Featured ArticleArticles, Cellular/Molecular

KV7/M Channels Mediate Osmotic Modulation of Intrinsic Neuronal Excitability

Anna Caspi, Felix Benninger and Yoel Yaari
Journal of Neuroscience 9 September 2009, 29 (36) 11098-11111; DOI: https://doi.org/10.1523/JNEUROSCI.0942-09.2009

Abstract

Modest decreases in extracellular osmolarity induce brain hyperexcitability that may culminate in epileptic seizures. At the cellular level, moderate hyposmolarity markedly potentiates the intrinsic neuronal excitability of principal cortical neurons without significantly affecting their volume. The most conspicuous cellular effect of hyposmolarity is converting regular firing neurons to burst-firing mode. This effect is underlain by hyposmotic facilitation of the spike afterdepolarization (ADP), but its ionic mechanism is unknown. Because blockers of KV7 (KCNQ) channels underlying neuronal M-type K+ currents (KV7/M channels) also cause spike ADP facilitation and bursting, we hypothesized that lowering osmolarity inhibits these channels. Using current- and voltage-clamp recordings in CA1 pyramidal cells in situ, we have confirmed this hypothesis. Furthermore, we show that hyposmotic inhibition of KV7/M channels is mediated by an increase in intracellular Ca2+ concentration via release from internal stores but not via influx of extracellular Ca2+. Finally, we show that interfering with internal Ca2+-mediated inhibition of KV7/M channels entirely protects against hyposmotic ADP facilitation and bursting, indicating the exclusivity of this novel mechanism in producing intrinsic neuronal hyperexcitability in hyposmotic conditions.

Introduction

Reduction in plasma osmolarity associated with a wide range of clinical syndromes can lead to epileptic seizures (Andrew, 1991). Likewise, lowering extracellular osmolarity induces neuronal network hyperexcitability and epileptiform discharges in hippocampal slices (Andrew et al., 1989; Traynelis and Dingledine, 1989; Dudek et al., 1990; Ballyk et al., 1991; Saly and Andrew, 1993). These effects of hyposmolarity have been attributed mainly to enhanced excitatory electric field (ephaptic) effects and ionic interactions (e.g., increases in the extracellular concentration of K+) consequent to cell swelling and concurrent shrinkage of the extracellular space (Traynelis and Dingledine, 1989; Ballyk et al., 1991). However, hyposmolarity was shown also to enhance the intrinsic excitability of principal hippocampal neurons. Thus, in CA1 pyramidal cells, lowering osmolarity markedly potentiated the spike afterdepolarization (ADP), causing regular firing neurons to convert to burst-firing mode (Azouz et al., 1997). Because bursting neurons play a pivotal role in neuronal recruitment and synchronization, this effect likely contributes to the hyposmotic enhancement of neuronal network excitability and induction of epileptic activity (Chagnac-Amitai and Connors, 1989; Jensen and Yaari, 1997; Sanabria et al., 2001; Yaari and Beck, 2002; Wittner and Miles, 2007; Yaari et al., 2007), yet the mechanisms responsible for hyposmotic spike ADP facilitation and bursting have not been elucidated.

The somatic spike ADP of adult CA1 pyramidal cells in normal physiological conditions is determined by interplay of two low voltage-activated, noninactivating currents in the perisomatic region: the persistent Na+ current (I NaP), which acts to redepolarize the neuron immediately after the fast spike (Azouz et al., 1996; Su et al., 2001; Yue et al., 2005), and the M-type K+ current (I M), which opposes I NaP and repolarizes the neuron (Yue and Yaari, 2004, 2006). The latter current is generated by a subset of KV7 (KCNQ) channels (KV7/M channels) (Wang et al., 1998; Shah et al., 2002) that are subjected to modulation by many chemical transmitters (Brown and Yu, 2000). Voltage-gated Ca2+ currents flowing during the spike enhance the ADP, but this effect is exerted indirectly via intracellular block of KV7/M channels, unleashing the depolarizing action of I NaP (Chen and Yaari, 2008). Recently, we have shown that, in marked resemblance to the effects of lowering osmolarity, selective pharmacological blockage of KV7/M channels strongly facilitates the spike ADP and induces bursting (Yue and Yaari, 2004, 2006). Given also that loss-of-function mutations in KV7/M channels cause epileptic seizures in man (Charlier et al., 1998; Singh et al., 1998) and animals (Peters et al., 2005), this similarity raises the intriguing possibility that lowering osmolarity enhances intrinsic neuronal excitability by inhibiting KV7/M channels. However, other mechanisms (e.g., hyposmotic enhancement of I NaP) can be entertained.

Here we have used intracellular current- and voltage-clamp recordings techniques and pharmacological manipulations in hippocampal slices to clarify how lowering osmolarity facilitates the spike ADP and induces intrinsic bursting in CA1 pyramidal cells. Our data show that this hyposmotic action is mediated exclusively by inhibition of I M via Ca2+ release from internal stores.

Materials and Methods

Hippocampal slice preparation.

All animal experiments were conducted in accordance with the guidelines of the Animal Care Committee of the Hebrew University. Male Sabra rats (125–150 g) were decapitated under isoflurane anesthesia, and transverse hippocampal slices (400 μm) were prepared with a Leica VT1000S vibrating microslicer and transferred to a storage chamber perfused with oxygenated (95% O2/5% CO2) normosmotic artificial CSF (aCSF) at room temperature.

Solutions.

Normosmotic (301 ± 0.7 mOsm; n = 10; measured with a Vapro-5520 vapor pressure osmometer; Wescor) aCSF contained the following (in mm): 100 NaCl, 3.5 KCl, 2 MgCl2, 1.6 CaCl2, 24 NaHCO3, 10 d-glucose, and 60 mannitol. Moderately (240.8 ± 2.2 mOsm; n = 5; a 20% reduction) or mildly (270 ± 1.5 mOsm; n = 5; a 10% reduction) hyposmotic aCSFs were prepared by omitting all or 30 mm mannitol, respectively, so that ion concentrations and ionic strength of all aCSFs were the same. High K+ aCSF was made by adding 26.5 mm KCl to normosmotic aCSF (final KCl concentration, 30 mm). The Ca2+-free aCSF was prepared by replacing CaCl2 with 2 mm MgCl2. High Ca2+ aCSF was made by increasing CaCl2 concentration to 3 mm. All aCSFs were titrated to pH 7.4. The aCSFs contained also the glutamate receptor antagonists 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) (15 μm) and 2-amino-5-phosphono-valeric acid (50 μm) to block fast EPSPs and the GABAA receptor antagonist picrotoxin (100 μm) to block fast IPSPs. In experiments designed to measure membrane chord resistance (R chord) or I M, tetrodotoxin (TTX) (1 μm), Ni2+ (1 mm), and Cd2+ (300 μm) were added to the aCSFs after cell impalement to block voltage-gated Na+ and Ca2+ channels, as well as Ca2+-gated K+ channels. Additional drugs used in specific experiments are indicated below.

Chemicals and drugs.

All chemicals and drugs were purchased from Sigma, except for CNQX (Research Biochemicals), TTX (Alomone Labs), XE991 [10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone] and ZD7288 (4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chloride) (Tocris Bioscience), and retigabine (a kind gift from Valeant). Stock solutions (10 mm) of linopirdine and retigabine were prepared in ethanol and dimethylsulfoxide, respectively, and diluted 1:1000 when added to the aCSF. All other drugs were added to the aCSF from aqueous stock solutions. Measurements of drug effects were usually performed after 30 min of slice perfusion with the drug-containing aCSF.

Electrophysiological recordings.

We used sharp glass microelectrodes filled with 3 m KCl (60–100 MΩ) and an Axoclamp 2B amplifier (Molecular Devices) for somatic intracellular recordings in the CA1 pyramidal layer. In some experiments, when indicated, 200 mm BAPTA was included in the microelectrode filling solution. The pyramidal cells included in this study had stable resting potentials more negative than −60 mV, apparent input resistances ≥25 MΩ, and overshooting action potentials. In current-clamp mode, the bridge balance was carefully monitored and adjusted before each measurement. Spikes were elicited by applying brief (4 ms) or long (180 ms) positive current pulses. In discontinuous voltage-clamp mode, the switching frequency between current injection and voltage sampling was 1.5–3.5 kHz. The current records were filtered at 0.3 kHz.

Measurement of membrane chord resistance (Rchord).

For exploring modulation of R chord by I M modifying treatments, slices were superfused with aCSFs containing 1 μm TTX, 1 mm Ni2+, and 300 μm Cd2+. Injecting a series of five 0.9-s-long positive current ramps, increasing from 0 to 1 nA in increments of 200 pA, produced corresponding incremental depolarization ramps (see Fig. 4 Aa). R chord in the voltage range of I M activation (greater than −60 mV) was measured from the slope of the second half of these voltage–current relationships.

Isolation and measurement of IM.

For pharmacological isolation of I M, the slices were superfused with aCSFs containing 1 μm TTX, 10 μm retigabine, and 50 μm ZD7288. We used an established voltage protocol to elicit I M in CA1 pyramidal cells (Halliwell and Adams, 1982; Schweitzer et al., 1990, 1998; Schweitzer, 2000). From a holding potential (V h) of approximately −40 mV, seven or nine hyperpolarizing voltage steps (duration 0.9 s) incrementing by −5 or −6 mV, as indicated, were delivered. These steps induced slow current “relaxations” after the instantaneous inward current drops (see Fig. 5 Aa), which represent the slow deactivation of I M. Current relaxations were fitted by single-exponential curves (starting after the capacitance artifact) and were extrapolated back to the beginning of the hyperpolarizing command pulses. I M amplitudes were derived as the differences between the instantaneous peak currents at command onset and the steady-state currents just before command offset.

Monitoring pyramidal cells size.

We used a high-sensitivity cooled CCD camera (Nikon DS-Qi1) mounted on a Nikon Eclipse FN1 light microscope equipped with a 60× objective to visualize CA1 pyramidal cells in situ. The cell of interest was continuously monitored during exposure to the different aCSFs. In case of upward shifts attributable to swelling of the slice, the plane of focus was adjusted, keeping the maximum extent of the neuron visualized. The area in the visual field occupied by pyramidal cell soma (cross-sectional area) was measured by outlining the soma borders and integrating the enclosed area using NIS-Elements Advanced Research software (Nikon).

Data analysis.

Offline analyses were performed with pClamp9 software (Molecular Devices). In current-clamp experiments, the size of the spike ADP was measured as the integrated “area under the curve” between the fast afterhyperpolarization (AHP) and the point at which membrane voltage returned to resting potential.

All results are presented as the mean ± SEM. Assessment of statistical significance of differences between means was performed with paired Student's t test or repeated-measures ANOVA, as appropriate.

Results

CA1 pyramidal cells do not swell in hyposmotic ACSF

Brain tissue swells under hyposmotic stress attributable to osmotic movement of water into the intracellular compartment (Somjen, 2004). However, recent studies suggest that cellular responses to low osmolarity are not uniform. In particular, it was found that neocortical pyramidal cells in situ (both in vivo and in neocortical slices) tenaciously maintain their volume when extracellular osmolarity is reduced by 40 mOsm (a 13.3% reduction in osmolarity) (Andrew et al., 2007; Risher et al., 2009). In contrast, astrocytes readily swell in this condition and remain hypovolemic for tens of minutes without displaying a regulatory volume decrease (Andrew et al., 1997; Risher et al., 2009).

To test how CA1 pyramidal cells in situ respond to a hyposmotic challenge, we monitored them visually during superfusion of hippocampal slices with moderately hyposmotic aCSF (−60 mOsm; a 20% reduction in osmolarity). Changes in soma volume were inferred from changes in maximal cross-sectional area of the soma (see Materials and Methods). A representative experiment is illustrated in Figure 1 A. The neuron in the center of the microscope field was monitored starting 10 min before (Fig. 1 Aa) until 30 min after changing from normosmotic to hyposmotic aCSF, and measurements were made at 5 min intervals (Fig. 1 Ab). No significant change in soma cross-sectional area was evident during this observation period. Similar results were obtained in eight additional experiments, as summarized in Figure 1, B and C (cross-sectional area before and 30 min after changing to hyposmotic aCSF: 102.2 ± 5.8 and 101.8 ± 7.1 μm2, respectively; n = 9). In contrast, raising K+ concentration in the aCSF to 30 mm consistently caused a progressive increase in soma cross-sectional area, as illustrated in Figure 1, D and E. On average, high-K+ aCSF caused an 18.5 ± 3.0% increase in soma cross-sectional area (before and 30 min after changing to high K+ aCSF: 98.5 ± 6.9 and 122.2 ± 8.4 μm2, respectively; p < 0.05; n = 9) (Fig. 1 F).

Figure 1.
Figure 1.

CA1 pyramidal cells do not swell in hyposmotic aCSF. A , Images of a representative CA1 pyramidal cell (in the center of the visual field) monitored before ( a ) and 30 min after superfusion with hyposmotic (20% reduction) aCSF ( b ). Note no change in soma cross-sectional area. B , Plots of maximal soma cross-sectional areas in nine neurons from separate experiments. Images were taken every 5 min, starting 10 min before and ending 30 min after changing from normosmotic to hyposmotic aCSF. The dark plot depicts the mean ± SEM values. C , Summary histogram depicting the data in B as percentage changes in soma cross-sectional area. No significant changes were observed during the hyposmotic challenge. D , Images of another neuron (in the center of the visual field) monitored before ( a ) and 30 min after superfusion with high-K+ (30 mm) aCSF ( b ). Note expansion of cell size. E , Plots of soma cross-sectional areas in nine neurons from separate experiments. Images were taken every 5 min, starting 10 min before and ending 30 min after changing from normal K+ to high K+ aCSF. The dark plot depicts the mean ± SEM values. F , Summary histogram depicting the data in E as percentage changes in soma cross-sectional area. Significant increases in soma cross-sectional area were observed already 10 min after beginning the high K+ challenge (*p < 0.05; n = 9).

These results suggest that, like their neocortical counterparts (Andrew et al., 2007; Risher et al., 2009), CA1 pyramidal cells do not swell in a hyposmotic milieu.

Hyposmolarity facilitates the ADP and induces bursting in CA1 pyramidal cells

In normosmotic aCSF, all impaled neurons (n = 56) fired a solitary spike followed by a distinct ADP when stimulated with brief (4 ms) positive current pulses (Fig. 2 Aa) (Jensen et al., 1996). It was shown previously in these neurons that lowering osmolarity by 40 mOsm (a 13% decrease) facilitates the spike ADPs, causing many neurons (∼43%) to fire high-frequency bursts of three to six spikes instead of solitary spikes (Azouz et al., 1997). Here we replicated these experiments using a stronger hyposmotic challenge (−60 mOsm; a 20% decrease). Changing to hyposmotic aCSF caused a slight depolarization of resting potential (from −66.7 ± 1.3 to −63.0 ± 1.5 mV; n = 7), which was counteracted by the injection of an appropriate steady negative current. A representative experiment is illustrated in Figure 2 A. Lowering osmolarity steeply increased ADP size by 69.5 ± 7.2% (from 280.1 ± 28.5 to 477.0 ± 68.5 mV/ms) (Fig. 2 Ab,Ac and overlaid traces in Ae). The facilitated ADP ultimately triggered three to five additional spikes, thus altering the firing mode of the neurons from regular to burst firing (Fig. 2 Ad). Similar results were consistently obtained in all seven experiments, as summarized in Figure 2, B and C.

Figure 2.
Figure 2.

Hyposmolarity facilitates the spike ADP, induces bursting, and enhances spike output in CA1 pyramidal cells. In each panel showing current-clamp recordings, here and in all figures below, the bottom traces depict the injected current stimuli, and the top traces represent the recorded voltage responses. Resting potentials are depicted to the left of the voltage responses. The neurons were maintained at their native resting potential throughout the recording session by injecting appropriate direct current. A , Lowering osmolarity by 20% caused progressive facilitation of the spike ADP and the conversion of the single spike responses to bursts of five spikes ( a–d ). Portions of traces in a–c are expanded and overlaid in e to allow comparison of the spike ADPs. B , Bar histogram showing ADP size in normosmotic versus hyposmotic aCSFs (n = 6; *p < 0.05). The ADPs were measured before the emergence of secondary spikes. C , As in B but showing the number of spikes evoked by the brief stimuli (n = 7; **p < 0.01). In hyposmotic aCSF, spike were counted after burst firing was established. D , The neuron shown in A was also stimulated with a set of long (180 ms) depolarizing current pulses. Bottom traces depict the minimal spike responses (evoked by 100 pA in this case). Top traces show the responses to larger stimulus intensities (400 pA). Changing from normosmotic ( a ) to hyposmotic aCSF ( b ) increased spike frequency and spike output at all stimulus intensities. E , Spike output plots showing the number of evoked spikes versus stimulus intensity in normosmotic (filled circles) and hyposmotic (open circles) aCSFs (n = 8; p < 0.05).

The neurons were also stimulated with long (180 ms) depolarizing current pulses, incrementing in steps of 50 pA (up to 400 pA). The number of spikes evoked by each current step increased with stimulus intensity (Fig. 2 Da,E). Lowering osmolarity enhanced spike output at all given intensities (Fig. 2 Db,E) (p < 0.05; n = 8).

Hyposmotic bursting is driven by INap

The spike ADP and associated bursting in adult CA1 pyramidal cells are normally driven by the depolarizing action of I NaP (Azouz et al., 1996; Su et al., 2001; Yue et al., 2005). Direct potentiation of these potentials by Ca2+ currents was found during postnatal development (Magee and Carruth, 1999; Chen et al., 2005; Metz et al., 2005), during epileptogenesis (Sanabria et al., 2001; Su et al., 2002; Yaari et al., 2007), and after pharmacological blockage of A-type K+ current in the apical dendrites (Magee and Carruth, 1999). To identify the inward currents driving hyposmotic bursting, we tested its sensitivity to Na+ and Ca2+ channel blockers. In the case of I NaP, we used the neuroprotective drug riluzole, which in CA1 pyramidal cells blocks I NaP completely in doses that only mildly reduce transient Na+ current (Yue et al., 2005). Adding 10 μm riluzole to the aCSF consistently abolished hyposmotic bursting, causing the neurons to fire single spikes in response to brief stimuli (Fig. 3 Aa–Ac) (p < 0.01; n = 11). These results are summarized in the bar histogram shown in Figure 3 B.

Figure 3.
Figure 3.

Hyposmotic bursting is driven by I NaP. A , In this neuron, lowering osmolarity by 20% invoked bursting (5 spikes; a , b ), which was suppressed by adding the I NaP blocker riluzole (10 μm) to the hyposmotic aCSF ( b , c ). B , Bar histogram summarizing the effects of riluzole on hyposmotic bursting (n = 11; **p < 0.01). C , In yet another neuron, hyposmotic bursting ( a , b ) was markedly prolonged by the Ca2+ channel blocker Ni2+ (1 mm; n = 6; b , c ). Again, 10 μm riluzole converted the prolonged bursts to single spikes ( c , d ). D , At a lower concentration (100 μm), Ni2+ only slightly increased the number of spikes in hyposmotic bursts (n = 2).

To test the potential contribution of Ca2+ currents, we used the Ca2+ channel blocker Ni2+. Adding 1 mm Ni2+ to the aCSF (in replacement of 1 mm Mg2+) enhanced hyposmotic bursting, eventually producing prolonged plateau potentials in all tested neurons (Fig. 3 Ca–Cc) (n = 6). This effect most likely is attributable to blocking Ca2+-gated K+ currents generating the medium and slow AHPs (Madison and Nicoll, 1984; Storm, 1989). The prolonged bursts and plateau potentials produced in hyposmotic Ni2+-containing aCSF were also converted to single spikes by subsequent addition of 10 μm riluzole (Fig. 3 Cd). At a lower concentration of Ni2+ (100 μm) that preferentially blocks T-type and R-type Ca2+ currents (Lee et al., 1999; Williams et al., 1999) and suppresses T-type Ca2+ current-driven bursting (Su et al., 2002; Yaari et al., 2007), Ni2+ only slightly enhanced hyposmotic bursting (Fig. 3 Dab) (n = 2).

Together, these findings indicate that hyposmotic bursting is driven predominantly by I NaP, whereas Ca2+ currents limit burst duration likely by activating Ca2+-gated K+ currents.

Hyposmotic increase in membrane chord resistance

The growth of the spike ADP and conversion to bursting in hyposmotic aCSF may be attributable to a genuine increase in I NaP. Alternatively, or additionally, it may result from a decrease in opposing K+ currents. In CA1 pyramidal cells, the main K+ current counteracting I NaP and curtailing the spike ADP is I M (Yue and Yaari, 2004). Inhibiting this current with KV7/M channel blockers, such as linopirdine or XE991 (Aiken et al., 1995; Wang et al., 1998), was shown to facilitate the spike ADP and induce bursting in striking resemblance to the effects of low osmolarity. Likewise, bursts induced by inhibiting I M were readily suppressed by inhibiting I NaP with riluzole and markedly prolonged by blocking Ca2+ currents with 1 mm Ni2+ (Yue and Yaari, 2006).

We first tested the hypothesis that hyposmotic facilitation of the spike ADP is attributable to inhibition of I M. We began by monitoring R chord in conditions of blocked voltage-gated Na+ and Ca2+ currents and Ca2+-gated K+ currents (see Materials and Methods). To that end, the aCSFs contained 1 μm TTX, 1 mm Ni2+, and 200 μm Cd2+. The resting potential of the neurons in this series of experiments was −67.0 ± 1.3 mV (n = 18). In most neurons, the voltage ramp depolarizations evoked by the injected current ramps displayed a conspicuous delayed rectification (decrease in R chord with depolarization), particularly at voltages above −60 mV (Fig. 4 Aa). This rectification likely results from activation of I M, whose threshold of activation is in this voltage range (Brown and Adams, 1980; Hu et al., 2007). Indeed, delayed rectification was reduced by the KV7/M channel blocker XE991 (20 μm) (Fig. 4 Cab and overlaid traced in Cd) and was accentuated by the KV7/M channel enhancer retigabine (10 μm) (Fig. 4 Eab and overlaid traces in Ed). In each tested neuron, we measured the average R chord for the second half of the voltage–current relationship. XE991 increased R chord by 120.7 ± 21.9% (from 13.6 ± 3.3 to 29.3 ± 4.3 MΩ; n = 6; p < 0.05; summary of results in Fig. 4 D), whereas retigabine decreased R chord by 59.9 ± 10.0% (from 8.2 ± 1.6 to 2.9 ± 0.9 MΩ; n = 5; p < 0.05) (summary of results in Fig. 4 F).

Figure 4.
Figure 4.

Hyposmotic modulation of membrane R chord. Current-clamp recordings in aCSFs containing 1 μm TTX, 1 mm Ni2+, and 300 μm Cd2+. A , In normosmotic aCSF, the neuron was injected with 0.9-s-long current ramps increasing up to 1 nA in steps of 200 pA. The depolarizing potentials evoked by the smaller ramps increased linearly, whereas those evoked by the larger ramps displayed delayed rectification, particularly during the second half of stimulation ( a ). Changing to hyposmotic aCSF (−20%) induced an increase in R chord ( a , b and overlaid traces in d depicting the responses to the largest ramp current). This effect reversed during restoring normal osmolarity ( b , c and overlaid traces in d ). B , Bar histogram summarizing the effects of lowering osmolarity on R chord measured during the second half of the largest ramp current stimulus (n = 8; *p < 0.05). C , Adding XE991 (20 μm) to normosmotic aCSF also increased R chord ( a , b and overlaid traces in d ). Subsequent lowering of osmolarity had no additional effect on R chord ( b , c and overlaid traces in d ), indicating that blocking I M occludes the effect of hyposmolarity. D , Bar histogram summarizing the effects of XE991 and of lowering osmolarity in the presence of XE991 on R chord (n = 5; *p > 0.05). E , Adding retigabine (10 μm) to normosmotic aCSF decreased R chord ( a , b and overlaid traces in d ). Changing to hyposmotic aCSF in the presence of retigabine reversed the effect of this drug and increased R chord beyond control value ( b , c and overlaid traces in d ). F , Bar histogram summarizing the effects of retigabine (n = 5; *p < 0.05) and hyposmolarity (n = 5; **p < 0.01) on R chord.

In all tested neurons, lowering osmolarity mimicked the effect of XE991 in reducing delayed rectification (Fig. 4 Aa,Ab and overlaid traces in Ad), causing R chord to increase by 123.3 ± 35.0% (from 9.5 ± 1.7 to 23.8 ± 4.1 MΩ; n = 8; p < 0.05) (summary of results in Fig. 4 B). This effect reversed during washing with normosmotic aCSF (n = 3) (Fig. 4 Ac and overlaid traces in Ad; summary of results in B). Pretreatment of the neurons with XE991 (which by itself increased R chord, as noted above) occluded this effect of hyposmolarity (n = 5) (Fig. 4 Cc and overlaid traces in Cd; summary of results in D). Conversely, when R chord was reduced by retigabine, changing to hyposmotic aCSF completely reversed its effect (n = 5) (Fig. 4 Ea–Ec and overlaid traces in Ed; summary of results in F). These data strongly suggest that lowering osmolarity inhibits I M.

Hyposmotic inhibition of IM

We next examined the effects of lowering osmolarity on I M directly, using the sharp microelectrode voltage-clamp technique (see Materials and Methods). The aCSFs in these experiments contained 1 μm TTX to block Na+, 10 μm retigabine to enhance I M (Tatulian et al., 2001), and 50 μm ZD7288 to eliminate activation of h-current at voltages below −70 mV (Halliwell and Adams, 1982; Gu et al., 2005). After impalement, the neurons were maintained at a holding potential (V h) of −42.2 ± 0.37 mV (n = 5) to activate I M and inactivate other classes of voltage-gated K+ channels. Hyperpolarizing voltage steps (duration, 0.9 s; applied at 0.2 Hz) incrementing by −6 mV (down to approximately −96 mV) induced slow current relaxations after the instantaneous inward current drops (Fig. 5 Aa). In each response, the current component undergoing relaxation represents the slow deactivation of I M (Brown and Adams, 1980; Halliwell and Adams, 1982; Schweitzer et al., 1990, 1998; Schweitzer, 2000). The reversal potential of these currents was approximately −90 mV (Fig. 5 B), approximating the reversal potential of K+ (−96.4 ± 6.2 mV) (Jensen et al., 1993).

Figure 5.
Figure 5.

Lowering osmolarity inhibits I M. Voltage-clamp recordings in aCSFs containing 1 μm TTX, 50 μm ZD7288, and 10 μm retigabine. A , In this neuron, I M amplitudes were measured from the outward current relaxations during nine sequential 0.9-s-long hyperpolarizing steps incrementing by −6 mV from a V h of −42 mV (the voltage protocol is shown in a , bottom). In a–d , the top traces depict the responses to the voltage steps. Shown below is the current relaxation obtained by stepping to −72 mV together with the fitted monoexponential line that was extrapolated to the beginning of the voltage step. Changing to hyposmotic aCSF (−20%) markedly inhibited I M ( a , b ). This effect reversed completely after returning to normosmotic aCSF ( c ). Subsequent addition of 10 μm linopirdine to the normosmotic aCSF mimicked the effect of hyposmolarity in inhibiting I M ( d ). B , Plots of I M amplitudes versus the hyperpolarizing command potentials in normosmotic (filled circles; n = 5) and hyposmotic (open circles; n = 5) aCSFs. C , Bar histogram showing I M inhibition by −20% (−60 mOsm) and −10% (−30 mOsm) reductions in osmolarity (*p < 0.05). D , In another neuron superfused with normosmotic aCSF, I M relaxations were recorded, as in A , before ( a ) and after adding 20 μm XE991 to the aCSF to block I M ( b ). The currents obtained after adding XE991 were subtracted from those obtained in control normosmotic aCSF ( c ). E , Another neuron was superfused with −20% (−60 mOsm) hyposmotic aCSF. Again, I M relaxations were recorded before ( a ) and after adding 20 μm XE991 to the aCSF ( b ). The currents obtained after adding XE991 were subtracted from those obtained in control hyposmotic aCSF ( c ). F , Plots of XE991-sensitive current amplitudes versus the hyperpolarizing command potentials in normosmotic (filled circles; n = 5) and hyposmotic (open circles; n = 5) aCSFs. G , Bar histogram comparing I M amplitudes (obtained from XE991-sensitive current relaxations at −72 mV) in normosmotic versus hyposmotic aCSFs (n = 5; **p < 0.01).

Hyposmotic aCSF reduced the holding current by 26.5 ± 6.8% (from 1.56 ± 0.07 to 1.14 ± 0.01 nA; n = 5) (Fig. 5 A, dotted line). Concomitantly, the current relaxations were markedly reduced at all step potentials (Fig. 5 Ab; the mean current–voltage relationship is depicted in B), indicating that I M was inhibited. Thus, at −72 mV (Fig. 5 Aab, bottom traces), I M was inhibited by 71.0 ± 8.9% (from 118.0 ± 15.8 to 35.6 ± 11.9 pA; n = 5; p < 0.05) (Fig. 5 C). All effects of hyposmotic aCSF on I M reversed during restoring normal osmolarity (Fig. 5 Ac) (n = 2). Adding 10 μm linopirdine to normosmotic aCSF decreased I M by 64.8 ± 17.4% (from 64.2 ± 18.7 to 15.13 ± 13.6 pA; n = 5), thus mimicking the effects of hyposmolarity (Fig. 5 Ad) (n = 5).

We tested also whether I M is modified by a milder decrease in osmolarity, which may be more pertinent to physiological changes in osmolarity (Somjen, 2004). Reducing aCSF osmolarity by 30 mOsm (a 10% reduction) decreased I M by 54.6 ± 17.6% (from 129.8 ± 15.8 to 61.5 ± 20.4 pA; n = 5; p < 0.05) (Fig. 5 C).

In another series of experiments, we used the same voltage protocol and aCSF compositions described above to record I M relaxations in CA1 pyramidal cells in two separate sets of slices: slices superfused with normosmotic aCSF (n = 5) and slices superfused with −20% (−60 mOsm) hyposmotic aCSF (n = 5). The mean holding potential in these experiments was −42.7 ± 0.39 mV (n = 10). Expectedly, as illustrated in Figure 5, A and E, I M relaxations were much smaller in hyposmotic aCSF (Fig. 5 Ea) than in normosmotic aCSF (Fig. 5 Da). We then applied 20 μm XE991 to the aCSFs for up to 30 min to block I M entirely. Application of XE991 reduced the holding current by 41.09 ± 9.5% (from 1.72 ± 0.15 to 1.01 ± 0.1 nA; n = 5; p < 0.05) in normosmotic aCSF and by 26.8 ± 2.9% (from 1.23 ± 0.03 to 0.90 ± 0.06 nA; n = 5; p < 0.05) in hyposmotic aCSF. No residual current relaxations were seen in XE991-treated neurons (Fig. 5 Db,Eb). Subtracting the current traces obtained in XE991-containing aCSFs from the respective currents obtained before XE991 applications provided a set of pure I M relaxations for each experimental condition (Fig. 5 Dc,Ec). As shown in the mean current–voltage relationship in Figure 5 F, I M amplitudes recorded in hyposmotic aCSF were markedly smaller than those obtained in normosmotic aCSF at all step potentials. Thus, at a step potential of −72 mV, I M in hyposmotic aCSF was 70.9 ± 6.7% smaller than in normosmotic aCSF (35.2 ± 6.5 vs 126.9 ± 7.5 pA, respectively; n = 5 in each group; p < 0.01) (Fig. 5 G). This result is very consistent with the hyposmotic I M inhibition estimated directly from I M relaxations (71.0 ± 8.9%) (Fig. 5 C).

Hyposmotic IM inhibition underlies ADP facilitation and induction of bursting

To examine whether the hyposmotic facilitation of the spike ADP and induction of bursting are exclusively or only partially attributable to I M inhibition, we examined how lowering osmolarity affects neurons in which I M was inhibited pharmacologically. Expectedly, pretreatment of the neurons with 20 μm XE991 in normosmotic aCSF converted single spikes to high-frequency bursts of three to six spikes (Yue and Yaari, 2004). To expose the underlying facilitated ADP, we suppressed the bursts by elevating extracellular Ca2+ concentration ([Ca2+]o) to 3 mm (Fig. 6 Aa,Ab). The decreased excitability in high [Ca2+]o is attributed to augmented screening of membrane surface potential by Ca2+ (Frankenhaeuser and Hodgkin, 1957; Su et al., 2001). A representative experiment is illustrated in Figure 6 A. In high-Ca2+ aCSFs, lowering osmolarity failed to increase the spike ADP in this (Fig. 6 Ab,Ac and overlaid traces in Ad), as well as in other four experiments (n = 5; p > 0.05) (Fig. 6 C).

Figure 6.
Figure 6.

Blocking I M pharmacologically occludes hyposmotic ADP facilitation. Current-clamp recordings of spike activity, as in Figure 1. A , In the first neuron, adding 20 μm XE991 to normosmotic aCSF to block I M induced bursting ( a ). The spike ADP was reduced by increasing aCSF Ca2+ content to 3 mm, converting bursting to single spiking ( b ). Reducing osmolarity of the high-Ca2+ aCSF had no effect on the spike ADP ( c and overlaid traces in d ). B , In another neuron also converted to bursting by 20 μm XE991 in normosmotic aCSF ( a ), bursting was suppressed by adding 10 μm riluzole to the aCSF ( b ). Reducing osmolarity in this condition had no effect on the spike ADP ( c and overlaid traces in d ). C , Bar histogram comparing ADP size in normosmotic versus hyposmotic aCSFs in XE991-treated neurons superfused with either high Ca2+ aCSF (n = 5; p > 0.05) or riluzole-containing aCSF (n = 5; p > 0.05).

In another set of experiments, we suppressed XE991-induced bursting in normosmotic aCSF with 10 μm riluzole (Fig. 6 Bab) (Yue and Yaari, 2006). A representative experiment is illustrated in Figure 6 B. In riluzole-containing aCSFs, lowering osmolarity again failed to further increase the spike ADP in this (Fig. 6 Bb,Bc and overlaid traces in Bd), as well as in other four neurons (n = 5; p > 0.05) (Fig. 6 C).

These data strongly suggest that I M inhibition is the sole mechanism underlying hyposmotic ADP facilitation and bursting.

Intracellular Ca2+ release mediates hyposmotic increase in Rchord

In most cell types examined, hyposmotic swelling elicits an increase in intracellular Ca2+ concentration ([Ca2+]i) by enhancing Ca2+ influx and by releasing Ca2+ from internal stores (Lang et al., 1998; Pasantes-Morales and Morales Mulia, 2000). Both mechanisms reportedly contribute to the hyposmotic increase of [Ca2+]i in dissociated hippocampal pyramidal cells, as well as in hippocampal slices (Borgdorff et al., 2000). As Ca2+ release from internal stores is a well characterized second-messenger cascade inhibiting KV7/M channels (Delmas and Brown, 2005; Hernandez et al., 2008), we hypothesized that hyposmotic I M inhibition may be mediated by an increase in [Ca2+]i.

To test this hypothesis, we first examined the effects of lowering osmolarity on R chord in neurons injected with the fast Ca2+ chelator BAPTA. To that end, BAPTA (200 mm in the recording microelectrode) was injected by 100-ms-long negative current pulses (−500 pA) delivered at 3 Hz for at least 10 min. A representative experiment is illustrated in Figure 7 A. Superfusing BAPTA-injected neurons with hyposmotic aCSF for up to 50 min consistently failed to change R chord (Fig. 7 Aa,Ab and overlaid traces in Ad). The same results were obtained in other six experiments (n = 7) (Fig. 7 C), yet R chord markedly increased with subsequent application of 20 μm XE991 (n = 4) (Fig. 7 Ab,Ac and overlaid traces in Ad). These observations confirm that hyposmotic increase in R chord is mediated by an increase in [Ca2+]i.

Figure 7.
Figure 7.

Preventing hyposmotic rise in [Ca2+]i impedes hyposmotic enhancement of R chord. Current-clamp recordings, as in Figure 2. A , This neuron was filled with the fast Ca2+ chelator BAPTA during bath perfusion with normosmotic aCSF. Changing to hyposmotic aCSF had no significant effect on R chord ( a , b and overlaid traces in d ), yet adding 20 μm XE991 to the hyposmotic aCSF markedly increased R chord ( c and overlaid traces in d ). B , In another neuron pretreated with 1 μm thapsigargin ( a ), changing to hyposmotic aCSF also had no effect on the voltage responses to injected current ramps ( a , b and overlaid traces in d ). Again, adding 20 μm XE991 to the hyposmotic aCSF markedly increased R chord ( c and overlaid traces in d ). C , Bar histogram comparing R chord in normosmotic versus hyposmotic aCSFs in BAPTA-injected (n = 7; p > 0.5) and thapsigargin-treated (n = 5; p > 0.05) neurons.

To assess the role of internal Ca2+ stores in the hyposmotic increase in R chord, slices were preincubated for at least 30 min with the endoplasmic Ca2+-ATPase inhibitor thapsigargin, known to deplete several types of internal Ca2+ stores (Thastrup et al., 1990; Takemura et al., 1991). As illustrated in Figure 7 B, hyposmolarity failed to modify R chord in this (Fig. 7 Ba,Bb and overlaid traces in Bd), as well as in other four experiments (n = 5) (Fig. 7 C), yet XE991 readily increased R chord in the thapsigargin neurons (n = 3) (Fig. 7 Bb,Bc and overlaid traces in Bd).

Together, these findings indicate that the augmentation of R chord by low osmolarity requires Ca2+ release from internal stores.

Intracellular Ca2+ release mediates hyposmotic IM inhibition

We next examined the consequences of BAPTA injection on hyposmotic inhibition of I M. In five BAPTA-injected neurons, hyperpolarizing steps incrementing by −5 mV from a V h of −44.2 ± 0.8 mV were applied to evoke I M relaxations (Fig. 8 Aa). Exposure of these neurons to hyposmotic aCSF for 40 min failed to modify I M (Fig. 8 Ab; the mean current–voltage relationship is shown in B).

Figure 8.
Figure 8.

Intracellular Ca2+ release mediates hyposmotic I M inhibition. Voltage-clamp recordings of I M, as in Figure 3. A , In this BAPTA-injected neuron, I M amplitudes were measured from outward current relaxations during 900-ms-long hyperpolarizing steps incrementing by −5 mV from a V h of −43 mV. Changing from normosmotic to hyposmotic aCSF had no effect on I M ( a , b ). B , Plots of mean I M amplitudes versus the hyperpolarizing command potentials in normosmotic (filled circles) and hyposmotic (open circles) aCSFs in BAPTA-injected neurons (n = 5; mean V h of −44.2 ± 0.8 mV). The two current–voltage relationships are similar. C , In a thapsigargin-treated neuron, I M amplitudes were measured from outward current relaxations during 0.9-s-long hyperpolarizing steps incrementing by −5 mV from a V h of −40 mV. Changing from normosmotic to hyposmotic aCSF had no effect on I M ( a , b ). D , Plots of mean I M amplitudes versus the hyperpolarizing command potentials in normosmotic (filled circles) and hyposmotic (open circles) aCSFs in thapsigargin-treated neurons (n = 5; mean V h of 41 ± 1 mV). Again, the two current–voltage relationships are similar. E , In a neuron bathed in Ca2+-free aCSF, I M amplitudes were measured from outward current relaxations during 0.9-s-long hyperpolarizing steps incrementing by −5 mV from a V h of −46 mV. Changing from normosmotic to hyposmotic aCSF markedly suppressed I M ( a , b ). F , Plots of mean I M amplitudes versus the hyperpolarizing command potentials in normosmotic (filled circles) and hyposmotic Ca2+-free (open circles; n = 5; mean V h of −42.8 ± 0.48 mV) aCSFs. G , Bar histogram comparing I M amplitudes in normosmotic versus hyposmotic aCSFs in BAPTA-injected neurons (V h of −69 mV; n = 5; p > 0.05), thapsigargin-treated neurons (V h of −66 mV; n = 5; p > 0.05), and neurons superfused with Ca2+-free aCSFs (V h of −68 mV; n = 5; *p < 0.05). Only in the latter condition lowering osmolarity reduced I M (by 63 ± 4%).

In another set of experiments, we similarly examined the consequences of thapsigargin pretreatment on hyposmotic inhibition of I M. The slices were preincubated for at least 30 min in aCSF containing 1 μm thapsigargin. In five neurons, slow I M relaxations were elicited by hyperpolarizing steps of 5 mV from a V h of 41.0 ± 1.0 mV (Fig. 8 Ca). Exposure to hyposmotic aCSF for up to 40 min failed to modify I M (Fig. 8 Cb; the mean current–voltage relationship is shown in D).

Although these results strongly implicate Ca2+ release from internal stores in hyposmotic I M inhibition, they do not exclude the participation of extracellular Ca2+ influx in this process, because Ca2+ release from internal stores may depend on Ca2+ influx (Chavis et al., 1996). The ramp stimulation experiments conducted in 1 mm Ni2+ and 300 μm Cd2+ containing aCSFs (Fig. 4) excluded a role for Ca2+ influx via voltage-gated channels. However, Ca2+ may enter through other routes, e.g., via TRPV4 channels that are Ca2+ permeable and open in hyposmotic aCSF (Liedtke et al., 2000, 2003). We therefore examined whether hyposmotic I M inhibition persists in Ca2+-free aCSFs. Slices were perfused with normosmotic Ca2+-free aCSFs for at least 30 min. Slow I M relaxations were elicited by hyperpolarizing steps of 5 mV from a V h of 42.8 ± 0.5 mV (n = 5) (Fig. 8 Ea). Changing to hyposmotic Ca2+-free aCSF consistently caused I M inhibition (Fig. 8 Eb; the mean current–voltage relationship is shown in F). Thus, at −68 mV, I M was inhibited by 63 ± 4% (from 129.7 ± 37.0 to 25.7 ± 7.0 pA; n = 5; p < 0.05). This is similar to hyposmotic inhibition of I M in Ca2+-containing aCSF (71 ± 8.9%) (Fig. 5 C).

These three sets of results, summarized in Figure 8 G, strongly indicate that Ca2+ release from internal stores mediates hyposmotic I M inhibition regardless of extracellular Ca2+ influx.

Intracellular Ca2+ release mediates hyposmotic ADP facilitation and bursting

As a final assessment of the role of intracellular Ca2+ release in hyposmotic ADP facilitation and bursting, we examined the consequences of interfering with this process. Injecting BAPTA significantly reduced the spike ADP by 18.7 ± 4.5% (from 316.7 ± 30.3 to 253.2 ± 23.3 mV/ms; n = 9; p < 0.01) (Fig. 9 Aa,Ab and overlaid traces in Ad), as reported recently (Chen and Yaari, 2008). However, the striking facilitatory effect of hyposmolarity on the ADP and bursting (Fig. 2 A) was completely abolished in BAPTA-injected neurons [Fig. 9 Ab,Ac and overlaid traces in Ad (n = 9; p > 0.05); summary of results in B]. To ensure that these results are not attributable to unspecific effects of BAPTA that may interfere with the propensity for bursting [e.g., enhancement of slow AHP current (Velumian and Carlen, 1999)], we also tested the effects of XE991 on BAPTA-injected neurons. Adding 20 μm XE991 to normosmotic aCSF facilitated the spike ADP to the point of bursting in the three tested neurons (Fig. 9 Ca–Cc).

Figure 9.
Figure 9.

Intracellular Ca2+ release mediates hyposmotic ADP facilitation and bursting. Current-clamp recordings of spike activity, as in Figure 1. A , In this neuron bathed in normosmotic aCSF, BAPTA injection suppressed the spike ADP ( a , b and overlaid traces in d ). In nine similar experiments, BAPTA-injection reduced ADP size by 18.7 ± 4.5% (p < 0.01). Changing to hyposmotic aCSF had no effect on ADP size in the representative neurons ( c and overlaid traces in d ), as well as in all other BAPTA-injected neurons (n = 9; p > 0.05). B , Bar histogram summarizing the experiments illustrated in A (**p < 0.01). C , In another BAPTA-injected neuron ( a , b ), blocking I M with 20 μm XE991 facilitated the spike ADP to the point of bursting. Similar results were obtained in another two neurons. D , In this neuron pretreated with 1 μm thapsigargin, changing from normosmotic to hyposmotic aCSF had no effect on the spike ADP ( a , b and overlaid traces in e ). Adding 20 μm XE991 to the hyposmotic aCSF caused ADP facilitation to the point of bursting ( c , d and overlaid traces in e ). E , Bar histogram showing ADP sizes in thapsigargin-treated neurons in normosmotic and hyposmotic aCSFs (n = 5; p > 0.05) and after XE991 application (n = 3). F , Another neuron was bathed with Ca2+-free normosmotic aCSF, which by itself caused ADP facilitation to the point of bursting ( a , b ). Changing to Ca2+-free hyposmotic aCSF caused marked enhancement of bursting. Similar results were obtained in six similar experiments.

We next examined how thapsigargin affects the hyposmotic enhancement of intrinsic neuronal excitability. The slices were incubated with 1 μm thapsigargin for at least 30 min and then challenged with hyposmotic aCSF for 30 min or more. As illustrated in Figure 9 D, hyposmotic ACSF had no effect on the spike ADP in thapsigargin-treated neurons [Fig. 9 Dab and overlaid traces in De (n = 5; p > 0.05); data are summarized in E]. Notwithstanding, subsequent addition of 20 μm XE991 to the hyposmotic aCSF consistently caused ADP facilitation and bursting in the thapsigargin-treated neurons [Fig. 9 Dc,Dd and overlaid traces in De (n = 3); summary of results in E].

Finally, we examined whether lowering osmolarity enhances intrinsic neuronal excitability in Ca2+-free aCSF (Fig. 9 F). Changing from normal to Ca2+-free aCSF by itself caused spike ADP facilitation and bursting (Fig. 9 Da,Db,Fa,Fb), as described previously (Su et al., 2001). The latter effect likely is attributable to enhancement of I NaP by low extracellular Ca2+ (Yue et al., 2005), yet lowering osmolarity in Ca2+-free aCSF caused a marked enhancement of burst discharge (in the neuron illustrated in Fig. 9 F, lowering osmolarity increased the number of intraburst spikes from 3 to 11; Fc). Similar results were obtained in six experiments. These findings are consistent with previously reported ADP facilitation in 13%-reduced hyposmotic Ca2+-free aCSF (Azouz et al., 1997).

Together, these data clearly demonstrate that hyposmotic facilitation of the spike ADP and induction of bursting are mediated exclusively by I M inhibition via Ca2+ release from internal stores.

Discussion

In this study, we sought to identify the mechanism by which lowering osmolarity facilitates the spike ADP, converting single spikes to high-frequency spike bursts. We show that this mechanism involves hyposmotic release of Ca2+ from internal stores, leading to inhibition of I M. Because I M normally antagonizes the depolarizing action of I NaP, its inhibition by low osmolarity unleashes I NaP, causing the regenerative growth of the spike ADP to the point of bursting. The overall result is a marked increase in intrinsic neuronal excitability and spike output.

Hyposmolarity modifies CA1 pyramidal cell excitability without concurrent swelling

Recent studies strongly indicate that hyposmotic swelling of neocortical tissue is attributable mostly to water movement into astrocytes, whereas principal neurons are osmo-resistant (Andrew et al., 2007; Risher et al., 2009). The main reason for this disparity is that astrocytes abundantly express aquaporins, whereas neurons lack these water-permeable channels (Nielsen et al., 1997). Our observations suggest that CA1 pyramidal cell somata also do not swell in hyposmotic aCSF (Fig. 1 Aab) (Zhang and Bourque, 2003), likely because they also lack aquaporins (Nielsen et al., 1997). A recent study in neocortical slices has shown that axons and dendrites are also resistant to hyposmotic challenges (Andrew et al., 2007). Intriguingly, as in neocortical slices (Andrew et al., 2007), progressive swelling of CA1 pyramidal cell somata occurred in high K+ aCSF (Fig. 1 Dab). This effect may be attributed to water influx through nonaquaporin water conduits opened during prolonged depolarization (Andrew et al., 2007).

Despite the lack of osmotic swelling, CA1 pyramidal cells were strongly modified by hyposmotic aCSF. Lowering osmolarity caused ADP augmentation and bursting and increased overall spike output of these neurons. These changes likely are triggered by mechanical and/or chemical stimuli arising from the swelling of tissue surrounding the pyramidal cells.

Ionic mechanism of hyposmotic bursting

Facilitation of the spike ADP to the point of bursting occurs when the depolarizing action of perisomatic I NaP (Yue and Yaari, 2006) or of dendritic Ca2+ currents (Magee and Carruth, 1999; Chen et al., 2005; Yaari et al., 2007) is enhanced attributable to increases in these currents and/or decreases in opposing K+ currents. We found that hyposmotic bursting is readily suppressed by blocking I NaP, whereas blockage of Ca2+ channels causes burst prolongation. The obvious conclusion is that hyposmotic bursting is driven predominantly by I NaP, whereas recruitment of Ca2+ currents mediates burst termination likely by activating Ca2+-gated K+ currents (Golomb et al., 2006).

Inhibition of IM underlies hyposmotic spike ADP facilitation and bursting

In CA1 pyramidal cells, the depolarizing action of I NaP is antagonized mainly by I M (Yue and Yaari, 2006). Therefore, I NaP-driven bursting can result from an increase in I NaP or, conversely, from a decrease in I M (Golomb et al., 2006). Our results strongly suggest that hyposmotic ADP facilitation and bursting is mediated by I M inhibition rather than by I NaP enhancement. First, lowering osmolarity inhibited I M. Second, blocking I M pharmacologically not only mimicked but also occluded all effects of hyposmolarity. Third, interfering with hyposmotic I M inhibition by chelating intracellular Ca2+ or depleting internal Ca2+ stores protected against the hyposmotic effects. Together, these observations point to the critical and exclusive role of I M inhibition in mediating hyposmotic enhancement of intrinsic neuronal excitability.

Role of internal Ca2+ stores

What mechanisms link hyposmolarity to I M inhibition in CA1 pyramidal cells? Direct obstruction of KV7/M channels via membrane tension associated with cell swelling is improbable for two reasons. First, these neurons do no swell in hyposmotic aCSF. Second, when KV7.2 and KV7.3 subunits (the main constituents of KV7/M channels) (Wang et al., 1998; Shah et al., 2002) are coexpressed with aquaporins in Xenopus oocytes, they are resistant to osmotic swelling of these cells (Grunnet et al., 2003). Therefore, an intracellular signaling cascade likely mediates hyposmotic inhibition of these channels. In CA1 pyramidal cells, lowering osmolarity was shown to increase [Ca2+]i by enhancing Ca2+ influx and Ca2+ release from internal stores (Borgdorff et al., 2000). In the same neurons, Ca2+ influx during single spikes was shown to inhibit KV7/M channels, causing significant spike ADP facilitation (Selyanko and Sim, 1998; Chen and Yaari, 2008). Furthermore, KV7/M channels in excised inside-out patches from sympathetic neurons were directly inhibited by Ca2+ with an IC50 of ∼100 nm (Selyanko and Brown, 1996). This inhibition is mediated by calmodulin bound to the C-terminal domain of KV7/M subunits (Yus-Najera et al., 2002; Gamper and Shapiro, 2003; Gamper et al., 2005; Shahidullah et al., 2005). We therefore hypothesized that lowering osmolarity inhibits I M by elevating [Ca2+]i. In support of our hypothesis, we found that injection of the fast Ca2+ chelator BAPTA prevents hyposmotic I M inhibition. Furthermore, we found that hyposmotic I M inhibition persists in Ca2+-free aCSF but is abolished by depleting internal Ca2+ stores with thapsigargin. Together, these findings strongly indicate that hyposmotic I M inhibition is achieved by the release of internally stored Ca2+ into the cytoplasm with subsequent block of KV7/M channels.

Our results do not illuminate the precise mechanisms by which hyposmolarity triggers the release of internally stored Ca2+ in pyramidal cells. Lowering osmolarity enhances the release of multiple transmitters from neurons (Schousboe and Pasantes-Morales, 1992) and glia (Darby et al., 2003; Takano et al., 2005), some of which inhibit I M by releasing Ca2+ from IP3-sensitive stores in the endoplasmic reticulum (Delmas and Brown, 2005). However, hyposmotic release of internally stored Ca2+ was observed also in dissociated CA1 pyramidal cells devoid of contacts with other neurons or glia (Borgdorff et al., 2000), as well as in many cell types other than neurons (Fischer et al., 1997; Missiaen et al., 1997). Thus, the nature of the physicochemical mechanisms coupling extracellular hyposmolarity to Ca2+ release from internal stores remains to be identified.

An increase in [Ca2+]i is expected not only to block KV7/M channels but also to activate Ca2+-gated K+ channels, an effect that would tend to oppose I M inhibition and suppress intrinsic neuronal excitability. However, in hippocampal neurons, the Ca2+ sensitivities of large-conductance (BK) Ca2+-gated K+ channels (EC50 of 2–4 μm) (Smart, 1987; Franciolini, 1988; Gong et al., 2001) and of small-conductance (SK) Ca2+-gated K+ channels (EC50 of 0.6 μm) (Hirschberg et al., 1999) are considerably smaller than that of KV7/M channels (EC50 of 100–120 nm) (Selyanko and Brown, 1996; Selyanko and Sim, 1998). Therefore, a hyposmotic increase in [Ca2+]i to ∼150 nm, as measured in acutely dissociated pyramidal cells (Borgdorff et al., 2000), may inhibit KV7/M channels without concurrently activating BK or SK channels. It is also possible that Ca2+ released from internal stores by hyposmotic stimulation is spatially targeted to a specific microdomain of KV7/M channels (Delmas and Brown, 2002). It is noteworthy that KV7/M controlling the spike ADP and bursting are localized to the axon initial segment (Devaux et al., 2004; Yue and Yaari, 2006; Shah et al., 2008), which has not been shown to express any type of Ca2+-gated K+ channels.

Functional implications

Small changes (1–3%) in brain osmolarity accompany normal activity and play an integral part in the control of brain-fluid homeostasis (Bourque, 2008). Reductions in osmolarity ≥10% used in this study are more relevant to pathophysiological conditions of impaired renal water excretion or excessive water intake, in which serum osmolarity can decrease to 230 mOsm (Adrogué and Madias, 2000; Verbalis, 2003). The clinical manifestations of these disorders are primarily neurological, including grand mal seizures (Andrew, 1991). Hyposmotic seizures and seizure-like discharges occur also in experimental animals and in brain tissue in vitro. They have been attributed mainly to enhanced nonsynaptic excitatory interactions consequent to cell swelling (Andrew et al., 1989; Traynelis and Dingledine, 1989; Dudek et al., 1990; Ballyk et al., 1991; Saly and Andrew, 1993). Here we provide an alternate or complementary mechanism: hyposmotic I M inhibition. Several lines of evidence indicate that I M inhibition strongly enhances brain excitability. First, mutations in KV7.2 and KV7.3 K+ channel genes leading to reductions in I M cause neonatal seizures (Charlier et al., 1998; Singh et al., 1998). Likewise, conditional transgenic suppression of KV7.2 channels in mouse brain produces an epileptic phenotype (Peters et al., 2005). Finally, blocking KV7/M channels with linopirdine or XE991 induces epileptiform seizures (Qiu et al., 2007), whereas enhancing KV7/M channel activity with retigabine exerts an anti-seizure action (Armand et al., 1999, 2000; Dost and Rundfeldt, 2000).

The cellular mechanisms by which I M inhibition causes neuronal network hyperexcitability likely include the induction of intrinsic bursting in principal neurons (Yue and Yaari, 2004), as well as reduction in early spike frequency adaptation (Gu et al., 2005; Otto et al., 2006). Furthermore, inhibiting I M presynaptically enhances the release of neurotransmitters (Peretz et al., 2007) and postsynaptically augments the temporal summation of EPSPs (Hu et al., 2007). All these effects would summate to enhance the frequency, spread, and synchronization of neuronal spike discharge, leading to epileptic seizures. Given that hyposmotic I M inhibition can be easily prevented (e.g., by pharmacological interference with the release of internally stored Ca2+), it would be important to determine in the future whether averting this effect of hyposmolarity exerts an anti-seizure effect in experimental and clinical hyposmotic conditions.

Footnotes

  • This research was supported by the Binational United States–Israel Science Foundation, the Deutsche Forschungsgemeinschaft Sonderforschungsbereich TR3, the Henri J. and Erna D. Leir Chair for Research in Neurodegenerative Diseases, and the Humboldt Foundation [Feodor Lynen Research Fellowship (F.B.)].

  • Correspondence should be addressed to Dr. Yoel Yaari, Department of Medical Neurobiology, Institute for Medical Research Israel–Canada, Hebrew University–Hadassah School of Medicine, P.O. Box 12272, Jerusalem 91120, Israel. yaari{at}md.huji.ac.il

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KV7/M Channels Mediate Osmotic Modulation of Intrinsic Neuronal Excitability
Anna Caspi, Felix Benninger, Yoel Yaari
Journal of Neuroscience 9 September 2009, 29 (36) 11098-11111; DOI: 10.1523/JNEUROSCI.0942-09.2009
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