Long-term blockade of serotonin reuptake affects synaptotagmin phosphorylation in the hippocampus.

Synaptic vesicle trafficking and transmitter release from presynaptic terminals are precisely regulated by a complex array of protein/protein interactions. Several of these proteins are substrates of endogenous protein kinases present in presynaptic terminals. The activity of Ca2+/calmodulin-dependent protein kinase II(CaMKII), one of the kinases involved in the modulation of transmitter release, was previously shown to increase in the hippocampus after long-term blockade of 5-hydroxytryptamine (5-HT) reuptake (a treatment known to elicit an increase in 5-HT release in this area). To investigate the changes induced in presynaptic protein phosphorylation by 5-HT reuptake blockade and concomitant CaMKII up-regulation, we analyzed two major CaMKII presynaptic substrates (synapsin I and synaptotagmin). All 5-HT reuptake blockers that we used, which induce an increase in CaMKII activity and autophosphorylation, also caused a large (2-3-fold) increase in the Ca2+/calmodulin-dependent post hoc phosphorylation of synaptotagmin. Conversely, the phosphorylation of synapsin I is much less affected. The change in synaptotagmin phosphorylation, as determined through immunoprecipitation and quantitative immunoblot analysis after fluvoxamine treatment, is due exclusively to increased phosphate incorporation (presumably caused by the increased kinase activity) and not to a change in the level of substrate protein after the treatment. Thus, drugs known to induce an increase in 5-HT release simultaneously induce an increase in the activity of presynaptic CaMKII and in the phosphate incorporation (post hoc) by a major CaMKII substrate in synaptic vesicles (synaptotagmin). This finding establishes a link between the facilitation of transmitter release induced by antidepressant drugs and the phosphorylation of synaptotagmin by CaMKII.

Presynaptic Ca2+/calmodulin-dependent protein kinase II: autophosphorylation and activity increase in the hippocampus after long-term blockade of serotonin reuptake.

It is known that long-term treatment with antidepressants induces an enhancement of neurotransmission in the pathway projecting from raphe nuclei to the hippocampus. In the case of selective serotonin (5-HT) reuptake inhibitors, this enhancement is due to a desensitization of presynaptic 5-HT autoreceptors and a concomitant increase in 5-HT release in terminal areas. To investigate whether this effect is accompanied by adaptive changes in the molecular machinery regulating transmitter release at serotonergic terminals, autophosphorylation and activity of Ca2+/calmodulin-dependent protein kinase II were measured in subsynaptosomal fractions from hippocampus and total cortex. Long-term treatment with two selective serotonin reuptake inhibitors (paroxetine and fluvoxamine) and with a nonselective reuptake inhibitor (venlafaxine) induces a large increase of kinase autophosphorylation in synaptic vesicles and synaptic cytosol in the hippocampus but not in synaptosomal membranes. No significant change was detected in total cortex. The change is not reproduced by the direct addition of the drugs to the phosphorylation system and is not elicited by acute treatment of the animals. The increase in autophosphorylation is not accounted for by neosynthesis or translocation of the kinase to synaptic terminals. The change is restricted to the kinase located inside the terminals and is not detected in synaptosomal membranes, containing predominantly postsynaptic kinase, suggesting that only presynaptic kinase is affected. In the same fractions, the kinase activity is increased. These results are in agreement with reports suggesting a presynaptic effect for the SSRIs and disclose a new putative site of action for psychotropic drugs.