Differential expression of HCN subunits alters voltage-dependent gating of h-channels in CA1 pyramidal neurons from dorsal and ventral hippocampus

Abstract

The rodent hippocampus can be divided into dorsal (DHC) and ventral (VHC) domains on the basis of behavioral, anatomical, and biochemical differences. Recently, we reported that CA1 pyramidal neurons from the VHC were intrinsically more excitable than DHC neurons, but the specific ionic conductances contributing to this difference were not determined. Here we investigated the hyperpolarization-activated current (I(h)) and the expression of HCN1 and HCN2 channel subunits in CA1 pyramidal neurons from the DHC and VHC. Measurement of Ih with cell-attached patches revealed a significant depolarizing shift in the V(1/2) of activation for dendritic h-channels in VHC neurons (but not DHC neurons), and ultrastructural immunolocalization of HCN1 and HCN2 channels revealed a significantly larger HCN1-to-HCN2 ratio for VHC neurons (but not DHC neurons). These observations suggest that a shift in the expression of HCN1 and HCN2 channels drives functional changes in I(h) for VHC neurons (but not DHC neurons) and could thereby significantly alter the capacity for dendritic integration of these neurons.

Fig. 1.

Pharmacological evidence for hyperpolarization-activated current (I_h) in dorsal (DHC) and ventral (VHC) hippocampal neurons. A: morphological reconstruction of a DHC CA1 pyramidal neuron depicting the somatic recording location for the data presented in this figure. B: resting membrane potential (RMP) was monitored over time as 10 μM ZD7288 was washed in for representative DHC and VHC neurons. Arrows indicate the approximate time when 10 μM ZD7288 was added to the perfusion system. C: RMP was significantly more depolarized for VHC neurons than DHC neurons before (Wilcoxon rank sum test, *P < 0.05) but not after (Wilcoxon rank sum test, P > 0.05) 10 μM ZD7288 wash-in. D: the change (Δ) in RMP in response to 10 μM ZD7288 wash-in was significantly larger for VHC neurons than DHC neurons (Wilcoxon rank sum test, *P < 0.05). E: voltage responses to 800-ms somatic step current injections starting from −73 mV for DHC and VHC neurons before (top) and after (bottom) 10 μM ZD7288 wash-in. F: in the presence of 10 μM ZD7288, input resistance (R_in) was significantly larger for VHC neurons than DHC neurons at all tested membrane potentials (Wilcoxon rank sum tests, all *P < 0.05; for voltages other than −73 mV, n = 3). Open triangles represent R_in (at −73 mV) before 10 μM ZD7288. Solid lines represent the voltage dependence of the somatic R_in in the absence of 10 μM ZD7288 (data from Dougherty et al. 2012). G: the change in R_in (at −73 mV) in response to 10 μM ZD7288 wash-in was significantly larger for VHC neurons than DHC neurons (Wilcoxon rank sum test, *P < 0.05). Unless otherwise stated, n = 5 for both DHC and VHC neurons.

Fig. 2.

Indirect electrophysiological evidence for I_h in DHC and VHC neurons. A: morphological reconstruction of a DHC CA1 pyramidal neuron depicting the somatic recording location for the data presented this figure. B: voltage responses for DHC (blue traces) and VHC (black traces) neurons elicited from −73 mV in response to 800-ms step current injections ranging from −200 pA to 0 pA in 40-pA increments. C: the rebound slope (RS) (at −73 mV) was significantly more negative in VHC neurons than DHC neurons (Student's t-test, *P < 0.05), whereas the RS (at RMP) was not significantly different (Wilcoxon rank sum test, P > 0.05). D: voltage responses for DHC (blue traces) and VHC (black traces) neurons elicited from −73 mV in response to the Chirp20 stimulus (see materials and methods). E: impedance amplitude (Z) profiles for traces in D. Vertical dashed lines indicate the resonance frequency (f_R) for the DHC (blue) and VHC (black) neurons, respectively. F: the f_R (at −73 mV) was significantly higher in VHC than DHC (Student's t-test, *P < 0.05) neurons, whereas f_R (at RMP) was not significantly different (Wilcoxon rank sum test, P > 0.05). n Values are listed in Table 1.

Fig. 3.

Voltage-dependent gating of h-channels from DHC and VHC neurons. A: currents evoked from DHC (left) and VHC (right) dendrites by 500-ms hyperpolarizing voltage steps from a holding potential 20 mV more positive than RMP. The dendritic recording location of these patches was ∼220 μm from the soma. Insets: close-up view of tail currents from the area bounded by the black dotted rectangles. Black vertical dashed lines indicate the approximate location for determining the peak tail current. Vertical scale bars represent 2 pA (DHC, left) and 4 pA (VHC, right), and horizontal scale bars represent 10 ms. B: the voltage dependence of activation determined from tail currents was described assuming a Boltzmann function with the following fit parameters: DHC membrane potential for 1/2 maximal conductance (V_1/2) = −113 mV, slope factor (k) = 9.7 mV; VHC V_1/2 = −95 mV, k = 7.9 mV. C–E: the V_1/2 value of the tail conductance-voltage (G-V) relationship (C) was significantly more depolarized for h-channels from VHC dendrites than DHC dendrites (Student's t-test, *P < 0.05), whereas the slope factor (D) and maximal conductance density (E) were not significantly different (slope factor: Student's t-test, P > 0.05; maximal conductance density: Student's t-test, P > 0.05). n Values are listed in Table 2.

Fig. 4.

Voltage-dependent kinetics of h-channels from DHC and VHC neurons. A: the time course of activation was described assuming a double-exponential function for DHC (blue, top) and VHC (black, bottom) neurons. Fitted curves are displayed superimposed on the current traces (red lines), and residuals (top red traces) are plotted separately for the indicated step hyperpolarizations. Example fits are from the traces displayed in Fig. 3A, and all kinetic measurements were made from dendritic recordings 200–300 μm from to soma. B and C: both fast and slow time constants describing activation (τ; B) were qualitatively similar across a range of membrane potentials for I_h measured from DHC and VHC neurons, as were their fractional amplitudes (FracA; C). D: tail currents were elicited by step depolarizations ranging from −20 to 20 mV (relative to RMP and in 10-mV increments) after a 200-ms step hyperpolarization to −70 mV (relative to RMP). Tail currents were described assuming an exponential function (fitted red curve), with the residual plotted separately (red trace, top). E: the time constant of deactivation (τ_deact) for I_h from DHC and VHC plotted across a range of membrane potentials.

Fig. 5.

Somatodendritic gradients of the functional properties of h-channels from DHC and VHC neurons. A–C: voltage dependence of activation for h-channels from somatic (0 μm; A), proximal stratum radiatum (pSR, 100–200 μm; B), and distal stratum radiatum (dSR, 200–300 μm; C) patches from DHC and VHC neurons were described assuming a Boltzmann function with the following fit parameters: soma: DHC V_1/2 = −113 mV, k = 9.5 mV, VHC V_1/2 = −115 mV, k = 8.8 mV; pSR: DHC V_1/2 = −115 mV, k = 8.5 mV, VHC V_1/2 = −104 mV, k = 11.3 mV; dSR: DHC V_1/2 = −116 mV, k = 9.3 mV, VHC V_1/2 = −97 mV, k = 9.0 mV. All conductance values for this figure were determined from the maximal current at each membrane potential. D: V_1/2 was significantly more depolarized for VHC neurons than DHC neurons in pSR (Wilcoxon rank sum test, *P < 0.05) and dSR (Student's t-test, *P < 0.05) but not the soma (Wilcoxon rank sum test, P > 0.05). E: slope factor (k) was significantly larger for VHC neurons than DHC neurons in pSR (Wilcoxon rank sum test, *P < 0.05) but not the soma (Wilcoxon rank sum test, P > 0.05) or dSR (Student's t-test, P > 0.05). F: conductance density was not significantly different between VHC and DHC neurons at the soma (Wilcoxon rank sum test, P > 0.05), pSR (Wilcoxon rank sum test, P > 0.05), or dSR (Wilcoxon rank sum test, P > 0.05). G–I: the V_1/2 value (G), the slope factor (H), and the maximal conductance density (I) were replotted on a normalized somatodendritic axis that accounts for the different lengths of the apical dendrites of DHC and VHC neurons. n Values are listed in Table 2.

Fig. 6.

Expression of HCN1 and HCN2 in dorsal and ventral CA1. A: HCN1, HCN2, and α-tubulin (tub) protein levels in microdissected slices of dorsal and ventral CA1 from 2 representative rats. Error bars represent SE. B–E: dorsal (B and C) and ventral (D and E) hippocampal slices immunostained for HCN1 (B and D) or HCN2 (C and E) with the preembedding silver-intensified ultrasmall immunogold electron microscopy technique. The darkened appearance of tissue in SLM is due to the presence of silver-intensified immunogold particles. s. pyr., CA1 stratum pyramidale; s. rad., CA1 stratum radiatum; s.l.-m., CA1 stratum lacunosum-moleculare. Scale bar, 500 μm. F–I: electron micrographs of hippocampal CA1 from dorsal (F and G) and ventral (H and I) hippocampus immunostained for HCN1 (F and H) or HCN2 (G and I). F–I, top left: electron micrographs of CA1 pyramidal neuron somata (arrows denote membrane-bound particles; nucl., nucleolus). Remaining subpanels are electron micrographs of immunopositive dendrites in SLM from each corresponding region. Scale bar in I, 1 μm and 0.5 μm for somata and dendrite subpanels, respectively.

Fig. 7.

Subunit-specific differences in HCN channel expression in dorsal and ventral CA1. A: 3-dimensional reconstructions from serial sections through dendrites immunopositive for HCN1 (left) or HCN2 (right) from dorsal (top) and ventral (bottom) CA1. Cube = 1 μm³. Green, dendrite; gray, spines; orange, postsynaptic density; black circles, membrane-bound immunogold particles; red circles, cytoplasmic immunogold particles. B: scatterplots of particle density as a function of dendritic diameter (HCN1, left; HCN2, right) for individual dendrites in pSR (gray) and SLM (red) of dorsal and ventral CA1. C, left: particle densities for HCN1 (left)- or HCN2 (right)-immunopositive somata and dendrites in pSR and SLM of dorsal and ventral hippocampal region CA1. Right: ratio of HCN1 to HCN2 particle densities at the soma and in pSR or SLM dendrites in dorsal and ventral CA1. D: histograms of % and cumulative % of dendrites in dorsal (blue lines; open bars) and ventral (black lines; gray bars) CA1 SLM harboring a given number of membrane-bound immunogold particles for HCN1 (left) or HCN2 (right).