Cholinergic modulation of pyramidal neuron excitability in the prefrontal cortex

S.B. Park, G.J. Stuart, C.E. Hill, A.T. Gulledge, Division of Neuroscience, JCSMR, Australian National University, Canberra, ACT, Australia

Muscarinic acetylcholine receptors (mAChRs) are important for normal cognitive function. We investigated the effects of acetylcholine on layer 5 pyramidal neurons in slices of prefrontal cortex obtained from anaesthetized and decapitated 3-6 week-old rats. Bath-application of the mAChR agonist carbachol (10 µM) depolarised neurons by 5.7 ± 1.8 mV (n = 13). In contrast, phasic application of ACh (100 µM; 5 – 1000 ms) resulted in transient membrane hyperpolarisation that inhibited action potential generation. Hyperpolarising responses had a mean amplitude of –6.4 ± 0.8 mV, an onset latency of 295 ± 34 ms, and a duration of 1.35 ± 0.12 s. ACh-induced hyperpolarisations were blocked by atropine (1 µM; n = 7) and the M1 antagonist pirenzepine (1 µM; n = 8), but not by the M2 antagonist methoctramine (1 µM; n = 8), indicating action via M1-type mAChRs. ACh-induced hyperpolarisations were associated with a 25 ± 5% increase in membrane conductance (n = 6) and reversed near the potassium equilibrium potential (-95.7 ± 1.7 mV, n = 6). The response was blocked by intracellular calcium chelation with BAPTA (10 mM; n = 10). Calcium imaging experiments revealed intracellular calcium increases associated with the hyperpolarisations (n = 5). Ryanodine (20 µM) blocked a significant proportion of the response (n = 6, df = 5, p < 0.05), implicating calcium release from intracellular stores. The inhibitory action of ACh was unaffected by bath applied TTX (500 nM, n = 4) or antagonists to GABA receptors (100 µM picrotoxin, 1µM CGP55845, and 10 µM SR95531, n = 6). Hyperpolarising responses were apamin sensitive (100 nM), suggesting the involvement of SK-type calcium activated potassium channels (n = 7, df = 6, p < 0.05). Importantly, phasic ACh application produced inhibition even during tonic depolarisation with bath-applied carbachol (5 µM, n = 4 of 4 neurons; 7 µM, n = 3 of 4; 10 µM, n = 9 of 14). Our data demonstrate that transient ACh application inhibits prefrontal pyramidal neurons via M1-type receptor-mediated calcium release from intracellular stores and subsequent SK channel activation.