PMC

Synaptic responses of M1 and M2 cells in Opn4^−/− mice to 500 and 360 nm light. Current-clamp recordings of light-evoked depolarization of M1 and M2 cells to 5 s 500 or 360 nm light stimulation of increasing intensities. A–D, Representative examples of light-evoked depolarization of M1 cell to 500 nm light stimulus at 15.2 log photons · cm⁻² · s⁻¹ (A), M2 cell to 500 nm light stimulus at 14.7 log photons · cm⁻² · s⁻¹ (B), M1 cell to 360 nm light stimulus at 13.4 log photons · cm⁻² · s⁻¹ (C), and M2 cell to 360 nm light stimulus at 12.9 log photons · cm⁻² · s⁻¹ (D). E, Average intensity (log photons · cm⁻² · s⁻¹) at which a synaptic response was detectable for M1 and M2 cells to 500 nm (black bars) and 360 nm (gray bars) light stimuli.

On pathway-evoked depolarization in M1 and M2 cells of Opn4^−/− mice. Current-clamp recordings in Opn4^−/− mouse line of M1 and M2 cell responses to a bright white light stimulus. A, M1 cell response in current-clamp mode to 10 s bright, full-field, white-light stimulus at intensity of −2 log_I in control solution (top), in the presence of 100 μm l-AP4 in the bath (middle), and after washout (bottom). Light responses of M1 cells in Opn4^−/− mouse were completely abolished in the presence of l-AP4. B, M2 cell responses in current-clamp mode to 10 s bright, full-field, white light stimulus at intensity of −2 log_I in control solution (top), in the presence of 100 μm l-AP4 in the bath (middle), and after washout (bottom). Light response of M2 cells in Opn4^−/− mouse were completely abolished in the presence of l-AP4. Gray lines indicate 0.1 s smoothing of membrane voltage.

On pathway-evoked currents in M1 and M2 cells of Opn4^−/− mice. Voltage-clamp recordings (V_Hold = −73 mV) of light-evoked current in M1 and M2 ipRGCs of the Opn4^−/− mouse line to a 10 s, full-field, bright white-light stimulus at −2 log_I before and after 100 μm l-AP4 application. A, B, Light-evoked inward current of M1 (A) and M2 (B) cell in control (top) and after 5 min of 100 μm l-AP4 application (bottom) (n = 5 M1; n = 4 M2). C, Maximum light-evoked current in M1 (gray) and M2 (black) cells before and after l-AP4 application. Light-evoked currents were completely abolished following blockade of the On-pathway input. Gray line indicates 0.1 s smoothing of membrane current.

Modulation of ipRGC V_m (in mV) by the On pathway. A, B, Whole-cell current-clamp recording of M1 (A) and M2 (B) cells where V_m was recorded continuously (each panel indicates 10 s of recording) in the absence (left) and presence (middle) of 100 μm l-AP4. Right panels show washout. C, V_m of individual M1 (gray, n = 8 cells) and M2 (black, n = 9 cells) cells in control solution and in the presence of 100 μm l-AP4. D, Mean ± SE V_m of M1 (n = 8 cells) and M2 (n = 9 cells) in control solution (black bars) and in the presence of 100 μm l-AP4 (white bars). Gray line indicates −58 mV. *p < 0.05, t test, # p < 0.06, t test.

Effects of light-evoked On-pathway input on light sensitivity of M1 and M2 cells. A, B, Representative responses of two M1 cells to 5 s, full-field, white light stimulus at −5 log_I in the absence (A) or presence (B) of 100 μm l-AP4. C, Normalized, averaged responses of M1 cells in the absence (black, n = 8) and presence (gray, n = 5) of 100 μm l-AP4 to increasing intensities of white light fit with logistic dose–response functions (see Materials and Methods). D, E, Representative responses of two M2 cells to 5 s, full-field, white light stimulus at −5 log_I in the absence (D) or presence (E) of 100 μm l-AP4. F, Normalized and averaged responses of M2 cells in the absence (black, n = 8) and presence (gray, n = 6) of 100 μm l-AP4 to increasing intensities of white light, fit with logistic dose–response functions (see Materials and Methods). Gray lines indicate 0.1 s smoothing of membrane voltage. p < 0.05, t test. Vertical scale bars: (A–E), 20 mV.

Light-evoked current in WT and Opn4^−/− ipRGCs. Voltage-clamp recordings (V_Hold = −73 mV) of light-evoked current to a 10 s, full-field, bright, white-light stimulus at −2 log_I in M1 and M2 cells of WT and Opn4^−/− mice. A, Representative examples of light-evoked inward current of M1 (top) and M2 (bottom) cells in a WT mouse. B, Representative examples of light-evoked inward current of M1 (top) and M2 (bottom) cells in an Opn4^−/− mouse. C, Mean ± SE maximum current measured during the 10 s light stimulus in M1 (n = 8 WT; n = 10 Opn4^−/−) and M2 (n = 6 WT; n = 8 Opn4^−/−) cells of WT (black bars) and Opn4^−/− (white bars). Notice the reduced current in the Opn4^−/− M1 but not M2 cells. D, Mean ± SE total charge measured during 10 s light stimulus in M1 (n = 8 WT; n = 10 Opn4^−/−) and M2 (n = 6 WT; n = 8 Opn4^−/−) cells of WT (black bars) and Opn4^−/− (white bars). Notice the reduced charge in the Opn4^−/− M1 but not M2 cells. Gray lines indicate 0.1 s smoothing of membrane voltage. *p < 0.05, t test.

Contribution of On channel input to light response of M1 and M2 ipRGCs in WT mouse. A, M1 cell response in current-clamp mode to 5 s bright, full-field, white-light stimulus at intensity of −2 log_I in control solution (top), in the presence of 100 μm l-AP4 (middle) and after washout (bottom). B, M2 cell response in current-clamp mode to 5 s bright, full-field, white-light stimulus at intensity of −2 log_I in control solution (top), with 100 μm l-AP4 in the bath (middle) and after washout (bottom). C, Mean ± SE maximum depolarization for M1 (n = 8 cells) and M2 (n = 9 cells) in control solution (black bars), 100 μm l-AP4 (white bars), and after washout (gray bars). Gray line in A, B indicates 0.1 s smoothing of membrane voltage. *p < 0.05, one-way ANOVA. Vertical scale bars (A, B) 20 mV.

ipRGC morphology in Opn4^−/− mice. A, B, Confocal stacks of whole-mount retinas in which an M1 and M2 cell have been filled with neurobiotin (green) and costained for ChAT (red), a cholinergic amacrine cell marker. A, M1 cell with dendrites stratifying near the border of the INL in the Off sublamina of the IPL. B, M2 cell with dendrites stratifying near the GCL in the On sublamina of the IPL. Note: Processes visible extending through IPL are those of Müller cells, which commonly take up dye during the patching procedure. C, D, Sholl analysis (15 μm steps from starting diameter of 10 μm) of M1 (C) and M2 (D) dendritic arbors in WT (black circles) and Opn4^−/− (open circles) mouse lines. GCL, Ganglion cell layer. Scale bars: A, B, 50 μm.