Serine/Threonine Protein Phosphatases and Synaptic Inhibition Regulate the Expression of Cholinergic-Dependent Plateau Potentials

Douglas D. Fraser,1,2 Daniel Doll,1 and Brian A. MacVicar1

 1Neuroscience Research Group, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1; and  2Departments of Paediatrics, Anatomy, and Cell Biology, Clinical Trials Group, Canadian Paediatric Epilepsy Network, Queen's University, Kingston, Ontario K7L 3N6, Canada


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Fraser, Douglas D., Daniel Doll, and Brian A. MacVicar. Serine/Threonine Protein Phosphatases and Synaptic Inhibition Regulate the Expression of Cholinergic-Dependent Plateau Potentials. J. Neurophysiol. 85: 1197-1205, 2001. We previously identified cholinergic-dependent plateau potentials (PPs) in CA1 pyramidal neurons that were intrinsically generated by interplay between voltage-gated calcium entry and a Ca2+-activated nonselective cation conductance. In the present study, we examined both the second-messenger pathway and the role of synaptic inhibition in the expression of PPs. The stimulation of m1/m3 cholinergic receptor subtypes and G-proteins were critical for activating PPs because selective receptor antagonists (pirenzepine, hexahydro-sila-difenidol hydrochloride, 4-diphenylacetoxy-N-methylpiperidine methiodide) and intracellular guanosine-5'-O-(2-thiodiphosphate) prevented PP generation in carbachol. Intense synaptic stimulation occasionally activated PPs in the presence of oxytremorine M, a cholinergic agonist with preference for m1/m3 receptors. PPs were consistently activated by synaptic stimulation only when oxytremorine M was combined with antagonists at both GABAA and GABAB receptors. These latter data indicate an important role for synaptic inhibition in preventing PP generation. Both intrinsically generated and synaptically activated PPs could not be elicited following inhibition of serine/threonine protein phosphatases by calyculin A, okadaic acid, or microcystin-L, suggesting that muscarinic-induced dephosphorylation is necessary for PP generation. PP genesis was also inhibited following irreversible thiophosphorylation by intracellular perfusion with ATP-gamma -S. These data indicate that the expression of cholinergic-dependent PPs requires protein phosphatase-induced dephosphorylation via G-protein-linked m1/m3 receptor(s). Moreover, synaptic inhibition via both GABAA and GABAB receptors normally prevents the synaptic activation of PPs. Understanding the regulation of PPs should provide clues to the role of this regenerative potential in both normal activity and pathophysiological processes such as epilepsy.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The cholinergic system has been implicated in pathological activities such as epileptogenesis (Lothman et al. 1991; Wasterlain et al. 1993). Elevations in endogenous acetylcholine are associated with seizure onset (Mizuno and Kimura 1996) and cholinergic agonists facilitate epileptogenesis in kindled animals (Buterbaugh et al. 1986). In tissue slice preparations, cholinergic receptor activation induces prolonged depolarizations (Bianchi and Wong 1994) and initiates ictal depolarizations (Nagao et al. 1996; Yaari and Jensen 1989). We have previously identified a novel plateau potential (PP) in hippocampal CA1 pyramidal neurons that has characteristics similar to ictal depolarizations (Fraser and MacVicar 1996a). This regenerative PP was observed in the presence of cholinergic (Haj-Dahmane and Andrade 1998; Klink and Alonso 1997) or metabotropic glutamate (Raggenbass et al. 1997; Svirskis and Hounsgaard 1998) agonists and requires interplay between calcium entry through high-voltage-activated (HVA) Ca2+ channels and a Ca2+-activated nonselective cation conductance (Congar et al. 1997; Crepel et al. 1994; Fraser and MacVicar 1996a). Direct enhancement of the Ca2+-activated nonselective cation conductance by muscarinic receptor stimulation was suggested to underlie PP expression; however, neither the muscarinic receptor subtype(s) nor the signal transduction pathway have been identified. Elucidating both the ionic mechanisms and the second-messenger pathways underlying PPs could be essential for determining the role of this PP in hippocampal epileptogenesis.

Muscarinic receptor activation modulates several voltage- and ligand-gated ion channels (Halliwell 1990; Krnjevic 1993; McCormick 1993) through a number of second-messenger pathways. These include the phosphoinositide cascade (Nicoll et al. 1990), protein kinase C (PKC) (Cantrell et al. 1996; Figenschou et al. 1996; Marsh et al. 1995; Toselli and Lux 1989; Zhang et al. 1992), Ca2+-calmodulin kinase II (CaMKII) (Muller et al. 1992; Pedarzani and Storm 1996), tyrosine kinase (Huang et al. 1993), and serine/threonine protein phosphastases (Herzig et al. 1995; Krause and Pedarzani 2000). Protein kinases catalyze the transfer of a phosphate from ATP to the side chain of an amino acyl residue, resulting in structural changes of the target protein (i.e., ion channels) (Hemmings et al. 1989). The degree of ion channel phosphorylation, however, depends not only on protein kinase activity, but also on protein phosphatases that catalyze dephosphorylation. Indeed, recent studies have demonstrated that the phosphorylation of ion channels results from the dynamic equilibrium between kinase and phosphatase activity (Bielefeldt and Jackson 1994; Pedarzani et al. 1998; Wang and Salter 1994; Wilson et al. 1998).

In the present study, we employed whole cell patch-clamp techniques in the hippocampal slice preparation to investigate the putative receptor subtype(s) and second messenger(s) underlying the expression of PPs in CA1 pyramidal neurons. We also investigated the role of synaptic inhibition on the synaptic activation of PPs. These results have been presented in abstract form (Fraser and MacVicar 1996b).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Hippocampal slice preparation and whole cell patch-clamp recording

These techniques have been described previously (Fraser and MacVicar 1996a). Sprague-Dawley rats, postnatal day 15-23, were decapitated and the brain immersed in chilled artificial cerebrospinal fluid containing (ACSF; in mM): 126 NaCl, 2.5 KCl, 2 MgCl2, 2 CaCl2, 1.25 NaH2PO4, 26 NaHCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and 10 D-glucose; pH 7.3. The hippocampi were isolated, sectioned perpendicular to their septotemporal axis (150-400 µM), and incubated in ACSF oxygenated with 5% CO2-95% O2 at room temperature.

Hippocampal slices were individually transferred to a recording chamber located on an upright microscope (Standard 14; Zeiss, Thornwood, NY) and submerged in rapidly flowing (1 ml/min) oxygenated ACSF (34-35°C). Patch electrodes (5-7 MOmega ) were pulled from 1.5-mm OD thin-walled glass (150F-4, World Precision Instruments) in two stages on a Narishige puller (PP-83; Tokyo, Japan) and filled with intracellular solution (in mM): 140 K-gluconate, 1.1 EGTA, 0.1 CaCl2, 10 HEPES, 2 Mg-ATP, and 0.3 Na-GTP, pH 7.2. Intracellular Ca2+ concentration was calculated to be 16 nM. Voltage recordings were obtained in bridge mode (Axoclamp-2A; Axon Instruments) and were low-pass filtered (4-pole Bessel) at 10 kHz (-3 dB). Capacitance neutralization was fully maximized, and series resistance was determined via a bridge circuit potentiometer by balancing the voltage drop across the patch in response to a negative current step (-30 pA; 10 ms). Data were digitized via a Tl-1 A/D interface (Axon Instruments) and analyzed using computer software (pCLAMP or Axotape). All data are presented as means ± SE. To determine statistical significance, data groups were prescreened for normality (Kolmogorov-Smirnov) and compared using a Student's paired t-test (SigmaStat, Jandel Scientific).

Chemicals

All salts were purchased from Fisher (Fair Lawn, NJ), Sigma (St. Louis, MO), or BDH (Toronto, ON). Carbachol (C-4832; Sigma), oxytremorine M (O-100; Sigma), atropine (A-0257; Sigma), pirenzipine (P-114; RBI; Natick, MA), 4-DAMP methiodide (D-104; RBI) and Na-orthovanadate (S-6508; Sigma) were dissolved in distilled H2O and added to the ACSF from concentrated stocks. Also added to the ACSF, but first dissolved in DMSO, were hexahydro-sila-difenidol hydrochloride (p-fluoro analogue; H-127; RBI), calyculin A (C-3987; LC Laboratories; Woburn, MA), and okadaic acid (O-2220; LC Laboratories). The final concentration of DMSO was always <= 0.1%; in control experiments, DMSO at these concentrations did not alter the cholinergic-dependent PP. Second-messenger inhibitors and analogues that were dissolved directly into the patch pipette solution included GDP-beta -S (G-7637; Sigma), H-7 dihydrochloride (371955; Calbiochem; La Jolla, CA), ADP-beta -S (A-8016; Sigma), 2,4-dinitrophenol (D-7004; Sigma), alkaline phosphatase (P-1153; Sigma), ATP-gamma -S (A-1388; Sigma), and PP2B 476-501 (C-6481; LC Laboratories). Also added to the patch pipette solution, but first dissolved in DMSO (<= 0.1%) was microcystin-LR (M-173; RBI).

During all pharmacological manipulations, control experiments were alternated with drug experiments to ensure the presence of cholinergic-dependent PPs in untreated matched slices. In addition, it was imperative that the protein phosphatase inhibitors okadaic acid and microcystin-LR were made fresh immediately before use, as these compounds demonstrated reduced inhibitory activity when taken from frozen concentrated stocks.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The results in this paper were obtained from 207 CA1 pyramidal neurons in the hippocampal slice preparation. The whole cell patch-clamp method was used since this technique allows for both long-duration recordings and intracellular perfusion of second-messenger inhibitors and analogues. The average access resistance obtained in control neurons was 15.3 ± 0.4 MOmega (mean ± SE, range 7-20; n = 110); recordings with series resistance >20 MOmega were discarded. The resting membrane potential, input resistance, membrane time constant, and action potential characteristics of the control cells were similar to our previous reports (Table 1) (Fraser and MacVicar 1996a).


                              
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Table 1. Membrane properties of CA1 pyramidal neurons following various pharmacological manipulations that abolished the cholinergic-dependent PP

Concentration dependence of the cholinergic-dependent PP

We investigated the activation of PPs in varying carbachol concentrations. In all neurons tested under control conditions, depolarizing current injection (>= 0.1 nA; 0.8 s) evoked repetitive action potential firing (Fig. 1A; n = 225), and afterward the membrane potential immediately returned to the prestimulation baseline. We tested the actions of different concentrations of carbachol, a nonhydrolyzable cholinergic agonist, on the afterpotentials following current-evoked action potential firing. Bath application at each dose lasted for >5 min. In 0.1 µM carbachol, as in control ACSF, the membrane potential returned to prestimulation level following cessation of the current-evoked action potentials (Fig. 1B; n = 6). However, in 0.3 µM carbachol, a slow afterdepolarization (sADP) of 4 ± 1 mV and 3.5 ± 0.4 s was observed immediately following the depolarizing current stimulus (Fig. 1B; n = 6/6). A larger amplitude sADP was evoked when the carbachol concentration was increased to 1 µM (7 ± 1 mV; 4.3 ± 0.4 s; n = 6/6). While action potential firing in 3 µM carbachol elicited a larger sADP in one-half the neurons (14 ± 3 mV; 6.5 ± 0.4 s; n = 3/6), a regenerative PP was observed in the remaining cells (n = 3/6). The membrane potential and duration of the PPs were -30 ± 8 mV and 2.8 ± 0.2 s, respectively. In the three remaining neurons exhibiting only a sADP at 3 µM, PPs were observed at a concentration of 10 µM. The membrane potential and duration of the PPs in 10 µM carbachol were -20 ± 5 mV and 6.2 ± 0.9 s, respectively (n = 6/6). At the highest concentration of carbachol tested (30 µM), PPs were elicited in all neurons (n = 6/6). These data demonstrate a clear concentration dependence of the cholinergic-dependent PP. A critical level of receptor stimulation must be obtained between 3 and 10 µM carbachol before a PP can be generated consistently. We therefore used concentrations of 20 µM to elicit PPs so that we could examine drug effects.



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Fig. 1. Increasing concentrations of carbachol revealed a cholinergic-dependent slow afterdepolarization (sADP) and plateau potential (PP). A: in control artificial cerebrospinal fluid (ACSF), depolarizing current injection elicited repetitive action potential firing in a CA1 pyramidal neuron. The membrane potential immediately returned to baseline levels following cessation of the current pulse. The inset illustrates the firing pattern of this neuron in response to a 0.2-s depolarizing current stimulus. The resting membrane potential of this neuron was -62 mV. B: bath application of variable concentrations of carbachol revealed a cholinergic-dependent sADP and PP. The sADP was observed following a 5-min incubation in 0.3 µM carbachol (arrow). The sADP elicited by burst firing increased in amplitude and duration following incubation in increasing concentrations of carbachol (1.0; 3.0 µM). Application of carbachol concentrations >10 µM revealed a cholinergic-dependent PP elicited by action potential firing (arrow).

Effects of muscarinic receptor antagonists

As reported previously, the PPs were abolished following co-application of 1 µM atropine with 20 µM carbachol; although a small sADP was still elicited (2 ± 1 mV; 2.9 ± 0.5 s; n = 5/5) (Fraser and MacVicar 1996a). Since atropine is a nonselective antagonist of all muscarinic receptors, we tested the ability of relatively selective muscarinic receptor antagonists to suppress the PP elicited in the presence of 20 µM carbachol (Fig. 2). Coapplication of either 1 µM pirenzipine (n = 3), an antagonist with greater affinity for m1 over m3 receptors (Dorje et al. 1991), or 1 µM 4-diphenylacetoxy-N-methylpiperidine methiodide (n = 3; 4-DAMP), an antagonist with equal affinity to both m1 and m3 receptors (Michel et al. 1989; Thomas et al. 1992), abolished cholinergic-dependent PPs. Coapplication of 1 µM hexahydro-sila-difenidol hydrochloride (HHSiD; p-fluoro analog), an antagonist with greater affinity for m3 over m1 receptors (Lambrecht et al. 1989), also inhibited cholinergic-dependent PPs (n = 3/4; data not shown). These data implicate m1 and/or m3 receptors in cholinergic-dependent PP genesis.



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Fig. 2. The cholinergic-dependent PP was abolished by m1/m3 receptor antagonists. A: a cholinergic-dependent PP was elicited by burst firing in a CA1 pyramidal neuron. B: in another neuron, coapplication of 1 µM pirenzepine with 20 µM carbachol (carb.) abolished the cholinergic-dependent PP. A small sADP, however, was still evoked in the presence of pirenzepine (arrow). C: coapplication of 1 µM 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) with 20 µM carbachol (carb.) abolished both the sADP and the PP in the illustrated neuron (arrow).

G-protein involvement in the cholinergic-dependent PP

Muscarinic receptors are coupled to effector systems via G-proteins (Brann et al. 1993), and some studies have shown that G-proteins are involved in activation of hippocampal cation currents (Crepel et al. 1994). However, other recent studies have excluded G-protein involvement in the muscarinic activation of a nonselective cation conductance in cultured neurons (Brown et al. 1993; Guerineau et al. 1995). To determine whether G-proteins are involved in the generation of the cholinergic-dependent PP, we substituted 2 mM guanosine-5'-O-(2-thiodiphosphate) (GDP-beta -S) for GTP in the patch pipette solution. GDP-beta -S is a hydrolysis-resistant guanine nucleotide that inhibits receptor-induced activation of G-proteins (Andrade 1994). Intracellular dialysis of hippocampal CA1 pyramidal neurons with this compound abolished both the initial membrane depolarization elicited by application of 20 µM carbachol (data not shown; n = 3/3) and the cholinergic-dependent PP (n = 3/3, Fig. 3). These data indicate that G-proteins are a necessary component of the cholinergic signal transduction pathway both in mediating the initial depolarization and in the expression of the PP.



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Fig. 3. The cholinergic-dependent PP was abolished by inclusion of GDP-beta -S in the patch pipette solution. A: in the absence of carbachol, depolarizing current injection elicits action potential firing in a CA1 pyramidal neuron. The membrane potential returned to baseline following cessation of current injection. The pipette solution contained 0.3 mM GTP. The inset illustrates the firing pattern of this neuron in response to a 0.2-s depolarizing current stimulus. In contrast to control, action potential firing in the presence of 20 µM carbachol elicited a sADP and PP following cessation of the current stimuli. The resting membrane potential of this neuron in control and carbachol was -66 and -61 mV, respectively. B: in this CA1 pyramidal neuron, 2 mM GDP-beta -S was substituted for GTP in the patch-pipette solution. As before, depolarizing current injection elicited action potential firing in control ACSF. The membrane potential returned to baseline following cessation of the current stimuli. The inset illustrates the firing pattern of this neuron in response to a 0.2-s depolarizing current stimulus. Application of 20 µM carbachol failed to depolarize this neuron from a membrane potential held at -60 mV (-0.04 nA) and the cholinergic-dependent PP could not be evoked by evoked action potential firing.

Role of dephosphorylation and protein phosphorylation

The regulation of ion channels by phosphatases has recently been demonstrated (Bielefeldt and Jackson 1994; Wang and Salter 1994; White et al. 1991), suggesting that dephosphorylation may be important in the modulation of neuronal responses. To test whether dephosphorylation plays a role in PP genesis, ATP in the pipette solution was replaced with 5 mM adenosine-5'-O-(3-thiotriphosphate) (ATP-gamma -S). We assumed that there was some basal degree of protein kinase activity, and therefore ATP-gamma -S was included in the patch pipette solution to irreversibly thiophosphorylate substrate proteins. Neurons were internally perfused with this compound for a minimum of 20 min prior to data collection. Following this intracellular perfusion, the initial depolarization induced by carbachol activation was significantly reduced to 48 ± 14% of control (P <=  0.038, n = 6). Furthermore, expression of the cholinergic-dependent PP was abolished (Fig. 4, A and B; n = 6/6). These data indicate that dephosphorylation was necessary for expression of cholinergic-dependent PPs.



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Fig. 4. Expression of the cholinergic-dependent PP required serine/threonine protein phosphatase-induced dephosphorylation. A: in control ACSF, depolarizing current injection elicited action potential firing in a CA1 pyramidal neuron. The membrane potential returned to baseline following cessation of current injection. The pipette solution contained 2 mM ATP. The inset illustrates the firing pattern of this neuron in response to a 0.2-s depolarizing current stimulus. In the presence of 20 µM carbachol, action potential firing elicited a sADP and PP following cessation of the current stimuli. The resting membrane potential of this neuron in control and carbachol was -64 and -58 mV, respectively. B: action potential firing elicited by depolarizing current injection in a CA1 pyramidal neuron. The membrane potential returned to baseline following cessation of the stimuli. In this cell, the recording pipette solution contained 5 mM ATP-gamma -S. Neurons were dialyzed with this compound for an obligatory 20-min period before data collection. Under these conditions, the cholinergic-dependent PP could not be elicited by burst firing. The resting membrane potential of this neuron in control and carbachol was -64 and -62 mV, respectively. C: cholinergic-dependent PPs could not be elicited in a hippocampal slice incubated in 1 µM of the cell-permeable protein phosphatase inhibitors calyculin A (cal A). The membrane potential of this neuron was held at -62 mV (-0.09 nA) and depolarized in the presence of carbachol to -60 mV. D: in another hippocampal neuron, action potential firing failed to elicit a cholinergic-dependent PP following incubation in 1 µM okadaic acid (OA) for >3 h prior to data collection. The membrane potential of this neuron was held at -60 mV (-0.1 nA) and depolarized in the presence of carbachol to -58 mV. E: cholinergic-dependent PPs could not be generated in neurons dialyzed with a recording pipette solution containing 10 µM microcystin-LR (m-LR). Neurons were dialyzed with this compound for an obligatory 20-min period before data collection. The membrane potential of this neuron was held at -60 mV (-0.17 nA) and depolarized in the presence of carbachol to -57 mV.

We tested whether inhibitors of protein phosphatases could block the expression of cholinergic PPs because the above data implicated that dephosphorylation was necessary. Several approaches were used to test for the involvement of serine/threonine protein phosphatases. First, hippocampal slices (150-450 µm) were incubated in either calyculin A (cal A) or okadaic acid (OA) at concentrations of 1-2 µM for >3 h. This experimental approach has been used successfully in previous studies, indicating that these inhibitors permeate neurons in tissue slices (Mulkey et al. 1993; Muller et al. 1992). Second, the cell-impermeable protein phosphatase inhibitor microcystin-LR (m-LR), was applied internally via the patch pipette at a concentration of 10 µM (Mulkey et al. 1993). In this latter experiment, data collection began only after 20 min of internal perfusion. The blockade of serine/threonine protein phosphatases, by either calyculin A, okadaic acid, or microcystin-LR, resulted in a depolarized resting membrane potential (Table 1) and a reduction in the depolarization induced by 20 µM carbachol (cal A, decreased to 33 ± 21% of control, P < 0.001, n = 7; OA, decreased to 44 ± 9% of control, P <=  0.003, n = 7; m-LR, decreased to 47 ± 25% of control, P <=  0.008, n = 6). Finally, the cholinergic-dependent PP could not be evoked in most neurons recorded from slices incubated in either cal A (Fig. 4C; n = 12/14 slices) or OA (Fig. 4D; n = 6/7 slices). The cholinergic-dependent PP was also abolished in 67% of neurons intracellularly loaded with m-LR (Fig. 4E; n = 4/6).

To examine the involvement of other protein phosphatase subtypes, we also tested specific inhibitors of either protein phosphatase 2B (calcineurin) or tyrosine phosphatase. To inhibit calcineurin, the selective peptide inhibitor 476-501 (100 µM) (Hendey et al. 1992) was internally perfused via the patch-clamp electrode. At 100 µM, peptide 476-501 does not inhibit serine/threonine protein phosphatases (Hendey et al. 1992). Dialysis of neurons for 20 min with this peptide inhibitor did not significantly depress the initial cholinergic-induced depolarization (decreased to 90 ± 24% of control, n = 4) and was also ineffective in inhibiting the cholinergic-dependent PP (data not shown, n = 4/4). To test for the involvement of tyrosine phosphatase, hippocampal slices were first incubated in 100 µM Na-orthovanadate for >2 h, and this same concentration of inhibitor was included in the patch pipette solution. At 100 µM, Na-orthovanadate has virtually no dephosphorylating effect on serine-threonine phosphoproteins (Swarup et al. 1982). This inhibitor neither affected the resting membrane potential nor significantly depressed the initial cholinergic-induced depolarization (decreased to 94 ± 16% of control, n = 4). In addition, the cholinergic-dependent PP was consistently elicited (data not shown, n = 4/4), suggesting that tyrosine phosphatase is not a component of this signal transduction cascade. Hence these data exclude the involvement of calcineurin or tyrosine phosphatase in expression of the cholinergic-dependent PP.

Lack of effect of protein kinase inhibition

Our experiments with ATP-gamma -S and serine/threonine protein phosphatase inhibitors indicated that dephosphorylation was critical for the expression of cholinergic-dependent PPs. Protein kinases are reported to activate protein phosphatases directly in some signal transduction pathways (Surmeier et al. 1995; Wilson and Kaczmarek 1993). Therefore we tested whether internal perfusion of 300 µM H-7, a potent, but nonselective kinase inhibitor (Hidaka et al. 1991; Ruegg and Burgess 1989), could abolish PPs elicited in 20 µM carbachol (Fraser et al. 1993; Malenka et al. 1989; Malinow et al. 1989; Zhang et al. 1992). The initial cholinergic-induced depolarization was significantly reduced by H-7 in the patch pipette solution (decreased to 64 ± 8% of control; P <=  0.032, n = 4). In contrast, H-7 did not block the expression of cholinergic-dependent PPs elicited by action potential firing (Fig. 5A; n = 4/4). These data imply that, although protein kinase(s) mediated some of the initial cholinergic-induced depolarization, there is no apparent involvement of protein kinases in expression of the cholinergic-dependent PP.



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Fig. 5. Cholinergic-dependent PPs were unaffected by intracellular perfusion of either a nonselective protein kinase inhibitor or a phosphorylation-inhibiting cocktail. A: action potential firing elicited by depolarizing current injection in a CA1 pyramidal neuron. The membrane potential returned to baseline following cessation of the stimuli. In this cell, the recording pipette solution contained 300 µM of the nonselective protein kinase inhibitor H-7. Despite intracellular dialysis with this compound, a PP was still evoked in the presence of 20 µM carbachol. The resting membrane potential of this neuron in control and carbachol was -63 and -60 mV, respectively. B: action potential firing elicited by depolarizing current injection in a CA1 pyramidal neuron. The membrane potential returned to baseline following cessation of the stimuli. In this cell, the recording pipette solution contained a phosphorylation-inhibiting cocktail (PIC; see RESULTS for contents). Despite intracellular dialysis with this compound, a PP was still evoked in the presence of 20 µM carbachol. The membrane potential of this neuron was held at -65 mV (-120 pA) and depolarized in the presence of carbachol to -63 mV.

We next determined the potential role for phosphorylation in the expression of PPs elicited in 20 µM carbachol by internally perfusing a phosphorylation-inhibiting cocktail (PIC) containing 0 Mg-ATP, 5 mM ADP-beta -S, 50 µM dinitrophenol, and 1 mg/ml alkaline phosphatase (Chen and Smith 1992; Chen et al. 1990; Hescheler et al. 1987; Shuba et al. 1990). The resting membrane potentials of neurons loaded with PIC were significantly more depolarized than control neurons (PIC -59 ± 2 mV, n = 9; see Table 1 for control values), and the initial cholinergic-induced depolarization was significantly depressed in neurons containing PIC (decreased to 40 ± 14% of control, P <=  0.017, n = 4). In contrast, the cholinergic-dependent PP was consistently elicited by evoked action potential firing in the presence of 20 µM carbachol (Fig. 5B; n = 4/4). This experiment further indicates that phosphorylation is unnecessary for the expression of cholinergic-dependent PPs.

Synaptic activation of PPs and GABA antagonists

The activation of PPs by current-evoked action potentials is an all-or-none event (Fraser and MacVicar 1996a). We investigated whether synaptic activation could also evoke all-or-none PPs in CA1 pyramidal neurons by stimulating the Schaffer collaterals while bath applying a cholinergic agonist (carbachol or oxotremorine M). Oxotremorine M was sometimes used because it has slight preference for m1/m3 as compared with m2 cholinergic receptors (Brann et al. 1993), and we showed above that m1/m3 receptors mediate PP genesis. In the presence of either carbachol (20 µM) or oxotremorine M (15 µM) alone, synaptic stimulation rarely activated PPs (n = 2/6). In contrast, intracellular current injection always evoked PPs in these same neurons (n = 6/6). Synaptic stimulation did not evoke PPs even though the synaptic-induced depolarization was equivalent to the depolarization elicited by intracellular current injection that resulted in PP genesis (Fig. 6A). As inhibitory circuits are also activated by synaptic stimulation, we perfused GABAergic antagonists to determine whether synaptic stimulation would elicit PPs when inhibition was depressed. Antagonists of GABAA receptors (30 µM bicuculline) and GABAB receptors (50 µM CGP 35348) alone did not unmask synaptically stimulated PPs (n = 0/6; Fig. 6B). Coapplication of oxytremorine M with either the GABAA antagonist bicuculline (30 µM; n = 5), or with the GABAB receptor antagonist CGP 35348 (50 µM; n = 6) also did not result in PP genesis with synaptic stimulation (data not shown). However, PPs were consistently evoked under these conditions by intracellular current injection (n = 8/11; oxytremorine M, 15 µM; data not shown). In contrast, when both GABAA and GABAB antagonists (bicuculline 30 µM and CGP 35348 50 µM) were bath applied in conjunction with oxotremorine M, synaptic stimulation consistently evoked PPs (n = 21/24; Fig. 6C). These results indicate that depression of both GABAA- and GABAB-mediated synaptic inhibition and activation of muscarinic receptors was necessary for the expression of synaptically driven PPs. In any one cell, the waveform of the synaptically evoked PP was remarkably similar to the PP evoked intrinsically by current injection (Fig. 6D). Similar to our previous report on intrinsically generated PPs (Fraser and MacVicar 1996a), PPs were not evoked by synaptic stimulation in oxotremorine M, bicuculline, and CGP 34358 when 10 mM bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid was included in the patch pipette to chelate intracellular calcium (n = 0/4; data not shown). Therefore synaptic stimulation appears to evoke an intrinsically generated all-or-none PP when muscarinic stimulation is combined with depression of synaptic inhibition.



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Fig. 6. Synaptic stimulation evoked PPs only when muscarinic agonists were coapplied with GABA receptor antagonists. A: PPs were consistently elicited by intracellular current injection in the presence of the cholinergic agonist oxytremorine M (15 µM). Under these same conditions, stimulation of the Schaffer collaterals failed to trigger PP genesis. B: application of both GABAA and GABAB receptor antagonists in the absence of cholinergic stimulation failed to unmask PP genesis by either intrinsic or synaptic stimulation. C: both intrinsic and synaptic stimulation consistently elicited PPs when the application of both GABAA and GABAB receptor antagonists was combined with the cholinergic agonist oxytremorine M. D: both an intrinsically activated PP and a synaptically activated PP are illustrated to demonstrate the similarities in waveform, despite the different methods of stimulation.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have previously identified a novel cholinergic-dependent PP in CA1 pyramidal neurons, which relies on the interaction between HVA Ca2+ channels and the Ca2+-activated nonselective cation conductance (Fraser and MacVicar 1996a). This PP is observed in the presence of cholinergic (or metabotropic glutamate) agonists and is similar to ictal depolarizations observed during cholinergic-induced seizures (Nagao et al. 1996; Yaari and Jensen 1989). In this paper, we have elucidated the muscarinic receptors and various components of the signal transduction pathway underlying the expression of cholinergic-dependent PPs. Our evidence suggests that m1/m3 receptors are coupled to serine/threonine protein phosphatases, either directly or indirectly via G-proteins. The expression of cholinergic-dependent PPs therefore requires phosphatase-induced dephosphorylation. We have also demonstrated that, similar to the generation of ictal depolarizations, the synaptic activation of the PP is facilitated by depression of inhibition.

The expression of the cholinergic-dependent PP appears to be mediated by m1 and/or m3 receptors. The presence of both m1/m3 receptor subtypes in hippocampal tissue is well-documented by anatomical studies (Vilaro et al. 1993), and m1/m3 receptor stimulation is implicated in the modulation of cellular excitability (Auerbach and Segal 1996; Cox et al. 1994). The binding of m1/m3 receptor antagonists, [3H]pirenzepine and [3H]4-DAMP, is maximal in the rat hippocampus relative to other structures (Aubert et al. 1996), and these antagonists inhibit phosphoinositide metabolism, suggesting that stimulation of m1/m3 receptors liberate this second messenger (Candura et al. 1995). The phosphoinositide signal transduction cascade activated by m1/m3 receptors is mediated by a G-protein (Gq/11) (Vilaro et al. 1993), and in agreement with this we have found that the effects of cholinergic stimulation were occluded by GDP-beta -S. Indeed, several studies have demonstrated that muscarinic or metabotropic glutamate receptor stimulation activates phospholipase C via G-proteins, resulting in IP3 production and elevated intracellular Ca2+ (Kostyuk and Verkhratsky 1994). Intracellular Ca2+ elevated by these neurotransmitters stimulates the Ca2+-activated nonselective cation conductance, thereby depolarizing the membrane potential (Congar et al. 1997; Crepel et al. 1994).

We have demonstrated critical roles for protein phosphatases and dephosphorylation in the expression of cholinergic-dependent PPs. The involvement of dephosphorylation was first tested using intracellular perfusion of ATP-gamma -S, which irreversibly thiophosphorylates substrate proteins. Intracellular dialysis of this compound depressed the initial cholinergic-induced depolarization and abolished the PPs elicited in carbachol. As dephosphorylation is mediated by protein phosphatases, we then investigated whether inhibitors of protein phosphatases prevented PP genesis. Both membrane-permeable (calyculin A and okadaic acid) and -impermeable (m-LR) inhibitors of serine/threonine protein phosphatases abolished the cholinergic-dependent PP. The effectiveness of these inhibtors in our study is similar to a previous study of protein phosphatases in hippocampal slices (Mulkey et al. 1993). Inhibitors of calcineurin (peptide 476-501) or tyrosine phosphatase (Na+-orthovanadate) did not, however, affect PP genesis. The expression of cholinergic-dependent PPs therefore required the activation of serine/threonine protein phosphatases, either directly or indirectly via G-proteins. A direct activation of protein phosphatases by G-proteins has been suggested previously (Bielefeldt and Jackson 1994). Recently, muscarinic modulation of calcium-activated potassium conductances has also been shown to rely on protein phosphatase activation (Krause and Pedarzani 2000).

Protein phosphatases are directly activated by protein kinases in some systems (Surmeier et al. 1995; Wilson and Kaczmarek 1993), and a variety of protein kinases are activated by muscarinic stimulation (PKC, Figenschou et al. 1996; Marsh et al. 1995; Zhang et al. 1992; CaMKII, Muller et al. 1992; Pedarzani and Storm 1996; tyrosine kinase, Huang et al. 1993). Although the nonselective protein kinase inhibitor H-7 depressed the initial cholinergic-induced depolarization in the present study, no inhibition of the cholinergic-dependent PP was observed. This finding suggested that PP genesis may be independent of protein kinase activity. To further test this possibility, we internally perfused neurons with PIC, a phosphorylation-inhibiting cocktail (Chen and Smith 1992; Chen et al. 1990; Hescheler et al. 1987; Shuba et al. 1990). Similar to the H-7 experiments, the initial cholinergic-induced depolarization was depressed by PIC. The cholinergic-dependent PP was still generated, however, again demonstrating that phosphorylation is not involved in PP genesis.

What is the target for phosphatase-induced dephosphorylation? We have previously demonstrated that the PP relies on interactions between calcium entry through HVA Ca2+ channels and the Ca2+-activated cation conductance (Fraser and MacVicar 1996a). We further postulated that this latter conductance is directly enhanced by cholinergic stimulation. Based on the data presented here, we postulate that the target for dephosphorylation is a serine/threonine site on the Ca2+-activated nonselective cation channel. This theory is consistent with previous reports demonstrating that the open channel time and Ca2+ sensitivity of nonselective cation channels are greatly reduced by PKA-induced phosphorylation in both invertebrate (Partridge et al. 1990) and vertebrate neurons (Razani-Boroujerdi and Partridge 1993). Elevations in the activity of either cAMP or PKA also depress the Ca2+-activated nonselective cation channel recorded from cochlea (Van den Abbeele et al. 1996) or insulinoma (Reale et al. 1995) cells. Consistent with these findings, we have shown that the PP could not be generated in the presence ATP-gamma -S or inhibitors of serine/threonine phophatases, indicating the importance of dephosphorylation in PP genesis. Interestingly, the conditions that reduce PP generation also enhance activity of HVA Ca2+ channels. For example, L-type Ca2+ currents in hippocampal neurons were enhanced by elevations in intracellular PKA activity (Chetkovich et al. 1991; Hell et al. 1995) or by protein phosphatase inhibitors (Mironov and Lux 1991). Given that PPs rely on both HVA Ca2+ channels and the Ca2+-activated nonselective cation conductance, and that Ca2+ influx was probably augmented by protein phosphatase inhibition, it is possible that the Ca2+-activated nonselective cation conductance was depressed to a much greater extent than is evident from our observations.

We have previously suggested that the PP is an excellent candidate for an intrinsic mechanism underlying ictal depolarizations observed during both experimental (Nagao et al. 1996; Yaari and Jensen 1989) and clinical (Lothman et al. 1991) epileptogenesis. We have found that synaptic stimulation can elicit PPs, but only when synaptic inhibition is depressed by GABA receptor antagonists. Depression of synaptic inhibition is also an important trigger for eliciting seizures in vivo (Lothman et al. 1991). Our results support the hypothesis that the ictal depolarization of epilepsy is an all-or-none PP that can be triggered by spike activity or synaptic inputs (Fraser and MacVicar 1996a). In addition, it has recently been demonstrated that a clinically relevant anticonvulsant (10-100 µM topiramate) depresses the cholinergic-dependent PP in subicular neurons (Palmieri et al. 2000). During interictal bursting, elevated levels of ACh and glutamate could stimulate muscarinic and metabotropic receptors, respectively. Activation of the protein phosphatase pathway via these receptors, in conjunction with increased intracellular calcium, may lead to PP genesis as described in this study. Recurrent seizures can, as a consequence, induce profound hypoglycemia (Wasterlain et al. 1993) and precipitous decreases in ATP and phosphocreatinine levels (DeFrance and McCandless 1991; Fujikawa et al. 1988). These conditions would favor dephosphorylation and possibly exacerbate existing seizure activity. As the PP represents a feed-forward regenerative potential that results in prolonged depolarization and maintained Ca2+ influx, these mechanisms may also represent a crucial component of excitotoxicity (Chen et al. 1997).


    ACKNOWLEDGMENTS

We are grateful to Drs. J. Armstrong, S. Kombian, S. Williams, and G. Spencer for comments on the manuscript.

This research was supported by grants from the Medical Research Council of Canada (MRC). D. D. Fraser received studentships from the MRC, the Alberta Heritage Foundation for Medical Research (AHFMR), and the Savoy Foundation for Epilepsy Research. D. Doll was supported by an MRC studentship. B. A. MacVicar is an AHFMR scientist and an MRC Senior Scientist.


    FOOTNOTES

Address for reprint requests: B. MacVicar, Dept. of Physiology and Biophysics, 3330 Hospital Drive N.W., University of Calgary, Alberta T2N 4N1, Canada (E-mail: macvicar{at}ucalgary.ca).

Received 10 July 2000; accepted in final form 22 November 2000.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society