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

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 microM) consistently suppressed intrinsic bursting in an atropine-sensitive (1 microM) manner. 3. Intrinsic bursting was suppressed by 4beta-phorbol 12,13-dibutyrate (PDBu; 5-10 microM), a potent PKC activator, but not by the inactive phorbol ester 4alpha-phorbol 12,13-didecanoate (PDC; 50 microM). Prior application of the PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7; 10 microM) 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 microM), 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.

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

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 (V_m, -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).

Figure 2. PDBu suppresses intrinsic bursting

Upper panels show the response of a neuron (resting V_m, -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).

Figure 3. PDBu suppresses intrinsic bursting through PKC activation

A, in control conditions, the neuron (V_m, -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 (V_m, -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).

Figure 4. H7 prevents CCh modulation of intrinsic bursting

A, the neuron was loaded with H7 (10 μM) as described in Fig. 3 In control conditions the H7-loaded neuron (V_m, -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 (V_m, -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).

Figure 5. CCh suppresses spike ADP

A spike was evoked from V_m (-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 V_m.

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

A, spikes were evoked from V_m (-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 (V_m, -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 (V_m, -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 V_m.

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

A, single spikes were evoked from V_m (-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 (V_m, -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 V_m.

Figure 8. PDBu blocks plateau ADPs

A, in a CA1 pyramidal cell (V_m, -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 (V_m, -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).