Luiz E. Mello

Temporal profile of neuronal injury following pilocarpine or kainic acid-induced status epilepticus

Systemic administration of pilocarpine and kainic acid (KA) has been extensively used to model temporal lobe epilepsy in rats. Here the regional distribution of selectively vulnerable neurons and the temporal evolution of such neuronal injury after status epilepticus (SE) are compared in both models. Using the silver staining technique of Gallyas, argyrophilic neurons were measured on a 0-3 (least-most) scale in 53 different brain areas. Few neurons were silver-stained 2.5 h after kainate-induced SE, but many silver-stained cells could be seen in most neocortical, hippocampal, amygdaloid and hypothalamic structures for pilocarpine group. In general, 8 or 24 h intervals between SE onset and perfusion times yielded the most intense neuronal silver-impregnation. Pilocarpine-induced neuronal silver impregnation was more prominent than that induced by kainate treatment for many areas in cortex, hippocampus, endopiriform nucleus, amygdaloid complex and hypothalamus. On the other hand, in the thalamus, some cortical areas, claustrum, lateral septum and caudoputamen, kainate-induced neuronal silver staining was also prominent, but occurred later than in pilocarpine-treated animals. Neuronal injury was found in almost the same brain areas in both models of SE but with different intensity levels and time course profiles. It was suggested that such differences in the temporal profile of cell damage should be taken into account when searching for neuroprotective agents.

Blockade of pilocarpine- or kainate-induced mossy fiber sprouting by cycloheximide does not prevent subsequent epileptogenesis in rats

Post-injury sprouting of hippocampal mossy fibers has been suggested to be a causal mechanism underlying the development of temporal lobe epilepsy. However, this hypothesis rests entirely on indirect correlational evidence. Here we demonstrate that cycloheximide, a protein synthesis inhibitor, blocked pilocarpine- and kainate-induced mossy fiber sprouting in rats, but did not prevent the subsequent development of spontaneous seizures or affect their frequency. These results provide direct evidence against a causal role for mossy fiber sprouting in temporal lobe epileptogenesis.

Cell damage and neurogenesis in the dentate granule cell layer of adult rats after pilocarpine- or kainate-induced status epilepticus.

Dentate granule cells are generally considered to be relatively resistant to excitotoxicity and have been associated with robust synaptogenesis after neuronal damage. Synaptic reorganization of dentate granule cell axons, the mossy fibers, has been suggested to be relevant for hyperexcitability in human temporal lobe epilepsy and animal models. A recent hypothesis suggested that mossy‐fiber sprouting is dependent on newly formed dentate granule cells. However, we recently demonstrated that cycloheximide (CHX) can block the mossy‐fiber sprouting that would otherwise be induced by different epileptogenic agents and does not interfere with epileptogenesis in those models. Here, we investigated cell damage and neurogenesis in the dentate gyrus of pilocarpine‐ or kainate‐treated animals with or without coadministration of CHX. Dentate granule cells were highly vulnerable to pilocarpine induced‐status epilepticus (SE), but were hardly damaged by kainate‐induced SE. CHX pretreatment markedly reduced the number of injured neurons after pilocarpine‐induced SE. Induction of SE dramatically increased the mitotic rate of KA‐ and KA + CHX‐treated animals. Induction of SE in animals injected with pilocarpine alone led to 2–7‐fold increases in the mitotic rate of dentate granule cells as compared to 5‐ and 30‐fold increases for pilocarpine + CHX animals. We suggest that such increased mitotic rates might be associated with a protection of a vulnerable precursor cell population that would otherwise degenerate after pilocarpine‐induced SE. We further suggest that mossy‐fiber sprouting and neurogenesis of granule cells are not necessarily linked to one another. Hippocampus 10:169–180, 2000

Supragranular mossy fiber sprouting is not necessary for spontaneous seizures in the intrahippocampal kainate model of epilepsy in the rat

In a previous study, we suggested a dissociation between spontaneous recurrent epileptic seizures (SRS) and hippocampal supragranular mossy fiber sprouting (MFS) in the pilocarpine model of epilepsy (PILO). One possible explanation, would be that SRS in the PILO model do not originate in the hippocampus and thus would not depend on MFS. In the present study, we investigated whether MFS is necessary for the SRS that develop after a small intrahippocampal dose of kainic acid (KA), a model where seizures are more likely to start in the hippocampus. Intrahippocampal injections of KA were performed in rats, with and without the concomitant administration of cycloheximide (CHX) (0.5 microg of KA and 6 microg of CHX). After injection, recording electrodes were positioned in the same stereotaxic location. Here again, CHX was able to completely block (5/8 animals) MFS, visualized by neo-Timm staining, without altering the frequency and intensity of spontaneous ictal and interictal EEG events. From these data, we can conclude that, in the intra-hippocampal KA model, MFS is not necessary for the occurrence of ictal events. We suggest that CHX can be used together with classic epileptogenic agents, as a means to study temporal lobe epilepsy (TLE) without the contributing effect of MFS--as seen in TLE patients with mass lesions in the lateral temporal lobe.

Meditation training increases brain efficiency in an attention task

Meditation is a mental training, which involves attention and the ability to maintain focus on a particular object. In this study we have applied a specific attentional task to simply measure the performance of the participants with different levels of meditation experience, rather than evaluating meditation practice per se or task performance during meditation. Our objective was to evaluate the performance of regular meditators and non-meditators during an fMRI adapted Stroop Word-Colour Task (SWCT), which requires attention and impulse control, using a block design paradigm. We selected 20 right-handed regular meditators and 19 non-meditators matched for age, years of education and gender. Participants had to choose the colour (red, blue or green) of single words presented visually in three conditions: congruent, neutral and incongruent. Non-meditators showed greater activity than meditators in the right medial frontal, middle temporal, precentral and postcentral gyri and the lentiform nucleus during the incongruent conditions. No regions were more activated in meditators relative to non-meditators in the same comparison. Non-meditators showed an increased pattern of brain activation relative to regular meditators under the same behavioural performance level. This suggests that meditation training improves efficiency, possibly via improved sustained attention and impulse control.

Neuroethological and morphological (Neo-Timm staining) correlates of limbic recruitment during the development of audiogenic kindling in seizure susceptible Wistar rats

Acute audiogenic seizures are a model of generalized tonic-clonic seizures, induced by high intensity acoustic stimulation in genetically susceptible rodents. The neural substrate are sensory motor brainstem nuclei. Recruitment of forebrain structures takes places upon repetition of acoustically evoked seizures. The term audiogenic kindling means forebrain kindling evoked by repeated brainstem seizures and has been described in several strains of genetically epilepsy-prone rats. Thus, the present work was conducted in order to test the hypothesis that audiogenic kindling recruits the forebrain, which may be behaviorally evaluated and associated with morphological changes as well. The behavioral sequences observed during the development of audiogenic kindling were assessed by neuroethological methods (cluster analysis), with the ETHOMATIC program. Seizure severity indexes (brainstem and limbic seizures) and latencies of wild running and tonic-clonic seizures were measured to quantify seizure evolution. Densitometric analysis of Neo-Timm staining was used for assessing morphological changes associated with audiogenic kindling. In group I, II resistant (R) and 16 susceptible (S) animals were stimulated (120 dB) 21 times, and allowed a 10 day recovery period prior to retesting. In group II, 22 R and 20 S were stimulated 60 times, and allowed a 2 month recovery period prior to retesting. Repetition of the acoustic stimulation in group I and group II susceptible animals led to a progressive and statistically significant attenuation of the behaviors associated with brainstem seizures and a concomitant increased expression of the behaviors associated with limbic seizures. After either a 10 day (group I) or 2 month (group II) recovery period, acoustic stimulation preferentially evoked brainstem-associated behaviors and seizures rather than limbic ones in the audiogenic susceptible animals, although in some animals overlapped brainstem and limbic seizures were detected. Latencies for the wild running and tonic seizures after acoustic stimulation significantly increased during audiogenic kindling for both group I and group II susceptible animals. The quantitative ethological evaluation in both group I and group II, illustrated by flowcharts, showed the evolution of the kindling installation by the presence of limbic seizure clusters, competing in time with the original tonic-clonic clusters. Expression of limbic seizures by group I animals, after acoustic stimulation, was not associated with changes in the mossy fiber Neo-Timm staining pattern of these animals. In group II however, Neo-Timm staining revealed mossy fiber sprouting in the ventral hippocampus (but not in the dorsal), and a significant change in the optical density of amygdaloid nuclei and perirhinal cortex in susceptible animals as compared to resistant ones. In conclusion, audiogenic kindling effectively recruits forebrain structures, responsible for the appearance of limbic seizures. It is possible that the paradigm used in group I was subthreshold for the development of clear-cut synaptic reorganization in the hippocampal mossy fiber system, since the behavioral patterns reverted ten days after the last seizure induction. In group II, however, an increased number of evoked seizures and a more prolonged time after the last chronic seizure showed structural re-arrangements in amygdala, perirhinal cortex and hippocampus, associated with permanence in terms of behavioral data (lack of regression of limbic seizures to control values).

Bilateral anterior thalamic nucleus lesions and high-frequency stimulation are protective against pilocarpine-induced seizures and status epilepticus.

OBJECTIVEThe thalamus is thought to play an important role in secondary generalization of seizures. The aim of the present study was to investigate the influence of anterior thalamic nucleus lesions and high-frequency stimulation in the pilocarpine model of secondarily generalized seizures in rats. METHODSAdult Wistar rats underwent unilateral (n = 7) or bilateral anterior nucleus thalamotomies (n = 10), or unilateral (n = 4) or bilateral (n = 9) anterior thalamic nucleus stimulation through implanted electrodes. Control animals (n = 9) received bilateral implants but no stimulation. Seven days after these procedures, animals were provided pilocarpine (320 mg/kg intraperitoneally) to induce seizures and status epilepticus (SE). Electrographic recordings from hippocampal and cortical electrodes were evaluated, and ictal behavior was assessed. RESULTSIn the control group, 67% of the animals developed SE 15.3 ± 8.8 minutes after pilocarpine administration. Neither unilateral anterior nucleus lesions nor stimulation significantly reduced the propensity or latency for developing seizures and SE. Bilateral thalamic stimulation did not prevent SE (observed in 56% of the animals), but it significantly prolonged the latency to its development (48.4 ± 17.7 min, P = 0.02). Strikingly, no animal with bilateral anterior nucleus thalamotomies developed seizures or SE with pilocarpine. CONCLUSIONBilateral anterior thalamic nuclear complex stimulation and thalamotomies were protective against SE induced by pilocarpine.

Deep brain stimulation of the anterior nucleus of the thalamus: Effects of electrical stimulation on pilocarpine-induced seizures and status epilepticus

PURPOSE Electrical stimulation of the anterior nucleus of the thalamus appears to be effective against seizures in animals and humans. As the optimal stimulation settings remain elusive, we studied the effects of different stimulation parameters against pilocarpine induced seizures and status epilepticus (SE). METHODS Adult rats had electrodes implanted bilaterally into the AN. Five days later, different groups of animals were stimulated with 1000 microA, 500 microA, or 200 microA and frequencies of either 20 Hz or 130 Hz. Pilocarpine (350 mg/kg i.p.) was injected 5 min after stimulation onset and seizures were monitored. Sham-treated controls had electrodes implanted but did not receive stimulation until they developed SE. After SE, these animals had the electrodes turned on to assess whether AN stimulation could arrest ongoing ictal activity. RESULTS Compared to sham-treated controls (n=8), stimulation at 500 microA (n=13) significantly increased the latency for seizures and SE by 1.9-2.2-fold. In contrast, stimulation at 1000 microA (n=8) produced a non-significant decrease in the latencies to these events. No major effect was observed with stimulation at 200 microA (n=11). Similar results were obtained for each current intensity, regardless of the stimulation frequency used (20 Hz and 130 Hz). In sham-treated controls that had the electrodes turned on after SE, stimulation was not able to arrest ongoing ictal activity. CONCLUSIONS The anticonvulsant effects of AN stimulation against pilocarpine-induced seizures were mainly determined by the current and not the frequency of stimulation. AN stimulation initiated after SE onset was ineffective.

Effects of aminophylline and 2-chloroadenosine on seizures produced by pilocarpine in rats: morphological and electroencephalographic correlates.

The effects of 2-chloroadenosine, aminophylline, bicuculline, beta-carboline-3-carboxylic acid methylester and Ro 15-1788 on seizures produced by pilocarpine were examined in rats. In animals pretreated with aminophylline at doses of 25-100 mg/kg, non-convulsant dose of pilocarpine, 100 mg/kg, resulted in severe motor limbic seizures, which rapidly developed into the status epilepticus. Electroencephalographic monitoring showed progressive evolution of seizure activity with initial high-voltage fast activity followed by high-voltage spiking and electrographic seizures. Morphological analysis of frontal forebrain sections with light microscopy demonstrated widespread damage to the hippocampal formation, thalamus, amygdala, olfactory cortex, substantia nigra and neocortex. Bicuculline, 2 mg/kg, beta-carboline-3-carboxylic acid methylester, 5 mg/kg, and Ro 15-1788, 50 mg/kg, did not augment seizures produced by pilocarpine, 100 mg/kg. 2-Chloroadenosine, 5 and 10 mg/kg, blocked the appearance of behavioral and electrographic seizures produced by pilocarpine, 380 mg/kg, and prevented the occurrence of brain damage. The results indicate that purinergic mechanisms are involved in the buildup of pilocarpine-induced convulsions and seizure-related brain damage in rats.

NMDA receptor-mediated excitability in dendritically deformed dentate granule cells in pilocarpine-treated rats

Membrane properties and synaptic responses were analyzed in dentate granule cells in hippocampal slices prepared from pilocarpine-treated, chronically epileptic rats. Perforant path stimulation evoked a long-lasting excitatory postsynaptic potential (EPSP) with multiple spikes in a stimulus intensity-dependent fashion. The response was strongly facilitated by paired-pulse stimulation. Application of N-methyl-D-aspartate (NMDA) receptor antagonist, D-2-amino-5-phosphonovalerate (APV), not only blocked the paired pulse facilitation but also reduced the amplitude of the EPSP, indicating the involvement of the NMDA-receptor in synaptic responses of pilocarpine-treated dentate granule cells. Dendrites of these neurons showed loss of spines and beaded branches. These findings suggest that a degenerating dendrite could be a morphological substrate of neuronal hyperexcitability mediated by NMDA receptors, implicating possible in vivo glutamate toxicity as an underlying mechanism of chronic epilepsy.