CaM kinase II
Neuroligins are postsynaptic cell adhesion molecules that are important for synaptic function through their trans-synaptic interaction with neurexins (NRXNs). The localization and synaptic effects of neuroligin-1 (NL-1, also called NLGN1) are specific to excitatory synapses with the capacity to enhance excitatory synapses dependent on synaptic activity or Ca(2+)/calmodulin kinase II (CaMKII). This study reports that CaMKII robustly phosphorylates the intracellular domain of NL-1. T739 was shown to be the dominant CaMKII site on NL-1, and it is phosphorylated in response to synaptic activity in cultured rodent neurons and sensory experience in vivo. Furthermore, a phosphodeficient mutant (NL-1 T739A) reduces the basal and activity-driven surface expression of NL-1, leading to a reduction in neuroligin-mediated excitatory synaptic potentiation. These results are the first to demonstrate a direct functional interaction between CaMKII and NL-1, two primary components of excitatory synapses (Bemben, 2014).
alpha and ßCaMKII are inversely regulated by activity in hippocampal neurons in culture: the alpha/ß ratio shifts toward alpha during increased activity and ß during decreased activity. The swing in ratio is ~5-fold and may help tune the CaMKII holoenzyme to changing intensities of Ca2+ signaling. The regulation of CaMKII levels uses distinguishable pathways, one responsive to NMDA receptor blockade that controls alphaCaMKII alone, the other responsive to AMPA receptor blockade and involving ßCaMKII and possibly further downstream effects of ßCaMKII on alphaCaMKII. Overexpression of alphaCaMKII or ßCaMKII results in opposing effects on unitary synaptic strength as well as mEPSC frequency that can account in part for activity-dependent effects observed with chronic blockade of AMPA receptors. Regulation of CaMKII subunit composition may be important for both activity-dependent synaptic homeostasis and plasticity (Thiagarajan, 2002).
Calcium/calmodulin-dependent protein kinase II (CaMKII) is expressed at high levels in the central nervous system, particularly in the hippocampus, where it constitutes ~2% of total protein. As a holoenzyme, neuronal CaMKII is made up of 6-12 subunits, primarily the 52 kDa alpha isoform and the 60 kDa ß isoform. The subunits of the holoenzyme are held together by association domains in their C-terminals, which form a central globular structure from which the N-terminals extend radially. The N-terminal contains the catalytic sites of the kinase as well as the autoinhibitory domains that bind to the catalytic sites in the basal state. The binding of Ca2+/calmodulin releases this autoinhibition, allowing phosphorylation to take place at a critical threonine residue, Thr286 in alpha and Thr287 in ß. This autophosphorylation allows the molecular memory of a transient Ca2+ signal to greatly outlast the duration of the Ca2+ transient itself, a property that endows CaMKII with the ability to decode Ca2+ signals in a frequency-dependent manner (Thiagarajan, 2002 and references therein).
Immunoprecipitation with subunit-specific antibodies indicates that the majority of the CaMKII holoenzymes are alpha/ß heteromers with variable subunit ratios, although some alpha homomers can also be found. Why have two isoforms? One significant distinction between the alpha and ß isoforms lies in their sharply different affinity for calmodulin. Half-maximal autophosphorylation is achieved at 130 nM calmodulin for alphaCaMKII and at 15 nM calmodulin for ßCaMKII. Due to this difference, the two isoforms have different sensitivities to Ca2+ signals under nonsaturating levels of calmodulin. alphaCaMKII is selective for higher levels of Ca2+ signals, while ßCaMKII has better sensitivity to lower levels of signal. When the two isoforms are combined in a heteromer, the response to Ca2+ signals has been found to depend on the ratio of alpha to ß subunits. Consequently, activity-dependent regulation of alpha- and ßCaMKII expression could provide a mechanism of tuning neuronal responses to different levels of activity. This is an intriguing possibility that raises several fundamental questions. Does the cell regulate the ratio of alpha to ß in an activity-dependent manner? And if so, what pathways of synaptic activity might control the regulation of alpha- and ßCaMKII? Could their regulation be coupled? What would be the consequence of such a regulation for synaptic transmission? This study uses a combination of immunotechniques and electrophysiology to address these issues. The data show that alpha- and ßCaMKII are inversely regulated by activity in a manner that may help tune CaMKII to changing levels of Ca2+ signal. Furthermore, tilting the ratio toward alpha or ß results in opposing effects on unitary synaptic strength and mEPSC frequency and has functional significance for both activity-dependent plasticity and homeostasis (Thiagarajan, 2002).
In excitatory neurons, treatment with TTX to block action potentials decreases the levels of alphaCaMKII and raises ßCaMKII, both in cell bodies and at synapses. Conversely, exposure to bicucculine to prevent inhibitory transmission and increase firing increases alphaCaMKII and decreased ßCaMKII. Thus, the changes in alphaCaMKII correlated positively with changes in electrical activity, while changes in ßCaMKII correlated negatively. This inverse regulation gives rise to ~5-fold changes in the alpha:ß ratio between the extremes of TTX or BIC treatment, while the sum total of these isoforms remained relatively unchanged, varying only 1.0- to 1.3-fold (for assumed values of the basal alpha/ß ratio ranging between 1:1 and 3:1). One may speculate that the inverse changes in isoform levels support the widest variation in isoform ratio consistent with holding fixed the total amount of enzyme (possibly important for structural reasons). That inhibitory neurons, which lack immunoreactivity for alphaCaMKII, fail to show regulation of ß with altered activity also fits with a pattern in which the overall level of CaMKII is tightly regulated (Thiagarajan, 2002).
Changes in the balance between alpha and ß isoforms predicts interesting functional consequences. ßCaMKII has been shown to exhibit a much higher affinity than alphaCaMKII for Ca2+/CaM. If the changes in the overall alpha:ß ratio between the opposite conditions of TTX and BIC are indicative of the subunit composition of the CaMKII holoenzyme, this would result in a much higher affinity of the holoenzyme for Ca2+/CaM. Assuming a ~9-fold difference in CaM affinity of ßCaMKII relative to alphaCaMKII, a rough calculation predicts a ~2-fold variation in the holoenzyme affinity for Ca2+/CaM. The increased sensitivity of the holoenzyme to CaM with decreased activity would serve as a homeostatic mechanism to confer responsiveness to weaker Ca2+ signals (Thiagarajan, 2002).
It first came as a surprise that activity-dependent changes in alpha and ß isoforms arose from largely distinct pathways, involving different glutamate receptors: levels of alphaCaMKII (but not ß) are strongly influenced by NMDAR activity; in contrast, ßCaMKII was strongly affected by AMPAR activity. These new findings make sense if put in context of previous studies on neuronal CaMKII. The strong reduction of alphaCaMKII but sparing of ß by blockade of NMDA receptors can be interpreted in light of several related observations: (1) multimeric CaMKII takes the form of alpha homomers as well as alpha/ß heteromultimers; (2) NMDAR-dependent changes in the abundance of alphaCaMKII can be detected as soon as 5 min after stimulation in hippocampal slices, consistent with a localized dendritic translation of alphaCamKII mRNA; (3) ßCaMKII mRNA is absent in dendrites, leaving alpha homomers as the only enzyme species that could be formed there. Taken together with these observations, these findings are consistent with a simple model wherein Ca2+ entry through NMDARs in the dendrites regulates alphaCaMKII homomers locally, on a fast time scale, with little or no control of ß (Thiagarajan, 2002).
The observation that levels of ßCaMKII strongly increased in response to blockade of AMPA receptors, not NMDARs, suggested that regulation of ß may be quite different than proposed for alpha alone. Under various pharmacological conditions, the pattern of changes observed in ßCaMKII was always consistent with an inverse relationship with AMPAR activity. Because ß transcripts are restricted to the cell body, and changes in ßCaMKII occur only slowly (evident only on time scales >1 hr), the regulation by AMPARs is likely to occur at the level of nuclear transcription. Thus, AMPAR-mediated depolarization could work through recruitment of voltage-gated Ca2+ channels and regulation of nuclear transcription factors. Regulation in or near the nucleus makes additional sense for the linkage between increased ßCaMKII and the downregulation of alpha that was observed in transfection studies. All considerations seem consistent with the following working hypothesis: ßCaMKII levels are regulated in the cell body, downstream of AMPA receptor activity, leading to a reciprocal regulation of alpha and thus the formation of alpha/ß heteromers of variable subunit ratio (Thiagarajan, 2002).
This scheme invokes the inter-relationship between levels of ß and alpha that was directly observed in the transfection experiments. Another possibility, not mutually exclusive, is that AMPAR block decreases postsynaptic depolarization and thereby reduces Ca2+ entry through NMDAR, leading to a fall in the alpha isoform (Thiagarajan, 2002).
Conceptual distinctions have been drawn between synaptic homeostasis, negative feedback regulation thought of as neuron wide, and forms of synaptic plasticity such as LTP, which can be self-reinforcing and synapse specific. Both kinds of regulation may be strongly impacted by inverse changes in the abundance of alpha- and ßCaMKII. For example, increased activity, and consequent elevation of the alpha/ß ratio would decrease the Ca2+/CaM sensitivity of CaMKII in a homeostatic manner. This can be viewed as 'input tuning', wherein the holoenzyme is adjusted appropriately to the ambient level of activity. The threshold for the induction of LTP, which already is high, would be further raised, thereby changing the rules governing synaptic plasticity . However, increasing the alpha/ß ratio may also change the cellular localization of CaMKII, promoting alphaCaMKII expression at specific subsynaptic sites where it could contribute to LTP. Understanding the full implications for plasticity and metaplasticity will become easier once more is known about how the subunit composition of CaMKII affects its cellular localization and degree of autophosphorylation and how alterations in the alpha/ß ratio and its downstream effects unfold over time scales ranging from minutes to days (Thiagarajan, 2002).
Altering the levels of alpha and ß causes striking changes in both mini size and frequency. Once again, the overall change in synaptic function cannot be neatly pigeonholed into strict categories of 'synaptic homeostasis' or 'synaptic plasticity' alone. Increases in the alpha/ß ratio accentuates the contribution of individual synaptic events (augmented mini area), while also tending to decrease the number of quanta received per unit time (lowered mini frequency), thus keeping the total synaptic drive within reasonable bounds. Evidently, simple biochemical changes can induce a powerful combination of self-reinforcing local changes, but negative feedback regulation over the neuron as a whole (Thiagarajan, 2002).
Calcium/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional enzyme that is very critical for synaptic plasticity and memory formation. Although significant progress has been made in understanding the role of postsynaptic CaMKII in synaptic plasticity, very little is known about its presynaptic function during plasticity changes. KN-93, a membrane-permeable CaMKII inhibitor, blocks glutamate-induced increases in the frequency of miniature excitatory postsynaptic currents (mEPSCs) and the number of presynaptic functional boutons in cultured hippocampal pyramidal neurons. In addition, presynaptic injection of the membrane-impermeable CaMKII inhibitor peptide 281-309 blocks synaptic plasticity induced by tetanus, glutamate, or NO/cGMP pathway activation as expressed by long-lasting increases in EPSC amplitude and functional presynaptic boutons. Presynaptic injection of CaMKII itself coupled with weak tetanus produces an immediate and long-lasting enhancement of EPSC amplitude. Thus, the present results conclusively prove that presynaptic CaMKII is essential for synaptic plasticity in cultured hippocampal neurons (Ninan, 2004).
Several lines of evidence indicating that presynaptic CaMKII activation is necessary for inducing synaptic plasticity in cultured hippocampal neurons. Application of glutamate or high-frequency stimulation of the presynaptic neuron should sharply increase autophosphorylation of presynaptic CaMKII leading to increased release of neurotransmitters. It is highly likely that presynaptic CaMKII activation is involved in the conversion of preexisting but silent presynaptic terminals to functional ones during glutamate-induced plasticity. Glutamate application in Mg2+ free culture (to allow the opening of NMDA receptor channels) induces clustering of the presynaptic proteins synaptophysin and synapsin I, suggesting the conversion of 'silent' presynaptic terminals to functional ones. It has been shown that there is persistent phosphorylation of synapsin I at its CaMKII sites during LTP. This model is supported by recent studies demonstrating that synapsin I dispersion after its phosphorylation controls synaptic vesicle mobilization. Moreover, autophosphorylated CaMKII binds to synthaxin and regulates exocytosis. The present study strongly indicates that activation of presynaptic CaMKII is necessary for induction of synaptic plasticity in cultured hippocampal neurons. Being critical in both pre- and post-synaptic mechanisms, CaMKII plays a unique role in the long-lasting potentiation of synaptic efficacy, which is most likely the basis of learning and memory (Ninan, 2004).
CaM kinase II and Long term potentiation (LTP) A constitutively active form of
CaMKII was introduced into neurons from hippocampal slices with a recombinant vaccinia virus to test
the hypothesis that increased postsynaptic activity of this enzyme is sufficient to produce long-term
synaptic potentiation (LTP), a cellular analog of learning. Postsynaptic expression of
constitutively active CaMKII increases CaMKII activity, enhances synaptic transmission, and prevents more
potentiation by an LTP-inducing protocol. These results suggest that
postsynaptic CaMKII activity is necessary and sufficient to generate LTP (Pettit, 1994).
CA2+-regulated protein kinases play critical roles in long-term potentiation. To better understand the role of
Ca2+/calmodulin (CaM) signaling pathways in synaptic transmission, Ca2+/CaM was injected into
hippocampal CA1 neurons. Ca2+/CaM induces significant potentiation of excitatory synaptic responses,
which is blocked by coinjection of a CaM-binding peptide and is not induced by injections of Ca2+ or
CaM alone. Reciprocal experiments demonstrate that Ca2+/CaM-induced synaptic potentiation and
tetanus-induced LTP occlude one another. Pseudosubstrate inhibitors or high-affinity substrates of
CaMKII or Protein kinase C (See Drosophila PKC) block Ca2/CaM-induced potentiation, indicating the requirement of CaMKII and PKC
activities in synaptic potentiation. It is thought that postsynaptic levels of free Ca2+/CaM are a rate limiting
factor and that functional cross-talk between Ca2+/CaM and PKC pathways occurs during the induction of
LTP (Wang, 1995).
To investigate the function of the autophosphorylated form of CaMKII in synaptic plasticity,
transgenic mice were generated that express a kinase that is Ca2+ independent as a result of a point mutation of Thr-286 to
aspartate, which mimics autophosphorylation. Mice expressing the mutant form of the kinase show an
increased level of Ca(2+)-independent CaMKII activity similar to that seen following LTP. The mice
nevertheless exhibit normal LTP in response to stimulation at 100 Hz. However, at lower frequencies, in the
range of 1-10 Hz, there is a systematic shift in the size and direction of the resulting synaptic change in the
transgenic animals that favors long term depression, a cellular analog of memory suppression. The regulation of this frequency-response function by
Ca(2+)-independent CaMKII activity seems to account for two previously unexplained synaptic phenomena,
the relative loss of LTD in adult animals compared with juveniles and the enhanced capability for depression
of facilitated synapses (Mayford, 1995).
Induction of long-term potentiation in the CA1 region of hippocampal slices is associated with increased
activity of Ca2+/calmodulin-dependent protein kinase II. Application of high but
not low frequency stimulation to two groups of afferents in the CA1 region of 32P-labeled slices results in
the phosphorylation of two major substrates of this enzyme, synapsin I and microtubule-associated protein
2, as well as in the autophosphorylation of CaM kinase II. Long term potentiation induction is associated with an increase in the amount of CaM kinase II in the
same region. All these changes are prevented when high frequency stimulation is applied in the presence
of the N-methyl-D-aspartate receptor antagonist, D-2-amino-5-phosphonopentanoate. These results indicate
that activation of CaM kinase II is involved in the induction of synaptic potentiation in both the
postsynaptic and presynaptic regions (Fukunaga, 1995).
Long-term potentiation (LTP) is an increase in synaptic responsiveness thought to be
involved in mammalian learning and memory. The localization (presynaptic and/or
postsynaptic) of changes underlying LTP has been difficult to resolve with current
electrophysiological techniques. A novel multiple-electrode stimulator was used to produce LTP in a substantial
portion of the synapses in a hippocampal CA1 minislice. The effects of such
stimulation were tested on the presynaptic protein synapsin I. Synapsin is a phosphoprotein that associates in the presynaptic terminal with synaptic vesicles. Synapsin I plays a key role in neurotransmitter release by regulating the availability of synaptic vesicles to exocytosis. It is thought that phosphorylation of synapsin I by CaM kinase II functions to increase the availability of vesicles for release. LTP-inducing stimulation produces a
long-lasting 6-fold increase in the phosphorylation of synapsin I at its
Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) sites without affecting
synapsin I levels. This effect was fully blocked by either the N-methyl-D-aspartate
receptor antagonist D(-)-2-amino-5-phosphonopentanoic acid (APV) or the CaM
kinase II inhibitor KN-62. These results indicate that LTP expression is accompanied by
persistent changes in presynaptic phosphorylation, and specifically that presynaptic
CaM kinase II activity and synapsin I phosphorylation may be involved in LTP
expression (Nayak, 1996).
To test the involvement
of Ca2+/calmodulin-dependent kinase II (CaM-K II) in LTP, a truncated, constitutively active form of this kinase was
directly injected into CA1 hippocampal pyramidal cells. Inclusion of CaM-K II in the recording pipette
results in a gradual increase in the size of excitatory postsynaptic currents (EPSCs). No change in evoked
responses occurs when the pipette contains heat-inactivated kinase. The effect of CaM-K II mimics
several features of LTP in that there is a decrease in synaptic failures, an increase in the size of
spontaneous EPSCs, and an increase in the amplitude of responses to iontophoretically applied
alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate. To determine whether the CaM-K II-induced
enhancement and LTP share a common mechanism, occlusion experiments were carried out. The enhancing
action of CaM-K II is greatly diminished by prior induction of LTP. In addition, following the increase in
synaptic strength by CaM-K II, tetanic stimulation fails to evoke LTP. These findings indicate that CaM-K
II alone is sufficient to augment synaptic strength and that this enhancement shares the same underlying
mechanism as the enhancement observed with LTP (Lledo, 1995).
The calcium-calmodulin-dependent kinase II (CaMKII) is required for hippocampal long-term potentiation (LTP) and spatial learning. In addition to its calcium-calmodulin (CaM)-dependent activity, CaMKII can undergo autophosphorylation, resulting in CaM-independent activity. A point mutation was introduced into the alphaCaMKII gene that blocks the autophosphorylation of threonine at position 286 (Thr286) of this kinase without affecting its CaM-dependent activity. The mutant mice have no N-methyl-D-aspartate receptor-dependent LTP in the hippocampal CA1 area and show no spatial learning in the Morris water maze. Thus, the autophosphorylation of alphaCaMKII at Thr286 appears to be required for LTP and learning (Giese, 1998).
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, the effects of AC activators on
activity-dependent changes in synaptic strength and on levels of autophosphorylated CaMKII
(Thr286) were investigated. In the CA1 region of hippocampal slices, briefly elevating extracellular Ca2+ induces 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 CaMKII autophosphorylation, electrical presynaptic
fiber stimulation was replaced 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 induces an LTP-like synaptic potentiation and a
persistent increase in autophosphorylated CaMKII levels. In the absence of forskolin or calyculin
A, treatment has no lasting effect on synaptic strength and induces a persistent decrease in autophosphorylated CaMKII levels. These results suggest that AC activation facilitates LTP induction by suppressing
protein phosphatases and enabling a persistent increase in the levels of autophosphorylated CaMKII (Makhinson, 1999).
The mammalian sensory neocortex exhibits experience-dependent plasticity such that neurons modify their
response properties according to changes in sensory experience. The synaptic plasticity mechanism of
long-term potentiation requiring calcium-calmodulin-dependent kinase type II (CaMKII) could underlie
experience-dependent plasticity. Plasticity in adult mice can be induced by changes in the patterns of tactile
input to the barrel cortex. This response is strongly depressed in adult mice that lack the gene encoding
alpha-CaMKII, although adolescent animals are unaffected. Thus, alpha-CaMKII is necessary either for the
induction or for the expression of plasticity in adult mice (Glazewski, 1996).
Phosphorylation of the transcription factor CREB is thought to be important in processes underlying
long-term memory. It is unclear whether CREB phosphorylation can carry information about the sign of
changes in synaptic strength, whether CREB pathways are equally activated in neurons receiving or
providing synaptic input, or how synapse-to-nucleus communication is mediated.
Ca(2+)-dependent nuclear CREB phosphorylation is rapidly evoked by synaptic stimuli, including (but not limited to) those that induce potentiation and depression of synaptic strength. In striking contrast, high
frequency action potential firing alone fails to trigger CREB phosphorylation. Activation of a
submembranous Ca2+ sensor, just beneath sites of Ca2+ entry, appears critical for triggering nuclear CREB
phosphorylation via calmodulin and a Ca2+/calmodulin-dependent protein kinase (Deisseroth, 1996).
The alpha-Ca2+/calmodulin kinase II (alpha CaMKII) is required for long-term potentiation in the CA1
region of the hippocampus. This kinase is also crucial for promoting presynaptic
plasticity. Paired-pulse facilitation is blunted in the CA1 region of mice heterozygous for a targeted
mutation of alpha CaMKII, confirming that this kinase can promote neurotransmitter release.
Unexpectedly, field and whole-cell recordings of posttetanic potentiation show that the synaptic
responses of mutants are larger than those of controls, indicating that alpha CaMKII can also inhibit
transmitter release immediately after tetanic stimulation. Thus, alpha CaMKII has the capacity either
to potentiate or to depress excitatory synaptic transmission depending on the pattern of presynaptic
activation (Chapman, 1995).
Hippocampal "place cells" fire selectively when an animal is in a specific location. The fine-tuning and
stability of place cell firing has been compared in two types of mutant mice with different long-term
potentiation (LTP) and place learning impairments. alpha calmodulin kinase II and CREBalphadelta mutants differ substantially in the severity of their defects in synaptic plasticity and learning. The alpha calmodulin kinase II mutant mice are severely impaired in spatial learning as well as in NMDAR-dependent LTP in the CA1 region of the hippocampus. In ontrast CREBalphadelta mutants have reduced LTP and mild spatial learning deficits. Place cells from both mutants showed decreased
spatial selectivity. Place cell stability is also deficient in both mutants and, consistent with the
severities in their LTP and spatial learning deficits, is more affected in mice with a point mutation
[threonine (T) at position 286 mutated to alanine (A)] in the alpha calmodulin kinase II
(alphaCaMKIIT286A) than in mice deficient for the alpha and Delta isoforms of cyclicAMP-responsive element binding proteins (CREBalphaDelta-). Thus, LTP appears to be
important for the fine tuning and stabilization of place cells, and these place cell properties may be
necessary for spatial learning (Cho, 1998).
The observed LTP deficits in the alphaCaMKII mutants are not due to abnormalities in GABAA inhibition, NMDAR currents, or synaptic function before and during the tetanus, suggesting that the deficits act downstream of Ca2+ influx through NMDARs. These findings are consistent with the observation that, after LTP induction, the autophosphorylated form of alpha CaMKII phosophorylates glutamate receptor subunits that may be required for LTP. In accordance with a model implicating the CaM-independent state of CaMKII in learning and memory, it was shown that the autophosphorylation of alphaCaMKII at Thr286 is required for spatial learning. Thus, the autophosphorylation of alphaCaMKII at Thr 286 is crucial for hippocampal LTP and hippocampus-dependent learning (Cho, 1998).
CaMKII is a calcium-activated kinase that is abundant in neurons and has been strongly implicated in memory and learning. Low-frequency stimulation of glutamatergic afferents in hippocampal slices from juvenile domestic chicks results in long-term depression of synaptic transmission. This reduction does not require activation of NMDA or metabotropic glutamate receptors and does not require a rise in postsynaptic calcium. However, buffering presynaptic calcium prevents the reduction of the excitatory postsynaptic potential or current that is induced by low-frequency stimulation. In addition, application of the calmodulin antagonist calmidazolium, or the specific CaMKII antagonist KN-93, completely blocks long-term depression. These findings demonstrate a newly discovered form of long-term synaptic depression in the avian hippocampus (Margrie, 1998).
Long-term potentiation (LTP) at the Schaffer collateral-CA1 synapse involves interacting signaling components, including
calcium (Ca2+)/calmodulin-dependent protein kinase II (CaMKII) and cyclic adenosine monophosphate (cAMP) pathways.
Postsynaptic injection of thiophosphorylated inhibitor-1 protein, a specific inhibitor of protein phosphatase-1 (PP1),
substitutes for cAMP pathway activation in LTP. Stimulation that induced LTP triggers cAMP-dependent phosphorylation of
endogenous inhibitor-1 and a decrease in PP1 activity. This stimulation also increases phosphorylation of CaMKII at Thr286
and Ca2+-independent CaMKII activity in a cAMP-dependent manner. The blockade of LTP by a CaMKII inhibitor is not
overcome by thiophosphorylated inhibitor-1. Thus, the cAMP pathway uses PP1 to gate CaMKII signaling in LTP (Blitzer, 1998).
To elucidate mechanisms that control and execute activity-dependent synaptic
plasticity, AMPA receptors
(AMPA-Rs) with an electrophysiological tag were expressed in rat hippocampal
neurons. Long-term potentiation (LTP) or increased activity of the
calcium/calmodulin-dependent protein kinase II (CaMKII) induce delivery of
tagged AMPA-Rs into synapses. This effect is not diminished by mutating the
CaMKII phosphorylation site on the GluR1 AMPA-R subunit, but is blocked by
mutating a predicted PDZ domain interaction site. These results show that LTP
and CaMKII activity drive AMPA-Rs to synapses by a mechanism that requires the
association between GluR1 and a PDZ domain protein (Hayashi, 2000).
Activation of mitogen-activated protein kinase (MAPK) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) are
required for numerous forms of neuronal plasticity, including long-term potentiation (LTP). LTP was induced in rat hippocampal
area CA1 using theta-pulse stimulation (TPS) paired with ß-adrenergic receptor activation [isoproterenol (ISO)], a
protocol that may be particularly relevant to normal patterns of hippocampal activity during learning. This stimulation results in a
transient phosphorylation of p42 MAPK, and the resulting LTP is MAPK dependent. In addition, CaMKII is regulated in two, temporally distinct ways after
TPS-ISO: a transient rise in the fraction of phosphorylated CaMKII and a subsequent persistent increase in CaMKII expression. The increases in MAPK and
CaMKII phosphorylation are strongly colocalized in the dendrites and cell bodies of CA1 pyramidal cells, and both the transient phosphorylation and delayed
expression of CaMKII are prevented by inhibiting p42/p44 MAPK. These results establish a novel bimodal regulation of CaMKII by MAPK, which may
contribute to both post-translational modification and increased gene expression (Giovannini, 2001).
An inducible pharmacogenetic approach has been developed where
pharmacological manipulations can be used to reveal recessive mutant phenotypes in a temporally controlled manner. This approach takes advantage of synergisms between pharmacological and genetic manipulations to alter the function of specific signaling pathways. For example, mice heterozygous for a point mutation (T286A) in the alpha-calcium/calmodulin-dependent kinase II (alphaCaMKII) gene show normal learning and memory. However, a concentration of an NMDA receptor antagonist (CPP) that does not affect learning in wild-type (WT) littermates, reveals learning deficits in this heterozygote [alphaCaMKIIT286A+/-]. Pretetanic application of a concentration of CPP (0.1 microM), ineffective in WT hippocampal slices, induces deficits in alphaCaMKIIT286A+/- slices in hippocampal long-term potentiation (LTP), a mechanism thought to be involved in learning and memory. Importantly, posttetanic application of CPP (0.1 microM) has no effect on the expression or maintenance of LTP in hippocampal slices from alphaCaMKIIT286A+/- mice. Thus, this pharmacogenetic approach allows a demonstration that NMDA receptor-dependent autophosphorylation of alphaCaMKII is required during the induction but not maintenance of LTP. This ability to temporally induce recessive mutant phenotypes could be applicable to a broad range of problems and genetic systems (Ohno, 2002)
Experience-dependent plasticity can be induced in the barrel cortex by removing all but one whisker, leading to potentiation of the neuronal response to the spared whisker. To determine whether this form of potentiation depends on synaptic plasticity, long-term potentiation (LTP) and sensory-evoked potentials were studied in the barrel cortex of alpha-calcium/calmodulin-dependent protein kinase II (alphaCaMKII)T286A mutant mice. Three different forms of LTP induction were studied: theta-burst stimulation, spike pairing, and postsynaptic depolarization paired with low-frequency presynaptic stimulation. None of these protocols produced LTP in alphaCaMKIIT286A mutant mice, although all three were successful in wild-type mice. To study synaptic plasticity in vivo, measured sensory-evoked potentials were measured in the barrel cortex, and it was found that single-whisker experience selectively potentiates synaptic responses evoked by sensory stimulation of the spared whisker in wild types but not in alphaCaMKIIT286A mice. These results demonstrate that alphaCaMKII autophosphorylation is required for synaptic plasticity in the neocortex, whether induced by a variety of LTP induction paradigms or by manipulation of sensory experience, thereby strengthening the case that the two forms of plasticity are related (Hardingham, 2003).
Mice deficient for the gene encoding alpha-calcium-calmodulin-dependent kinase II (alpha-CaMKII
knockout mice) provide a promising tool to link behavioral and cellular abnormalities with a specific
molecular lesion. The heterozygous mouse exhibit a well-circumscribed syndrome of behavioral
abnormalities, consisting primarily of a decreased fear response and an increase in defensive
aggression, in the absence of any measured cognitive deficits. Unlike the heterozygote, the homozygote
displays abnormal behavior in all paradigms tested. At the cellular level, both extracellular and
whole-cell patch clamp recordings indicate that serotonin release in putative serotonergic neurons of
the dorsal raphe is reduced. Thus, alpha-CaMKII knockout mice, in particular the heterozygote, may
provide a model for studying the molecular and cellular basis underlying emotional disorders involving
fear and aggression (Chen, 1994).
Memory is not a unified system but occurs in multiple forms. For example, one may distinguish between explicit memory (concerned with memories about places, objects and people), and implicit memory. Explicit memory is deficient in amnesia victims. Implicit memory, a second type of memory, is memory of skills (motor skills for example), and is often found intact in amnesia patients. Motor skills, perceptuo-motor skills, perceptual skills, and early-stage cognitive skill learning can remain intact when amnesia occurs; such skills appear to be independent of the brain structures damaged in amnesia. The skills retained or acquired by post-amnesic patients can include highly specific information (Squire, 1992 and references).
CaMKII, found in the cells of the hippocampus, is implicated in explicit memory. It is thought that the hippocampus can form cognitive maps. Hippocampal "place cells" are thought to carry, or be involved in forming, explicit memory. Spatial location is encoded in the pattern of firing of individual hippocampal pyramidal cells. When an animal moves around a familiar environment, different place cells in the hippocampus fire as the animal enters different regions of space. A given cell fires only when the head of the animal is in a certain part of the environment called the "place field" or "firing field" of the cell. As the animal moves around, some cells start firing and others cease, so that firing of many cells signals the momentary location of the animal. Any given environment involves only about half of the million pyramidal cells (Rotenberg, 1996 and references).
Recordings were made in place cells in transgenic mice that express a mutated Ca++-independent form of CaM Kinase II. These mice show a severe deficit in spatial memory, although they retain the ability to perform nonspatial tasks (Bach, 1995). These mice have normal long-term potentiation (LTP) when slices of hippocampus are tested at 100 Hz, but they lack LTP in response to stimulation of 5-10 Hz and are impaired in spatial memory tasks. In these transgenic mice, the place cells in the CA1 region of the hippocampus exhibit three important differences from those of wild type cells: there are fewer place cells, they are less precise in their representation of location, and they are less stable over time. These findings suggest that LTP in the 5-10 Hz range may be important for the maintenance of place-field stability and that this stability maybe essential for the storage of spatial memory (Rotenberg, 1996).
Bidirectional changes in the efficacy of neuronal synaptic transmission, such as hippocampal long-term potentiation (LTP) and long-term depression (LTD), are thought
to be mechanisms for information storage in the brain. LTP and LTD may be mediated by the modulation of AMPA
receptor phosphorylation. LTP and LTD reversibly modify the
phosphorylation of the AMPA receptor GluR1 subunit. However, contrary to the hypothesis that LTP and LTD are the functional inverse of each other, LTP and LTD
are associated with the phosphorylation and dephosphorylation, respectively, of distinct GluR1 phosphorylation sites. Moreover, the site modulated depends on the
stimulation history of the synapse. LTD induction in naive synapses dephosphorylates the major cyclic-AMP-dependent protein kinase (PKA) site, whereas in
potentiated synapses the major calcium/calmodulin-dependent protein kinase II (CaMKII) site is dephosphorylated. Conversely, LTP induction in naive synapses and
depressed synapses increases phosphorylation of the CaMKII site and the PKA site, respectively. LTP is differentially sensitive to CaMKII and PKA inhibitors
depending on the history of the synapse. These results indicate that AMPA receptor phosphorylation is critical for synaptic plasticity, and that identical stimulation
conditions recruit different signal-transduction pathways depending on synaptic history (Lee, 2000).
To investigate the function of the alpha calcium-calmodulin-dependent kinase II (alphaCaMKII) inhibitory autophosphorylation at threonines 305 and/or 306, knockin mice were generated that express alphaCaMKII that cannot undergo inhibitory phosphorylation. In addition, mice were generated that express the inhibited form of alphaCaMKII, which resembles the persistently phosphorylated kinase at these sites. The data demonstrate that blocking inhibitory phosphorylation increases CaMKII in the postsynaptic density (PSD), lowers the threshold for hippocampal long-term potentiation (LTP), and results in hippocampal-dependent learning that seems more rigid and less fine-tuned. Mimicking inhibitory phosphorylation dramatically decreases the association of alphaCaMKII with the PSD and blocks both LTP and learning. These data demonstrate that inhibitory phosphorylation has a critical role in plasticity and learning (Elgersma, 2002).
Local protein translation in dendrites could be a means for delivering synaptic proteins to their sites of action, perhaps in a spatially regulated fashion that could contribute to plasticity. To directly test the functional role of dendritic translation of calcium/calmodulin-dependent protein kinase IIalpha (CaMKIIalpha) in vivo, the endogenous gene was mutated to disrupt the dendritic localization signal in the mRNA. Targeted mutagenesis of the CaMKII 3'UTR was carried out by substituting sequences from the 3'UTR of bovine growth hormone mRNA, a message that is not dendritically localized but which contains an identical polyadenylation hexanucleotide as the endogenous CaMKII gene. In this mutant mouse, the protein-coding region of CaMKII is intact, but mRNA is restricted to the soma. Removal of dendritic mRNA produces a dramatic reduction of CaMKII in postsynaptic densities (PSDs), a reduction in late-phase long-term potentiation (LTP), and impairments in spatial memory, associative fear conditioning, and object recognition memory. These results demonstrate that local translation is important for synaptic delivery of the kinase and that local translation contributes to synaptic and behavioral plasticity (Miller, 2002).
Wang, H., et al. (2008). CaMKII activation state underlies synaptic labile phase of LTP and short-term memory formation. Curr. Biol. 18(20): 1546-54. PubMed Citation: 18929487
The labile state of short-term memory has been known for more than a century. It has been frequently reported that immediate postlearning intervention can readily disrupt newly formed memories. However, the molecular and cellular mechanisms underlying the labile state of new memory are not understood. Using a bump-and-hole-based chemical-genetic method (Wang, 2003), alpha CaMKII activity levels were rapidly and selectively manipulated in the mouse forebrain during various stages of the short-term memory processes. A rapid shift in the alpha CaMKII activation status was found within the immediate 10 min after learning severely disrupts short-term memory formation. The same manipulation beyond the 15 min after learning has no effect, suggesting a critical time window for CaMKII action. During this same 10 min time window only, shifting in CaMKII activation state is capable of altering newly established synaptic weights and/or patterns. It is concluded that the initial 10 min of memory formation and long-term potentiation are sensitive to inducible genetic upregulation of alphaCaMKII activity. The results suggest that molecular dynamics of CaMKII play an important role in underlying synaptic labile state and representation of short-term memory during this critical time window (Wang, 2008).
To investigate the role of the entorhinal cortex in memory at a molecular level, transgenic mice were developed in which transgene expression was inducible and limited to the superficial layers of the medial entorhinal cortex, pre- and para-subiculum. Expression of a constitutively active mutant form of CaMKII in these structures disrupted spatial memory formation. Immediate post-training activation of the transgene disrupted previously established memory while transgene activation 3 weeks following the training was ineffective. These results demonstrate that, similar to the hippocampus, the entorhinal cortex plays a time-limited role in spatial memory formation but is not a final cortical repository of long-term memory. Moreover, these results suggest that the indiscriminate activation of CaMKII is able to disrupt preexisting memories, possibly by altering the pattern of synaptic weight changes that are thought to form the basis of the memory trace (Yasuda, 2006).
Lesion studies in both humans and experimental animals have identified structures in the medial temporal lobe as important for the ability to form new explicit memories -- i.e., memories for people, places, and events. The medial temporal lobe consists of the hippocampus, entorhinal cortex (EC), peri- and post-rhinal cortex, subiculum, and pre- and parasubiculum (Pr-PaS). While the role of the hippocampus is well established, the function of the surrounding cortical structures is less well characterized. While patients with damage restricted to the hippocampus show explicit memory impairments, larger medial temporal lobe lesions that encompass both the hippocampus and surrounding cortex lead to more severe memory deficits. In Alzheimer's disease, the EC seems to be involved in the very earliest stages of the disease, suggesting a critical role for this structure in human memory (Yasuda, 2006 and references therein).
The EC is known to be the primary interface between the hippocampus and neocortex. This interface is layer specific; superficial layers provide major input to the hippocampus, while deeper layers receive output from hippocampus. Although superficial layers are input layers to hippocampus in all mammals, there are species differences in projections of layer II and layer III. In the mouse, layer II provides input directly to the hippocampal trisynaptic circuit via the perforant path to the dentate gyrus, while layer III provides direct CA1, CA3, and subiculum input. Electrophysiological studies indicate a modular arrangement to the EC with neurons of the medial entorhinal cortex (MEC) encoding spatial information similar to the hippocampus while lateral entorhinal cortex neurons lack spatial content (Yasuda, 2006 and references therein).
The MEC has been shown to contain cells (termed grid cells) that fire in multiple locations that are equally spaced to form a grid-like representation of the animal's environment. Lesion studies have demonstrated a role for MEC and for the TA connections in CA1 in spatial memory (Yasuda, 2006 and references therein).
In the hippocampus, it is thought that long-term potentiation (LTP) or a related form of synaptic plasticity underlies the formation of long-term memory. The induction of LTP requires the stimulation of NMDA receptors and activation of CaMKII, which in turn facilitates synaptic transmission by the potentiation of AMPA-type glutamate receptors and the insertion of new receptors. Genetic studies have demonstrated a critical role for NMDA receptors and CaMKII in both LTP induction and long-term memory. One of the difficulties with these studies has generally been a lack of anatomical specificity and temporal control over the genetic changes making the assignment of function to a specific brain structure or phase of memory difficult. This limitation has been overcome in studies of the NMDA receptor by using Cre recombinase and tetracycline transactivator (tTA) technology. These studies have demonstrated a requirement for hippocampal CA1-dependent NMDA receptor function in the formation of long-term spatial memory. While the molecular mechanisms of memory have been studied extensively in the hippocampus itself, little is known about the molecular mechanisms in the parahippocampal input structures (Yasuda, 2006 and references therein).
In order to directly assess the role of these structures in memory at a molecular level, a line of transgenic mice was examined in which expression of an activated form of CaMKII was limited to the parahippocampal region and regulated by the tetracycline system. Expression of activated CaMKII in the superficial layer II and III neurons of the MEC and Pr-PaS disrupted both spatial and recognition memory. Post-training activation of the kinase could disrupt previously encoded memories; however, following a 6 week consolidation period, spatial memory was insensitive to CaMKII activation (Yasuda, 2006).
In several songbird species, a specialized anterior forebrain pathway (AFP) that includes part of the avian basal ganglia has been implicated specifically in song learning. To further elucidate cellular mechanisms and circuitry involved in vocal learning, quantitative immunoblot analysis was used to determine if early song tutoring promotes within the AFP phosphorylation of calcium/calmodulin-dependent kinase II, a multifunctional kinase whose phosphorylation at threonine 286 is critical for many forms of neural plasticity and behavioral learning. In young male zebra finches likely to have begun the process of song acquisition, brief tutoring by a familiar conspecific adult promotes a dramatic increase in levels of phosphorylated CaMKII (pCaMKII) in Area X, the striatal/pallidal component of the AFP. In contrast, pCaMKII levels in this region are not elevated if 1) the tutor does not sing, 2) the tutor sings but is visually isolated from the pupil, or 3) the tutor is an unfamiliar adult. In young males that have not previously heard any conspecific song, first exposure to a song tutor produces a more modest, but significant rise in pCaMKII levels. Young females (who do not develop song behavior) exhibit no effect of tutoring on pCaMKII levels in that portion of the basal ganglia that corresponds to Area X in males. These data are consistent with the hypothesis that Area X participates in encoding and/or attaching reward value to a representation of tutor song that is accessed later to guide motor learning (Singh, 2006).
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