Abstracts

Rationale: Abnormal neuronal activity during development has been shown to alter important aspects of neuronal maturation. Our laboratory is interested in understanding the effects of network hyperactivity upon the developing hippocampus. Using cultures of hippocampal slices, we have previously shown that with time in culture dendrites of CA1 pyramidal neurons increase in length and branching complexity. In addition, synaptic proteins such as NR2A and PSD95 concurrently increase in expression. When chronic (4-7 days), spontaneous network bursting is induced in sister slices via the GABAa receptor antagonist bicuculline methiodide the expression of the postsynaptic proteins was reduced and the CA1 basilar dendrites failed to grow. These results motivated us to examine the time-course of these effects and attempt to identify the underlying molecular mechanisms. Methods: We utilized hippocampal slices prepared from postnatal day 4 rats and maintained in vitro for up to ten days. Quantitative immunoblotting was done to examine changes in the expression of post-synaptic proteins. Recordings of extracellular field potentials were undertaken to assay changes in network activity. Morphological studies of dendrites were performed in rat slices following gene-gun transfection of GFP vector or in slices from Thy1-GFP-M mice. CA1 hippocampal pyramidal cells were confocally imaged and the basilar dendrites were reconstructed using Neurolucida.Results: We were surprised to find that after only four hours of treatment there was a 40% and 70% reduction in the protein concentration of PSD95 and NR2A respectively. Additionally, when slices were exposed to epileptiform activity for one hour, there was a 20% reduction in burst duration compared to slices acutely treated with bicuculline. More prolonged network bursting further reduced the burst phenotype to 58%. Four hours of epileptiform activity also produced a 26% and 16% decrease in the number of branch points and total dendritic length of the basilar dendrites of CA1 pyramidal neurons compared to control slices. An emerging mediator of activity-dependent plasticity as well as dendrite growth is calcineurin. When slices were pretreated with the calcineurin antagonist FK506 for one hour prior to bursting, the mean burst duration was only reduced by 20.4% compared to slices not treated with FK506 (69%). Similarly, suppression of calcineurin activity blocked bicuculline-induced reductions in dendritic arborization both in total dendrite length and number of branch points. Moreover, calcineurin inhibitors completely suppressed effects on the glutamatergic synaptic markers NR2A, NR1 and the PSD95. Conclusions: These results suggest that short time treatment (4 hours) of bicuculline reduces dendritic arborization, the expression of post-synaptic proteins and the duration network bursting of CA1 pyramidal neurons. These apparent compensatory responses of the hippocampus to prolonged network hyperexcitability appear to be mediated by calcineurin-dependent signaling mechanisms.