Bidirectional control of BK channel open probability by CAMKII and PKC in medial vestibular nucleus neurons

Large conductance K(+) (BK) channels are a key determinant of neuronal excitability. Medial vestibular nucleus (MVN) neurons regulate eye movements to ensure image stabilization during head movement, and changes in their intrinsic excitability may play a critical role in plasticity of the vestibulo-ocular reflex. Plasticity of intrinsic excitability in MVN neurons is mediated by kinases, and BK channels influence excitability, but whether endogenous BK channels are directly modulated by kinases is unknown. Double somatic patch-clamp recordings from MVN neurons revealed large conductance potassium channel openings during spontaneous action potential firing. These channels displayed Ca(2+) and voltage dependence in excised patches, identifying them as BK channels. Recording isolated single channel currents at physiological temperature revealed a novel kinase-mediated bidirectional control in the range of voltages over which BK channels are activated. Application of activated Ca(2+)/calmodulin-dependent kinase II (CAMKII) increased BK channel open probability by shifting the voltage activation range towards more hyperpolarized potentials. An opposite shift in BK channel open probability was revealed by inhibition of phosphatases and was occluded by blockade of protein kinase C (PKC), suggesting that active PKC associated with BK channel complexes in patches was responsible for this effect. Accordingly, direct activation of endogenous PKC by PMA induced a decrease in BK open probability. BK channel activity affects excitability in MVN neurons and bidirectional control of BK channels by CAMKII, and PKC suggests that cellular signaling cascades engaged during plasticity may dynamically control excitability by regulating BK channel open probability.     

Dopamine D1/5 receptor-mediated long-term potentiation of intrinsic excitability in rat prefrontal cortical neurons: Ca2+-dependent intracellular signaling

Prefrontal cortex (PFC) dopamine D1/5 receptors modulate long- and short-term neuronal plasticity that may contribute to cognitive functions. Synergistic to synaptic strength modulation, direct postsynaptic D1/5 receptor activation also modulates voltage-dependent ionic currents that regulate spike firing, thus altering the neuronal input-output relationships in a process called long-term potentiation of intrinsic excitability (LTP-IE). Here, the intracellular signals that mediate this D1/5 receptor-dependent LTP-IE were determined using whole cell current-clamp recordings in layer V/VI rat pyramidal neurons from PFC slices. After blockade of all major amino acid receptors (V(hold) = -65 mV) brief tetanic stimulation (20 Hz) of local afferents or application of the D1 agonist SKF81297 (0.2-50 microM) induced LTP-IE, as shown by a prolonged (>40 min) increase in depolarizing pulse-evoked spike firing. Pretreatment with the D1/5 antagonist SCH23390 (1 microM) blocked both the tetani- and D1/5 agonist-induced LTP-IE, suggesting a D1/5 receptor-mediated mechanism. The SKF81297-induced LTP-IE was significantly attenuated by Cd(2+), [Ca(2+)](i) chelation, by inhibition of phospholipase C, protein kinase-C, and Ca(2+)/calmodulin kinase-II, but not by inhibition of adenylate cyclase, protein kinase-A, MAP kinase, or L-type Ca(2+) channels. Thus this form of D1/5 receptor-mediated LTP-IE relied on Ca(2+) influx via non-L-type Ca(2+) channels, activation of PLC, intracellular Ca(2+) elevation, activation of Ca(2+)-dependent CaMKII, and PKC to mediate modulation of voltage-dependent ion channel(s). This D1/5 receptor-mediated modulation by PKC coexists with the previously described PKA-dependent modulation of K(+) and Ca(2+) currents to dynamically regulate overall excitability of PFC neurons.     

Cholecystokinin facilitates neuronal excitability in the entorhinal cortex via activation of TRPC-like channels

Cholecystokinin (CCK) is one of the most abundant neuropeptides in the brain, where it interacts with two G protein-coupled receptors (CCK-1 and CCK-2). Activation of both CCK receptors increases the activity of PLC, resulting in increases in intracellular calcium ion (Ca(2+)) release and activation of PKC. Whereas high density of CCK receptors has been detected in the superficial layers of the entorhinal cortex (EC), the functions of CCK in this brain region have not been determined. Here, we studied the effects of CCK on neuronal excitability of layer III pyramidal neurons in the EC. Our results showed that CCK remarkably increased the firing frequency of action potentials (APs). The effects of CCK on neuronal excitability were mediated via activation of CCK-2 receptors and required the functions of G proteins and PLC. However, CCK-mediated facilitation of neuronal excitability was independent of inositol trisphosphate receptors and PKC. CCK facilitated neuronal excitability by activating a cationic channel to generate membrane depolarization. The effects of CCK were suppressed by the generic, nonselective cationic channel blockers, 2-aminoethyldiphenyl borate and flufenamic acid, but potentiated by gadolinium ion and lanthanum ion at 100 μM. Depletion of extracellular Ca(2+) also counteracted CCK-induced increases in AC firing frequency. Moreover, CCK-induced enhancement of neuronal excitability was inhibited significantly by intracellular application of the antibody to transient receptor potential channel 5 (TRPC5), suggesting the involvement of TRPC5 channels. Our results provide a cellular and molecular mechanism to help explain the functions of CCK in vivo.     

Receptor-stimulated phospholipase A(2) liberates arachidonic acid and regulates neuronal excitability through protein kinase C

Type B photoreceptors in Hermissenda exhibit increased excitability (e.g., elevated membrane resistance and lowered spike thresholds) consequent to the temporal coincidence of a light-induced intracellular Ca(2+) increase and the release of GABA from presynaptic vestibular hair cells. Convergence of these pre- and postsynaptically stimulated biochemical cascades culminates in the activation of protein kinase C (PKC). Paradoxically, exposure of the B cell to light alone generates an inositol triphosphate-regulated rise in diacylglycerol and intracellular Ca(2+), co-factors sufficient to stimulate conventional PKC isoforms, raising questions as to the unique role of synaptic stimulation in the activation of PKC. GABA receptors on the B cell are coupled to G proteins that stimulate phospholipase A(2) (PLA(2)), which is thought to regulate the liberation of arachidonic acid (AA), an "atypical" activator of PKC. Here, we directly assess whether GABA binding or PLA(2) stimulation liberates AA in these cells and whether free AA potentiates the stimulation of PKC. Free fatty-acid was estimated in isolated photoreceptors with the fluorescent indicator acrylodan-derivatized intestinal fatty acid-binding protein (ADIFAB). In response to 5 microM GABA, a fast and persistent increase in ADIFAB emission was observed, and this increase was blocked by the PLA(2) inhibitor arachidonyltrifluoromethyl ketone (50 microM). Furthermore, direct stimulation of PLA(2) by melittin (10 microM) increased ADIFAB emission in a manner that was kinetically analogous to GABA. In response to simultaneous exposure to the stable AA analogue oleic acid (OA, 20 microM) and light (to elevate intracellular Ca(2+)), B photoreceptors exhibited a sustained (>45 min) increase in excitability (membrane resistance and evoked spike rate). The excitability increase was blocked by the PKC inhibitor chelerythrine (20 microM) and was not induced by exposure of the cells to light alone. The increase in excitability in the B cell that followed exposure to light and OA persisted for > or =90 min when the pairing was conducted in the presence of the protein synthesis inhibitor anisomycin (1 microm), suggesting that the synergistic influence of these signaling agents on neuronal excitability did not require new protein synthesis. These results indicate that GABA binding to G-protein-coupled receptors on Hermissenda B cells stimulates a PLA(2) signaling cascade that liberates AA, and that this free AA interacts with postsynaptic Ca(2+) to synergistically stimulate PKC and enhance neuronal excitability. In this manner, the interaction of postsynaptic metabotropic receptors and intracellular Ca(2+) may serve as the catalyst for some forms of associative neuronal/synaptic plasticity.     

NMDA receptor regulation of nNOS phosphorylation and induction of neuron death

Stimulation of NMDA receptors activates neuronal nitric oxide synthase (nNOS) and the production of nitric oxide (NO). Dephosphorylation of nNOS increases nNOS enzymatic activity. We have examined the regulation of nNOS phosphorylation in rat cortical neurons following NMDA receptor activation. We show that nNOS is constitutively phosphorylated and that NMDA receptor activation decreases the level of nNOS phosphorylation by a mechanism that is blocked specifically by NMDA receptor antagonists and inhibitors of the Ca²⁺-regulated phosphatases calcineurin and PP1/PP2A. Using quantitative digital microscopy, we show that NMDA receptor activation induces the accumulation of nitrotyrosine, a measure of nNOS activity, and TdT-mediated fluorescein-dUTP nick end labeling (TUNEL) positivity, a measure of cell death. A calcineurin inhibitor blocked the increase in both TUNEL and nitrotyrosine positivity. Notably, TUNEL was increased in those neurons that were most strongly positive for nitrotyrosine. We conclude that NMDA receptor activation induces death of neurons by a cell autonomous pathway involving nNOS dephosphorylation by a calcineurin-dependent mechanism.     

Calcium-dependent regulation of cholinergic cell phenotype in the hypothalamus in vitro

Glutamate is a major fast excitatory neurotransmitter in the CNS including the hypothalamus. Our previous experiments in hypothalamic neuronal cultures showed that a long-term decrease in glutamate excitation upregulates ACh excitatory transmission. Data suggested that in the absence of glutamate activity in the hypothalamus in vitro, ACh becomes the major excitatory neurotransmitter and supports the excitation/inhibition balance. Here, using neuronal cultures, fura-2 Ca(2+) digital imaging, and immunocytochemistry, we studied the mechanisms of regulation of cholinergic properties in hypothalamic neurons. No ACh-dependent activity and a low number (0.5%) of cholinergic neurons were detected in control hypothalamic cultures. A chronic (2 wk) inactivation of N-methyl-D-aspartate (NMDA) ionotropic glutamate receptors, L-type voltage-gated Ca(2+) channels, calmodulin, Ca(2+)/calmodulin-dependent protein kinases II/IV (CaMK II/IV), or protein kinase C (PKC) increased the number of cholinergic neurons (to 15-24%) and induced ACh activity (in 40-60% of cells). Additionally, ACh activity and an increased number of cholinergic neurons were detected in hypothalamic cultures 2 wk after a short-term (30 min) pretreatment with bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid tetrakis(acetoxy-methyl) ester (BAPTA AM; 2.5 microM), a membrane permeable Ca(2+)-chelating agent that blocks cytoplasmic Ca(2+) fluctuations. An increase in the number of cholinergic neurons following a chronic NMDA receptor blockade was likely due to the induction of cholinergic phenotypic properties in postmitotic noncholinergic neurons, as determined using 5-bromo-2'-deoxyuridine (BrdU) labeling. In contrast, a chronic inactivation of non-NMDA glutamate receptors or cGMP-dependent protein kinase had little effect on the expression of ACh properties. The data suggest that Ca(2+), at normal intracellular concentrations, tonically suppresses the development of cholinergic properties in hypothalamic neurons. However, a decrease in Ca(2+) influx into cells (through NMDA receptors or L-type Ca(2+) channels), inactivation of intracellular Ca(2+) fluctuations, or downregulation of Ca(2+)-dependent signal transduction pathways (CaMK II/IV and PKC) remove the tonic inhibition and trigger the development of cholinergic phenotype in some hypothalamic neurons. An increase in excitatory ACh transmission may represent a novel form of neuronal plasticity that regulates the activity and excitability of neurons during a decrease in glutamate excitation. This type of plasticity has apparent region-specific character and is not expressed in the cortex in vitro; neither increase in ACh activity nor change in the number of cholinergic neurons were detected in cortical cultures under all experimental conditions.     

Activity-dependent decrease of excitability in rat hippocampal neurons through increases in I(h)

Hippocampal long-term potentiation (LTP) induced by theta-burst pairing of Schaffer collateral inputs and postsynaptic firing is associated with localized increases in synaptic strength and dendritic excitability. Using the same protocol, we now demonstrate a decrease in cellular excitability that was blocked by the h-channel blocker ZD7288. This decrease was also induced by postsynaptic theta-burst firing alone, yet it was blocked by NMDA receptor antagonists, postsynaptic Ca2+ chelation, low concentrations of tetrodotoxin, omega-conotoxin MVIIC, calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitors and a protein synthesis inhibitor. Increasing network activity with high extracellular K+ caused a similar reduction of cellular excitability and an increase in h-channel HCN1 protein. We propose that backpropagating action potentials open glutamate-bound NMDA receptors, resulting in an increase in I(h) and a decrease in overall excitability. The occurrence of such a reduction in cellular excitability in parallel with synaptic potentiation would be a negative feedback mechanism to normalize neuronal output firing and thus promote network stability.     

Slow afterhyperpolarization governs the development of NMDA receptor-dependent afterdepolarization in CA1 pyramidal neurons during synaptic stimulation

CA1 pyramidal neurons from animals that have acquired a hippocampus-dependent task show a reduced slow postburst afterhyperpolarization (sAHP). To understand the functional significance of this change, we examined and characterized the sAHP activated by different patterns of synaptic stimuli and its impact on postsynaptic signal integration. Whole cell current-clamp recordings were performed on rat CA1 pyramidal neurons, and trains of stratum radiatum stimuli varying in duration, frequency, and intensity were used to activate the AHP. At -68 mV, a short train of subthreshold stimuli (20-150 Hz) generated only the medium AHP. In contrast, just two suprathreshold stimuli >50 Hz triggered a prominent sAHP sensitive to bath-applications of isoproterenol, carbachol, or intracellularly applied BAPTA, suggesting that the underlying current is the Ca2+-activated K+ current, the sIAHP. The sAHP magnitude was positively related to stimulus train duration and frequency, consistent with its dependence on intracellular Ca2+ accumulation for activation. About 20% of neurons recorded did not have a sAHP. In response to high-frequency suprathreshold stimuli, these neurons developed a pronounced afterdepolarization (ADP) and multiple action potential firing. The ADP magnitude increased with successive stimuli and was positively related to stimulus intensity and frequency. It was sensitive to bath-applications of thapsigargin and nitrendipine, and abolished by d-AP5, indicating that it is supported by intracellular Ca2+ release, the L-type Ca2+ influx, and N-methyl-D-aspartate (NMDA) receptor-mediated influx. In the presence of D-AP5, we were unable to trigger an ADP with maximal stimulus intensity. Pharmacologically eliminating the sAHP allowed neurons to develop an ADP with the original stimulus train. We propose that the slow AHP acts to facilitate Mg2+ re-block of the activated NMDA receptors, thereby reducing temporal summation and preventing an NMDA receptor-dependent ADP during intense synaptic events. Neuromodulation of the sAHP may thus affect information throughput and regulate NMDA receptor-mediated plasticity.     

Endogenous PGE2 regulates membrane excitability and synaptic transmission in hippocampal CA1 pyramidal neurons

The significance of cyclooxygenases (COXs), the rate-limiting enzymes that convert arachidonic acid (AA) to prostaglandins (PGs) in the brain, is unclear, although they have been implicated in inflammatory responses and in some neurological disorders such as epilepsy and Alzheimer's disease. Recent evidence that COX-2, which is expressed in postsynaptic dendritic spines, regulates PGE2 signaling in activity-dependent long-term synaptic plasticity at hippocampal perforant path-dentate granule cell synapses, suggests an important role of the COX-2-generated PGE2 in synaptic signaling. However, little is known of how endogenous PGE2 regulates neuronal signaling. Here we showed that endogenous PGE2 selectively regulates fundamental membrane and synaptic properties in the hippocampus. Somatic and dendritic membrane excitability was significantly reduced when endogenous PGE2 was eliminated with a selective COX-2 inhibitor in hippocampal CA1 pyramidal neurons in slices. Exogenous application of PGE2 produced significant increases in frequency of firing, excitatory postsynaptic potentials (EPSP) amplitude, and temporal summation in slices treated with the COX-2 inhibitor. The PGE2-induced increase in membrane excitability seemed to result from its inhibition of the potassium currents, which in turn, boosted dendritic Ca2+ influx during dendritic-depolarizing current injections. In addition, the PGE2-induced enhancement of EPSPs was blocked by eliminating both PKA and PKC activities. These findings indicate that endogenous PGE2 dynamically regulates membrane excitability, synaptic transmission, and plasticity and that the PGE2-induced synaptic modulation is mediated via cAMP-PKA and PKC pathways in rat hippocampal CA1 pyramidal neurons.     

NMDA-mediated activation of the tyrosine phosphatase STEP regulates the duration of ERK signaling

The intracellular mechanism(s) by which a cell determines the duration of extracellular signal-regulated kinase (ERK) activation is not well understood. We have investigated the role of STEP, a striatal-enriched tyrosine phosphatase, in the regulation of ERK activity in rat neurons. Glutamate-mediated activation of NMDA receptors leads to the rapid but transient phosphorylation of ERK in cultured neurons. Here we show that activation of NMDA receptors led to activation of STEP, which limited the duration of ERK activity as well as its translocation to the nucleus and its subsequent downstream nuclear signaling. In neurons, STEP is phosphorylated and inactive under basal conditions. NMDA-mediated influx of Ca(2+), but not increased intracellular Ca(2+) from other sources, leads to activation of the Ca(2+)-dependent phosphatase calcineurin and the dephosphorylation and activation of STEP. We have identified an important mechanism involved in the regulation of ERK activity in neurons that highlights the complex interplay between serine/threonine and tyrosine kinases and phosphatases.     

Long-term potentiation of intrinsic excitability in LV visual cortical neurons

Neuronal excitability has a large impact on network behavior, and plasticity in intrinsic excitability could serve as an important information storage mechanism. Here we ask whether postsynaptic excitability of layer V pyramidal neurons from primary visual cortex can be rapidly regulated by activity. Whole cell current-clamp recordings were obtained from visual cortical slices, and intrinsic excitability was measured by recording the firing response to small depolarizing test pulses. Inducing neurons to fire at high-frequency (30-40 Hz) in bursts for 5 min in the presence of synaptic blockers increased the firing rate evoked by the test pulse. This long-term potentiation of intrinsic excitability (LTP-IE) lasted for as long as we held the recording (>60 min). LTP-IE was accompanied by a leftward shift in the entire frequency versus current (F-I) curve and a decrease in threshold current and voltage. Passive neuronal properties were unaffected by the induction protocol, indicating that LTP-IE occurred through modification in voltage-gated conductances. Reducing extracellular calcium during the induction protocol, or buffering intracellular calcium with bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid, prevented LTP-IE. Finally, blocking protein kinase A (PKA) activation prevented, whereas pharmacological activation of PKA both mimicked and occluded, LTP-IE. This suggests that LTP-IE occurs through postsynaptic calcium influx and subsequent activation of PKA. Activity-dependent plasticity in intrinsic excitability could greatly expand the computational power of individual neurons.     

Auxiliary Hyperkinetic beta subunit of K+ channels: regulation of firing properties and K+ currents in Drosophila neurons.

Auxiliary Hyperkinetic beta subunit of K+ channels: regulation of firing properties and K+ currents in Drosophila neurons. Molecular analysis and heterologous expression have shown that K+ channel beta subunits regulate the properties of the pore-forming alpha subunits, although how they influence neuronal K+ currents and excitability remains to be explored. We studied cultured Drosophila "giant" neurons derived from mutants of the Hyperkinetic (Hk) gene, which codes for a K+ channel beta subunit. Whole cell patch-clamp recording revealed broadened action potentials and, more strikingly, persistent rhythmic spontaneous activities in a portion of mutant neurons. Voltage-clamp analysis demonstrated extensive alterations in the kinetics and voltage dependence of K+ current activation and inactivation, especially at subthreshold membrane potentials, suggesting a role in regulating the quiescent state of neurons that are capable of tonic firing. Altered sensitivity of Hk currents to classical K+ channel blockers (4-aminopyridine, alpha-dendrotoxin, and TEA) indicated that Hk mutations modify interactions between voltage-activated K+ channels and these pharmacological probes, apparently by changing both the intra- and extracellular regions of the channel pore. Correlation of voltage- and current-clamp data from the same cells indicated that Hk mutations affect not only the persistently active neurons, but also other neuronal categories. Shaker (Sh) mutations, which alter K+ channel alpha subunits, increased neuronal excitability but did not cause the robust spontaneous activity characteristic of some Hk neurons. Significantly, Hk Sh double mutants were indistinguishable from Sh single mutants, implying that the rhythmic Hk firing pattern is conferred by intact Shalpha subunits in a distinct neuronal subpopulation. Our results suggest that alterations in beta subunit regulation, rather than elimination or addition of alpha subunits, may cause striking modifications in the excitability state of neurons, which may be important for complex neuronal function and plasticity.     

Serotonergic inhibition of action potential evoked calcium transients in NOS-containing mesopontine cholinergic neurons

Nitric oxide synthase (NOS)-containing mesopontine cholinergic (MPCh) neurons of the laterodorsal tegmental nucleus (LDT) are hypothesized to drive the behavioral states of waking and REM sleep through a tonic increase in firing rate which begins before and is maintained throughout these states. In principle, increased firing could elevate intracellular calcium levels and regulate numerous cellular processes including excitability, gene expression, and the activity of neuronal NOS in a state-dependent manner. We investigated whether repetitive firing, evoked by current injection and N-methyl-D-aspartate (NMDA) receptor activation, produces somatic and proximal dendritic [Ca(2+)](i) transients and whether these transients are modulated by serotonin, a transmitter thought to play a critical role in regulating the state-dependent firing of MPCh neurons. [Ca(2+)](i) was monitored optically from neurons filled with Ca(2+) indicators in guinea pig brain slices while measuring membrane potential with sharp microelectrodes or patch pipettes. Neither hyperpolarizing current steps nor subthreshold depolarizing steps altered [Ca(2+)](i). In contrast, suprathreshold currents caused large and rapid increases in [Ca(2+)](i) that were related to firing rate. TTX (1 microM) strongly attenuated this relation. Addition of tetraethylammonium (TEA, 20 mM), which resulted in Ca(2+) spiking on depolarization, restored the change in [Ca(2+)](i) to pre-TTX levels. Suprathreshold doses of NMDA also produced increases in [Ca(2+)](i) that were reduced by up to 60% by TTX. Application of 5-HT, which hyperpolarized LDT neurons without detectable changes in [Ca(2+)](i), suppressed both current- and NMDA-evoked increases in [Ca(2+)](i) by reducing the number of evoked spikes and by inhibiting spike-evoked Ca(2+) transients by approximately 40% in the soma and proximal dendrites. This inhibition was accompanied by a subtle increase in the spike repolarization rate and a decrease in spike width, as expected for inhibition of high-threshold Ca(2+) currents in these neurons. NADPH-diaphorase histochemistry confirmed that recorded cells were NOS-containing. These findings indicate the prime role of action potentials in elevating [Ca(2+)](i) in NOS-containing MPCh neurons. Moreover, they demonstrate that serotonin can inhibit somatic and proximal dendritic [Ca(2+)](i) increases both indirectly by reducing firing rate and directly by decreasing the spike-evoked transients. Functionally, these data suggest that spike-evoked Ca(2+) signals in MPCh neurons should be largest during REM sleep when serotonin inputs are expected to be lowest even if equivalent firing rates are reached during waking. Such Ca(2+) signals may function to trigger Ca(2+)-dependent processes including cfos expression and nitric oxide production in a REM-specific manner.     

FGF-2 potentiates Ca(2+)-dependent inactivation of NMDA receptor currents in hippocampal neurons

Peptide growth factors such as the neurotrophins and fibroblast growth factors have potent effects on synaptic transmission, development, and cell survival. We report that chronic (hours) treatment with basic fibroblast growth factor (FGF-2) potentiates Ca(2+)-dependent N-methyl-D-aspartate (NMDA) receptor inactivation in cultured hippocampal neurons. This effect is specific for the NMDA-subtype of ionotropic glutamate receptor and FGF-2. The potentiated inactivation requires ongoing protein synthesis during growth factor treatment and the activity of protein phosphatase 2B (PP2B or calcineurin) during agonist application. These results suggest a mechanism by which FGF-2 receptor signaling may regulate neuronal survival and synaptic plasticity.     

Modulation of excitability in Aplysia tail sensory neurons by tyrosine kinases

Tyrosine kinases have recently been shown to modulate synaptic plasticity and ion channel function. We show here that tyrosine kinases can also modulate both the baseline excitability state of Aplysia tail sensory neurons (SNs) as well as the excitability induced by the neuromodulator serotonin (5HT). First, we examined the effects of increasing and decreasing tyrosine kinase activity in the SNs. We found that tyrosine kinase inhibitors decrease baseline SN excitability in addition to attenuating the increase in excitability induced by 5HT. Conversely, functionally increasing cellular tyrosine kinase activity in the SNs by either inhibiting opposing tyrosine phosphatase activity or by direct injection of an active tyrosine kinase (Src) induces increases in SN excitability in the absence of 5HT. Second, we examined the interaction between protein kinase A (PKA), which is known to mediate 5HT-induced excitability changes in the SNs, and tyrosine kinases, in the enhancement of SN excitability. We found that the tyrosine kinases function downstream of PKA activation since tyrosine kinase inhibitors reduce excitability induced by activators of PKA. Finally, we examined the role of tyrosine kinases in other forms of 5HT-induced plasticity in the SNs. We found that while tyrosine kinase inhibitors attenuate excitability produced by 5HT, they have no effect on short-term facilitation (STF) of the SN-motor neuron (MN) synapse induced by 5HT. Thus tyrosine kinases modulate different forms of SN plasticity independently. Such differential modulation would have important consequences for activity-dependent plasticity in a variety of neural circuits.     

Defective place cell activity in nociceptin receptor knockout mice with elevated NMDA receptor-dependent long-term potentiation

There is growing evidence that NMDA receptor-dependent long-term potentiation (LTP) in the hippocampus mediates the synaptic plasticity that underlies spatial learning and memory. LTP deficiencies correlate well with spatial memory deficits and LTP enhancements may improve spatial memory. In addition, LTP deficiencies are associated with abnormal place cells as expected from the spatial mapping hypothesis of hippocampal function. In contrast, nothing is known on how enhanced NMDA receptor-dependent LTP affects place cells. To address this question we recorded place cells from mice lacking the nociceptin receptor (NOP₁/ORL₁/OP4) that have enhanced hippocampal LTP. We found that the enhanced LTP was mediated by NMDA receptors, did not require L-type calcium channels, and occurred only when high frequency tetanizing stimulus trains were used. Place cells in nociceptin receptor knockout mice were abnormal in several ways: they were less stable, had noisier positional firing patterns, larger firing fields and higher discharge rates inside and outside the firing fields. Our results suggest that excessive LTP can cause subnormal hippocampal place cell function. The effects of LTP enhancement on place cell function may therefore also depend on molecular details of synaptic plasticity, including the relationship between stimulus frequency and synaptic strength, and not merely on the magnitude of synaptic strength increases. The data have important clinical implications on development of strategies to improve cognitive function.     

Nerve growth factor enhances the excitability of rat sensory neurons through activation of the atypical protein kinase C isoform, PKMζ

Our previous work showed that nerve growth factor (NGF) increased the excitability of small-diameter capsaicin-sensitive sensory neurons by activating the p75 neurotrophin receptor and releasing sphingolipid-derived second messengers. Whole cell patch-clamp recordings were used to establish the signaling pathways whereby NGF augments action potential (AP) firing (i.e., sensitization). Inhibition of MEK1/2 (PD-98059), PLC (U-73122, neomycin), or conventional/novel isoforms of PKC (bisindolylmaleimide I) had no effect on the sensitization produced by NGF. Pretreatment with a membrane-permeable, myristoylated pseudosubstrate inhibitor of atypical PKCs (aPKCs: PKMζ, PKCζ, and PKCλ/ι) blocked the NGF-induced increase in AP firing. Inhibitors of phosphatidylinositol 3-kinase (PI3K) also blocked the sensitization produced by NGF. Isolated sensory neurons were also treated with small interfering RNA (siRNA) targeted to PKCζ. Both Western blots and quantitative real-time PCR established that PKMζ, but neither full-length PKCζ nor PKCλ/ι, was significantly reduced after siRNA exposure. Treatment with these labeled siRNA prevented the NGF-induced enhancement of excitability. Furthermore, consistent with the high degree of catalytic homology for aPKCs, internal perfusion with active recombinant PKCζ or PKCι augmented excitability, recapitulating the sensitization produced by NGF. Internal perfusion with recombinant PKCζ suppressed the total potassium current and enhanced the tetrodotoxin-resistant sodium current. Pretreatment with the myristoylated pseudosubstrate inhibitor blocked the increased excitability produced by ceramide or internal perfusion with recombinant PKCζ. These results demonstrate that NGF leads to the activation of PKMζ that ultimately enhances the capacity of small-diameter capsaicin-sensitive sensory neurons to fire APs through a PI3K-dependent signaling cascade.     

ERK integrates PKA and PKC signaling in superficial dorsal horn neurons. II. Modulation of neuronal excitability

Protein kinases belonging to the protein kinase A (PKA), protein kinase C (PKC), and extracellular signal-related kinase (ERK) families have been identified as key players in modulating nociception at the level of the spinal cord dorsal horn, yet little is known about the effects of these kinases on membrane properties of the dorsal horn neurons. PKA, PKC, and ERK exert inhibitory effects on transient potassium currents (A-type currents or IA) in mouse superficial dorsal horn neurons (Hu et al. 2003). Here we aimed to determine the effects of these kinases on action potential firing and membrane properties of these neurons to evaluate the impact of the modulation of IA (and other conductances) in these neurons. We found that activating PKC and PKA has dramatic effects on action potential firing, reflecting an increase in the excitability of superficial dorsal horn neurons. In addition, we found that inhibitors of both PKC and ERK signaling decrease the excitability of dorsal horn neurons, suggesting that these kinases exert a tonic excitation of these cells. Consistent with our findings that these kinases inhibit A-type currents, we found that PKA, PKC, and ERK act to shorten the first-spike latency after depolarization induced by current injection. In addition, activation of these kinases increases spike frequency and action potential amplitude of dorsal horn neurons. Interestingly, we found that the effects of PKA and PKC activators are blocked by inhibitors of ERK signaling, suggesting that PKA and PKC may exert their actions by activation of ERKs.     

Kinase-dependent modification of dendritic excitability after long-term potentiation

Patterns of presynaptic activity properly timed with postsynaptic action potential output can not only increase the strength of synaptic inputs but can also increase the excitability of dendritic branches of adult CA1 pyramidal neurons. Here, we examined the role of protein kinase A (PKA) and mitogen-activated protein kinase (MAPK) in the enhancement of dendritic excitability that occurs during theta-burst pairing of presynaptic and postsynaptic firing activity. Using dendritic and somatic whole-cell recordings in rat hippocampal slices, we measured the increase in the amplitude of back-propagating action potentials in the apical dendrite that occurs in parallel with long-term potentiation (LTP) of synaptic inputs. We found that inhibition of the MAPK pathway prevents this enhancement of dendritic excitability using either a weak or strong LTP induction protocol, while synaptic LTP can still be induced by the strong protocol. Both forms of plasticity are blocked by inhibition of PKA and occluded by interfering with cAMP degradation, consistent with a PKA-mediated increase in MAPK activity following induction of LTP. This provides a signalling mechanism for plasticity of dendritic excitability that occurs during neuronal activity and demonstrates the necessity of MAPK activation. Furthermore, this study uncovers an additional contribution of kinase activation to plasticity that may occur during learning.     

Substrates for coincidence detection and calcium signaling for induction of synaptic potentiation in the neonatal visual cortex

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.