Repetitive treatment with serotonin modifies protein synthesis and protein phosphorylation in the central nervous system of Hirudo medicinalis

Serotonin (5HT) is involved in the modulation of several behaviors of the leech . In addition application of 5HT mimics some elementary forms of non associative learning such as short term sensitization and dishabituation; moreover, seasonal variation of 5HT contenent are correlated with changes in learning capabilities (Catarsi et al 1990 J. Comp. Physiol.167A 469-474). 5HT mimics short term learning by activating cAMP dependent protein kinases. Behavioral experiments. Behavioral experiments have shown that long term dishabituation requires new protein synthesis, but the mechanism of transformation of short -term to long term ones is unknow. In order to study this issue, we have labeled leech segmental ganglia with 32 P ortophoshoric acid or 35 S methionine and we have mimicked short term learning by 5 min application of 50 uM 5HT after 2 hr labeling, while long term learning has been mimicked by repetitive stimulation (5 min 50 uM 5HT for 4 times, every 90 min for 24 hr total labeling incubation. labeled proteins have been separed by two dimensional electrophoresis and detected by autoradiography. A single 5 min treatment wit 5HT was found to produce a general increase of protein phosphorylation. In the repetitive treatment experiments, in which samples were solubilized 18 after the last 5 min incubation with 5HT it was found that group of spots exhibited an increase in 32P incorporation (molecular weights 174, 105, 79, 74, 71 and 37 kDa while spots with molecular weight of 52, 52, 34 and 32 exhibited a decrease of label. Therefore repetitive treatment with 5HT result in persistence of the phosphorylation of some proteins. In addition, repetitive 5HT stimulationmodified 25S L methionine incorportion into several proteins: increasein spot of 91, 54, 30, 29, 25 and 20 kDa and a decrease in spots of 47, 36 and 30 kDa. in order to know whether protein phoshoryltion is necessary to trigger protein synthesis following 5HT application, segmental ganglia were incubate in the presence of both 5HT and such an inhibitor staurosporine; the presence of such inhibitor resulted in a 20-50% inhibition of 35 S incorporation into proteins. these reults suggest that repetitive treatments with 5HT induces chnges of protein synthesis and that this process mediated by protein phosphoyilation.