Figure 1.
Light responses in isolated ipRGCs and evidence for expression of the hyperpolarization-activated inwardly-rectifying current (Ih).
A, B) Light-evoked responses recorded from dissociated ipRGCs, retrolabeled by injection of rhodamine labeled latex microspheres into the suprachiasmatic nucleus. A) Voltage clamp recording of a typical ipRGC. Cell was held at −73 mV and given a 4 s flash of white light (black bar), triggering a large inward current. B) Current clamp recording from a different ipRGC. A 1 s light flash (black bar) depolarized the cell, causing spiking that persisted several minutes after termination of the light stimulus. C–F) Evidence for the presence of Ih. C) Hyperpolarizing the membrane in 4 s steps evoked an instantaneous current response that, at membrane potentials negative to −83 mV, was followed by a slowly activating inward current. Bath-application of 3 mM Cs+ to block Ih abolished the slow component. D) Group data (N = 6) plotting the total current (squares; Itot), the Cs+-insensitive current (circles; ICs), and the difference between them (triangles; Itot-ICs), which isolates the Cs+-sensitive, presumptive Ih current. E, F) Bath-application of the alternative Ih blocker 100 µM ZD7288 likewise abolished the slow component (N = 4).
Figure 2.
The depolarizing sag in ipRGCs and its mediation by Ih.
A–D) Analysis of the hyperpolarizations evoked in ipRGCs by current injections of 0, −10, −20, −40 and −60 pA (500 ms). For larger current steps, membrane potential sagged from its peak hyperpolarization back toward resting potential (Panels A and C; ‘Control’). A) Depolarizing sag in an example cell (top) and its blockade (bottom) by bath application of the Ih blocker ZD7288 (100 µM). B) Current-voltage plots of data in A drawn from the end of the current pulse, at the time marked by symbols in A (squares: control conditions; triangles: in presence of drug). Input resistance (RN), calculated as the slope of the linear fit to these current-voltage data, was increased by the Ih antagonist. C, D) Extracellular Cs+ (3 mM), an alternative Ih blocker, reproduced the effects of ZD7288 in another ipRGC, blocking the depolarizing sag (C) and increasing RN (D). Resting potential was maintained at −72 mV in both this cell and that in A (see methods). E, F) Group data showing that both ZD7288 and Cs+ dramatically attenuate the depolarizing sag (E; measured as the peak-to-steady state potential difference during −60 pA current injections) and increase RN (F; measured as in B and D). * P<0.05.
Figure 3.
Differing effects of Ih blockers ZD7288 and Cs+ on spiking evoked by depolarizing current injections.
A, B) Depolarizing current injections (20 pA) evoke trains of action potentials in an ipRGC. Spike frequency is lower in the presence of ZD7288 (B) relative to control (A). C) Group data for the effect shown in A and B. The difference is significant (* P<0.05) for all intensities of current injection (10–60 pA; N = 7 cells). D–F) Failure of the alternative Ih blocker Cs+ (3 mM) to cause a similar reduction in evoked action potentials. The treatment even caused a small increase in spiking for a 20 pA current injection (N = 4; * P<0.05). Vm was maintained near −72 mV throughout for all cells (see Methods).
Figure 4.
Current-voltage relationship of the fully activated Ih as measured by tail current analysis.
A) Ih was activated by a step to −123 mV and tail currents were recorded at a variety of test potentials ranging from −103 to −63 mV (left). This voltage protocol was repeated in the presence of the 100 µM ZD7288 (middle). The ZD7288 traces were subtracted from the control traces to isolate Ih (right) and these tail currents were fit with a single exponential to extrapolate the amplitude at the termination of the −123 mV step. B) Tail current amplitudes as a function of test potential for the cell in A. A linear fit was used to extrapolate the reversal potential of Ih, approximately −43 mV for this cell. C) Tail current amplitudes as a function of test potential and the linear fit for all cells included in this analysis (N = 8).
Figure 5.
Activation and deactivation of Ih.
A) Activation of Ih in an example recording in response to a −123 mV voltage step fit with a single exponential used to calculate the time constant of activation. B) Deactivation of Ih as estimated from a single exponential fit to the tail current from the same recording as in A after the membrane potential was stepped back to −73 mV. C) The activation curve reflecting tail current amplitude measured at the end of steps to hyperpolarized test potentials and normalized to the tail current amplitude following a step to −123 mV. D) Plot of sag amplitude (steady-state minus peak hyperpolarization, inset) from current-clamp recordings in which the membrane was hyperpolarized by current injections of −80 to −10 pA.
Figure 6.
Blockade of Ih does not affect ipRGC membrane potential.
Membrane potential was maintained near −72 mV with DC injection before the addition of the pharmacological agent. Neither ZD7288 (A; 100 µM; N = 6) nor extracellular Cs+ (B; 3 mM; N = 4) had a significant effect on membrane potential. n.s. P>0.05.
Figure 7.
Blockade of Ih does not affect the ipRGC light response.
Light responses were evoked by a 1 s flash of light (black bar; intensity = −1 log I) before (A) and after (B) bath application of 3 mM CsCl. Blockade of Ih was confirmed by the loss of a depolarizing sag during 500 ms hyperpolarizing current injections. Cs+ had no effect on either the depolarization (C; N = 5) or the number of spikes (D; N = 3) evoked by the light flash. n.s. P>0.05.