Tyrosine phosphorylation during synapse formation between identified leech neurons.

Abstract

1. We have examined whether tyrosine phosphorylation is required for synapse formation between identified neurons from the central nervous system of the leech in culture. 2. Within a few hours of contact with the cell body of the serotonergic Retzius neuron (R cell), the soma of the postsynaptic pressure-sensitive neuron (P cell), but not the R cell, could be labelled intracellularly with an antibody against phosphotyrosine residues. The labelling seemed specific for P cells contacted by R cells, as it was greatly reduced in pairs of either R or P cells and in single cells. Genistein (20,C4M) and lavendustin A (10 /sM), selective inhibitors of tyrosine kinases, blocked the labelling of contacted P cells, whereas their ineffective analogues (genistin and lavendustin B) had no effect on labelling. 3. R cell contact also induced the loss of an extrasynaptic, depolarizing response (due to modulation of cation channels) to serotonin (5-HT) in the P cell within a few days of juxtaposing cell bodies and within an hour of contact with growth cones. Treatment of the neurons with the tyrosine kinase inhibitors (but not the ineffective analogues) prevented the loss of the depolarizing response and of single cation channel modulation by 5-HT. 4. R cells formed inhibitory, CF-dependent synapses with P cells. Synapse formation was prevented by the tyrosine kinase inhibitors but not by their ineffective analogues. These compounds had no obvious effect on neurite outgrowth or cell adhesion. We conclude that tyrosine phosphorylation is a signal during the formation of this synapse.

In VitroSynaptogenesis between the Somata of IdentifiedLymnaeaNeurons Requires Protein Synthesis But Not Extrinsic Growth Factors or Substrate Adhesion Molecules

“…Various researchers interested in synapse formation have resorted, therefore, to simple cell culture techniques in which individually definable neurons can be extracted from the nervous system and studied in isolation under controlled experimental conditions (Dagan and Levitan, 1981;Bulloch and Syed, 1992;Haydon and Drapeau, 1995). Notwithstanding the fact that these cell culture conditions are artificial and may not reflect those that occur naturally, these studies have provided insights into cellular mechanisms that are fundamental to synapse formation (Chow and Poo, 1985;Bulloch and Syed, 1992;Hawver and Schacher, 1993;Catarsi et al, 1995;Haydon and Drapeau, 1995;Mennerick et al, 1995).…”

Channel modulation by tyrosine phosphorylation in an identified leech neuron.

“…the P cell patch was at its resting potential of about - To investigate whether the cationic channel is regulated by endogenous tyrosine kinases in P cells, we examined the effect of inhibiting tyrosine kinases as this should result in a progressive dephosphorylation of the substrate by endogenous phosphatases. P cells were exposed to genistein, an inhibitor of tyrosine kinases ( phosphorylation in P cells (Catarsi et al 1995). After a 3-5 min control period, 30 uM genistein was applied, resulting in a progressive increase in the probability of opening of the channel (PO) reaching its maximal effect 2-3 min after the drug application ( Fig.…”

Synapse formation and function: Insights from identified leech neurons in culture

“…Interestingly, the cytoplasm of contacted P cells (but not Retzius cells) is immunoreactive for tyrosine phosphorylation and exposure of the neurons to specific inhibitors of tyrosine kinases prevents the loss of cation channel modulation [ Fig. 8(D)] and synapse formation (Catarsi et al, 1995). These results suggest that tyrosine phosphorylation of an unknown target protein is an important signal during the formation of this synapse.…”