SYNAPTIC CONNECTIVITY IN CULTURED HYPOTHALAMIC NEURONAL NETWORKS

We have developed a novel approach to analyze the synaptic connectivit y of spontaneously active networks of hypothalamic neurons in culture. Synaptic connections were identified by recording simultaneously from pairs of neurons using the whole cell configuration of the patch-clam p technique and testing for evoked postsynaptic current responses to e lectrical stimulation of one of the neurons. Excitatory and inhibitory responses were distinguished on the basis of their voltage and time d ependence. The distribution of latencies between presynaptic stimulati on and postsynaptic response showed multiple peaks at regular interval s, suggesting that responses via both monosynaptic and polysynaptic pa ths were recorded. The probability that an excitatory event is transmi tted to another excitatory neuron and results in an above-threshold st imulation was found to be only one in thr ee to four. This low value i ndicates that In addition to evoked synaptic responses other sources o f excitatory drive must contribute to the spontaneous activity observe d in these networks. The Various types of synaptic connections (excita tory and Inhibitory, monosynaptic. and polysynaptic) were counted, and the observations analyzed using a probabilistic model of the network structure. This analysis provides estimates for the ratio of inhibitor y to excitatory neurons in the network (1:1.5) and for the ratio of po stsynaptic cells receiving input from a single GABAergic or glutamater gic neuron (3:1). The total number of inhibitory synaptic connections was twice that of excitatory connections. Cell pairs mutually connecte d by an excitatory and an inhibitory synapse occurred significantly mo re often than predicted by a random process. These results suggests th at the formation of neuronal networks in vitro is controlled by cellul ar mechanisms that favor inhibitory connections in general and specifi cally enhance the formation of reciprocal connections between pairs of excitatory and inhibitory neurons. These mechanisms may contribute to network formation and function in vivo.