This project deploys a range of structural techniques to examine normal synaptic plasticity and development. These approaches have in common their dependence on rapid-freezing and direct visualization of living brain by light microscopic techniques. This project has been engaged in exploring various live brain preparations suitable for these purposes. Success has been achieved with a new approach to culturing hippocampal slices. Their typical laminar organization and most of their original thickness can be maintained for up to 12 weeks by culturing them at the interface between air and culture medium. After ten days in culture CA1 dendrites have almost no spines, but have large, filopodia-like structures containing smooth endoplasmic reticulum. Extracellular field EPSP (fEPSP) amplitude was 3.5 + 1.1 mV and tetanic stimulation (100Hz, 1sec) caused no long-term potentiation (LTP). At 3 weeks, spines were present but very heterogeneous, and fEPSP amplitude was 5.6 + 0.9. Thus a significant increase in fEPSP amplitude and ability to produce LTP occurred in second week, so the ability to express LTP was correlated with the appearance of the spines. Spine density increased in CA1 neurons in the cultures on a schedule similar to that of in vivo hippocampus. Chronic exposure of slices to drugs which interact with synaptic activity caused changes in dendritic spine density. Blockade of NMDA receptors with 2-APV, or blockade of action potential discharges with tetrodotoxin (TTX) prevented dendritic spine development. Enhancing synaptic activity by blockade of inhibition with picrotoxin did not affect spine density to a significant degree. Expression of glutamate receptors in slices were examined with RT-PCR and immunoblotting methods. Their expression increased in the course of culture period similar to in vivo hippocampus. When the slices were chronically exposed to TTX, expression of NMDA receptors was suppressed.
