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. 2012 Mar 12;590(Pt 11):2645–2658. doi: 10.1113/jphysiol.2012.228486

Kv1.1-dependent control of hippocampal neuron number as revealed by mosaic analysis with double markers

Shi-Bing Yang 1, Kellan D Mclemore 1,3, Bosiljka Tasic 2,4, Liqun Luo 2, Yuh Nung Jan 1, Lily Yeh Jan 1
PMCID: PMC3424722  PMID: 22411008

Abstract

Megencephaly, or mceph, is a spontaneous frame-shift mutation of the mouse Kv1.1 gene. This mceph mutation results in a truncated Kv1.1 channel α-subunit without the channel pore domain or the voltage sensor. Interestingly, mceph/mceph mouse brains are enlarged and – unlike wild-type mouse brains – they keep growing throughout adulthood, especially in the hippocampus and ventral cortex. We used mosaic analysis with double markers (MADM) to identify the underlying mechanism. In mceph-MADM6 mice with only a small fraction of neurons homozygous for the mceph mutation, those homozygous mceph/mceph neurons in the hippocampus are more abundant than wild-type neurons produced by sister neural progenitors. In contrast, neither mceph/mceph astrocytes, nor neurons in the adjacent dorsal cortex (including the entorhinal and parietal cortex) exhibited overgrowth in the adult brain. The sizes of mceph/mceph hippocampal neurons were comparable to mceph/+ or wild-type neurons. Our mosaic analysis reveals that loss of Kv1.1 function causes an overproduction of hippocampal neurons, leading to an enlarged brain phenotype.

Key points

  • The classical function of potassium channels in electrical signaling is to regulate nerve conduction, muscle contraction and hormone secretion.

  • Certain types of potassium channels are also involved in regulating cell proliferation, as in the case of Kv1.1 mutant mice, which exhibit overgrowth of neurons and astrocytes thus leading to the phenotype of megencephaly, or enlarged brain, particularly in the hippocampus.

  • We used a novel mouse genetic tool, Mosaic Analysis with Double Markers (MADM), to test whether Kv1.1 function is required cell-autonomously for megencephaly. We found that in the adult hippocampus, neurons but not astrocytes lacking Kv1.1 are more numerous than their counterparts with two functional alleles of Kv1.1.

  • Our study reveals that loss of Kv1.1 function causes an overproduction of hippocampal neurons in a cell-autonomous manner.

  • This study raises the prospect that targeting Kv1.1 potassium channel may help to induce neuron production.

Introduction

More than a century ago, the German physiologist Julius Bernstein proposed that potassium channels are the fundamental ionic conductances which determine the membrane potential in neurons (Bernstein, 1902). In their seminal paper six decades ago, Hodgkin and Huxley proposed that potassium channels set the resting potential and drive the repolarization and hyperpolarization phase of an action potential (Hodgkin & Huxley, 1952b). With the advance of molecular cloning, genetics and biophysical instrumental breakthrough, the biological functions of potassium channels uncovered include not only the orchestration of electrical activity in excitable tissues such as neurons and muscles but also the control of vital physiological functions including hormone secretion in pancreatic cells and electrolyte transport in kidney tubules (Shieh et al. 2000). Since the cloning of the Drosophila Shaker potassium channel gene (Papazian et al. 1987), more than 200 genes encoding various potassium channels have been characterized. The molecular architecture of a typical potassium channel is composed of four α-subunits, the main pore-forming protein, and several accessory β-subunits that often modify channel gating and trafficking properties of the α-subunits (Stühmer et al. 1989). Potassium channels have a pore structure well suited for efficient potassium flow close to the free diffusion limit (∼108 ions s−1) as well as high selectivity to potassium over other cations such as sodium and calcium (Zhou & MacKinnon, 2003).

Voltage-gated potassium channels (Kv) are diverse (Jan & Jan, 1997). Hodgkin and Huxley formulated a simple yet elegant operating model for Kv channels to describe gating properties such as activation, inactivation and deactivation (Hodgkin & Huxley, 1952a). There are 12 voltage-gated potassium channel families (Kv1 to Kv12) with a range of channel properties (Gutman et al. 2005). The first four transmembrane segments of the α-subunits form the voltage sensing domain and control the gate of the pore formed by the pore loop and the flanking fifth and sixth transmembrane segments (Long et al. 2007). Kv channels are either homotetrameric or heterotetrameric; the N-terminal T1 domain is the molecular barcode for potassium channel tetramerization within each family (Covarrubias et al. 1991; Li et al. 1992; Sheng et al. 1992). Kv1 channels are highly expressed in the central nervous system and cardiovascular system, and in the nervous system, Kv1 channels are mainly located at axons and nerve terminals of a nerve cell (Wang et al. 1994; Campomanes et al. 2002), while some dendritic Kv1 channels are regulated by local activity and provide feedback regulation to its excitability (Shen et al. 2004; Guan et al. 2006). Physiological studies have also revealed that the Kv1 channels control action potential propagation and repolarization, set the resting membrane potential and modulate neurotransmitter release from nerve terminals (Glazebrook et al. 2002; Heeroma et al. 2009). Functions other than regulation of excitability have been suggested for different members of the Kv1 family, for Kv1.1 and Kv1.2 in the modulation of neuronal myelination by oligodendrocytes (Herrero-Herranz et al. 2007), and for Kv1.3, Kv1.4 and Kv1.5 in the control of glial cell function and glial precursor cell division (Preussat et al. 2003).

Thus, other than its well-known role in controlling cell excitability, potassium channels also control cell proliferation possibly by regulating membrane potentials that indirectly manipulate intracellular calcium, potassium and chloride, vital ions which regulate cell proliferation by controlling intracellular signalling or cell volume (Wonderlin & Strobl, 1996; Pardo, 2004). Interestingly, a recessive mutant megencephaly (mceph), so named for the phenotype of an enlarged brain, has arisen spontaneously (Donahue et al. 1996). Positional cloning revealed that this mceph mutant results from a deletion of 11 nucleotides in the Kv1.1 gene, the Shaker-like voltage-gated potassium channel in mouse, and this deletion leads to a frame-shift resulting in a truncation of Kv1.1 to include only the N-terminus and the first transmembrane segment (Petersson et al. 2003). Since the pore region (formed by the P-loop and the flanking 5th and 6th transmembrane segments) is missing, this MCEPH protein cannot form functional channel by itself or when it is co-assembled with other Kv1 channel α-subunits. However, since this truncated version of Kv1.1 contains the N-terminal T1 domain, an in vitro study has revealed that the MCEPH protein still can co-assemble with other α-subunits in the Kv1 channel family and suppresses Kv1 channel activity (Persson et al. 2005). The brain of the homozygous mceph mutant is significantly larger than wild-type brain starting from 3 weeks of age (Donahue et al. 1996). However, the mceph/mceph brain is not uniformly expanded: the hippocampus and ventral cortex are significantly increased in volume while other parts of the brain structure such as the adjacent dorsal cortex (including the entorhinal and parietal cortex), cerebellum and midbrain are comparable in size to the wild-type brain regions (Diez et al. 2003). Stereological and histological analysis has suggested that hyperplasia rather than hypertrophy causes this phenotype since the enlarged mceph hippocampus and ventral cortex contain more neurons and glial cells but the individual cell size is not abnormally large (Almgren et al. 2007). Indeed, the mceph/mceph brains continue to grow throughout adulthood, unlike wild-type brains that stop growing soon after birth. Previous studies have proposed multiple mechanisms to account for this megencephalic phenotype, including non-cell autonomous mechanisms such as an elevated level of trophic factors like the brain-derived neurotrophic factor (BDNF) (Lavebratt et al. 2006) and cell-autonomous mechanisms such as reduced apoptosis and increased survival (Almgren et al. 2007).

We used a novel mouse genetic tool, mosaic analysis with double markers (MADM), to resolve cell-autonomous versus non-cell autonomous mechanisms for the megencephalic phenotypes due to an overabundance of neurons and glial cells. We found many more neurons homozygous for the mceph mutation in the hippocampus, especially in the dentate gyrus, a region with neurogenesis from embryogenesis to adulthood in mammals, but not in the adjacent dorsal cortex (including the entorhinal and parietal cortex), indicating the mceph mutation increases neuron number in a cell-autonomous fashion. Moreover, glial cells homozygous for the mceph mutation did not show any increase in number, indicating the increased gliogenesis in the mceph/mceph brain is caused by a non-cell-autonomous mechanism.

Methods

Animals

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and used protocols approved by the Institutional Animal Care and Use Committee of the University of California–San Francisco. Both female and male mice were used for experiments and those mice were fed with chow diet and subjected to a 12/12 h day–night cycle. mceph and Nestin-cre mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and MADM6-GT and MADM6-TG mice were generated as described (Tasic et al. submitted). All mice were crossed to CD1 strain (Charles River, MA, USA) and maintained in the mixed genetic background.

Immunostaining

Mice, which were fed ad libitum, were anaesthetized by intraperitoneal Avertin injection and perfused transcardially with saline followed by 4% paraformaldehyde. Brains were removed and post-fixed overnight in 4% paraformaldehyde. Brains were then cryoprotected overnight in saline containing 30% sucrose at 4°C until they sank. Brain sections (50 μm) were washed in blocking medium containing 0.1% Triton X-100 and 5% donkey serum (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA), and incubated overnight (4°C) with primary antibodies against green fluorescent protein (GFP) (chicken 1:200, Aves, Tigard, OR, USA) c-myc (rabbit 1:200, Novus, Littleton, CO, USA), NeuN (rabbit, 1:400, Millipore, Billerica, MA, USA) and glial fibrillary acidic protein (GFAP) (rabbit, 1:400, Sigma-Aldrich, St Louis, MO, USA) followed by Alexa dye-tagged secondary antibodies (donkey 1:500, Invitrogen, Carlsbad, OR, USA). The slides were mounted using Fluoromount G mounting medium containing DAPI (Southern Biotech, Birmingham, AL, USA) and images were acquired using a confocal microscope (Zeiss, Germany).

Statistical analysis

We counted all the fluorescent neurons within the region of interest (such as the hippocampus or the adjacent dorsal cortex (including the entorhinal and parietal cortex) and quantified the percentage of red (mceph/mceph), green (wild-type Kv1.1) or yellow (mceph/+) in all fluorescent (red + green + yellow) neurons or astrocytes. Statistical analyses were performed with Prism software (GraphPad Software Inc., La Jolla, CA, USA) using Student's t test for pair-wise comparisons. P < 0.05 was considered statistically significant. The soma volume was measured using Zeiss LSM Image Browser and Imaris (Bitplane, South Windsor, CT, USA).

Results

Paradigm of generating mceph-MADM6 mice

We generated mceph-MADM6 mice to study the cell-autonomous and non-cell-autonomous mechanisms for the megencephalic phenotype (Fig. 1) (Zong et al. 2005; Tasic et al. submitted). In heterozygous mceph/+ mice with cre-recombinase active during the proliferative phase, somatic recombination may cause homozygosity of the mceph mutation distal to the MADM locus so that cells are marked with different colours according to their respective genotypes. Because homozygous mutant and homozygous wild-type cells are simultaneously generated as siblings and their progeny are labelled with different colours, the MADM mice have built-in control cells within the same brain region for phenotypic analysis (Espinosa et al. 2009; Liu et al. 2011). The mouse Kv1.1 gene is located about 8.8 centimorgan distal to the ROSA26 locus on chromosome 6 where the MADM gene cassette has been introduced (Petersson et al. 2003; Zong et al. 2005; Tasic et al. submitted). To generate homozygous mceph/mceph neurons throughout the entire nervous system in the mceph/+ genetic background, we crossed mceph/+;MADM6-TG/TG (MADM6-tdTomato(loxP)GFP) mice to nestin-cre;MADM6-GT/GT mice (MADM6-GFP(loxP)tdTomato). We chose the nestin-cre recombindase to induce interchromosomal recom-bination because nestin is an intermediate filament protein in the precursor cells of the nervous system and the cre-recombinase under the nestin promoter has been used to induce loxP recombination throughout the nervous system early in development (Dubois et al. 2006). With the MADM scheme, occasionally cells in the nervous system expressing the nestin driven cre-recombinase will undergo mitotic recombination and functional GFP and tdTomato sequences are restored during this interchromosomal recombination, which are then segregated and expressed in different progeny. The result is the specific labelling of clones of cells expressing GFP (green), or tdTomato (red), or both GFP and tdTomato (yellow), or neither (colourless). Because mceph is on the MADM6-TG chromosome and the wild-type gene for Kv1.1 is on the MADM6-GT chromosome, homozygous mceph/mceph mutant progeny cells are labelled with red colour, homozygous wild-type progeny cells are labelled with green colour and heterozygous mceph/+ progeny cells are either yellow or colourless (Fig. 1). Hence, we can identify homozygous mceph/mceph cells as the red cells for quantitative analyses. The low frequency of recombination events (Zong et al. 2005) allows us to characterize the cell-autonomous phenotype of homozygous mceph/mceph cells surrounded predominantly by phenotypically normal heterozygous cells carrying one mceph mutation.

Figure 1. Paradigm for generating the mceph-MADM6 mice.

Figure 1

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MADM6 markers were bred into mceph/+ mice, with the mceph mutation distal to the MADM6-TG insertion. During S phase, DNA is replicated and, in the presence of cre-recombinase (in this case cre-recombinase expression is controlled under the nestin promoter, which turns on early in neural progenitor cells), infrequent interchromosomal recombination takes place and the functional GFP and tdTomato genes are restored. During chromosome segregation, there will be equal numbers of homozygous mceph/mceph (red) and wild-type (green) precursor cells, and equal numbers of cells with duo colour (yellow), or colourless, which are heterozygous mceph/+. Lower diagram illustrates interchromosomal recombination in the presence of cre-recombinase in non-dividing cells. Recombination happening at this stage will generate more yellow mceph/+ cells and hence there are more yellow cells than red and green cells.

mceph/mceph hippocampal neurons are hyperplastic

We began by counting neurons with different colours in the hippocampus from 3-month-old mceph-MADM6 mice, at a developmental stage when mceph/mceph mutant mice have already developed enlarged brains (Donahue et al. 1996). In the hippocampus from the nestin-cre;mceph-MADM6 mouse, there were two morphologically distinguishable types of cells (Fig. 2A): one group of cells have pyramidal or round cell body with thin and elongated processes and most of those cells were immuno-positive for NeuN, a specific marker for neurons (Mullen et al. 1992) (Fig. 2B–D). The second group of cells have a bushy appearance with diffuse processes, and those cells were positive for GFAP, a specific marker for astrocytes, the predominant glial cells in the hippocampus (Zhou et al. 2007) (Fig. 2F–H). We counted the number of florescent cells in the hippocampus and identified them as neurons or astrocytes based on their distinct morphology. We next asked if mceph induced hyperplasia is cell-autonomous, since only a very small percentage of the total cell population in mceph-MADM6 hippocampus are mceph/mceph neurons. As we described in the previous section, homozygous mceph/mceph neurons are labelled with red fluorescence whereas wild-type neurons arising from sibling progenitors that have undergone somatic recombination are green, so we divided the number of red, green and yellow neurons by the total number of fluorescent neurons to determine the respective percentage. We found that in the mceph-MADM6 hippocampus there were many more red mceph/mceph neurons than green wild-type neurons in the hippocampal region (Fig. 2E). In contrast, there were comparable numbers of red (mceph/mceph) and green (wild-type) astrocytes in the mceph-MADM6 hippocampus (Fig. 2I).

Figure 2. MADM analysis of the mceph/+ hippocampus.

Figure 2

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A, a representative overview image of a hippocampal section from an adult mceph-MADM6 mouse. DAPI counterstain (blue signals) was used for revealing brain structures. Scale bar = 500 μm. B–D and F–H, cells that have a round or pyramidal cell body and carry distinct thin and long processes are neurons (white arrows), since most of the cells with this type of morphology are positive for NeuN (C), a specific marker for neurons, and negative for GFAP (G), a specific marker for astrocytes (D and H); cells that exhibited a bushy appearance (white arrowheads) were negative for NeuN (C) but were positive for GFAP (G) and therefore those cells with such bushy appearance were defined as astrocytes (D and H). E, statistical analysis of red mceph/mceph neurons, green wild-type neurons and yellow mceph/+ neurons in hippocampus. There were more mceph/mceph neurons than green wild-type neurons in the hippocampus (P < 0.001, Student's t test, n = 47). I, in contrast, there were equal numbers of red mceph/mceph astrocytes and green wild-type astrocytes in the hippocampus (P > 0.05, Student's t test, n = 36). Scale bar = 20 μm in B–D and 40 μm in F–H.

Neurogenesis is known to persist in the hippocampus, especially in the dentate gyrus in adult mammals (Ming & Song, 2005). We next separately analysed the neurons in the CA1 region and dentate gyrus (Fig. 2A). We found that in the CA1 region of the hippocampus from the mceph-MADM6 mice there is a slight but significant increase of mceph/mceph pyramidal neurons (Fig. 3). Moreover, in the dentate gyrus region, the red mceph/mceph granule cells were dramatically increased as compared to wild-type green cells (Fig. 4). We also have noticed that those red mceph/mceph granule cells were often clustered and close to one another (Fig. 4B and C), indicating they may share common ancestry progenitor cells. To control for the possibility that the excessive red neuron counts in mceph-MADM6 mice are due to a difference in fluorescent protein expression or detection sensitivity, we also generated wild-type-Kv1.1-MADM6 mice. We found there were equal numbers of red and green neurons in such wild-type hippocampus (Fig. 5), indicating that deleting Kv1.1 is the primary cause of the increase of red mceph/mceph neurons in mceph-MADM6 mice. Meanwhile, we have also noticed that there were more yellow neurons than red and green neurons and this might be caused by cre-mediated recombination in non-dividing neurons (Fig. 1). This phenomenon has been reported previously (Zong et al. 2005). Unlike the hippocampus, the size of other parts of the homozygous mceph/mceph brain seem to be relatively normal (Almgren et al. 2007). We asked whether the increase in mceph/mceph neuron number is restricted to the hippocampus by counting fluorescent neurons in the dorsal cortex (including the entorhinal and parietal cortex), which is adjacent to the hippocampus. We found this part of the cerebral cortex did not exhibit an overgrowth phenotype; in the dorsal cortex there are equal numbers of red mceph/mceph neurons and green wild-type neurons (Fig. 6).

Figure 3. MADM analysis of the CA1 region in the mceph-MADM6 hippocampus.

Figure 3

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A, GFP signals representing the chromosome with the wild-type Kv1.1 gene. B, tdTomato signals representing the chromosome with the mceph mutation. C, overlay of signals in A and B. D, statistical analysis of red neurons, green neurons and yellow neurons in hippocampal CA1 region. There were more red mceph/mceph neurons than green wild-type neurons in the CA1 hippocampus (P < 0.05, Student's t test). n = 14 for each group. Scale bar = 100 μm.

Figure 4. MADM analysis of the dentate gyrus in the mceph-MADM6 hippocampus.

Figure 4

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A, GFP signals representing the chromosome with the wild-type Kv1.1 gene. B, tdTomato signals representing the chromosome with the mceph mutation. C, overlay of signals in A and B. D, statistical analysis of red neurons, green neurons and yellow neurons in hippocampus. There were more red mceph/mceph neurons than green wild-type neurons in the hippocampus (P < 0.001, Student's t test). n = 16 for each group. Scale bar = 200 μm.

Figure 5. MADM analysis of the wild-type-MADM6 hippocampus.

Figure 5

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A, GFP signals representing one chromosome 6 with the wild-type Kv1.1 gene. B, tdTomato signals representing the other chromosome 6 with the wild-type Kv1.1 gene. C, overlay of signals in A and B. Arrowheads indicate red neurons, arrows indicate green neurons. D, statistical analysis of red neurons, green neurons and yellow neurons in hippocampus. There were equal numbers of red and green neurons in the hippocampus (P > 0.05, Student's t test). n = 20 for each group. Scale bar = 100 μm.

Figure 6. MADM analysis of the mceph/+ entorhinal cortex.

Figure 6

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A, GFP signals representing the chromosome with the wild-type Kv1.1 gene. B, tdTomato signals representing the chromosome with the mceph mutation. C, overlay of signals in A and B. D, statistical analysis of red mceph/mceph neurons, green wild-type neurons and yellow mceph/+ neurons in the entorhinal cortex. There were equal numbers of red mceph/mceph neurons and green wild-type neurons in the entorhinal cortex (P > 0.05, Student's t test). n = 4 for each group. Scale bar = 200 μm.

In addition to hyperplasia, an enlarged brain may also result from hypertrophy such as an increase in the individual cell size. Since we have found that the mceph/mceph granule cells in the dentate gyrus are the most numerous among the whole hippocampus, indicating the Kv1.1 gene may have greater effect in the granule cells and their progenitors. We measured the soma volume of homozygous, red, mceph/mceph and heterozygous, yellow, mceph/+ neurons, as well as wild-type green granule cells in the dentate gyrus. Similar to the earlier stereological study, although mceph/mceph granule cells are hyperplastic, we found that those red mceph/mceph granule cells had comparable soma volume as compared to heterozygous mceph/+ and wild-type granule cells in the dentate gyrus (Fig. 7).

Figure 7. Red mceph/mceph hippocampal neurons were comparable in size as compared to green wild-type neurons and yellow mceph/+ neurons.

Figure 7

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A, representative 3-demensional reconstruction of granule cells in the dentate gyrus from a mceph-MADM6 mouse. mceph/mceph, wild-type and mceph/+ neurons were outlined in red, green and red/green contour, respectively. B, statistical analysis of the soma volume from red mceph/mceph neurons, green wild-type neurons and yellow mceph/+ neurons in hippocampus. Red mceph/mceph neurons had soma size comparable to that of yellow mceph/+ neurons and green wild-type neurons in the hippocampus (P > 0.05, Student's t test). n = 3, 13 and 10 for wild-type, mceph/mceph and mceph/+ neurons, respectively. Each grid in Arepresents 10 × 10 μm.

Discussion

Using the MADM method for mosaic analysis, we have discovered a cell-autonomous hyperplastic effect of the Kv1.1 mutation in a region-specific and cell type-specific manner, because mceph/mceph neurons but not mceph/mceph glial cells in the hippocampus nor mceph/mceph cortical neurons in the adjacent dorsal cortex (including the entorhinal and parietal cortex) exhibited such an abnormal overgrowth phenotype in adult brains (Figs 2–6). It seems possible that the enlarged brain results from a combination of neural progenitor overproliferaction and a reduction of apoptosis of neurons and possibly their progenitors.

Kv1.1 exhibits an uneven temporal and spatial distribution pattern in the mouse brain. Kv1.1 mRNA has two expression periods during development. The first wave of Kv1.1 expression peaks during embryogenesis around E14.5 and then gradually fades away. The second wave of Kv1.1 mRNA expression peaks around P15 and then maintains a relatively high level throughout adulthood (Hallows & Tempel, 1998; Prüss et al. 2010). This tightly regulated temporal expression of Kv1.1 – taken together with our findings – implies an important role of Kv1.1 channel protein in suppressing neurogenesis, since the Kv1.1 mRNA temporal expression pattern coincides with that for neurogenesis: most of the neuronal precursor cells are born between E12 and E19 and the mouse brain reaches its final size around the 3rd week of age (Wullimann, 2009); in between these two time periods the Kv1.1 mRNA expression is relatively low and creates a time window for neurogenesis. At the adult stage when the Kv1.1 mRNA is at high levels, it is not evenly distributed throughout the brain, but is especially high in the dentate gyrus and the CA2/3 regions in the hippocampus where neurogenesis is known to take place in adulthood (Grosse et al. 2000; Prüss et al. 2010). As we described earlier, the mceph mutation results in a truncated Kv1.1 protein with an intact N-terminal T1 tetramerization domain. This truncated protein does not form a functional Kv channel; rather, since the N-terminal T1 domain is still intact, this truncated Kv1.1 protein could still co-assemble with other α-subunits in the Kv1 family such as Kv1.2 and Kv1.3; in vitro heterologous expression study has shown that co-expression of the MCEPH protein in Xenopus oocytes suppresses potassium currents mediated by other Kv1 channels (Persson et al. 2005). Because the Kv1.1 null mutant mice also exhibit megencephalic phenotype with especially enlarged hippocampus and ventral cortex (Persson et al. 2007), this phenotype is likely to be the result of abolished Kv1.1 channel function. It thus appears that Kv1.1-containing channels are important in restricting the neuronal number, since the sparsely generated mceph/mceph neurons without Kv1.1-containing channels are much more numerous (Figs 2–4).

In mceph/mceph mice, the enlarged hippocampus also contains an excessive number of astrocytes (Almgren et al. 2007). However, in our mceph-MADM6 mice, those mceph/mceph astrocytes did not hold an advantage in cell proliferation and/or survival (Fig. 2). In contrast to its expression in neurons, the Kv1.1 expression level in astrocytes is relatively low (Smart et al. 1997; Bekar et al. 2005; Beraud et al. 2006). It has been shown that newborn neurons recruit astrocytes migrating into their newly established territories (Kaneko et al. 2010); additionally, increased neuronal activity is also known to stimulate glial cell proliferation (Ongür et al. 2007). Moreover, over-reactive astrocytes have been detected at the inferior colliculus and hippocampus in mceph/mceph mice after acoustic startle stimulation (Fisahn et al. 2011). Based on those findings, we hypothesize that the increase of astrocytes observed in mceph/mceph mice may be caused by non-cell-autonomous mechanisms such as increased astrocyte proliferation induced by hyperexcitable mceph/mceph neurons with reduced Kv1 channel activity (Smart et al. 1998) and/or increased proliferation following hyperplasia of mceph/mceph neurons to keep neuron-glia ratio constant (Geisert et al. 2002).

Potassium channels are important regulators in cell growth and cell proliferation. Altered expression of potassium channels has been found in highly proliferative cells such as cancers and certain potassium channels can control cancer cell proliferation, migration and metastasis (Pardo, 2004). It is well-established that the EAG1 potassium channel is oncogenic, since pharmacological blockade or genetic silencing of EAG1 can effectively reduce tumour size in vivo (Pardo et al. 1999). As for the Kv1 family, Kv1.3 and Kv1.5 transcripts have been detected at high levels in human cancer biopsies (Bielanska et al. 2009). In contrast, our study indicates that Kv1.1 is likely to be anti-proliferative in the progenitors for pyramidal neurons in the CA1 and granule cells in the dentate gyrus of the hippocampus (Figs 2–4), presumably because their precursors without Kv1.1 are more proliferative though it is also possible that without Kv1.1 neurons and/or their precursors refrain from undergoing apoptosis. Kv1.1-containing channels operate at more negative membrane potential than most other potassium channels; in addition, these channels inactivate much more slowly than other channels in the Kv1 family (Storm, 1988). Whether and how Kv1.1 suppresses neurogenesis in the hippocampus is currently unknown. Based on the unique biophysical property of Kv1.1, it is conceivable that hippocampal neural progenitors without Kv1.1-containing channels are more excitable and hyperexcitability may increase intracellular calcium, a vital intracellular ion to trigger cell proliferation (Apáti et al. 2011). Moreover, removal of Kv1.1 may cause a reduction in the intracellular potassium loss, which has the potential to promote cell survival and avert the initiation of the cell signalling cascade that leads to apoptosis (Bortner & Cidlowski, 2007). Indeed, homozygous mceph/mceph mice have a reduced apoptotic rate in the hippocampus (Almgren et al. 2007, 2008). Along the same line, an astrocyte specific sodium channel has been shown to control the viability of astrocytes in the spinal cord independent of the electrogenic role of sodium channels. Sontheimer and colleagues (1994) demonstrated that this astrocyte specific sodium channel controls intracellular sodium concentration, which leads to the activation of the Na+,K+-ATPase. Blocking this sodium channel by tetrodotoxin causes a depletion of intracellular sodium, slows down the Na+,K+-ATPase, and, as a consequence, results in excessive depolarization and massive calcium influx which may lead to cell death and apoptosis. It will be important to pursue further studies such as double labelling with TUNEL and BrdU at the subventricular zone (Ming & Song, 2005) or the subgranular zone in the dentate gyrus (Li & Pleasure, 2007), two regions with robust adult neurogenesis, in mceph-MADM6 mice. Extending studies such as the MADM analysis to mice of older ages will also be informative in assessing the relative contribution of reduced apoptosis and increased proliferation to the ever growing brain of mceph mutant mice.

In summary, using the innovative technology provided by the MADM mice, we have revealed a novel cell-autonomous function of Kv1.1 in the control of neuron numbers, thereby attributing Kv1 channel functions beyond what Hodgkin and Huxley proposed for hyperpolarization and repolarization of the action potential. It seems likely that there are other unexpected functions of potassium channels waiting to be discovered in the future.

Acknowledgments

We thank Drs Woo-Ping Ge, An-Chi Tien, Jamsine Chen and Grant Li at UCSF for discussions and Dr Hui Zong (Institute of Molecular Biology, University of Oregon, Eugene) for assistance in producing the MADM6 mice. This work was supported by American Diabetes Association Mentor-Based Fellowship 7-06-MN-29 (to S.B.Y.), HHMI Summer Student Program (to K.D.M.), NIH grant R01-NS050835 (to L.L.) and NIMH grant MH065334 (to L.Y.J.). L.L, Y.N.J. and L.Y.J. are investigators of the Howard Hughes Medical Institute.

Glossary

Abbreviations

MADM

mosaic analysis with double markers

Author contributions

S.B.Y. and K.D.M. performed experiments and data analysis. B.T and L.L. provided experimental materials. S.B.Y., Y.N.J. and L.Y.J. prepared the manuscript. All authors have reviewed and edited the manuscript.

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