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Conditional transgenic suppression of M channels in mouse brain reveals functions in neuronal excitability, resonance and behavior

Abstract

In humans, mutations in the KCNQ2 or KCNQ3 potassium-channel genes are associated with an inherited epilepsy syndrome. We have studied the contribution of KCNQ/M-channels to the control of neuronal excitability by using transgenic mice that conditionally express dominant-negative KCNQ2 subunits in brain. We show that suppression of the neuronal M current in mice is associated with spontaneous seizures, behavioral hyperactivity and morphological changes in the hippocampus. Restriction of transgene expression to defined developmental periods revealed that M-channel activity is critical to the development of normal hippocampal morphology during the first postnatal weeks. Suppression of the M current after this critical period resulted in mice with signs of increased neuronal excitability and deficits in hippocampus-dependent spatial memory. M-current-deficient hippocampal CA1 pyramidal neurons showed increased excitability, reduced spike-frequency adaptation, attenuated medium afterhyperpolarization and reduced intrinsic subthreshold theta resonance. M channels are thus critical determinants of cellular and neuronal network excitability, postnatal brain development and cognitive performance.

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Figure 1: Generation of IM-deficient transgenic mice.
Figure 2: Spontaneous epileptiform activity in IM-deficient mice.
Figure 3: Home-cage and open-field activity.
Figure 4: Morphological analyses in hippocampus.
Figure 5: Comparison of spike-frequency adaptation in CA1 pyramidal neurons.
Figure 6: Comparison of the medium afterhyperpolarizations (mAHPs) following spike trains in CA1 pyramidal cells.
Figure 7: Resonance behavior of CA1 pyramidal neurons at depolarized membrane potentials (M-resonance).
Figure 8: Morris water-maze test.

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Acknowledgements

We thank S.B. Prusiner, UCSF, for providing the tg(Prnp-tTA) mouse line and H. Voss for animal caretaking and execution of doxycycline application protocols. We also thank F. Morellini for help with behavioral testing and statistical analysis, S. Fehr and I. Hermans-Borgmeyer for help with in situ hybridizations, F. Kutschera for developing the cage activity recorder, and S. Schillemeit and C. Petterson Oksvold for technical assistance. This study was supported by grants of the German Federal Ministry of Education and Research, as a part of the National Genome Research Network (NGFN), to D.I. and O.P. Research by H.H. and J.F.S. was supported by the European Commission (Contract No. QLG3-1999-00827), and by the Norwegian Research Council (NFR) Medicine & Health (MH) and Norwegian Centre of Excellence (SFF) programs.

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Correspondence to Johan F Storm or Dirk Isbrandt.

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Supplementary information

Supplementary Fig. 1

In situ hybridization analyses in hippocampus. In situ hybridization (ISH) experiments using a 35S-labeled hKCNQ2-specific antisense probe in male control (a), mutant (b), mutant on dox (c), WDW mutant (d), and female mutant mice (e). The smaller images illustrate magnifications of the CA1 area marked by a dotted rectangle. In order to visualize cell nuclei in ISH experiments, sections were stained with hemalaun. Scale bar: 500 µm (JPG 65 kb)

Supplementary Fig. 2

Identification of a critical period for phenotype development using the Tet-Off system. Mutant animals were administered doxycycline during different periods of pre- and postnatal development, thus giving rise to dox-water (DW), water-dox-water (WDW), or water-dox (WD) mutants. During the period from conception until weaning, doxycycline was added to the drinking water of the mothers. After weaning doxycycline was added to the drinking water of the offspring. Periods of doxycycline application (transgene expression off) are marked in red, periods of pure water application (transgene expression on) are marked in blue. The morphological hippocampal phenotype of the animals was assessed at 12 to 16 weeks of age. The dotted line marks the critical period during which transgene expression was associated with markedly altered hippocampal morphology and severe behavioral abnormalities. # Three females out of more than 100 animals occasionally showed hyperactivity and circling behavior. * The phenotype of DW mice treated with doxycycline until P0 was variable. This was probably due to residual doxycycline in the brains of the neonates and to a delayed increase in transgene expression. (GIF 10 kb)

Supplementary Fig. 3

Comparison of resonance behavior of CA1 pyramidal neurons after blocking INaP with TTX. Representative voltage responses to ZAP current injection into CA1 pyramidal cells from mutants on dox (a, n = 7 cells from 4 mice) and mutant mice (b, n = 10 cells from 5 mice). The cells were depolarized to –48 mV by current injection. Note that mutant cells showed little resonance behavior. (c) and (d) Effect of XE991 application (10 µM) on the resonance behavior of the same cells as shown in (a) (XE991 was tested in 5 of the 7 mutant on dox cells) and (b) (XE991 was tested on 8 of the 10 mutant cells). Note that XE911 suppressed the resonance in mutant on dox cells (c), but it had little effect on the resonance in mutant cells (d). (e and (f) Plots of impedance magnitude as a function of input frequency before (black) and after (blue) application of XE911 (10 µM) from the same cells shown in (a)-(d). (g) and (h) Summary plot of the resonance frequency and Q factors from mutant and mutant on dox cells before and after application of XE991 in the presence of TTX. (GIF 23 kb)

Supplementary Fig. 4

Comparison of resonance behavior of CA1 pyramidal neurons at hyperpolarized membrane potentials (H-resonance). Resonance at hyperpolarized membrane potentials was tested by injecting an oscillating current with linearly increasing frequency (ZAP current) into the cell, starting from a holding level of 7-9 mV. Typical membrane potential response to a ZAP current injection in cells from mutants on dox (a, n=5 cells from 2 mice) and mutants (b, n=7 cells from 2 mice). (c, d) Impedence plotted as a function of input frequency calculted from the data shown in (a) and (b). Mutant on dox cells (c, e) and mutant cells (d, e) showed a resonance peak between 2 and 3 Hz. (f) As indicated by the Q factors, the strength of the observed resonance was unchanged in mutant on dox and in mutant cells. (GIF 11 kb)

Supplementary Fig. 5

Comparison of I/V plots in CA1 pyramidal neurons from mutant on dox and mutant mice. Current-voltage (I/V) plots from >mutant on dox cells (filled circles, >n = 6 cells from 2 mice) and mutant cells (open circles, n = 8 cells from 3 mice). The cells were kept in a medium with 3.5 mM [K+] at their natural resting membrane potential (–72.31 ± 2.32 mV for mutant on dox cells, and –74.72 ± 1.54 mV for mutant cells), and 400-ms-long current pulses of different intensities were injected into the cells. The membrane potential responses to the current pulses (measured at the end of each pulse) were plotted against the amplitude of the current pulses (which were all subthreshold for spike generation). Note that current pulses larger than 0.15 nA evoked significantly less depolarization in mutant on dox than in mutant cells (* - P < 0.05). (GIF 6 kb)

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Peters, H., Hu, H., Pongs, O. et al. Conditional transgenic suppression of M channels in mouse brain reveals functions in neuronal excitability, resonance and behavior. Nat Neurosci 8, 51–60 (2005). https://doi.org/10.1038/nn1375

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