Introduction

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Linopirdine is a putative cognitive enhancing drug that increases stimulus-evoked release of a number of neurotransmitters, including ACh (Nickolson et al., 1990; Zaczek et al., 1995; Aiken et al., 1996). Although the exact mechanism by which linopirdine enhances ACh release is unknown, Maciag et al. (1994) showed that its effects were insensitive to 4-aminopyridine, atropine and Na⁺, Cl and Ca⁺⁺ channel antagonists. In addition, they found no apparent role for cholinergic autoreceptors. TEA was found to have actions similar to linopirdine on ACh release suggesting the involvement of a K⁺ channel in the action of linopirdine. Recently, Aiken et al. (1995) demonstrated that linopirdine reduced spike frequency adaptation and blocked I_M in rat hippocampal CA₁ neurons in vitro. Because the concentration-response curves for I_M block and ACh release have similar slopes and IC_50/EC₅₀ values, it has been proposed that M-current block may represent the mechanism underlying linopirdine-induced neurotransmitter release enhancement (Aiken et al., 1996).

Before ascribing pharmacological relevance to its M-current blocking activity, however, it is necessary to determine the effects of linopirdine on other K⁺ channels. Many K⁺ channel blockers have been shown to be non-selective. For example, TEA blocks I_C, I_K, I_M, I_K(IR) and I_K(ATP), 4-AP blocks I_A, I_D, some types of I_K and I_K(ATP) and charybdotoxin blocks an intermediate-, as well as, the large-conductance calcium-activated K⁺ channel (Cook and Quast, 1990; Halliwell, 1990). Thus, there may be other K⁺ channels more sensitive than the M channel to the blocking action of linopirdine. In addition, a number of other potassium currents have been implicated in the control of neurotransmitter release. 4-AP and -dendrotoxin, at concentrations that inhibit I_A, increase the release of glutamate from guinea-pig cerebrocortical synaptosomes (Tibbs et al., 1989). Amoroso et al. (1990) demonstrated enhanced release of -aminobutyric acid by block of an adenosine triphosphate-sensitive K⁺ channel in substantia nigra, and Robitaille et al. (1993) reported an increase in transmitter release at the neuromuscular junction produced by block of Ca⁺⁺-gated K⁺ channels.

Our study was undertaken to determine the selectivity of linopirdine for I_M by determining its effects on the voltage-gated K⁺ currents I_M, I_A and I_K, the afterhyperpolarization currents, I_mAHP and I_sAHP, the leak current, I_L, and the inward rectifier, I_Q, recorded from rat pyramidal CA₁ neurons in the hippocampal slice. Portions of this work have previously been published in a preliminary form (Schnee and Brown, 1995).

	Methods

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Studies in this report were carried out in accordance with the Declaration of Helsinki and with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health (Rockville, MD).

Tissue preparation. Pathogen-free male CD rats from Charles River (Wilmington, MA) weighing 30 to 80 g (15-25 days old) were anaesthetized with halothane. After decapitation, the brain was rapidly excised (<1 min) and submerged in an ice-cold oxygenated physiological solution. The brain was bisected and transverse slices containing the hippocampus were prepared on a Vibratome tissue slicer. Slices were transferred to a Perspex holding chamber filled with chilled saline and allowed to reach room temperature (23°C). For recording, slices were placed on a nylon mesh in a submersion-type chamber (Medical Systems, Greenvale, NY), pinned to a Sylgard base and perfused with an oxygenated physiological saline solution at room temperature at a rate of 3 ml min¹. The physiological solution for both dissection and recording was (mM) NaCl (127.0), NaHCO₃ (26.0), KCl (3.0), CaCl₂ (2.5), NaH₂PO₄ (1.25), MgSO₄ (1.0) and glucose (10.0), gassed with 5% CO₂ in O₂ (pH 7.35). TTX (0.1 µM) or Cd⁺⁺ (0.3 mM) were added to the perfusion solution to block Na⁺ and Ca⁺⁺ currents, respectively.

Electrophysiological recording. Microelectrodes were pulled from borosilicate glass (1.5 mm OD/1.0 mm ID; World Precision Instruments, Sarasota, FL) using a Sutter P-80/PC electrode puller (Sutter Instruments, Novato, CA). Electrode resistances were 2-2.5 Mohms when filled with intracellular solution. Tight-seal (1-5 Gohm) whole-cell voltage-clamp recordings, with access resistances of  20 Mohms, were obtained from neurons in the CA₁ pyramidal cell body layer using the "blind" patch technique. Current recordings were obtained by means of an Axopatch 200A amplifier (Axon Instruments, Foster City, CA). Signals were filtered at 5 KHz and recorded with pClamp software (version 6.0.1, Axon Instruments). Series resistance compensation was not used in order to minimize noise and "ringing" of the amplifier. In preliminary studies, little difference in current amplitudes was noted between the presence and absence of series resistance compensation when using 2 to 2.5 Mohm resistance electrodes. Except where stated, application time of drugs was approximately 20 min. Internal solutions for whole cell recording were (in mM) Kgluconate (140), KCl (10), HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (10), EGTA (ethylene glycol-bis(-aminoethylether)-N,N,N',N'-tetraacetic acid) (10), MgCl₂ (2.0), CaCl₂ (1.0) and MgATP (2.0), pH adjusted to 7.4 with KOH; later experiments used KMethylsulfate (140) or KAspartate (130), KCl (10), HEPES (10), BAPTA (1,2-bis(2-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid) (10), K₂ATP (adenosine 5'-triphosphate, dipotassium salt) (5.0), MgCl₂ (2.0) and CaCl₂ (1.0), pH adjusted to 6.7 (to avoid rundown; Brown et al., 1989; Cloues and Marrion, 1996) with KOH to record I_M and pH 7.3 to record other currents.

Drugs. Linopirdine (free base) was synthesized at the DuPont Merck Pharmaceutical Company (Wilmington, DE). A stock solution in 0.1 N HCl was prepared immediately before use and added to the superfusing solution. All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).

Data analysis. All calculated data are expressed as mean ± S.E.M. In cumulative dose-response experiments on the effect of linopirdine on I_L amplitude over a range of voltage steps, a two-factor analysis of variance model (SuperANOVA, version 1.11, Abacus Concepts, Berkeley, CA) was used to test the hypothesis that mean control (pretreatment) values were unchanged with drug treatment. If there was evidence of a statistically significant treatment effect, Duncan's New Multiple Range test (SuperANOVA) was used to identify the significant dose effects. Statistical significance level was set at P < .05.

	Results

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Voltage-Gated Currents

I_M. M-current activates at potentials positive to 70 mV and is fully activated at 30 mV (Brown, 1988). To record I_M, hippocampal CA₁ neurons were stepped to 30 mV for 30 sec from a holding potential of 50 mV, repolarized by 20 mV for 2 sec and then stepped to 30 mV for 10 sec. Using this protocol, M-current deactivated in a mono-exponential manner during the 20 mV repolarization step. The amplitude of I_M was measured by fitting a single exponential function (Clampfit, Axon Instruments) to the outward deactivation tail current and extrapolating to the onset of the repolarization step. Each measure of I_M was the mean of nine repetitions of this protocol. Time constants for current deactivation from 30 mV were in the range of 110 to 220 msec, which were comparable to previously published reports (Brown, 1988). I_M in this preparation was sensitive to 50 µM carbachol and 1 mM Ba⁺⁺ (figs. 1A and B) and was stable for >60 min after breaking into whole cell mode (amplitude was 106 ± 5, 104 ± 6 and 104 ± 9% of control after 20, 40 and 60 min, respectively).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Representative traces of the effect of 50 µM carbachol (A), 1 mM barium (B) and 3 µM linopirdine (C) on M-current recorded from the soma of a CA1 neuron in a rat hippocampal slice. Each trace illustrates the deactivation tail current during a 2 sec voltage step from 30 to 50 mV; the amplitude of which is equivalent to the M-current. (D) Concentration-response curve for the inhibition of M-current by linopirdine (n = 5, 11, 5 and 2 at 1, 3, 10 and 30 µM linopirdine, respectively). Recordings were made in the presence of 0.1 µM TTX and 0.3 mM Cd++ using a patch pipette with an internal pH of 6.7.

I_A. The transient outward potassium current, I_A, observed in these studies was initially characterized by determining its electrophysiological and pharmacological properties. Steady-state inactivation was studied by applying, from a holding potential of 50 mV, 100 msec prepulses to potentials of 20 to 110 mV followed by a 300 msec voltage step to +50 mV. Steady state activation was examined by clamping to potentials between 40 and +50 mV from a 50 mV holding potential in 10 mV increments. These studies showed that >90% of I_A inactivation was removed by prepulses to potentials more negative than 75 mV and that I_A activated positive to 30 mV (fig. 2A). These characteristics, along with the observed block of I_A by 4-AP (fig. 2B), are consistent with known properties of this current.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   A, Mean steady-state inactivation and activation curves for the peak of the transient outward current, IA, rat hippocampal CA1 neurons (n = 6). B and C, Representative traces of the effect of 4-AP (5 mM) and linopirdine (10, 100 µM) on IA in neurons held at 50 mV, clamped to 100 mV for 100 msec to remove inactivation, then stepped to +50 mV to activate IA. D, Concentration-response curve for the inhibition of IA amplitude and inactivation rate constant () by linopirdine (n = 6, 5 and 5 at 10, 30 and 100 µM linopirdine, respectively). Recordings were made in the presence of 0.1 µM TTX, 10 mM TEA (except for B) using a patch pipette with an internal pH of 7.3.

I_K. The delayed rectifier current, I_K, observed in these studies was evoked by 2000 msec depolarizing steps to +60 mV from a holding potential of 50 mV in 10 mV increments without a hyperpolarizing prepulse. I_K, measured as the steady-state current amplitude at the end of each depolarizing step (baseline values were 5416 ± 654 pA at +60 mV; n = 13), activated at potentials positive to 30 mV (fig. 3A) and only slowly inactivated during the command pulse (fig. 3B). These characteristics, along with the blocking effects of TEA (fig. 3B), are established properties of I_K.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   A, Mean steady-state activation curve for the peak of the delayed rectifier outward current, IK, rat hippocampal CA1 neurons. B and C, Representative traces of the effect of TEA (10 mM) and linopirdine (10, 100 µM) on IK in neurons held at 50 mV and stepped to +50 mV to activate IK. D, Concentration-response curve for the inhibition of IK amplitude by linopirdine (n = 9, 7 and 5 at 10, 30 and 100 µM linopirdine, respectively) Recordings were made in the presence of 0.1 µM TTX using a patch pipette with an internal pH of 7.3.

Afterhyperpolarization Currents

The outward tail current recorded from hippocampal CA₁ neurons after 10 mV incremental depolarizing steps from a holding potential of 50 mV to +60 mV in the absence of calcium channel blockers (figs. 4A and 6B) appeared to be a composite of two currents which could be differentiated kinetically (medium and slow) as well as pharmacologically. Voltage steps of 3.5 to 108 msec duration to 10 to +60 mV elicited tail currents which decayed in a biexponential manner (figs. 4, B, C and D). The first component was not well resolved under the experimental conditions used. Results of the second exponential fit (as performed by the Clampex portion of pCLAMP; mixed method) were used to measure I_mAHP parameters. Maximum I_mAHP amplitude (744 ± 129 pA; n = 7) with a deactivation rate constant of 33 ± 7 msec (n = 7) was evoked by a 108 msec step to +60 mV. The slowest tail current component (referred to as I_sAHP) was activated by long (1.5 sec) depolarizing steps to 30 mV and above (fig. 6A). Typically, I_sAHP peaked 3 to 5 sec after termination of the depolarizing step (at an amplitude of 228 ± 45 pA; n = 14) and deactivated over the next several seconds (fig. 6B and C). The effect of linopirdine on I_mAHP and I_sAHP was determined using a step duration and amplitude at or near maximum for each current component; i.e., a step to +60 mV for 54 ms for I_mAHP and 1.5 sec for I_sAHP.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   A, Representative response of a rat hippocampal CA1 neuron held at 50 mV, stepped to + 50 mV for 54 msec and returned to 50 mV. Upon repolarization to 50 mV, a biexponentially decaying tail current (an expansion of which is shown in B) can be visualized. The amplitude of the second component, measured at the time indicated by the solid triangle, was recorded as ImAHP/IC. The effect of step potential and duration of the preceding depolarizing pulse on the mean activation of ImAHP/IC (n = 8) is shown in C and D, respectively. Recordings were made in the presence of 0.1 µM TTX using a patch pipette with an internal pH of 7.3.

I_mAHP. To characterize I_mAHP pharmacologically, the effects of TEA and cobalt, inhibitors of hippocampal I_C, were evaluated. TEA (1.5 mM) was effective in reducing the amplitude of I_mAHP (by 57 ± 4.5%; n = 6) (fig. 5A) and had no effect on I_sAHP. Cobalt (2 mM) also reduced I_mAHP amplitude (72%, n = 2), whereas cholinergic agonists (carbachol and muscarine, 5-50 µM) and norepinephrine (3-10 µM) had no consistent effect on I_mAHP. Because I_mAHP was blocked by TEA and cobalt, not blocked by carbachol and had a deactivation time constant of approximately 30 msec, it was assumed that I_mAHP is largely comprised of what is commonly referred to as I_C (Storm, 1990).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   A and B, Representative traces of the effect of TEA (1.5 mM) and linopirdine (3, 30 µM) on ImAHP/IC (measured as described in the legend to fig. 4) in neurons held at 50 mV, stepped to + 50 mV for 54 msec and returned to 50 mV. C, Concentration-response curve for the inhibition of ImAHP by linopirdine after a 54 msec depolarization to +50 mV. (n values for ImAHP were 5, 8, 8, 5, respectively, at 3, 10, 30 and 100 µM linopirdine.) Recordings were made in the presence of 0.1 µM TTX using a patch pipette with an internal pH of 7.3.

I_sAHP. The current underlying the slow afterhyperpolarization in hippocampal pyramidal neurons can be blocked by several neurotransmitters, including norepinephrine and acetylcholine (Storm, 1990). To verify the identity of I_sAHP as the current commonly referred to in the literature as I_AHP, the effects of norepinephrine, muscarine and TEA were evaluated. In agreement with published reports, 10 µM norepinephrine (fig. 6B) and 10 µM muscarine markedly attenuated I_sAHP, whereas 1.5 mM TEA had no effect on I_sAHP in the same preparations in which it blocked I_mAHP by an average of 57 percent (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 6.   A, Mean steady-state activation curve for the peak of the slow afterhyperpolarization tail current, IsAHP, which occurs in rat hippocampal CA1 neurons after a 1.5-sec depolarization step from a holding potential of 50 mV (n = 8). B and C, Representative traces of the effect of norepinephrine (10 µM) and linopirdine (3, 30 µM) on the slow afterhyperpolarization tail current. D, Concentration-response curve for the inhibition of IsAHP by linopirdine (n = 12, 13, 11 and 7 at 3, 10, 30 and 100 µM linopirdine, respectively). Recordings were made in the presence of 0.1 µM TTX using a patch pipette with an internal pH of 7.3.

Inward Rectifier and Leak Current

I_Q. I_Q, a mixed Na⁺/K⁺ current, was recorded in CA₁ neurons in response to 10 mV incremental hyperpolarizing voltage steps from a holding potential of 30 mV (fig. 7A). I_Q slowly activates ( = 241 ± 7.5 msec at 100 mV) at potentials negative to 60 mV, is noninactivating and is sensitive to 5 mM cesium (fig. 7B). Linopirdine, at 30 µM, had no effect on I_Q (fig. 7C) and, at concentrations as high as 300 µM, only slightly reduced I_Q (by 26%). Thus, the inwardly rectifying current examined in this study was even less sensitive to linopirdine than were the weakly inhibited I_A, I_K and I_sAHP.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   A, Representative traces of the voltage-dependent activation of IQ in rat hippocampal CA1 neurons upon incremental 10 mV hyperpolarization steps to 100 mV from a holding potential of 30 mV. B and C, Effect of cesium (5 mM) or linopirdine (30 µM) on IQ and IL in neurons stepped to 100 from 30 mV. D, Concentration-dependent effect of linopirdine on IL from 40 to 100 mV.  Baseline,  10 µM,  30 µM and  100 µM linopirdine. For the purpose of clarity, standard error bars were not included on the 10 and 30 µM linopirdine lines. Recordings were made in the presence of 0.1 µM TTX using a patch pipette with an internal pH of 7.3.

I_L. I_L, a leak potassium current, was measured in CA₁ neurons as the instantaneous current component evoked by the voltage protocol utilized to activate I_Q (fig. 7C). In agreement with Benson et al. (1988), the instantaneous current was nonrectifying over the range of 40 to 100 mV (fig. 7D). In four cells, linopirdine induced a small, concentration-dependent reduction of I_L which was statistically significant at both 30 and 100 µM. Under control conditions, a voltage step from 30 to 100 mV induced an instantaneous current of 892 ± 108 pA which was reduced by 9 ± 5, 17 ± 8 and 28 ± 8% in the presence of 10, 30 and 100 µM linopirdine, respectively. These results indicate that, like I_A, I_K, I_sAHP and I_Q, I_L is weakly inhibited by linopirdine.

	Discussion

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Our objective was to determine the selectivity of linopirdine for I_M by measuring its effects on other voltage-dependent and calcium-activated K⁺ currents. The order of sensitivity to linopirdine among the seven currents recorded from hippocampal CA₁ neurons was I_M > I_mAHP  I_K = I_sAHP > I_L = I_A  I_Q. Thus, linopirdine showed approximately seven times more selectivity for I_M than the next most sensitive current, I_mAHP, and a more than 50-fold selectivity for I_M over the other five measured currents. In addition, I_M and I_mAHP were the only K⁺ currents that could be completely blocked by linopirdine.

The inhibition of I_M by linopirdine observed in our study confirms previous reports on its effects in rat hippocampal CA₁ neurons (Aiken et al., 1995) and rat superior cervical ganglia (Lamas et al., 1997; Costa and Brown, 1997). The IC₅₀ of 2.4 µM determined in this study agrees well with reported values in the range of 3.4 to 8.5 µM. In addition, in all four studies, linopirdine, in a concentration range of 30 to 100 µM, inhibited I_M by 100%. This, together with the lack of effect of internal GTPS, GDPS or BAPTA on I_M inhibition (Costa and Brown, 1997) and the block of M channels in outside-out membrane patches (Lamas et al., 1997), strongly indicate that linopirdine is a direct M channel blocker as opposed to working through a G-protein- or calcium-coupled second messenger system. The ability of linopirdine to block M channels is, as previously discussed (Aiken et al., 1995), likely to account for its voltage-dependent depolarization of resting membrane potential and reduction of spike frequency adaptation.

Given the putative role of I_C in mediating spike repolarization (Storm, 1990), the block of I_mAHP (I_C) observed in the present study by linopirdine may account for its effects on action potential duration in hippocampal CA₁ neurons. In studies performed at room temperature, a single concentration of linopirdine (10 µM) had no effect on action potential duration (Aiken et al., 1995). Under these conditions, action potential duration was already prolonged relative to physiologic temperature and, based on the IC₅₀ of 16.3 µM determined in this study, I_C inhibition was likely to be less than 50%. However, in earlier studies performed at 37°C using a concentration range of 5 to 100 µM (Lampe and Brown, 1991), linopirdine exerted a concentration-dependent prolongation of action potential duration, with small effects (20-30% increase) noted at 10 µM and a more than 100% increase seen at 30 µM. In addition, the weak inhibition of I_A and I_K observed in this study (and by Lamas et al., 1997 in rat superior cervical ganglia) may also contribute to the prolongation of action potential duration exerted by  30 µM linopirdine.

In current clamp studies using sharp microelectrodes, we observed that 10 µM linopirdine had no significant effect on: 1) normal resting membrane potential, 2) the voltage sag during a prolonged hyperpolarizing pulse from resting potential (Lampe and Brown, 1991) and 3) the slow afterhyperpolarization after a train of action potentials evoked by a prolonged depolarizing pulse (Aiken et al., 1995). Because these potentials can, at least in part, be accounted for by I_L, I_Q and the I_sAHP, respectively, our findings of a weak or absent effect of 10 µM linopirdine on these currents are in agreement with the current clamp results. At 30 µM, we have seen a depolarization of normal resting potential induced by linopirdine (B.J. Lampe, P.A. Murphy and B.S. Brown, unpublished observation) which may be due to its small but significant inhibition of I_L.

Using cultured rat sympathetic ganglia, Lamas et al. (1997) also studied the effects of linopirdine on a variety of ionic currents. In general, there is good agreement between their results and those of our study in that linopirdine potently inhibited I_M, weakly inhibited I_A and I_K and, at 10 µM, had no significant effect on I_AHP or I_Q. One notable difference between the two studies, however, is the effect of linopirdine on I_C. In sympathetic ganglia, 10 µM linopirdine had no effect on I_C whereas in hippocampal CA₁ neurons, I_mAHP/I_C was inhibited at an IC₅₀ of 16.3 µM and was one of two currents completely suppressed by linopirdine. The difference in the pharmacology of I_C between ganglia and CA₁ neurons may represent a difference in the expression of BK channel subtypes, because the activation of BK channels is believed to correspond to the I_C macroscopic current (Sah, 1996). A similar explanation has been proposed to account for the well known difference in the pharmacology of the small-conductance, calcium-activated K⁺ current in sympathetic ganglia and hippocampus (Lancaster and Adams, 1986; Storm, 1989; Sah, 1996) where the ganglionic current is apamin-sensitive but the hippocampal current is apamin-insensitive. This pharmacological difference may be related to the expression of different SK channel subtypes in the two tissues (Kohler et al., 1996).Similarly, because there are at least two major subtypes of BK channels (for review, see Gribkoff et al., 1997), a difference in BK expression between sympathetic ganglia and hippocampal CA₁ neurons could explain the apparent difference in sensitivity of I_C to linopirdine in the two cell types.

Previous studies into the mechanism whereby linopirdine enhances neurotransmitter release indicated an inhibition of M-current as the most likely site of action (Aiken et al., 1995, 1996). Our results support this conclusion and, in addition, suggest that the inhibition of I_C may also play an important role in the action of linopirdine because there was only a 7-fold separation between the IC₅₀ values for the two currents. Accordingly, both an inhibition of I_C/BK channels and the resultant action potential broadening have been associated with enhancement of transmitter release (Robitaille et al., 1993; Jackson et al., 1991). Furthermore, although the mechanism of M-current block has been studied in some detail, with results indicating a direct channel interaction (Lamas et al., 1997; Costa and Brown, 1998), similar studies have not been performed with BK channels. However, because most, if not all, known activators and blockers of BK channels are direct channel blockers (Gribkoff et al., 1997), the same may also be true of linopirdine.

We have shown that linopirdine is a selective blocker of M-current at concentrations below 10 µM in hippocampal CA₁ neurons. At higher concentrations, it will first block I_C, then I_K, I_sAHP, I_L, I_A and I_Q. This progression of ion channel effects, as well as its interaction with peripheral ligand-gated channels (Lamas et al., 1997), must be considered when interpreting the functional and toxicological properties of linopirdine.