Adenylyl cyclase-dependent form of chemical long-term potentiation triggers translational regulation at the elongation step.

The persistent maintenance of long-term potentiation requires both messenger RNA and protein synthesis. While there is mounting evidence for an active role of protein synthesis in hippocampal long-term potentiation, the nature of mechanisms underlying its regulation has not yet been established. We used a previously described chemical long-term potentiation protocol [J Neurosci 19 (1999) 2500] to address the hypothesis that signaling mechanisms, involved in long-lasting long-term potentiation, directly regulate protein synthesis. Chemical long-term potentiation is an N-methyl-D-aspartate receptor-dependent form of plasticity, which relies on both synaptic activity, in the form of spontaneous bursting induced by high concentrations of K(+) and Ca(2+), and cyclic AMP/adenylyl cyclase signaling. We found that chemical long-term potentiation in CA1 of the mouse hippocampus lasts for at least 3 hours and requires both messenger RNA and protein synthesis. However, surprisingly de novo total protein synthesis was paradoxically decreased at 1 hour after long-term potentiation induction. Consistent with the decrease in total protein synthesis in potentiated CA1, phosphorylation of eukaryotic elongation factor 2 was increased and is likely responsible for inhibition of translation at the elongation step. Increased phosphorylation of eukaryotic elongation factor 2 was dependent on coincident cyclic AMP/adenylyl cyclase activation and synaptic activity and required N-methyl-D-aspartate receptor activation. Despite the inhibition in total protein synthesis, the level of the immediate early gene protein Arc (activity regulated cytoskeleton-associated protein) increased at 1 hour after chemical long-term potentiation induction. Taken together, the results suggest that regulation at the elongation step of protein synthesis contributes to persistent forms of long-term potentiation.

Adenylyl cyclase activation modulates activity-dependent changes in synaptic strength and Ca2+/calmodulin-dependent kinase II autophosphorylation.

Activation of the Ca2+- and calmodulin-dependent protein kinase II (CaMKII) and its conversion into a persistently activated form by autophosphorylation are thought to be crucial events underlying the induction of long-term potentiation (LTP) by increases in postsynaptic Ca2+. Because increases in Ca2+ can also activate protein phosphatases that oppose persistent CaMKII activation, LTP induction may also require activation of signaling pathways that suppress protein phosphatase activation. Because the adenylyl cyclase (AC)-protein kinase A signaling pathway may provide a mechanism for suppressing protein phosphatase activation, we investigated the effects of AC activators on activity-dependent changes in synaptic strength and on levels of autophosphorylated alphaCaMKII (Thr286). In the CA1 region of hippocampal slices, briefly elevating extracellular Ca2+ induced an activity-dependent, transient potentiation of synaptic transmission that could be converted into a persistent potentiation by the addition of phosphatase inhibitors or AC activators. To examine activity-dependent changes in alphaCaMKII autophosphorylation, we replaced electrical presynaptic fiber stimulation with an increase in extracellular K+ to achieve a more global synaptic activation during perfusion of high Ca2+ solutions. In the presence of the AC activator forskolin or the protein phosphatase inhibitor calyculin A, this treatment induced a LTP-like synaptic potentiation and a persistent increase in autophosphorylated alphaCaMKII levels. In the absence of forskolin or calyculin A, it had no lasting effect on synaptic strength and induced a persistent decrease in autophosphorylated alphaCaMKII levels. Our results suggest that AC activation facilitates LTP induction by suppressing protein phosphatases and enabling a persistent increase in the levels of autophosphorylated CaMKII.

Isolation and characterization of synaptoneurosomes from single rat hippocampal slices.

A technique for recovering functional synaptoneurosomes containing vesicularized elements of the presynapse and postsynapse into an enriched fraction has been modified to allow for small amounts of starting brain tissue. Single 400 microm rat hippocampal slices were homogenized and sequentially filtered in a 1 cc tuberculin syringe to produce an enriched synaptoneurosome fraction. Data from Western immunoblots for specific synaptic proteins suggest that these fractions are neurochemically similar to synaptosome fractions generated by sucrose gradients. Electron micrographs show that the 'small scale' preparations contain an abundant population of fused presynaptic and postsynaptic vesicularized bodies as previously published for synaptoneurosome fractions prepared from relatively large amounts of starting tissue. The single slice synaptoneurosome preparation is a quick, easy and reliable method for use in the study of synaptic function.