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. Author manuscript; available in PMC: 2020 Jan 16.
Published in final edited form as: Neuron. 2018 Dec 10;101(2):260–273.e6. doi: 10.1016/j.neuron.2018.11.028

Figure 1. CaV2.1 α1 OE Results in Increased CaV2.1 Currents and Almost Complete Loss of CaV2.2 Currents at the P7 Calyx.

Figure 1.

(A) Schematic of auditory brainstem. Globular bushy cells (GBC) which give rise to the calyx of Held are depicted for clarity.

(B) (Top) Developmental transition of calyx of Held from multiple CaV2 subtype synapse to CaV2 exclusive at onset of hearing (P12). (Bottom) Experimental timeline from virus injection into VCN at P1 to electrophysiological recordings at P7.

(C) Schematic of HdAd constructs expressing either CaV2.1 or CaV2.2 cDNAs (light blue) driven by the Punisher overexpression cassette and mEGFP marker (green) driven by a 470 bp human synapsin promoter; arrows indicate viral inverted terminal repeat sequences; J indicates the viral genome packaging signal sequence.

(D) Pharmacological isolation of CaV2 isoforms expressed in the presynaptic terminal at P7 in control (n = 5), CaV2.1 α1 OE (n = 4), or CaV2.2 α1 OE (n = 6). Average traces before application of blockers (black), after applying 200 nM ω-agatoxin IVA to specifically block CaV2.1 (Aga, brown), after 2 μM ω-conotoxin GVIA to block CaV2.2 (Cono, blue) and 50 μM Cd2+ to block the remaining Ca2+ currents (gray).

(E–H) Ca2+ current amplitudes before blocker application (CaV2.1 α1 OE versus control, p = 0.0328 Mood’s median test and post hoc Bonferroni test), Aga-sensitive Ca2+ current amplitudes (CaV2.1 α1 OE versus control, p = 0.0328), Cono-sensitive Ca2+ current amplitudes (CaV2.1 α1 OE versus control, p = 0.0054), and Cd2+-sensitive Ca2+ current amplitudes (n.s., Kruskal Wallis and post hoc Dunn’s test, n = 5/4/6 for control, CaV2.1 α1 OE, and CaV2.2 α1 OE, respectively).

(I) Relative Ca2+ current fractions sensitive to respective blockers.

(J and K) Average Ca2+ current traces to 10 ms step depolarizations from −80 mV holding to voltages between −50 and +40 mV for control (J and K, left, black) and CaV2.1 α1 OE (J, right, brown) or CaV2.2 α1 OE (K, right, blue).

(L–O) Current-voltage relationships of either steady-state Ca2+ currents (L and N) or tail Ca2+ currents (M and O, n = 10 for control, CaV2.1 α1 OE and CaV2.2 α1 OE). All data are shown as mean ± SEM. See also Table S1.