Fig. 2.

Membrane depolarization induces Ca²⁺ influx and spine loss. (A) Membrane depolarization of D₂ MSNs in response to elevated extracellular potassium concentration (mM, n=5 for each concentration). The membrane potentials measured correlate to those predicted by Nernst equation. (B-E) Membrane depolarization induces L-type Ca²⁺ channel-dependent Ca²⁺ elevation in D₂ MSNs. Images of Fura-2 AM loaded D₂ MSNs in Corticostriatal co-culture were captured using two-photon microscopy. Images of two EGFP-labeled cells stimulated with 35 mM KCl in presence of ionotropic receptor blockers are shown at excitation wavelength 950 nm (B), and 700 nm (C) and 780 nm (D). Scale bar, 10 μm. Ca²⁺ concentration in the somas of D₂ MSNs was determined by computing the ratio 700/780 images. (E) Changes of Ca²⁺ concentration relative to baselines are shown as a function of time for D₂ MSNs stimulated by membrane depolarization (n=4, black traces) or D₂ MSNs stimulated in the presence of 10 μM nimodipine (n=6, red traces). (F) A D₂ MSN in corticostriatal co-cultures treated with 35 mM KCl for 24 hours in presence of ionotropic receptor blockers at 20 DIV. Bar: upper panel 10 μm; lower panel, 5 μm. (G) Time course of the change of spine density in D₂ MSNs after membrane depolarization. Spine density is shown in mean±standard deviation (p<0.001, one way ANOVA; Ctrl, 11.69±1.66, n=12; 8 hrs, 10.41±1.13, n=16; 16 hrs, 8.42±1.99, n=14; 24 hrs, 5.79±0.96, n=14). (H) Spine losses in D₂ and D₁ MSNs after 24 hours of membrane depolarization. (35 mM KCl treated groups are shown in shadows. D₂ MSNs control, median=11.3, n=21; D₂ MSNs with 35 mM KCl, median=5.3, n=23; D₁ MSNs, median=10.8, n=21; D₁ MSNs with 35 mM KCl, median=9.5, n=24. *p<0.05, ***p<0.001, Mann-Whitney Rank Sum Test)