Local Inhibition of PERK Enhances Memory and Reverses Age-Related Deterioration of Cognitive and Neuronal Properties.

Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is one of four known kinases that respond to cellular stress by deactivating the eukaryotic initiation factor 2 α (eIF2α) or other signal transduction cascades. Recently, both eIF2α and its kinases were found to play a role in normal and pathological brain function. Here, we show that reduction of either the amount or the activity of PERK, specifically in the CA1 region of the hippocampus in young adult male mice, enhances neuronal excitability and improves cognitive function. In addition, this manipulation rescues the age-dependent cellular phenotype of reduced excitability and memory decline. Specifically, the reduction of PERK expression in the CA1 region of the hippocampus of middle-aged male mice using a viral vector rejuvenates hippocampal function and improves hippocampal-dependent learning. These results delineate a mechanism for behavior and neuronal aging and position PERK as a promising therapeutic target for age-dependent brain malfunction.SIGNIFICANCE STATEMENT We found that local reduced protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) expression or activity in the hippocampus enhances neuronal excitability and cognitive function in young normal mice, that old CA1 pyramidal cells have reduced excitability and increased PERK expression that can be rescued by reducing PERK expression in the hippocampus, and that reducing PERK expression in the hippocampus of middle-aged mice enhances hippocampal-dependent learning and memory and restores it to normal performance levels of young mice. These findings uncover an entirely new biological link among PERK, neuronal intrinsic properties, aging, and cognitive function. Moreover, our findings propose a new way to fight mild cognitive impairment and aging-related cognitive deterioration.

Variations in Acetylcholinesterase Activity within Human Cortical Pyramidal Neurons Across Age and Cognitive Trajectories.

We described an extensive network of cortical pyramidal neurons in the human brain with abundant acetylcholinesterase (AChE) activity. Emergence of these neurons during childhood/adolescence, attainment of highest density in early adulthood, and virtual absence in other species led us to hypothesize involvement of AChE within these neurons in higher cortical functions. The current study quantified the density and staining intensity of these neurons using histochemical procedures. Few faintly stained AChE-positive cortical pyramidal neurons were observed in children/adolescents. These neurons attained their highest density and staining intensity in young adulthood. Compared with the young adult group, brains of cognitively normal elderly displayed no significant change in numerical density but a significant decrease in staining intensity of AChE-positive cortical pyramidal neurons. Brains of elderly above age 80 with unusually preserved memory performance (SuperAgers) showed significantly lower staining intensity and density of these neurons when compared with same-age peers. Conceivably, low levels of AChE activity could enhance the impact of acetylcholine on pyramidal neurons to counterbalance other involutional factors that mediate the decline of memory capacity during average aging. We cannot yet tell if elderly with superior memory capacity have constitutively low neuronal AChE levels or if this feature reflects adaptive neuroplasticity.

Rapid regulation of sialidase activity in response to neural activity and sialic acid removal during memory processing in rat hippocampus.

Sialidase cleaves sialic acids on the extracellular cell surface as well as inside the cell and is necessary for normal long-term potentiation (LTP) at mossy fiber-CA3 pyramidal cell synapses and for hippocampus-dependent spatial memory. Here, we investigated in detail the role of sialidase in memory processing. Sialidase activity measured with 4-methylumbelliferyl-α-d-N-acetylneuraminic acid (4MU-Neu5Ac) or 5-bromo-4-chloroindol-3-yl-α-d-N-acetylneuraminic acid (X-Neu5Ac) and Fast Red Violet LB was increased by high-K+-induced membrane depolarization. Sialidase activity was also increased by chemical LTP induction with forskolin and activation of BDNF signaling, non-NMDA receptors, or NMDA receptors. The increase in sialidase activity with neural excitation appears to be caused not by secreted sialidase or by an increase in sialidase expression but by a change in the subcellular localization of sialidase. Astrocytes as well as neurons are also involved in the neural activity-dependent increase in sialidase activity. Sialidase activity visualized with a benzothiazolylphenol-based sialic acid derivative (BTP3-Neu5Ac), a highly sensitive histochemical imaging probe for sialidase activity, at the CA3 stratum lucidum of rat acute hippocampal slices was immediately increased in response to LTP-inducible high-frequency stimulation on a time scale of seconds. To obtain direct evidence for sialic acid removal on the extracellular cell surface during neural excitation, the extracellular free sialic acid level in the hippocampus was monitored using in vivo microdialysis. The free sialic acid level was increased by high-K+-induced membrane depolarization. Desialylation also occurred during hippocampus-dependent memory formation in a contextual fear-conditioning paradigm. Our results show that neural activity-dependent desialylation by sialidase may be involved in hippocampal memory processing.

Corticosteroid-induced dendrite loss and behavioral deficiencies can be blocked by activation of Abl2/Arg kinase.

Stressor exposure induces neuronal remodeling in specific brain regions. Given the persistence of stress-related illnesses, key next steps in determining the contributions of neural structure to mental health are to identify cell types that fail to recover from stressor exposure and to identify "trigger points" and molecular underpinnings of stress-related neural degeneration. We evaluated dendrite arbor structure on hippocampal CA1 pyramidal neurons before, during, and following prolonged exposure to one key mediator of the stress response - corticosterone (cortisol in humans). Basal dendrite arbors progressively simplified during a 3-week exposure period, and failed to recover when corticosterone was withdrawn. Corticosterone exposure decreased levels of the dendrite stabilization factor Abl2/Arg nonreceptor tyrosine kinase and phosphorylation of its substrates p190RhoGAP and cortactin within 11days, suggesting that disruption of Arg-mediated signaling may trigger dendrite arbor atrophy and, potentially, behavioral abnormalities resulting from corticosterone exposure. To test this, we administered the novel, bioactive Arg kinase activator, 5-(1,3-diaryl-1H-pyrazol-4-yl)hydantoin, 5-[3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl]-2,4-imidazolidinedione (DPH), in conjunction with corticosterone. We found that repeated treatment corrected CA1 arbor structure, otherwise simplified by corticosterone. DPH also corrected corticosterone-induced errors in a hippocampal-dependent reversal learning task and anhedonic-like behavior. Thus, pharmacological compounds that target cytoskeletal regulators, rather than classical neurotransmitter systems, may interfere with stress-associated cognitive decline and mental health concerns.

Transient Cerebral Ischemia Alters GSK-3ß and p-GSK-3ß Immunoreactivity in Pyramidal Neurons and Induces p-GSK-3ß Expression in Astrocytes in the Gerbil Hippocampal CA1 Area.

Glycogen synthase kinase 3ß (GSK-3ß) is a key downstream protein in the PI3K/Akt pathway. Phosphorylation of serine 9 of GSK-3ß (GSK-3ß activity inhibition) promotes cell survival. In this study, we examined changes in expressions of GSK-3ß and phosphorylation of GSK-3ß (p-GSK-3ß) in the gerbil hippocampal CA1 area after 5 min of transient cerebral ischemia. GSK-3ß immunoreactivity in the CA1 area was increased in pyramidal cells at 6 h after ischemia-reperfusion. It was decreased in CA1 pyramidal cells from 12 h after ischemia-reperfusion, and hardly detected in the CA1 pyramidal cells at 5 days after ischemia-reperfusion. p-GSK-3ß immunoreactivity was slightly decreased in CA1 pyramidal cells at 6 and 12 h after ischemia-reperfusion. It was significantly increased in these cells at 1 and 2 days after ischemia-reperfusion. Five days after ischemia-reperfusion, p-GSK-3ß immunoreactivity was hardly found in CA1 pyramidal cells. However, p-GSK-3ß immunoreactivity was strongly expressed in astrocytes primarily distributed in strata oriens and radiatum. In conclusion, GSK-3ß and p-GSK-3ß were significantly changed in pyramidal cells and/or astrocytes in the gerbil hippocampal CA1 area following 5 min of transient cerebral ischemia. This finding indicates that GSK-3ß and p-GSK-3ß are closely related to delayed neuronal death.

SCYL2 Protects CA3 Pyramidal Neurons from Excitotoxicity during Functional Maturation of the Mouse Hippocampus.

Neuronal death caused by excessive excitatory signaling, excitotoxicity, plays a central role in neurodegenerative disorders. The mechanisms regulating this process, however, are still incompletely understood. Here we show that the coated vesicle-associated kinase SCYL2/CVAK104 plays a critical role for the normal functioning of the nervous system and for suppressing excitotoxicity in the developing hippocampus. Targeted disruption of Scyl2 in mice caused perinatal lethality in the vast majority of newborn mice and severe sensory-motor deficits in mice that survived to adulthood. Consistent with a neurogenic origin of these phenotypes, neuron-specific deletion of Scyl2 also caused perinatal lethality in the majority of newborn mice and severe neurological defects in adult mice. The neurological deficits in these mice were associated with the degeneration of several neuronal populations, most notably CA3 pyramidal neurons of the hippocampus, which we analyzed in more detail. The loss of CA3 neurons occurred during the functional maturation of the hippocampus and was the result of a BAX-dependent apoptotic process. Excessive excitatory signaling was present at the onset of degeneration, and inhibition of excitatory signaling prevented the degeneration of CA3 neurons. Biochemical fractionation reveals that Scyl2-deficient mice have an altered composition of excitatory receptors at synapses. Our findings demonstrate an essential role for SCYL2 in regulating neuronal function and survival and suggest a role for SCYL2 in regulating excitatory signaling in the developing brain. Significance statement: Here we examine the in vivo function of SCYL2, an evolutionarily conserved and ubiquitously expressed protein pseudokinase thought to regulate protein trafficking along the secretory pathway, and demonstrate its importance for the normal functioning of the nervous system and for suppressing excitatory signaling in the developing brain. Together with recent studies demonstrating a role of SCYL1 in preventing motor neuron degeneration, our findings clearly establish the SCY1-like family of protein pseudokinases as key regulators of neuronal function and survival.

Effect of Combined Stress on Morphological Changes and Expression of NO Synthases in Rat Ventral Hippocampus.

Adult rats were subjected to 7-day combined stress with stochastic changes of stressors of different modalities (noise, vibration, pulsating bright light) along with mobility restriction and elevated temperature in the chamber during stress exposures (daily 30-min sessions). Circulatory disorders, inhibition of endothelial NO-synthase expression in endothelial cells of the microcirculatory bed, perivascular edema, pronounced degenerative changes, and enhanced expression of inducible NO synthase in CA3 pyramidal neurons in the ventral hippocampus of stressed 12-month-old rats were observed. These findings can attest to the involvement NOdependent mechanisms and different contribution of NO synthase isoforms into the formation of hippocampal neuronal damage.

Decreased NOS1 expression in the anterior cingulate cortex in depression.

Decreased function of the anterior cingulate cortex (ACC) is crucially involved in the pathogenesis of depression. A key role of nitric oxide (NO) has also been proposed. We aimed to determine the NO content in the cerebrospinal fluid (CSF) and the expression of NO synthase (NOS) isoforms, that is, NOS1, NOS2, and NOS3 in the ACC in depression. In depressive patients, CSF-NOx levels (the levels of the NO metabolites nitrite and nitrate) were significantly decreased (P = 0.007), indicating a more general decrease of NO production in this disorder. This agreed with a trend toward lower NOS1-mRNA levels (P = 0.083) and a significant decrease of NOS1-immunoreactivity (ir) (P = 0.043) in ACC. In controls, there was a significant positive correlation between ACC-NOS1-ir cell densities and their CSF-NOx levels. Furthermore, both localization of NOS1 in pyramidal neurons that are known to be glutamatergic and co-localization between NOS1 and GABAergic neurons were observed in human ACC. The diminished ACC-NOS1 expression and decreased CSF-NOx levels may be involved in the alterations of ACC activity in depression, possibly by affecting glutamatergic and GABAergic neurotransmission.

Down-regulation of MET in hippocampal neurons of Alzheimer's disease brains.

We found that mRNA of MET, the receptor of hepatocyte growth factor (HGF), is significantly decreased in the hippocampus of Alzheimer's disease (AD) patients. Therefore, we tried to determine the cellular component-dependent changes of MET expressions. In this study, we examined cellular distribution of MET in the cerebral neocortices and hippocampi of 12 AD and 11 normal controls without brain diseases. In normal brains, MET immunoreactivity was observed in the neuronal perikarya and a subpopulation of astrocytes mainly in the subpial layer and white matter. In AD brains, we found marked decline of MET in hippocampal pyramidal neurons and granule cells of dentate gyrus. The decline was more obvious in the pyramidal neurons of the hippocampi than that in the neocortical neurons. In addition, we found strong MET immunostaining in reactive astrocytes, including those near senile plaques. Given the neurotrophic effects of the HGF/MET pathway, this decline may adversely affect neuronal survival in AD cases. Because it has been reported that HGF is also up-regulated around senile plaques, ß-amyloid deposition might be associated with astrocytosis through the HGF signaling pathway.

Anoxia-induced NMDA receptor activation opens pannexin channels via Src family kinases.

Anoxic depolarization of pyramidal neurons results from a large inward current that is activated, in part, by excessive glutamate release during exposure to anoxia/ischemia. Pannexin-1 (Panx1) channels can be activated both by ischemia and NMDA receptors (NMDARs), but the mechanisms of Panx1 activation are unknown. We used whole-cell recordings to show that pharmacological inhibition or conditional genetic deletion of Panx1 strongly attenuates the anoxic depolarization of CA1 pyramidal neurons in acute brain slices from rats and mice. Anoxia or exogenous NMDA activated Src family kinases (SFKs), as measured by increased phosphorylation of SFKs at Y416. The SFK inhibitor PP2 prevented Src activation and Panx1 opening during anoxia. A newly developed interfering peptide that targets the SFK consensus-like sequence of Panx1 (Y308) attenuated the anoxic depolarization (AD) without affecting SFK activation. Importantly, the NMDAR antagonists, D-APV and R-CPP, attenuated AD currents carried by Panx1, and the combined application of D-APV and (10)panx (a Panx1 blocker) inhibited AD currents to the same extent as either blocker alone. We conclude that activation of NMDARs during anoxia/ischemia recruits SFKs to open Panx1, leading to sustained neuronal depolarizations.

Inhibition of protein kinase Mζ disrupts the stable spatial discharge of hippocampal place cells in a familiar environment.

It is widely held that spatial computations in the rodent hippocampus require the location-specific discharge of place cells that together form a stable cognitive map used to solve and perform spatial tasks. It is not known, however, if map stability requires persistent hippocampal synaptic strength changes that are vulnerable to blockade of protein kinase Mζ (PKMζ) phosphorylation activity, a manipulation that reverses hippocampal LTP and disrupts multiple forms of long-term memory. Here we report that acute intrahippocampal inhibition of PKMζ disrupts place cell activity in a familiar environment, where the map is expected to be stable. After this disruption, new, stable spatial firing patterns can later form, but the new and original maps are unrelated even though the rat is exposed to a constant environment. We therefore propose that the previously demonstrated erasure of stored spatial memory and the disruption of place cell firing are parallel effects of PKMζ blockade. We similarly propose that the known sparing of new spatial memory formation depends on the sparing of new map formation. On these bases, we argue that the loss of the map used to perform a practiced spatial task leads to behavioral performance deficits, and that synaptic plasticity maintained by PKMζ, which stabilizes the map, is essential for the proper expression of spatial memory.

Functional proteomics related to energy metabolism of synaptosomes from different neuronal systems of rat hippocampus during aging.

The effect of aging on hippocampus is often confounded by diseases that commonly occur in the elderly. In this research, functional proteomics was used to characterize age-related changes in energy metabolism of different neuronal pathways within the hippocampus of Wistar rats aged 2, 6, 12, 18, and 24 months. The "large" synaptosomes, derived from glutamatergic mossy fiber endings connecting granule cells of dentate gyrus with apical dendrites of CA3 pyramidal cells, and the "small" synaptosomes, derived from the cholinergic small nerve endings of septo-hippocampal fibers, whose projections reach CA1 pyramidal cells, were isolated. Because most brain disorders are associated with bioenergetic changes, the maximum rate (V(max)) of selected enzymes of glycolysis, Krebs cycle, glutamate and amino acids metabolism, and acetylcholine catabolism were evaluated. The results show that "large" and "small" synaptosomes possess specific and independent metabolic features coherently with the selective vulnerability of the respective hippocampal subfields to Alzheimer's disease and cerebral ischemia. This study represents a reliable model to study in vivo (i) the physiopathological molecular mechanisms of some brain diseases dependent on energy metabolism, (ii) the responsiveness to noxious stimuli, and (iii) the effects of drugs, discriminating their action sites at subcellular level.

Ceramide levels regulated by carnitine palmitoyltransferase 1C control dendritic spine maturation and cognition.

The brain-specific isoform carnitine palmitoyltransferase 1C (CPT1C) has been implicated in the hypothalamic regulation of food intake and energy homeostasis. Nevertheless, its molecular function is not completely understood, and its role in other brain areas is unknown. We demonstrate that CPT1C is expressed in pyramidal neurons of the hippocampus and is located in the endoplasmic reticulum throughout the neuron, even inside dendritic spines. We used molecular, cellular, and behavioral approaches to determine CPT1C function. First, we analyzed the implication of CPT1C in ceramide metabolism. CPT1C overexpression in primary hippocampal cultured neurons increased ceramide levels, whereas in CPT1C-deficient neurons, ceramide levels were diminished. Correspondingly, CPT1C knock-out (KO) mice showed reduced ceramide levels in the hippocampus. At the cellular level, CPT1C deficiency altered dendritic spine morphology by increasing immature filopodia and reducing mature mushroom and stubby spines. Total protrusion density and spine head area in mature spines were unaffected. Treatment of cultured neurons with exogenous ceramide reverted the KO phenotype, as did ectopic overexpression of CPT1C, indicating that CPT1C regulation of spine maturation is mediated by ceramide. To study the repercussions of the KO phenotype on cognition, we performed the hippocampus-dependent Morris water maze test on mice. Results show that CPT1C deficiency strongly impairs spatial learning. All of these results demonstrate that CPT1C regulates the levels of ceramide in the endoplasmic reticulum of hippocampal neurons, and this is a relevant mechanism for the correct maturation of dendritic spines and for proper spatial learning.

Effects of transient cerebral ischemia on the expression of DNA methyltransferase 1 in the gerbil hippocampal CA1 region.

DNA methylation is a key epigenetic modification of DNA that is catalyzed by DNA methyltransferases (Dnmt). Increasing evidences suggest that DNA methylation in neurons regulates synaptic plasticity as well as neuronal network activity. In the present study, we investigated the changes in DNA methyltransferases 1 (Dnmt1) immunoreactivity and its protein levels in the gerbil hippocampal CA1 region after 5 min of transient global cerebral ischemia. CA1 pyramidal neurons were well stained with NeuN (a neuron-specific soluble nuclear antigen) antibody in the sham-group, Four days after ischemia-reperfusion (I-R), NeuN-positive ((+)) cells were significantly decreased in the stratum pyramidale (SP) of the CA1 region, and many Fluro-Jade B (a marker for neuronal degeneration)(+) cells were observed in the SP. Dnmt1 immunoreactivity was well detected in all the layers of the sham-group. Dnmt1 immunoreactivity was hardly detected only in the stratum pyramidale of the CA1 region from 4 days post-ischemia; however, at these times, Dnmt1 immunoreactivity was newly expressed in GABAergic interneurons or astrocytes in the ischemic CA1 region. In addition, the level of Dnmt1 was lowest at 4 days post-ischemia. In brief, both the Dnmt1 immunoreactivity and protein levels were distinctively decreased in the ischemic CA1 region 4 days after transient cerebral ischemia. These results indicate that the decrease of Dnmt1 expression at 4 days post-ischemia may be related to ischemia-induced delayed neuronal death.

Activity of succinate dehydrogenase in the neocortex and hippocampus of Mongolian gerbils with ischemic and reperfusion brain injury.

We analyzed changes in activity of SDH, one of the most important enzymes of the Krebs cycle, in the cytoplasm of hippocampal and cortical neurons of Mongolian gerbils (Meriones unguiculatus) at the early and delayed reperfusion period after global brain ischemia. The data indicate that SDH activity in pyramidal neurons of various hippocampal areas and in neurons of II, III and V layers of cerebral cortex after 7-min forebrain ischemia depends on both the localization of these neurons and duration of the postischemic reperfusion. SDH activity in neurons significantly increased on days 2 and 7 after reperfusion.

Regulation of hippocampal long-term potentiation and long-term depression by diacylglycerol kinase ζ.

Diacylglycerol (DAG) is an important signaling molecule at neuronal synapses. Generation of synaptic DAG is triggered by the activation of diverse surface receptors including N-methyl-D-aspartate (NMDA) receptors and metabotropic glutamate receptors. The action of DAG is terminated by enzymatic conversion of DAG to phosphatidic acid (PA) by DAG kinases (DGKs). DGKζ, one of many mammalian DGKs, is localized to synapses through direct interaction with the postsynaptic scaffolding protein PSD-95, and regulates dendritic spine maintenance by promoting DAG-to-PA conversion. However, a role for DGKζ in the regulation of synaptic plasticity has not been explored. We report here that Schaffer collateral-CA1 pyramidal synapses in the hippocampus of DGKζ-knockout (DGKζ(-/-) ) mice show enhanced long-term potentiation (LTP) and attenuated long-term depression (LTD). The attenuated LTD at DGKζ(-/-) synapses involves both NMDA receptors and metabotropic glutamate receptors. These changes in LTP and LTD were reversed by phospholipase C inhibition, which blocks DAG production. Similar reversals in both LTP and LTD were also induced by inhibition of protein kinase C, which acts downstream of DAG. These results suggest that DGKζ regulates hippocampal LTP and LTD by promoting DAG-to-PA conversion, and establish that phospholipase C and protein kinase C lie upstream and downstream, respectively, of DGKζ-dependent regulation of hippocampal LTP and LTD.

Hippocalcin mediates calcium-dependent translocation of brain-type creatine kinase (BB-CK) in hippocampal neurons.

Hippocalcin (Hpca) is a Ca(2+)-binding protein that is expressed in neurons and contributes to neuronal plasticity. We purified a 48 kDa Hpca-associated protein from rat brain and identified it to be the creatine kinase B (CKB) subunit, which constitutes brain-type creatine kinase (BB-CK). Hpca specifically bound to CKB in a Ca(2+)-dependent manner, but not to the muscle-type creatine kinase M subunit. The N-terminal region of Hpca was required for binding to CKB. Hpca mediated Ca(2+)-dependent partial translocation of CKB (approximately 10-15% of total creatine kinase activity) to membranes. N-myristoylation of Hpca was critical for membrane translocation, but not for binding to CKB. In cultured hippocampal neurons, ionomycin treatment led to colocalization of Hpca and CKB adjacent to the plasma membrane. These results indicate that Hpca associates with BB-CK and that together they translocate to membrane compartments in a Ca(2+)-dependent manner.

Mice lacking doublecortin and doublecortin-like kinase 2 display altered hippocampal neuronal maturation and spontaneous seizures.

Mutations in doublecortin (DCX) are associated with intractable epilepsy in humans, due to a severe disorganization of the neocortex and hippocampus known as classical lissencephaly. However, the basis of the epilepsy in lissencephaly remains unclear. To address potential functional redundancy with murin Dcx, we targeted one of the closest homologues, doublecortin-like kinase 2 (Dclk2). Here, we report that Dcx; Dclk2-null mice display frequent spontaneous seizures that originate in the hippocampus, with most animals dying in the first few months of life. Elevated hippocampal expression of c-fos and loss of somatostatin-positive interneurons were identified, both known to correlate with epilepsy. Dcx and Dclk2 are coexpressed in developing hippocampus, and, in their absence, there is dosage-dependent disrupted hippocampal lamination associated with a cell-autonomous simplification of pyramidal dendritic arborizations leading to reduced inhibitory synaptic tone. These data suggest that hippocampal dysmaturation and insufficient receptive field for inhibitory input may underlie the epilepsy in lissencephaly, and suggest potential therapeutic strategies for controlling epilepsy in these patients.

[Morpho-functional changes of hippocampal CA1 area in Mongolian gerbils after ischemic postconditioning].

The aim of this study was to determine the effect of ischemic postconditioning on hippocampal CA1 neuronal survival and cytoplasmic activity of lactate dehydrogenase (LDH) in the gerbil model of cerebral ischemia-reperfusion injury. Ischemia was induced by bilateral common carotid artery occlusion (for 7 min) in male Mongolian gerbils (Meriones unguiculatus). Ischemic postconditioning protocol comprised 3 cycles of 15 s reperfusion/15 s ischemia. After 48 h of reperfusion, CA1 neuronal death was detected by Nissl staining and the cytoplasmic LDH was demonstrated histochemically in CA1 area of the hippocampus with a quantitative cytophotometric assessment of the enzyme activity. The results have shown that 7 min ischemia resulted in a significant decrease in the number of viable neurons (up to 24%) in the CA1 area of hippocampus; in addition, it reduced the activity of LDH in these neurons (from 0.260 +/- 0.009 to 0.190 +/- 0.006 relative units). The application of ischemic postconditioning significantly increased the number of viable neurons (up to 52.9%, P < 0.01) in the CA1 area of hippocampus, and it was accompanied by an increase in the activity of LDH (0.240 +/- 0.008 relative units, P < 0.001).

Protein Kinase C Controls the Excitability of Cortical Pyramidal Neurons by Regulating Kv2.2 Channel Activity.

The family of voltage-gated potassium Kv2 channels consists of the Kv2.1 and Kv2.2 subtypes. Kv2.1 is constitutively highly phosphorylated in neurons and its function relies on its phosphorylation state. Whether the function of Kv2.2 is also dependent on its phosphorylation state remains unknown. Here, we investigated whether Kv2.2 channels can be phosphorylated by protein kinase C (PKC) and examined the effects of PKC-induced phosphorylation on their activity and function. Activation of PKC inhibited Kv2.2 currents and altered their steady-state activation in HEK293 cells. Point mutations and specific antibodies against phosphorylated S481 or S488 demonstrated the importance of these residues for the PKC-dependent modulation of Kv2.2. In layer II pyramidal neurons in cortical slices, activation of PKC similarly regulated native Kv2.2 channels and simultaneously reduced the frequency of action potentials. In conclusion, this study provides the first evidence to our knowledge that PKC-induced phosphorylation of the Kv2.2 channel controls the excitability of cortical pyramidal neurons.