Short-term CaMKII inhibition with tatCN19o does not erase pre-formed memory in mice and is neuroprotective in pigs.

The Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a central regulator of learning and memory, which poses a problem for targeting it therapeutically. Indeed, our study supports prior conclusions that long-term interference with CaMKII signaling can erase pre-formed memories. By contrast, short-term pharmacological CaMKII inhibition with the neuroprotective peptide tatCN19o interfered with learning in mice only mildly and transiently (for less than 1 h) and did not at all reverse pre-formed memories. These results were obtained with ≥500-fold of the dose that protected hippocampal neurons from cell death after a highly clinically relevant pig model of transient global cerebral ischemia: ventricular fibrillation followed by advanced life support and electrical defibrillation to induce the return of spontaneous circulation. Of additional importance for therapy development, our preliminary cardiovascular safety studies in mice and pig did not indicate any concerns with acute tatCN19o injection. Taken together, although prolonged interference with CaMKII signaling can erase memory, acute short-term CaMKII inhibition with tatCN19o did not cause such retrograde amnesia that would pose a contraindication for therapy.

Activity-dependent regulation of synaptic strength by PSD-95 in CA1 neurons.

CaMKII and PSD-95 are the two most abundant postsynaptic proteins in the postsynaptic density (PSD). Overexpression of either can dramatically increase synaptic strength and saturate long-term potentiation (LTP). To do so, CaMKII must be activated, but the same is not true for PSD-95; expressing wild-type PSD-95 is sufficient. This raises the question of whether PSD-95's effects are simply an equilibrium process [increasing the number of AMPA receptor (AMPAR) slots] or whether activity is somehow involved. To examine this question, we blocked activity in cultured hippocampal slices with TTX and found that the effects of PSD-95 overexpression were greatly reduced. We next studied the type of receptors involved. The effects of PSD-95 were prevented by antagonists of group I metabotropic glutamate receptors (mGluRs) but not by antagonists of ionotropic glutamate receptors. The inhibition of PSD-95-induced strengthening was not simply a result of inhibition of PSD-95 synthesis. To understand the mechanisms involved, we tested the role of CaMKII. Overexpression of a CaMKII inhibitor, CN19, greatly reduced the effect of PSD-95. We conclude that PSD-95 cannot itself increase synaptic strength simply by increasing the number of AMPAR slots; rather, PSD-95's effects on synaptic strength require an activity-dependent process involving mGluR and CaMKII.

Reversal of synaptic memory by Ca2+/calmodulin-dependent protein kinase II inhibitor.

Long-term potentiation (LTP) is an activity-dependent strengthening of synapses that is thought to underlie memory storage. Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been a leading candidate as a memory molecule because it is persistently activated after LTP induction and can enhance transmission. Furthermore, a mutation that blocks persistent activation blocks LTP and forms of learning. However, direct evidence for a role of the kinase in maintaining synaptic strength has been lacking. Here, we show that a newly developed noncompetitive inhibitor of CaMKII strongly reduces synaptic transmission in the CA1 region of the hippocampal slice. This occurs through both presynaptic and postsynaptic action. To study the role of CaMKII in the maintenance of LTP, inhibitor was applied after LTP induction and then removed. Inhibition occurred in both LTP and control pathways but only partially recovered. The nonrecovering component was attributable primarily to a postsynaptic change. To test whether nonrecovery was attributable to a persistent reversal of LTP, we first saturated LTP and then transiently applied inhibitor. This procedure allowed additional LTP to be induced, indicating a reversal of an LTP maintenance mechanism. This is the first procedure that can reverse LTP by chemical means and suggests that a component of synaptic memory is attributable to CaMKII. The procedure also enhanced the LTP that could be induced in the control pathway, consistent with the idea that CaMKII is involved in controlling basal synaptic strength, perhaps as a result of LTP that occurred in vivo.

Testing the role of calmodulin in the excitation of Limulus photoreceptors.

The phototransduction cascade in Limulus ventral photoreceptors involves multiple second messengers, including Ca(2+) and cGMP. Light-induced Ca(2+) release from intracellular stores is an intermediate step, but the subsequent Ca(2+)-activated reaction remains to be determined. The possibility that Ca(2+)/calmodulin (Ca(2+)/CaM) might be involved is suggested by the high calmodulin content of the transducing lobe. To test whether CaM can excite the transduction cascade we injected a 25 microM Ca(2+)/CaM solution. This produced a rapid, brief depolarization similar to that produced by light, suggesting a role for CaM in the cascade. However, an important caveat is that Ca(2+) dissociating from the Ca(2+)/CaM complex might excite this process. Several control experiments argue against, but do not entirely eliminate this possibility. To test whether endogenous CaM has a function in excitation, trifluoperazine was pressure injected into the rhabdomeric region. The response to brief flashes was not affected, but the response to steady illumination was transiently attenuated by each injection. We conclude that calmodulin should be considered a candidate to couple intermediate and late stages of the transduction cascade.

The excitation cascade of Limulus ventral photoreceptors: guanylate cyclase as the link between InsP3-mediated Ca2+ release and the opening of cGMP-gated channels.

BACKGROUND: Early stages in the excitation cascade of Limulus photoreceptors are mediated by activation of Gq by rhodopsin, generation of inositol-1,4,5-trisphosphate by phospholipase-C and the release of Ca2+. At the end of the cascade, cGMP-gated channels open and generate the depolarizing receptor potential. A major unresolved issue is the intermediate process by which Ca2+ elevation leads to channel opening. RESULTS: To explore the role of guanylate cyclase (GC) as a potential intermediate, we used the GC inhibitor guanosine 5'-tetraphosphate (GtetP). Its specificity in vivo was supported by its ability to reduce the depolarization produced by the phosphodiesterase inhibitor IBMX. To determine if GC acts subsequent to InsP3 production in the cascade, we examined the effect of intracellular injection of GtetP on the excitation caused by InsP3 injection. This form of excitation and the response to light were both greatly reduced by GtetP, and they recovered in parallel. Similarly, GtetP reduced the excitation caused by intracellular injection of Ca2+. In contrast, this GC inhibitor did not affect the excitation produced by injection of a cGMP analog. CONCLUSION: We conclude that GC is downstream of InsP3-induced Ca2+ release and is the final enzymatic step of the excitation cascade. This is the first invertebrate rhabdomeric photoreceptor for which transduction can be traced from rhodopsin photoisomerization to ion channel opening.

Postsynaptic application of a cAMP analogue reverses long-term potentiation in hippocampal CA1 pyramidal neurons.

The molecular mechanisms that underlie the maintenance of long-term potentiation (LTP) remain unclear. We have examined the influence of postsynaptic cAMP-dependent processes on LTP maintenance in CA1 hippocampal cells. After LTP induction, drugs affecting cAMP-dependent processes were perfused into the cell through a patch pipette. A cAMP analogue, Rp-cAMPS (4 mM), dramatically decreased the amplitude of potentiated synaptic responses. The amplitude of responses in the control pathway was also decreased but to a lesser extent, indicating a specific effect on the potentiation process. This specific effect was not due to the larger amplitude of potentiated responses, was not use-dependent and, unlike other factors that affect LTP maintenance, did not depend on the delay (2, 10, or 25 min) of drug application after LTP induction. Lower concentrations of Rp-cAMPS (1.0 and 0.4 mM) also produced an inhibitory effect but reduced the LTP and control pathways comparably. One possible action of Rp-cAMPS is competitive inhibition of protein kinase A (PKA). Surprisingly, a potent and noncompetitive PKA inhibitor, regulatory type II subunit of PKA, produced only a weak depression of potentiated and control responses indicating there must be other targets for Rp-cAMPS. Moreover, Sp-8-OH-cAMPS, which is an activator of PKA, and Rp-8-OH-cAMPS, which is a weak inhibitor of PKA, both produced effects similar to those of Rp-cAMPS. We conclude that there are postsynaptic cyclic nucleotide-dependent processes that can specifically alter the mechanisms that maintain LTP and that are not primarily dependent on PKA.

Simultaneous roles for Ca2+ in excitation and adaptation of Limulus ventral photoreceptors.

The ventral photoreceptors of Limulus have been one of the main preparations for the study of invertebrate phototransduction. The study of ventral photoreceptors has revealed that they have remarkable performance characteristics, most notably the very large amplification of the transduction process. This amplification is critically dependent upon the coupling of photoactivated rhodopsin to the phosphoinositide cascade, resulting in the release of Ca2+ from intracellular stores. The consequent elevation of Ca2+ within the photoreceptor's cytosol is amongst the most rapid and dramatic known to be activated by the phosphoinositide cascade. This review summarizes the evidence that intracellular Ca2+ is a key regulator of transduction in Limulus photoreceptors. The mechanisms that regulate Ca2+ as well as the possible targets of the action of Ca2+ are reviewed. Ca2+ elevation is critical for triggering both excitation and adaptation processes in the photoreceptor. The question of how a single second messenger can produce these two opposing effects is of obvious interest and is a topic dealt with throughout this review.