RAPID COMMUNICATION Activation of Protein Kinase C Increases Neuronal Excitability by Regulating Persistent Na Current in Mouse Neocortical Slices

Astman, Nadav, Michael J. Gutnick, and Ilya A. Fleidervish. duction in dendritic action-potential propagation (Colbert Activation of protein kinase C increases neuronal excitability by and Johnston 1998; Tsubokawa and Ross 1997). regulating persistent Na current in mouse neocortical slices. J. NeuIn the present study, we sought to determine the effect rophysiol. 80: 1547–1551, 1998. Effects of the protein kinase C of PKC activation on neocortical neurons. We found that, activating phorbol ester, phorbol 12-myristate 13-acetate (PMA), although exposure to phorbol esters causes a decrease in were studied in whole cell recordings from layer V neurons in slices persistent Na current (INaP) at depolarized voltages, it also of mouse somatosensory neocortex. PMA was applied intracellularly leads to a marked decrease in threshold for action-potential (100 nM to 1 mM) to restrict its action to the cell under study. In generation. This paradoxical effect is related to a hyperpolarcurrent-clamp recordings, it enhanced neuronal excitability by inducizing shift in the voltage dependence of INaP activation. Poring a 10to 20-mV decrease in voltage threshold for action-potential generation. Because spike threshold in neocortical neurons critically tions of this work were previously presented in abstract form depends on the properties of persistent Na current (INaP), effects of (Fleidervish et al. 1997). PMA on this current were studied in voltage clamp. After blocking K and Ca currents, INaP was revealed by applying slow depolarizM E T H O D S ing voltage ramps from 070 to 0 mV. Intracellular PMA induced a decrease in INaP at very depolarized membrane potentials. It also CD1 mice of either sex, postnatal day 7 to postnatal day 28, shifted activation of INaP in the hyperpolarizing direction, however, were anesthetized with pentobarbital sodium (60 mg/kg) and desuch that there was a significant increase in persistent inward current capitated. Brains were removed, and 400-mm-thick coronal slices at potentials more negative than 045 mV. When tetrodotoxin (TTX) of somatosensory cortex were prepared as previously described was added to the bath, blocking INaP and leaving only an outward (Fleidervish et al. 1996; Fleidervish and Gutnick 1996). Renonspecific cationic current (Icat ), PMA had no apparent effect on cordings were made in a small (300 ml ) interface-type recording responses to voltage ramps. Thus PMA did not affect Icat , and it did chamber, where slices were continuously perfused with artificial not induce any additional current. Intracellular application of the cerebrospinal fluid whose composition was (in mM) 124 NaCl, 3 inactive PMA analogue, 4a-PMA, did not affect INaP. The specific KCl, 2 CaCl2 , 2 MgSO4, 1.25 NaH2PO4, 26 NaHCO3, and 10 protein kinase C inhibitors, chelerythrine (20 mM) and calphostin C glucose (pH 7.3 at 32 { 0.57C when bubbled with a 95% O2-5% (10 mM), blocked the effect of PMA on INaP. The data suggest that CO2 gas mixture) . PMA enhances neuronal excitability via a protein kinase C–mediated Whole cell recordings from 108 layer V neurons were made using increase in INaP at functionally critical subthreshold voltages. This the patch-clamp technique (Hamill et al. 1981) as modified for novel effect would modulate all neuronal functions that are influenced ‘‘blind’’ recording in slices (Blanton et al. 1989). Patch pipettes, by INaP, including synaptic integration and active backpropagation of pulled from borosilicate glass capillaries (Hilgenberg, Germany) on action potential from the soma into the dendrites. a Narishige PP83 puller, had resistances of 1.5–3.5 MV. The pipette solution for persistent sodium current recording consisted of (in mM) 135 CsCl, 4 NaCl, 2 MgCl2, and 10 N-2-hydroxyethylpiperazineI N T R O D U C T I O N N*-2-ethansulfonic acid (HEPES, cesium salt), pH 7.25. Care was taken to maintain membrane access resistance as low as possible The dynamics of neocortical circuit function depend on (usually 3–4 MV and always õ10 MV); series resistance was 80% the excitability of individual neurons as well as on the synapcompensated using the built-in circuitry of an Axopatch-1D amplifier tic interactions between them. Neocortical neuronal excit(Axon Instruments). Command voltage protocols were generated, ability may be modulated by a variety of ligands that regulate and data were acquired on-line with an Axolab 1100 A/D interface. voltage-gated channels through activation of protein kinase Data were low-pass filtered at 2 kHz (03 dB, 4-pole Bessel filter) and sampled at 5–10 kHz digitalization frequency. Before data acquiC (PKC). Although most attention in this regard has focused sition, linear leak current was subtracted using the built-in circuits on K channel regulation (Krnjevic et al. 1971; McCormick of the amplifier. et al. 1993), there is considerable evidence that PKC-mediAn Axoclamp-2A amplifier in a Bridge mode was used for curated phosphorylation of the Na channel may also be of rent-clamp experiments. The pipette solution contained (in mM) considerable functional significance. Studies at the channel 135 K gluconate, 2 MgCl2 , and 10 HEPES (potassium salt) , pH level have shown that this regulation results in a decrease 7.25. Data were low-pass filtered at 10 kHz (03 dB, 4-pole Bessel in Na channel availability (Cantrell et al. 1996; Lotan et filter) and sampled at 20 kHz digitalization frequency. Voltages al. 1990; Numann et al. 1991); this would be expected to in Fig. 1 have not been corrected for liquid junction potential. reduce neuronal excitability. Recent reports, however, indiData were analyzed using PClamp 5.5 and Microcal Origin 3.78 cate that in hippocampus, PKC activation increases dendritic software. Spike amplitudes were measured from threshold to peak. Apparent input resisitance (Rin ) was determined as the slope of excitability and thereby reverses the activity-dependent re-