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. 2001 Sep 1;21(17):6657-65.
doi: 10.1523/JNEUROSCI.21-17-06657.2001.

Alcohol hypersensitivity, increased locomotion, and spontaneous myoclonus in mice lacking the potassium channels Kv3.1 and Kv3.3

F Espinosa  1 , A McMahon, E Chan, S Wang, C S Ho, N Heintz, R H Joho
Affiliations

Alcohol hypersensitivity, increased locomotion, and spontaneous myoclonus in mice lacking the potassium channels Kv3.1 and Kv3.3

F Espinosa et al. J Neurosci. .

Abstract

The Shaw-like potassium (K(+)) channels Kv3.1 and Kv3.3 are widely coexpressed in distinct neuronal populations in the CNS, possibly explaining the relatively "mild" phenotypes of the Kv3.1 and the Kv3.3 single mutant. Kv3.1-deficient mice show increased cortical gamma- and decreased delta-oscillations (Joho et al., 1997, 1999); otherwise, the Kv3.1-mutant phenotype is relatively subtle (Ho et al., 1997; Sánchez et al., 2000). Kv3.3-deficient mice display no overt phenotype (Chan, 1997). To investigate whether Kv3.1 and Kv3.3 K(+) channels are functionally redundant, we generated the Kv3.1/Kv3.3 double mutant. Kv3.1/Kv3.3-deficient mice were born at the expected Mendelian frequencies indicating that neither Kv3.1 nor Kv3.3 K(+) channels are essential for embryonic development. Although there are no obvious changes in gross brain anatomy, adult Kv3.1/Kv3.3-deficient mice display severe ataxia, tremulous movements, myoclonus, and hypersensitivity to ethanol. Mice appear unbalanced when moving, whereas at rest they exhibit whole-body jerks every few seconds. In spite of the severe motor impairment, Kv3.1/Kv3.3-deficient mice are hyperactive, show increased exploratory activity, and display no obvious learning or memory deficit. Myoclonus, tremor, and ethanol hypersensitivity are only seen in the double-homozygous Kv3.1/Kv3.3-deficient mice, whereas increased locomotor and exploratory activity are also present in double-heterozygous mice. The graded penetrance of mutant traits appears to depend on the number of null alleles, suggesting that some of the distinct phenotypic traits visible in the absence of Kv3.1 and Kv3.3 K(+) channels are unrelated and may be caused by localized dysfunction in different brain regions.

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Figures

Fig. 1.
Fig. 1.
Generation of the Kv3.1/Kv3.3 double mutant by recombination. A, A double-heterozygous male with the null alleles for Kv3.1 and Kv3.3 on two different chromosomes 7 was crossed to a Kv3.1−/− female. Recombination in the male germ line (after ∼100 offspring) linked the two null alleles on the same chromosome. B, By the use of double-heterozygous (+/−,+/−) breeding pairs, the two null alleles segregate together and yield offspring at the expected Mendelian ratios of 1:2:1 (indicated below genotypes), indicating that Kv3.1 and Kv3.3 K+ channels are not required for embryonic development. C, In comparison with wild-type mice (WT), immunoblot analyses of cerebellar protein extracts show reduced levels of Kv3.1 and Kv3.3 in double-heterozygous mice (HET) and no detectable Kv3.1 or Kv3.3 protein in double-homozygous mice (DKO).
Fig. 2.
Fig. 2.
A, Kv3.1 and Kv3.3 K+ channels are required for postnatal development. In this litter of seven pups, two double mutants began to loose weight at ∼P15 and died by P20 and P21. Wild-type and heterozygous littermates gained weight normally and grew to adulthood.Inset, The drop in body temperature of the double mutants is shown. The Kv3.1 and Kv3.3 genotypes and the gender [female (f), male (m)] are shown on the right. B, Adult Kv3.1/Kv3.3-deficient mice are smaller than are wild-type and double-heterozygous mice. At 1.5 months (45 d) and 3.5 months (100 d) of age, Kv3.1−/−Kv3.3−/−(DKO), Kv3.1+/−Kv3.3+/−(HET), and Kv3.1+/+Kv3.3+/+(WT) male mice differ from each other in body weight [mean ± SEM and number of mice (above eachvertical bar) shown; one-factor ANOVA; *p < 0.05; **p < 0.01; ***p < 0.001].
Fig. 3.
Fig. 3.
No obvious alterations in gross brain anatomy in the Kv3.1/Kv3.3 double mutant. A, Hematoxylin- and eosin-stained parasagittal sections (4 μm thick) of a wild type and a Kv3.1/Kv3.3 double mutant show no changes in gross brain anatomy. Cerebellar foliation is unchanged in the mutant brains.B, When examined at higher magnification, both brains display the same characteristic layering of the molecular (m), Purkinje (p), and granule (g) cell layers (top). Hippocampal cytoarchitecture also displays no obvious alterations (bottom).
Fig. 4.
Fig. 4.
Kv3.1/Kv3.3-deficient mice have a severe motor-skill deficit. In contrast to wild-type or heterozygous mice, Kv3.1−/−Kv3.3−/− mice cannot stay on a rotating rod (one-factor ANOVA; ***p < 0.001). DKO mice show no difference between the rotating and the stationary rod (paired t test,p = 0.36). Male mice were placed on an accelerating, rotating rod (diameter, 3.8 cm), and the time until fall was measured [mean ± SEM and number of mice (above eachvertical bar) shown]. At time 0, the rod turned at 5 rpm and accelerated at 10 rpm/min. Each animal was subjected to five trials during an ∼1 hr test period.
Fig. 5.
Fig. 5.
Kv3.1/Kv3.3-deficient mice are hypersensitive to ethanol. Ethanol (in 0.9% saline) was injected intraperitoneally in male mice, and sideways falls were counted for the first 10 min after ethanol injection [mean ± SEM and number of mice (above eachvertical bar) shown]. The ethanol effect was fully visible within 2 min of injection and lasted for ∼20 and ∼45 min for 1 and 2 mg/gm, respectively.
Fig. 6.
Fig. 6.
Kv3.1/Kv3.3-deficient mice show increased spontaneous locomotion and center-field occupancy. Top left, Spontaneous locomotor activity of male mice was monitored for 60 min and plotted in 15 min intervals [mean ± SEM and number of mice (in parentheses) shown]. Heterozygous and homozygous Kv3.1 and Kv3.3 mutants were significantly more active than were wild-type mice. For mice of all three genotypes, locomotor activity decreased during the 60 min interval (two-factor ANOVA). Top right, The increased distance traveled (shown on the top left) is caused by increased ambulatory activity (one-factor ANOVA; *p < 0.05; ***p < 0.001). Bottom, The open field was divided into 64 squares (8 × 8), and occupancy in each square was determined. Center occupancy (4 × 4 squares) was significantly increased for +/−,+/− and −/−,−/− mice (one-factor ANOVA). The test was conducted in an open field (44 × 44 cm) bounded by Plexiglas walls where the movement of the mouse was monitored along thex- and y-axes by infrared beams ∼2.5 cm apart (Opto-Varimex and Auto-Track-System software; Columbus Instruments). Low and high occupancy (seconds/60 min) is indicated bydark and light gray shades, respectively.
Fig. 7.
Fig. 7.
Kv3.1/Kv3.3-deficient mice display normal active avoidance learning. There are no differences between WT,HET, and DKO male mice in learning an avoidance task (days 1–5) and in recalling it 2 weeks later (day 19) (mean ± SEM and number of mice shown).
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