Journal: Frontiers in Endocrinology

Article Title: Acetylcholine regulation of GnRH neuronal activity: A circuit in the medial septum

doi: 10.3389/fendo.2023.1147554

Figure Lengend Snippet: ACh-induced increase in [Ca 2+] i occurs through nAChR. (A) Bright field image (left) was used to identify GnRH neurons (arrows). Cells maintained 8–11 days in vitro were loaded with calcium-sensitive dye Calcium Green-1 AM (middle) and imaged. Identity of cells as GnRH neurons was verified post hoc using immunocytochemistry (right). Scale bar, 25 µm. (B) Averaged trace from cells in single explant showing a transient increase in intracellular calcium levels ([Ca 2+ ]i) with acetylcholine (ACh, 100 µM) in the presence in the presence of amino acid blockers (AAB), (BIC, 20 µM; CNQX, 10 µM; d-AP5, 20 µM), (n=155, N=6). Calcium responses were quantified by calculating the area under the curve (AUC, grey area) for the stimulation of nAChRs. Y-axis: the optical density (OD) expressed as ΔF/F 0 (%), was normalized to 50% OD baseline over the 3 minutes prior to agonist application; X-axis: 2 min. (C) Blocking muscarinic receptors (mAChRs) with scopolamine (10 nM) did not prevent ACh-induced increase in [Ca 2+ ]i (n=169, N=5). (D) Blocking nAChRs with mecamylamine (10 µM) did prevent ACh-induced calcium response (n=73, N=5). (E) Application of nicotine (100 µM) mimicked ACh-induced increase in [Ca 2+ ]i, indicating that the calcium increase relies upon nAChRs activation (n=132, N=5). (F) Summary data showing the average AUC for (B–E) . Asterisks represent statistical significance between ACh (control) and other compounds ( p <0.05, one-way ANOVA test with Dunnett’s multiple comparisons). All recordings display the mean of all explants (N) . Average number of GnRH cells analyzed/explant ranged from 15-34. (n = number of cells, N = number of explants).

Article Snippet: (−)-Bicuculline (BIC) chloride (20 µM, an A-type γ-aminobutyric acid [GABA] receptor antagonist), D-(−)-2-amino-5-phosphonopentanoic acid (AP5, 10 µM, a N-methyl-D-aspartate receptor antagonist), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 µM, AMPA/kainate receptor antagonist), tetrodotoxin citrate (TTX, 0.5 µM, a voltage-gated sodium channel blocker), acetylcholine (ACh, 100 µM, non-selective AChR agonist), scopolamine hydrobromide (10 nM, non-selective mAChR antagonist), mecamylamine hydrochloride (10 nM, non-selective nAChR antagonist), (−)-nicotine tartrate (100 µM, nAChR agonist), PNU282987 (10 µM, alpha7 nAChR agonist), TC2559 difumarate (10 µM, alpha4beta2 nAChR agonist), and CC4 (10 uM, alpha6beta2 nAChR agonist) were all purchased from Cayman Chemical (Ann Arbor, MI).

Techniques: In Vitro, Immunocytochemistry, Blocking Assay, Activation Assay, Control