Figure 3
Functional properties of cultured rod precursors. (a) The Y-axis plots the rod precursor generation rate in arbitrary units. The scheme illustrates the generation of retinal cultures at 1 × and 4 × cell densities from PN0 mice. Horizontal arrows indicate the use of DIV2, DIV4, DIV6, and DIV8 cultures for patch-clamp recordings (E-phys). (b–d) Sweeps plot membrane currents activated in response to 2 s-long voltage steps at − 60, − 80, − 100, and − 120 mV from a holding voltage of − 40 mV in GFP+ cells isolated on the day of birth (PN0) and recorded after 2 (PN0/DIV2) (b), 4 (PN0/DIV4) (c), 6 (PN0/DIV6) (d) and 8 (PN0/DIV8) (e) days in vitro (DIV). (f) Protocol for measuring the Cs-sensitive current (IHYP) at steady-state (see “Materials and methods”). The double arrows line indicates the current activated by membrane hyperpolarisation (IHYP), computed by the difference between current amplitudes in saline with either 30 mM KCl or 30 mM KCl + 3 mM CsCl. (g) Filled symbols plot membrane conductance (GHYP) values generated by diving IHYP for the driving force (see “Materials and methods”). GHYP is plotted, after normalisation to membrane capacitance, as a function of activating voltage. The continuous line plots the best-fitting Boltzmann function used to estimate activation parameters (see “Materials and methods”) for the PN0/DIV4 cell above. (h–j) Symbols plot parameters generated by best-fits to individual cells voltage-dependent activation curves similar to panel (f). Circles plot GHYP (h) normalised to membrane capacitance, the inverse slope factor SHYP (i), and the half-activation voltage V0.5HYP (j) for PN0/DIV2 (N = 6), PNO/DIV4 (N = 11), PN0/DIV6 (N = 6), and PN0/DIV8 (N = 13) cells cultured at 1 × (open circles); PN0/DIV4 (N = 3) and PN0/DIV8 (N = 1) cells cultured at 4 × cell density (filled circles). Two-way ANOVA indicated non-significant effects of DIV (F = 1.42953 with 3, 32 df: P = 0.25233) and cell density (F = 1.11282 with 1 and 32 df: P = 0.29937) on GHYP.
