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. 2019 May 2;4(9):e126418.
doi: 10.1172/jci.insight.126418.

Abolishing cAMP sensitivity in HCN2 pacemaker channels induces generalized seizures

Verena Hammelmann  1 , Marc Sebastian Stieglitz  1 , Henrik Hülle  1 , Karim Le Meur  1 , Jennifer Kass  1 , Manuela Brümmer  1 , Christian Gruner  1 , René Dominik Rötzer  1 , Stefanie Fenske  1 , Jana Hartmann  2 , Benedikt Zott  2 , Anita Lüthi  3 , Saskia Spahn  1 , Markus Moser  4 , Dirk Isbrandt  5 , Andreas Ludwig  6 , Arthur Konnerth  2 , Christian Wahl-Schott  1   7 , Martin Biel  1
Affiliations

Abolishing cAMP sensitivity in HCN2 pacemaker channels induces generalized seizures

Verena Hammelmann et al. JCI Insight. .

Abstract

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are dually gated channels that are operated by voltage and by neurotransmitters via the cAMP system. cAMP-dependent HCN regulation has been proposed to play a key role in regulating circuit behavior in the thalamus. By analyzing a knockin mouse model (HCN2EA), in which binding of cAMP to HCN2 was abolished by 2 amino acid exchanges (R591E, T592A), we found that cAMP gating of HCN2 is essential for regulating the transition between the burst and tonic modes of firing in thalamic dorsal-lateral geniculate (dLGN) and ventrobasal (VB) nuclei. HCN2EA mice display impaired visual learning, generalized seizures of thalamic origin, and altered NREM sleep properties. VB-specific deletion of HCN2, but not of HCN4, also induced these generalized seizures of the absence type, corroborating a key role of HCN2 in this particular nucleus for controlling consciousness. Together, our data define distinct pathological phenotypes resulting from the loss of cAMP-mediated gating of a neuronal HCN channel.

Keywords: Behavior; Epilepsy; Ion channels; Neuroscience.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Impaired modulation of Ih by cAMP in thalamocortical neurons expressing HCN2EA.
(A) Structural model of the CNBD of HCN channels. The 2 key residues R591 (yellow) and T592 (pink) that are crucial for binding of cAMP are highlighted. (B) Horizontal brain slices of WT and HCN2EA mice. The position of the dLGN (red) and the VB (blue) is indicated. (C) Detection of HCN2 and HCN4 in Western blot analysis of punched dLGN and VB regions. Images are representatives of n = 3/group. (D) Western blot analysis of membrane preparations of HCN2EA and WT mice probed for HCN2 and a loading control (Na+/K+-ATPase). Images are representatives of n = 3/group. (E) Quantification of HCN2 expression level in relation to the Na+/K+-ATPase (n = 3).
Figure 2
Figure 2. HCN2 and HCN4 staining in the thalamus.
(A) Distribution of HCN2 and HCN4 in the VB region of mouse thalamus. Scale bar: 200 μm. (B) Overlay of anti-HCN2 (green), anti-HCN4 (red), and Hoechst (blue). Scale bars: 200 μm. Upper: Single channels for HCN2 (green) and HCN4 (red) staining. Lower: Magnification (scale bars: 25 μm) of the dLGN (left) and VB (middle). (C) Magnified HCN2 stainings of WT (upper), HCN2EA litters (middle), and HCN2-KO mice (lower panel) in the VB region. Scale bars: 20 μm. (D) Analysis of the mean intensity of the HCN2 fluorescence in WT and HCN2EA soma and dendrites (WT: gray squares, soma [n = 20], dendrites [n = 26]; EA: blue squares, soma [n = 17], dendrites [n = 26]). (E) Magnified HCN4 stainings of WT (upper) and HCN2EA litters (middle). Staining in the absence of primary antibody is shown in the bottom panels. Scale bars: 20 μm. (F) Analysis of the mean intensity of the HCN4 fluorescence in WT and HCN2EA soma and dendrites (WT: gray circles, soma [n = 23], dendrites [n = 25]; EA: orange circles, soma [n = 25], dendrites [n = 25]) ***P < 0.001 by 1-way ANOVA with Bonferroni’s post hoc test. NS, not significant.
Figure 3
Figure 3. Impaired modulation of Ih by cAMP in thalamocortical neurons expressing HCN2EA.
(A) Representative Ih traces from thalamocortical WT (upper) and HCN2EA neurons (lower) in the VB. Below, voltage protocol used to evoke current traces. (B) Ih density of thalamocortical neurons in the dLGN (HCN2EA, red squares [n = 13]; WT, gray squares [n = 11]; P = 0.3524 by Mann-Whitney test) and the VB (HCN2EA, blue squares [n = 4]; WT, gray squares [n = 6]; P = 0.6021 by Mann-Whitney test). NS, not significant. (C) Normalized current-voltage relationships of Ih in thalamocortical neurons of WT and HCN2EA mice in the absence (–cAMP) and presence of 1 μM cAMP (Boltzmann fit). Upper left: dLGN, HCN2EA: solid red line, –cAMP (n = 6); dashed red line, +cAMP (n = 4). Upper right: dLGN, WT: solid gray line, –cAMP (n = 6); dashed gray line, +cAMP (n = 5). Lower left: VB, HCN2EA: solid blue line, –cAMP (n = 4); dashed blue line, +cAMP (n = 5). Lower right: VB, WT: solid gray line, –cAMP (n = 6); dashed gray line, +cAMP (n = 8). The cAMP-dependent shift of the midpoint potentials (V0.5) of activation is indicated as dashed black lines.
Figure 4
Figure 4. Lack of cAMP modulation in HCN2 alters firing properties of thalamocortical neurons in the dLGN.
(A) Firing properties of WT and HCN2EA dLGN neurons in the absence (–cAMP, left) or presence of 1 μM cAMP (right). Current injections (300 ms) are indicated above the traces. Insets: Enlarged traces elicited at 150 pA/+cAMP for WT (I) and HCN2EA (II). Traces are representatives of measurements shown in B. (B) Firing frequencies of TC neurons at different injected currents. HCN2EA (solid red line, –cAMP [n = 5]; dashed red line, +cAMP [n = 3]); WT (solid gray line, –cAMP [n = 4]; dashed gray line, +cAMP [n = 3]). Insets: Representative examples of burst and tonic firing at 200 pA. When injecting 400 pA, neurons fired in tonic mode independently of genotype and cAMP concentration. (C) Resting membrane potential (RMP) of TC neurons in the absence and presence of 1 μM cAMP. HCN2EA (red squares, –cAMP [n = 9]; red circles, +cAMP [n = 8]). WT (gray squares, –cAMP [n = 11]; gray circles, +cAMP [n = 7]). NS, P = 0.7052 (EA), ***P = 0.0004 (WT) by unpaired t test. (D) Voltage-sag ratios (peak vs. steady state) at different negative current injections in the absence and presence of 1 μM cAMP. HCN2EA (red squares, –cAMP [n = 9]; red circles, +cAMP [n = 8]); WT (gray squares, –cAMP [n = 11]; gray circles, +cAMP [n = 7]). Inset: Example measurement of the voltage sag and calculation of the sag ratio.
Figure 5
Figure 5. Lack of cAMP modulation in HCN2 impairs visual learning.
(A) Scheme of the geniculate pathway. The dLGN receives input directly from the retina and projects towards the primary visual cortex (V1). (B) Left: Design of the visual discrimination task. CS+, conditioned stimulus; CS–, nonconditioned stimulus. Right: Learning curve of HCN2EA (red) and WT (gray) animals (n = 8). *P < 0.05 by 1-way ANOVA with Bonferroni’s post hoc test. Dashed line indicates chance level. (C) Number of transitions over the virtual cliff for HCN2EA (red, n = 5), WT (gray, n = 5), and Cnga3–/– Rho–/– Opn4–/– triple-KO mice (black, n = 4). **P < 0.01 by 1-way ANOVA with Bonferroni’s post hoc test. NS, not significant. (D) Swimming traces of WT (upper left) and HCN2EA mice (lower left) on day 1 and day 4 in a Morris water maze. Right: Latency of WT (gray, n = 9) and HCN2EA mice (red, n = 9) to find a hidden platform.
Figure 6
Figure 6. Analysis of neuronal network activity in the thalamic VB region.
(A) Firing properties of WT and HCN2EA TC neurons from the VB region. Shown are action potentials elicited by 300-ms depolarizing current pulses at resting membrane potential of WT and HCN2EA TC neurons under control conditions (–cAMP) and in the presence of 1 μM cAMP. Unlike WT neurons, HCN2EA neurons fail to switch from burst to tonic firing in the presence of cAMP within the current range from 50–200 pA. The insets show representative examples of tonic (I) and burst (II) firing activity induced by injecting 150 pA in a WT and HCN2EA neuron, respectively. Traces are representatives of measurements shown in B. (B) Frequency of action potential generation as a function of the injected current. HCN2EA: solid blue line, –cAMP (n = 6); dashed blue line, +cAMP (n = 6). WT: solid gray line, –cAMP (n = 4); dashed gray line, +cAMP (n = 5). (C) Resting membrane potential (RMP) of TC neurons from the VB of HCN2EA (blue squares, –cAMP [n = 11]; blue circles, +cAMP [n = 7]) and WT (gray squares, –cAMP [n = 13]; gray circles, +cAMP [n = 10]).**P < 0.01 (P = 0.0083 [EA], P = 0.0013 [WT]) by Mann-Whitney test. (D) Voltage-sag ratios of TCs in response to injected current. HCN2EA (blue squares, –cAMP [n = 11]; blue circles, +cAMP [n = 7]); WT (gray squares, –cAMP [n = 13]; gray circles, +cAMP [n = 10]). (E) Original traces of spontaneous activity of individual thalamic neurons visualized using 2-photon calcium imaging. The raster plots under the traces (gray) indicate the detected peaks. All active cells from WT mice show irregular transients (left, n = 74), whereas more than one-third of the active cells from HCN2EA mice show oscillatory transients (right, n = 25). (F) Autocorrelograms of the traces shown in E. Gray, original autocorrelation function (ACF); black, smoothed ACF; red, cubic spline interpolation of the smoothed ACF.
Figure 7
Figure 7. Absence epilepsy in HCN2EA mice.
(A) EEG traces of WT (left), HCN2EA (middle), and global HCN2-KO mice (right panel). Spike and wave discharges (SWDs) in the EEG of HCN2EA and global HCN2-KO are marked by asterisks. SWDs concur with episodes of low activity in the EMG. Higher magnifications of SWD traces are displayed below the EEG of HCN2EA and HCN2-KO mice. Representative traces of animals analyzed in B. (B) SWD characteristics of HCN2EA (light blue, n = 5) and global HCN2-KO (dark blue, n = 6) animals compared with WT traces (gray, n = 6) regarding the time between 2 SDWs (left), mean SWD duration (middle), and the time per day in SWDs (right). NS, not significant (1-way ANOVA, Bonferroni’s post hoc test). (C) Time spent in different vigilance states (wake, NREM and REM sleep) during light and dark conditions (HCN2EA, blue; WT, gray; n = 7). (D) Power spectra of NREM sleep in HCN2EA (blue, n = 7) and WT animals (gray, n = 7). **P < 0.01 by 2-way ANOVA. (E) Mean sigma power in HCN2EA (blue, n = 7) and WT animals (gray, n = 7). *P = 0.0175 by Mann-Whitney test.
Figure 8
Figure 8. Absence epilepsy in VB-specific HCN2-KO mice.
(A) Strategy for specific deletion of HCN channels in the VB. Left panel: Scheme of the AAV2/8-hSyn-Cre-EGFP vector that was injected stereotactically into the VB of HCNfl/fl mice. The schematic brain shows the injected region in the thalamus. Only cells in the VB show the green EGFP signal due to viral transfection. (B) Staining with anti-HCN2 antibody (red) in the VB region shows that Cre/EGFP–positive neurons (green) lack HCN2, while nontransduced (EGFP-negative) cells express HCN2 in the plasma membrane (marked by white arrows). Scale bar: 5 μm. VM, ventromedial region. (C) VB-specific HCN2-KO mice show SWDs (marked with an asterisk) in EEG traces (representative trace, n = 3). The inset shows a magnification of the SWD. (D) Representative EEG (green) and EMG (black) traces of an HCN2fl/fl mouse that was injected with a control AAV vector that expresses only EGFP (n = 3). (D) Representative EEG (green) and EMG (black) traces of a VB-specific HCN4-KO animal that was generated by injecting the same vector as displayed in Figure 7A into the VB of an HCN4fl/fl mouse (n = 3).
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