Cell and Molecular Biology

Regulation of glutamate transporters often accompanies glutamatergic synaptic plasticity. We investigated the mechanisms responsible for the increase in glutamate uptake associated with increased glutamate release at the Aplysia sensorimotor synapse during long-term sensitization (LTS) and long-term facilitation. An increase in the V max of transport, produced by LTS training, suggested that the increased glutamate uptake was due to an increase in the number of transporters in the membrane. We cloned a high-affinity, Na +dependent glutamate transporter, ApGT1, from Aplysia central nervous system that is highly enriched in pleural sensory neurons, and in pleural-pedal synaptosome and cell/glial fractions. ApGT1, expressed in Xenopus oocytes, demonstrated a similar pharmacological profile to glutamate uptake in Aplysia synaptosome and cell/glial fractions (strong inhibition by threo-beta-benzyloxyaspartate and weak inhibition by dihydrokainate) suggesting that ApGT1 may be the primary glutamate transporter in pleural-pedal ganglia. Levels of ApGT1 and glutamate uptake were increased in synaptosomes 24 h after induction of LTS by electrical stimulation or serotonin. Regulation of ApGT1 during LTS appears to occur post-transcriptionally and results in an increased number of transporters in synaptic membranes. These results suggest that an increase in levels of ApGT1 is responsible, at least in part, for the long-term increase in glutamate uptake associated with long-term memory.

Regulation of the efficacy of synaptic transmission by activity-dependent processes has been implicated in learning and memory as well as in developmental processes. We previously described transient potentiation of excitatory synapses onto layer 2/3 pyramidal neurons in the visual cortex that is induced by coincident presynaptic stimulation and postsynaptic depolarization. In the adult visual cortex, activation of N-methyl-d-aspartate (NMDA) glutamate receptors is necessary to induce this plasticity. These receptors act as coincidence detectors, sensing presynaptic glutamate release and postsynaptic depolarization, and cause an influx of Ca(2+) that is necessary for the potentiation. In the neurons of the neonatal visual cortex, on the other hand, coincident presynaptic stimulation and postsynaptic depolarization induce stable long-term potentiation (LTP). In addition, reduced but significant LTP can be induced in many neurons in the presence of the NMDA receptor (NMDAR) antagonist, 2-amino-5-phosphonovaleric acid despite the Ca(2+) requirement. Therefore there must be an alternative postsynaptic Ca(2+) source and coincidence detection mechanism linked to the LTP induction mechanism in the neonatal cortex operating in addition to NMDARs. In this study, we find that in layer 2/3 pyramidal neurons, release of Ca(2+) from inositol trisphosphate (InsP(3)) receptor-mediated intracellular stores and influx through voltage-gated Ca(2+) channels (VGCCs) provide alternative postsynaptic Ca(2+) sources. We hypothesize that InsP(3)Rs are coincidence detectors, sensing presynaptic glutamate release through linkage with group I metabotropic glutamate receptors (mGluRs), and depolarization, through VGCCs. We also find that the downstream protein kinases, PKA and PKC, have a role in potentiation in layer 2/3 pyramidal neurons of the neonatal visual cortex.

The cerebral cortex is able to undergo dramatic adaptive functional changes in response to a variety of challenges such as sensory experience, patterned vs. chaotic environmental cues, training, learning, and peripheral or central nervous system injury. The period of early postnatal development is particularly conducive to such changes although the adult cortex can also exhibit robust plasticity. A major site of these forms of adaptive changes is the cortical synapse. In this study, we examine the ability of cerebral cortical synapses, particularly in the visual cortex, to undergo changes in their functional strength in response to a particular model of synaptic learning-the covariance or BCM model. This model incorporates the demand for incoming synaptic activity and the level of activation of a target or postsynaptic neuron to correlate or covary. A dramatic switch in the underlying cellular signaling mechanism for this form of synaptic plasticity occurs during postnatal development. The neurotransmitter receptor mechanism that triggers the plasticity switches from one mediated by metabotropic glutamate receptors to a NMDA glutamate receptor-mediated process. The implications of this switch for developmentally regulated cortical synaptic plasticity are considered.

Calcium-calmodulin-dependent kinase II (CaMKII) has a long history of involvement in synaptic plasticity, yet little focus has been given to potassium channels as CaMKII targets despite their importance in repolarizing EPSPs and action potentials and regulating neuronal membrane excitability. We now show that Kv4.2 acts as a substrate for CaMKII in vitro and have identified CaMKII phosphorylation sites as Ser438 and Ser459. To test whether CaMKII phosphorylation of Kv4.2 affects channel biophysics, we expressed wild-type or mutant Kv4.2 and the K ϩ channel interacting protein, KChIP3, with or without a constitutively active form of CaMKII in Xenopus oocytes and measured the voltage dependence of activation and inactivation in each of these conditions. CaMKII phosphorylation had no effect on channel biophysical properties. However, we found that levels of Kv4.2 protein are increased with CaMKII phosphorylation in transfected COS cells, an effect attributable to direct channel phosphorylation based on site-directed mutagenesis studies. We also obtained corroborating physiological data showing increased surface A-type channel expression as revealed by increases in peak K ϩ current amplitudes with CaMKII phosphorylation. Furthermore, endogenous A-currents in hippocampal pyramidal neurons were increased in amplitude after introduction of constitutively active CaMKII, which results in a decrease in neuronal excitability in response to current injections. Thus CaMKII can directly modulate neuronal excitability by increasing cell-surface expression of A-type K ϩ channels.

Memory acquisition and synaptic plasticity are accompanied by changes in the intrinsic excitability of CA1 pyramidal neurons. These activity-dependent changes in excitability are mediated by modulation of intrinsic currents which alters the responsiveness of the cell to synaptic inputs. The afterhyperpolarization (AHP), a major contributor to the regulation of neuronal excitability, is reduced in animals that have acquired several types of hippocampus-dependent memory tasks and also following synaptic potentiation by high frequency stimulation. BK channels underlie the fast AHP and contribute to spike repolarization, and this AHP is reduced in animals that successfully acquired traceeyeblink conditioning. This suggests that BK channel function is activity-dependent, but the mechanisms are unknown. In this study, we found that blockade of BK channels with paxilline (10 µM) decreased I AHP amplitude and increased spike half-width and instantaneous frequency in response to a +100 pA depolarization. In addition, induction of long term potentiation (LTP) by theta burst stimulation (TBS) in CA1 pyramidal neurons reduced BK channel's contribution to I AHP , spike repolarization, and instantaneous frequency. This result indicates that BK channel activity is decreased following synaptic potentiation. Interestingly, blockade of mammalian target of rapamycin (MTORC1) with rapamycin (400 nM) following synaptic potentiation restored BK channel function, suggesting a role for protein translation in signaling events which decreased postsynaptic BK channel activity following synaptic potentiation.

Kv4.2 is the primary pore-forming subunit encoding A-type currents in many neurons throughout the nervous system, and it also contributes to the transient outward currents of cardiac myocytes. A-type currents in the dendrites of hippocampal CA1 pyramidal neurons are regulated by activation of ERK/MAPK, and Kv4.2 is the likely pore-forming subunit of that current. We showed previously that Kv4.2 is directly phosphorylated at three sites by ERK/MAPK (T602, T607, and S616). In this study we determined whether direct phosphorylation of Kv4.2 by ERK/MAPK is responsible for the regulation of the A-type current observed in neurons. We made site-directed mutants, changing the phosphosite serine (S) or threonine (T) to aspartate (D) to mimic phosphorylation. We found that the T607D mutation mimicked the electrophysiological changes elicited by ERK/MAPK activation in neurons: a rightward shift of the activation curve and an overall reduction in current compared with wild type (WT). Surprising...

Transient outward K+ currents are particularly important for the regulation of membrane excitability of neurons and repolarization of action potentials in cardiac myocytes. These currents are modulated by protein kinase C (PKC) activation, and the K+ channel subunit, Kv4.2, is a major contributor to these currents. Furthermore, the current recorded from Kv4.2 channels expressed in oocytes is reduced by PKC activation. The mechanism underlying PKC regulation of Kv4.2 currents is unknown. In this study, we determined that PKC directly phosphorylates the Kv4.2 channel protein. In vitro phosphorylation of the intracellular amino (N)- and carboxyl (C)-termini of Kv4.2 glutathione S-transferase (GST) fusion protein revealed that the Kv4.2 C-terminal was phosphorylated by PKC, while the N-terminal was not. Amino acid mapping and site-directed mutagenesis revealed that the phosphorylated residues on the Kv4.2 C-terminal were Serine (Ser) 447 and Ser537. A phospho-site specific antibody showed that phosphorylation at the Ser537 site increased in the hippocampus in response to PKC activation. Surface biotinylation experiments revealed that alanine mutation to block phosphorylation at both of the PKC sites increased surface expression compared to wildtype Kv4.2. Electrophysiological recordings of the wildtype and both the alanine and aspartate mutant Kv4.2 channels expressed with KChIP3 revealed no significant difference in the half activation or inactivation voltage of the channel. Interestingly, the Ser537 site lies within a possible extracellular regulated kinase (ERK)/mitogen activated protein kinase (MAPK) recognition (docking) domain in the Kv4.2 C-terminal sequence. We found that phosphorylation of Kv4.2 by PKC enhanced ERK phosphorylation of the channel in vitro. These findings suggest the possibility that Kv4.2 is a locus for PKC and ERK cross-talk.

Transient outward K+ currents are particularly important for the regulation of membrane excitability of neurons and repolarization of action potentials in cardiac myocytes. These currents are modulated by PKC (protein kinase C) activation, and the K+- channel subunit Kv4.2 is a major contributor to these currents. Furthermore, the current recorded from Kv4.2 channels expressed in oocytes is reduced by PKC activation. The mechanism underlying PKC regulation of Kv4.2 currents is unknown. In the present study, we determined that PKC directly phosphorylates the Kv4.2 channel protein. In vitro phosphorylation of the intracellular N- and C-termini of Kv4.2 GST (glutathione transferase) tagged fusion protein revealed that the C-terminal of Kv4.2 was phosphorylated by PKC, whereas the N-terminal was not. Amino acid mapping and site-directed mutagenesis revealed that the phosphorylated residues on the Kv4.2 C-terminal were Ser447 and Ser537. A phospho-site-specific antibody showed that phosp...

Background: Exposure to chronic stress produces negative effects on mood and hippocampus-dependent memory formation. Alterations in signaling cascades and histone acetylation present a mechanism of modulation of transcription that may underlie stressdependent processes in the hippocampus critical to learning and memory and development of depressive behaviors. Methods: The rat model of chronic variable stress (CVS) was used to investigate the role of changes in protein acetylation and other molecular components of hippocampus-dependent memory formation and anhedonic behavior in response to CVS. Results: Chronic variable stress treatment decreased both extracellular signal-regulated protein kinases 1 and 2 activation and Bcl-2 expression in all three regions of the hippocampus that corresponded behaviorally with a decrease in memory for the novel object location task and increased anhedonia. Extracellular signal-regulated protein kinases 1 and 2 activation was not significantly affected in the amygdala and increased in the medial prefrontal cortex by CVS. Chronic variable stress had no significant effect on activation of Akt in the hippocampus. We investigated molecular and behavioral effects of infusion of the sirtuin inhibitor, sirtinol, into the dentate gyrus (DG). Sirtinol infusion into the DG prevented the CVS-mediated decrease in extracellular signal-regulated protein kinases 1 and 2 activity and Bcl-2 expression, as well as histone acetylation in the DG previously observed following CVS. This corresponded to enhanced performance on the novel object location memory task, as well as reduced anhedonic behavior. Conclusions: These results suggest that changes in sirtuin activity contribute to changes in molecular cascades and histone acetylation within the hippocampus observed following CVS and may represent a novel therapeutic target for stress-induced depression.

Evidence suggests that when presented with novel acute stress, animals previously exposed to chronic homotypic or heterotypic stressors exhibit normal or enhanced hypothalamic-pituitary-adrenal (HPA) response compared with animals exposed solely to that acute stressor. The molecular mechanisms involved in this effect remain unknown. The extracellular signal-regulated kinase (ERK) is one of the key pathways regulated in the hippocampus in both acute and chronic stress. The aim of this study was to examine the interaction of prior chronic stress, using the chronic variable stress model (CVS), with exposure to a novel acute stressor (2,5-dihydro-2,4,5-trimethyl thiazoline; TMT) on ERK activation, expression of the downstream protein BCL-2, and the glucocorticoid receptor co-chaperone BAG-1 in control and chronically stressed male rats. TMT exposure after chronic stress resulted in a significant interaction of chronic and acute stress in all 3 hippocampus subregions on ERK activation an...

Small conductance, Ca 2؉-activated voltage-independent potassium channels (SK channels) are widely expressed in diverse tissues; however, little is known about the molecular regulation of SK channel subunits. Direct alteration of ion channel subunits by kinases is a candidate mechanism for functional modulation of these channels. We find that activation of cyclic AMP-dependent protein kinase (PKA) with forskolin (50 M) causes a dramatic decrease in surface localization of the SK2 channel subunit expressed in COS7 cells due to direct phosphorylation of the SK2 channel subunit. PKA phosphorylation studies using the intracellular domains of the SK2 channel subunit expressed as glutathione S-transferase fusion protein constructs showed that both the amino-terminal and carboxylterminal regions are PKA substrates in vitro. Mutational analysis identified a single PKA phosphorylation site within the amino-terminal of the SK2 subunit at serine 136. Mutagenesis and mass spectrometry studies identified four PKA phosphorylation sites: Ser 465 (minor site) and three amino acid residues Ser 568 , Ser 569 , and Ser 570 (major sites) within the carboxyl-terminal region. A mutated SK2 channel subunit, with the three contiguous serines mutated to alanines to block phosphorylation at these sites, shows no decrease in surface expression after PKA stimulation. Thus, our findings suggest that PKA phosphorylation of these three sites is necessary for PKA-mediated reorganization of SK2 surface expression. The small conductance, Ca 2ϩ-activated K ϩ (SK) 2 channels are found in both neuronal and non-neuronal tissue (1). Functionally, the SK channels are best characterized in the central nervous system. Three genes encode the SK channel subunits (SK1, SK2, and SK3) in mamma

Kv4 potassium channels regulate action potentials in neurons and cardiac myocytes. Co-expression of EF hand-containing Ca 2؉-binding proteins termed KChIPs with pore-forming Kv4 ␣ subunits causes changes in the gating and amplitude of Kv4 currents (An, W. F.,

The hippocampus is a brain region that is particularly susceptible to structural and functional changes in response to chronic stress. Recent literature has focused on changes in gene transcription mediated by post-translational modifications of histones in response to stressful stimuli. Chronic variable stress (CVS) is a rodent model that mimics certain symptoms of depression in humans. Given that stress exhibits distinct effects on the cells of the sub-regions of the hippocampus, we investigated changes in histone acetylation in the CA1, CA3, and dentate gyrus (DG) of the hippocampus in response to CVS. Western blotting revealed a significant decrease in acetylation of histone 4 (H4) at Lys12 in CA3 and DG of CVS animals compared to control animals. Furthermore, phospho-acetyl H3 (Lys9/Ser10) was also decreased in the CA3 and DG regions of the hippocampus of CVS animals. In addition, since histone deacetylases (HDACs) contribute to the acetylation state of histones, we investigated the effects of two HDAC inhibitors, sodium butyrate, a class I and II global HDAC inhibitor, and sirtinol, a class III sirtuin inhibitor, on acetylation of histone 3 (H3) and histone 4 (H4). Application of HDAC inhibitors to hippocampus slices from control and CVS animals revealed increased histone acetylation in CVS animals, suggesting that levels of histone deacetylation by HDACs were higher in the CVS animals compared to control animals. Interestingly, histone acetylation in response to sirtinol was selectively increased in the slices from the CVS animals, with very little effect of sirtuin inhibitors in slices from control animals. In addition, sirtuin activity was increased specifically in CA3 and DG of CVS animals. These results suggest a complex and regionally-specific pattern of changes in histone acetylation within the hippocampus which may contribute to stress-induced pathology.

Corticosterone (CORT) release from the adrenal glands in response to acutely stressful stimuli is well-characterized, however several non-experimental, environmental stressors can also engender a CORT response. The aim of this study was to investigate an acute activation of the HPA axis in pair-housed animals in response to separation. We observed a rapid significant increase in CORT in the animal remaining in the home cage following cage mate removal, that was not caused by cage opening and transient removal of cage mate. In addition, we examined this response in both control, non-stressed animals and in animals subjected to chronic variable stress (CVS) and found that although basal levels of CORT differed between control and CVS animals, there was no significant difference in the acute CORT levels between the control and CVS animals after separation, indicating that this environmental event is perceived as acutely stressful in both conditions. Furthermore, we examined the time course of CORT activation and found that CORT levels rapidly rise within minutes of separation peaking at 15 minutes and returning to baseline by 90 minutes. The results of this study demonstrate that separation can induce an acute stress response in the remaining cage mate measured by increased CORT and should be considered in molecular, behavioral, and electrophysiological studies.