Abstracts

Rationale: A seizure consisting  of sustained repetitive firing of a group of neurons leads to intracellular calcium accumulation, and activation of cfos gene.  We tested whether excitability of CA1 pyramidal neurons expressing cfos in response to a single seizure is enhanced. Methods: Transgenic mice that express EGFP under the control of tetracycline repressor and early immediate gene, cfos (Mayford) were maintained on high doxycycline diet until 48 hours prior to a single seizure, induced by pentyleneterazol (40 mg/kg, i.p.). Hippocampal slices appropriate for patch clamp electrophysiology were prepared 30 minutes after the seizure. Whole-cell patch clamp recordings of passive and active membrane properties, EPSCs, IPSCS were made from GFP+ve and neighboring GFP-ve cells in the same slice. Results: A small number of GFP+veCA1 pyramidal neurons dispersed among GFP-ve neurons were visualized from hippocampal slices under fluorescent/DIC microscope. Passive membrane properties (resting membrane potential, membrane time constant and membrane resistance) of GFP +ve and neighboring GFP-ve were similar (16 pairs, from 16 animals). Although action potential threshold, amplitude, and width were similar, 11/20 GFP +ve cells fired >1 action potential in response to depolarizing current steps, only 3/20 GFP –ve  (p +ve and neighboring GFP-ve neurons. To evaluate AMPA transmission onto those GFP+ve neurons, miniature EPSCS were recorded after blocking GABA-A receptors (picrotoxin), NMDA receptors (APV) and action potentials (TTX) under voltage clamp. The amplitude of mEPSCs from GFP +ve neurons (19.37 mV +/- 1.88, mean +/- SD) was larger than that recorded from neighboring GFP –ve neurons (16.85 mV +/- 2.12) from the same slice and animal (P = 0.0002, paired t-test for 15 pairs). We also evaluated spontaneous EPSCs (sEPSCs) without applying TTX. Both amplitude and frequency of sEPSCs from GFP+ve cells were larger than neighboring GFP-ve cells from the same slices (recorded from 13 pairs, mean +/- SD. amplitude: 18.63 mV +/- 2.21 vs. 14.47 mV +/- 2.45, P < 0.0001; frequency: 1.84 Hz +/- 2.23 vs. 0.40 Hz +/- 0.28, P < 0.05). To investigate whether AMPAR properties were altered during seizure-induced enhancement of transmission, we investigated rectification of evoked EPSCs. There was inward rectification of eEPSCs recorded from GFP+ve neurons, which was not observed in GFP-ve neurons. To investigate the balance between excitatory and inhibitory synaptic inputs, miniature IPSCs were recorded from 5 pair GFP+ve neurons and surrounding GFP-ve neurons (within 100 µm distance) after blocking AMPA transmission with DNQX and APV, blocking action potential with TTX. Both amplitude and frequency of mIPSCs from GFP+ve neurons were less than that in surrounding  GFP-ve neurons (mean +/- SD, amplitude: 24.68 mV +/- 2.20 vs. 38.05 mV +/- 5.45, P < 0.05; frequency: 0.57 Hz +/- 0.16 vs. 1.89 Hz +/- 0.53, P < 0.05). Morphological study of the neurons performed by 3D reconstruction of biocytin filled neurons is in progress. Conclusions: These studies suggest that a single seizure potentiates AMPAR-mediated transmission, alters excitability and diminishes GABA-A receptor mediated  inhibition in a small number of CA1 pyramidal neurons, identifiable by the activation of cfos gene. Seizures cause calcium entry into neurons, which entry could also trigger plasticity of synaptic transmission,altered  excitability and expression of cfos gene and protein.   Funding: NIH NINDS, RO1 NS044370 NIH-NINDS, RO1 NS040337