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Department of Physiology, Northwestern University Feinberg School of Medicine, Chicago, Illinois
Submitted 9 March 2008; accepted in final form 8 August 2008
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ABSTRACT |
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INTRODUCTION |
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; Moyer et al. 1996; Oh et al. 2003; Saar et al. 1998). This type of intrinsic plasticity reflects modulation of ion channels and affects not only synaptic throughput but also pattern, frequency, and timing of action potentials that are pivotal for encoding information within the neural network. Different forms of activity-dependent intrinsic plasticity have been described in cortical and hippocampal pyramidal neurons in recent years (Cudmore and Turrigiano 2004; Misonou et al. 2004; Xu et al. 2005). Despite the differences in the underlying mechanisms and time scales of expression, two themes have emerged: a rise in intracellular Ca2+ is required, and modulation of K+ channels is often involved.
Here we describe a novel, plastic behavior of CA1 pyramidal neurons: a brief spike train only seconds in duration induced a persistent increase in spike frequency adaptation—a gradual reduction in the firing frequency in response to sustained excitation—that lasted for 
60 min. This form of intrinsic plasticity does not require synaptic activation and depends on the availability of the KV7/KCNQ (M-type) channels, a unique class of voltage-dependent K+ channels that exhibit subthreshold-active, slowly activating, and noninactivating gating properties (Brown and Adams 1980). In response to an increase in neuronal activity, as encoded by an enhanced Ca2+ influx through the CaV1/L-type Ca2+ channels and subsequent activation of protein kinase A (PKA), CA1 pyramidal neurons dynamically regulate their output by potentiating the KV7/KCNQ current. Such a mechanism thus confers an activity-dependent stabilization of firing during sustained excitation—a homeostatic intrinsic plasticity in CA1 pyramidal neurons.
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METHODS |
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Five- to 10-wk-old, F344XBN male rats (Harlan; Indianapolis, IN) were used in the present study. All experiments were conducted in strict accordance with a protocol approved by the Animal Care and Use Committee of Northwestern University.
Hippocampus slice preparation
Rats were anesthetized with a mixture of ketamine and xylazine and transcardially perfused with ice-cold artificial cerebrospinal fluid (ACSF, see following text) followed by decapitation. The brain was rapidly removed, and a block containing the left hippocampus and surrounding structures was dissected out, attached to a mounting tray with cyanoacrylate glue, and immersed in ice-cold ACSF consisting of the following (in mM): 119 NaCl, 26 NaHCO3, 2.5 KCl, 1 NaH2PO4*H2O, 1.3 MgCl2*6H2O, 2 CaCl2*2H2O, and 25 glucose and saturated with carbogen (95% O2-5% CO2). Transverse hippocampus slices (300 µm) were cut along the dorsal-ventral axis using a vibrating microtome (VT 1000s; Leica Instrument, Leitz, Nussloch, Germany). Slices were transferred to fresh ACSF and equilibrated at 34°C for 30 min and then maintained at room temperature (
22°C) for 
30 min prior to electrophysiological recording and acute dissociation.
Perforated-patch recording in slice
Only slices from the middle third of the hippocampus were used in this study. Slices were transferred to a small volume (<0.5 ml) recording chamber that was mounted on a fixed-stage, upright microscope (Axioskop; Carl Zeiss, Thornwood, NY or BX51; Olympus, Melville, NY) equipped with infrared differential interference contrast (IR-DIC) optics. The recording chamber was superfused with carbogen-saturated ACSF with a flow rate of 2–3 ml/min. Experiments were performed at room temperature.
Patch electrodes were fabricated from filamented, thick-walled borosilicate glass pipettes and heat-polished to a resistance of 34 M
when filled with an internal solution consisting of the following (mM): 140 KMeSO4, 10 KCl, 10 HEPES, 2 Mg2ATP, 0.4 Na3GTP, and 10 Tris-phosphocreatine; pH adjusted to 7.25 with KOH. The final osmolarity of this solution was 290 mosM. Liquid junction potential (7 mV) was not corrected for. Alexa594 (Invitrogen, Carlsbad, CA) was included in the initial experiments to verify the integrity of the membrane under perforated-patch configuration and identification of the recorded neuron (Fig. 1, A–C).
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. Membrane rupturing was accompanied by an instantaneous appearance of fluorescent signal inside the cytoplasm (Fig. 1, A and B), and a sudden jump in the series resistance and whole cell capacitance, followed by a rapid decrease in the holding current and the KV7/KCNQ current relaxation in voltage-clamp configuration. As abrupt changes in the series resistance, whole cell capacitance, and holding current were predictive of the ensuing current rundown, these parameters were used as indicators of intactness of the perforated-patch, and Alexa594 was omitted in the subsequent experiments.
Electrophysiological records were acquired at 5 or 10 kHz with a Digidata 1322A interface (Molecular Devices) in conjunction with a PC, and filtered at 1 or 2 kHz, respectively, with a low-pass Bessel filter. Stimulus generation and data acquisition was performed using pClamp9 (Molecular Devices). Only data gathered from neurons with resting membrane potential less than –60 mV (RMP = –69 ± 1 mV; n = 107); input resistance >150 M (Rinput = 228 ± 7 M; data from all drug treatments combined), series resistance <40 M (32.0 ± 0.7 M), spike height >110 mV from baseline potential of –65 mV were accepted for further analysis.
To examine the gating properties of KV7/KCNQ channels, voltage-clamp recordings were performed in the presence of Cs+ (2 mM) to block inwardly rectifying KIR2 channels and HCN channels, 4-aminopyridine (4-AP, 2.5–5 mM) to block KV1-4 channels, and TTX (0.5 µM) to block NaV channels and spontaneous synaptic activity. Unless otherwise mentioned, current-clamp recordings were performed in a modified ACSF containing the following compounds to suppress synaptic activity and currents with voltage dependence known to overlap with that of KV7/KCNQ current: Cs+ (1 mM) to suppress inwardly rectifying KIR2 channels and HCN channels, 4-AP (100 µM) to suppress KV1 channels, SR95531 (5 µM) to block GABAA receptors, CGP55845 (1 µM) to block GABAB receptors, D-2-amino-5-phosphonopentanoic acid D-(AP5, 50 µM), and CNQX (20 µM) to suppress excitatory synaptic transmission. Experiments performed in this modified ACSF without further addition of pharmacological agents are referred to as "controls" in this study. A 1-s test pulse with stimulus intensity that reliably elicited the same number of action potentials (range, 7–10 action potentials for neurons included in this study; Table 1) was delivered every 60-s for 10–20 min to establish a measure for baseline excitability. A brief, suprathreshold conditioning pulse train (30 Hz, 3-s; each pulse within the train was 1.5 nA in amplitude and 2 ms in duration) was then delivered to generate the corresponding number of action potentials. Subsequently, membrane excitability was monitored once every 30-s for as long as the recordings remained in the perforated patch configuration. TEA, linopirdine, XE991, BAPTA-AM, SR33805, calciseptine, nimodipine, and 
-conotoxin MVIIC were added to the modified ACSF for 15 min prior to CS presentation to ensure steady-state blockade. -conotoxin MVIIC was added to the modified ACSF along with cytochrome c (1 mg/ml). For experiments involving H-89, slices were preincubated in H-89 for 30 min prior to recording.
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Acute dissociation was done with our standard procedure (Chan et al. 2004). Isolated, individual hippocampal CA1 neurons were aspirated into sterilized glass pipettes containing 1–2 µl of diethyl pyrocarbonate (DEPC)-treated water and 1.5 U/ml SUPERase-In (Ambion, Austin, TX). After aspiration of the neuron, the electrode tip was broken in a 0.6-ml presiliconized tube (Midwest Scientific, Valley Park, MO) containing 1.9 µl of DEPC-treated water, 0.7 µl of SUPERase-In (20 U/ml), 0.7 ml of oligo-dT (50 mM), 0.7 µl of BSA (143 mg/ml), 1.0 ml of dNTPs (10 mM), and the contents were ejected. The mixture was heated to 65°C for 5 min to denature the nucleic acids and then placed on ice for 1 min. Single-strand cDNA was synthesized from the cellular mRNA by adding 1.2 µl of SuperScript II reverse transcriptase (RT) (200 U/ml), 1 ml of 10x RT buffer, 2 ml of MgCl2 (25 mM), 1 ml of DTT (0.1 M), and 0.5 ml of RNase Out (40 U/ml) and then incubating the mixture at 42°C for 90 min. The reaction was terminated by heating the mixture to 70°C for 15 min. The RNA strand in the RNA-DNA hybrid was then removed by adding 0.5 ml of RNase H (2 U/ml) and incubating at 37°C for 20 min. All reagents except SUPERase-In were obtained from Invitrogen. PCR primers were developed from GenBank sequences with commercially available OLIGO 6.7.1 software (Molecular Biology Insights, Cascade, CO). After amplification, PCR products were labeled with ethidium bromide and separated by electrophoresis on agarose gels. Amplicons were of the expected size and sequence. RT-PCR was performed using procedures designed to minimize the chance of cross-contamination. Negative controls for contamination from extraneous and genomic DNA were run for every batch of neurons. To verify that genomic DNA did not contribute to the PCR products, neurons were aspirated and processed in the normal manner, except that the reverse transcriptase was omitted. Contamination from extraneous sources was checked by replacing the cellular template with buffer solution. Both controls were consistently negative in these experiments.
Immunohistochemistry
Two KV7.2/KCNQ2 antibodies (2n1-929, Affinity BioReagents, Golden, CO; 2c578-593, Alomone Laboratories, Jerusalem, Israel) and two KV7.3/KCNQ3 antibodies (3n1-930, Affinity BioReagents, Golden, CO; 3c668-686, Alomone Laboratories) that were raised against nonoverlapping epitopes were used in the present study. For each KV7/KCNQ subtype, the immunostaining patterns we obtained these antibodies were virtually identical. Thus for clarity, only labelings with antibodies from Affinity BioReagents [raised against the same immunogenic peptides that Roche et al. (2002) used in their study; n-Q2 and n-Q3] were presented. In brief, rats were anesthetized with a mixture of ketamine and xylazine and perfused transcardially first with 0.9% saline followed by ice-cold fixative containing 2% paraformaldehyde, 15% saturated picric acid in 0.1 M phosphate buffer, pH 7.3–7.4. Brains were cryoprotected in 30% sucrose in 0.1 M PB overnight at 4°C. Sections (40 µm) were cut with a sliding microtome and incubated with 0.2–5.0 µg/ml antibodies for primary antibodies in phosphate-buffered saline (PBS) containing 10% normal goat serum (NGS) and 0.1% Triton X-100 (Tx) for 24 h at 4°C. After washes in PBS, the sections were incubated (at room temperature for 2 h) with biotinylated donkey anti-guinea pig or anti-rabbit IgGs (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1:200 in PBS-Tx containing 1% NGS. The sections were then washed and reacted with avidin-biotin peroxidase complex (ABC-Elite kit, Vector Laboratories, Burlingame, CA) at room temperature for 2 h. Bound peroxidase enzyme activity was revealed using Tris-buffered saline (pH 7.3) containing 0.025% 3-3'-diaminobenzidine tetrahydrochloride (DAB), 0.05% nickel chloride and 0.003% hydrogen peroxide. No specific immunostaining for respective molecules was observed for controls (data not shown) as sections were incubated without the primary antibodies.
Data analysis and statistics
Curve-fitting (using a least-squares criterion), data and statistical analyses, and plotting were done using Clampfit9 (Molecular Devices), IgorPro 5.05 (WaveMetrics, Lake Oswego, OR), and Statview (SAS Institute, Cary, NC). Data were presented as mean ± SE and compared statistically using Mann-Whitney or Wilcoxon signed-rank test as appropriate. P 0.05 indicates statistical significance. Activation curves for KV7/KCNQ channels were fit with a single Boltzmann function of the form: g(V) = 1/{1 + exp[(V – V1/2)/k]}, where V1/2 is the half voltage of activation and k is the slope factor. The time courses of current deactivation/activation were fit with a double-exponential function.
Solutions and channel ligands
KMeSO4 was purchased from MP Biomedicals (Solon, OH); d-AP5, CGP55845, nimodipine, XE991 hydrochloride, linopirdine hydrochloride, SR95531, and SR33805 were purchased from Tocris Cookson (Ballwin, MO); TTX, calciseptine, and -conotoxin MVIIC from Alomone Laboratories; BAPTA-AM from Invitrogen; and H-89 from Calbiochem (San Diego, CA). All other drugs were purchased from Sigma (St. Louis, MO). Drugs were dissolved as stock solutions in either water or DMSO, aliquoted, and frozen at –30°C before use. Each drug was diluted in the perfusate immediately before the experiment. When used, the final concentration of DMSO was always <0.1%.
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RESULTS |
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All recordings were performed using amphotericin-perforated patches (Fig. 1, A–C) to prevent disruption of intracellular milieu and signaling. Synaptic blockers (see METHODS) were included in all the experiments except where noted to eliminate changes in membrane properties arising from synaptic activation and recurrent network activity. A brief conditioning spike train (CS, 30 Hz for 3 s) was applied to mimic sustained firing in CA1 pyramidal neurons (Fig. 1D). Membrane excitability was monitored periodically before and after CS presentation (METHODS). Under the control condition, CS reliably induced a gradual and persistent suppression of membrane excitability (n = 10/12; Fig. 2, A and B). In contrast, the firing pattern of neurons in the absence of CS was stable across time (Fig. 2B). Input resistance was monitored regularly and did not show significant change before and after CS presentation (pre CS: 168.0 ± 19.8 M; 25 min post CS: 189.8 ± 26.2 M; P > 0.05). Neither spike threshold nor rate of rise of the first action potential—indices of NaV channel availability—was altered immediately following CS (Fig. 1E). Thus it is unlikely that this form of intrinsic plasticity is due to a slowing of recovery from accumulated NaV channel inactivation that developed during CS. To quantify the changes in the firing rate and pattern induced by CS, we formulated an index for spike frequency adaptation for all action potentials generated during each depolarizing step that accounts for changes in the interspike interval and the time that the neurons reach maximal adaptation (i.e., cease to fire as represented by an index of 1; Fig. 2C, legend). The plot of adaptation index against time revealed a leftward shift in the curve following CS (Fig. 2C), illustrating that CA1 pyramidal neurons adapted more readily and fired fewer action potentials following CS. This increase in spike frequency adaptation was associated with a slight elevation in spike threshold, particularly for action potentials generated later in the depolarizing step (Fig. 2D). Altogether, these measurements suggest that an activity-dependent modulation of a slowly or noninactivating subthreshold active conductance is responsible for mediating a long-lasting suppression of membrane excitability.
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KV7/KCNQ channels are necessary for CS-induced intrinsic plasticity
One possibility for the change in firing pattern following CS is a progressive enhancement of a K+ conductance. In support, when TEA (10 mM), a broad-spectrum K+ channel antagonist, was included in the bath solution, CS presentation did not induce a change in spike output (number of spikes generated 25 min post CS as compared with control, TEA: 91.9 ± 6.0%, P < 0.01, n = 7. Fig. 3 E). Based on the gating properties, pharmacological criteria, and known channel function, the primary candidate for this form of intrinsic plasticity were the KV7/KCNQ channels (Fig. 4, A and B, and Supplementary Fig. S2, A–C) (Marrion 1997). To test this hypothesis, linopirdine and XE991, selective KV7/KCNQ channel blockers, were separately included in the bath solution. Both compounds dose-dependently blocked CS-induced intrinsic plasticity (25 min post CS, linopirdine 1 µM: 84.6 ± 4.3%, n = 4, P = 0.06; 3 µM: 110.9 ± 7.7%, n = 3; P < 0.05. XE991 1 µM: 85.2 ± 6.3%, n = 8, P < 0.05; 3 µM: 114.5 ± 10.9%, n = 12; P < 0.001. Fig. 3, A–C and E), suggesting that KV7/KCNQ channels are involved in mediating a change in spike output following CS. However, another interpretation is that rather than occluding CS-induced intrinsic plasticity, linopirdine and XE991 were masking it through nonspecific effects on membrane excitability. This latter point is of particular concern because although linopirdine and XE991 exhibit high affinity to KV7/KCNQ channels, their specificity in native neurons has not been extensively tested. To discern between these two possibilities, we monitored membrane excitability in the presence of higher concentrations of XE991. In the absence of CS, prolonged application of XE991 (3 µM, n = 2; 10 µM, n = 4; pooled data) did not lead to a slow increase in the membrane excitability (Fig. 3D), arguing against the likelihood that linopirdine or XE991 was masking CS-induced intrinsic plasticity by slowly exerting a nonspecific effect on channels other than KV7/KCNQ channels.
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; Wang et al. 1998). Fitting the TEA dose-response relationship with a single-component Hill-Langmuir function yielded an IC50 of 1.3 mM and a slope factor of 0.35 (Fig. 4B, legend), the shallowness of which suggests that the KV7/KCNQ current in adult CA1 pyramidal neurons is unlikely mediated by channels of a single stoichiometry (Hadley et al. 2000, 2003). Further analysis indicates that while much of the current is mediated by heteromeric KV7.2/KCNQ2 and KV7.3/KCNQ3 channels, a small portion (30%) could be attributed to homomeric KV7.2/KCNQ2 channels (Fig. 4B, legend).
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; Wang et al. 1998), CS presentation did not change the spike output (25 min post CS, TEA: 105.2 ± 11.6%, P < 0.0005, n = 5. Fig. 8, A, C, and D). This surprising finding lends support for the presence of functional homomeric KV7.2/KCNQ2 channels and further highlights their importance in governing the output of adult CA1 pyramidal neurons.
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Ca2+ plays a pivotal role in inducing various forms of activity-dependent intrinsic plasticity (Cudmore and Turrigiano 2004; Fan et al. 2005; Frick et al. 2004; Misonou et al. 2004; Xu et al. 2005). We first assessed the dependence on Ca2+ of CS-induced intrinsic plasticity by including BAPTA-AM (20 µM), a membrane-permeable Ca2+ chelator, in the bath solution. BAPTA-AM prevented CS-induced suppression of membrane excitability (25 min post CS, BAPTA-AM: 115.6 ± 5.3%; P < 0.005, n = 5. Fig. 6, A–C), suggesting that Ca2+ influx associated with CS is involved in initiating this form of plasticity. In hippocampal neurons, CaV1/L-type and CaV2/N- and P, Q-type Ca2+ channels constitute a major source for activity- and spike-induced Ca2+ influx (Christie et al. 1995; Eliot and Johnston 1994). When SR33805 (5 µM) or calciseptine (1 µM), two selective nondihydropyridine CaV1/L-type Ca2+ channel blockers (Avery and Johnston 1996; de Weille et al. 1991; Romey and Lazdunski 1994), were included in the bath solution, CS presentation did not induce a change in spike output (25 min post CS, SR33805: 97.7 ± 3.1%; P < 0.005, n = 5. Calciseptine: 104.0 ± 3.9%; P < 0.005, n = 4. Fig. 7, B and C, left; Supplementary Fig. S3, A and B). Similar results were obtained when nimodipine (1 µM), the classical dihydropyridine CaV1/L-type Ca2+ channel blocker, was included in the bath solution (25 min post CS, nimodipine: 102.3 ± 8.6%; P < 0.005; n = 4. Fig. 7, A–C, left). In contrast, -conotoxin MVIIC (1 µM), a blocker of CaV2/N- and P, Q-type Ca2+ channels, reduced but failed to prevent CS-induced intrinsic plasticity (25 min post CS, -conotoxin MVIIC: 75.3 ± 2.4%, n = 4; P > 0.05. Fig. 7, B and C, right; Supplementary Fig. S3C; see also Fig. 8 ). These data indicate that an elevation of intracellular Ca2+ level mediated by influx preferentially through the CaV1/L-type Ca2+ channels during CS serves as a trigger for the induction of this sustained reduction in membrane excitability.
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; Xu and Lipscombe 2001). Bath application of BayK8644 (10 µM), a CaV1/L-type Ca2+ channel agonist, led to a potentiation of the KV7/KCNQ current that was readily suppressed by XE991 (10 µM; Fig. 9, A and B). In the presence of BayK8644, the XE991-sensitive current showed no significant change in the voltage dependence of steady-state activation (Fig. 9E; cf. Supplementary Fig S2C). In contrast, bath applications of 300 nM and 10 µM, nimodipine suppressed the KV7/KCNQ current by 46.9 ± 7.8 and 58.9 ± 9.0%, respectively (Fig. 9, C, D, and F). Using acutely dissociated CA1 pyramidal neurons, we verified that XE991 (10 µM) did not affect Ba2+ current through the CaV1/2 class Ca2+ channels (Supplementary Fig. S4, A–C). Altogether, our experiments illustrate a form of intrinsic plasticity in response to repetitive firing that requires both the CaV1/L-type Ca2+ channels and the KV7/KCNQ channels.
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) and subsequent phosphorylation of the KV7.2/KCNQ2 subunits (Schroeder et al. 1998). To test the hypothesis that a PKA-dependent pathway is involved in CS-induced intrinsic plasticity, we examined the effect of H-89, a potent and selective PKA inhibitor, on plasticity induction. Preincubation of slices in H-89 (5 µM) blocked CS-induced suppression of membrane excitability (25 min post CS, H-89: 119.2 ± 13.0%, P < 0.0001, n = 6. Fig. 8, B–D). The most parsimonious explanation for our data is a model in which Ca2+ influx through CaV1/L-type Ca2+ channels during CS leads to a long-lasting potentiation of the KV7/KCNQ current via PKA phosphorylation, thereby resulting in a persistent suppression of membrane excitability. |
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DISCUSSION |
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KV7/KCNQ channels are well suited to regulate intrinsic excitability
KV7/KCNQ channels are subthreshold-active K+ channels that give rise to the muscarine-sensitive, noninactivating M-current (Supplementary Fig. S2, B–D). Mutations in the genes that encode the KV7/KCNQ channel subunits cause congenital epilepsy and dominant hereditary deafness (Jentsch 2000), indicating a key role in the normal functioning of various neural circuits. With scRT-PCR, we demonstrated that hippocampal CA1 pyramidal neurons from adult rats express mRNAs for KV7.2/KCNQ2 and KV7.3/KCNQ3 (Fig. 4, C and D), subunits that encode a major portion of the neuronal KV7/KCNQ current. We also confirmed the presence of corresponding proteins with immunohistochemistry (Fig. 5). Unfortunately, our immunoperoxidase staining performed on fixed tissue sections does not allow for accurate estimation of the putative localization of the membrane-associated KV7/KCNQ channels. Immunolabelings performed on unfixed tissue preparations have shown that KV7.2/KCNQ2 and sometimes KV7.3/KCNQ3 proteins are concentrated at the axon initial segments and nodes of Ranvier, colocalizing with NaV channels (Devaux et al. 2004; Pan et al. 2006). I n many neurons, these sites correspond to the action potential initiation zones (Clark et al. 2005; Colbert and Johnston 1996; Colbert and Pan 2002; Khaliq and Raman 2006; Stuart and Hausser 1994). Corroborating with this immunolocalization pattern, two recent studies have demonstrated that functional KV7/KCNQ channels are concentrated near the perisomatic region: focal application of XE991 to the perisomatic region but not to the distal dendrites enhanced temporal summation of excitatory postsynaptic potentials (Hu et al. 2007) and spike output (Hu et al. 2007; Yue and Yaari 2006) in CA1 pyramidal neurons. Furthermore, enhancing KV7/KCNQ channel function with retigabine decreased the amplitude of population spikes in CA1 that represents the synchronous discharge of a number of CA1 pyramidal neurons in response to repetitive synaptic stimulation (Hu et al. 2007). Modulation of the KV7/KCNQ channel function will thus profoundly affect the global integrative properties of hippocampal pyramidal neurons, altering information coding and throughput within the CA1 subfield by gating the timing and generation of action potentials (Fig. 2, A–C, and Supplementary Fig. S2A) (Hu et al. 2007).
Most of our experiments were carried out in a modified ACSF containing synaptic blockers, 4-AP, and Cs+. We have also verified that CS-induced intrinsic plasticity occurs in plain ACSF without additional pharmacology (Supplementary Fig. S1). The reduction in membrane excitability observed in plain ACSF was smaller than that in modified ACSF (number of spikes 25 min following CS: plain ACSF, 84.7 ± 1.7%, n = 5; modified ACSF, 65.8 ± 5.7%)—a quantitative difference that is not surprising and could be attributed to at least two factors. First, in plain ACSF, CS may have induced additional forms of plasticity that partially obscured the effects we saw in modified ACSF. For example, HCN current, which was blocked in our modified ACSF with Cs+, has been shown to undergo activity-dependent long-term changes (Brager and Johnson 2007; Fan et al. 2005; van Welie et al. 2004). Likewise, expressions of KV1.1 and KV1.2 channels, the functions of which were suppressed by 4-AP in our modified ACSF, were also shown to be regulated by neuronal activity or a signaling pathway that is known to be involved in long-term synaptic plasticity (Raab-Graham et al. 2006; Tsaur et al. 1992). Second, the difference in magnitude of CS-induced intrinsic plasticity may reflect the amount of Ca2+ influx triggered in plain and modified ACSF.
Functional homomeric KV7.2/KCNQ2 channels in CA1 pyramidal neurons
In many neuronal types, it is thought that the KV7/KCNQ current is largely mediated by heteromeric channels of KV7.2/KCNQ2 and KV7.3/KCNQ3 subunits (Hadley et al. 2003; Shen et al. 2005; Wang et al. 1998). Our TEA dose-response profile of the KV7/KCNQ current is compatible with this idea but further implicates the presence of a highly TEA-sensitive component (30%), likely mediated by homomeric KV7.2/KCNQ2 channels (Fig. 4, A and B; corresponding figure legend). Surprisingly, CS-induced intrinsic plasticity was sensitive to TEA at a concentration that preferentially blocks these homomeric channels (Fig. 8, A, C, and D). It is tempting to think that this form of intrinsic plasticity reflects an activity-dependent modulation of homomeric KV7.2/KCNQ2 channels given that KV7.2/KCNQ2 proteins are more prevalent at the axon initial segments and nodes of Ranvier than KV7.3/KCNQ3 proteins in certain neuronal types (Devaux et al. 2004; Pan et al. 2006; Schwarz et al. 2006). However, linopirdine and XE991 dose-dependently suppressed CS-induced intrinsic plasticity (Fig. 3, A–C, and E) but do not discriminate between KV7/KCNQ channels of different subunits. Thus the linkage between homomeric KV7.2/KCNQ2 channels and CS-induced intrinsic plasticity still requires further investigation.
CaV1.2 channels account for the properties of Ca2+ influx that modulate KV7/KCNQ channels
Hippocampal pyramidal neurons express both high- (CaV1.2) and low (CaV1.3)-threshold activated L-type Ca2+ channels (Hell et al. 1993). Compared with other Ca2+ channel subtypes, CaV1/L-type Ca2+ channels are relatively insensitive to voltage-dependent inactivation (Lipscombe et al. 2004), allowing them to reliably report ongoing neuronal activity in the form of sustained Ca2+ influx. As CaV1.2 channels are far more sensitive to nimodipine than CaV1.3 channels (IC50 for nimodipine: CaV1.2 140 nM; CaV1.3 3 µM) (Xu and Lipscombe 2001), our finding that CS-induced intrinsic plasticity could be blocked by a low concentration of nimodipine (Fig. 7, A–C) suggests that CaV1.2 channels are the primary source of Ca2+ influx that mediates KV7/KCNQ channel modulation. This preferential coupling may reflect the abundance of CaV1.2 over CaV1.3 channels in pyramidal neurons as well as their close spatial proximity to the KV7/KCNQ channels (Davare et al. 2001; Hell et al. 1993) as CaV1.2 proteins have been reported to cluster in the perisomatic region of pyramidal neurons.
Could KV7/KCNQ channels and CaV1/L-type Ca2+ channels belong to a multiprotein complex that includes key elements of signaling transduction pathways such as kinases? Several pieces of evidence suggest so. First, components of PKA have been co-purified with the KV7.2/KCNQ2 subunit by affinity chromatography (Cooper et al. 2000), implicating a physical association between these two proteins. Second, both the KV7/KCNQ channels and the CaV1/L-type Ca2+ channels are known to associate with A-kinase anchoring proteins (AKAP) (Hoshi et al. 2003; Hulme et al. 2003). In particular, CaV1.2 channels have been demonstrated to associate with AKAP79/150 (Gao et al. 1997; Oliveria et al. 2007)—the same AKAP that interacts with the KV7.2/KCNQ2 subunit (Hoshi et al. 2003). AKAP79/150 is present at extremely high levels within the central nervous systems, including the hippocampus (Glantz et al. 1992). Thus there lies a distinct possibility that the KV7/KCNQ channels and the CaV1.2 channels are organized into a macromolecular complex that includes PKA, protein kinase C (PKC), and protein phosphatase 2B (PP2B)—enzymes that bind to AKAP79/150 (Carr et al. 1992; Coghlan et al. 1995; Klauck et al. 1996) that are also known to regulate the KV7/KCNQ channels (Hoshi et al. 2003; Marrion 1996; Schroeder et al. 1998)—to allow for rapid, localized, and efficient modulation of the KV7/KCNQ current following Ca2+ entry during neuronal activity. In addition to potentiating the KV7/KCNQ current, PKA has also been shown to increase Ca2+ transient through the CaV1/L-type Ca2+ channels in CA1 pyramidal neurons (Hoogland and Saggau 2004)—an effect that could be mediated by both the CaV1.2 (Davare et al. 2001) and CaV1.3 (Qu et al. 2005) channels. This synergistic action would result in a positive feedback loop that amplifies the amount of Ca2+ influx to further modulate the KV7/KCNQ channels during sustained neuronal activity.
Plausible signaling cascade in the Ca2+-dependent potentiation of KV7/KCNQ channels
KV7/KCNQ channels are not Ca2+ dependent in the sense that Ca2+ binding is not obligatory for channel opening (Adams et al. 1982). However, they are exquisitely sensitive to changes in the level of intracellular Ca2+ with reports of both an enhancement and a suppression (Cruzblanca et al. 1998; Kirkwood and Lisman 1992; Marrion 1996, 1997; Marrion et al. 1991; Tokimasa et al. 1996; Yu et al. 1994). Ca2+-dependent modulation of the KV7/KCNQ channels is thought to occur in spatially restricted domains (Delmas and Brown 2005), and the distinct effects likely reflect the interactions of Ca2+ with different Ca2+-sensing molecules present either on the KV7/KCNQ channels themselves or within the KV7/KCNQ channel macromolecular complex. Ca2+-mediated suppression of the KV7/KCNQ current arises through Ca2+ binding to the calmodulin associated with the KV7/KCNQ channels (Gamper and Shapiro 2003; Wen and Levitan 2002; Yus-Najera et al. 2002). On the other hand, two intracellular signaling pathways have been implicated in the potentiation of the KV7/KCNQ current. The first is mediated by arachidonic acid (AA) or its related metabolites (Moore et al. 1988; Schweitzer et al. 1990); the second, activation of PKA by cyclic AMP (Sims et al. 1988) and subsequent phosphorylation of the KV7.2/KCNQ2 subunits (Schroeder et al. 1998). Ca2+ influx through the CaV1/L-type Ca2+ channels can stimulate both pathways by activating the Ca2+-stimulated form of phospholipase A2 (cPLA2) and adenylyl cyclases (AC1 and AC8) to produce AA and cAMP, respectively. Both classes of enzymes are known to be present in the hippocampus (Kishimoto et al. 1999; Xia et al. 1991).
We suspect that multiple processes are involved in the induction and maintenance of CS-induced intrinsic plasticity, judging by its time course and kinetic features (Fig. 2B). The onset of CS-induced suppression of membrane excitability occurs quite rapidly, and in all cases, the decrease in spike output was apparent in the first few voltage traces acquired immediately following CS presentation (Fig. 1A). This again reinforces the notion that one molecular mechanism involved (e.g. phosphorylation) can be rapidly deployed. While our results support an involvement of the cAMP-PKA pathway (Fig. 8, B–D), CS-induced intrinsic plasticity nevertheless took 20 min to reach plateau (Fig. 2B). Therefore we cannot rule out the possibility that other processes are recruited on a different and perhaps overlapping time scale to maintain such long-lasting suppression of membrane excitability. Enhancing Ca2+ influx through the CaV1/L-type Ca2+ channels with BayK8644 increases the KV7/KCNQ conductance without shifting its voltage dependence (Fig. 9E; cf. Supplementary Fig. S2C). At the moment, we cannot rule out the possibility that such increase in KV7/KCNQ conductance is mediated by an increase in the functional KV7/KCNQ channel density.
Functional relevance to learning and memory
Enduring changes in neuronal excitability following periods of activity or learning a behavioral task are features of central neurons thought to encode information at the cellular level. Reduction in the capacity to regulate intrinsic excitability, in fact, has been correlated to impaired learning in aging animals (Moyer et al. 2000; Tombaugh et al. 2005). Cholinergic agonists and CaV1/L-type Ca2+ channel blockers increase the excitability of hippocampal pyramidal neurons in vitro (Moyer et al. 1992; Weiss et al. 2000) and facilitate learning of hippocampus-dependent tasks in vivo (Deyo et al. 1989; Weiss et al. 2000). An attractive hypothesis waiting to be tested is that the facilitating effect of cholinergic or Ca2+ treatment on learning is mediated in part by modulation of the KV7/KCNQ current. In support of this idea, a recent study showed that transgenic mice expressing dominant-negative KV7.2/KCNQ2 subunits are impaired in learning hippocampus-dependent spatial tasks (Peters et al. 2005). Given the widespread and overlapping expression patterns of the KV7/KCNQ channels and the CaV1/L-type Ca2+ channels, the interactions between them may serve as a generalized principle in fine tuning the activity of various neural circuits in a history-dependent manner. Understanding the ways in which neurons adjust their output in response to prior activity will be a pivotal step in advancing our knowledge of information storage at the cellular and network level, and ultimately provide a key to addressing higher brain functions such as learning and memory.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 The online version of this article contains supplemental data.
Present address and address for reprint requests and other correspondence: W. W. Wu, Vollum Institute, Oregon Health and Science University, 3181 SW Sam Jackson Park Rd., Mail Code L-474, Portland, OR 97239 (E-mail: wuwendy{at}ohsu.edu)
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of –31mV and a slope factor of 7.5 mV. F: maximal percentage inhibition of the KV7/KCNQ current in the presence of 300 nM and 10 µM nimodipine.