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. 1999 Jul 1;518(Pt 1):71–79. doi: 10.1111/j.1469-7793.1999.0071r.x

Protein kinase C mediates muscarinic block of intrinsic bursting in rat hippocampal neurons

Gil Alroy 1, Hailing Su 1, Yoel Yaari 1
PMCID: PMC2269419  PMID: 10373690

Abstract

  1. Acetylcholine released from basal forebrain cholinergic fibres suppresses intrinsic bursting in cortical pyramidal cells through activation of muscarinic receptors. The signal transduction pathway mediating this action is not known. We used intracellular recordings from CA1 pyramidal cells in hippocampal slices to investigate the involvement of protein kinase C (PKC) in this cholinergic function.

  2. Bath-applied carbachol (CCh; 5 μM) consistently suppressed intrinsic bursting in an atropine-sensitive (1 μM) manner.

  3. Intrinsic bursting was suppressed by 4β-phorbol 12,13-dibutyrate (PDBu; 5-10 μM), a potent PKC activator, but not by the inactive phorbol ester 4α-phorbol 12,13-didecanoate (PDC; 50 μM). Prior application of the PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7; 10 μM) extracellularly or intracellularly prevented the PDBu effect.

  4. Pretreatment with H7, but not with the broad-spectrum kinase inhibitor N-(2-guanidino-ethyl)-5-isoquinoline-sulfonyl hydrochloride (HA1004; 10 μM), prevented the CCh-induced suppression of bursting.

  5. The active component of the spike after-depolarization (ADP) was reduced by CCh in an atropine-sensitive manner. This effect was mimicked by PDBu, but not by PDC. It was prevented by pretreatment with H7, but not with HA1004.

  6. Blocking most K+ currents with Ca2+-free, TEA-containing saline induced large TTX-sensitive plateau potentials lasting > 150 ms, driven by a persistent Na+ current. These potentials were suppressed by PDBu, but not by PDC. Pretreatment with H7 prevented the PDBu-induced suppression of the plateau potentials.

  7. We conclude that cholinergic suppression of intrinsic bursting in hippocampal CA1 pyramidal cells is mediated by muscarinic activation of PKC, which down-regulates the persistent Na+ current underlying slow depolarizing potentials in these neurons.

Neurons in the mammalian brain vary with respect to their intrinsic firing patterns. In cortical (Agmon & Connors, 1989; Chagnac Amitai et al. 1990; Jensen et al. 1994) and subcortical structures (McCormick & Prince, 1988; McCormick & Feeser, 1990; Silva et al. 1991), a subgroup of neurons exhibits intrinsic bursting. An intrinsic burst in these neurons consists of three to seven closely spaced action potentials, capping a distinct slow depolarizing envelope. The spontaneous generation of intrinsic bursts is a probable driving force for physiological and pathophysiological brain rhythms (Wong & Prince, 1979; Jensen & Yaari, 1997). We have previously shown that muscarinic receptor activation by endogenously released acetylcholine or exogenously applied carbachol (CCh) converts intrinsically bursting hippocampal CA1 pyramidal cells into regularly firing (i.e. non-bursting) neurons (Azouz et al. 1994). A similar muscarinic suppression of intrinsic bursting was seen in neocortical pyramidal neurons (Metherate et al. 1992). This effect was independent of an associated muscarinic depolarization, suggesting that muscarinic receptors directly modulate the membrane currents that generate the burst (Azouz et al. 1994). However, the nature of the signal transduction pathway that mediates this muscarinic effect is not known.

Central muscarinic receptors are a heterogeneous group of metabotropic receptors that interact with various membrane-associated guanine nucleotide-binding proteins (G-proteins). These, in turn, are negatively coupled to adenylyl cyclase or positively coupled to guanylyl cyclase or to phospholipase C (for review see Brown et al. 1997). Activation of the m1, m3 and m5 muscarinic receptor subtypes induces the breakdown of inositol phospholipids, which leads to the production of inositol trisphosphate and diacylglycerol. Diacylglycerol, in turn, activates protein kinase C (PKC; Nishizuka, 1984b). In the hippocampus, CCh acting on muscarinic receptors has been shown to elicit the breakdown of inositol phospholipids (Downes, 1982), suggesting that this pathway may mediate muscarinic action in this region. Indeed, several muscarinic effects in CA1 pyramidal cells were shown to involve PKC activation, including block of the slow after-hyperpolarization (slow AHP) and the associated spike frequency accommodation (Malenka et al. 1986), enhancement of NMDA receptor-mediated response (e.g. Segal, 1992), and induction of cholinergic long-term potentiation (Auerbach & Segal, 1996).

In hippocampal CA1 (Azouz et al. 1996) and neocortical pyramidal cells (Franceschetti et al. 1995) alike, intrinsic bursting is generated by a persistent Na+ current (INa,P; French et al. 1990; Alzheimer et al. 1993). It was recently shown that muscarinic activation of PKC reduces INa,P (Cantrell et al. 1996; Mittmann & Alzheimer, 1998). This mechanism may constitute the link between muscarinic receptor activation and block of intrinsic bursting. We tested this hypothesis in rat hippocampal slices using pharmacological manipulations of PKC activity. Our results suggest that muscarinic receptor activation blocks intrinsic bursting by a PKC-dependent action, most probably by suppression of INa,P.

A preliminary report of these findings has appeared in a recent abstract (Alroy & Yaari, 1997).

METHODS

Slice preparation and solutions

Experiments were performed in accordance with institutional and national guidelines for animal experiments. Transverse hippocampal slices were prepared from 5-week-old (100-150 g) Sabra rats. Animals were anaesthetized with ether before decapitation. Both hippocampi were dissected out and kept in cold (4°C) saline solution. Slices (450 μm thick) were cut with a vibratome, placed on a nylon mesh support in an interface chamber at 33°C and perfused from below with oxygenated (95 % O2-5 % CO2) saline solution. The upper surface of the slices was exposed to the humidified gas mixture. The slices were allowed to recover for 1 h. The viability of the slices was tested with extracellular recordings of evoked field potentials. For antidromic and orthodromic stimulation, bipolar stimulation electrodes (50 μm Teflon-coated platinum wires) were positioned in CA1 alveus and stratum pyramidale, respectively. Single-shock (5-10 V, 50 μs) stimuli were delivered by a stimulator (Master 8, AMPI) via isolation units. Slices with multiple population spike responses in control conditions were discarded.

The standard saline solution contained (mM): NaCl, 124; KCl, 3.5; NaH2PO4, 1.25; MgSO4, 2; CaCl2, 2; NaHCO3, 26; and D-glucose, 10. In most experiments the KCl concentration was raised to 7.5 mM in order to increase the fraction of bursting pyramidal cells (Jensen et al. 1994). The glutamate receptor antagonists 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX; 15 μM) and 2-amino-5-phosphonovaleric acid (APV; 50 μM) were added to the saline solutions in order to block fast excitatory postsynaptic potentials. In some saline solutions CaCl2 was replaced with 2 mM MnCl2 and tetraethylammonium (TEA; 10 mM) was added to maximally block K+ currents. APV, TEA, carbachol (CCh), atropine and the phorbol esters 4β-phorbol 12,13-dibutyrate (PDBu) and 4α-phorbol 12,13-didecanoate (PDC) were purchased from Sigma. CNQX and the protein kinase inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) and N-(2-guanidino-ethyl)-5-isoquinoline-sulfonyl hydrochloride (HA1004) were purchased from RBI. In most experiments drugs were applied in the bath. In some experiments H7 was added to the microelectrode filling solution at a concentration of 10 μM and injected into cells by negative current pulses.

Intracellular recordings

Intracellular recordings were made with potassium acetate-filled (4 M) glass microelectrodes (50-80 MΩ). An active bridge circuit in the amplifier (Axoclamp 2A, Axon Instruments) allowed simultaneous injection of current and measurement of membrane potential. The bridge balance was carefully monitored and adjusted before each measurement. The intracellular signals were recorded on a digital tape recorder (VT-100, Instrutech), digitized and stored by a personal computer using a data acquisition system (Digidata 1200, Axon Instruments). Off-line data analysis was performed using pCLAMP (Axon Instruments) and SigmaPlot (Jandel Scientific).

All recordings were made in the CA1 pyramidal layer. Neurons were identified as pyramidal cells if they responded with short latency spikes to antidromic stimulation and manifested strong spike frequency accommodation during sustained depolarization. Pyramidal cells accepted for this study had a stable resting potential of at least -55 mV and overshooting action potentials. Moderate changes in membrane potential (Vm; < 2 mV) during drug application were compensated by DC current injection.

Data measurement and analysis

To measure passive membrane properties, pyramidal cells were injected with small (ca 100 pA) 200 ms negative current pulses. The input resistance (Rin) was provided by the slope of the linear regression line fitted through the linear portion of the steady-state voltage versus current plot. Rin values thus obtained are most probably underestimated due to somatic leak produced by the impaling microelectrode (Spruston & Johnston, 1992).

In pyramidal cells responding with a solitary spike to a brief pulse, the following parameters were measured (Jensen et al. 1996): (i) the fast after-hyperpolarization (fast AHP; taken as the membrane potential measured at the end-point of the fast spike repolarization); (ii) the amplitude of the active, re-depolarizing after-depolarization (ADP) component (if present); (iii) the ADP duration (measured from the trough of the fast AHP to the time point when membrane potential returns to, or crosses, resting potential).

Averaged data are expressed as means ± standard deviation. The significance of the differences between the measured parameters was evaluated using Wilcoxon's paired-sample test, with a significance level of 0.05, using Statistica (Statistica StatSoft, Inc., Tulsa, OK, USA).

RESULTS

Muscarinic block of intrinsic bursting in CA1 pyramidal cells

The firing patterns of hippocampal CA1 pyramidal cells were characterized by injecting long (150-200 ms) and brief (3-5 ms) depolarizing current pulses through the recording microelectrode. As previously shown (Jensen et al. 1994, 1996), these neurons display a gradient of bursting characteristics, ranging from non-bursters to neurons that burst only in response to long stimuli (grade I bursters), or in response to both long and brief stimuli (grade II bursters), or even burst spontaneously (grade III bursters). The propensity to burst is upgraded by modest increases in the extracellular K+ concentration (Jensen et al. 1994).

The effects of CCh on intrinsic bursting were examined in 32 intrinsically bursting pyramidal cells (21 grade I and 11 grade II bursters). Bath application of CCh (5 μM) for 30 min suppressed intrinsic bursting in all cases. A typical experiment is illustrated in Fig. 1. In control saline solution the neuron (a grade II burster) generated a burst of four to five spikes in response to brief or long depolarizing current pulses (Fig. 1a). Adding CCh to the saline solution converted the burst responses to regular firing (Fig. 1b). This effect was reversed within 10-15 min after adding 1 μM atropine to the CCh-containing saline solution (Fig. 1c), confirming its muscarinic nature (Azouz et al. 1994).

Figure 1. CCh suppresses intrinsic bursting in a CA1 pyramidal cell.

Figure 1

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In this and subsequent figures, each record shows membrane potential (top trace) and current pulse injected into the neuron (bottom trace). Upper panels show the response of a neuron (Vm, -63 mV) to 150 ms positive current pulses. In control conditions, the neuron generated a burst of 5 spikes at the onset of depolarization (a). After 10 min of perfusion with saline solution containing 5 μM CCh, the neuron generated a train of non-accommodating single spikes in response to the same stimulus (b). This effect was reversed within 15 min after adding 1 μM atropine to the saline solution (c). Lower panels, CCh similarly converted the response of the neuron to a brief (5 ms) stimulation from a burst (a) into a single spike (b). This effect was also reversed upon adding atropine (c).

PKC activation suppresses intrinsic bursting

Muscarinic receptor activation may suppress intrinsic bursting through the stimulation of PKC. To test this notion, we first examined the effect of PDBu, a potent activator of PKC (Castagna et al. 1982; Nishizuka, 1984a), on 29 bursting pyramidal cells (20 grade I and 9 grade II bursters). In control conditions, the mean resting membrane potential, input resistance, apparent membrane time constant and spike threshold were -64.7 ± 5.5 mV, 26.4 ± 7.3 MΩ, 18.4 ± 5.2 ms and -55.8 ± 3.8 mV, respectively. The mean fast AHP was -57.1 ± 5.1 mV. Bath application of PDBu (5-10 μM) for 45-60 min caused a mild depolarization of resting membrane potential (by 2.1 ± 1.7 mV) and reduced spike frequency accommodation, as previously reported (Malenka et al. 1986). PDBu did not cause any significant change in input resistance, apparent membrane time constant, spike threshold or fast AHP. In all neurons tested, as illustrated in Fig. 2, PDBu converted intrinsic bursting into regular spiking. This effect was not a consequence of the PDBu-induced depolarization, as it persisted when the pyramidal cells were repolarized by current injection to control resting potential.

Figure 2. PDBu suppresses intrinsic bursting.

Figure 2

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Upper panels show the response of a neuron (resting Vm, -63 mV) to a 150 ms positive current pulse. In control conditions, the neuron generated a stereotyped burst of 4-5 spikes (a). After 30 min of perfusion with standard saline solution containing PDBu (10 μM) the neuron generated a train of non-accommodating single spikes in response to the same current pulse (b). Lower panels, application of PDBu similarly converted the response to a brief (5 ms) stimulus from a burst (a) into a single spike (b).

Phorbol esters may exert effects through actions other than PKC activation (Doerner et al. 1990). To test the specificity of the action of PDBu on PKC, we first examined in four intrinsic bursters the effects of PDC (50 μM), which does not activate PKC (Castagna et al. 1982). As shown in Fig. 3A, application of PDC for 1 h had no effect on bursting in these neurons.

Figure 3. PDBu suppresses intrinsic bursting through PKC activation.

Figure 3

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A, in control conditions, the neuron (Vm, -67 mV) generated a burst of 5 spikes at the onset of depolarization (a). Application of PDC (50 μM) for more than 40 min did not alter the firing pattern of the neuron (b). B, another neuron was loaded with H7 (10 μM) by applying positive and negative current pulses for at least 10 min prior to the beginning of the recordings. In control conditions the H7-loaded neuron (Vm, -69 mV) generated 3-4 spikes at the onset of depolarization in response to a 150 ms positive current pulse (a). Application of PDBu (10 μM) for over 60 min failed to alter the firing pattern of the neuron (b).

We then tested the consequences of pretreating pyramidal cells with the PKC inhibitor H7 on the effects of PDBu. H7 (10 μM) was bath applied for 30 min prior to PDBu application. H7 by itself did not produce any changes in resting potential, input resistance or firing pattern of the pyramidal cells (cf. Szente et al. 1990). However, under these conditions PDBu failed to change the firing pattern of any of the three bursting pyramidal cells tested (Fig. 3B).

Muscarinic block of intrinsic bursting requires PKC activation

The results thus far have indicated that PKC activation by PDBu mimics the muscarinic suppression of intrinsic bursting and that this effect is sensitive to H7. To test whether PKC activation mediates the muscarinic effect, we examined in eleven bursters whether the action of CCh is also sensitive to H7. The PKC inhibitor was either bath applied (10 μM; n = 7) or injected into single neurons through the recording microelectrode (n = 4). In nine of these eleven neurons, H7 precluded the CCh-induced suppression of intrinsic bursting (Fig. 4A). By contrast, pretreatment with bath-applied HA1004 (5 μM), a broad-spectrum kinase inhibitor with low affinity to PKC (Hidaka et al. 1984), did not impede the CCh-induced suppression of intrinsic bursting in four of five bursters (Fig. 4B). Taken together, these data suggest that PKC activation is involved in the muscarinic suppression of intrinsic bursting.

Figure 4. H7 prevents CCh modulation of intrinsic bursting.

Figure 4

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A, the neuron was loaded with H7 (10 μM) as described in Fig. 3 In control conditions the H7-loaded neuron (Vm, -67 mV) generated a burst of 3 spikes in response to a 5 ms positive current pulse (a). Bath application of CCh (5 μM) for over 30 min did not alter the firing pattern of the neuron (b). B, a second neuron was loaded with the broad-spectrum kinase inhibitor HA1004 (10 μM) using the same method. In control conditions, the HA1004-loaded neuron (Vm, -66 mV) generated a burst of 4-5 spikes in response to a brief (5 ms) positive current (a). Bath application of CCh (5 μM) for 15 min converted the neuron into a regularly firing cell, generating two spikes in response to the brief current pulse (b).

PKC activation reduces active spike ADPs

Solitary spikes evoked by brief stimuli in grade I bursters and in some non-bursters are followed by a protracted ADP, which often re-depolarizes before declining slowly towards resting membrane potential (Kandel & Spencer, 1961; Jensen et al. 1996). This ADP has been designated ‘active’ ADP, to differentiate it from the rapidly declining (‘passive’) ADPs typically seen in non-bursting CA1 pyramidal cells (Jensen et al. 1994). It has been shown that large active ADPs underlie intrinsic bursting (Jensen et al. 1996) and that the two potentials share a common ionic mechanism (Schwartzkroin, 1975; Azouz et al. 1994, 1996).

The effect of CCh on the active spike ADPs was tested in 21 CA1 pyramidal cells. As illustrated in Fig. 5, CCh (5 μM) reduced the active ADP (from 14.1 ± 2.1 to 8.1 ± 1.3 mV, a reduction of 6.2 ± 1.5 mV). This effect was reversed by atropine (1 μM).

Figure 5. CCh suppresses spike ADP.

Figure 5

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A spike was evoked from Vm (-65 mV) by brief (5 ms) depolarizing current pulses. In control conditions, the neuron displayed a distinct post-spike re-depolarization (ADP) (a), which was suppressed after 15 min of CCh (5 μM) application (b). The residual passive ADP appeared to decline monotonically. The effect of CCh on ADP amplitude was reversed by 1 μM atropine (c). The ADP portions of the traces are superimposed on an expanded time scale in panel d. Dashed line indicates Vm.

Application of PDBu (10 μM) reduced active spike ADPs in all five pyramidal cells tested (from 11.2 ± 1.5 to 5.1 ± 1.1 mV, a reduction of 5.7 ± 1.3 mV; Fig. 6A). This effect was not mimicked by PDC (50 μM; n = 3; Fig. 6B). Moreover, it was prevented by pretreatment with H7 (n = 3; Fig. 6C), indicating that PKC activation attenuates the active spike ADP.

Figure 6. PDBu suppresses spike ADP in a PKC-dependent manner.

Figure 6

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A, spikes were evoked from Vm (-64 mV) by brief (3-4 ms) depolarizing current pulses. In control conditions, the neuron displayed a distinct post-spike re-depolarization (ADP) (a), which was suppressed after 40 min of PDBu (10 μM) application (b). The residual passive ADP appeared to decline monotonically. B, another pyramidal cell (Vm, -62 mV) displayed a similar ADP in response to a brief depolarization (a). Application of PDC (50 μM) for over 40 min failed to attenuate this ADP (b). C, in control saline solution containing 10 μM H7, a third pyramidal cell (Vm, -69 mV) displayed a distinct ADP as in A and B (a). Application of PDBu (10 μM) did not cause any change in the amplitude or the duration of the ADP (b). The ADP portions of the traces in A-C are superimposed on an expanded time scale in the right-hand panels (c). Dashed lines indicate Vm.

Muscarinic suppression of active ADPs is mediated by PKC

To examine whether CCh reduces active ADPs through PKC activation, we pretreated the hippocampal slice with H7 or HA1004. In the presence of H7, CCh did not affect the ADPs in the four neurons tested (Fig. 7A). In the presence of HA1004, the active ADPs in the three pyramidal cells tested were reduced by CCh (Fig. 7B). Taken together, the data suggest that muscarinic suppression of the active ADP is mediated by PKC.

Figure 7. CCh block of the spike ADP is PKC dependent.

Figure 7

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A, single spikes were evoked from Vm (-61 mV) by brief (5 ms) depolarizing current pulses. In control saline solution containing 10 μM H7, the neuron displayed a distinct post-spike re-depolarization (ADP) (a). Application of CCh (5 μM) did not cause any change to the amplitude or duration of the ADP (b). B, a second neuron (Vm, -63 mV) also displayed post-spike ADPs in response to brief depolarizing pulses in saline solution containing 10 μM HA1004 (a). CCh (5 μM) reduced the ADP (b). The ADP portions of the traces in A and B are superimposed on an expanded time scale in the right-hand panels (c). Dashed lines indicate Vm.

PKC activation abates TTX-sensitive plateau potentials

The simplest explanation for the PKC-mediated muscarinic suppression of intrinsic bursting and active ADPs is suppression of INa,P, the main current generating these potentials in CA1 pyramidal cells (Azouz et al. 1996). To test whether PKC activation suppresses INa,P in these neurons, the slices were perfused with Ca2+-free saline solution containing 2 mM Mn2+ and 10 mM TEA to block Ca2+ and most K+ currents. This procedure induces very large (40-50 mV) and prolonged (> 150 ms) TTX-sensitive plateau potentials following the fast spike, which are thought to be generated by INa,P (García-Muñoz et al. 1993; Azouz et al. 1996, 1997). The effects of phorbol esters on these plateau potentials are illustrated in Fig. 8. Application of the inactive phorbol ester PDC (50 μM) for over 50 min was ineffective (n = 5; Fig. 8Ab), whereas application of PDBu (5-10 μM) greatly attenuated the plateau potentials (n = 8; Fig. 8Ac). Treating the slices with the PKC inhibitor H7 (10 μM) precluded the effect of PDBu (n = 4; Fig. 8B). These data are consistent with the notion that protein phosphorylation by PKC suppresses INa,P.

Figure 8. PDBu blocks plateau ADPs.

Figure 8

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A, in a CA1 pyramidal cell (Vm, -66 mV), suppression of K+ and Ca2+ currents by bath application of TEA (10 mM) in Ca2+-free saline solution (containing 2 mM Mn2+), induced a TTX-sensitive plateau potential (a). Application of the inactive phorbol ester PDC (50 μM) for over 40 min did not suppress this plateau potential (b), while application of PDBu (5 μM) markedly attenuated it (c). The effect of PDBu was essentially irreversible. B, plateau potentials recorded from another neuron (Vm, -68 mV; a). Application of the PKC inhibitor H7 (10 μM) had no effect on this potential (b). In the presence of H7, PDBu had no effect on the amplitude or the duration of the plateau (c).

DISCUSSION

Experimental data obtained in recent years suggest that muscarinic receptor activation may modify brain rhythms by changing the intrinsic firing pattern of individual neurons from phasic to tonic discharge (Metherate et al. 1988; McCormick, 1992). This effect may be achieved indirectly by muscarinic depolarization (McCormick, 1992) or directly by suppression of the burst mechanism (Metherate et al. 1992; Azouz et al. 1994). Here we show that PKC activation mediates the latter effect in hippocampal pyramidal cells, through which it may play a critical role in muscarinic modulation of normal and abnormal brain rhythms.

Muscarinic activation blocks intrinsic bursting in CA1 pyramidal cells

In the present study we have confirmed that 5 μM CCh blocks intrinsic bursting in CA1 pyramidal cells (Azouz et al. 1994) and further have shown that CCh blocks the active spike ADP in these neurons. Several lines of evidence suggest that large active ADPs underlie the generation of intrinsic bursts in hippocampal pyramidal cells (Kandel & Spencer, 1961; Wong & Prince, 1981; Jensen et al. 1994). First, the propensity to burst increases with ADP magnitude (Jensen et al. 1996). Second, procedures which augment ADP amplitude, such as increasing extracellular K+ concentration (Jensen et al. 1994) or reducing extracellular osmolarity (Azouz et al. 1997), also induce or enhance intrinsic bursting. Finally, both the active spike ADP and the depolarizing envelope of the burst are generated primarily by INa,P (Jensen et al. 1996). Therefore, it is likely that the muscarinic suppression of intrinsic bursting is a consequence of the muscarinic block of the active spike ADP and may involve reduction of INa,P.

Muscarinic suppression of intrinsic bursting was previously also found in neocortical pyramidal cells (Metherate et al. 1992). In contrast, other studies in hippocampal (Benardo & Prince, 1982b) and subicular pyramidal cells (Kawasaki & Avoli, 1996) have shown muscarinic enhancement of burst activity. However, the latter effect was brought about only with high concentrations of muscarinic agonists (drop application of 1-200 mM acetylcholine: Benardo & Prince, 1982a; or bath application of 30-100 μM CCh: Kawasaki & Avoli, 1996). Such high muscarinic agonist concentrations may promote bursting by effectively suppressing repolarizing K+ currents (for review see Brown et al. 1997) and thereby enhancing inward Ca2+ currents, though potentiation of a sustained K+ current at high CCh concentrations (50 μM) also was described (Zhang et al. 1992). Alternatively, they may do so by directly activating a voltage-dependant cationic non-selective current (Haj Dahmane & Andrade, 1996). Whatever the mechanism, it appears that muscarinic receptor activation may exert a dual action on intrinsic bursting in hippocampal pyramidal cells, suppressing native bursters at low, and inducing bursting at high agonist concentrations. It seems, however, that the effect obtained at low agonist concentrations more closely resembles the physiological muscarinic effect of acetylcholine, because repetitive stimulation of cholinergic fibres in hippocampal (Azouz et al. 1994) or neocortical slices (Metherate et al. 1992) suppresses, rather than enhances, intrinsic bursting in an atropine-sensitive manner.

Involvement of PKC in the muscarinic effects

In hippocampal neurons, activation of muscarinic receptors leads to phospholipase C-dependent activation of PKC (Nishizuka, 1986). Activation of PKC by phorbol esters was shown to mimic several muscarinic actions in hippocampal neurons, suggesting that they are mediated by muscarinic receptors coupled to phospholipase C (e.g. Malenka et al. 1986).

Our data suggest that the muscarinic suppression of intrinsic bursting is mediated by the phospholipase C cascade. First, specific activation of PKC by PDBu mimicked the muscarinic effect. Second, blocking PKC activity with H7 prevented the muscarinic effect. Finally, blocking the activity of other kinases, such as the cAMP- and cGMP-dependent protein kinases, with HA1004 did not occlude the muscarinic effect.

Mechanism by which PKC activation suppresses intrinsic bursting

In CA1 pyramidal cells (Azouz et al. 1996), as well as in neocortical pyramidal cells (Franceschetti et al. 1995), the depolarizing drive for the active spike ADP and intrinsic bursting is provided by INa,P. This current is subjected to muscarinic modulation; in acutely dissociated hippocampal (Cantrell et al. 1996) and neocortical pyramidal cells (Mittmann & Alzheimer, 1998), CCh (albeit at 20 μM or more) reduced INa,P in an atropine-sensitive manner. This effect readily explains the muscarinic suppression of intrinsic bursting and active spike ADP. The involvement of protein phosphorylation by PKC in this chain of events is supported by the finding that PKC activation markedly curtailed the TTX-sensitive plateau potentials, which are presumed to be generated by INa,P.

In neocortical slices, activating PKC with the phorbol ester phorbol 12-myristate 13-acetate (PMA) applied intracellularly affected INa,P in a complex fashion, reducing maximal INa,P while shifting the current-voltage relationship in a hyperpolarizing direction (Astman et al. 1998). Consequently, INa,P was reduced only at very depolarized potentials; at near threshold potentials INa,P increased, leading to enhanced pyramidal cell excitability. Activation of different PKC isoforms (Nishizuka, 1988) by CCh versus PMA may account for the incongruent effects of CCh and PMA on INa,P in neocortical neurons. Also, PMA was shown to cause not only activation but also degradation of PKC in non-neural cells (e.g. Krug et al. 1987).

In summary, our data indicate that PKC activation links muscarinic receptor stimulation to the suppression of intrinsic bursting in hippocampal pyramidal cells. The final step in this process may involve direct phosphorylation of Na+ channels by PKC, causing a reduction in the persistent Na+ channel activity that normally sustains large spike ADPs and burst potentials. Through this modulatory action PKC may regulate physiological (Lisman, 1997) and pathophysiological (Connors, 1984; Chagnac Amitai & Connors, 1989; Jensen & Yaari, 1997) brain functions executed by intrinsic bursters in the hippocampus and neocortex.

Acknowledgments

This work was supported by the USA-Israel Binational Science Foundation (BSF) and the Israeli Ministry of Science (MOS).

References

  1. Agmon A, Connors BW. Repetitive burst-firing neurons in the deep layers of mouse somatosensory cortex. Neuroscience Letters. 1989;99:137–141. doi: 10.1016/0304-3940(89)90278-4. 10.1016/0304-3940(89)90278-4. [DOI] [PubMed] [Google Scholar]
  2. Alroy G, Yaari Y. PKC mediates muscarinic block of intrinsic burst firing by suppression of persistent sodium currents in rat hippocampal neurons. Neuroscience Letters. 1997;48:S3. 10.1016/S0304-3940(97)90009-4. [Google Scholar]
  3. Alzheimer C, Schwindt PC, Crill WE. Modal gating of Na+ channels as a mechanism of persistent Na+ current in pyramidal neurons from rat and cat sensorimotor cortex. Journal of Neuroscience. 1993;13:660–673. doi: 10.1523/JNEUROSCI.13-02-00660.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Astman N, Gutnick MJ, Fleidervish IA. Activation of protein kinase C increases neuronal excitability by regulating persistent Na+ current in mouse neocortical slices. Journal of Neurophysiology. 1998;80:1547–1551. doi: 10.1152/jn.1998.80.3.1547. [DOI] [PubMed] [Google Scholar]
  5. Auerbach JM, Segal M. Muscarinic receptors mediating depression and long-term potentiation in rat hippocampus. The Journal of Physiology. 1996;492:479–493. doi: 10.1113/jphysiol.1996.sp021323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Azouz R, Alroy G, Yaari Y. Modulation of endogenous firing patterns by osmolarity in rat hippocampal neurones. The Journal of Physiology. 1997;502:175–187. doi: 10.1111/j.1469-7793.1997.175bl.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Azouz R, Jensen MS, Yaari Y. Muscarinic modulation of intrinsic burst firing in rat hippocampal neurons. European Journal of Neuroscience. 1994;6:961–966. doi: 10.1111/j.1460-9568.1994.tb00590.x. [DOI] [PubMed] [Google Scholar]
  8. Azouz R, Jensen MS, Yaari Y. Ionic basis of spike after-depolarization and burst generation in adult rat hippocampal CA1 pyramidal cells. The Journal of Physiology. 1996;492:211–223. doi: 10.1113/jphysiol.1996.sp021302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Benardo LS, Prince DA. Cholinergic excitation of mammalian hippocampal pyramidal cells. Brain Research. 1982a;249:315–331. doi: 10.1016/0006-8993(82)90066-x. [DOI] [PubMed] [Google Scholar]
  10. Benardo LS, Prince DA. Ionic mechanisms of cholinergic excitation in mammalian hippocampal pyramidal cells. Brain Research. 1982b;249:333–344. doi: 10.1016/0006-8993(82)90067-1. [DOI] [PubMed] [Google Scholar]
  11. Brown DA, Abogadie FC, Allen TG, Buckley NJ, Caulfield MP, Delmas P, Haley JE, Lamas JA, Selyanko AA. Muscarinic mechanisms in nerve cells. Life Sciences. 1997;60:1137–1144. doi: 10.1016/s0024-3205(97)00058-1. [DOI] [PubMed] [Google Scholar]
  12. Cantrell AR, Ma JY, Scheuer T, Catterall WA. Muscarinic modulation of sodium current by activation of protein kinase C in rat hippocampal neurons. Neuron. 1996;16:1019–1026. doi: 10.1016/s0896-6273(00)80125-7. [DOI] [PubMed] [Google Scholar]
  13. Castagna M, Takai Y, Kaibuchi K, Sano K, Kikkawa U, Nishizuka Y. Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. Journal of Biological Chemistry. 1982;257:7847–7851. [PubMed] [Google Scholar]
  14. Chagnac Amitai Y, Connors BW. Synchronized excitation and inhibition driven by intrinsically bursting neurons in neocortex. Journal of Neurophysiology. 1989;62:1149–1162. doi: 10.1152/jn.1989.62.5.1149. [DOI] [PubMed] [Google Scholar]
  15. Chagnac Amitai Y, Luhmann HJ, Prince DA. Burst generating and regular spiking layer 5 pyramidal neurons of rat neocortex have different morphological features. Journal of Comparative Neurology. 1990;296:598–613. doi: 10.1002/cne.902960407. [DOI] [PubMed] [Google Scholar]
  16. Connors BW. Initiation of synchronized neuronal bursting in neocortex. Nature. 1984;310:685–687. doi: 10.1038/310685a0. [DOI] [PubMed] [Google Scholar]
  17. Doerner D, Abdel Latif M, Rogers TB, Alger BE. Protein kinase C-dependent and -independent effects of phorbol esters on hippocampal calcium channel current. Journal of Neuroscience. 1990;10:1699–1706. doi: 10.1523/JNEUROSCI.10-05-01699.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Downes CP. Receptor-stimulated inositol phospholipid metabolism in the central nervous system. Cell Calcium. 1982;3:413–428. doi: 10.1016/0143-4160(82)90027-6. 10.1016/0143-4160(82)90027-6. [DOI] [PubMed] [Google Scholar]
  19. Franceschetti S, Guatteo E, Panzica F, Sancini G, Wanke E, Avanzini G. Ionic mechanisms underlying burst firing in pyramidal neurons: intracellular study in rat sensorimotor cortex. Brain Research. 1995;696:127–139. doi: 10.1016/0006-8993(95)00807-3. 10.1016/0006-8993(95)00807-3. [DOI] [PubMed] [Google Scholar]
  20. French CR, Sah P, Buckett KJ, Gage PW. A voltage-dependent persistent sodium current in mammalian hippocampal neurons. Journal of General Physiology. 1990;95:1139–1157. doi: 10.1085/jgp.95.6.1139. 10.1085/jgp.95.6.1139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. García-Muñoz A, Barrio L C, Buño W. Membrane potential oscillations in CA1 hippocampal pyramidal neurons in vitro: intrinsic rhythms and fluctuations entrained by sinusoidal injected current. Experimental Brain Research. 1993;97:325–333. doi: 10.1007/BF00228702. [DOI] [PubMed] [Google Scholar]
  22. Haj Dahmane S, Andrade R. Muscarinic activation of a voltage-dependent cation nonselective current in rat association cortex. Journal of Neuroscience. 1996;16:3848–3861. doi: 10.1523/JNEUROSCI.16-12-03848.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hidaka H, Inagaki M, Kawamoto S, Sasaki Y. Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry. 1984;23:5036–5041. doi: 10.1021/bi00316a032. [DOI] [PubMed] [Google Scholar]
  24. Jensen MS, Azouz R, Yaari Y. Variant firing patterns in rat hippocampal pyramidal cells modulated by extracellular potassium. Journal of Neurophysiology. 1994;71:831–839. doi: 10.1152/jn.1994.71.3.831. [DOI] [PubMed] [Google Scholar]
  25. Jensen MS, Azouz R, Yaari Y. Spike after-depolarization and burst generation in adult rat hippocampal CA1 pyramidal cells. The Journal of Physiology. 1996;492:199–210. doi: 10.1113/jphysiol.1996.sp021301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Jensen MS, Yaari Y. Role of intrinsic burst firing, potassium accumulation, and electrical coupling in the elevated potassium model of hippocampal epilepsy. Journal of Neurophysiology. 1997;77:1224–1233. doi: 10.1152/jn.1997.77.3.1224. [DOI] [PubMed] [Google Scholar]
  27. Kandel ER, Spencer WA. Electrophysiology of hippocampal neurones: II. after-potentials and repetitive firing. Journal of Neurophysiology. 1961;24:243–259. doi: 10.1152/jn.1961.24.3.243. [DOI] [PubMed] [Google Scholar]
  28. Kawasaki H, Avoli M. Excitatory effects induced by carbachol on bursting neurons of the rat subiculum. Neuroscience Letters. 1996;219:1–4. doi: 10.1016/s0304-3940(96)13175-x. 10.1016/S0304-3940(96)13175-X. [DOI] [PubMed] [Google Scholar]
  29. Krug E, Biemann HP, Tashjian AH., Jr Evidence for increased synthesis as well as increased degradation of protein kinase C after treatment of human osteosarcoma cells with phorbol ester. Journal of Biological Chemistry. 1987;262:11852–11856. [PubMed] [Google Scholar]
  30. Lisman JE. Bursts as a unit of neural information: making unreliable synapses reliable. Trends in Neurosciences. 1997;20:38–43. doi: 10.1016/S0166-2236(96)10070-9. 10.1016/S0166-2236(96)10070-9. [DOI] [PubMed] [Google Scholar]
  31. McCormick DA. Cellular mechanisms underlying cholinergic and noradrenernic modulation of neuronal firing mode in the cat and guinea pig dorsal lateral geniculate nucleus. Journal of Neuroscience. 1992;12:278–289. doi: 10.1523/JNEUROSCI.12-01-00278.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. McCormick DA, Feeser HR. Functional implications of burst firing and single spike activity in lateral geniculate relay neurons. Neuroscience. 1990;39:103–113. doi: 10.1016/0306-4522(90)90225-s. 10.1016/0306-4522(90)90225-S. [DOI] [PubMed] [Google Scholar]
  33. McCormick DA, Prince DA. Noradrenergic modulation of firing pattern in guinea pig and cat thalamic neurons, in vitro. Journal of Neurophysiology. 1988;59:978–996. doi: 10.1152/jn.1988.59.3.978. [DOI] [PubMed] [Google Scholar]
  34. Malenka RC, Madison DV, Andrade R, Nicoll RA. Phorbol esters mimic some cholinergic actions in hippocampal pyramidal neurons. Journal of Neuroscience. 1986;6:475–480. doi: 10.1523/JNEUROSCI.06-02-00475.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Metherate R, Cox CL, Ashe JH. Cellular bases of neocortical activation: modulation of neural oscillations by the nucleus basalis and endogenous acetylcholine. Journal of Neuroscience. 1992;12:4701–4711. doi: 10.1523/JNEUROSCI.12-12-04701.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Metherate R, Tremblay N, Dykes RW. Transient and prolonged effects of acetylcholine on responsiveness of cat somatosensory cortical neurons. Journal of Neurophysiology. 1988;59:1253–1276. doi: 10.1152/jn.1988.59.4.1253. [DOI] [PubMed] [Google Scholar]
  37. Mittmann T, Alzheimer C. Muscarinic inhibition of persistent Na+ current in rat neocortical pyramidal neurons. Journal of Neurophysiology. 1998;79:1579–1582. doi: 10.1152/jn.1998.79.3.1579. [DOI] [PubMed] [Google Scholar]
  38. Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumor promotion. Nature. 1984a;308:693–698. doi: 10.1038/308693a0. [DOI] [PubMed] [Google Scholar]
  39. Nishizuka Y. Turnover of inositol phospholipids and signal transduction. Science. 1984b;225:1365–1370. doi: 10.1126/science.6147898. [DOI] [PubMed] [Google Scholar]
  40. Nishizuka Y. Studies and perspectives of protein kinase C. Science. 1986;233:305–312. doi: 10.1126/science.3014651. [DOI] [PubMed] [Google Scholar]
  41. Nishizuka Y. The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature. 1988;334:661–665. doi: 10.1038/334661a0. 10.1038/334661a0. [DOI] [PubMed] [Google Scholar]
  42. Schwartzkroin PA. Characteristics of CA1 neurons recorded intracellularly in the hippocampal in vitro slice preparation. Brain Research. 1975;85:423–436. doi: 10.1016/0006-8993(75)90817-3. 10.1016/0006-8993(75)90817-3. [DOI] [PubMed] [Google Scholar]
  43. Segal M. Acetylcholine enhances NMDA-evoked calcium rise in hippocampal neurons. Brain Research. 1992;587:83–87. doi: 10.1016/0006-8993(92)91430-m. 10.1016/0006-8993(92)91430-M. [DOI] [PubMed] [Google Scholar]
  44. Silva LR, Amitai Y, Connors BW. Intrinsic oscillations of neocortex generated by layer 5 pyramidal neurons. Science. 1991;251:432–435. doi: 10.1126/science.1824881. [DOI] [PubMed] [Google Scholar]
  45. Spruston N, Johnston D. Perforated patch-clamp analysis of the passive membrane properties of three classes of hippocampal neurons. Journal of Neurophysiology. 1992;67:508–529. doi: 10.1152/jn.1992.67.3.508. [DOI] [PubMed] [Google Scholar]
  46. Szente MB, Baranyi A, Woody CD. Effects of protein kinase C inhibitor H-7 on membrane properties and synaptic responses of neocortical neurons of awake cats. Brain Research. 1990;506:281–286. doi: 10.1016/0006-8993(90)91262-f. 10.1016/0006-8993(90)91262-F. [DOI] [PubMed] [Google Scholar]
  47. Wong RK, Prince DA. Dendritic mechanisms underlying penicillin-induced epileptiform activity. Science. 1979;204:1228–1231. doi: 10.1126/science.451569. [DOI] [PubMed] [Google Scholar]
  48. Wong RK, Prince DA. Afterpotential generation in hippocampal pyramidal cells. Journal of Neurophysiology. 1981;45:86–97. doi: 10.1152/jn.1981.45.1.86. [DOI] [PubMed] [Google Scholar]
  49. Zhang L, Weiner JL, Carlen PL. Muscarinic potentiation of IK in hippocampal neurons: Electrophysiological characterization of the signal transduction pathway. Journal of Neuroscience. 1992;12:4510–4520. doi: 10.1523/JNEUROSCI.12-11-04510.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

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