Abstracts

Rationale: We have reported that adult rats have enhanced mGluR mediated long-term depression (mLTD) following a single early life seizure (ELS). This enhanced mLTD is at least partially due to alterations in the FMRP/PP2A/S6K complex. It is still unknown how changes in other mLTD related signalling may impact mLTD. Herein we further explore alterations in mLTD following ELS using selective inhibitors of S6 Kinase (S6K), proteasome degradation, type 2 voltage gated calcium channels, and CaMKII . Using these methods we also gain crucial insight into the mechanisms of mLTD in the normal (control) animal. We hypothesized that S6K, CaMKII, voltage gated calcium channels (type II) and proteasome mediated degradation are all crucial to the expression of mLTD. Furthermore we hypothesize that these processes are altered following ELS.Methods: ELS was induced in rats at P7 with kainate (2 mg/kg, s.c.). Hippocampal slices were prepared at P60+. Induction paradigms designed to isolate mLTD were utilized in area CA1 of the hippocampus and LTD was measured using field excitatory postsynaptic potentials . Pf-4708671 (25 M) was used to probe the dependence of mLTD on S6K signaling. MG-132 (10 M) was used to probe the dependence of mLTD on proteasome mediated protein degradation. Isradipine (10 M) was used probe role of type II voltage gated calcium channels in mediating mLTD. TatCN21 was used to assess the involvement of CaMKII (2 M) in mediating mLTD. Mediators of the observed changes in mLTD were assessed using semi-quantitative western blot expression assays of subcellular fractionated and whole CA1 homogenates.Results: Blocking S6K with PF-4708671 completely blocked mLTD in control slices, while having no significant effect in ELS slices. Similarly, MG-132 blocked mLTD in controls, but had no significant effect in ELS slices. Both Isradipine and tatCN21 completely blocked mLTD in control slices and produced a slight reduction of mLTD in ELS tissue. Expression of total CaMKII was not changed in ELS animals, compared to controls. However phosphorylated (Threonine 286) CaMKII was significantly elevated in ELS tissue, specifically in the synaptic (membrane bound) fraction. Conclusions: ELS results in multiple long-term signaling changes resulting in enhanced mLTD. Following ELS, mLTD is no longer dependent on S6K function and proteasome mediated degradation. ELS also reduces the requirement of CAMKII and type II voltage gated calcium channels in mediating mLTD. We speculate that these changes in mLTD may underlie the phenotype we observe following ELS resembling intellectual disability and autism. The results reported herein also identify potential targets for therapeutic intervention.