Agonist‐induced depression of cholinergic current in <i>Aplysia</i> bag cell neurons requires ionotropic acetylcholine receptor trafficking

Cholinergic input initiates or regulates prolonged neuronal activity implicated in executive attention, learning and neuroendocrine control. In the hermaphroditic sea snail, Aplysia, ionotropic acetylcholine receptors on neuroendocrine bag cell neurons trigger an ∼30-min afterdischarge that leads to egg-laying hormone secretion and reproductive behaviour. The afterdischarge is followed by an ∼18-h refractory period, where acetylcholine fails to cause further bursting. Here, we report on a similar long-term depression of acetylcholine receptors in cultured bag cell neurons. Under whole-cell voltage-clamp, the current evoked by 2-s pressure-ejections of 1 mM acetylcholine exhibited persistent rundown, with partial recovery by 6 h after the first ejection and complete recovery at 24 h. Consecutive applications at 3-, 10-, 30-, 60- or 90-min intervals reliably yielded ∼two-thirds of current remaining at the second ejection. Similar rundown was elicited using the cholinergic agonist tretramethylammonium, albeit at 10 mM. Rundown was independent of channel gating, as the acetylcholine receptor pore-blocker hexamethonium failed to alter progression. Neither the protein kinase A blocker KT5720 nor the muscarinic receptor antagonist phenyltrimethylammonium affected rundown. However, the proteasome inhibitor lactacystin attenuated rundown, while disrupting the trans-Golgi network with brefeldin A or protein synthesis with anisomcyin prevented recovery 24 h later, consistent with receptor trafficking underlying both rundown and recovery. A decrease in receptor membrane density following acetylcholine was confirmed by confocal microscopy of neurons stained with the dye-conjugated ionotropic antagonist <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:semantics><mml:mi>α</mml:mi> <mml:annotation>${{\alpha}}$</mml:annotation></mml:semantics> </mml:math> -conotoxin ImI-ATTO Fluor-590. Hence, retrieval of receptors from the membrane mediates rundown and, analogous to the refractory period, may control the timing of reproduction. KEY POINTS: Hormone secretion from neuroendocrine cells typically requires a prolonged burst of action potentials; for example, bag cell neurons from the marine snail Aplysia trigger reproduction by the neurohaemal release of egg-laying hormone during a lengthy afterdischarge. The bag cell neuron afterdischarge is initiated by cholinergic synaptic input onto ionotropic acetylcholine receptors, but following an afterdischarge, the neurons become unresponsive to acetylcholine for many hours. We now demonstrate that, after an initial exposure to acetylcholine, a proportion of these ionotropic receptors are internalized and made unavailable, without any influence from a metabotropic counterpart. This prolonged depression of cholinergic receptors also lasts many hours and likely serves to prevent disruption of ongoing bursting and/or the amount of hormone released. Therefore, changes in the number of ionotropic receptors at the membrane may influence the timing of neuroendocrine cell activity.