CaMKII inhibitor 1 (CaMK2N1) mRNA is upregulated following LTP induction in hippocampal slices.

CaMK2N1 and CaMK2N2 (also known as CaMKIINα and ß) are endogenous inhibitors of calcium/calmodulin-dependent kinase II (CaMKII), an enzyme critical for memory and long-term potentiation (LTP), a form of synaptic plasticity thought to underlie learning. CaMK2N1/2 mRNAs are rapidly and differentially upregulated in the hippocampus and amygdala after acquisition or retrieval of fear memory. Moreover, CaMK2N2 protein levels increase after contextual fear conditioning. Therefore, it was proposed that CaMK2N1/2 genes (Camk2n1/2) could be immediate-early genes transcribed promptly (30-60 min) after training. As a first approach to explore a role in synaptic plasticity, we assessed a possible regulation of Camk2n1/2 during the expression phase of LTP in hippocampal CA3-CA1 connections in rat brain slices. Quantitative PCR revealed that Camk2n1, but not Camk2n2, is upregulated 60 min after LTP induction by Schaffer collaterals high-frequency stimulation. We observed a graded, significant positive correlation between the magnitude of LTP and Camk2n1 change in individual slices, suggesting a coordinated regulation of these properties. If mRNA increment actually resulted in the protein upregulation in plasticity-relevant subcellular locations, CaMK2N1 may be involved in CaMKII fine-tuning during LTP maintenance or in the regulation of subsequent plasticity events (metaplasticity).

Fast Decay of CaMKII FRET Sensor Signal in Spines after LTP Induction Is Not Due to Its Dephosphorylation.

Because CaMKII is the critical Ca(2+) sensor that triggers long-term potentiation (LTP), understanding its activation and deactivation is important. A major advance has been the development of a FRET indicator of the conformational state of CaMKII called Camui. Experiments using Camui have demonstrated that the open (active) conformation increases during LTP induction and then decays in tens of seconds, with the major fast component decaying with a time-constant of ~ 6 sec (tau1). Because this decay is faster if autophosphorylation of T286 is prevented (the autophosphorylation prolongs activity by making the enzyme active even after Ca(2+) falls), it seemed likely that the fast decay is due to the T286 dephosphorylation. To test this interpretation, we studied the effect of phosphatase inhibitors on the single-spine Camui signal evoked by two-photon glutamate uncaging. We applied inhibitors of PP1 and PP2A, two phosphatases that are present at synapses and that have been shown to dephosphorylate CaMKII in vitro. The inhibitors increased the basal Camui activation state, indicating their effectiveness in cells. However, in no case did we find that tau1 was prolonged, contrary to what would be expected if the decay was phosphatase-dependent. This could either mean that decay was due to some unknown phosphatase or that the decay was not due to dephosphorylation. To distinguish between these possibilities, we expressed pseudo-phosphorylated Camui (T286D) (plus additional mutations [T/A] that prevented inhibitory 305/306 phosphorylation). This form had an elevated basal activation state, but was further activated during glutamate uncaging; importantly the activation state decayed with tau1 nearly the same as that of WT Camui. Therefore, the data strongly indicate that tau1 is not due to T286 dephosphorylation. We conclude that, although Camui is an excellent tool for observing CaMKII signaling, further experimentation is needed to determine how CaMKII is turned off by its dephosphorylation.

Excitotoxic insult results in a long-lasting activation of CaMKIIα and mitochondrial damage in living hippocampal neurons.

Over-activation of excitatory NMDA receptors and the resulting Ca2+ overload is the main cause of neuronal toxicity during stroke. CaMKII becomes misregulated during such events. Biochemical studies show either a dramatic loss of CaMKII activity or its persistent autonomous activation after stroke, with both of these processes being implicated in cell toxicity. To complement the biochemical data, we monitored CaMKII activation in living hippocampal neurons in slice cultures using high spatial/temporal resolution two-photon imaging of the CaMKIIα FRET sensor, Camui. CaMKII activation state was estimated by measuring Camui fluorescence lifetime. Short NMDA insult resulted in Camui activation followed by a redistribution of its protein localization: an increase in spines, a decrease in dendritic shafts, and concentration into numerous clusters in the cell soma. Camui activation was either persistent (> 1-3 hours) or transient (~20 min) and, in general, correlated with its protein redistribution. After longer NMDA insult, however, Camui redistribution persisted longer than its activation, suggesting distinct regulation/phases of these processes. Mutational and pharmacological analysis suggested that persistent Camui activation was due to prolonged Ca2+ elevation, with little impact of autonomous states produced by T286 autophosphorylation and/or by C280/M281 oxidation. Cell injury was monitored using expressible mitochondrial marker mito-dsRed. Shortly after Camui activation and clustering, NMDA treatment resulted in mitochondrial swelling, with persistence of the swelling temporarily linked to the persistence of Camui activation. The results suggest that in living neurons excitotoxic insult produces long-lasting Ca2+-dependent active state of CaMKII temporarily linked to cell injury. CaMKII function, however, is to be restricted due to strong clustering. The study provides the first characterization of CaMKII activation dynamics in living neurons during excitotoxic insults.

Measuring CaMKII concentration in dendritic spines.

Here, we present a method for measuring the concentration of endogenous protein in cellular compartments. Importantly, the method is applicable to compartments such as dendritic spines with dimensions often close to the resolution limit of optical microscopy. To our knowledge, a method with such capabilities has not yet been described. The method utilizes overexpression of the protein of interest, which is tagged with fluorescent protein. This is followed by immunostaining of both overexpressed and endogenous proteins. Expression of a volume marker is also required. We applied this method to measure the concentration of Ca/calmodulin kinase II (CaMKII) in different cellular compartments of hippocampal pyramidal neurons. It was found that the concentrations of CaMKIIα subunits in cell bodies, proximal dendrites, and spines on these dendrites are 71, 46, and 103 µM, respectively. Considering the 3:1 ratio of α to ß CaMKII subunits in the hippocampus, the concentrations of total (α+ß) CaMKII subunits in these compartments are 94, 61, and 138 µM, respectively.

Role of the CaMKII/NMDA receptor complex in the maintenance of synaptic strength.

During long-term potentiation (LTP), synapses undergo stable changes in synaptic strength. The molecular memory processes that maintain strength have not been identified. One hypothesis is that the complex formed by the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and the NMDA-type glutamate receptor (NMDAR) is a molecular memory at the synapse. To establish a molecule as a molecular memory, it must be shown that interfering with the molecule produces a persistent reversal of LTP. We used the CN class of peptides that inhibit CaMKII binding to the NR2B subunit in vitro to test this prediction in rat hippocampal slices. We found that CN peptides can reverse saturated LTP, allowing additional LTP to be induced. The peptide also produced a persistent reduction in basal transmission. We then tested whether CN compounds actually affect CaMKII binding in living cells. Application of CN peptide to slice cultures reduced the amount of CaMKII concentrated in spines, consistent with delocalization of the kinase from a binding partner in the spine. To more specifically assay the binding of CaMKII to the NMDAR, we used coimmunoprecipitation methods. We found that CN peptide decreased synaptic strength only at concentrations necessary to disrupt the CaMKII/NMDAR complex, but not at lower concentrations sufficient to inhibit CaMKII activity. Importantly, both the reduction of the complex and the reduction of synaptic strength persisted after removal of the inhibitor. These results support the hypothesis that the CaMKII/NMDAR complex has switch-like properties that are important in the maintenance of synaptic strength.

CaMKII control of spine size and synaptic strength: role of phosphorylation states and nonenzymatic action.

CaMKII is an abundant synaptic protein strongly implicated in plasticity. Overexpression of autonomous (T286D) CaMKII in CA1 hippocampal cells enhances synaptic strength if T305/T306 sites are not phosphorylated, but decreases synaptic strength if they are phosphorylated. It has generally been thought that spine size and synaptic strength covary; however, the ability of CaMKII and its various phosphorylation states to control spine size has not been previously examined. Using a unique method that allows the effects of overexpressed protein to be monitored over time, we found that all autonomous forms of CaMKII increase spine size. Thus, for instance, the T286D/T305D/T306D form increases spine size but decreases synaptic strength. Further evidence for such dissociation is provided by experiments with the T286D form that has been made catalytically dead. This form fails to enhance synaptic strength but increases spine size, presumably by a structural process. Thus very different mechanisms govern how CaMKII affects spine structure and synaptic function.

Autonomous CaMKII can promote either long-term potentiation or long-term depression, depending on the state of T305/T306 phosphorylation.

Ca(2+)/calmodulin-dependent kinase II (CaMKII) is a key mediator of long-term potentiation (LTP). Whereas acute intracellular injection of catalytically active CaMKII fragments saturates LTP (Lledo et al., 1995), an autonomously active form (T286D) of CaMKII holoenzyme expressed in transgenic mice did not saturate potentiation (Mayford et al., 1995). To better understand the role of the holoenzyme in the control of synaptic strength, we transfected hippocampal neurons with constructs encoding forms of CaMKII mimicking different phosphorylation states. Surprisingly, T286D not only failed to potentiate synaptic strength, but produced synaptic depression through an long-term depression (LTD)-like process. T305/T306 phosphorylation was critical for this depression because overexpression of the pseudophosphorylated form (T286D/T305D/T306D) caused depression that occluded LTD, and overexpression of an autonomous form in which T305/T306 could not be phosphorylated (T286D/T305A/T306A) prevented LTD (instead producing potentiation). Therefore, autonomous CaMKII can lead to either LTP or LTD, depending on the phosphorylation state of the control point, T305/T306.

Synaptic strength of individual spines correlates with bound Ca2+-calmodulin-dependent kinase II.

Both synaptic strength and spine size vary from spine to spine, but are strongly correlated. This gradation is regulated by activity and may underlie information storage. Ca2+-calmodulin-dependent kinase II (CaMKII) is critically involved in the regulation of synaptic strength and spine size. The high amount of the kinase in the postsynaptic density has suggested that the kinase has a structural role at synapses. We demonstrated previously that the bound amount of CaMKIIalpha in spines persistently increases after induction of long-term potentiation, prompting the hypothesis that this amount may correlate with synaptic strength. To test this hypothesis we combined two recently developed methods, two-photon uncaging of glutamate for determining the EPSC of individual spines (uEPSC) and quantitative microscopy for measuring bound CaMKIIalpha in the same spines. We found that under basal conditions the relative bound amount of CaMKIIalpha varied over a 10-fold range and positively correlated with the uEPSC. Both the bound amount of CaMKIIalpha in spines and uEPSC also positively correlated with spine size. Interestingly, the bound CaMKIIalpha fraction (bound/total CaMKIIalpha in spines) remained remarkably constant across all spines. The results are consistent with the hypothesis that bound CaMKII serves as a structural organizer of postsynaptic molecules and thereby may be involved in maintaining spine size and synaptic strength.

Persistent accumulation of calcium/calmodulin-dependent protein kinase II in dendritic spines after induction of NMDA receptor-dependent chemical long-term potentiation.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a leading candidate for a synaptic memory molecule because it is persistently activated after long-term potentiation (LTP) induction and because mutations that block this persistent activity prevent LTP and learning. Previous work showed that synaptic stimulation causes a rapidly reversible translocation of CaMKII to the synaptic region. We have now measured green fluorescent protein (GFP)-CaMKIIalpha translocation into synaptic spines during NMDA receptor-dependent chemical LTP (cLTP) and find that under these conditions, translocation is persistent. Using red fluorescent protein as a cell morphology marker, we found that there are two components of the persistent accumulation. cLTP produces a persistent increase in spine volume, and some of the increase in GFP-CaMKIIalpha is secondary to this volume change. In addition, cLTP results in a dramatic increase in the bound fraction of GFP-CaMKIIalpha in spines. To further study the bound pool, immunogold electron microscopy was used to measure CaMKIIalpha in the postsynaptic density (PSD), an important regulator of synaptic function. cLTP produced a persistent increase in the PSD-associated pool of CaMKIIalpha. These results are consistent with the hypothesis that CaMKIIalpha accumulation at synapses is a memory trace of past synaptic activity.

Forskolin-induced LTP in the CA1 hippocampal region is NMDA receptor dependent.

Chemically induced long-term potentiation (cLTP) could potentially work by directly stimulating the biochemical machinery that underlies synaptic plasticity, bypassing the need for synaptic activation. Previous reports suggested that agents that raise cAMP concentration might have this capability. We examined the cLTP induced in acute slices by application of Sp-cAMPS or a combination of the adenylyl cyclase activator, forskolin, and the phosphodiesterase inhibitor, rolipram. Under our conditions, cLTP was induced but only if inhibition was reduced. We found that this form of cLTP was blocked by a N-methyl-d-aspartate receptor (NMDAR) antagonist and required the low-frequency test stimulation typically used to monitor the strength of synapses. Interestingly, similar LTP could be induced by lowering the Mg(2+) concentration of the ACSF during forskolin/rolipram or Sp-cAMPS application or even by just lowering Mg(2+) concentration alone. This LTP was also NMDAR dependent and required only a few ( approximately 5) low-frequency stimuli for its induction. The finding that even low-frequency synaptic stimulation was sufficient for LTP induction indicates that a highly sensitized plasticity state was generated. The fact that some stimulation was required means that potentiation is probably restricted to the stimulated axons, limiting the usefulness of this form of cLTP. However, when similar experiments were conducted using slice cultures, potentiation occurred without test stimuli, probably because the CA3-CA1 connections are extensive and because presynaptic spontaneous activity is sufficient to fulfill the activity requirement. As in acute slices, the potentiation was blocked by an NMDAR antagonist. Our general conclusion is that the induction of LTP caused by elevating cAMP requires presynaptic activity and NMDA channel opening. The method of inducing cLTP in slice cultures will be useful when it is desirable to produce NMDAR-dependent LTP in a large fraction of synapses.

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.

Pathway-specific properties of AMPA and NMDA-mediated transmission in CA1 hippocampal pyramidal cells.

CA1 pyramidal cells receive glutamatergic input from the entorhinal cortex through the perforant path (PP) and from CA3 through Schaffer collaterals (SC). The PP input terminates in the stratum lacunosum molecular approximately 300 microm from the cell body, whereas SC synapses have a more proximal location in the stratum radiatum. We compared the properties of AMPA- and NMDA-mediated transmission at these two inputs. The AMPA-mediated components have linear voltage dependence in both inputs. The reversal potential in the PP is only slightly more positive than in the SC, indicating that distal membrane voltage could be effectively set. The NMDA-mediated responses in the two pathways, however, are very different. The PP exhibits inward rectification, as evidenced by very low outward currents. The rectification persists in the absence of extracellular Mg2+. It cannot be attributed to clamping problems, because large outward AMPA currents can be observed even when conditions are modified to have the AMPA currents kinetically match the NMDA currents. Thus, it appears that the PP NMDA channels have novel properties. A second difference between the PP and SC pathways is that the PP has a larger NMDA/AMPA charge ratio. This difference could be observed under many conditions, including block of all voltage-dependent conductances and elimination of the negative resistance of NMDA channels by removing extracellular Mg2+. The difference in ratio thus cannot be attributed to regenerative currents. The higher NMDA component of the distal PP synapses could help to make these synapses more powerful under depolarizing conditions.