Wednesday 25 February 2004 Plenary lecture 25 A 1 Growth Factor Gene Therapy in the Central Nervous System

s of the 2nd Scientific Conference "Restauración Neurológica 2004" 218 shown, but resulted controversial in pharmacological experiments using antagonists (atropine) or agonists (oxotremorine). MC requires protein synthesis as is completely blocked by anisomycin. Microdialysisbiochemical studies have shown an increase in acetyl choline and a reduction in norepinephrine and serotonine release at the dentate gyrus after amygdala stimulation; while behavioral reinforcing paradigms showed changes in glutamate and glycine, suggesting that different sub-systems might mediate different forms of reinforcement. Finally we have shown that aging, associated with cognitive impairments have a profound effect impairing mechanisms of motivational consolidation, induced both by a behavioral reinforce or the stimulation of the amygdala 25A4 The contribution of the basal forebrain cholinergic system to cortical plasticity associated with normal learning and functional recovery following brain injury James M. Conner and Mark H. Tuszynski Department of Neurosciences, University of California San Diego, La Jolla, CA, USA Cortical plasticity, and the reorganization of cortical sensorimotor representations, has been proposed as a substrate for normal learning and recovery of function following brain injury. The physiological and cellular mechanisms responsible for cortical reorganization have not been fully elucidated. In our recent studies, we investigated the contribution of the basal forebrain cholinergic system in modulating plasticity associated with cortical motor representations. We first investigated the effects of specific basal forebrain cholinergic lesions upon cortical reorganization associated with learning a skilled motor task and addressed the functional/behavioral consequences of blocking cortical map reorganization. Results from this study demonstrate that disrupting basal forebrain cholinergic function significantly impairs, but does not abolish, acquisition of a new motor skill. Electrophysiological mapping techniques indicated that skilled motor learning was associated with a significant 30.3 ± 7.7% expansion of the caudal forelimb representation in the cortex controlling the trained limb. In animals with selective lesions of the basal forebrain cholinergic system, the expected expansion is completely blocked. Additional experiments demonstrated that the basal forebrain cholinergic lesions did not lead to deficits in i) global attention associated with performing the reaching task, ii) other forms of learning such as associative fear conditioning or iii) general sensorimotor function. Taken together, these results support the hypothesis that the basal forebrain cholinergic system may be specifically implicated in forms of learning requiring plasticity of cortical representations. 25A5 The development and function of mammalian motoneurons following their rescue from programmed cell death by deletion of the proapoptotic gene Bax Ronald Oppenheim, David Prevette, Sharon Vinsant and Thomas Gould Department of Neurobiology and Anatomy, Wake Forest University Medical Center, Winston-Salem, North Carolina, USA Genetic deletion of the pro-apoptotic gene Bax (Bax KO), a member of the Bcl-2 family, permanently rescues many populations of developing neurons, including motoneurons (MNs) from apoptotic programmed cell death (PCD). Because normally 5060% of all post-mitotic embryonic MNs undergo PCD, Bax deletion results in the survival of thousands of excess neurons into postnatal and adult stages. Although this sub-population of “undead” MNs initially differentiate normally and innervate peripheral muscle targets, by perinatal/early postnatal stages, two populations of MNs can be identified: approximately one-half of all MNs appear cytologically normal and maintain innervation of target muscles, whereas the remaining sub-population (“undead” cells) have small atrophied cell bodies and peripheral axons that appear to reach no further than the ventral root or proximal sciatic nerve. We reasoned that the failure of the sub-population of “undead” MNs to grow normally and sustain muscle innervation may be due to limiting amounts of target-derived neurotrophic factors (NTFs). To test this idea, we have provided Bax KO mice with excess amounts of one potent NTF, GDNF, during either embryonic, postnatal or adult stages. GDNF but not BDNF treatment was able to prevent the atrophy and loss of innervation in Bax KO mice when available to the embryo and reversed the atrophy and denervation of the “undead” MNs when GDNF treatment occurred postnatally or in the adult. Preliminary studies suggest that following postnatal treatment with GDNF, the rescued “undead” MNs in the Bax KO mice may contribute to motor behavior. Studies are in progress to examine disease progression in a mouse model of ALS (the SOD1 mutant) when crossed with the Bax Abstracts of the 2nd Scientific Conference "Restauración Neurológica 2004" 219s of the 2nd Scientific Conference "Restauración Neurológica 2004" 219 KO (i.e. will Bax deletion or Bax deletion plus GDNF rescue the disease phenotype). 25A6 Involvement of DNA damage and repair systems in neurodegenerative process Daniela Uberti, Giulia Ferrari-Toninelli, Maurizio Memo Department of Biomedical Sciences and Biotechnologies. University of Brescia Medical School, Brescia, Italy Preservation of genomic stability is an essential biological function. Cells engage very efficiently mechanisms involving DNA surveillance/repair proteins that work to maintaining inherited nucleotide sequence of genomic DNA over time. After DNA damage, that can arise during duplication or after genotoxic stimuli, cells activate intracellular pathways which are able to recognize the damage, to arrest cell cycle, to recruit DNA repair factors, to repair the damage or induce apoptosis. This definitely relevant process is finalized to prevent the generation and the persistence of impaired cells which may ultimately be detrimental to the organism. Very little is known about the role of DNA damage sensors and repair factors in terminally differentiated, not proliferating cells, like neurons. It is well recognized that mutation of genes related with DNA damage repair are associated with specific cancer-prone syndromes. Interestingly, many human pathological conditions with genetic defects in DNA damage responses are also characterized by neurological deficits. These neurological deficits can manifest themselves during many stages of development, suggesting an important role for DNA repair during the development and maintenance of the brain. Here I will present recent data from my group underlining the contribution to neurodegeneration of at least two transcription factors known to be involved in DNA damage sensing and repairing: the tumour suppressor gene p53 and the component of the DNA repair system MSH2. Both proteins participate in the cancer prevention machinery for the body as well as in the neurodegenerative process. Moreover, they interact with each others to orchestrate DNA repair functions. 25A7 The injection of the fraction 25-35 of amyloid-ß into hippocampus of neonatal rats produces changes on cognitive, morphological and biochemical tests E-M Cuevas, J-F Guevara, L-J Solis, A-E Osorio, F-A Diaz, I-M García, and I-D Limón Instituto Nacional de Neurología y Neurocirugía México D.F. and Laboratorio de Neurofarmacología FCQ-BUAP. Puebla, México In Alzheimer’s disease (AD), the neuropathologycal hallmarks includes extracellular deposits of bamyloid in senile plaques, reactive astrocytes and intracellular deposits formation of neurofibrillary tangles, the loss of neuronal cells and synapses. Histopathological, behavioral and biochemical research suggest that the amyloid-ß protein produce a toxic effect and start the injuries. It’s unclear how or when this protein is aggregated into hippocampus and cortex.The aim of this work were evaluated the effect of 25-35 fraction Aß injected into neonatal hippocampus and cortex, on learning, memory, histopathological and biochemical test. The animals used, were PD7 male and female Wistar rats pups. The pups were treated uni and bilateral with 1 μL of 2535Aß (100μM) into hippocampus by stereotaxic surgery (AP=+1.5, L=±2.3, P=-2.0). After twelve and fourteen weeks were evaluated spatial learning and memory in the radial maze, respectively. The program was (number trial /number opportunities. 1st day 3/8, 3/7 y 3/6, 2nd day 1/6, 3/5, 3⁄4 y 2/3, 3st.day 3/5, 3⁄4, 3/3, and for memory test 8/3. We found in female rats deficits 18% and 32% uni-bilatral lesion on spatial memory, respectively. In the hye, PASS and Bielschowsky stained we found a decrease 40% in the neuron number in CA1 region. We found until 25% reactivity antibodies to GFAP. Moreover, we found a 23% of increase for NO levels at four hours after deposit the 25-35 fraction Aß. However, we didn’t found amyloid deposits using the antibody to Aß. Those results suggest that 25-35 Aß fraction produce neurotoxicity in CA1 neuron of hippocampus and decrease the spatial memory since neonatal stage, maybe NO way. 25A8 Cerebellar and pontine norepinephrine contents after motor recovery in rats Rigoberto González-Piña, Antonio Bueno-Nava, Carmen Escalante-Membrillo, Sergio Montes, Angélica González-Maciel, Fructuoso Ayala-Guerrero Laboratorio de Plasticidad Cerebral y Proliferación Celular, InCH-CNR, SSA México, Instituto Nacional de Neurología y Neurocirugía MVS, SSA México, Laboratorio de Microscopia Electrónica, INP SSA México, Laboratorio de Neurociencias, Fac. de Psicología, UNAM, México Abstracts of the 2nd Scientific Conference "Restauración Neurológica 2004" 220s of the 2nd Scientific Conference "Restauración Neurológica 2004" 220 Motor cortex ablation induce motor deficits such as hemiplegia in rats. A spontaneous recovery that can be enhanced by either cerebellar infusion of norepinephrine (NE) or administration of NE agonists has been reported. These suggest that cerebellum (C) and pons (P), that contains the NEergic locus coeruleus, can play an important role in the recovery after motor brain injury. However, there are not data that relate directly the cerebellar and pontine NE contents with motor recovery. In order to assess the status of the NE contents in P and C after functional recovery, we trained 21 male wistar rats (280-320 g) to obtain their basal gait prints. Then animals were allocated into two groups: sham operated (n=10) and injured by right motor cortex ablation (n=11). After 6 hours post-surgery, gait prints were recorded every 6 hours during 48 hours. Subsequently, animals were decapitated and the left and right hemispheres of P and C were processed for NE extraction and HPLC analysis. The remained brain was fixed in formalin-buffer 10% in order to be stained with Nissl's method to verify the extension of the lesion. Length, width and angle were measured in the gait prints. Results showed that gait parameters were fully recovered 48 hours after performance of the lesion, while NE contents were increased in left and right P and decreased in left and right C. We conclude that recovery observed could be a result of a reorganization in the NE pathways, which also could involve to other cerebral structures. 25A9 Proechimys guyannensis rat: an animal model of resistance to epilepsy Esper Cavalheiro Neurologia Experimental-Escola Paulista de MedicinaSão Paulo Brazil The potential interest of Proechimys Guyannensis (PG), a spiny rat living in the Amazonian region, as an animal model of anti-convulsant mechanisms prompted the investigation of the susceptibility of PG to different epileptogenic paradigms. The findings pointed out a remarkable resistance of these animals to different models of experimental epilepsy: 1) Amygdala kindling development – Proechimys animals demonstrated a striking resistance to reach the stage 5 of kindling. From 43 Proechimys rats submitted to the kindling process only 3 animals reached the stage 5. From 40 animals that did not reach the kindled state, 16 did not extended beyond stage 1, 15 from stage 2, 7 from stage 3 and 3 from stage 4. Amygdala electrical stimulations were followed by very long after-discharges, mainly in stages 1-4. 2) Intrahippocampal kainic acid (KA) – A remarkable sensibility to intrahippocampal KA was noticed in PG. One-tenth of the KA dose usually used in Wistar rats elicited self-sustained electrographic status epileptus in PG animals which lasted for more than 48h with increased mortality rate. On the other hand, none of the surviving animals presented spontaneous seizures in the long-term observation period (up to 120 days). Neuropathological examinations of the hippocampus of Proechimys animals after KA injection showed a complete neuronal destruction at the injected hippocampal formation, more pronounced in CA1/CA3 areas, and with less marked changes in the contralateral hippocampus. 3) Pilocarpine – Pilocarpine (350-380 mg/kg – doses regularly used in Wistar rats), when administered to PG induced severe tonic seizures followed by death of all animals. Dose slightly lower (300 mg/kg) than those previously mentioned was able to induce repetitive electrographic and behavioural seizures that culminated in status epilepticus 20-30 min following pilocarpine. However, pilocarpine-induced SE in PG had a shorter duration, rarely exceeding 2 hours, clearly in contrast to the 8-12 h long SE in the Wistar rat.From 60 animals injected with pilocarpine, 48 presented SE and only 2 presented some spontaneous seizures (approximately 1/week for 8 weeks) after silent periods of 60 and 66 days.The histological analysis of the brain of these 2 animals revealed neuronal loss in the CA3 area, in the hilus of the dentate gyrus (DG) and mossy fibers sprouting in the supragranular layer of DG and in the stratum radiatum of CA3. Altogether these data indicate that PG, although extremely sensitive to chemical convulsants (ie, KA and pilocarpine) and presented longer afterdischarge following electrical stimulation of the amygadala, is unable to establish a circuitry appropriated to the elaboration of spontaneous seizures, ie, epilepsy. In other terms, it seems probable that limbic circuitries, such as those found in the amygdala or the hippocampus, is highly affected by excitatory stimulation. However some “external factors” such as inputs originating in extralimbic areas seem to prevent or inhibit the formation of a true epileptic focus. Some behavioural aspects observed in PG during the 3 experimental situations described above could give some hints to justify this hypothesis. During amygdala kindling stimulation and also during the seizures observed after systemic pilocarpine or intrahippocampal KA, a cataleptic behaviour was clearly observed in PG animals characterized by opistotonus and S tail suggesting the participation of the opiate system in this process.These findings indicate that PG rats may have natural enAbstracts of the 2nd Scientific Conference "Restauración Neurológica 2004" 221s of the 2nd Scientific Conference "Restauración Neurológica 2004" 221 dogenous antiepiletic mechanisms and further investigations (anatomical, biochemical, etc) need to be carried out to clarify this phenomenon. 25A10 Subacute electrical stimulation of parahippocampus and gaba system of patients with temporal lobe epilepsy Luisa Rocha Depto. Farmacobiología. Centro de Investigación y de Estudios Avanzados. México, D.F. We previously reported that subacute electrical stimulation of the parahippocampal cortex (PHC) reduces electrographic and clinical seizures in patients with intractable mesial temporal lobe epilepsy (MTLE). The present study was carried out to evaluate the GABA system and neuronal loss in PHC of patients with MTLE who received subacute electrical stimulation and presented antiepileptic effects. GABA tissue content, GABAA and benzodiazepine (BDZ) receptor levels as well as cell density were determined in PHC of patients with MTLE who received subacute electrical stimulation (130 Hz, 450 μs, 200-400 μA, continuously during 16-20 days) in this brain area and demonstrated a significant decrease of interictal spikes and seizures (ESAE group, n=5). Values were compared with those obtained from patients with MTLE plus electrical stimulation but without antiepileptic effects (ESWAE group, n=4) and those with MTLE in whom no electrical stimulation was applied (MTLE group, n=4). Autopsy material acquired from subjects without history of epilepsy was considered control PHC (C group, n=4 obtained from 3 subjects). ESAE group demonstrated high GABA tissue levels (219%), as well as a significantly higher cell count (58.5%) when compared with the MTLE group. Both the MTLE and ESAE groups demonstrated decreased GABAA receptor values compared to controls with no significant differences in BDZ receptor values. The present results suggest that subacute electrical stimulation of PHC electrical stimulation is more effective in patients with less severe epilepsy, and effect associated with a high GABA tissue content and a low rate of cell loss. 25A11 Localization of the epileptogenic zone in temporal lobe epilepsy by ictal V-EEG compressed spectral arrays analysis L. Morales Chacon, Carlos S Catasus, Rolando Palmero, J.Bosch Bayard , L. Lorigados, M.E. Garcia, J. Bender, A. Sanchez International Center for Neurological Restoration. Havana Cuba Objective: Localizing the seizure onset and temporal evolution of ictally dominant frequencies in patients with temporal lobe epilepsy submitted to successful Standard Anterior Lobectomy and evaluate the role of this methodology in lateralization diagnosis when Resonance Magnetic Imaging shows normal or bilateral abnormalities. Methods: The onset of the ictally dominant frequency and its temporal evolution was studied in eight patients using Compressed Spectral Arrays Analysis. Five clinically and electroencephalographic representative seizures were selected, the time period chosen was 20 s before and 16s after the electroencephalographic seizure onset. Sources of EEG activity were determined with an inverse solution method called Variable Resolution Electrical Tomography (VARETA), which provided a splinedistributed solution. We also compared the lateralization diagnosis provided by this methodology with the information showed by neurofunctional techniques. Results: In all patient an ictally dominant and consistent frequency of 5.90± 1.31 Hz could be determined, however in 6 of 8 patients a second dominant frequency peak between 4.24-8 Hz was found. The use of linear inverse solution will allow to control that the analyzed frequency is generated approximately within the brain region whose resection rendered all patients seizure free. The lateralization and localization of the main dominant frequency during the evaluated period around the ictal EEG onset corresponds well with the lateralization proposed by other neuroimaging techniques such a interictal/ictal SPECT, and Cho/Cr ratio measured by MRS (x2 (18)=42.5 p=0.009. Conclusions: Our data suggest, that the ictally dominant frequency analysis and inverse solutions can be reliably used to detect ictal seizures onset arising in mesial temporal lobe epilepsy. Combination of ictal EEG using spectral analysis, SPECT and ERM improved the reliability of the localization and lateralization diagnosis in Temporal Lobe Epilepsy patients much more important when the Resonance Magnetic Imaging shows normal or bilateral abnormalities. 25A12 Excitatory Amino Acid Transporter Activity and Oxidative Stress J. G. Ortiz and N. Berríos-Cartagena Abstracts of the 2nd Scientific Conference "Restauración Neurológica 2004" 222s of the 2nd Scientific Conference "Restauración Neurológica 2004" 222 Dept. of Pharmacology & Toxicology, Univ. of Puerto Rico School of Medicine, San Juan, Puerto Rico The activity of high-affinity, sodium-dependent, glutamate transporters (EAAT 1-5) terminates synaptic transmission, prevents extracellular glutamate (Glu) from reaching excitotoxic levels, and controls synaptic spillover. EAAT regulation is rather complex involving phosphorylation/dephosphorylation states, internalization/surface expression and redox status. The possible reversal of EAAT activity has been the focus of the last decade. However such approach is impractical as it would require precise knowledge of EAAT status in orde to be of theapeutic use. In addition to excessive Glu receptors activation, elevated [Glu] levels are likely to interfere with the cystineglutamate exchange interfering with glutathione homeostasis. Riluzole, used for amyolateral sclerosis (ALS) directly increases EAAT activity and partially prevents veratridine-induced EAAT inhibition.. Moreover, chlorpheg, a cystine glutamate exchange inhibitor, significantly increases EAAT activity in rat hippocampal slices in the presence of 50 μM Glu and partially prevents the inhibitory effects of veratridine on EAAT activity. These results suggest that modulation of EAAT activity may be accomplished by separate mechanisms; directly as with riluzole and indirectly via cystine glutamate exchange (and glutathione) dynamics. 25A13 Post-traumatic epilepsy Pierre Jallon Epilepsy Unit, Geneva University Hospital, CH.1211 Geneva 14, Switzerland Objectives: to evaluate the frequency, the clinical presentations and the therapeutic management of post traumatic seizures and post traumatic epilepsy . Methods: we analysed and compared the recent studies concerning early post traumatic seizures and late post traumatic epilepsy in children and adults. Results: Early seizures were more prevalent among subjects 0-15 years of age with severe head trauma than among individuals over 15 years of age. A majority of early seizures are focal and of those 75 % are partial motor seizures. The fits may be single or repetitive. Status epilepticus or acute repetitive seizures are more common in children than in adults. The occurrence of early seizures is an indication for repeating imaging even in case of mild head injury. EEG plays only a limited role in the evaluation of early seizures. The incidence of late post traumatic seizures differs largely with age, type of trauma, neurological or neuroradiological signs attesting the severity of the trauma. Incidence varies from 7 % in civilian injuries to 35 % in military injuries. Conversely to early seizures, late seizures are less common and more delayed in children than in adults. The spectrum of all types of partial seizures as well as generalised seizures can be observed except bilateral myoclonias and absences. The issue of using antiepileptic drugs (AEDs) to prevent post traumatic seizures remains very controversial. Phenytoin remains the drug of choice for preventing and treating early post traumatic seizures because its efficacy and the possibility to use intravenous administration. All other AEDs can be used in treating late seizures. 25A14 Preliminar study of the anticonvulsant and anxiolytic like effects of GABA-amides derivated from n-GABA-3-(R-fenil)-2-E-propenamide in mice Ma.Eva González-Trujano, Teresa Zapatero, Magadalena Briones Velasco, Lino Joel Reyes Trejo, Andrés Navarrete Castro Instituto Nacional de Psiquiatría “Ramón de la Fuente Muñíz”, Av. México-Xochimilco No. 101. Col. San Lorenzo Huipulco. C.P. 14370, México, D.F. Facultad de Química de la Universidad Nacional Autónoma de México. Ciudad Universitaria Coyoacán. C.P. 04510, México, D.F. México Both etiology and treatment of anxiety and epilepsy have been associated with the GABAergic system. It is well known that GABA can not cross the bloodbrain barrier. A direct substitution therapy in neurological diseases attendant on GABA deficiency is therefore not possible. This report concerns the relationship between ten GABA-AMIDES synthesized from GABA and R-propenamide group in order to get major liposolubility as a fundamental strategy to develop therapeutic alternatives as anticonvulsant and anxiolytic drugs. Taconic mice maintained at standard conditions were used. GABA-AMIDES were obtained by synthesis and diacepam was tested as positive drug. Mice were injected with pentylenetetrazol, strychnine and 4-aminopyridine to record occurrence of the first episode of clonic or tonic seizures and mortality. Anxiolytic like effect was carry out on a exploration model. Effects on the coordination motor and miorelaxation were also assayed. The R-propenamide group increased liposolubility of GABA, important factor to produce effect on the central nervous system, but not a determinant factor to Abstracts of the 2nd Scientific Conference "Restauración Neurológica 2004" 223s of the 2nd Scientific Conference "Restauración Neurológica 2004" 223 produce anxiolytic like effect, because it was not observed a direct relation between pharmacological effect and log P data. Number and position of constituents appears to play an interesting roll in these pharmacological properties. It is because, two GABA-AMIDES with the chemical group metoxi on positions 2,4 or 3,4 were the most active. Agree with LD50 data, all active GABA-AMIDES were no toxic. One of them showed anticonvulsant activity. These results suggest that GABA-AMIDES have anxiolytic like effects without adverse effects, it is an advantage over drugs clinically used. Differential effect of these GABA-AMIDES could involve different mechanism of action to the GABAergic system. 25A15 CaM kinase II-Mediated Modulation of GABAA Receptor Phosphorylation in Status Epilepticus A. Rana, P.D. Franks, J.T. Parsons, A. de Blase, S.B. Churn Departments of Neurology, Anatomy and Neurobiology, Pharmacology and Toxicology, and Physiology, Virginia Commonwealth University, Richmond, VA USA. e Physiology and Neurobiology, University of Conneticut, Storrs, CT USA Calmodulin-dependent kinase II (CaM kinase II) activation has been shown to positively modulate both agonist and allosteric modulator binding, and agonistinduced anion currents. Prolonged status epilepticus (SE) has been shown to result in both inhibition of CaM kinase II activity and loss of γ-amino butyric acid (GABA) receptor function. This study was designed to determine if the SE-induced inhibition of CaM kinase II activity also resulted in loss of GABAA receptor phosphorylation. The pilocarpine rat model of SE was utilized, and SE level was determined both behaviorally and by EEG measurements by surface electrodes. Following SE or sham treatments, animals were decapitated, and crude synaptic membrane fractions isolated by differential centrifugation. Standard CaM kinase II assays were performed, GABAA receptor subunits isolated away from synaptic proteins by detergent solublization, immunoprecipitation and resolution onto SDSPAGE. Phosphate incorporation into GABAA receptor subunits was quantified by direct counting of resolved protein bands on either SDS-PAGE or western blots (Instant Imager, Packard Instruments).There was poor correlation between behavioral seizures and loss of kinase activity. However, all animals that progressed into continuous seizure activity measured by EEG recordings, displayed a significant loss of kinase activity in brain fraction homogenates. In crude synaptic membrane fractions SE resulted in an approximate 41% inhibition of CaM kinase II activity towards exogenously added substrates (Autocamtide 3, Calbiochem). In parallel reactions, SE resulted in approximately 40% inhibition of CaM kinase IImediated phosphorylation of GABAA receptor α1 subunit (27.8 vs. 16.6 amol/mm2/min). Furthermore, SE resulted in approximately 37% inhibition of GABAA receptor β 2/3 subunits that co-precipitated with α1 subunit protein. Since CaM kinase II activation has been shown augment both GABAA receptor agonist and allosteric modulator binding and agonistinduced current, the data suggest a mechanism whereby SE results in loss of GABAA receptor operation. RO1-NS39970, P50-NS25630 25A16 The hyperthermia induced seizures produce transient gabaergic cell death in the hippocampus of the rat Sandra A. Orozco, Misael González , Sebastián Castillo and Alfredo Feria Unidad de Investigación Medica en Enfermedades Neurológicas, Hospital de Especialidades, Unidad de Investigación Medica en Enfermedades Oncológicas, Hospital de Oncología, CMN, Siglo XXI, IMSS, México D.F. y Unidad de morfología de Alta Resolución,CUCBA, Universidad de Guadalajara, Guadalajara, Jal. México The febrile seizure is the most prevalent age-specific seizures in infant and young children. Whether they result in long term sequels such neuronal lost and lobe epilepsy, it is controversial. Some studies of human febrile seizure have found no adverse effects on the developing brain. However, adults with temporal lobe epilepsy have a history of prolonged febrile seizures in early life. This study is aimed to know if hyperthermia-induced seizures produced neuronal death in infant and developing brain. Infant rats were subject to hyperthermic seizures (a model of prolonged febrile seizure). Neuronal death was assessed in sections of forebrain, stained by DAPI nuclear stain, in situ terminal deoxynucleotidyl tranferase dTUP nick-end labeling (TUNEL stain), annexin V antibody and electronic microscopy. We used two neuron marker, NeuN antibody by neurons and GAD65/67 antibody by GABAergic cells. The studies were carried at 24 hrs after the seizure and 5, 15, 20 and 30 days after seizures. The results showed TUNEL and annexin positive cells in hippocampal formation mainly in the CA1, CA3, and dentate Abstracts of the 2nd Scientific Conference "Restauración Neurológica 2004" 224s of the 2nd Scientific Conference "Restauración Neurológica 2004" 224 gyrus, 24 hrs after seizures and few TUNEL and annexin positive cells at 5, 15 and 20 days after seizures. The 100% tunnel positive cells, 30% colocalized with GAD65/67 positive cells and 20% colocalized with NeuN positive cells. The electronic pictures showed some apoptotic neurons with dispersed chromatin and some neurons with necrotic characteristics. These results showed the prolonged febrile seizures resulted in transient neuronal death in immature hippocampus involved death of GABAergic neurons. The loss of these neurons could be enhanced susceptibility to further limbic seizures. 25A17 Anticonvulsant valproic acid induces differentiation in human neuroblastoma cells A.M. Ferreri, P. Rocchi Department of Experimental Pathology ,University of Bologna, Bologna, Italy Valproic acid (vpa,2-propylpentanoic acid) is an established drug in the long-term therapy of epilepsy During the past years ,it has become evident that vPA is also associated with anti-cancer activity .the present study was aimed at evaluating the effect of sodium valproate on differentiation and proliferation in the human neuroblastoma cell lines AF8 and TS12.Neuronal differentiation was assessed by means of morphological and cytochemical parameters,i.e. neurite outgrouth and acetylcholinesterase specific activity Growth curves and colony-forming assay were performed in order to determine cell growth inhibition Sodium valproate induced inhibition off cell growth (demonstrated by growth curves) and reduced the colony forming ability in a dose dependent manner in both cell lines. Inhibition of cell proliferation was accompanied by morphological features of neuronal differentiation: on both cell lines valproate induced neuritogenesis in a dose dependent fashion. Biochemical differentiation was demonstrated by the increase in the acetylcholinesterase specific activity,an enzime widely used as biochemical marker for neuronal differentiation in neuroblastoma cells.these results underline the role of HDACs inhibitors as new cancer drugs and suggest the evaluation of sodium valproate for cytodifferentiation therapy in the treatment of neuroblastoma. Posters 25P1 Subacute electrical stimulation with antiepileptic effects modifies GABAergic system in patients with intractable temporal lobe seizures Manola Cuéllar, Marcos Velasco, Francisco Velasco, Ana Luisa Velasco, Sandra Orozco and Luisa Rocha Farmacobiologia,CINVESTAV-IPN. Hospital General de México. Hospital de Especialidades, México, D.F, Mexico Experimental studies demonstrated that high frequency stimulation (HFS) of cerebral regions reduces seizures in patients with refractory temporal lobe epilepsy (TLE). We suggest that HFS in these patients induces the activation of Gabaergic system. Methods: Tissue cerebral were obtained from 3 groups: control (C, n=3), TLE (n=6) and TLE with HFS (HFS, n=6). HFS consisted of continuous stimulation with biphasic pulses (duration: 0.45 sec; frequency: 130 Hz; amplitude: 0.2-0.4 mA). Ammon ́s horn (AH) in hippocampus and parahippocampal cortex (PC) GABAA and BZD receptors system were evaluated using autoradiographic technique. GABA and Glu content were determinated by high performance liquid chromatographic analysis. Neuronal population count was performed using Nissl staining technique. Results: Concerning hippocampus TLE showed a decrease for GABAA and BZD receptor binding, tissue content of amino acids and neuronal count were similar to previous reports. HFS exhibited increased for GABAA receptor levels in AH (1086%) compared with TLE, BZD receptor binding was increased in AH compared with C (339%) and TLE (207%). GABAA receptor binding in PC (85%) exhibited decreased values compared with C, whereas BZD receptor binding did not show changes in this region. The regions AH (46%) and PC (44%) exhibited increased neuronal population count compared to TLE. There was a direct correlation between increased levels of the GABA and Glu in PC with decreased percentage of interictal spikes from the HFS. Conclusions: These results suggest that the antiepileptic effects of HFS involve increase GABAA and BZD receptors in AH, enhanced GABA and Glu amino acid levels and less neuronal loss in PC. 25P2 Inhibitory effect of 5-HT1A receptors on experimental limbic seizures in rats Abstracts of the 2nd Scientific Conference "Restauración Neurológica 2004" 225s of the 2nd Scientific Conference "Restauración Neurológica 2004" 225 María-Leonor López-Meraz, Leticia Neri-Bazán, María Eva González Trujano & Luisa Rocha Centro de Investigación y de Estudios Avanzados del I.P.N., México D.F., Instituto Nacional de Psiquiatría “Ramón de la Fuente”, México, D.F. The purpose of this investigation was to evaluate the participation of 5-HT1A receptors on pentylenetetrazol (PTZ, 60 mg/Kg i.p.), kainic acid (KA, 10 mg/Kg i.p.) and amygdala kindling (daily 1s train of 1ms pulses at 60 Hz and 400 mA)induced seizures in male Wistar rats, employing two agonists (8-OHDPAT 0.01, 0.1, 1 mg/Kg and indorenate 1, 3, 10 mg/Kg) and an antagonist (WAY100635 1 mg/Kg) of these receptors. Neither of these 5-HT1A ligands modified the latency to the PTZinduced clonic-tonic seizures. However, 8-OH-DPAT enhanced twice the number of clonus induced by PTZ, effect associated with decreased mortality. On the other hand, 8-OHDPAT and indorenate augmented the latency to the first wet dog shake (WDS) (44-58% and 67%, respectively) induced by KA, effect that was blocked with the antagonist. Additionally, 8-OH-DPAT diminished the frequency of WDS (44-59%) and generalized seizures (GS) (78-84%) during the KAinduced status epilepticus (SE). Similarly, Indorenate decreased in 69-75% the number of GS. Interestingly, WAY100635 decreased non significantly the number of WDS and GS. In addition, 8-OH-DPAT and indorenate diminished the percentage of animals with SE (63-83%). In contrast, the evaluated drugs did not alter the kindled seizure expression. Nevertheless, indorenate decreased the seizure susceptibility during the postictal depression (34%), effect reverted by WAY100635. These results suggest that 5-HT1A receptors induce inhibitory effects on the KAinduced SE and during the postictal depression associated with the amygdala kindled seizures. Supported by CONACyT (scholarship 153240). 25P3 Increase P-glycoprotein expression in several limbic brain regions in the phenytoin resistant amygdala-kindled rats Graciela Jiménez-Rubio, Sandra Orozco-Suárez, M. Eva González-Trujano, Luisa Rocha Instituto Nacional de Psiquiatría "Ramón de la Fuente" and Centro de Investigación y Estudios Avanzados, Sede Sur, Farmacobiología, Instituto Politécnico Nacional. México, D.F., Unidad de Inv. Médica en Enfermedades Neurológicas, H de Especialidades, CMN, Siglo-XXI, IMSS. México, D.F., Centro de Investigación y Estudios Avanzados, Sede Sur, Farmacobiología, Instituto Politécnico Nacional. México, D.F. Studies have shown that multidrug transporter Pglycoprotein (PGP-1) is overexpressed in endothelial cells (EC) from brain blood vessels of patients with refractory temporal lobe epilepsy (TLE), suggesting the role of PGP-1 in pharmacoresistance to antiepileptic drugs (AEDs). In this study we used the amygdala-kindled rats, a refractory model to AEDs, to evaluate the PGP-1 expression in limbic brain regions thought to be involved in TLE. Male Wistar rats (250-350 g) were kindled by basolateral amygdala stimulation. After kindling acquisition, four independent acute phenytoin (75 mg/kg i.p.) trials were done. Response to phenytoin was determined using a threshold after-discharge; the response was sensitive (KSEN), variable (KVAR) or resistant kindled rats (KRES). PGP-1-expression was analysed 24 h after the last kindled seizure by immunohistochemistry. Cerebral cortex, hippocampus and amygdala were examined by confocal and fluorescence microscopy. In kindled rats PGP-1 staining was observed in EC, astrocytes in hippocampus and cortex, and neurons in amygdala. In KSEN rats PGP-1 staining was observed in EC and astrocytes, whereas in KVAR rats it was found in EC, glia and neurons both in all testing areas. The KRES rats showed an increased PGP-1 expression in EC and astrocytes in cortex and hippocampus; neurons and glia in amygdala. The data indicate that PGP-1 over-expression in KVAR and KRES rats mainly in capillary endothelial and glial cells of the blood brain barrier is likely to reduce the penetration of AEDs into brain parenchyma, which could explain the variability of the response and the phenytoin resistant in this epilepsy model. 25P4 Febrile seizures modify the benzodiazepine receptor binding in the developing rat brain González Ramírez M, Orozco Suárez S, Salgado Ceballos H,Briones Velasco M, Rocha Arrieta L Unidad de Investigación Médica en Enfermedades Neurológicas H. Especialidades CMN SXXI IMSS. División de Neurociencias, INP “Ramón de la Fuente” y Depto. De Farmacobiología, CINVESTAV del IPN, México DF. It has been described that immature brain is more susceptible to seizures than the mature one and that febrile seizures early in life may lead to epilepsy. The present study was carried out to evaluate the benzodiazepine (BDZ) receptor binding in specific brain areas of immature rats at different times. So, pups of 10 Abstracts of the 2nd Scientific Conference "Restauración Neurológica 2004" 226s of the 2nd Scientific Conference "Restauración Neurológica 2004" 226 days old were placed on the floor of a 3 l glass container and an air stream was directed ~50 cm above them. Hyperthermia was maintained for 30 min, aiming for a core temperature of 41°C. Thereafter, rats were placed on a cool surface, monitored for 15 min, and then returned to their home cages for rehydratation by the mothers. The control group was normothermic temperature. Animals were sacrificed by decapitation 30 min, 24 h and 20 days after seizures and their brains were used for autoradiography assay with the purpose to determine BDZ receptor binding in different brain areas. Animals sacrificed at 24 h increased BDZ receptor levels in cingulated cortex (79.55%), frontal cortex (70.51%), parietal cortex (70.53%), striatum (23.94%), basolateral amygdaloid nuclei (51.29%), central amygdaloid nuclei (32.16%), medial amygdaloid nuclei (49.88%), entorhinal cortex (35.09%) and substantia nigra, compact part (33.41%). In contrast, the groups sacrificed at 30 min and 20 days after febrile seizures did not present significant changes as compared to the control ones. The results indicate that the febrile seizures do not induce permanent BDZ changes in the immature rat brain. Moreover, it is possible that the enhanced BDZ receptor levels 24 h after febrile seizures may play a protective effect in the immature brain. 25P5 In vivo study of transgene expresión of pgfa2GAD67 into rat hippocampus Cecilia Zavala, Paula Vergara, José Segovia and Luisa Rocha Farmacobiología. Fisiología, Biofísica y Neurociencias, CINVESTAV-IPN, México, D.F., México Decreased GABAergic inhibition has been suggested to induce hyperexcitability. The present study evaluates the potential for preventing seizure generalization by increasing local GABAergic inhibitory activity by applying of a new technique: to express the GABA-synthesizing enzyme GAD67 in the host’s own astrocytes. Using a transgene in which the activity of GAD67 is under the control of a glial fibrillary acidic protein (GFAP) promoter, it confers astrocytespecific expression. The rats were transfected with 1,2 and 4 μg of gfa2-GAD67 (GAD) or pgfa2-Lac-Z (LZ) into hippocampus. Sham control rats (CSh) were not transfected. Three days following microinjection of the transgene, the rats received a single administration of pentylenetetrazol (PTZ, 70 mg/kg i.p.). The latency and duration of the seizures were evaluated. The hippocampal tissue samples were obtained 24 hours after of administration of PTZ and expression of transgene was detected by RT-PCR. In the GAD group the latency to the first clonic seizure was decrease in 76% compared to CSh. Nevertheless, an increase of duration of postictal period was observed in the GAD groups. The duration of mioclonic seizures was significantly decreased in the GAD group (2 μg) (4.8 ± 0.6 sec) compared to CSh (16.14 ± 5.2 sec). The duration of tonic seizures was decreased in the GAD group (1μg) compared to CSh (28.67 ± 16.29 sec and 52.67 ±18.8 sec, no significant). Increased expression of GAD67 was found in animals transfected with pGfa2-GAD67 transgene. The present results suggest that transfection with pGfa2-GAD67 have inhibitory effects on the PTZinduced seizures. 25P6 Immunological disorders in epileptic patients are associated to the epilptogenic focus localization Lourdes Lorigados, Lilia Morales, Nancy Pavón, Teresa Serrano, María de los Angeles Robinson, María Eugenia García, Juan Bender Centro Internacional de Restauración Neurológica (CIREN), Havana, Cuba Clinical and experimental data support the role of immune mechanisms in the pathogenia of epilepsy. The purpose of this work is to study the immunologic aspects in 30 epileptic patients with complex partial crisis resistant to antiepileptic drugs. The patients were evaluated by EEG-Video and they were grouped attending to epileptogenic focus localization in: temporal (n=16), lateralized (n=6) and extratemporal (n=4). We also studied a group with psychogenic epilepsy (n=4); this group was diagnosed after EEG-Video evaluation. The following immunologic evaluations have been carried out: levels of serum immunoglobulins (IgG, IgM e IgA) by radial immuno-diffusion test and lymphocyte subpopulations using immuno-cytochemical methods. We measured the percent of lymphocytes T and B (CD3 and CD20), lymphocyte T helper/inductor (CD4), suppresor/cytotoxic (CD8), interleukine-2 receptor (CD25) and human leukocyte antigen (HLA-DR). The results show a significant increase of CD8+ lymphocytes (p<0.05) and in the activation markers (CD25+ and HLA-DR+ cells). The evaluation of immunologic parameters applied to different group of epileptogenic focus localization showed that the increase of CD8+ lymphocytes is limited to temporal and lateralized patients (P<0.01). The patients with extratemporal localization of focus and the psychogenic cases showed normal values for the evaluated immunologic Abstracts of the 2nd Scientific Conference "Restauración Neurológica 2004" 227s of the 2nd Scientific Conference "Restauración Neurológica 2004" 227 lymphocytes markers. We did not find a deficit in the humoral immunologic aspects. Taking into account that patients diagnosed as psychogenic received an antiepileptic drug treatment identical to that of the other group, the observed immunologic changes might be related with the pathogenia of certain epilepsy variants associated with the focus localization and not with the medication. 25P7 Biochemical and morphological parameters in temporal lobectomised patients Lourdes Lorigados, Lilia Morales, Sandra Orozco, Leticia Nery, Ivan García, Barbara Estupiñan, Barbara Paula, Luisa Rocha Centro Internacional de Restauración Neurológica, Havana, Cuba,Centro Médico, DF, México, CINVESTAV, SedeSur, DF, México, f Hosp. Carlos J Finlay, Cuba Temporal lobe epilepsy (TLE) is one of the most frequent types of human focal epilepsy. The main goal of the present work was to examine some biochemical markers in cerebrospinal fluid (CSF) and tissue from 8 lobectomized patients (TLE) and to determine the contribution of apoptotic mechanism to epileptic process in these patients. Patients and methods: levels of amino acids were measured by HPLC method in tissue and CSF. We studied the evolutive levels of amino acids in CSF; the samples were taken before and after (1 and 6 months) surgical treatment. The CSF control group (n=10) was obtained from surgical patients who did not present neurological diseases and the tissue control group (n=4) was obtained from death not-neurological patients. The immunehistochemical evaluation was carried up using the following cellular markers: enolase, Neun, GABA and GAD 67/65. The apoptosis study was carried out for the occurrence of terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL) and Anexin-V markers. The final evaluation of cerebral tissue was done by confocal microscopy. The results showed increased levels of glutamate in cerebral tissue. There are significant decreased of glutamate levels between pre-treatment and before (6 months) treatment. Numerous TUNELpositive cells were observed whereas Anexin Vpositive cells were not significant. We found a decrease of enolase, Neun, GABA and GAD 67/65 positive cells. The results evidence there is death but not only by apoptotic phenomena but also by necrosis. The surgery process can restore the amino acids levels. 25P8 Effect of carbamazepine on convulsive activivity and brain damage induced by administration of kainic Acid Fructuoso Ayala-Guerrero, Alfonso Alfaro, Rosa María Romero, Graciela Mexicano-Medina and Angélica González-Maciel Facultad de Psicología, Universidad Nacional Autónoma de México; Instituto de la Comunicación Humana-CNR and Instituto Nacional de Pediatría, México Several animal models of epilepsy have been used in order to evaluate drugs with anticonvulsant efficacy. Since administration of Kainic Acid (KA) causes physiological and anatomical disturbances similar to those in temporal lobe epilepsy of humans, the kainate treated rat has been considered as an experimental model of temporal lobe epilepsy. Carbamazepine, an antiepileptic drug, has been utilized in clinical for controlling certain types of epileptic attacks. The objective of this work was to analize the action of carbamazepine on convulsive crisis and brain damage induced by KA administration. Experiments were carried out on chronically implanted wistar white rats. One group of five rats was treated with KA (10 mg/kg ) alone, while other similar group was pretreated with carbamazepine (25 mg/kg) 30 minutes before administration of KA. Ten hour polygraphic recordings were continuously obtained under the two mentioned conditions. Under general anesthesia,, brain were removed from the skull for histological analysis ten days after administration of drugs. Animals receiving administration of KA alone presented convulsive activity and brain damage. Carbamazepine pretreatment induced a slight decrement of convulsive activity but brain damage was still present. 25P9 Epilepsy surgery. A preliminary Study Iván García Maeso, Juan E. Bender, Lilia Morales Chacon, Margarita Báez, Lourdes Lorigados Pedre, Bárbara Estupiñán, Maria Eugenia García, Carlos Sánchez Catasús, Rolando Palmero Group of Epilepsy Surgery Project. International Center of Neurological Restoration, Havana, Cuba Epilepsy surgery dates back to more than two millennia and the first intervention was cranial trepanation. This pathology occurs in 0.1% of the population. Its initial medical treatment only achieves control of crisis in a 50% to 80%; the remaining 20% is considAbstracts of the 2nd Scientific Conference "Restauración Neurológica 2004" 228s of the 2nd Scientific Conference "Restauración Neurológica 2004" 228 ered medically untreatable and surgically remediable. Method and Material: 8 patients with refractory temporal lobe epilepsy, of 23 candidates for surgical treatment were operated in the second semester of last year. The surgical technique used was the adjusted temporal lobectomy. Transoperatory Electrocorticographic recordings before and after resection were performed. A follow-up of the cases took place at one, three and six months for a posterior evaluation in a year time. Results: 88 % of operated patients are free of crisis and only one has suffered an occasional crisis. The complications were as follows: three patients with hemosiderotic meningitis, one with a transitory dysfunction of the type III cranial pair; one a diminishing of the verbal memory whereas another patient presented an inadequate syndrome of the antidiuretic hormone. Conclusions: Resective surgery of temporal lobe is the technique that we had selected for the treatment of refractory temporal epilepsy and the patient had a satisfactory evolution. 25P10 Antiepileptic effect of an extract of Ambrosia paniculata (Willd.) O. E. Schultz (mug-wort) in several models of experimental epilepsy María T. Buznego, Héctor Pérez-Saad Department of Experimental Epilepsy, Institute of Neurology and Neurosurgery, Cuba The acute effect of Ambrosia paniculata in several animal models of epilepsy was studied. Intraperitoneal injections (0.01 mL x g of body weight) of a decoction of the dry leaves significantly enhanced the latency of the first convulsion and the survival time in mice injected with picrotoxin (7mg/kg) or isoniazid (210 mg/kg). Epileptic spikes were induced by topical application of penicillin -a glass electrode filled with penicillin-agar-saline mixtureand recorded in sensorimotor and occipital cortices. The plant decoction reduced significantly the spike amplitude in both sites. The same decoction was not effective against the electroshock-induced convulsions in mice. Since convulsions induced by isoniazid, picrotoxin and penicillin -differing from electroshockimplicate a disruption of the GABAergic neurotransmission, these results suggest that Ambrosia paniculata, like several conventional antiepileptic drugs, has a selective effect on this neurotransmitter system, that explain the traditional use of the plant in epilepsy. 25P11 Therapeutic effect of ozone in the brain ischemia: mechanism of action seems to involve hydrogen peroxide J.D. García-Salman, M. García, A. Cuba, R.M. Coro, H. Perez-Saad Department of Experimental Neurology, Institute of Neurology and Neurosurgery, 29st and D, Vedado, Havana 10400, Cuba Brain can be considered as a main target organ for active oxygen species. It is the highest oxygendemanding organ, has a high content of unsaturated fatty acids, a poor antioxidant defense and a high ferrum ion content. During brain ischemia, free radical reactions increase. There are some evidences suggesting that SOD activity reduces edema, mortality and infarct volumes. Other findings found links between hydrogen peroxide and selective gene expression, differentiation and neuronal plasticity. On the other hand, therapy with ozone is based in the generation of ozonids, including hydrogen peroxide. Then, ozonids could be carried by body fluids to the target tissue and exert therapeutic effects. It has been suggested that maximal effects be obtained after a period of preconditioning. In that sense, we treated Mongolian gerbils with intrarectal ozone (1 mg/Kg) for three weeks before ligation of the right carotid artery. Treatment prevents impairment of spontaneous exploratory activity, observed 7 days after ligation in oxygen-treated gerbils. In these animals a significant damage in CA1 pyramidal neurons of the right hippocampus was found, compared with the contralateral hemisphere. In another experiment, the spontaneous exploratory activity before and 7 days after 5 min bilateral carotid occlusion was tested. Ozone was administered in the six subsequent days of reperfusion. The results show that ozone treatment also prevents impairments of the spontaneous exploratory activity. These findings suggest that ozonids particularly hydrogen peroxide are protecting memory and learning-related synapses through improving survival of brain cells. 25P12 Three-synaptic hippocampal response during repeated electrical stimulation of entorhinal cortex in the Mongolian gerbil. Effect of clonidine Héctor Pérez-Saad, Margarita Bu Wong and Nora Sánchez Rodríguez Department of Experimental Neurology, Institute of Neurology and Neurosurgery, 29 and D st. Vedado, Havana 10400, Cuba Abstracts of the 2nd Scientific Conference "Restauración Neurológica 2004" 229s of the 2nd Scientific Conference "Restauración Neurológica 2004" 229 The Mongolian gerbil (Meriones unguiculatus) has been considered as a model of temporal lobe epilepsy, which involves the hipocampal formation. In the present work the effect of clonidine and fenitoin on the electrophysiological response of dentate gyrus to electrical stimulation of entorrinal cortex was studied in anaesthetized gerbils (20 % urethane, 0,01 mL x g of body weight). Intracerebral electrodes following the stereotaxic atlas of Loskotawere used. The amplitude of the population spike was obtained after averaging 20 stimuli (500 μA, 1 Hz and 100 μs). Clonidine (100 μg/kg ip.) and fenitoin (10 mg/kg ip.) reduced significantly the spike amplitude 1 h after injection. In a group of animals the entorrinal cortex was stimulated with a 7 Hz frequency: a second and third population spikes appeared about 20 seconds after the starting the stimulation. These two spikes immediately disappear after coming back to the initial 1 Hz frequency and reappear again with 7 Hz stimulation; this process can be repeated several times with similar results. These spikes also disappear if the 7 Hz stimulation persists for the next 30 to 60 seconds. Fifteen minutes after clonidine injection (100 μg/kg ip.) the primary spike was not modified, but the second spike was retarded to about 20 seconds, with an increased latency and decreased amplitude; the third spike –with very low voltagecompletely disappeared. These results show a particular hipocampal hyper excitability of the seizure-sensitive gerbils and new data about the mode of action of coniine in epilepsy.