The Journal of Neurophysiology Vol. 78 No. 6 December 1997, pp. 2999-3007
Copyright ©1997 by the American Physiological Society

Kinetics of Muscarinic Reduction of I_sAHP in Hippocampal Neurons: Effects of Acetylcholinesterase Inhibitors

Y. Zhang, P. L. Carlen, and L. Zhang

Playfair Neuroscience Unit, Departments of Medicine (Neurology), Toronto Hospital Research Institute, Bloorview Epilepsy Program, University of Toronto, Toronto, Ontario M5T 2S8, Canada

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Zhang, Y., P. L. Carlen, and L. Zhang. Kinetics of muscarinic reduction of I_sAHP in hippocampal neurons: effects of acetylcholinesterase inhibitors. J. Neurophysiol. 78: 2999-3007, 1997. The present experiments were designed to elucidate the time frame in which an evoked cholinergic impulse decreases the Ca²⁺-dependent K⁺ current (I_sAHP) in hippocampal CA1 neurons, and to determine to what extent acetylcholinesterase (AChE) inhibitors enhance the efficacy of the cholinergic impulse. Whole cell voltage-clamp recordings were performed on hippocampal CA1 neurons of rat brain slices and I_sAHPs were evoked by constant depolarizing pulses. Cholinergic afferent fibers in stratum oriens were stimulated electrically and the time interval between the afferent stimulus and the depolarizing pulse was varied from 1 to 30 s. In slices perfused with the standard external medium, the afferent stimulus caused a profound decrease in the following I_sAHP only when the stimulus preceded the depolarizing pulse by 1-2 s. The stimulus was without effects on the I_sAHP when applied 5s before the depolarizing pulse. The effects of the afferent stimulus were greatly enhanced in CA1 neurons exposed to the catalytic AChE inhibitors neostigmine, physostigmine, or 9-amino-1,2,3,4-tetrahydro-acridine. A substantial decrease in the I_sAHP was observed even when the stimulus preceded the depolarizing pulse by 30 s. However applications of peripheral site AChE inhibitors decamethonium and propidium caused only minor or no enhancement of the I_sAHP reduction after the afferent stimulus. We suggest in physiological conditions that muscarinic modulation of ionic conductances of CNS neurons has a limited time course after a cholinergic impulse and that the modulation is greatly enhanced and prolonged when catalytic activities of AChEs are suppressed pharmacologically.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

The central muscarinic cholinergic system plays a critical role in learning and memory processes (Winkler et al. 1995) and impairment and/or alteration of this system was implicated in some pathological conditions (Whitehouse et al. 1981; Wurtman 1992). Despite intensive studies on muscarinic modulations of CNS neurons, we know little about the kinetics of these modulations, particularly in physiological conditions where the receptor activation and downstream second messenger cascades are elicited by stimulating cholinergic synapses. To improve our understanding in this regard, we studied in these experiments the temporal profile of muscarinic synaptic modulation of a slow Ca²⁺-dependent K⁺ current in hippocampal neurons. Specifically we estimated the time frame in which an evoked cholinergic impulse alters this current, and we examined to what extent this time frame is expanded after treatments with acetylcholinesterase (AChE) inhibitors.

Studying the above issues are important for several reasons. Muscarinic stimulation is known to have great influences on synaptic and intrinsic ionic activities of CNS neurons (Auerback and Segal 1994; Brown et al. 1990; Brunner and Misgeld 1994; Cantrell et al. 1996; Dutar et. 1995; Hasselmo and Barkai 1995; Huerta and Lisman 1995; Knipper et al. 1994; Krnjevi 1993; Zhang et al. 1992, 1996) and knowing the kinetics of muscarinic modulation is crucial to understand when or how these modulations may occur. This study may provide a convenient experimental protocol to examine possible dysfunction in central cholinergic synapses (Hsu et al. 1997; Taylor and Griffith 1993) or the effects of AChE inhibitors designed to treat patients with Alzheimer's disease (Eagger and Harvey 1995; Parnetti 1995; Poirier et al. 1995; Wagstaff and McTavish 1994). Furthermore the signal transduction pathway is common in principle to members of the G protein-coupled receptor family and the data about kinetics of muscarinic cholinergic synapses may have general implications for other neurotransmitter systems.

The hippocampus of mammals receives dense cholinergic innervation originated from medial septum and diagonal band of Broca (Lewis and Shute 1967) and expresses high levels of muscarinic receptors (Levey et al. 1995). Muscarinic stimulation is known to produce robust effects on intrinsic ionic conductances of hippocampal neurons (see reviews by Dutar et al. 1995; Krnjevi 1993), particularly the inhibition of the Ca²⁺-dependent K⁺ current (I_sAHP) that underlies the slow afterhyperpolarization (sAHP) after repetitive discharges (reviewed by Storm 1990). In hippocampal CA1 neurons of brain slices, the I_sAHP is readily reduced either after external application of cholinergic agonists at submicro molar concentrations (Madison et al. 1987), or by electrical stimulation of cholinergic afferents (Cole and Nicoll 1983, 1984; Zhang et al. 1995, 1996). The I_sAHP reduction is mediated by kinase-dependent processes (Abdul-Ghani et al. 1996; Baskys et al. 1990; Malenka et al. 1986; Müller et al. 1992; Pedarzani and Storm 1993) without decreasing the corresponding intracellular Ca²⁺ signals (Knöpfel et al. 1990; Müller and Connor 1991; Zhang et al. 1996), likely because of a direct modulation of ionic channels underlying the I_sAHP (Sah and Isaacson 1995). Thus the muscarinic reduction of the I_sAHP after the afferent stimulation represents a sensitive and biological measurement for the efficacy of cholinergic synapses.

We show here that the afferent stimulus decreased the following I_sAHP only when it preceded the depolarizing pulse by 1-2 s. Perfusion of slices with AChE inhibitors greatly enhanced the effect of the afferent stimulus, such that it decreased the following I_sAHP even when it was given at 30 s before the generation of this current. We suggest that in physiological conditions, intrinsic ionic conductances of CNS neurons may be modulated for a limited time after a cholinergic impulse, and this time course is greatly extended when the catalytic activities of AChEs are suppressed pharmacologically.

	METHODS

Abstract Introduction Methods Results Discussion References

Whole cell recordings of the I_sAHP in brain slices and cholinergic afferent stimulation were described previously (Zhang et al. 1994-1996). Briefly male Wistar rats (150-250 g) were anesthetized with halothane and decapitated. The brain was immediately removed and maintained in an ice-cold artificial cerebrospinal fluid (ACSF), for 3-15 min, before slicing. Transverse brain slices were obtained using a vibrotome (series 1000, Tech. Prod. Int., St. Louis, MO) and eight to nine sections of 400 µm thickness were collected from each half brain. After slicing, the slices were kept in the oxygenated ACSF at 22-23°C, for at least 1 h before further manipulations. To promote choline uptake and ACh synthesis, slices were incubated with the ACSF containing 1 mM choline chloride (Sigma, St. Louis, MO), for up to seven hours until transferred to the recording chamber. To reduce muscarinic effects of choline on membrane conductances (Krnjevi and Reinhardt 1979), these slices were perfused with the standard ACSF for 20 min in the recording chamber before doing the whole cell recording. With these arrangements, stable I_sAHPs were recorded with amplitudes comparable to those observed previously from nontreated slices (Zhang et al. 1994, 1995), suggesting a minor, if any, residual effect of choline incubation.

The components of the ACSF are the following (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 2 CaCl₂, 2 MgCl₂, 26 NaHCO₃, and 10 glucose, with osmolarity of 300 ± 5 (SE) mOsm. When aerated with 5% CO₂-95% O₂ the pH value of the ACSF is 7.4. To block fast glutamatergic transmission, slices were perfused with ionotropic glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20 µM) [Research Biochemical International (RBI), Natick, MA] and D-2-amino-5-phosphonopentanoic acid (D-AP5, 50 µM, RBI) or the more general antagonist kynurenic acid (1.5-2 mM, RBI). -aminobutyric acid-A (GABA_A)-mediated responses were blocked by bicuculline methiodide (10 µM, RBI). The following AChE inhibitors were directly added to the ACSF when required: physostigmine sulfate (RBI), 9-amino-1,2,3,4-tetrahydroacridine (THA, RBI), neostigmine (Sigma), decamethonium bromide (Sigma), or propidium iodide (Sigma). When required, atropine (Sigma) was added to the ACSF to block muscarinic receptors.

Electrical stimulation of synaptic afferents was done by a Grass stimulator (S88, Grass Medical Instruments, Quincy, MA), and constant current pulses (duration of 0.1-0.3 ms, 10-80 µA) were delivered via a photoelectric stimulation isolation unit (Grass Medical Instruments). A single afferent stimulus was applied before the depolarizing pulse that triggered the I_sAHP and the time interval between the stimulus and the subsequent depolarizing pulse was varied from 1 to 30 s. The strength of the afferent stimulation was adjusted to ensure a substantial decrease in the following I_sAHP without spiking. In some experiments, a train of stimuli (20 pulses, 1 s duration) was also used to simulate repetitive discharges of cholinergic neurons (Dutar et al. 1995).

In initial experiments, two tungsten stimulating electrodes were placed in stratum radiatum and stratum oriens, respectively, and the extent of the I_sAHP reduction after an afferent stimulus at these two sites was compared in the same neurons. The amplitudes of the I_sAHPs were measured at 500 ms after the end of depolarizing pulses. When the stimulus was given at 1 s before the depolarizing pulse in the presence of 2 µM neostigmine, the I_sAHP was inhibited by 50.3 ± 9.1% or 73.4 ± 7.7% after the stimulus in stratum radiatum or stratum oriens, respectively (9 measurements from 5 neurons). The I_sAHP reduction by the oriens stimulus is significantly greater than that after the radiatum stimulus (P = 0.039), consistent with earlier studies by Cole and Nicoll (1983, 1984). Therefore the oriens stimulation was used throughout the following experiments.

Whole cell recordings of CA1 neurons were made from submerged slices at a bath temperature of 32-32°C. Humidified, warmed 5% CO₂-95% O₂ was also applied over the perfusate to ensure a warm, oxygen-enriched local environment. We used a standard patch pipette solution that contained 150 mM potassium methylsulfate (ICN, New York), 2 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES; Fluka, NY), and 100 µM K-ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA; Fluka) (Zhang et al. 1994). This solution had a pH of 7.25 adjusted with KOH and osmolality of 280 ± 10 mOsm. We did not add ATP or guanosine 5-triphosphate (GTP) to the solution in an attempt to avoid interruption of native second messenger systems. Instead, we used patch pipettes with relatively high tip resistances (4-5 M) to reduce the current rundown (cf. Zhang et al. 1994). Patch pipettes were pulled from borosilicate thin wall glass tubes (TW150F-4, World Precision Instruments, Sarasota FL) by using a two-stage Narishige puller (Tokyo).

Signals were recorded using an Axopatch amplifier 200 B (Axon Instruments, Foster City, CA). The low-pass Bessel filter was set at 2-5 KHz and series resistance compensation was near 80%. Data were acquired, stored, and analyzed with PCLAMP software (version 6.3) through an IBM compatible computer. Digitization was performed using a 12-bit A/D interface (Digidata 1200, Axon Instruments). The I_sAHPs were evoked by constant positive voltage pulses (200-300 ms, 60 mV) from holding potentials of 50 to 60 mV. Because only one scaled output is availably from the Axopatch 200B amplifier, we did not monitor the voltage output in the present experiment. It is possible that the membrane voltage was not precisely controlled during the depolarizing pulse, because of the space clamp limitation and/or large current amplitude. However the voltage control during the I_sAHP signal generated after the depolarizing pulse could be well maintained (see Constanti and Sim 1987; Sah and McLachlan 1991; Zhang et al. 1995).

All solutions were made using deionized sterile water (resistivity18.2 M/cm) from a Milli-Q UV Plus system (Millipore). Mean ±SE is presented throughout the text.

	RESULTS

Abstract Introduction Methods Results Discussion References

Muscarinic reduction of the I_sAHP after afferent stimulation

Examined in the voltage-clamp mode at 50 to 60 mV, the outward tail current after the depolarizing pulse displayed two components, i.e., an early transient that decayed in 100 ms and a sustained portion lasting several seconds (Fig. 1). The early transient is referred as to I_mAHP composed by several currents, including a fast Ca²⁺-dependent I_C and slowly inactivating I_M and I_Q (I_H) (Alger et al. 1994; Maccaferri et al. 1993; Storm 1990). The sustained tail current represents the I_sAHP, which is highly sensitive to muscarinic stimulation (Madison et al. 1987; Zhang et al. 1994-1996). Electrical stimulation of cholinergic afferents in stratum oriens, by either a single pulse or a train of stimuli (see METHODS), caused a reversible reduction in the following I_sAHP, but not the I_mAHP (Fig. 1, A and B). Perfusion of slices with 5 µM atropine fully abolished the I_sAHP reduction after the similar stimulus and the mean decrease in the I_sAHP was 59.1 ± 8.3% or 2.5 ± 1.9% measured before or after atropine respectively (n = 7, P = 0.003, Student's t-test, Fig. 1C), in keeping with our previous results (Zhang et al. 1995, 1996). However the I_sAHP reduction by the stimulus train was only partly attenuated by atropine in the same neurons recorded (n = 3, Fig. 1D), suggesting the involvement of other neurotransmitter systems. Thus the single stimulus was used throughout the following experiments to focus on the muscarinic-mediated decrease of the I_sAHP.

View larger version (29K): [in this window] [in a new window]   FIG. 1. Muscarinic reduction of Ca2+-dependent K+ current (IsAHP) after afferent stimulation. All records were collected from a CA1 neuron before (A and B) and after perfusion of slice with 5 µM atropine (C and D). Neuron was clamped at 50 mV, and IsAHPs were evoked by depolarizing pulses of 60 mV and 300 ms every 30 s. Superimposed currents for each panel were collected before, immediately after, and 30 s after afferent stimulation. Baseline currents (· · ·) were aligned horizontally for comparison. : stimulation artifacts and decreased currents after stimulus. A single stimulus was applied at stratum oriens in A and C and a train of stimuli (20 pulses in 1 s) of same intensity were used in B and D. Inward responses during stimulation train were truncated for demonstration. Note that application of 5 µM atropine prevent IsAHP reduction after single stimulus (C), but attenuated only partly the current evoked following train of stimuli (D).

When applied at 0.1-0.2 or 1 s before the depolarizing pulse, the afferent stimulus caused a similar decrease in the following I_sAHP in the same neuron (n = 4), with a mean decrease of 55 ± 10% or 48 ± 8% respectively. Because the stimulus often induced large inward currents after application of an AChE inhibitor, which may interfere with the generation of the I_sAHP, the minimum time interval between the stimulus and the depolarizing pulse was then set at 1 s in most of the following experiments.

Time course of I_sAHP decrease after the afferent stimulation

Two paradigms were used to determine the time course in which a decreased I_sAHP was observed after the afferent stimulus. First, we evoked the I_sAHPs repeatedly by using four depolarizing pulses over a period of 10 s. The time intervals separating the first pulse and following pulses were 2, 5, and 10 s respectively (Fig. 2). Once the stable I_sAHPs were recorded such that the currents evoked by the first, third, or fourth pulses were comparable in their amplitudes (Fig. 2A), an afferent stimulus was given 200 ms before the first depolarizing pulse (Fig. 2B). CNQX (20 µM), D-AP5 (50 µM), and bicuculline (10 µM) were added to the perfusate to reduce the polysynaptic activities induced by the afferent stimulation and repetitive depolarizing stimulation. One example is shown in Fig. 2 where the I_sAHPs evoked after the afferent stimulus (Fig. 2B) were markedly decreased in comparison with controls (Fig. 2A). The differences could be clearly revealed by subtracting the I_sAHPs recorded before and after the stimulus. The subtracted current (Fig. 2C) showed a rapid decline in its amplitude, i.e., it is large after the first depolarizing pulse and is barely detectable in response to the fourth pulse. In a set of four neurons examined, the reduction in the I_sAHP evoked right after the afferent stimulus was 43.0 ± 2.8%, whereas the reduction was21.5 ± 3.2%, 4.8 ± 3.1, or 2.4 ± 1.3% for I_sAHPs evoked 2, 5, or 10 s after the afferent stimulus respectively.

View larger version (20K): [in this window] [in a new window]   FIG. 2. Effects of afferent stimulus on IsAHPs evoked by multiple depolarizing pulses. All currents were evoked from a CA1 neuron at holding potential of 50 mV, and 4 constant depolarizing pulses of +60 mV were generated in 10 s. Interpulse intervals between 1st and subsequent pulses are 2, 5, or 10 s respectively. 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 20 µM), D-2-amino-5-phosphonopentanoic acid (D-AP5; 50 µM), and bicuculline (10 µM) were applied throughout recording period. A and B: IsAHPs evoked by train of pulses before and immediately after an afferent stimulus in stratum oriens. , stimulation artifact. This recording paradigm is illustrated below panel B. C: net decreased current was obtained by subtracting currents of A and B. Note that net decreased current is large in response to 1st depolarizing pulse and that it is barely detectable in response to 4th pulse.

By using the above paradigm (Fig. 2), the changes in the I_sAHPs after the afferent stimulation can be examined sequentially in a short period. The above observations suggest a time frame of ~5 s in which the I_sAHP could be decreased after an afferent stimulus. One concern about this paradigm is that the intracellular Ca²⁺ signal corresponding to the I_sAHP generation may not recover fully during frequent activation, thereby interfering with the muscarinic cascades responsible for the I_sAHP reduction. To clarify this issue, we used another paradigm in the following experiments. We evoked the I_sAHPs by constant depolarizing pulses every 30 s to ensure the recovery of intracellular Ca²⁺ signals and when required the afferent stimulus was given at 1-30 s before the depolarizing pulse. The I_sAHPs recorded before and after the stimuli were compared in the same neurons (Fig. 3).

View larger version (12K): [in this window] [in a new window]   FIG. 3. Afferent stimulus decreased the IsAHP in a limited time. All records were collected from a CA1 neuron and recordings were performed in presence of 20 µM CNQX, 50 µM AP-5, and 10 µM bicuculline methiodide (BMI). Neuron was clamped at 50 mV and IsAHPs were evoked by constant depolarizing pulses of +60 mV every 30 s. Superimposed currents in each panel were taken 30 s before, immediately after, and 30 s after stimulus at stratum oriens. During these recordings, holding current changed within 50 pA; baseline currents of these records were aligned horizontally for comparison. Each panel was collected about 3 min apart and in sequence as illustrated. Time intervals between stimulus and depolarizing pulse are 1 s (A and D), 2 s (B), and 5 s (C), respectively. , stimulation artifacts and decreased currents after stimulus; recording paradigm is illustrated at bottom. Note that afferent stimulus, when applied about 1 s before depolarizing pulse, induced a profound reduction in IsAHP (A and D); whereas similar stimulus, when applied 5 s before depolarizing pulse, was without effect on following IsAHP.

Examined under the second paradigm, the I_sAHP reduction after the afferent stimulus dropped steeply with an increase in the time between the stimulus and the depolarizing pulse that triggered the I_sAHP. When a similar stimulus was given at 1, 2, or 5 s before the depolarizing pulse, the following I_sAHP was decreased by 49.3 ± 6.8%, 14.3 ± 1.9%, or6.8 ± 1.9%, respectively, from the control amplitude of 176.6 ± 13.7 pA, 216 ± 18.2 pA, or 194.2 ± 12.7 pA measured 30 s before the stimulus (Fig. 3). The afferent stimulus caused no decrease (by 2.3 ± 1.2%) in the following I_sAHP when it was given at 10 s before the depolarizing pulse. The relation between I_sAHP reduction and the time separating the stimulus and the following depolarizing pulses could be described by a single exponential fit (Fig. 4A, ), with a time constant of 1.45 s. These observations suggest that without any applied AChE inhibitors, muscarinic cascades responsible for the I_sAHP reduction have a limited time course after a cholinergic impulse.

View larger version (15K): [in this window] [in a new window]   FIG. 4. Enhancement of synaptic reduction of IsAHP by AChE inhibitors. A: relationship between IsAHP reduction and time interval separating stimulus and following depolarizing pulses. IsAHP reduction was normalized as percentage decrease compared with controls measured before stimulation and time intervals between stimulus () and depolarizing pulses varied from 1 to 30 s (insert). , data collected from nontreated neurons;  and  represent data collected from physostigmine- and 9-amino-1,2,3,4-tetrahydroacridine (THA)-treated neurons. Line through  is a single exponential fit, with =1.45 s (Table Curve software, Jandel Scientific, San Rafael, CA). Lines through  and  are linear regression fits, with slopes of 1.1 or 1.9 respectively. Note slow decline in effects of afferent stimulation in presence of THA or physostigmine. B: concentration-dependent enhancement by THA on synaptic reduction of IsAHP. Stimulus was applied 5 s before depolarizing pulses that triggered IsAHP and percentage reduction in following IsAHP was plotted against concentrations of THA. Line through data points was a computed semilogarithmic dose-response fit (Table Curve Software), with a coefficient factor R2 = 0.98 and an apparent EC50 of 0.40 µM. Number of neurons examined are indicated in parentheses.

Enhancement of the I_sAHP reduction by AChE inhibitors THA and physostigmine

We examined the I_sAHP reduction in the presence of9-amino-1,2,3,4-tetrahydro-acridine (THA) and physostigmine, which are centrally acting agents known to suppress catalytic activities of AChEs (Eagger and Harvey 1995; Parnetti 1995; Poirier et al. 1995; Taylor and Radic 1994; Wagstaff and McTavish 1994). To ensure a sufficient inhibition of AChEs in our experimental conditions, we first examined the concentration-dependent effects of THA on the I_sAHP reduction, by measuring the I_sAHP reduction after the afferent stimulus applied 5 s before the depolarizing pulses. As we described above (Figs. 3 and 4), this stimulus was ineffective on the following I_sAHP before application of THA (change by 3.5 ± 1.2%), but attenuated the following I_sAHP after perfusion of THA for 10-15 min. The I_sAHP reduction was significant at ~0.3 µM and plateaued at ~2 µM of THA, with an apparent EC₅₀ of 0.4 µM (Fig. 4B). The enhanced I_sAHP reduction by THA was long-lasting and no clear reversal was observed after washing THA for >50 min (n = 2, not shown), suggesting a powerful inhibition of THA on the AChEs that control synaptically released ACh.

Having determined the effective concentrations of THA, we then tested how the THA treatment extends the effective time frame of the afferent stimulus. To do so, an afferent stimulus with constant intensity was given 1 to 30 s before the depolarizing pulse that triggered the I_sAHP and the resulting changes in the following I_sAHP were measured before and after perfusion of 2 µM THA. In a set of five CA1 neurons examined before the THA application, the I_sAHP was significantly decreased after the stimulus delivered at 1 s, but not 5 s, before the depolarizing pulse (reduction by 43.2 ± 4.5% and 3.5 ± 1.8%, respectively). After the THA perfusion, the similar stimulus was highly effective in decreasing the following I_sAHP in the same neurons, such that a significant reduction (18%) in the following I_sAHP was observed when the stimulus was given at 30 s before the depolarizing pulse (Fig. 5F).

View larger version (27K): [in this window] [in a new window]   FIG. 5. Enhancement of IsAHP reduction by THA. All records were collected from a CA1 neuron and IsAHPs were evoked by constant depolarizing pulses of +60 mV every 30 s, from holding potential of 60 mV. Superimposed currents in each panel were evoked 30 s before, immediately after, and 30 or 60 s after afferent stimulus. : stimulation artifacts and decreased currents after stimulus. A and B: currents were recorded during perfusion of slice with standard artificial cerebrospinal fluid (ACSF). Note that IsAHP is greatly attenuated after stimulus applied at 1 s, but not 5 s, before depolarizing pulse. C and D: records were collected from same neuron after ~10 min perfusion of 2 µM THA. For comparison of actions on IsAHP, traces were aligned arbitrarily to baseline current level (· · ·) just before depolarizing pulses. An inward shift in holding current after stimulus was demonstrated. Note in D markedly decreased IsAHP after similar stimulus as used in B. E and F: records were collected after 20 min perfusion of THA. Note that similar stimulus, when applied at 10 or 30 s before depolarizing pulse, caused a substantial decrease in following IsAHPs. F, insert: expanded IsAHP recorded before and after stimulus. In presence of THA, afferent stimulus induced also a decrease of ImAHP (open arrow in D) and an inward current.

Similar results were also obtained in slices treated with 2 µM physostigmine (Table 1). In five CA1 neurons perfused with the standard ACSF, the baseline reduction of the I_sAHPs was 60.4 ± 7.9% or 1.9 ± 2.4% after the afferent stimulus applied at 1 or 5 s before the depolarizing pulse. After the physostigmine application for 8-15 min, the similar stimulus decreased the following I_sAHP by 45.6 ± 9.0% and24.0 ± 1.3% when it was applied 5 or 30 s before the depolarizing pulse, respectively.

The I_sAHP reduction measured in the presence of THA or physostigmine was plotted versus the time interval separating the stimulus and the following depolarizing pulse in Fig. 4A ( and ). The I_sAHP reduction declined slowly over time, with a linear slope of 1.1 or 1.9 for the THA- or physostigmine-group respectively. These values mean that for every second increase in the time interval, the effectiveness of the afferent stimulation drops by only 1-2%. This is in sharp contrast to the data observed in the nontreated neurons (Fig. 4A, ).

To control polysynaptic activities possibly involved in the enhanced I_sAHP reduction, we further examined the effect of THA in the presence of 1.5 mM kynurenic acid and 10 µM bicuculline, antagonists for ionotropic glutamate and GABA_A receptors respectively. In CA1 neurons perfused with the standard ACSF, an afferent stimulus decreased the following I_sAHP by 70.1 ± 11.5% or 4.1 ± 3.6% when it preceded the depolarizing pulse by 1 or 5 s respectively(n = 5, Fig. 6, A and B). Following coapplications of 2 µM THA, kynurenic acid, and bicuculline, while the fast synaptic currents were greatly attenuated, the I_sAHP reduction after the similar stimulus was still enhanced, with a mean reduction by 70.0 ± 14.6 or 44.8 ± 12.4%, respectively (Fig. 6, C and D). Adding 5 µM atropine to the above perfusate prevented the I_sAHP reduction after the afferent stimulus, with a mean change by only 1.6 ± 3.3% (Fig. 6, E and F). These results indicate that the enhanced I_sAHP reduction in the presence of AChE inhibitors is also mediated by muscarinic receptors and independent of polysynaptic activities.

View larger version (25K): [in this window] [in a new window]   FIG. 6. Enhanced IsAHP reduction by THA is independent of polysynaptic activity. All records were collected from a CA1 neuron and IsAHPs were evoked by constant depolarizing pulses of +60 mV every 30 s, from holding potential of 60 mV. Superimposed currents in each panel were evoked 30 s before, immediately after, and 30 s after afferent stimulus. : stimulation artifacts and decreased currents evoked after stimulus. A and B: currents were recorded during perfusion of slice with standard ACSF. Note that IsAHP is greatly attenuated after stimulus applied at 1 s, but not 5 s, before depolarizing pulse. C and D: currents were evoked from same neuron after perfusion of 1.5 mM kynurenic acid (KA), 10 µM bicuculline (BMI), and 2 µM THA for ~10 min. Note that fast outward IPSC was abolished but IsAHP was still decreased after stimulus applied 5 s before depolarizing pulse. E and F: records were taken after adding 5 µM atropine to above perfusate. Note that IsAHPs were larger in presence of atropine than before, but they were not decreased by similar stimulus.

We noticed that sometimes the I_sAHP became larger after atropine applications than that recorded in the presence of THA alone (Fig. 6, E and F). This may reflect a reversal of the tonically depressed I_sAHP by endogenous ACh, presumably resulting from promoted ACh synthesis and/or retarded ACh hydrolysis by AChE inhibitors. We did not quantify these changes in this study because of their infrequent occurrence.

Effects of peripheral site AChE inhibitors on the I_sAHP reduction

We also examined the effects of decamethonium bromide and propidium iodide on the I_sAHP reduction after the afferent stimulation. These two agents are known as peripheral site inhibitors (Eichler et al. 1994; Radic et al. 1991; Taylor and Radic 1994), with K_i values of 15 µM or 3 µM measured in biochemical assays, respectively (Berman et al. 1981; Hallek and Szinics 1988). The I_sAHP reduction was enhanced significantly after perfusion of slices with 10 µM decamethonium for about 8-15 min, as judged by the changes in the I_sAHP after the stimulus applied 5 s before the depolarizing pulse (Fig. 7,B and C). Whereas applications of propidium (10-100 µM) for 10-15 min caused no enhancement in the I_sAHP reduction after the afferent stimulus (Fig. 7, E and F). The effects of several AChE inhibitors examined were summarized in Table 1. Although only one or two concentrations of the above agents were examined, AChE inhibitors acting on catalytic sites of the enzyme are highly efficient in enhancing the effect of the afferent stimulus, in comparison with peripheral site inhibitors. These observations are agreement with the view that the strength of central muscarinic cholinergic synapses is effectively regulated by the catalytic activities of AChEs.

View larger version (26K): [in this window] [in a new window]   FIG. 7. Differential effects of AChE inhibitors on stimulus-induced IsAHP reduction. Records were collected from 3 individual CA1 neurons and IsAHPs were evoked by constant depolarizing pulses of +60 mV every 30 s, from holding potential of 60 mV. Superimposed currents in each panel were evoked 30 s before, immediately after, and 30 s after afferent stimulus. : stimulation artifacts and decreased currents after stimulus. Time interval between stimulus and depolarizing pulse was 5 s for all recordings. A and B: IsAHPs were recorded from a neuron before and after perfusion with 2 µM of neostigmine. CNQX (20 µM), 10 µM bicuculline, and 50 µM D-AP5 were applied throughout recording period. Note decreased IsAHP after stimulus in presence of 2 µM neostigamine. C and D: records were collected from another neuron and illustrated as above. Note small decrease in IsAHP after stimulus in presence of 10 µM decamethonium bromide. B and D (···): baseline holding current before stimulus. E and F: records were collected from another CA1 neuron. Perfusion of 10 µM propidium iodide did not enhance IsAHP reduction after stimulus.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

We show in hippocampal CA1 neurons of rat brain slices, a stimulus of cholinergic afferents produced a profound reduction in the Ca²⁺-dependent K⁺ current, I_sAHP, with the constraint that the stimulus precedes the generation of the I_sAHP by 1-2 s. The I_sAHP reduction after the afferent stimulus was greatly enhanced in the presence of AChE inhibitors, such that even when applied 30 s before, the similar stimulus decreased substantially the following I_sAHP.

The I_sAHP was chosen as a biological indicator for the muscarinic cascades based on several considerations. First, the I_sAHP is highly sensitive to cholinergic agonists, with an EC₅₀ value of 0.5 µM for carbachol (Madison et al. 1987). Thus the decrease in the I_sAHP may closely reflect the activation state of high-affinity muscarinic receptors in CNS neurons. Secondly, the I_sAHP is an intrinsic ionic conductance existing in a variety of CNS neurons and muscarinic reduction of the I_sAHP is associated with enhanced neuronal excitability (Andreasen and Lambert 1995; Baskys et al. 1990; Bernardo and Prince 1982; Cole and Nicoll 1984; Dodd et al. 1981; Krnjevi 1993; Sah and McLachlan 1991). Thirdly, the muscarinic reduction of the I_sAHP is mediated by kinase-dependent processes (Malenka et al. 1986; Müller et al. 1992), without a decrease in the corresponding intracellular Ca²⁺ signals (Knöpfel et al. 1990; Müller and Connor 1991; Zhang et al. 1996), suggesting a direct modulation of I_sAHP channels (Sah and Isaacson 1995) by receptor-mediated second messengers. Furthermore we have established experimental protocols allowing us to record the stable I_sAHP in the whole cell mode and to stimulate cholinergic afferents in brain slices (Zhang et al. 1994-1996, see also METHODS). By using the experimental protocol presented here, the entire process of receptor-mediated intracellular cascades can be assessed in the native form, i.e., from synaptic release of ACh to the kinase-dependent modulation of ionic channels.

The afferent stimulus is highly effective in decreasing the following I_sAHP when the stimulus is given 0.1-1 s before the depolarizing pulse that triggered the I_sAHP. A decrease in the I_sAHP is also noticeable even when the stimulus is applied during the depolarizing pulse (Zhang et al. 1996). These observations suggest a quick onset <100 ms of the muscarinic cascades that mediate the I_sAHP reduction. However we cannot determine presently the minimum time needed for the I_sAHP reduction after the stimulus, because the I_sAHP displays a rising phase of 50-100 ms after the depolarizing pulses (Zhang et al. 1995) and its generation may overlap in time with the muscarinic cascades. The present experiments were aimed at exploring the effective time frame of the afferent stimulus. We showed that the efficacy of the stimulus dropped steeply with an increase in the time interval from 1 to 5 s, with an apparent time constant of 1.5 s. No reduction in the I_sAHP was observed if the stimulus preceded the depolarizing pulse by >5 s. These results imply that under our experimental conditions (bath temperature of 32-33°C), the muscarinic cascades responsible for the I_sAHP reduction terminate in 5 s after an evoked cholinergic impulse. The onset and termination may be faster in the physiological state because of higher temperatures (37-38°C) and an intact cytoplasmic environment. It is conceivable that a cholinergic impulse in vivo could produce a robust reduction of the sAHP, but only when it arrives shortly before the activation of this conductance.

ACh is a labile transmitter and its ambient level in mammalian cerebrospinal fluid is in the low-nM range (Flentge et al. 1992). It is generally thought that the low level of ACh in the brain results from the high rate of AChE hydrolysis of ACh (K_cat = 1.6 × 10⁴ s¹) (Massoulié et al. 1993; Taylor and Radic 1994). In agreement with this view, we show here in nontreated CA1 neurons, that an evoked cholinergic impulse decreased the following I_sAHP in a limited time frame of 1.5 s. Whereas in the presence of AChE inhibitors, the impulse decreased the following I_sAHP even when it was applied 30 s before the depolarizing pulses. In addition, the stimulus decreased the I_mAHP and induced a long-lasting inward current (Fig. 5). Previous studies indicate that the generation of the I_mAHP involves multiple conductances including a Ca²⁺-dependent I_C and Ca²⁺-independent I_M and I_Q (I_H) (Alger et al. 1994; Halliwell and Adams 1982; Maccaferri et al. 1993; Storm 1990) and that the I_mAHP is less sensitive than the I_sAHP to muscarinic stimulation (Madison et al. 1987). An inward current (or EPSP in current clamp mode) was shown previously after tetanic stimulation of cholinergic afferents in hippocampal neurons and it results from a muscarinic blockade of the resting conductance (Cole and Nicoll 1983, 1984; Madison et al. 1987; see review by Krnjevi 1993). The present data suggest that in standard conditions, the function of central cholinergic synapses is effectively regulated by the catalytic activity of AChEs, such that only the I_sAHP is affected in a short time frame after an evoked cholinergic impulse. Retardation of ACh hydrolysis by AChE inhibitors remarkably strengthens central cholinergic synapses, causing the cholinergic impulse to induce strong muscarinic excitation in the innervated neurons.

In the present experiments, brain slices were incubated in the choline-containing ACSF until they were transferred to the recording chamber. While held in the recording chamber, these slices were washed for at least 20 min before the whole cell recordings. With these arrangements, stable I_sAHPs were recorded and the cholinergic afferent stimulus of stratum oriens decreased the following I_sAHP consistently and reproducibly. The present observations are consistent with the notions that choline uptake is important for ACh synthesis in the mammalian CNS and that newly formed ACh molecules are largely concentrated in the release pool (Collier et al. 1993; Jope 1979; Parsons et al. 1993). We suggest that incubation of slices with choline-containing medium may promote choline uptake and ACh synthesis, thus providing sufficient levels of releasable ACh in preserved cholinergic terminals. This experimental protocol may be considered in future studies designed to examine the function of central cholinergic synapses, such as during aging (Taylor and Griffith 1993) or after ischemic insults (Hsu et al. 1997).

The cholinergic system has been implicated in Alzheimer's disease for some time (Perry et al. 1978; Whitehouse et al. 1981; see also review by Geula and Mesulam 1994). Despite a degeneration of cholinergic neurons (Whitehouse et al. 1981), accumulating evidence suggests the involvement of AChEs in the deposition of amyloid plaques in the brain of Alzheimer patients (Beeri et al. 1995; Inestrosa et al. 1996; Smith and Guello 1984). Interestingly, the acceleration of amyloid formation by AChEs in vitro is suppressed by the inhibitors acting on peripheral sites of this enzyme (Inestrosa et al. 1996). If a similar trend exists in vivo, it will be of great interest to develop AChE inhibitors that display minimal effects on the catalytic activity of this enzyme. Within this context, the experimental protocol presented here may be useful in future to test newly developed AChE inhibitors.

We conclude that in physiological conditions, muscarinic modulation of ionic conductances of CNS neurons persists only a short time after cholinergic impulses. This modulation can be greatly enhanced by strengthening cholinergic transmission via inhibition of AChEs.

	ACKNOWLEDGEMENTS

  This work was supported by the Medical Research Council of Canada and the Heart and Stroke Foundation of Canada and Ontario. L. Zhang is a scholar of the Heart and Stroke Foundation of Canada.

	FOOTNOTES

  Address for reprint requests: L. Zhang, Playfair Neuroscience Unit, Room 13-411, Toronto Hospital (Western Division), 399 Bathurst St., Toronto, Ontario M5T 2S8, Canada.

  Received 7 March 1997; accepted in final form 29 August 1997.

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