Ionotropic acetylcholine receptors control neuroendocrine cell excitability in Aplysia

Abstract

In the hermaphroditic sea snail, Aplysia californica, activation of ionotropic acetylcholine receptors on neuroendocrine bag cell neurons triggers an ~30-min afterdischarge that leads to egg-laying hormone secretion and reproductive behavior. Nevertheless, how this brief input leads to such prolonged bursting is incompletely understood. Here, using cultured bag cell neurons and cellular electrophysiology, I hypothesized that: i) triggering acetylcholine receptors recruits persistent, voltage-gated Ca2+ current; ii) following repeated activation, the cholinergic response depresses due to receptor internalization; iii) acetylcholine receptors are redox- regulated and interact with H2O2-sensitive, non-selective cation channels. Initially, the depolarization (n=7) induced by a 2-s pressure-ejection of 1 mM acetylcholine took significantly longer to return to baseline than the similarly evoked current (n=6) (165.50±64.4 s vs 25.21±4.21 s time to 75% recovery). Moreover, the depolarization was significantly shortened to 59.06±14.53 s (n=8) by the Ca2+ channel antagonist, Co2+, and extended to 279.4±147.4 s (n=5) by activating protein kinase C, previously found to insert additional voltage-gated Ca2+ channels into the membrane. Next, the current evoked by two, successive pressure-ejections of acetylcholine exhibited persistent and significant depression to 59.15±6.27% (n=7), with partial recovery to 87.63±7.72% (n=5) by 6 hr, and complete recovery (110.3±25.02%; n=15) at 24 hr. Pre-treatment for 12 hr with the proteasome inhibitor, lactacystin, significantly attenuated depression, such that the second current was 84.83±15.24% (n=9), 10 min after the first, while disrupting the trans-Golgi network with brefeldin A prevented the 24 hr recovery, leaving the response at 65.22±11.46% (n=7). A significant decrease in receptor membrane density following acetylcholine (6246±1980 AU; n=6) vs control (9232±2513 AU; n=11) was confirmed by confocal microscopy of neurons stained with 𝛼-conotoxin ImI-ATTO Fluor-590. Finally, prior acetylcholine receptor activation led to a significantly greater H2O2-induced, non-selective cationic current (-267.3±56.2 pA; n=7 vs -204.0±17.2 pA; n=6 in control) and bursting (1.399±0.270 Hz; n=4 vs 0.5611±0.3346 Hz; n=6 in control). Hence, ionotropic acetylcholine receptors in bag cell neurons appear to regulate reproductive timing not only by initiating the afterdischarge but also, controlling receptor density, and engaging diverse channels. This work extends the canonical role of ionotropic acetylcholine receptors beyond fast neurotransmission, emphasizing their importance in long-term neuromodulation.