ANALYSIS OF THE HYPEREXCITABILITY OF CA3 PYRAMIDAL NEURONS IN A MOUSE-MODEL PRESENTING THE INACTIVATION OF RAC1 AND RAC3 GTP ASES Francesca Talpo 1) , Paolo Spaiardi 1) , Mauro Toselli 1) , Ivan De Curtis 2) , Gerardo Biella 1) 1) University of Pavia, Dept. of Physiology, via Forlanini 6-27100 Pavia, Italy; 2)Cell Adhesion Unit, San Raffaele Scientific Institute and University Vita-Salute San Raffaele, Via Olgettina 58-20132 Milano, Italy INTRODUCTION Rac1 and Rac3 are small GTPases, belonging to the Rho family. These proteins regulate the actin cytoskeleton and are co-expressed in the developing mammalian brain. Recently double knockout mice for Rac1 and Rac3 (Rac1 N /Rac3 KO mice) were produced and characterized (Corbetta et al., 2009). Rac1 N /Rac3 KO mice showed significative cytoarchitectonic modifications and an evident epileptic phenotype. These animals died approximately thirteen days after birth. The main cytoarchitectonic modification revealed by the anatomical analysis of Rac1 N /Rac3 KO mice was a drastic bilateral reduction of hilar mossy cells and of parvalbumine-positive GABAergic interneurons in the dorsal hippocampus. A reduction of parvalbumine-positive GABAergic interneurons was also observed in neocortex. These anatomical modifications appeared to result in a marked increase in excitability of the nervous tissue. The aim of this work was to test electrophysiologically which neuronal elements and/or which synaptic connections were impaired in Rac1 N /Rac3 KO mice to produce the epileptic phenotype. MATERIALS AND METHODS Animal model. In this study two groups of animals (P11-P15) were used. The experimental group consisted of 9 Rac1 N /Rac3 KO mice. These animals did not express Rac1 and Rac3 at neuronal level. The control group consisted of 10 Rac3 KO mice, littermates of Rac1 N /Rac3 KO mice. These animals did not express Rac3, but they expressed Rac1. Electrophysiological methods . Whole-cell patch-clamp experiments in current clamp mode were performed on hippocampal CA3 pyramidal neurons (Rac1 N /Rac3 KO mice, n=15; Rac3 KO mice, n=17 ) in submerged slice preparation (350 μm thickness). Patch pipettes: 4-7 M . Cutting solution (in mM): Sucrose 70; NaCl 80; KCl 2.5; NaHCO 3 26; Glucose 15; MgCl 2 7; CaCl 2 1; NaH 2 PO 4 1.25 (bubbled with 95% O 2 and 5% CO 2 gas mixture). Bath solution (in mM): NaCl 125; KCl 2.5; NaHCO 3 26; Glucose 15; MgCl 2 1.3; CaCl 2 2.3; NaH 2 PO 4 1.25 (bubbled with 95% O 2 and 5% CO 2 gas mixture). Internal solution (in mM): K-gluconate 130; NaCl 4; MgCl 2 2; EGTA 1; Hepes 10; CPP 5; Na 2 ATP 2; Na 3 GTP 0.3 (pH 7.4, adjusted with KOH). Drugs (added to the bath solution): 4-Aminopyridine (4-AP, 50-100-500μM). CONCLUSIONS • The epileptogenic threshold of CA3 pyramidal neurons is lower in Rac1 N /Rac3 KO mice than in Rac3 KO control animals. • The lack of neuronal Rac1 and Rac3 proteins impairs the migration of parvalbumine-positive GABAergic interneurons during the late phases of hippocampal development. The reduced number of GABAergic interneurons at hippocampal level in Rac1 N /Rac3 KO mice produces an unbalance of the hippocampal excitability, that gives rise to an evident epileptic phenotype. Bibliography CORBETTA S., GUALDONI S., CICERI G., MONARI M., ZUCCARO E., TYBULEWICZ V. L. J., DE CURTIS I. (2009). Essential role of Rac1 and Rac3 GTPases in neuronal development. FASEB J. 23, 1347–1357. RESULTS Following 4-AP perfusion spontaneous EPSPs frequency increases more in Rac1 N /Rac3 KO than in Rac3 KO mice (control mice) Rac1 N /Rac3 KO mice are more susceptible to induction of ictal-like activity than Rac3 KO mice (control mice) Representative voltage traces recorded from CA3 pyramidal neurons at potential of -70 mV. In A spontaneous EPSPs (sEPSPs) occurring in a Rac3 KO animal in basal conditions (top left) and during 4-AP [50 M] application (top right) are shown. In B sEPSPs occurring in a Rac1 N /Rac3 KO animal in basal conditions (top left) and during 4-AP [50 M] application (top right) are shown. A significative portion of each trace is shown over an expanded time scale in the bottom part of both figures. Time of 4-AP [100 M] perfusion and percentage of cells presenting ictal-like activity in Rac3 KO control mice (n=6) vs Rac1 N /Rac3 KO mice (n=6). The time of 4-AP [100 M] perfusion is similar between Rac3 KO and Rac1 N /Rac3 KO mice (left histogram). However, the ictal-like epileptiform activity is evident in the large majority of Rac1 N /Rac3 KO mice but is never present in Rac3 KO mice (right histogram). Representative voltage traces recorded from CA3 pyramidal neurons, that show the effects elicited by the application of the convulsant 4-AP [100 M]. In Rac3 KO control mice (n=6, top trace) the application of 4-AP [100 M] (black lines) induces a preictal-like activity, but it fails to induce ictal-like epileptiform activity. In Rac1 N /Rac3 KO mice (n=6, bottom trace) the same pharmacological protocol induces ictal-like epileptiform activity, showing a clear paroxismal depolarizing shift (PDS). Time of 4-AP [500 M] perfusion and percentage of cells presenting ictal-like activity in Rac3 KO control mice (n=9) vs Rac1 N /Rac3 KO mice (n=5). All the recorded cells present ictal-like activity in response to 4-AP [500 M] perfusion, both in Rac3 KO control mice and Rac1 N /Rac3 KO mice (right histogram). However, the time of 4-AP perfusion which is necessary to induce the discharge is statistically lower (t-test, p<0.005) in Rac1 N /Rac3 KO mice than in Rac3 KO control mice (left histogram). Representative voltage traces recorded from CA3 pyramidal neurons, that show the effects elicited by the application of the convulsant 4-AP [500 M]. The application of 4-AP [500 M] (black lines) induces ictal-like epileptiform activity both in Rac3 KO control mice (n=9; top trace) and in Rac1 N /Rac3 KO mice (n=5; bottom trace). Analysis of EPSPs amplitude. The percentage increase of sEPSPs amplitude induced by 4-AP was calculated compairing sEPSPs amplitude before and following 4-AP [50 M] perfusion, both in Rac3 KO and Rac1 N /Rac3 KO animals. The percentage increase of sEPSPs amplitude is not statistically different between Rac1 N /Rac3 KO mice (n=7) and Rac3 KO control mice (n=6). Percentage increase of EPSPs frequency from basal conditions to 4-AP perfusion Percentage increase of sEPSPs frequency induced by 4-AP perfusion: comparative analysis of Rac3 KO and Rac1 N /Rac3 KO mice Analysis of EPSPs frequency. The percentage increase of sEPSPs frequency induced by 4-AP was measured compairing sEPSPs frequency before and following 4-AP [50 M] perfusion, both in Rac3 KO and Rac1 N /Rac3 KO animals. The percentage increase of sEPSPs frequency is statistically higher (t-test, p<0.05) in Rac1 N /Rac3 KO mice (n=7) than in Rac3 KO control mice (n=6). Percentage increase of sEPSPs amplitude induced by 4-AP perfusion: comparative analysis of Rac3 KO and Rac1 N /Rac3 KO mice cted. F1000 Posters. Co opyright protected. F1000 Posters. Copyright protec 00 Posters. Copyright protected. F1000 Posters. Copyright protected. F1000 P otected. F1000 Posters. Copyright protected. F1000 Posters. Copyright protected. F1000 Posters. Copyri . Copyright protected. F1000 Posters. 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