Postsynaptic Inhibitors of Calcium/Calmodulin-Dependent Protein Kinase Type II Block Induction But Not Maintenance of Pairing-Induced Long-Term Potentiation

Abstract

The role of postsynaptic kinases in the induction and maintenance of long-term potentiation (LTP) was studied in the CA1 region of the rat hippocampal slice. A peptide inhibitor for the catalytic domain of calcium/calmodulin-dependent protein kinase type II (CaM-kinase) was applied through a perfused patch pipette. The inhibitor completely blocked both the shortterm potentiation and LTP induced by a pairing protocol. This indicates that the kinase or kinases affected by the peptide are downstream from depolarization in the LTP cascade. The ability to block LTP required that measures be taken to interfere with degradation of the peptide kinase inhibitor by endogenous proteases; either addition of protease inhibitors or modifications of the peptide itself greatly enhanced the effectiveness of the peptide. Protease inhibitors by themselves or control peptide did not block LTP induction. To study the effect of kinase inhibitor on LTP maintenance, we induced LTP in one pathway. Subsequent introduction of the kinase inhibitor blocked the induction of LTP in a second pathway, but it did not affect maintenance of LTP in the first. The implications for the role of kinases in LTP maintenance are discussed. Key words: long-term potentiation; calcium/calmodulindependent kinase; peptide inhibitors; hippocampal slices; whole-cell recording; intracellular perfusion; protease inhibitors; fluorescent imagingLong-term potentiation (LTP) in the CA1 region of the hippocampus is the best-studied example of the activity-dependent synaptic modifications that may underlie learning and memory (Bliss and Collingridge, 1993;Malenka and Nicoll, 1993) There is now considerable biochemical, physiological, and genetic evidence for the involvement of protein kinases in LTP induction (Colley and Routtenberg, 1993;Suzuki, 1994;Roberson et al., 1996). Some experiments have shown specifically that blocking different postsynaptic kinases is sufficient to block LTP induction (Malenka et al., 1989;Malinow et al., 1989;O'Dell et al., 1991;Wang and Feng, 1992;Hvalby et al., 1994;Blitzer et al., 1995;Feng, 1995;Wang and Kelly, 1996). Furthermore, introduction of protein kinase C (C -kinase) or calcium /calmodulin-dependent protein kinase type II (C aM-kinase) (Hu et al., 1987; McGladeMcCulloh et al., 1993;L ledo et al., 1995) or their activators (Wang and Kelly, 1995) into the postsynaptic cell results in synaptic potentiation that may occlude tetanus-induced LTP (Wang and Kelly, 1995).Although there is general agreement that postsynaptic kinases are involved in LTP induction, their exact role remains unclear. LTP is initiated by C a 2ϩ entry through the NMDA channels (Bliss and Collingridge, 1993), an entry that requires depolarization (Mayer et al., 1984;Nowak et al., 1984). The depolarization may be provided by dendritic action potentials (Jaffe et al., 1992;Magee and Johnston, 1997) and the EPSP, both of which depend on voltage-dependent channels Sakmann, 1994, 1995;Andreasen and Lambert, 1995), which themselves may be regulated by phosphorylation ...

A large sustained Ca2+ elevation occurs in unstimulated spines during the LTP pairing protocol but does not change synaptic strength

“…Acute hippocampal slices were prepared from 17-21-day-old Long-Evans rats according to the methods described by Otmakhov et al(1997). Rats were decapitated under anesthesia (5% urethane).…”

“…To investigate possible other effects of D-890 that might impair LTP, we tested the effect of the drug on a molecule that is known to be required for pairing-induced LTP induction, CaMKII (Otmakhov et al, 1997). These tests were performed by the laboratory of Thomas Soderling and showed that the drug inhibited CaMKII activity in vitro.…”

“…7). When using CaMKII inhibitors, Otmakhov et al (1997) had to use a concentration in the intracellular electrode 1,000-fold higher than the IC 50 measured in vitro to obtain significant effects on LTP induction, probably because dendritic concentration is much lower than the electrode concentration. The actual concentration of D-890 at the synapse is probably much lower than 1 mM.…”

“…Unfortunately the interpretation of this effect was made unclear by a subsequent control experiment showing that D-890 inhibits CaMKII activity in vitro. Since CaMKII is required for LTP induction (Otmakhov et al, 1997), it is possible that the inhibition of LTP by D-890 resulted from an inhibition of the kinase rather than inhibition of VDCC. However, given the fact that the concentration of D-890 was equal to IC 50 , whereas with other CaMKII inhibitor levels much higher than IC 50 are required to inhibit LTP, probably because of diffusion gradients (Otmakhov et al, 1997), it remains possible that D-890 in fact inhibited LTP as a result of inhibiting VDCC.…”

Propagation of CaMKII translocation waves in heterogeneous spiny dendrites

“…Second, once activated, CaMKII can transition into a Ca 2+ /CaM-independent, hyper-activated state via the autophosphorylation of neighboring enzymatic subunits (Hanson et al 1994; Rich and Schulman 1998), and thus continue to phosphorylate its substrates even after the plasticity-inducing Ca 2+ signal has ended (Saitoh and Schwartz 1985;Miller and Kenney 1986;Lou et al 1986;Yang and Schulman 1999). Third, suppression of CaM-KII using chemical antagonists blocks all known forms of NMDA receptor-dependent long-term potentiation (LTP) (Malinow et al 1989;Fukunaga et al 1993;Pettit et al 1994;Lledo 1995;Barria et al 1997b;Otmakhov et al 1997;Malenka and Bear 2004).…”