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, 32 (42), 14835-48

TRIP8b-independent Trafficking and Plasticity of Adult Cortical Presynaptic HCN1 Channels

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TRIP8b-independent Trafficking and Plasticity of Adult Cortical Presynaptic HCN1 Channels

Zhuo Huang et al. J Neurosci.

Abstract

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are subthreshold activated voltage-gated ion channels. In the cortex, these channels are predominantly expressed in dendrites where they significantly modify dendritic intrinsic excitability as well synaptic potential shapes and integration. HCN channel trafficking to dendrites is regulated by the protein, TRIP8b. Additionally, altered TRIP8b expression may be one mechanism underlying seizure-induced dendritic HCN channel plasticity. HCN channels, though, are also located in certain mature cortical synaptic terminals, where they play a vital role in modulating synaptic transmission. In this study, using electrophysiological recordings as well as electron microscopy we show that presynaptic, but not dendritic, cortical HCN channel expression and function is comparable in adult TRIP8b-null mice and wild-type littermates. We further investigated whether presynaptic HCN channels undergo seizure-dependent plasticity. We found that, like dendritic channels, wild-type presynaptic HCN channel function was persistently decreased following induction of kainic acid-induced seizures. Since TRIP8b does not affect presynaptic HCN subunit trafficking, seizure-dependent plasticity of these cortical HCN channels is not conditional upon TRIP8b. Our results, thus, suggest that the molecular mechanisms underlying HCN subunit targeting, expression and plasticity in adult neurons is compartment selective, providing a means by which pre- and postsynaptic processes that are critically dependent upon HCN channel function may be distinctly influenced.

Figures

Figure 1.
Figure 1.
Enhancement of spontaneous excitatory synaptic transmission by presynaptic HCN1 channel inhibition is unaffected by TRIP8b deletion. A(i), Example morphologies of wild-type (Wt) and TRIP8b−/− EC layer III neurons. The scale indicates 50 μm and is applicable to both images. A(ii), Sholl analysis of wild-type and TRIP8b−/− EC layer III pyramid neurons (see Materials and Methods). The proximal dendritic tree represents apical, oblique, and basal dendrites counted within a particular radius. B(i), C(i), Typical mEPSC recordings obtained from wild-type and TRIP8b−/− EC layer III neurons before and after 20 min bath application of the HCN channel blocker, ZD7288 (ZD; 15 μm). The outward holding values at −70 mV are shown above the traces. The cumulative probability curves for each of the examples are shown on the right. The average, normalized mEPSCs from the traces under control conditions (black) and after ZD7288 (red) are shown in the insets. The scale shown in B(i) and C(i) applies to all traces within the respective panels. B(ii), C(ii), Graphs depicting the individual (open squares) and the mean (filled squares) mEPSC frequency obtained from wild-type and TRIP8b−/− neurons in the absence and presence of ZD7288. The numbers of observations are indicated above the graphs. Significance at *p < 0.05. B(iii), C(iii), Amplitude histograms of mEPSCs recorded from wild-type and TRIP8b−/− neurons under control conditions and following 20 min external application of ZD7288.
Figure 2.
Figure 2.
Comparable modulation of PPR by HCN channel inhibitors in wild types and TRIP8b-null neurons. A(i), B(i), Example pairs of EPSCs evoked at a frequency of 20 Hz in wild-type (Wt) and TRIP8b−/− EC layer III pyramids by external stimulation of distal dendrites before (control) and after 20 min external application of ZD7288 (ZD, 15 μm). The outward holding currents at −70 mV are stated above the traces. Between each paired pulse, a single stimulus was used to elicit an EPSC. By subtracting this EPSC from the paired EPSCs, the amplitude and shapes of the individual EPSCs were obtained. The insets show the overlaid first EPSC (black) and second EPSC (gray) subtracted traces in the absence and presence of external ZD7288. The time course in the change in PPR caused by 20 min bath application of ZD7288 in 6 wild-type and 4 TRIP8b−/− neurons is shown below. The scale bars shown for the control pair of EPSCs in A(i) and B(i) apply to those obtained after application of ZD7288. The vertical and horizontal scale bars for the insets in A(i) are 50 pA and 100 ms, respectively, while those for B(i) represent 40 pA and 100 ms. A(ii), B(ii), Graphs showing the individual (open squares) and mean (filled squares) PPR values obtained from 8 wild-type and 9 TRIP8b−/− neurons under control conditions and following 20 min application of 15 μm ZD7288. C–E, Bar graphs depicting the amplitudes, decay time constants (τ), and rise time constants (τ) of single evoked EPSCs in wild types and TRIP8b−/− neurons in the absence and presence of external ZD7288. The numbers of observations for each group are indicated above the bar. Significance at *p < 0.05.
Figure 3.
Figure 3.
Altered postsynaptic properties of TRIP8b−/− EC layer III neurons A(i), A(ii), Representative recordings made from wild-type (Wt) and TRIP8b−/− soma and dendrites, respectively, at the normal RMP when a series of 400 ms hyperpolarizing and depolarizing current steps were applied. The RMP values are indicated on the traces. The average numbers of spikes produced in response to a given depolarizing current injection at the normal RMP are shown on the right. The scale bar shown applies to both traces. B(i), Example wild-type and TRIP8b−/− somatic and dendritic traces obtained when a 400 ms, 100 pA hyperpolarizing pulse was applied from a potential of −70 mV. Such traces were used to calculate the RN. B(ii), Bar graph showing the average somatic and dendritic RN values in wild-type and TRIP8b−/− neurons. The numbers of observations are indicated above the bars. Significance at *p < 0.05.
Figure 4.
Figure 4.
Enhanced dendritic excitability of TRIP8b−/− neurons is due to reduced HCN channel function. A(i), A(iii), Representative recordings from wild-type (Wt) and TRIP8b−/− EC layer III pyramid soma in response to a series of 400 ms hyperpolarizing and depolarizing steps from −150 pA to +200 pA. The RMP values are stated adjacent to the recordings. Traces were obtained under control conditions and following 20 min bath application of ZD7288 (ZD; 15 μm). ZD7288 caused hyperpolarization of the RMP in wild types and thus recordings were made at the hyperpolarized RMP (HRMP) as well as at the original RMP (ORMP). The scale bars for the control traces in A(i) and A(iii) apply to the other traces shown in those panels. A(ii), A(iv), Graphs depicting the mean and standard error of action potential numbers (AP No.) recorded with a given 400 ms depolarizing current injection in the absence and presence of ZD7288 in wild-type and TRIP8b−/− soma. As ZD7288 hyperpolarized the RMP in wild types, average numbers of spikes produced by a given current injection at the HRMP and ORMP are shown. B, C, Bar graphs to show the RMP and RN values of wild-type and TRIP8b−/− soma before and after 20 min application of ZD7288. The numbers of observations for each group are indicated above the bars. D(i), D(iii), Example traces obtained from wild-type and TRIP8b−/− EC layer III dendrites at a distance of 150–200 μm from the soma in the absence and presence of ZD7288. To obtain the recordings 400 ms square pulses from −300 pA to +200 pA were applied. As application of ZD7288 resulted in hyperpolarization of the wild-type RMP, traces were obtained at the HRMP and ORMP. The RMP values are stated beside the traces. The scale bars shown for the controls apply to all traces within the panel. D(ii), D(iv), Average spike numbers recorded in wild-type and TRIP8b−/− dendrites, respectively, in response to a given 400 ms depolarizing step from the ORMP under control conditions and following application of ZD7288. E, F, The mean RMP and RN values obtained in wild-type and TRIP8b−/− dendrites under control conditions and following the application of ZD7288. The number of observations for each group are indicated above the bar. Significance at *p < 0.05.
Figure 5.
Figure 5.
Enhanced postsynaptic EPSP integration due to reduced HCN channel function in TRIP8b null dendrites. A(i), Example single dendritic αEPSPs obtained at −70 mV in wild-type (Wt) and TRIP8b−/− dendrites at a distance of 100–150 μm from the soma under control (con) conditions and following the application of 15 μm ZD7288 (ZD). A(ii), Graphs showing the amplitude and decay time constants (τ) of single αEPSPs before and after ZD7288 in wild types and TRIP8b−/− dendrites. The numbers of observations for each group are indicated above the bars. B(i), Representative recordings at −70 mV of a train of αEPSPs at a frequency of 50 Hz in wild-type and TRIP8b−/− dendrites in the absence (con) and presence of ZD7288 (ZD). B(ii), The summation ratios obtained from trains of αEPSPs at frequencies of either 50 Hz or 20 Hz under control conditions and after ZD7288 in 6 wild-type and 6 TRIP8b−/− dendrites. Each symbol represents the mean and the SEM. Summation ratios were calculated by dividing the amplitude of fifth αEPSP by that of the first. The wild-type and TRIP8b−/− summation ratios were compared for significance. The ratios obtained in the absence and presence of ZD7288 were also evaluated for significance. In A and B, significance at *p < 0.05.
Figure 6.
Figure 6.
Altered dendritic but not presynaptic HCN1 subunit distribution in TRIP8b EC tissue. A, B, Immunoreactivity for HCN1 in EC superficial layers in wild-type (Wt) dendrites and synaptic terminals, respectively, as revealed using a preembedding immunogold method. Immunoparticles for HCN1 were located at postsynaptic sites along the extrasynaptic plasma membrane (arrows) of dendritic spines (s) and shafts (Den) establishing synapses with axon terminals (b), as well as associated with intracellular membranes (crossed arrows). A significant proportion of immunoparticles for HCN1 were also detected at presynaptic sites (arrowheads) in axon terminals (b). C, D, HCN1 immunoreactivity in TRIP8b−/− EC superficial dendrites and synaptic terminals, respectively. In contrast to wild-type tissue, more particles were located intracellularly (crossed arrows) than along the extrasynaptic plasma membrane (arrows) of dendritic spines (s) and shafts (Den). The scale bar in A represents 0.2 μm and applies to all examples shown in A–D. E, The number of immunogold particles counted from dendrites and synaptic terminals present in ultra-thin sections obtained from 3 wild-type and 3 TRIP8b−/− mice. Although fewer particles were present in TRIP8b−/− dendrites compared with wild types, this was not significant (ns). F, G, Quantification of immunogold particles present on the plasma membrane and in the cytosol of dendrites and axons found in ultra-thin sections produced from 3 wild-type and 3 TRIP8b−/− mice. Significance at *p < 0.05.
Figure 7.
Figure 7.
Reduced HCN protein expression following epileptogenesis. A, HCN1 subunit immunohistochemical analysis using a preembedding immunoperoxidase method in the entorhinal cortex (EC) of animals treated with either SP (30 mg/kg, s.c.) or kainic acid (20 mg/kg, i.p.) and SP (SE animals) 24 h prior. Staining patterns are representative of data obtained from 3 different SP and 3 SE mice. Immunoreactivity for HCN1 is strong in EC superficial layers including layers I, II and II from SP mice while it is significantly reduced in the same layers of the SE mice. Scale bar, 500 μm. B(i), C(i), Example electrophysiological recordings before and after 20 min application of ZD7288 (15 μm) from EC layer III dendrites present in acute slices obtained from 24 h SP and 24 h SE mice, respectively. The traces were obtained by applying 400 ms hyperpolarizing and depolarizing current steps from −300 pA to + 200 pA in increments of 50 pA. The RMP of the neurons is indicated next to each recording. Bath application of ZD7288 resulted in hyperpolarization of the RMP in SP neurons and hence the traces obtained at the hyperpolarized RMP (HRMP) as well as the original RMP (ORMP) are shown. The scale bar shown with the first trace applies to all traces within this panel. B(ii), C(ii), Graphs depicting average action potential numbers (AP No.) recorded from 24 h SP, 24 SE, 1 wk SP, and 1 wk SE neurons in response to depolarizing current injection (I) steps in the absence and presence of ZD7288. D, E, Bar graphs demonstrating the RMP and RN values of SP and SE neurons with and without ZD7288. The number of observations for each treatment group are shown above each bar. RN values were obtained by applying a 400 ms hyperpolarizing pulse from a fixed potential of −70 mV in all neurons. Significance at *p < 0.05.
Figure 8.
Figure 8.
A decrease in presynaptic HCN channel function contributes to enhanced spontaneous excitatory synaptic transmission following epileptogenesis. A(i), (iv), B(i), (iv), Example mEPSC recordings made from EC layer III neurons obtained from mice induced with SE or control (SP) either 24 h or 1 week prior. The recordings were obtained under control conditions and following 20 min treatment with 15 μm ZD7288 (ZD). The values above the traces represent the outward holding currents at −70 mV. The cumulative probability curves for each of the recordings are presented on the right. The average, normalized mEPSC before (black) and after (red) ZD7288 application are shown in the inset. Each horizontal and vertical scale bar for the traces in A(i), A(iv), B(i), and B(iv) denote 100 ms and 10 pA, respectively, and applies to both recordings within each of the panels. A(ii), (v), B(ii), (v), Graphs depicting the individual (open squares) and mean (filled squares) mEPSC frequency in the absence and presence of ZD7288 in 24 h SP, 24 h SE, 1 wk SP, and 1 wk SE neurons, respectively. Numbers of observations for each group are indicated above the graph. A(iii), (vi), B(iii), B(vi), Amplitude histograms of mEPSCs before and after application of ZD7288 in 24 h SP, 24 h SE, 1 wk SP and 1 wk SP neurons, respectively. A and B, significance at *p < 0.05.
Figure 9.
Figure 9.
Reduced presynaptic HCN channel function following induction of epileptogenesis results in increased evoked synaptic transmission. A(i), (ii), Typical pair of evoked EPSCs at 20 Hz recorded from EC layer III pyramids following stimulation of the distal dendrites after either 24 h SP treatment only or induction of SE under control conditions or after 20 min application of ZD7288 (15 μm; ZD). The values above the traces are the outward holding currents at −70 mV. Single EPSCs were also elicited between pairs and these were subtracted from the pairs to obtain the amplitude and kinetics of the individual EPSCs within the pair. The inset illustrates the overlay of the first (black) and second (gray) EPSC in the absence and presence of external ZD7288. The graphs on the right of the traces depict the individual (open squares) and mean (filled squares) PPRs before and after application of ZD7288 in 24 h SP and SE neurons. The numbers of observations are indicated above the symbols. The scale bars shown for the control pair and individual (insets) in A(i) and A(ii) apply to those obtained after application of ZD7288. A(iii), Time course of the effects of ZD7288 on PPR in 3 SP and 3 SE neurons. B(i), (ii), Example pairs and single (inset) evoked EPSCs in EC layer III pyramids obtained from 1 wk SP and SE mice. The recordings before and after 20 min application of ZD7288 are shown. The individual (open squares) and mean (filled square) PPR with and without ZD7288 are graphed on the right. The numbers of observations for each treatment group are indicated on the graph. The scale bars displayed for the pairs and single EPSCs under control conditions apply to the traces illustrating the effects of ZD7288. Significance at *p < 0.05.

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