title: A unifying hypothesis for M1 muscarinic receptor signalling in pyramidal neurons.
abstract: Phasic release of acetylcholine (ACh) in the neocortex facilitates attentional processes. Acting at a single metabotropic receptor subtype, ACh exerts two opposing actions in cortical pyramidal neurons: transient inhibition and longer-lasting excitation. Cholinergic inhibitory responses depend on calcium release from intracellular calcium stores, and run down rapidly at resting membrane potentials when calcium stores become depleted. We demonstrate that cholinergic excitation promotes calcium entry at subthreshold membrane potentials to rapidly refill calcium stores, thereby maintaining the fidelity of inhibitory cholinergic signalling. We propose a 'unifying hypothesis' for M1 receptor signalling whereby inhibitory and excitatory responses to ACh in pyramidal neurons represent complementary mechanisms governing rapid calcium cycling between the endoplasmic reticulum, the cytosol and the extracellular space.
  
  
  
  Gq-coupled M1-type muscarinic acetylcholine (ACh) receptors (mAChRs) mediate two distinct electrophysiological responses in cortical pyramidal neurons: transient inhibition driven by calcium-dependent small conductance potassium ('SK') channels, and longer-lasting and voltage-dependent excitation involving non-specific cation channels. Here we examine the interaction of these two cholinergic responses with respect to their contributions to intracellular calcium dynamics, testing the 'unifying hypothesis' that rundown of inhibitory SK responses at resting membrane potentials (RMPs) reflects depletion of intracellular calcium stores, while mAChR-driven excitation acts to refill those stores by promoting voltage-dependent entry of extracellular calcium. We report that fidelity of cholinergic SK responses requires the continued presence of extracellular calcium. Inhibitory responses that diminished after repetitive ACh application at RMPs were immediately rescued by pairing mAChR stimulation with subthreshold depolarization (∼10 mV from RMPs) initiated with variable delay (up to 500 ms) after ACh application, but not by subthreshold depolarization preceding mAChR stimulation. Further, rescued SK responses were time-locked to ACh application, rather than to the timing of subsequent depolarizing steps, suggesting that cholinergic signal transduction itself is not voltage-sensitive, but that depolarization facilitates rapid cycling of extracellular calcium through the endoplasmic reticulum to activate SK channels. Consistent with this prediction, rescue of SK responses by subthreshold depolarization required the presence of extracellular calcium. Our results demonstrate that, in addition to gating calcium release from intracellular stores, mAChR activation facilitates voltage-dependent refilling of calcium stores, thereby maintaining the ongoing fidelity of SK-mediated inhibition in response to phasic release of ACh.
authors.affiliation: Department of Molecular and Systems Biology, Geisel School of Medicine at Dartmouth College, Hanover, NH, 03755, USA.
authors.forename: Sameera
authors.initials: S
authors.lastname: Dasari
authors.affiliation: Department of Molecular and Systems Biology, Geisel School of Medicine at Dartmouth College, Hanover, NH, 03755, USA.
authors.forename: Corey
authors.initials: C
authors.lastname: Hill
authors.affiliation: Department of Molecular and Systems Biology, Geisel School of Medicine at Dartmouth College, Hanover, NH, 03755, USA.
authors.forename: Allan T
authors.identifier: 0000-0002-5721-374X
authors.initials: AT
authors.lastname: Gulledge
mesh_terms: D000109:Acetylcholine / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000502:physiology; D005260:Female; D008297:Male; D008810:Mice, Inbred C57BL; D017397:Prefrontal Cortex / Q000502:physiology*; D017966:Pyramidal Cells / Q000502:physiology*; D043643:Receptor, Muscarinic M1 / Q000502:physiology*; D015398:Signal Transduction
chemical_list: D043643:Receptor, Muscarinic M1; D000109:Acetylcholine; D002118:Calcium
keywords: SK channel; acetylcholine; calcium; muscarinic receptor; neocortex; pyramidal Neuron