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Keywords:

  • Cellular specificity;
  • Electron microscopy;
  • Excitatory synapses;
  • Isoforms;
  • Phosphorylation;
  • Plasticity;
  • Postsynaptic density

Summary

  1. Top of page
  2. Summary
  3. Structure and Organization of CaMKII Subunits and Variants
  4. Anatomic Distribution of CaMKII Isoforms
  5. Cellular and Subcellular Specificity of CaMKII Expression
  6. Neuronal Excitability and CaMKII
  7. CaMKII and Epilepsy
  8. Acknowledgments
  9. Disclosure
  10. References

Calcium/calmodulin-dependent protein kinase type II (CaMKII) is a highly abundant serine/threonine kinase comprising a significant fraction of total protein in mammalian forebrain and forming a major component of the postsynaptic density. CaMKII is essential for certain forms of synaptic plasticity and memory consolidation and this is mediated through substrate binding and intramolecular phosphorylation of holoenzyme subunits. CaMKII is multifunctional; it targets a variety of cellular substrates, and this diversity depends on holoenzyme subunit composition. CaMKII comprises homooligomeric and heterooligomeric complexes generated from four subunits (α, β, δ, and γ) encoded by separate genes that are further expanded by extensive alternative splicing to more than 30 different isoforms. Much attention has been paid to understanding the regulation of CaMKII function through its structural diversity and/or substrate specificity. However, given the importance of subunit composition to holoenzyme activity, it is likely that specificity of cellular expression of CaMKII isoforms also plays a major role in regulation of enzyme function. Herein we review the cellular colocalization of CaMKII isoforms with special regard to the cell-type specificity of isoform expression in brain. In addition, we highlight the remarkable specificity of subcellular localization by the CaMKIIα isoform. In addition, we discuss the role that this cellular specificity of expression might play in propagating the type of recurrent neuronal activity associated with disorders such as temporal lobe epilepsy.

A well-functioning nervous system relies on the ability of neurons to efficiently transmit impulses to other neurons and support cells. For >60 years the focus of neuronal communication has been synaptic junctions, and tremendous advances have been made in our understanding of the molecular composition of these specialized structures (Margeta & Shen, 2010). In addition to the myriad receptors and membrane-bound proteins responsible for binding of chemical transmitters, a host of other proteins that are intracellular juxtaposed to neuronal membranes (particularly at synaptic junctions) have been identified as critical regulators of neuronal transmission. The serine/threonine kinase, calcium/calmodulin-dependent protein kinase type II (CaMKII) has emerged as a key regulator of synaptic function. CaMKII is an extremely abundant neuronal protein that is a major component of the postsynaptic density (Kennedy et al., 1983; Lisman et al., 2002; Okabe, 2007; Bayés et al., 2011) and is critical for regulating forms of synaptic plasticity, including long-term potentiation (LTP) (Lisman, 2003; Okamoto et al., 2009). CaMKII is multifunctional and regulated by intracellular concentrations of calcium; therefore, subcellular localization is a key factor in regulating enzyme function. The molecular and cellular mechanisms of CaMKII function have previously been summarized in numerous excellent reviews (Okamoto et al., 2009; Wayman et al., 2011). Herein, we focus on the anatomic specificity of CaMKII localization and how this is a key factor in regulating neuronal excitability.

Structure and Organization of CaMKII Subunits and Variants

  1. Top of page
  2. Summary
  3. Structure and Organization of CaMKII Subunits and Variants
  4. Anatomic Distribution of CaMKII Isoforms
  5. Cellular and Subcellular Specificity of CaMKII Expression
  6. Neuronal Excitability and CaMKII
  7. CaMKII and Epilepsy
  8. Acknowledgments
  9. Disclosure
  10. References

Multiple substrates of CaMKII activity have been identified, and it is generally regarded that proper targeting of cellular function is dependent on holoenzyme composition. The CaMKII holoenzyme exists as a dodecomeric complex of subunits arranged in a characteristic ring formation that is critical for the enzyme’s ability to decipher both amplitude and frequency of calcium signaling (Chao et al., 2011). Multiple subunits of CaMKII have been identified, and both homomeric and heteromeric complexes have been described (Schulman, 2004). In mammals, CaMKII subunits are encoded by four separate genes (α, β, δ, and γ), and each gene undergoes alternative splicing to generate >30 individual isoforms, which in theory can associate in a holoenzyme complex, thereby creating a wealth of diversity in potential enzymatic function (Tombes et al., 2003). The proteins coded by these genes are generally 50–60 kDa and contain highly conserved N-terminal catalytic (approximately 95%) and C-terminal association (approximately 80%) domains flanking a divergent regulatory linker domain (Hudmon & Schulman, 2002). This variability underlies the multifunctional nature of CaMKII holoenzyme activity and suggests that subunit composition is an important regulator of function. Changes in the size of the regulatory domain have been associated with the calcium frequency response of the enzyme (Bayer et al., 2002).

Anatomic Distribution of CaMKII Isoforms

  1. Top of page
  2. Summary
  3. Structure and Organization of CaMKII Subunits and Variants
  4. Anatomic Distribution of CaMKII Isoforms
  5. Cellular and Subcellular Specificity of CaMKII Expression
  6. Neuronal Excitability and CaMKII
  7. CaMKII and Epilepsy
  8. Acknowledgments
  9. Disclosure
  10. References

Despite the fact that CaMKII activity is dependent on subunit composition, colocalization studies in brain have been lacking. All four CaMKII isoforms (α, β, δ, and γ) have been localized in brain (Takaishi et al., 1992). In addition, CaMKIIδ and CaMKIIγ have been localized within multiple tissues outside the central nervous system (Tobimatsu & Fujisawa, 1989). CaMKIIα and CaMKIIβ are essentially brain specific, although an isoform of CaMKIIβ has been described in rodent pancreatic islet cells (Urquidi & Ashcroft, 1995). Nonetheless CaMKIIα and CaMKIIβ are the predominant isoforms in brain and are heavily expressed throughout the forebrain, with the highest level of expression found within neocortical and hippocampal structures (Benson et al., 1991, 1992). In general, subunit expression is both overlapping and distinct. For example, all four subunits are expressed throughout the principal cell layers of the hippocampus proper and the dentate gyrus (Sakagami & Kondo, 1993; Murray et al., 2003). In contrast, CaMKIIβ and CaMKIIγ subunits are expressed throughout most layers of the neocortex, whereas CaMKIIα is expressed in superficial (II, III) and deep (V, VI) layers and CaMKIIδ is expressed predominantly in layer II and a group of cells just superficial to the cortical white matter (Sakagami & Kondo, 1993; Murray et al., 2003).

A more detailed analysis of subunit expression at the cellular and subcellular level has been hampered by a lack of quality antibody reagents for protein detection. The vast majority of work has focused on CaMKIIα and CaMKIIβ, which are more widely expressed in brain. Existing evidence suggests that there is differential cellular specificity in colocalization of isoforms that has profound effects on holoenzyme function. In hippocampus, virtually all neurons of the pyramidal cell layer as well as the granule cells of the dentate gyrus are immunopositive for both CaMKIIα and CaMKIIβ, suggesting a high degree of colocalization (Ochiishi et al., 1994). Consistent with this observation, the subcellular localization of CaMKIIα in hippocampal neurons is modulated by expression of CaMKIIβ subunits (Shen et al., 1998). Specifically, CaMKIIα is shifted from a cytosolic cellular compartment to neuronal spines in CA1 pyramidal neurons through binding of CaMKIIβ to F-actin. In contrast, the localization of CaMKIIα in spines in hippocampal neurons is drastically reduced in CaMKIIβ knockout mice (Borgesius et al., 2011). Concomitantly, these mice are deficient in N-methyl-d-aspartate (NMDA)–dependent long-term potentiation (LTP) and perform poorly in a conditional learning paradigm, activities that have previously been associated with synaptic CaMKIIα expression (Okamoto et al., 2009; Lucchesi et al., 2011). These observations illustrate clearly the capacity for CaMKII-subunit heteroligomerization in vivo and highlight the notion that cellular specificity of subunit expression can profoundly influence enzyme function and subsequent behavior. Therefore, knowing the cellular specificity of subunit expression is critical for understanding CaMKII influence on brain function.

Cellular and Subcellular Specificity of CaMKII Expression

  1. Top of page
  2. Summary
  3. Structure and Organization of CaMKII Subunits and Variants
  4. Anatomic Distribution of CaMKII Isoforms
  5. Cellular and Subcellular Specificity of CaMKII Expression
  6. Neuronal Excitability and CaMKII
  7. CaMKII and Epilepsy
  8. Acknowledgments
  9. Disclosure
  10. References

Of the four CaMKII isoforms, CaMKIIα is unique in that it displays a highly specific cellular and subcellular pattern of expression (Fig. 1). The first hint of cell-specific expression came from studies on the mRNA expression in monkey forebrain. A distinct complementarity in the expression pattern between CaMKIIα mRNA and mRNA for glutamic acid decarboxylase (GAD), the rate-limiting enzyme for γ-aminobutyric acid (GABA) production was observed throughout nuclei of the diencephalon as well as basal ganglia and certain midbrain regions (Benson et al., 1991). Notably, cells in nuclei that were predominantly GABAergic, such as the reticular thalamic nucleus or the globus pallidus, were devoid of CaMKIIα mRNA. Subsequent immunolabeling of CaMKIIα and GAD expression in monkey sensorimotor cortex confirmed that these molecules were localized in mutually exclusive cells and that CaMKIIα expression was localized to excitatory neuronal populations (Jones et al., 1994). In other studies in rodent, a similar segregation of CaMKIIα and GABAergic neurons was reported (Benson et al., 1992; Ochiishi et al., 1994; Sík et al., 1998). Here, colocalization with immunostaining using antibodies against CaMKIIα and GAD or GABA clearly illustrated that CaMKIIα was absent from inhibitory GAD/GABA positive cells throughout the forebrain as well as nuclei of the brainstem and spinal cord. This was most evident in hippocampus, cerebral cortex, and thalamus, where CaMKIIα was expressed in cells giving rise to excitatory projections and absent in local inhibitory interneurons. Two exceptions to this rule were noted. First, cells within the commissural nucleus of the stria terminalis were positive for both GABA and CaMKIIα. Second, cerebellar Purkinje cells also coexpressed GABA and CaMKIIα, although at lower levels than did forebrain neurons. Although the significance of this coexpression is unknown, it is interesting that in contrast to local interneurons throughout the forebrain whose projections terminate in a relatively confined area close to the cell of origin, Purkinje cells are the output neurons of the cerebellum and their terminals end on deep cerebellar nuclei. These observations suggest that the regulation of cellular specificity is related to the organization of neuronal circuitry and not simply transmitter phenotype. In addition, like the sequence and structural organization of CaMKII itself (Tombes et al., 2003), the cell specify of CaMKIIα expression is conserved across species.

image

Figure 1.   The α subunit of CaMKII is specifically localized to excitatory synaptic contacts onto excitatory forebrain neurons. (A) Cartoon drawings depicting a GABAergic inhibitory neuron from reticular thalamic nucleus (RTN) and a glutamatergic excitatory dorsal thalamic neuron from rodent ventrobasal thalamic nucleus (VB). Only the VB neurons express CaMKIIα. Synaptic input from local interneurons (brown), thalamocortical (green), corticothalamic (red), and lemniscal (blue) projections are depicted as well. (B) Section from thalamic VB stained by preembedding immunostaining for CaMKIIα (arrowheads) and by postembedding immunostaining for glutamate (black grains) illustrating the localization of CaMKIIα at excitatory synapses (red arrowheads) on a glutamatergic thalamic VB neuron. Abbreviations; Glu(+)T, typical large lemniscal glutamatergic axon terminal; AP, thalamic relay cell spiny appendage. (C) Cartoon drawings depicting synaptic contacts onto an excitatory VB neuron. CaMKIIα is localized to excitatory synaptic contacts but not inhibitory synapses, even when they are observed on the same CaMKIIα-expressing neuron. Note that CaMKIIα is found both presynaptically and postsynaptically at corticothalamic synapses but only postsynaptically at lemniscal inputs.

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CaMKIIα is known to be a major component of the postsynaptic density, which raised the intriguing possibility that synaptic localization of the enzyme might also show specificity. The original identification of CaMKIIα as the major component of postsynaptic densities came from protein sedimentation methods that resulted in fractions enriched for type I, or excitatory, synaptic junctions (Kennedy et al., 1983). Subsequent immunoelectron microscopy confirmed that CaMKII was present at excitatory synapses in hippocampal neurons, but the antibody used did not distinguish between CaMKIIα and CaMKIIβ, and no attempt at examining the specificity of synaptic localization was made (Ouimet et al., 1984). However, using a combination of preembedding and postembedding immunoelectron microscopy, Liu and Jones (1996) provided the first clear evidence that CaMKIIα was localized to excitatory but absent from inhibitory synaptic junctions in rodent cerebral cortex and thalamus. CaMKIIα was associated with only asymmetric postsynaptic thickenings in which presynaptic terminals contained glutamate. CaMKIIα was also found in certain presynaptic terminals, and these were always glutamatergic and associated with cell bodies known to express CaMKIIα mRNA. Notably, not all asymmetric synapses were CaMKIIα positive. However, all symmetric synaptic contacts associated with presynaptic GABA terminals were devoid of CaMKIIα, even when terminating on a CaMKII-positive cell (Fig. 1). Similar observations were made in the CA1 region of rat hippocampus, suggesting that specific excitatory synaptic localization is a general feature of CaMKIIα (Liu & Jones, 1997).

Although in vivo evidence from fixed brain sections indicates that CaMKIIα is specifically localized to excitatory synapses exclusively in excitatory neurons, in certain circumstances, CaMKIIα may translocate to inhibitory synapses where it interacts with GABA-receptor complexes (Marsden et al., 2010). In vitro phosphorylation studies have identified intracellular conserved CaMKII phosphorylation domains on several GABAA-receptor subunits (McDonald & Moss, 1994, 1997; Houston et al., 2007) expressed in multiple brain regions including cerebral cortex and thalamus. In cultures of dissociated hippocampal neurons, surface membrane expression of GABAA receptors can be regulated by a chemically induced form of long-term depression, and this is dependent of Ca2+and CaMKII (Marsden et al., 2007). Furthermore, in cultured hippocampal neurons, stimulation with NMDA or glutamate induced clustering of endogenous CaMKIIα as well as transfected green fluorescent protein–tagged CaMKIIα (Marsden et al., 2010). This clustering occurred on dendritic shafts and the accumulated puncta colocalized with inhibitory synaptic markers. Therefore, similar to the activity-dependent translocation of CaMKIIα to excitatory synapses, the kinase can be induced to cluster at inhibitory synapses as well. The fact that CaMKIIα clustering at inhibitory synapses has been detected in only in vitro preparations to date suggests this occurs only under rare circumstances or at levels far below those detectable by immunoreactivity with existing antibodies. Therefore, until evidence is provided of in vivo inhibitory synaptic clustering, these observations remain an anomaly.

Neuronal Excitability and CaMKII

  1. Top of page
  2. Summary
  3. Structure and Organization of CaMKII Subunits and Variants
  4. Anatomic Distribution of CaMKII Isoforms
  5. Cellular and Subcellular Specificity of CaMKII Expression
  6. Neuronal Excitability and CaMKII
  7. CaMKII and Epilepsy
  8. Acknowledgments
  9. Disclosure
  10. References

The selective expression of CaMKIIα at excitatory synapses onto excitatory neurons could have profound effects on synaptic plasticity and on the propagation of neuronal activity. The nature of CaMKII activation makes it a natural candidate for converting transient synaptic activation into longer term signals. In response to transient influxes of Ca2+ and in the presence of calmodulin and ATP, CaMKII subunits undergo rapid autophosphorylation at characteristic residues (threonine 286 in CaMKIIα) through intramolecular and/or intermolecular interactions, which enable CaMKII to be active independent of Ca2+/calmodulin (Hudmon & Schulman, 2002; Schulman, 2004). Ca2+/calmodulin-independent enzyme activity can continue to phosphorylate substrates, including CaMKII itself and subsequent Ca2+ influx, leads to increasing gradients of independent CaMKII activation. This ability of CaMKII to remain active beyond brief bursts of Ca2+ underlies its ability to detect frequency and amplitude of Ca2+ flux and thereby encode spike frequency at synapses (De Koninck & Schulman, 1998; Bayer et al., 2002). Consistent with this idea, perhaps the most well-characterized function of CaMKII is its role in synaptic plasticity associated with LTP, which is a sustained increase in synaptic transmission resulting from high-frequency stimulation. CaMKII is necessary and sufficient for the induction of LTP and inhibitors of CaMKII or genetic ablation block LTP induction (Malenka et al., 1989; Malinow et al., 1989; Silva et al., 1992). The mechanism of this regulation is not known but in short is thought to involve Ca2+ influx through NMDA receptors and the subsequent translocation of CaMKIIα containing holoenzyme complexes to synaptic sites (Merrill et al., 2005; Skelding & Rostas, 2009). Subsequent docking of CaMKII to NMDA receptors (in particular NR1 and NR2B subunits), and possibly other synaptic-associated molecules, retains activated kinase which in turn leads to enhanced surface expression of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptors and potentiation of transmission. Introduction of activated kinase in vivo occludes LTP and induces surface expression of AMPA receptors in hippocampus (Boehm & Malinow, 2005).

Most studies on synaptic plasticity and CaMKII have focused on CaMKIIα subunit, although recent efforts have identified a novel nonenzymatic role for CaMKIIβ in regulating F-actin stabilization at activated synapses (Shen et al., 1998; Borgesius et al., 2011). Therefore, these studies have all centered on excitatory synaptic contacts onto CaMKIIα-positive excitatory neurons. Although traditionally thought not to express forms of activity-dependent synaptic plasticity like those seen on excitatory projection neurons (McBain et al., 1999), it is now established that aspiny interneurons can indeed display forms of prolonged activity-dependent plasticity (Laezza & Dingledine, 2011). The rules for induction of synaptic plasticity differ from those in principal excitatory neurons, however, possibly reflecting a lack of CaMKIIα. For example, NMDA receptors are not necessary for induction of LTP in hippocampal interneurons and the composition of many AMPA receptors lack a GluA2 subunit leading to a highly Ca2+ permeable AMPA receptor that operates more efficiently at hyperpolarizing membrane potentials. Kinase-mediated signaling is more heterologous in interneurons, and a variety have been implicated in synaptic plasticity including protein kinase A and protein kinase C. CaMKII isoforms other than CaMKIIα might play a role in synaptic plasticity in inhibitory interneurons, since general inhibitors of CaMKII function have been shown to block induction of plasticity (Houston et al., 2009). Whether the absence of CaMKIIα precipitates separate rules for synaptic plasticity in interneurons remains to be investigated. It would be intriguing to see whether overexpression of CaMKIIα in interneurons shifted plasticity requirements to those typical for principal relay neurons and indeed whether spine formation could be induced in these cells as has been reported for other synaptic molecules associated with plasticity in principal excitatory neurons (Passafaro et al., 2003). But a recent report showing that genetic ablation of GluA2 AMPA receptors in CA1 pyramidal neurons of the hippocampus induced a Ca2+-dependent LTP that was independent of NMDA-receptor activation and CaMKII function, suggesting that synaptic plasticity in interneurons can be completely independent of CaMKII (Asrar et al., 2009).

CaMKII and Epilepsy

  1. Top of page
  2. Summary
  3. Structure and Organization of CaMKII Subunits and Variants
  4. Anatomic Distribution of CaMKII Isoforms
  5. Cellular and Subcellular Specificity of CaMKII Expression
  6. Neuronal Excitability and CaMKII
  7. CaMKII and Epilepsy
  8. Acknowledgments
  9. Disclosure
  10. References

Recent hypotheses on the origins of neurologic disease have focused on the notion of dysfunctional molecular mechanisms of synaptic transmission that result in widespread changes in the function of neuronal circuits. In particular, disorders of recurrent seizure activity, epilepsies, have been associated with alterations of ligand and/or voltage-gated ion channels including NMDA and AMPA receptors as well as intracellular signaling molecules at synapses, and these changes are thought to contribute to plasticity of the epileptic brain and the propagation of recurrent neuronal activity in these disorders (McNamara et al., 2006; Jefferys, 2010). It is not surprising that CaMKII, the major component of excitatory synapses, has been implicated in the etiology of seizure activity. A decrease in CaMKII activity has been consistently reported following the onset of epileptiform activity in a variety of models. Reduced autophosphorylation of CaMKII, a measure of kinase activation, has been reported following status epilepticus brought on by administration of GABA-receptor antagonists (Bronstein et al., 1988; Perlin et al., 1992), as well as in kindled rats (Wasterlain & Farber, 1984; Goldenring et al., 1986; Wu et al., 1990), following pilocarpine-induced seizures (Churn et al., 2000a; Kochan et al., 2000) and in a kainic acid–induced model of epilepsy (Yamagata et al., 2006). Reduced phosphorylation of CaMKII substrates has also been reported following onset of seizure activity (Singleton et al., 2005; Yamagata et al., 2006). Consistent with a role for reduced CaMKII activity in the etiology of seizures, mice lacking CaMKIIα exhibit epileptiform activity in response to normally subconvulsive brain stimuli (Butler et al., 1995). What’s more, in dissociated cultures of hippocampus or cerebral cortex, inhibition of CamKIIα expression (Churn et al., 2000b) or a generalized reduction in CaMKII activity (Ashpole et al., 2012) produced eplileptic-like activity. Recently, it was shown that inhibiting autophosphorylation of CaMKIIα reduced the afterhyperpolarization following repetitive spike firing leading to prolonged neuronal excitability, suggesting a possible mechanistic link between loss of CaMKII and epileptogenesis (Liraz et al., 2009; Sametsky et al., 2009).

These data strongly suggest that loss of CaKMII activity at synapses is linked with onset of epileptogenesis. It is likely that loss of CaMKII activation is not the only factor, but instead is linked with other changes in the synapse related to glutamate receptor activation and downstream signaling. But what are the mechanisms leading to reduced CaMKII activation? We and others have shown that following seizure onset in a variety of epilepsy models, CaMKIIα messenger RNA is transiently downregulated (Murray et al., 1995, 2003). The peak in decreased mRNA coincides with reduced CaMKII activation following seizure activity, suggesting that the two are causally linked. Contrary to this notion, however, is the observation that total protein levels of CaMKII were not decreased following the onset of recurrent seizure activity (Fig. 2) (Solà et al., 1998; Kochan et al., 2000; Yamagata et al., 2006). In fact, at prolonged timepoints following seizure onset (>48 h), we found an increase in CaMKIIα total protein (Fig. 2). Similar increases in CaMKIIα were observed in cultured cortical slices following treatment with the GABA-receptor antagonist bicuculline and the potassium channel antagonist 4-AP, and this could be blocked by inhibiting NMDA-receptor function (Fig. 2). Currently the reasons for discordant observations of decreased CaMKII activity and mRNA and increased protein expression following induction of seizure activity remain unknown. However, a plausible explanation could involve two independent regulatory mechanisms. First, reduced activity of CaMKII could result from enhanced dephosphorylation. Kinase activity is balanced throughout the nervous system by the activity of phosphatases, which selectively dephosphorylate proteins (MacDonald et al., 2006). Two protein phosphatases, PP1 and PP2A, have been shown to regulate phosphorylation of CaMKII (Strack et al., 1997). Although little attention has been paid to the regulation of phosphatase activity in epilepsy, at least one study suggests that increased PP1/PP2A activity leads to loss of CaMKII phosphorylation and subsequent activity following seizure onset (Dong & Rosenberg, 2004). Second, the decreased expression of CaMKIIα mRNA following seizures may reflect a transient deregulation in mRNA stability. Inhibitors of transcription increase basal levels of CaMKIIα mRNA suggest there is an ongoing active degradation of CaMKII mRNA in the basal state (Murray et al., 2003). Although the proteins responsible for this degradation are unknown, onset of seizure activation could increase their abundance, thereby leading to decreased CaMKII mRNA levels. Translation of CaMKII protein from remaining mRNA may also be elevated following seizures, which would offset reduced mRNA levels and maintain protein at a constant level. At later times, when CaMKII mRNA has returned to normal (36–48 h), the elevated translation of CaMKII would lead to increased protein levels. Evidence for such a mechanism is lacking, but this presents a plausible hypothesis. Regardless of the mechanism, the fact remains that CaMKII activity is reduced in the relative short term (24 h) following seizure onset and that this is likely to be an important factor in the development of recurrent seizure activity.

image

Figure 2.   Increased expression of CaMKIIα following seizure activity. (A) Bright-field images taken from frontal sections of rat somatosensory cortex show increased immunoreactivity for CaMKIIα 48 h following the onset of recurrent seizures induced by intraperitoneal injection of kainic acid (10 mg/kg). Control animals received vehicle injection. (B) Western blot analysis of total protein homogenate from somatosensory cortex confirmed the increase in CaMKIIα following kainaite-induced seizures. (C) CaMKIIα protein expression was also increased by seizure induction in vitro following treatment of cultured cortical slices by coapplication of the GABA-receptor antagonist bicuculline methiodide and the potassium channel antagonist 4-AP. The lower band (arrow) indicates immunoreactivity against β-Actin, used as a control for normalization of protein loading. (D) Quantification of seizure-induced changes in normalized (relative to β-Actin expression) CaMKIIα protein expression in cultured cortical slices shows increased expression 6 and 24 h after onset of drug application. Increases in CaMKIIα protein expression were blocked when the NMDA-receptor antagonist (2R)-amino-5-phosphonovaleric acid was present in the media. Scale bar in A = 100 μm.

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Acknowledgments

  1. Top of page
  2. Summary
  3. Structure and Organization of CaMKII Subunits and Variants
  4. Anatomic Distribution of CaMKII Isoforms
  5. Cellular and Subcellular Specificity of CaMKII Expression
  6. Neuronal Excitability and CaMKII
  7. CaMKII and Epilepsy
  8. Acknowledgments
  9. Disclosure
  10. References

I was privileged to have had the opportunity to work closely with Jürgen Wenzel over the last 5 or so years. As it turns out we crossed paths more than 20 years ago while I was an undergraduate student at UC Irvine. I regret deeply that we never formally met back then. The time we had to collaborate has been all too brief. Jürgen is a rare breed of neuroanatomist that I fear is quickly becoming extinct. His knowledge of anatomy goes well beyond what can be trapped in books, and his technical expertise is unsurpassed. I only hope he continues to educate those of us struggling to understand the structure of the brain.

Disclosure

  1. Top of page
  2. Summary
  3. Structure and Organization of CaMKII Subunits and Variants
  4. Anatomic Distribution of CaMKII Isoforms
  5. Cellular and Subcellular Specificity of CaMKII Expression
  6. Neuronal Excitability and CaMKII
  7. CaMKII and Epilepsy
  8. Acknowledgments
  9. Disclosure
  10. References

None of the authors has any conflicts of interest to disclose.

We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

References

  1. Top of page
  2. Summary
  3. Structure and Organization of CaMKII Subunits and Variants
  4. Anatomic Distribution of CaMKII Isoforms
  5. Cellular and Subcellular Specificity of CaMKII Expression
  6. Neuronal Excitability and CaMKII
  7. CaMKII and Epilepsy
  8. Acknowledgments
  9. Disclosure
  10. References