Kv1.1 null mice have enlarged hippocampus and ventral cortex
© Persson et al; licensee BioMed Central Ltd. 2007
Received: 18 July 2006
Accepted: 24 January 2007
Published: 24 January 2007
Mutations in the Shaker-like voltage-gated potassium channel Kv1.1 are known to cause episodic ataxia type 1 and temporal lobe epilepsy. Mice that express a malfunctional, truncated Kv1.1 (BALB/cByJ-Kv1.1mceph/mceph) show a markedly enlarged hippocampus and ventral cortex in adulthood.
To determine if mice lacking Kv1.1 also develop a brain enlargement similar to mceph/mceph, we transferred Kv1.1 null alleles to the BALB/cByJ background. Hippocampus and ventral cortex was then studied using in vivo 3D-magnetic resonance imaging and volume segmentation in adult Kv1.1 null mice, BALB/cByJ-Kv1.1mceph/mceph, BALB/cByJ-Kv1.1mceph/+, BALB.C3HeB -Kv1.1-/+ and wild type littermates. The Kv1.1 null brains had dramatically enlarged hippocampus and ventral cortex. Mice heterozygous for either the null allele or the mceph allele had normal-sized hippocampus and ventral cortex.
Total absence of Kv1.1 can induce excessive overgrowth of hippocampus and ventral cortex in mice with a BALB/cByJ background, while mice with one wild type Kv1.1 allele develop normal-sized brains.
The megencephaly BALB/cByJ-Kv1.1mceph/mcephmice (mceph/mceph),  show dramatic and progressive increase in brain size  after birth. The phenotype results from an 11 base-pair deletion in the gene encoding the Shaker-like voltage-gated potassium channel subunit Kv1.1 . Kv1.1 is expressed in axons and dendrites of neurons and forms tetramers with other Kv1 subunits creating channels that regulate neuronal excitability and nerve signaling. The mceph/mceph mice express a truncated Kv1.1 protein in the endoplasmatic reticulum (ER), MCEPH, consisting of 230 out of 495 amino acids. MCEPH has a dominant negative effect on Kv1.2 and Kv1.3 currents when overexpressing MCEPH in Xenopus oocytes. However, in brain, no ER-retention of Kv1.2 was seen probably due to rapid MCEPH degradation and low MCEPH levels . mceph/mceph mice behave normally at birth but from postnatal week 4 they begin to display progressive motor disturbances and typical behavior of complex partial seizures. The progressive brain overgrowth is significant at 8 weeks of age and affects primarily hippocampus and ventral cortex, whereas thalamus, olfactory bulb and cerebellum have normal size [2, 5]. The enlarged areas have marked disturbances in expression of signaling components of brain derived neurotrophic factor (BDNF), Insulin like Growth factor (IGF) system, and several neuropeptides [3, 5–7]. The hippocampal enlargement appears to result from both more and larger neurons and astrocytes [1, 7]. BALB/cByJ mice heterozygous for the mceph mutation appear to behave normally but their brain has not been studied.
The link between this Kv1.1 truncation and brain overgrowth is not yet fully understood.
To establish if lack of functional Kv1.1 or the presence of the malfunctioning and truncated protein itself is the reason for the brain enlargement in mceph/mceph, we studied and compared hippocampus and ventral brain size using MRI in (1) animals with a complete lack of Kv1.1 (Kv1.1 null homozygotes), (2) animals that were lacking Kv1.1 in one allele but carried a functional Kv1.1 on the other allele (Kv1.1 null heterozygotes), (3) animals that had the truncating deletion in Kv1.1 in both (Kv1.1mceph/mceph, homozygotes) or only one allele (mceph heterozygotes). The wild type littermates of Kv1.1 null and mceph/mceph animals served as control groups. The mceph alleles and Kv1.1 null alleles were on genetic backgrounds of 100% and 94% BALB/cByJ, respectively. While the mceph mutation originated on BALB/cByJ, Kv1.1 null alleles were transferred through breeding from a C3HeB/FeJ background onto the background of theoretically 94% BALB/cByJ and 6% C3HeB/FeJ.
Results and discussion
Behavior and appearance of the Kv1.1 null mice were similar to that of the mceph/mceph mice and markedly different from that of the wild type littermates. The phenotypes include body tremor, a hyperstartle response, jittering, forelimb paddling and cramps, a sitting posture, audio sensitivity, porphyria, as well as inflammation and crusting of eye lid and the outer ear. These pathologic signs were present in all mceph/mceph and all Kv1.1 null mice, but not in heterozygous or wild type mouse. Kv1.1 mice had a milder epileptic behavior than mceph/mceph mice. Nine of 9 mceph/mceph mice had class IV, and 7 of 9 mceph/mceph mice had class V seizures while only 4 out of the 6 Kv1.1 null mice with enlarged brain regions had class IV seizures and 2 of these 4 Kv1.1 null mice had class V seizures. The 3 Kv1.1 null mice with normal-sized brains had no class IV or V seizures.
Hence, lack of Kv1.1 can cause a significant enlargement of ventral cortex and hippocampus, while one Kv1.1 allele was enough for normal brain region size. The variability in brain size was larger among the Kv1.1 null mice than among the mceph/mceph mice (6 of 13 Kv1.1 compared to 1 of 12 mceph/mceph mice had brain sizes within the wild type range). A likely explanation for this is the presence of 6% C3HeB/FeJ genomic background in the Kv1.1 null mice, whereas the mceph/mceph mice have a pure BALB/cByJ background. We know that the mceph trait has reduced penetrance in intercrosses to other strains . The Kv1.1 null mice are epileptic on all genetic backgrounds tested including 129/SvJ, N:NIH-BC, C57BL/6J, C3HeB/FeJ, Swiss Black and hybrids thereof, but has not previously been reported to have excessive brain growth . Investigation of brain morphology in 3 months old Kv1.1 null mice on 129/SvJ × Swiss black hybrid background revealed only the typical effects of seizures in the hippocampus: neuronal loss and mossy fiber sprouting . Hence, we here report for the first time that Kv1.1.null mice can display excessive brain growth. A factor possibly increasing the severity of the brain overgrowth and the seizure behavior in mceph/mceph mice is heteromultimerization with, and hence retention of, other Kv1 subunits in ER by the MCEPH protein expressed in mceph/mceph mice . We can only speculate about how lack of Kv1.1 and overgrowth of hippocampus and ventral cortex could be linked on BALB/cByJ and what factors that modify this link on other genetic backgrounds. One possible explanation would be seizure-induced upregulation of trophic factors affecting cellular growth, proliferation and survival. This is supported by our observation that enlargement and elevated levels of trophic factors co-occurred spatially in mceph/mceph brain . Also, the mceph/mceph mice display, at least in hippocampal dentate gyrus, increased proliferation, neurogenesis and enhanced cell survival, as well as more neurons and astrocytes in several hippocampal regions . It is known that there are marked differences between mouse strains in cell proliferation as well as survival rate of the new cells in the hippocampus under normal conditions . Also, seizures are known to induce cell proliferation and apoptosis [12, 13], with the intensity of seizures being of importance for the balance between generation of neurons and neuronal loss . Importantly, there are significant differences between mouse strains in the sensitivity and resistance of neurons to seizures [15, 16]. Thus, the different steps linking lack of Kv1.1 with brain overgrowth need to be further elucidated. Nevertheless, the results in this study propose the possibility that some patients with epilepsy and enlarged brain (megalencephaly) might suffer from lack of functional Kv1.1 or related protein. Hence, screening for mutations in the genes encoding Kv1 subunits should maybe be considered for these patients.
We were here able to demonstrate that absence of Kv1.1 can lead to an increased growth of the brain. Also, the genetic background is critical and the BALB/c is suitable for this study.
The BALB/cByJ-Kv1.1mceph/mceph(mceph/mceph), BALB/cByJ-Kv1.1+/+ (wild type) and C3HeB/FeJ-Kcna1tm1Tem (C3HeB/FeJ-Kv1.1-/-, Kv1.1 null). (Smart et al., 1998) mouse strains were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). The mice were kept in a barrier animal facility at 12 h light: 12 h darkness, a temperature of 21–22°C and a relative humidity of 40–50%. Rodent breeding diet R70 (Lactamin AB, Stockholm, Sweden) and water were provided ad libitum. All studies were approved by the Northern Stockholm Ethical Committee on Experimental Animal Care and performed in accordance with guidelines from the Swedish National Board for Laboratory Animals.
Generation of a BALB.C3HeB-Kv1.1-/-semicongenic strain
C3HeB/FeJ-Kv1.1-/- mice were outcrossed to BALB/cByJ-Kv1.1+/+. F1 males/females heterozygous for the Kv1.1 null locus were backcrossed to the recipient strain BALB/cByJ-Kv1.1+/+. Heterozygous mice from the N4 generation were mated to give homozygous Kv1.1 null offspring. These Kv1.1 null mice (((C3HeB/FeJ-Kv1.1-/- × BALB/cByJ-Kv1.1+/+) × BALB/cByJ-Kv1.1+/+) N4F2) as well as their Kv1.1 null heterozygous and wild type littermates were studied.
Presence of the mceph mutation was determined through amplification of a fragment covering the 11 base pair deletion. The PCR products were size separated on 3% agarose gels and visually inspected for determination of genotype. Presence of the Kv1.1 null allele was determined using the NEOTD protocol provided by The Jackson Laboratory. This protocol can not distinguish between homozygotes and heterozygotes so this was done by checking for the presence of a Kv1.1 gene fragment.
3-dimensional Magnetic Resonance Imaging (3D-MRI)
Mice studied were 12 weeks old and include 12 BALB/cByJ-Kv1.1mceph/mceph(mceph/mceph), 8 BALB/cByJ-Kv1.1+/+ (wild type), 8 BALB/cByJ-Kv1.1mceph/+ (mceph heterozygots), 13 semicongenic BALB.C3HeB-Kv1.1-/- (Kv1.1 null), 10 semicongenic BALB.C3HeB-Kv1.1+/+ (wild type), and 9 semicongenic BALB.C3HeB-Kv1.1-/+(Kv1.1 null heterozygots). For MRI, mice were anesthetized with 1.5–2.0 % isofluran in air delivered via a mouthpiece allowing spontaneous respiration. The mouse was positioned in supine position on an acrylic cradle and the head was placed in the center of the coil. To reduce motion artifacts, the head of the mouse was fixed to the rig. Body temperature was measured with a rectal probe and maintained at 36 to 37°C using an MRI-compatible air temperature control system.
MRI was performed using a 4.7 T, 40 mm bore horizontal magnet (Bruker Biospec Avance 47/40, Bruker, Karlsruhe, Germany) fitted with a 12 cm inner diameter self-shielded gradient system (max. gradient strength 200 mT/m). A volume coil (Bruker) with 25 mm inner diameter was used for excitation and signal detection. The sequence employed for 3D-imaging was an inversion recovery (IR) spin echo sequence with rapid acquisition with relaxation enhancement (RARE) imaging . The sequence was optimized for maximum contrast of the hippocampus with parameters: repetition time (TR) 2566.8 ms, echo time (TE) 35.6 ms, RARE-factor 8 with RARE-maximum 4, inversion time 450 ms, matrix size 64 × 64 × 128 and 2 averages. Field of view (FOV) for the 3D was 0.90 × 1.20 × 1.80 cm resulting in a resolution of 0.14 × 0.18 × 0.14 mm in dorso-ventral, left-right and rostro-caudal directions, respectively.
3D-MR images were analyzed by using Amira 3.0 software (Mercury Computer Systems, GmbH). The border of the hippocampus was drawn at the border with the fimbria and corpus callosum and checked in three orthogonal planes to ensure accuracy. Measurements of ventral cortex area were performed as previously detailed  in four coronal sections spanning the interval between 1.2 and 2.5 mm posterior to Bregma (every second or third consecutive section, ) using Bruker standard software (Paravision 3.0.2). The rhinal fissure defined the dorsal boundary of the ventral cortex  and the dark fimbria defined the medial boundary of the ventral cortex. The amygdala was included in the region defined as ventral cortex. Measurements were performed twice and the mean value calculated. Ventral cortex volume was expressed as measured area (mm2) × slice thickness and presented for one hemisphere. Intra-rater reliability was assessed by measuring right ventral cortex area ten times in 10 mceph/mceph mice, and by measuring the hippocampus volume twice in five mice. For the ventral cortex, the average correlation coefficient (r) between the repeated measurements was 0.99. The mean ventral cortex area of the 10 mice was then compared between any two repeated measurements. The mean and SEM of the difference between any two repeated measurements was 2.9% and 2.3%, respectively. For the hippocampus volume, mean and SEM of the set of five mice differed between the two repeated measurements with 5.9% and 15.2%, respectively, and r was 0.99 between repeated measurements. Inter-rater reliability was assessed by calculating r between the measurements of two independent investigators as described previously  and included measurements of ventral cortex volume from five mceph/mceph and five wild type mice. The mean and SEM of measured volumes differed between investigators with 3.1% and 13.8%, respectively, and the correlation coefficientbetween the investigators was r = 0.98.
Presence of class IV and V epileptiform activities (according to classification by Racine ) was determined in 9 mceph/mceph and 9 Kv1.1 null mice aged 11–12 weeks by visual scoring of records from video camera (SSC-M183CF, Sony Corporation, Tokyo, Japan), and time lapse security recorder (HS-1024, Mitsubishi Electric, Tokyo, Japan). Class IV is defined as rearing, and class V as rearing followed by loss of postural control and falling. These classes are considered as models of generalized complex partial seizures. Class V is considered a full motor seizure. The mice were videotaped during both the night and the day for 3 h each, starting 3 h before and 6 h after the turn off of light.
Sizes of brain regions were compared using two-tailed non-paired t-tests, after checking for equality in variance in the compared groups using Bartlett's test. The null hypothesis was rejected if P < 0.017 (Bonferroni correction for 4 comparisons). The statistical analyses were performed using STATA version 8.2 (Stata Corporation, College Station, Texas).
magnetic resonance imaging
This study was supported by grants from the Swedish Medical Research Council, the Swedish Society for Medical Research, the Magnus Bergvall Foundation, and the Karolinska Institutet Foundation.
- Donahue LR, Cook SA, Johnson KR, Bronson RT, Davisson MT: Megencephaly: a new mouse mutation on chromosome 6 that causes hypertrophy of the brain. Mamm Genome. 1996, 7: 871-876. 10.1007/s003359900259.PubMedView ArticleGoogle Scholar
- Diez M, Schweinhardt P, Petersson S, Wang FH, Lavebratt C, Schalling M, Hokfelt T, Spenger C: MRI and in situ hybridization reveal early disturbances in brain size and gene expression in the megencephalic (mceph/mceph) mouse. Eur J Neurosci. 2003, 18: 3218-3230. 10.1111/j.1460-9568.2003.02994.x.PubMedView ArticleGoogle Scholar
- Petersson S, Persson AS, Johansen JE, Ingvar M, Nilsson J, Klement G, Århem P, Schalling M, Lavebratt C: Truncation of the Shaker-like voltage-gated potassium channel, Kv11, causes megencephaly. Eur J Neurosci. 2003, 18: 3231-3240. 10.1111/j.1460-9568.2003.03044.x.PubMedView ArticleGoogle Scholar
- Persson AS, Klement G, Almgren M, Sahlholm K, Nilsson J, Petersson S, Århem P, Schalling M, Lavebratt C: A truncated Kv11 protein in the brain of the megencephaly mouse, expression and interaction. BMC Neurosci. 2005, 6: 65-10.1186/1471-2202-6-65.PubMedPubMed CentralView ArticleGoogle Scholar
- Lavebratt C, Trifunovski A, Persson AS, Wang FH, Klason T, Ohman I, Josephsson A, Olson L, Spenger C, Schalling M: Carbamazepine protects against megencephaly and abnormal expression of BDNF and Nogo signaling components in the mceph/mceph mouse. Neurobiol Dis. 2006, 24: 374-383. 10.1016/j.nbd.2006.07.018.PubMedView ArticleGoogle Scholar
- Petersson S, Sandberg Nordqvist A, Schalling M, Lavebratt C: The megencephaly mouse has disturbances in the insulin-like growth factor (IGF) system. Brain Res Mol Brain Res. 1999, 72: 80-88. 10.1016/S0169-328X(99)00211-9.PubMedView ArticleGoogle Scholar
- Petersson S, Lavebratt C, Schalling M, Hokfelt T: Expression of cholecystokinin, enkephalin, galanin and neuropeptide Y is markedly changed in the brain of the megencephaly mouse. Neuroscience. 2000, 100: 297-317. 10.1016/S0306-4522(00)00285-2.PubMedView ArticleGoogle Scholar
- Smart SL, Lopantsev V, Zhang CL, Robbins CA, Wang H, Chiu SY, Schwartzkroin PA, Messing A, Tempel BL: Deletion of the Kv11 potassium channel causes epilepsy in mice. Neuron. 1998, 20: 809-819. 10.1016/S0896-6273(00)81018-1.PubMedView ArticleGoogle Scholar
- Rho JM, Szot P, Tempel BL, Schwartzkroin PA: Developmental seizure susceptibility of kv11 potassium channel knockout mice. Dev Neurosci. 1999, 21: 320-327. 10.1159/000017381.PubMedView ArticleGoogle Scholar
- Almgren M, Persson AS, Witgen BM, Schalling M, Nyengaard J, Lavebratt C: Lack of potassium channel induces neurogenesis and survival causing 2-fold hippocampal enlargement. Hippocampus Accepted.
- Kempermann G, Kuhn HG, Gage FH: Genetic influence on neurogenesis in the dentate gyrus of adult mice. Proc Natl Acad Sci USA. 1997, 94: 10409-10414. 10.1073/pnas.94.19.10409.PubMedPubMed CentralView ArticleGoogle Scholar
- Bengzon J, Kokaia Z, Elmer E, Nanobashvili A, Kokaia M, Lindvall O: Apoptosis and proliferation of dentate gyrus neurons after single and intermittent limbic seizures. Proc Natl Acad Sci USA. 1997, 94: 10432-10437. 10.1073/pnas.94.19.10432.PubMedPubMed CentralView ArticleGoogle Scholar
- Parent JM, Yu TW, Leibowitz RT, Geschwind DH, Sloviter RS, Lowenstein DH: Dentate granule cell neurogenesis is increased by seizures and contributes to aberrant network reorganization in the adult rat hippocampus. J Neurosci. 1997, 17: 3727-3738.PubMedGoogle Scholar
- Parent JM, Janumpalli S, McNamara JO, Lowenstein DH: Increased dentate granule cell neurogenesis following amygdala kindling in the adult rat. Neurosci Lett. 1998, 247: 9-12. 10.1016/S0304-3940(98)00269-9.PubMedView ArticleGoogle Scholar
- McKhann GM, Wenzel HJ, Robbins CA, Sosunov AA, Schwartzkroin PA: Mouse strain differences in kainic acid sensitivity, seizure behavior, mortality, and hippocampal pathology. Neuroscience. 2003, 122: 551-561. 10.1016/S0306-4522(03)00562-1.PubMedView ArticleGoogle Scholar
- Schauwecker PE, Steward O: Genetic determinants of susceptibility to excitotoxic cell death: implications for gene targeting approaches. Proc Natl Acad Sci USA. 1997, 94: 4103-4108. 10.1073/pnas.94.8.4103.PubMedPubMed CentralView ArticleGoogle Scholar
- Hennig J, Nauerth A, Friedburg H: RARE imaging: a fast imaging method for clinical MR. Magn Reson Med. 1986, 3: 823.PubMedView ArticleGoogle Scholar
- Paxinos G, Franklin KBJ: The mouse brain in stereotaxic coordinates. 2001, London: Academic Press, 2Google Scholar
- Racine RJ: Modification of seizure activity by electrical stimulation II Motor seizure. Electroencephalogr Clin Neurophysiol. 1972, 32: 281-294. 10.1016/0013-4694(72)90177-0.PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.