Cellular and synaptic mechanisms underlying epileptiform activity in the kainic acid lesioned hippocampus of the rat

Simpson, Louise Helen (1991) Cellular and synaptic mechanisms underlying epileptiform activity in the kainic acid lesioned hippocampus of the rat. University of Southampton, Doctoral Thesis.

Abstract

Experiments were carried out on hippocampal slices from control rats and those pretreated with a unilateral intracerebroventricular injection of kainic acid (KA). The KA produced a lesion in the CA3/CA4 area of the ipsilateral hippocampus. There was no significant difference in resting membrane potential, input resistance or time constant between CA1 pyramidal cells from control and lesioned slices. However, in cells from KA lesioned hippocampal slices, the spike frequency acommodation usually seen during a depolarising current pulse (+0.5 nA for 1 s) was significantly reduced, as was the hyperpolarisation to follow a shorter current pulse (+0.5 nA for 100 ms). Just subthreshold stimulation in the stratum radiatum (SR) region of control slices led to an excitatory postsynaptic potential (EPSP) followed by a biphasic inhibitory postsynaptic potential (IPSP). The control EPSP 10-90% rise time (VRT), half width (VHW), time to peak (TPK) and amplitude (SPK) increased with hyperpolarisation from -60 to -85 mV. 6-Cyano-7-nitro-quinoxaline-2,3-dione (CNQX) (2-5 μM) blocked the control EPSP by between 85 and 100% indicating that it was mainly mediated by non-NMDA receptors. Just subthreshold stimulation of lesioned slices evoked a long lasting intracellular EPSP which decreased in VRT, VHW, TPK and SPK with hyperpolarisation. This EPSP was followed by a small, seemingly voltage independent, hyperpolarisation. Experiments using CNQX in conjunction with D-2-amino-5-phosphonovalerate (D-APV) and 7-Cl-kynurenate (7-Cl-KYN) showed that lesioned slice EPSPs were mediated by both NMDA and non-NMDA receptors cells. In control slices stimulation of pyramidal cells at levels above threshold for firing led to a single intracellular action potential and extracellular population spike. CNQX (2-5 μM) attenuated the majority of this response thus it was thought to be mainly mediated by non-NMDA receptor subtypes. Suprathreshold stimulation of lesioned slices triggered graded bursting activity in the form of between 2 and 7 intracellular action potentials/extracellular population spikes. A proportion of the intra- and extracellular burst was blocked by CNQX (2-5 μM) and therefore mediated by non-NMDA receptors. The CNQX insensitive component could be blocked by D-APV (10 μM) or 7-Cl-KYN (40 μM) indicating the involvement of both NMDA and non-NMDA receptor subtypes in the burst response. However, the relative contribution of EAA receptor subtypes to the burst varied between cells/slices. The block by 7-Cl-KYN showed that the NMDA receptor mediated component of the epileptiform burst response depends upon activation of the glycine site of the NMDA receptor. D-serine (200 μM), a strychnine insensitive glycine site agonist, partially reversed the 7-Cl-KYN block. D-serine (200 μM) was applied to the extracellular burst response but had no significant effect, and so exogenous glycine levels in the slice were thought to be sufficient to saturate the glycine site of the NMDA receptor channel complex. Two intracellular marking techniques using biocytin and 5,6-carboxyfluorescein were also studied to explore the feasibility of incorporating cell labelling into the normal routine of electrophysiological experients. Both methods provided high quality morphological data to complement the electrophysiological recordings.

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