Functional synapses in synchronized bursting of neocortical neurons in culture

Spontaneous electrical activities in pairs of neocortical neurons in culture were simultaneously recorded using a whole cell current clamp technique. Synchronous bursting activities were observed in all 59 pairs tested. In 52 pairs of neurons electrically stimulated, EPSPs were recorded in 20 pairs (39%), among which 3 pairs (6%) showed bidirectional coupling. The response latency observed was 4. 05+/-0.61 ms (mean+/-S.E.M.). The synaptic delay was estimated at 1. 5-1.9 ms, suggesting the response latency is derived from a polysynaptic connection. The burst latency which was defined as the time difference of the onset of bursting in each neuron was 5.87+/-0. 47 ms (mean+/-S.E.M.), and was weakly correlated with the spatial distance between the neurons (37.5-600 micro(m) apart) (Rs=0.362, tied P value=0.0065). No synchronized bursting was observed in bathing solution with a low Ca2+ concentration (0.4 mM) or in bathing solution containing 50 microM D-AP5 and 15 microM CNQX. No dye-coupling between bursting neurons was observed on injection of the small molecule dye Lucifer yellow or the neurotracer neurobiotin. Disrupting neural connections completely by cutting the cell layer, caused disappearance of synchronized bursting with each neuron bursting independently. In conclusion, these results are consistent with the hypothesis that synchronized bursting in cultured neocortical neurons is attributed to connections by way of several synapses rather than by way of gap junctions and/or diffusible factors.