Article Figures & Data
Figures
- Figure 1.
HCN3 is densely expressed in the IGL. A, A representative image of the IGL in a HCN2−/− mouse brain slice (30 μm); the IGL is marked by white dashed lines. NPY staining (green) as a marker for the IGL. B, C, Merged images showing immunolabeling for NPY (green) and HCN (red) in IGL neurons in the mouse (B, C) and the rat (Di, Dii), as indicated. Dii, HCN3-IR in a thick section (200 μm) from the rat. Images in B, C, and Di are from thin sections (30 μm). Note that dense labeling for HCN3 was present on the soma of both NPY-positive and -negative IGL cells in sections from both mice and rats; labeling for other 3 isoforms was essentially absent. Scale bar: 10 μm for all images in B–D.
- Figure 2.
Deletion of HCN2 does not alter properties of Ih recorded from IGL neurons. A (mouse), B (rat), Representative images for the IGL in live brain slices, as marked by white dotted lines; images for IGL neurons that were intracellularly biocytin-filled and fluorescently labeled (green). Scale: horizontal bars, 0.5 mm; vertical bars, 10 μm. C–E, Exemplar Ih current traces were recorded from regions as indicated, and were blocked by ZD7288 (50 μm). The same voltage protocol (−130 mV, 10 s, VH = −50 mV) was used for recordings in all neurons here. F, Bar graph for current density. *p < 0.001, HCN2−/− versus HCN2+/+ for vLGN neurons. G, Bar graph for activation time constant (Tauactivation). *p < 0.001, one-way ANOVA with pairwise comparisons, n = 10/each.
- Figure 3.
Ih in IGL neurons exhibits the hallmark of HCN3 channels. A–C, Families of current traces from an HCN2−/− mouse IGL neuron (A), rat IGL neuron (B), and a rat vLGN neuron (C). The same protocol was used for all recordings (−50 to −130 mV, −10 mV/step, 10 s, VH = −50 mV). 8-Br cAMP (200 μm) was applied by superfusion in this and following figures. Color for all panels: Dark gray for control; orange for 8-Br cAMP. Normalized tail currents (Itail) are plotted as a function of voltage steps and are fit with the Boltzmann equation (right). Symbols for all panels: triangles, HCN2−/− IGL; squares, rat IGL; circles, rat vLGN. D, Group data showing effects of 8-Br cAMP on V1/2 (mV). *p < 0 0.05, 8-Br cAMP vs control for rat vLGN neurons. E, Time constants of activation (Tauactivation) and deactivation (Taudeactivation) in the absence and presence of 8-Br cAMP. *p < 0 0.05, cAMP versus control in rat vLGN neurons; one-way ANOVA, n = 15/each.
- Figure 4.
Depletion of endogenous PIP2 produces a hyperpolarizing shift in activation of native and recombinant HCN3 channels. A, Exemplar Ih traces recorded from IGL neurons preincubated for 40–50 min in (Ai, control) or wortmannin (Aii, wort) before or after superfusion with 200 μm 8-Br cAMP. B, Group data showing steady-state activation curves. Lines are fit using a Boltzmann function to normalized tail currents, yielding the V1/2 and slope factor (both in mV) of −89.2 ± 2.4 and 9.4 ± 1.1 for control (squares); −106.3 ± 3.5 and 8.2 ± 1.1 for wortmannin alone (circles); −105.6 ± 2.6 and 9.4 ± 0.9 for wortmannin + 8-Br cAMP (diamonds). Colored symbols used here represent the same conditions in subsequent panels for IGL neurons. There is a significant difference in V1/2 values between control and wortmannin groups, p < 0.001, t test, n = 22. C, D, Effect of the drugs on the activation time constant (at −110 mV (C) and the current density at −130 mV (D). Almost identical effects are observed in IGL neurons from rats (n = 8) and HCN2−/− mice (n = 10), and the two datasets were pooled. *p < 0.05, compared to control. E, HCN3 current traces recorded from intact oocytes following incubation in either control solution (which contains DMSO) (left) or wortmannin (right). F, Effects of wortmannin on steady-state activation curves; inset shows representative tail currents (left, wortmannin and right, control); red traces at −75 mV are for comparison. G, Negative shift in the V1/2 of HCN3 channel activation following incubation with vehicle DMSO (control, n = 10) or wortmannin for 40 (n = 10) or 60 (n = 4) min. Vehicle controls were indistinguishable from the controls in the absence of DMSO (data not shown) in all experiments. The wortmannin-treated populations were significantly different from vehicle control and each other (*p < 0.05, vs control, one-way ANOVA with a Holm–Sidak post hoc test).
- Figure 5.
PIP2 enhances opening of Ih channels in IGL neurons. A, B, Current traces recorded from rat (A) and HCN2−/− mouse (B) IGL neurons in normal (control, dark gray) and PIP2 (10 μm, green)-containing intracellular solution. Activation curves for each neuron type are shown to the right. PIP2 shifts the V1/2 to a more depolarized potential and increases current density at physiological relevant membrane potentials (*p < 0.05). Symbols: squares for rat IGL and triangles for HCN2−/− IGL in the absence (gray) and presence of PIP2 (green) here and in subsequent panels. C, Time course for changes in V1/2 values in control and PIP2. Time 0 represents time immediately after break-in, and the dash line indicates 0 mV shift; n = 4/each point. D, Group data summarizing drug-induced changes in V1/2 values. 8-Br cAMP (200 μm) was added by superfusion ∼15 min after PIP2 dialysis through the recording pipette. Symbols for each treatment are indicated at bottom. E, F, Group data showing effects of drugs on activation and deactivation kinetics. Data from rat IGL neurons are shown in E, while data from HCN2−/− mice are shown in F. Symbols are the same as in D. *p < 0.05, one-way ANOVA (* for each condition, as compared to corresponding control), n = 10/each.
- Figure 6.
PIP2 increases Ih-dependent burst firing in IGL neurons. A, Intrinsic temporal summation producing burst firing was initiated by intracellular injection of a train containing five current pulses (100 pA); the protocol shown at bottom was previously described (Ying et al., 2005). EPSC-shaped current pulses were generated with the following function: I(t) = A × (1 − e−t/τrise)n e−t/τdecay, where A is the infinite time amplitude of the current (in picoamperes), n is an integer, and τrise (0.5 ms) and τdecay (5 ms) are rising and falling time constants, respectively (Magee, 1998). The latency was measured as indicated by vertical arrows. The horizontal arrow indicates the holding membrane potential of −80 mV in control before injection of the train in all panels. Bi, Overlay showing comparison of response to ZD7288 (ZD, 30 μm) to control in the same neuron as in A. Bii, Overlay showing effects of ZD7288 and ZD7288 plus DC compensation. Ci, In a different neuron, the control response was recorded within 50 s of obtaining the whole-cell configuration [before dialysis of PIP2 (10 μm) into the cell]. Cii, Overlay showing comparison of control, PIP2 (after 15 min dialysis) and PIP2 plus ZD7288 (30 μm). D, Bar graph showing effects of drugs on the latency of burst onset. E, Plot showing dependence of the latency on membrane potential. *p < 0.01, versus control, Tukey test; n = 14, 6, and 6 for control, ZD7288, and PIP2, respectively. Horizontal error bars are not shown for clarity. Symbol colors correspond to bar colors in D.
- Figure 7.
Upregulation of HCN channel function facilitates rebound burst firing in HCN2−/− IGL neurons. A–C, Rebound bursts were elicited in IGL neurons using a hyperpolarizing current pulse (see Materials and Methods) in the absence (gray) and presence of ZD7288 (ZD, red), wortmannin (wort, cyan), or PIP2 (green); drugs were applied as in Figure 6. The membrane potential was held at −60 mV by DC injection before the current pulse was applied; traces are overlaid for comparison between control and drug. A short horizontal bar indicates the segment shown on an expanded time scale for better viewing of rebound delay. Calibration: 500 ms for main traces and 30 ms for insets. D, Bar graph summarizing effects of drugs on the number of action potentials/burst. E, The sag amplitude is plotted against rebound delay. Symbols: gray circle, control; green triangle, PIP2; cyan square, wortmannin (wort); red circle, ZD7288. SEs for rebound delay are small (±2.2 to ±3 ms) and are omitted for clarity. *p < 0.05, one-way ANOVA versus control (n = 24), n = 8/each for drug.
- Figure 8.
PIP2 is required for spontaneous LTS oscillations in HCN2−/− IGL neurons. A, Rhythmic burst firing of low-threshold spikes (LTSs) was recorded in IGL neurons in the absence (dark gray) and presence of wortmannin (15 μm, cyan). The last burst in each trace is shown on an expanded time scale (right). Membrane potential (in millivolts) is shown to the left of each trace. B, PIP2 (10 μm, green) in the recording pipette. C, Bar graph summarizing effects of drugs on IBI. ZD7288 (30 μm) was applied by superfusion. *p < 0.05, one-way ANOVA, versus control, n = 6/each. D, The number of bursts is plotted against IBI to show the regularity of burst firing; each trace is the average of 6 cells. SEs are omitted for clarity.
