Supplementary Information

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1 Hyperpolarization-activated cation channels inhibit EPSPs by interactions with M-type K + channels Meena S. George, L.F. Abbott, Steven A. Siegelbaum Supplementary Information Part 1: Supplementary Figures 1-5 Part 2: Supplementary Methods

2 Supplementary Figure 1 a Vpeak (mv) Eh V 1/2 = 90 mv V 1/2 = 80 mv V 1/2 = 70 mv b Synaptic strength g syn (µs) Vpeak (mv) Synaptic strength g syn (µs) Supplementary Figure 1. Relation between V pea k and synaptic strength when I h properties were altered in simple model where I h was the only voltage-gated conductance. (a) Depolarizing shifts in the mid-point voltage of activation (V 1/2 ) of I h resulted in a depolarizing effect on V peak. (b) When the RMP was held fixed at 70 mv by adjusting the passive leak reversal potential, increases in I h had a solely inhibitory effect on V peak.

3 Supplementary Figure 2 a) = 0 V m (mv) b) c) V m (mv) Time (ms) = Time (ms) = V m (mv) Time (ms)

4 Supplementary Figure 2. The effects of Ih on temporal summation during a burst of EPSPs. Membrane voltage response to five synaptic stimuli delivered at 100 Hz in the absence of Ih (black) and in the presence of a fixed level of Ih (blue; = 0.01 S cm-2). (a) Responses in the absence of M-current ( g syn = µs). Note depolarizing effect of Ih. (b) Responses in the presence of M-current for a weak synaptic input ( g syn = of M-current for a µs). Note depolarizing effect of Ih. (c) Responses in the presence strong synaptic input ( g syn = 0.01 µs). Note inhibitory effect of Ih for EPSPs early in burst. In all panels: M-current V1/ 2 = 35 mv; M-current maximal conductance = S cm. In these models, the synaptic time constant (τsyn ) was slowed to 10 ms.

5 Supplementary Figure 3 a) 0 10 = b) Vpeak (mv) Vpeak (mv) = c) 45 = Vpeak (mv) Synaptic strength of each input g syn (µs)

6 Supplementary Figure 3. Effects of Ih on peak voltage during a burst of EPSPs in the absence and presence of M-current. Plots of peak voltage during a burst of five EPSPs elicited at 100 Hz as a function of synaptic strength for differing levels of Ih in the absence (a) or presence of two different levels of M-current maximal conductance (b and c). τsyn = 10 ms. M-current V1/ 2 = 35 mv.

7 Supplementary Figure 4 a RMP (mv) (S cm 2 ) b 60 V EPSP (mv) c Synaptic strength g syn (µs) Vpeak (mv) Synaptic strength g syn (µs)

8 Supplementary Figure 4. Ih exerted a purely inhibitory effect on VEPSP and on Vpe ak when RMP was held constant in model also containing a voltage-gated Hodgkin-Huxley K+ conductance. (a) Increasing Ih (V1/ 2 = 90 mv) in the presence of delayed rectifier K+ conductance depolarized the RMP. (b) Increasing Ih diminished the EPSP amplitude ( VEPSP = V pe ak RMP) for all synaptic strengths. Relation of VEPSP and synaptic strength plotted for four different levels of (S cm-2). V1/2 = 90 mv. (c) When the RMP was held fixed at 70 mv by adjusting the passive leak reversal potential, Ih exerted a purely inhibitory effect on Vpeak.

9 Supplementary Figure 5 50 I h somato-dendritic gradient Proximal excitatory input (250 µm) 20 µm Somatic response gradient (a) (b) (c) 20 µm Soma no 1000 µm 250 µm proximal No in soma or dendrite Uniform level of in soma and dendrite Only somatic No in dendrite Electrodes Local response gradient no 1.5 µm a Vpeak (mv) b Vpeak (mv) c Vpeak (mv) = 0 (S cm 2 ) = (S cm 2 ) = (S cm 2 ) Synaptic strength g syn (µs)

10 Supplementary Figure 5. Effects of Ih on V pe ak in a multicompartment neuronal model without or with M-type K+ channels. Multicompartment neuronal model with a 50-fold increasing linear dendritic gradient of Ih, a passive leak conductance, and a dendritic excitatory synaptic input located 250 µm from the soma. Effects of Ih on Vpeak shown in the presence of the Ih gradient (red) or in the absence of Ih (black). Dashed lines depict Vpe ak at the dendritic site of synaptic input and solid lines depict Vpe ak at the soma. V1/ 2 = 90 mv for Ih and 35 mv for M-conductance. (a) In the absence of Mconductance, Ih was excitatory for all synaptic input strengths for Vpe ak, both in the dendrite and soma. (b) With a uniform level of maximal M-conductance ( S cm-2) in the soma and dendrite, Ih produced inhibitory effects on Vpe ak at the dendrite and soma. (c) With M-conductance only present at the soma ( S cm-2 ), Ih produced inhibitory effects on Vpeak at the soma (solid lines) but only excitatory effects at the dendritic site of synaptic input (dashed lines).

11 Supplementary Methods Tissue Preparation Horizontal brain slices were prepared from P28 P40 mice. Mice were rapidly decapitated following spinal dislocation. Their brains were rapidly removed and placed in cold (2 C 3 C) modified ACSF containing (in mm): NaCl (10), NaH 2 PO 4 (1.25), KCl (2.5), NaHCO 3 (25), glucose (25), CaCl 2 (0.5), MgCl 2 (7), sucrose (190), and Na-pyruvate (2), continuously bubbled with 95%/5% O 2 /CO 2. The hemisected brain was submerged in cold ACSF and cut into 300 µm sections with a Vibratome Slices were transferred to standard ACSF at 35 C for min and then stored at room temperature (21 C 22 C). Experiments were performed hr after slice preparation. Electrophysiology Recordings and Solutions The standard ACSF had the following composition (mm): NaCl (125), NaH 2 PO 4 (1.25), KCl (2.5), NaHCO 3 (25), glucose (25), CaCl 2 (2), and MgCl 2 (1). In all experiments, inhibitory transmission was blocked by the GABA A and GABA B receptor antagonists gabazine (2 µm) and CGP (1 µm), respectively. Whole-cell recordings were obtained from hippocampal CA1 pyramidal cells in submerged slices at 31 C 33 C. Patch pipettes (2.5 5 MΩ) were filled with intracellular solution containing (mm): KMeSO 4 KCH 3 SO 4 (130), KCl (10), HEPES (10), NaCl (4), MgATP (4), Na 2 GTP (0.3), phosphocreatine (10), and EGTA (0.5). Series resistance was less than 40 MΩ and capacitance was fully compensated throughout the experiment. Focal stimulating electrodes (patch pipettes coated with AgCl paint and filled with 1 M NaCl) were used to

12 apply single, unipolar shocks of ms in duration with a constant current stimulator. For graded stimulation, shock amplitude was adjusted to evoke a response in control conditions and then incremented until spike threshold was reached. These same shock amplitudes were reapplied after addition of ZD7288 and in the same order. Stimuli were separated by 15 seconds. All drugs were obtained from Tocris-Cookson and used at the following concentrations (µm): gabazine (2), CGP (1), ZD7288 (10 µm), and XE991 (10 µm). Electrophysiological Data Acquisition and Analysis Recordings were obtained using a two-channel Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA). Data were digitized on a Windows PC using an ITC-18 A/D board (Instrutech Instruments, Port Washington, NY) controlled by custom routines written in Igor Pro (Wavemetrics, Eugene, OR). All current-clamp data were acquired at 20 khz and low-pass filtered at 4 khz using the Multiclamp 700B Bessel filter. Analysis was performed using custom routines written in Igor Pro. Statistical tests were performed using Excel (Microsoft, Redmond, WA) and Igor Pro. Hyperpolarizing current injections were used to measure the sag. The sag ratio was measured as (1 ΔV ss /ΔV min ) x 100%, where ΔV ss is the steady-state hyperpolarization (relative to resting potential) at the end of the hyperpolarizing pulse and ΔV min is the peak hyperpolarization near the beginning of the current step. The amplitude of the current step was adjusted so that a constant value of V min (between 90 and 95 mv) was

13 achieved, to assure uniform activation of I h during the hyperpolarization in different cells and under different conditions. Input resistance was determined by injecting a small hyperpolarizing current step of 50 pa for 500 ms from the resting potential and dividing the change in steady-state voltage (ΔV ss ) by the injected current. Computational Modeling A single compartment model (SCM) was implemented and run in NEURON 50 (version 5.9; available at The compartment for the NEURON models had a diameter of 20 µm and a length of 20 µm. The membrane capacitance was set to 1 µf/cm 2. The temperature was set to 33 C. All models contained passive leak conductance, I h -conductance, and a synaptic input. The passive leak had a conductance of 0.3 ms cm -2 and a reversal potential of 70 mv (results were qualitatively similar for a leak reversal potential of 80 mv). The I h -conductance reversal potential was fixed to 30 mv but the maximal conductance was varied as was the V 1/2. The I h - conductance model was taken from previously published models based on experimental data 10, 17. The synaptic input was modeled as an alpha function using the AlphaSyapse feature in NEURON (τ = 1 ms and reversal potential of 0 mv; similar results were obtained with τ = 3 ms) to mimic excitatory synaptic input. Five models were studied containing different K + conductances or altered K + conductance properties. Model 1 contained only a passive leak and I h. Model 2 also included the Hodgkin-Huxley delayed-rectifier K + conductance 35, 37 (K dr ). Model 3 had

14 the same components as model 2 but with the K dr conductance fixed to its steady-state value at the RMP for the duration of each simulation; this created an infinitely slow K dr (τ Kdr = ). Model 4 also had the same components as model 2 but the K dr conductance reached its steady-state voltage-dependent value without any delay throughout the simulation (τ Kdr = 0); this effectively creates an instantaneous voltage-dependent leak conductance. Model 5 contained a passive leak, I h, and M-type K + conductance. The maximal M-conductance and V 1/2 were varied. The M-conductance model was derived from previously published models 42, 43. The temperature-dependence of the M- conductance model was removed to improve consistency with recent studies 40. The original model used a temperature of 23 C, but the time constant (τ M ) at this temperature was already faster than the kinetics at a temperature near 33 C (the temperature used in our models) indicated by those studies 40. A dimensionless (point process) singlecompartment model using the same conductance parameters was created in C++ and confirmed the results from the NEURON models. Models 1 and 5 were used to study temporal summation. In these models, 5 synaptic conductances (τ = 1 ms and reversal potential of 0 mv) were activated at a frequency of 100 Hz. Each synapse had the same synaptic conductance, which was varied. In addition, the synaptic time constants were also slowed (τ = 10 ms) to assess increased temporal summation, defined as the ratio of the amplitudes of the fifth EPSP to the first EPSP (ΔV EPSP5 / ΔV EPSP1 ). Various synaptic frequencies were also assessed.

15 Multicompartment models were also implemented and run in NEURON 50 (version 5.9). The soma had the same dimensions as in the SCM. A dendritic cable was added to the soma of length 1000 µm and diameter 1.5 µm. The dendrite was divided into 10 segments. An axial resistance of 150 Ω cm and dendritic membrane capacitance of 1 µf/cm 2 were used. The parameters for the passive leak were the same as used in the SCM. The maximal I h -conductance at the soma was set to S cm -2 and a 50-fold increasing linear dendritic gradient of maximal I h -conductance extended to the distal end of the dendrite. The V 1/2 of I h was set to 90 mv. An excitatory synaptic input modeled as an alpha function with the same parameters as in the SCM was placed 250 µm away from the soma, mimicking a proximal input. At that dendritic distance, the maximal I h - conductance was 12.5 that of the soma. Any M-conductance placed in the dendrite was maintained at a uniform level of maximal conductance throughout the dendrite. Three models were created in total: (1) with no M-conductance in any compartment, (2) with only somatic M-conductance, and (3) with a uniform level of somatic and dendritic M- conductance. The M-conductance V 1/2 was set to 35 mv. The maximal M-conductance level was varied and results were qualitatively similar for different levels.