Neo-Timm Staining Research Articles

Long-term monotherapy treatment with vitamin E reduces oxidative stress, but not seizure frequency in rats submitted to the pilocarpine model of epilepsy

The imbalance between antioxidant system and reactive oxygen species (ROS) generation is related to epileptogenesis, neuronal death, and seizure frequency. Treatment with vitamin E has been associated with neuroprotection and control of seizures. In most experimental studies, vitamin E treatment has short duration. Therefore, the aim of this study was to verify the role of long-term treatment with vitamin E in rats submitted to the pilocarpine model of epilepsy. Rats were divided into two main groups: control (Ctr) and pilocarpine (Pilo). Each one was subdivided according to treatment: vehicle (Ctr V and Pilo V) or vitamin E at dosages of 6 IU/kg/day (Ctr E6 and Pilo E6) or 60 IU/kg/day (Ctr E60 and Pilo E60). Treatment lasted 120 days from status epilepticus (SE). There were no statistical differences concerning treatment in the Ctr group for all variables, so the data were grouped. Carbonyl content in the hippocampus of Pilo V and Pilo E6 was higher compared with that of the Ctr group (8 ± 1.5, 7.1 ± 1, and 3.1 ± 0.3 nmol carbonyl/mg protein, respectively for Pilo V, Pilo E6, and Ctr; p < 0.05). Carbonyl content was restored to control values in Pilo E60 rats (4.2 ± 1.1 and 3.1 ± 0.3 nmol carbonyl/mg protein, respectively for Pilo E60 and Ctr; p > 0.05). The volume of the hippocampal formation (6.5 ± 0.3, 6.6 ± 0.4, 6.3 ± 0.3, and 7.4 ± 0.2, respectively for Pilo V, Pilo E6, Pilo E60, and Ctr) and subfields CA1 (1.6 ± 0.1, 1.4 ± 0.2, 1.5 ± 0.1, and 2 ± 0.05, respectively for Pilo V, Pilo E6, Pilo E60, and Ctr) and CA3 (1.7 ± 0.1, 1.5 ± 0.2, 1.4 ± 0.1, and 2 ± 0.1, respectively for Pilo V, Pilo E6, Pilo E60, and Ctr) was reduced in the Pilo group regardless of treatment. Parvalbumin immunostaining was increased in the hilus of the Pilo E60 group compared with that in the Ctr group (26 ± 2 and 39.6 ± 8.3 neurons, respectively for Ctr and Pilo E60). No difference was found in seizure frequency and Neo-Timm staining. Therefore, long-term treatment with 60 IU/kg/day of vitamin E prevented oxidative damage in the hippocampus and increased hilar parvalbumin expression in rats with epilepsy without a reduction in seizure frequency.

Protein-caloric dietary restriction inhibits mossy fiber sprouting in the pilocarpine model of TLE without significantly altering seizure phenotype

Given the known effects of undernutrition over protein synthesis, we promoted neonatal undernutrition to evaluate its effect over the neuroplasticity induced by the pilocarpine model of epilepsy and also over spontaneous seizure expression. A well-nourished group (WN), fed ad libitum rat chow diet, and an undernourished group (UN), fed 60% of the amount of diet consumed by a WN group, were submitted to status epilepticus (SE) through pilocarpine injection at 45 days of age. Thereafter, animals were behaviorally monitored for 6h daily to quantify seizures. On the 120th day, electroencephalography (EEG) was recorded and rats were sacrificed to measure proteins and glutamate release from hippocampus. Neo-Timm staining was used to detect mossy fiber sprouting. The results indicate no statistical difference in the latency for the first spontaneous recurrent seizure (SRS), in the number of daily SRS, or in EEG epileptiform activity duration between groups. However, PILO promoted more K+-stimulated glutamate release in the hippocampus slices from WN animals when compared to the UN group. It was also found a lower degree of mossy fibers sprouting in UN group. Data from this work, thus, indicate that the decreased neuroplasticity as currently measured does not directly impact on the manifestation of spontaneous seizures.

Topographical Analysis of Reactive Zinc in the Central Nervous System of Adult Zebrafish (Danio Rerio)

Reactive zinc (Zn) is crucial for neuronal signaling and is largely distributed within presynaptic vesicles of some axon terminals of distinct vertebrates. However, the distribution of reactive Zn throughout the central nervous system (CNS) is not fully explored. We performed a topographical study of CNS structures containing reactive Zn in the adult zebrafish (Danio rerio). Slices of CNS from zebrafish were stained by Neo-Timm and/or cresyl violet. The Zn specificity of Neo-Timm was evaluated with Zn chelants, N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), sodium diethyldithiocarbamate (DEDTC), Zn sulfide washing solution, and hydrochloric acid (HCl). Unfixed slices were also immersed in the fluorescent Zn probe (zinpyr-1). Yellow-to-brown-to-black granules were revealed by Neo-Timm in the zebrafish CNS. Telencephalon exhibited slightly stained regions, while rhombencephalic structures showed high levels of staining. Although stained granules were found on the cell bodies, rhombencephalic structures showed a neuropil staining profile. The TPEN produced a mild reduction in Neo-Timm staining, while HCl and mainly DEDTC abolished the staining, indicating a large Zn content. This result was also confirmed by the application of a Zn probe. The present topographical study revealed reactive Zn throughout the CNS in adult zebrafish that should be considered in future investigation of Zn in the brain on a larger scale.

Modeling epileptogenesis and temporal lobe epilepsy in a non-human primate

Here we describe a new non-human primate model of temporal lobe epilepsy (TLE) to better investigate the cause/effect relationships of human TLE. Status epilepticus (SE) was induced in adult marmosets by pilocarpine injection (250mg/kg; i.p.). The animals were divided in 2 groups: acute (8h post-SE) and chronic (3 and 5 months post-SE). To manage the severity of SE, animals received diazepam 5min after the SE onset (acute group: 2.5 or 1.25mg/kg; i.p.; chronic group/; 1.25mg/kg; i.p). All animals were monitored by video and electrocorticography to assess SE and subsequent spontaneous recurrent seizures (SRS). To evaluate brain injury produced by SE or SRS we used argyrophil III, Nissl and neo-Timm staining techniques. Magnetic resonance image was also performed in the chronic group. We observed that pilocarpine was able to induce SE followed by SRS after a variable period of time. Prolonged SE episodes were associated with brain damage, mostly confined to the hippocampus and limbic structures. Similar to human TLE, anatomical disruption of dentate gyrus was observed after SRS. Our data suggest that pilocarpine marmoset model of epilepsy has great resemblance to human TLE, and could provide new tools to further evaluate the subtle changes associated with human epilepsy.

Study of spontaneous recurrent seizures and morphological alterations after status epilepticus induced by intrahippocampal injection of pilocarpine

Epileptic seizures are clinical manifestations of neuronal discharges characterized by hyperexcitability and/or hypersynchrony in the cortex and other subcortical regions. The pilocarpine (PILO) model of epilepsy mimics temporal lobe epilepsy (TLE) in humans. In the present study, we used a more selective approach: microinjection of PILO into the hilus of the dentate gyrus (H-PILO). Our main goal was to evaluate the behavioral and morphological alterations present in this model of TLE. Seventy-six percent of all animals receiving H-PILO injections had continuous seizures called status epilepticus (SE). A typical pattern of evolution of limbic seizures during the SE with a latency of 29.3±16.3minutes was observed using an analysis of behavioral sequences. During the subsequent 30days, 71% of all animals exhibited spontaneous recurrent seizures (SRSs) during a daily 8-hour videotaping session. These SRSs had a very conspicuous and characteristic pattern detected by behavioral sequences or neuroethological analysis. Only the animals that had SE showed positive Neo-Timm staining in the inner molecular layer of the dentate gyrus (sprouting) and reduced cell density in Ammon's horn pyramidal cell subfield CA1. However, no correlation between the intensity of sprouting and the mean number and total number of SRSs was found. Additionally, using Fluoro-Jade staining, we observed neurodegeration in the hilus and pyramidal cell subfields CA3 and CA1 24hours after SE. These data indicate that H-PILO is a reliable, selective, efficient, low-mortality model that mimics the acute and chronic behavioral and morphological aspects of TLE.

Hippocampal Nogo-A and neo-Timm's staining in amygdala kindling rats

Purpose: Nogo-A, one of the axon regeneration inhibitors, has been shown to be up-regulated in both the experimental and human temporal lobe epilepsy. However, the role of Nogo-A in mossy fiber sprouting (MFS) relative to epileptogenesis is unknown. This work was designed to examine the relationship of the hippocampal Nogo-A protein expression with MFS during the development of amygdala kindling.Methods: Using immunohistochemistry and neo-Timm's histological procedures, we evaluated the distribution and density of Nogo-A and Nogo-66 receptor (Ng-R) expression and MFS in the bilateral hippocampus of amygdala kindling rats.Results: Nogo-A expression in the ipsilateral hippocampus gradually increased with the development of kindling in the sector CA2–3. In contrast, no increased Nogo-A expression was observed in the contralateral hippocampus as the rats advanced to stage 5 kindled seizures. Furthermore, poorer Nogo-A and Nogo-66 receptor (Ng-R) expression were observed in the dentate granule cells as aberrant MFS occurred.Conclusions: In amygdala kindling rats, generalized stage 5 seizures were not associated with increased Nogo-A expression in the contralateral hippocampus supporting the concept that seizures by themselves do not induce Nogo-A expression. Furthermore, in the ipsilateral hippocampus, the expression of Nogo-A relative to MSF suggests that this protein may partially control aberrant synaptic reorganization during epileptogenesis.

A proliferação de neurônios granulares hipocampais aumenta e os dendritos dos novos neurônios são anormais no modelo experimental de ELT induzida por pilocarpina

Hippocampal doublecortin-positive granular neurons have abnormal dendritic morphology in the temporal lobe epilepsy pilocarpine model To quantify the number and morphology of doublecortin-positive (DCX+) granular neurons in a temporal lobe epilepsy model, Wistar rats were treated with pilocarpine and, 90 min after status epilepticus (SE) onset, with diazepam. Controls received saline instead of pilocarpine. Rats were monitored 8 h per day to record spontaneous recurrent seizures (SRS) and perfused 30 days after SE. A series of sections containing the hippocampus was immunolabeled for DCX. The stereological optical fractionator method was used to count DCX+ neurons in granular cell layer (GCL) and subgranular zone of the dentate gyrus (DG). DCX+ neurons (n = 40) were digitally reconstructed and their dendritic distribution measured with Sholl’s concentric spheres analysis. The first sphere has the radius equal to the GCL thickness followed by spheres with increments of 50 μm in their radii. Neo-Timm staining was performed to evaluate the mossy fiber sprouting. Statistical difference in Student and Mann-Whitney tests at p < 0.05, values are mean ± SEM. Each animal had 8.4 ± 3.7 SRS recorded. The DCX+ counting revealed the presence of 8,987 ± 1,986 neurons in controls DG (n = 7) and 19,337 ± 4,715 neurons in epileptic DG (n = 7). Basal dendrites were observed in 85% of DCX+ neurons in epileptics and in 30% of DCX+ neurons in controls with a length of 63.9 ± 9.7 μm and 19.8 ± 5.3 μm, respectively. Apical dendrites of DCX+ neurons had ~500 μm in both groups, but the epileptics had more nodes (3.9 ± 0.3) and dendrites emerging from GCL (4.1 ± 0.4) than controls (2.47 ± 0.2 nodes and 2.95 ± 0.3 dendrites). The epileptic rats had more dendritic endings (4.55 ± 0.5) than controls (1.7 ± 0.4) in the first 50 μm of the molecular layer (GCL + 50). Epileptic rats had more DCX+ dendritic length in GCL (168 ± 14 μm against 126 ± 11 μm in controls) and GCL + 50 (231 ± 26 μm against 163 ± 17 μm in controls); the contrary occurred in GCL + 100 (94 ± 23 μm against 160 ± 23 μm in controls). Neo-Timm staining revealed a confluent band of silver granules in epileptic DG with a mean thickness of 48 μm (equivalent to GCL + 50) and no band in controls. Hilar basal dendrites were more frequent, long and ramified and the apical dendrites presented intense ramification in the inner molecular layer of epileptic rats. Dendrites were more abundant in regions with mossy fiber sprouting. Formation of synapses between these new dendrites and axons could contribute to the pathological process of hippocampal epileptogenesis.

Effects of herbimycin A in the pilocarpine model of temporal lobe epilepsy

Pilocarpine-induced status epilepticus (SE) causes widespread tyrosine phosphorylation in the brain. It has been postulated that this intracellular signal may mediate potentially epileptogenic changes in the morphology and physiology of particular brain regions, including the hippocampus. The present study evaluated the effects of herbimycin A, a protein tyrosine kinase (PTK) inhibitor, over the acute (during which intense biochemical and electrophysiological activation occurs) and the chronic phase (characterized by spontaneous and recurrent epileptic seizures and the presence of synaptic reorganization, e.g., mossy fiber sprouting) of the pilocarpine model of epilepsy. The administration of a single dose of 1.74 nmol of herbimycin A (i.c.v., 5 μL) 5 min after the onset of SE did not change the acute behavioral manifestation of seizures despite significantly decreasing c-Fos immunoreactivity in different areas of the hippocampus and of the limbic cortex. Herbimycin-treated animals developed spontaneous recurrent seizures, as did control animals, with a similar latency for the appearance of the first seizure and similar seizure frequency. Neo-Timm staining revealed that all animals experiencing SE, regardless of whether or not injected with herbimycin, showed aberrant mossy fiber sprouting in the supragranular region of the dentate gyrus. Herbimycin did not obviously affect neuronal cell death as evaluated in Nissl-stained sections. These results indicate that the PTK blockade achieved with the current dose of herbimycin reduced the acute c-Fos expression but failed to alter the spontaneous seizure frequency or to attenuate the morphological modifications triggered by the SE.

Neo-Timm staining in the thalamus of chronically epileptic rats

The thalamus is an important modulator of seizures and is severely affected in cholinergic models of epilepsy. In the present study, chronically epileptic rats had their brains processed for neo-Timm and acetylcholinesterase two months after the induction of status epilepticus with pilocarpine. Both controls and pilocarpine-treated animals presented neo-Timm staining in the anterodorsal nucleus, laterodorsal nucleus, reticular nucleus, most intralaminar nuclei, nucleus reuniens, and rhomboid nucleus of the thalamus, as well as in the zona incerta. The intensity of neo-Timm staining was similar in control and pilocarpine-treated rats, except for the nucleus reuniens and the rhomboid nucleus, which had a lower intensity of staining in the epileptic group. In animal models of temporal lobe epilepsy, zinc seems to modulate glutamate release and to decrease seizure activity. In this context, a reduction of neo-Timm-stained terminals in the midline thalamus could ultimately result in an increased excitatory activity, not only within its related nuclei, but also in anatomical structures that receive their efferent connections. This might contribute to the pathological substrate observed in chronic pilocarpine-treated epileptic animals.

Estudo qualitativo da formação hipocampal de animais hipertensos com epilepsia

The aim of our study was to investigate the hippocampus and dentate gyrus neuropathological features of spontaneous hypertensive rats (SHR) with epilepsy. Animals were randomly divided into 4 groups: control Wistar, Wistar with epilepsy, control SHR and SHR with epilepsy. The pilocarpine model of epilepsy was used in this experiement. After spontaneous recurrent seizures, all animals were perfused and their brains processed for histological analysis through Nissl and neo-Timm methods. In the Wistar rats with epilepsy we observed cell loss in hippocampal subfields CA1, CA3 and hilus of the dentate gyrus when compared with control animals. In the SHR with epilepsy we observed hippocampal formation atrophy with ventricular dilatation. No morphological alterations were observed in SHR and Wistar control rats. The neo-Timm staining of hippocampal formation has shown supragranular sprouting in Wistar and SHR with epilepsy. CONCLUSION We found neuropathological alterations in hippocampal formation in Wistar with epilepsy and SHR with epilepsy, suggesting that epilepsy per se or associated to hypertention are able to cause neuronal damage.

Sprouting of mossy fibers and the vacating of postsynaptic targets in the inner molecular layer of the dentate gyrus

Aberrant mossy fiber sprouting, which presumably results from hilar mossy cell death after status epilepticus (SE), is a frequently studied feature of temporal lobe epilepsy. Although mossy fiber sprouting can be suppressed by the protein synthesis inhibitor cycloheximide, spontaneous seizures remain unaltered. We have investigated the mechanisms underlying the ability of cycloheximide to block SE-induced mossy fiber sprouting in the inner molecular layer of dentate gyrus (IML). Pilocarpine-induced SE in the presence of cycloheximide resulted in a reduced number of injured hilar cells compared to rats not pretreated with cycloheximide. Presumed mossy cells, identified by calcitonin gene related peptide (CGRP) immunohistochemistry, were not significantly reduced in either group 60 days after SE. Whereas controls had a strong band of CGRP-positive fibers (putative mossy cell axons) and no neo-Timm stained fibers in the IML, pilocarpine-treated rats had no CGRP fibers and strong neo-Timm staining. Cycloheximide–pilocarpine-treated animals, in contrast, had CGRP and neo-Timm staining similar to controls. Cycloheximide might protect hilar CGRP-positive cells during SE and, by allowing those cells to retain their normal axonal projection, prevent mossy fiber sprouting. The recently suggested “irritable” mossy cell hypothesis relies on the survival of mossy cells for network hyperexcitability. We hypothesized that CGRP may be a marker for a subpopulation of relatively resistant mossy cells in rats, which, if they survive injury, may become irritable and contribute to hyperexcitability. We suggest that cycloheximide prevents SE-induced mossy fiber sprouting by preventing the loss of hilar CGRP-positive cells (putative mossy cells).

Electrophysiologic abnormalities of the hippocampus in the pilocarpine/cycloheximide model of chronic spontaneous seizures.

Mossy fiber sprouting (MFS) and synaptic reorganization in the dentate gyrus (DG) is considered one of the physiopathologic mechanisms in temporal lobe epilepsy. Supragranular MFS can be blocked by cycloheximide (CHX) without interfering with the genesis of spontaneous recurrent seizures. The aim of this study was to investigate electrophysiologic properties of the hippocampus in the CHX/pilocarpine (CHX/PILO) model as compared with the conventional PILO model. In vitro electrophysiology was performed 2 months after status epilepticus (SE) induction using extracellular recordings in hippocampal slices from PILO (n = 8) and CHX/PILO animals (n = 10). Field potential responses were evoked in the CA1 and DG regions during perfusion with normal artificial cerebrospinal fluid (aCSF) and aCSF containing 3.5, 5, or 8 mM K+ without or with bicuculline added. Neo-Timm staining was used for the assessment of supragranular MFS. Evoked potentials in PILO- and CHX/PILO-treated rats displayed small-amplitude polyspiking activity (epileptiform responses) in CA1 and an apparently normal isolated population spike in DG. More important, PILO and CHX/PILO animals did not differ regarding electrophysiologic abnormalities, even under high K+ or high K+/bicuculline. Analysis of the neo-Timm staining revealed strong supragranular MFS in PILO-injected rats and significantly less staining in CHX/PILO rats. Thus, occurrence of abnormal stimulus responses and high K+- or high K+/bicuculline-induced epileptiform activities did not depend on the degree of MFS. We therefore suggest that other mechanisms such as anomalous intrinsic bursting and disinhibition rather than MFS might account for the increased hippocampal hyperexcitability in this model.

Behavioral, morphologic, and electroencephalographic evaluation of seizures induced by intrahippocampal microinjection of pilocarpine.

We studied, by means of video-EEG and neo-Timm histochemistry, the behavioral, electrophysiologic, and structural characteristics of seizures induced by intrahippocampal microinjection of pilocarpine (HIP-PILO), a selective model of temporal lobe epilepsy (TLE). We investigated the behavioral and electrophysiologic (hippocampus and amygdala EEG) evaluation of status epilepticus (SE) induced by HIP-PILO and the consequent spontaneous recurrent seizures (SRSs). We evaluated hippocampal structural rearrangements after SE by means of neo-Timm staining. HIP-PILO induced SE in 17 (71%) of 24 animals. Although three animals displayed spontaneous remission of SE (not used in analysis) before the established SE duration (90 min), none of those undergoing SE died. Of SE animals, 10 (71%) of 14 had SRSs. All animals with SE had clear-cut mossy fiber sprouting (MFS) in the inner molecular layer of the dentate gyrus and epileptiform activity in hippocampus and amygdala. HIP-PILO rats displayed SE, SRS, MFS, and limbic epileptiform activity, without animal loss after SE. Thus, our data support this as a selective and efficient model of TLE.

Correlations between granule cell dispersion, mossy fiber sprouting, and hippocampal cell loss in temporal lobe epilepsy.

Correlations between granule cell dispersion (GCD), collateral mossy fiber (MF) sprouting, and hippocampal cell loss were studied to assess the relation between GCD and synaptic reorganization in the dentate gyrus of patients with epilepsy. Twenty specimens from patients with medically intractable temporal lobe epilepsy (TLE) were studied along with two control specimens. GCD was considered to be present when the stratum granulosum was wider than 120 microm, the close apposition between the granule cell (GC) soma was lost, and GCs were scattered in the molecular layer (ML). Patterns of MF sprouting were differentiated as wide or narrow according to the area of neo-Timm's staining in the ML. GC loss and volumetric cell-density decreases in the different subfields were assessed. MF sprouting was observed in 16 (80%) and GCD in nine (45%) cases. A significant correlation was found between MF sprouting and cell loss in all the subfields except the cornu Ammonis field 2 (CA2). A wide band of MF sprouting was associated with severe cell loss. Cases with GCD had a wide band of MF sprouting and also a higher degree of cell loss than cases without GCD. GCD is associated with a specific pattern of MF sprouting, but cell loss was found to be a major determinant for MF reorganization.

Hippocampal AMPA and NMDA mRNA levels correlate with aberrant fascia dentata mossy fiber sprouting in the pilocarpine model of spontaneous limbic epilepsy.

There is considerable controversy whether aberrant fascia dentata (FD) mossy fiber sprouting is an epiphenomena related to neuronal loss or a pathologic abnormality responsible for spontaneous limbic seizures. If mossy fiber sprouting contributes to seizures, then reorganized axon circuits should alter postsynaptic glutamate receptor properties. In the pilocarpine-status rat model, this study determined if changes in alpha amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) and n-methyl-D-aspartic acid (NMDA) receptor subunit mRNA levels correlated with mossy fiber sprouting. Sprague-Dawley rats were injected with pilocarpine (320 mg/kg; i.p.) and maintained in status epilepticus for 6 to 8 hours (pilocarpine-status). Rats were killed during the: (1) latent phase after neuronal loss but before spontaneous limbic seizures (day 11 poststatus; n = 7); (2) early seizure phase after their first seizures (day 25; n = 7); and (3) chronic seizure phase after many seizures (day 85; n = 9). Hippocampi were studied for neuron counts, inner molecular layer (IML) neo-Timm's staining, and GluR1-3 and NMDAR1-2b mRNA levels. Compared with controls, pilocarpine-status rats in the: (1) latent phase showed increased FD GluR3, NMDAR1, and NMDAR2b; greater CA4 and CA1 NMDAR1; and decreased subiculum GluR1 hybridization densities; (2) early seizure phase showed increased FD GluR3, increased CA1 NMDAR1, and decreased subiculum NMDAR2b densities; and (3) chronic seizure phase showed increased FD GluR2; increased FD and CA4 GluR3; decreased CA1 GluR2; and decreased subiculum GluR1, GluR2, NMDAR1, and NMDAR2b levels. In multivariate analyses, greater IML neo-Timm's staining: (1) positively correlated with FD GluR3 and NMDAR1 and (2) negatively correlated with CA1 and subiculum GluR1 and GluR2 mRNA levels. These results indicate that: (1) hippocampal AMPA and NMDA receptor subunit mRNA levels changed as rats progressed from the latent to chronic seizure phase and (2) certain subunit alterations correlated with mossy fiber sprouting. Our findings support the hypothesis that aberrant axon circuitry alters postsynaptic hippocampal glutamate receptor subunit stoichiometry; this may contribute to limbic epileptogenesis.

Hippocampal AMPA and NMDA mRNA levels and subunit immunoreactivity in human temporal lobe epilepsy patients and a rodent model of chronic mesial limbic epilepsy.

This study compared temporal lobe epilepsy patients, along with kindled animals and self sustained limbic status epilepticus (SSLSE) rats for parallels in hippocampal AMPA and NMDA receptor subunit expression. Hippocampal sclerosis patients (HS), non-HS cases, and autopsies were studied for: hippocampal AMPA GluR1-3 and NMDAR1&2b mRNA levels using in situ hybridization: GluR1, GluR2/3, NMDAR1, and NMDAR2(a&b) immunoreactivity (IR); and neuron densities. Similarly, spontaneously seizing rats after SSLSE, kindled rats, and control animals were studied for: fascia dentata neuron densities: GluR1 and NMDAR2(a&b) IR; and neo-Timm's staining. In HS and non-HS cases, the mRNA hybridization densities per granule cell, as well as molecular layer IR, showed increased GluR1 (relative to GluR2/3) and increased NMDAR2b (relative to NMDAR1) compared to autopsies. Likewise, the molecular layer of SSLSE rats with spontaneous seizures demonstrated more neo-Timm's staining, and higher levels of GluR1 and NMDAR2(a&b) IR compared to kindled animals and controls. These results indicate that hippocampal AMPA and NMDA receptor subunit mRNAs and their proteins are differentially increased in association with spontaneous, but not kindled, seizures. Furthermore, there appears to be parallels in fascia dentata AMPA and NMDA receptor subunit expression between HS (and non-HS) epileptic patients and SSLSE rats. This finding supports the hypothesis that spontaneous seizures in humans and SSLSE rats involve differential alterations in hippocampal ionotrophic glutamate receptor subunits. Moreover, non-HS hippocampi were more like HS cases than hippocampi from kindled animals with respect to glutamate receptors; therefore, hippocampi from kindled rats do not accurately model human non-HS cases, despite some similarities in neuron densities and mossy fiber axon sprouting.

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.

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).

P-21-1 - Changes of the lymphocyte subset in major depressive disorder and schizophrenia

This study compared temporal lobe epilepsy patients, along with kindled animals and self sustained limbic status epilepticus (SSLSE) rats for parallels in hippocampal AMPA and NMDA receptor subunit expression. Hippocampal sclerosis patients (HS), non-HS cases, and autopsies were studied for: hippocampal AMPA GluR1-3 and NMDAR1&2b mRNA levels using in situ hybridization; GluR1, GluR2/3, NMDAR1, and NMDAR2a&b immunoreactivity (IR); and neuron densities. Similarly, spontaneously seizing rats after SSLSE, kindled rats, and control animals were studied for: fascia dentata neuron densities; GluR1 and NMDAR2a&b IR; and neo-Timm's staining. In HS and non-HS cases, the mRNA hybridization densities per granule cell, as well as molecular layer IR, showed increased GluR1 (relative to GluR2/3) and increased NMDAR2b (relative to NMDAR1) compared to autopsies. Likewise, the molecular layer of SSLSE rats with spontaneous seizures demonstrated more neo-Timm's staining, and higher levels of GluR1 and NMDAR2a&b IR compared to kindled animals and controls. These results indicate that hippocampal AMPA and NMDA receptor subunit mRNAs and their proteins are differentially increased in association with spontaneous, but not kindled, seizures. Furthermore, there appears to be parallels in fascia dentata AMPA and NMDA receptor subunit expression between HS (and non-HS) epileptic patients and SSLSE rats. This finding supports the hypothesis that spontaneous seizures in humans and SSLSE rats involve differential alterations in hippocampal ionotrophic glutamate receptor subunits. Moreover, non-HS hippocampi were more like HS cases than hippocampi from kindled animals with respect to glutamate receptors; therefore, hippocampi from kindled rats do not accurately model human non-HS cases, despite some similarities in neuron densities and mossy fiber axon sprouting.

Childhood generalized and mesial temporal epilepsies demonstrate different amounts and patterns of hippocampal neuron loss and mossy fibre synaptic reorganization.

In this study, we determined whether childhood seizures were associated with hippocampal neuron loss and mossy fibre synaptic reorganization and if hippocampal sclerosis evolved from longer seizure histories. Children undergoing surgical treatment for catastrophic epilepsy were grouped into the following pathology categories: (i) those with generalized seizures and extra-hippocampal congenital pathologies (i.e. prenatal cortical dysplasia; n = 17); (ii) cases of generalized seizures and extra-hippocampal acquired lesions. (i.e. postnatal ischaemic injuries and encephalitis; n = 7); (iii) children with complex partial hippocampal epilepsy (n = 4). Further, to determine whether the epileptogenic location influenced hippocampal pathology, the seizure focus was classified as (i) hippocampal, (ii) temporal (n = 13) or (iii) extra-temporal (n = 11). Surgical and autopsy (n = 23) hippocampi were studied for (i) fascia-dentata (FD) and Ammon's horn (AH) neuron densities; (ii) thickness; height or length of the FD molecular layer, stratum granulosum (SG) and stratum pyramidale; and (iii) grey value (GV) densities of supragranular neo-Timm's staining. Statistically significant results (P < 0.05) showed the following. (i) Autopsy hippocampal neuron densities for the hilus (H), AH and prosubiculum (Pro) decreased logarithmically at the same time as the thickness of the stratum pyramidale and Pro increased. By contrast, autopsy granule cell densities and thickness did not significantly change with age; however, the SG lengthened-expanding around the enlarging H. Further, the supragranular molecular layer height increased logarithmically, and took longer than the increase in stratum pyramidale thickness. (ii) Compared with age-matched autopsies, young children with a history of hippocampal seizures showed decreased granule cell, hilar and regio superior neuron densities similar to adults with hippocampal sclerosis (average loss 70%). By contrast, children with extra-hippocampal congenital or acquired pathologies showed only decreased granule cell densities, along with a thinner and shorter SG. Compared with extra-temporal locations, those with temporal lobe lesions showed decreased hilar and AH neuron densities, but averaged 20-30% less than autopsies and not in the pattern typical of hippocampal sclerosis. (iii) The neo-Timm's GV densities, when compared with autopsies, showed supragranular mossy fibre sprouting in children with congenital pathologies and temporal lobe lesions; however, the greatest GVs were in children with hippocampal seizures. (iv) Of the children with extra-hippocampal congenital or acquired pathologies there were no statistical correlations between longer duration of seizures with changes in neuron densities, hippocampal heights, or mossy fibre sprouting. These results indicate the following. (i) In the human there is anatomical evidence for postnatal maturation of the hippocampus and our results are consistent with the notion that AH pyramids are a stable population; however, there are probably increases in granule cell numbers. Further, compared with the AH, dendritic maturation of the FD granule cells appears to take longer. (ii) Extra-hippocampal childhood seizures whether from prenatal or postnatal aetiologies are associated with moderate FD and minimal AH neuron losses and signs of aberrant mossy fibre sprouting. (iii) By contrast, young children with the syndrome of mesial temporal epilepsy show patterns of neuron loss and mossy fibre sprouting, typical of hippocampal sclerosis. (iv) Repeated extra-hippocampal childhood seizures are not associated with progressive evolution of hippocampal damage or mossy fibre sprouting. These findings support the hypothesis that childhood seizures can damage or alter the postnatally developing granule cells of the human hippocampus, and that early neuron loss and aberrant axon circuits may contribute to chronic hippocampal seizures. However, repeated childhood generalized seiz