n = 2). Hence, despite the significant loss of principal cells (50-90% loss) GABA terminals (GAD-IR puncta) were normal, which suggests GABA hyperinnervation of the remnant pyramidal cells and/or dendrites in human epileptic hippocampus. The apparent increase in puncta ranged from 2 (fascia dentata) to 3.3 (CA1) times normal puncta densities. These findings would suggest increased inhibition and less excitability; however, those regions were epileptogenic. We suggest that GABA terminal sprouting or hyperinnervation of the few remnant projection cells may serve to synchronize their membrane potentials so that subsequent excitatory inputs will trigger a larger population of neurons for seizure onset in the hippocampus and propagation out to undamaged regions of subiculum and neocortex.

Recovery of decreased glutamate decarboxylase immunoreactivity after rat hippocampal kindling.

The present study was designed to test the hypothesis that chronic gamma-aminobutyric acid (GABA) disinhibition of granule cells could explain permanent kindled epileptogenicity. Quantitative and statistical comparisons of glutamate decarboxylase immunoreactivity (GAD-IR), the synthesizing enzyme for GABA, were made of GAD-IR cells and puncta in stratum granulosum of the fascia dentata. The use of GAD immunocytochemistry in kindled and control tissue was used to allow direct anatomic confirmation that we were measuring changes in GAD-IR which would represent GABA synthesis for release by the recurrent inhibitory system of the fascia dentata. Immediately after the last kindled seizure, optically detected GAD-IR puncta densities were significantly reduced in stratum granulosum. At 3 or 7 days after the last kindled seizure, GAD-IR was normal in puncta, indicating that the transient GAD-IR loss was probably a metabolic response to the recent seizure represented by over-use of GAD needed for synthesis of GABA after a prolonged kindled seizure. When the prolonged kindled seizures were discontinued GAD-IR recovered in the puncta. This transient effect did not occur in other areas such as Ammon's horn (CA3) or substantia nigra. The extent of the GAD-IR loss showed no correlation with the severity of the final behavioral seizure (R = 0.23), or the final afterdischarge (AD) duration in entorhinal cortex (R = 0.17) or motor cortex (R = 0.53). A massed stimulation control group given 19 shorter-duration ADs every 10 min (non-kindling) did not reduce GAD-IR. These findings support the hypothetical model that prolonged kindled seizures release excessive GABA which depletes GAD in axon terminals for 1 day after the seizure. However, such a transient suppression of GAD-IR provides no evidence that disinhibition contributes to the kindling process, because kindling proceeds normally with inter-seizure intervals as long as 1 week. The finding of full recovery of GAD-IR within 1 week does not support the model of loss of GABA inhibition to explain the permanency of kindled epileptogenesis.

Distribution of glutamate-decarboxylase-immunoreactive neurons and synapses in the rat and monkey hippocampus: light and electron microscopy.

We have studied the distribution of gamma-aminobutyric acid (GABA) neurons, axons, and synapses in the rat and monkey hippocampal formation by using glutamate decarboxylase (GAD) immunocytochemistry together with Nissl stains, electron microscopy, and double-labeled retrograde transport of horseradish peroxidase. The numbers of GAD-containing (putative GABA) neurons and their percentages compared to all Nissl-stained neurons were calculated throughout all the various fields and strata of the mammalian hippocampus. Although their numbers are greatest in the polymorph region of the fascia dentata (FD) and in the principal cell layers stratum pyramidale (SP) and stratum granulosum (SG), GAD immunoreactive (GAD-IR) cells are numerous in other strata that contain mostly dendrites and scattered cells. These GAD-IR (putative GABA) neurons in dendritic regions may be involved in feedforward dendritic inhibition or may directly inhibit nearby neurons. We used a postmortem delay technique, which resulted in apparent diffusion of GAD into dendrites and axons and allowed better visualization of the extensive dendritic domain of GAD-IR neurons. Computerized image analysis of GAD-IR puncta indicated that putative GABA terminals were numerous on apical and basilar dendrites of all pyramidal cells but unexpectedly highest in the monkey presubiculum. In the rat, GAD-IR neurons projected axons ipsilaterally from every region to the fascia dentata and CA1; however, commissural GAD-IR axons to the fascia dentata arose from GAD-IR neurons in only the contralateral fascia dentata and subiculum. Electron microscopy of GAD-stained hippocampus identified GAD-IR neurons with non-GAD-IR (possibly excitatory) synapses and GAD-IR terminals on somata and dendrites, 80% being the symmetric type and 20% the asymmetric type. In contrast, non-GAD-IR terminals were asymmetric 80% of the time.