THE FORMATION OF GLUTAMATERGIC SYNAPSES IN CULTURED CENTRAL NEURONS -SELECTIVE INCREASE IN MINIATURE SYNAPTIC CURRENTS

The formation of synapses between cultured rat thalamic neurons was st udied with electrophysiological and immunocytochemical methods. Thalam ic neurons in culture form predominantly glutamatergic synapses. Alrea dy after 3 days in vitro glutamatergic miniature EPSCs occurred sponta neously and their frequency was strongly increased after KC depolariza tion, while GABAergic mIPSCs were found after K+ depolarization at low er frequency. This demonstrates that both, excitatory glutamatergic an d inhibitory GABAergic synapses were functional in close succession to initial neurite outgrowth. Synapses formed independent of spontaneous electrical activity, which was absent during the first week in cultur e. Spontaneous action potentials appeared during the second week and c hronic action potential blockade by addition of tetrodotoxin reduced n euronal survival and the number of glutamatergic synapses per neuron. During in vitro differentiation the number of synapsin I immunoreactiv e presynaptic terminals and the frequency of spontaneous glutamatergic miniature EPSCs increased closely correlated, while the frequency of GABAergic mIPSCs after K+ depolarization did not increase. Thus, the c ontinous formation of presynaptic terminals, including possible matura tion of transmitter release, appeared to underlie the increase in mEPS C frequency. Analysis of miniature EPSC amplitudes at different stages in vitro revealed an increase in amplitudes, suggesting synaptic diff erentiation after initial establishment of functional transmission in glutamatergic synapses. This process was synapse specific as amplitude s of GABAergic mIPSCs were invariant.