Mouse brain expression of Kv3 subunits in development, adulthood, and aging
Enrica Boda1,2
*
, Eriola Hoxha1,2*
, Alessandro Pini2,#, Francesca Montarolo1,2, Filippo Tempia1,2,31 Neuroscience Institute Cavalieri Ottolenghi (NICO), University of Turin, Regione Gonzole, 10,
10043 Orbassano (Turin) ITALY
2 Dept. of Neuroscience, Section of Physiology, University of Turin, Corso Raffaello, 30, 10125
Turin ITALY
3 National Institute of Neuroscience-Italy (INN), University of Turin, Corso Raffaello, 30, 10125
Turin ITALY
#Current address: Department of Anatomy, Histology and Forensic Medicine, University of Firenze,
Viale Pieraccini 6, 50139 Firenze, Italy
*
These two authors contributed equally to the workRunning title: Mouse brain expression of Kv3 subunits
Corresponding author : Filippo Tempia
Neuroscience Institute Cavalieri Ottolenghi (NICO) University of Turin
Regione Gonzole, 10
10043 Orbassano (Turin) ITALY Telephone:
+39011-670-6609
Telefax: +39011-670-8174 filippo.tempia@unito.it
The experiments were supported by grants (to F.T.) from: MIUR (PRIN-2005 and 2007), Regione Piemonte (Ricerca Scientifica Applicata 2004 projects A183 and A74 and Ricerca Sanitaria Finalizzata 2006 and 2007), Compagnia di San Paolo and CRT (Bando Alfieri 2007). E.B. is recipient of a CRT fellowship (Progetto Lagrange).
ABSTRACT
In neurons, voltage-dependent Kv3 potassium channels are essential for the generation of action potentials at high frequency. The pattern of expression of Kv3 channels has been described in the adult, but scarce information is available on the time course of expression during brain development and aging. We analysed the profile of expression of the four Kv3 subunits by quantitative RT-PCR and Western blot in the whole mouse brain and in dissected brain regions (olfactory bulb, septum, neocortex, hippocampus, brainstem, cerebellum) from 14 days after conception to 18 months after birth. Although all Kv3 transcripts were significantly expressed in embryonic age in whole brain extracts, only Kv3.1, Kv3.2 and Kv3.4 subunit proteins were present, suggesting a novel role for Kv3 channels at this developmental stage. With the exception of Kv3.4, during postnatal development, Kv3 transcripts and proteins showed a progressive increase of expression and reached an asymptote in adulthood. Conversely, during aging Kv3 expression was rather stable. Our results suggest that the increase in Kv3 levels during development might participate to the maturation of electrical activity of neurons, while they likely do not contribute to neuronal senescence.
INTRODUCTION
By assuring a brief action potential duration and a short-lasting fast afterhyperpolarisation (fAHP), potassium currents mediated by the voltage-dependent channels of the Kv3 subfamily enable the generation of action potentials at high frequency in neurons (Rudy and McBain, 2001). As a consequence, Kv3 currents were detected in almost all fast spiking neurons (FSNs) and their suppression causes a reduced rate of action potential firing (Rudy and McBain, 2001). Mammalian and human Kv3 channels are tetramers composed by the assemblage of subunits coded by KCNC genes (Kv3.1: KCNC1; Kv3.2: KCNC2; Kv3.3: KCNC3; Kv3.4: KCNC4). Homomeric channels expressed in a heterologous system have distinct functional properties, including different rates of inactivation. Heteromeric channels can be formed by the assemblage of different Kv3 subunits, as happens also with other subfamilies of voltage-dependent potassium channels: in particular, the Kv3.3 and Kv3.4 subunits can assemble with Kv3.1 in vitro (Baranauskas et al., 2003; Weiser et al., 1994), while Kv3.1 and Kv3.2 co-immunoprecipitate in vivo (Hernández-Pineda et al., 1999). The pattern of expression of the four Kv3 subunits is also distinct (Weiser et al., 1994; Chang et al., 2007), so that FSNs in a nucleus express either only one or, more often, two subunits. Only in a few cases three or all four subunits are co-expressed. The subunit with the most diffuse pattern of expression is Kv3.1, while Kv3.3 is considered as the main subunit in the hindbrain, often together with Kv3.1. The overlapping expression of Kv3.1 and Kv3.3 is likely the reason of the fact that the genetic deletion of either subunit causes milder symptoms compared to the double knock-out of both (Ho et al., 1997; Espinosa et al., 2001; McMahon et al., 2004). Recently, the human spino-cerebellar ataxia SCA13 has been shown to be due to KCNC3 gene mutations (Waters et al., 2006; Figueroa et al., 2011), which have a dominant negative effect on channel function, with the consequence that functional alterations are present also where the expression of Kv3.3 overlaps with Kv3.1.
Although the pattern of expression has been described in detail, at present there are no quantitative studies about the abundance of the four subunits of the Kv3 subfamily in the different
brain regions. Furthermore, the developmental profile of expression has been investigated only for the Kv3.1 (Perney et al., 1992; Parameshwaran et al,. 2003; Feng and Morest, 2006; Bortone et al., 2006; Goldberg et al., 2011), and in part for the Kv3.2 subunits (Tansey et al., 2002; Goldberg et al., 2011). Nothing is known either about the age when the other subunits begin to be expressed or about the time course followed during development. Therefore, our study was aimed at showing the developmental and regional profile of expression of the four Kv3 subunits in the mouse brain
MATERIALS AND METHODS
Tissue collection and RNA isolation
Kv3 subunit quantification was performed on wild type CD-1 mouse (Harlan, Corezzana, Italy) brains, manually divided in two parts (hemibrains) by a sagittal cut. For each individual one hemibrain was used to assess Kv3 transcript levels and the contralateral one was used to quantify Kv3 proteins.
The analysis of Kv3 transcripts was performed on hemibrains at different ages from embryonic day 14 (E14) to 6 months, including the birth day (P0) and the postnatal days 7 (P7), 14 (P14) and 60 (P60) (5-6 individuals per time point). The experiments on dissected brain regions were performed at P7, P14 and P60, 6 and 18 months of age (4-6 individuals per time point). Animal procedures were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). Brains were placed in an ice-cold artificial liquor containing (in mM)125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, 20 glucose, bubbled with 95% O2
-5% CO2. and manually divided in two parts. For the analysis on separated brain regions,
cerebellum, brain stem and olfactory bulbs were manually dissected. Coronal sections from neocortex, septum (two 400 m thick slices starting from Bregma 1.10 mm) and hippocampus (two 400 m thick slices starting from Bregma -1.50) were cut using a vibratome (Leica Microsystems GmbH, Wetzlar, Germany). Stereotaxic coordinates were obtained from (Paxinos and Franklin, 2001). All samples were rapidly frozen and stored at -80°C. Total RNA was isolated by extraction
with the TRIzol Reagent (Invitrogen Life Technologies Inc., Grand Island, NY, USA), in accordance with the manufacturer’s instructions. Genomic DNA contamination was prevented by treating the extracted RNA with Deoxyribonuclease I (Sigma-Aldrich). RNA samples were stored at -80°C.
Real Time RT-PCR and data analysis
One g of total RNA was reverse-transcribed to cDNA using the High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. cDNA samples were stored at -20°C.
Quantitative Real Time PCR was carried out using the ABI Prism 7000 Sequence Detection System instrumentation (Applied Biosystems). Taqman Gene Expression Assays were purchased from Applied Biosystems to determine the amount of the four target genes (Kv3.1, cod. Mm00657708_m1; Kv3.2, cod. Mm01234232_m1; Kv3.3, cod. Mm00434614_m1; Kv3.4 cod. Mm00521443_m1) and the housekeeping genes phosphoglycerate kinase 1 (Pgk1, cod. Mm00435617_m1) and beta-actin (b-Act; cod. Mm00607939_s1). PCR was performed according to the following reaction conditions: 50°C for 2 minutes, 95°C for 10 minutes, followed by 50 cycles 95°C for 15 seconds alternating with 60°C for 1 minute.
Data extracted from each real time RT-PCR run were analysed by means of the 7000 v1.1 SDS instrument software (Applied Biosystems). The baseline of the run was automatically determined. The threshold value was manually set to the value of 0.2. The CT (Cycle Threshold) was automatically calculated. A relative quantification approach was used: the amount of the target RNA copies were normalized to the endogenous reference Pgk1 (Boda et al., 2009) for the analysis of the developmental and regional profile of Kv3 transcripts in CD1 mice. Data concerning Kv3 and housekeeping gene transcripts expression were analysed as in Livak and Schmittgen (2001). For each time point, Kv3 expression data in CD1 hemibrains were normalized to the value at E14. Further, data obtained in dissected brain regions were normalized to Kv3 levels in the entire
hemibrain at P7. Best fitting curves to linear and exponential functions were obtained by IgorPro built-in functions, with the method of Chi-square minimization. Data were statistically evaluated by means of a two ways ANOVA test. A P value < 0.05 was considered significant.
Western blot analysis
The analysis of Kv3 subunit proteins was performed on hemibrains collected from CD-1 mouse at E14, P7 and P60. Kv3 protein quantification was also carried out onto adult (P60) forebrain and hindbrain portions, obtained by manually separating the telencephalic hemispheres and the diencephalon from the brain stem and the cerebellum, by applying a coronal cut at the level of the midbrain. Brain portions were rapidly frozen at -80°C. Protein extracts were obtained by tissue homogenization in lysis buffer (50 mM Tris HCl, pH 8, 150 mM NaCl, 1% Triton X-100 and protease inhibitor cocktail, Sigma). The homogenate was centrifuged at 13.000 rpm for 25 min at 4°C. Protein concentration was determined using Pierce BCA™ (bicinchoninic acid) protein assay kit and a BioPhotometer (Eppendorf). Bovine serum albumin (BSA) was used as standard.
Briefly, equal amounts of proteins (50 g) for each sample were loaded on 12% SDS-PAGE and then blotted onto polyvinylidene difluoride (PVDF) membranes (GE Healthcare) using Mini Trans-Blot electrophoresis apparatus (Bio-Rad Laboratories). After blocking with 5% non-fat dry milk in TBS-T buffer (20 mM Tris pH 7.4, 137 mM NaCl, 1% Tween 20) for 1 h at room temperature, membranes were then probed overnight at 4°C with primary antibodies (anti-Kv3.1,
anti-Kv3.2, anti-Kv3.3, anti-Kv3.4, Alomone, Jerusalem, Israel) (1:200) diluted in 2.5 % milk in TBS-T buffer. As reference (loading control), membranes were probed with primary antibody against Actin (1:5000) (Sigma Aldrich, Saint Louis, USA). PVDF membranes were washed in TBS-T buffer and incubated for 1 h at room temperature in appropriate peroxidase-coupled secondary antibodies (Vector Labs). Finally, specific peroxidase signals were detected using a chemiluminescent substrate (ECL) (Pierce, Rockford, IL) exposed to Kodak X-MAX film, and developed using the Curix 60 AGFA Film Processor. Anti-Kv3 antibody specificity was assessed
by pre-adsorbing antibodies with their specific control peptide antigens (supplied together with anti-Kv3 antibodies by Alomone; see Suppl. Material and Suppl. Fig. 1). Levels of protein immunoreactivity were quantified by measuring the optical density of the 110kDa reactive bands (corresponding to the glycosylated form of Kv3 monomers; McMahon et al., 2004; Cartwright et al., 2007) by means of ImageJ software. Background optical density levels were taken for each image of a blot and were subtracted from the optical density obtained for each individual immunoreactive band. Experiments were run in triplicate. Data were statistically evaluated by means of a one way ANOVA test. A P value < 0.05 was considered significant.
RESULTS
Time course analysis of Kv3 mRNAs and proteins in whole mouse brain
The average level of Kv3 mRNA expression in the whole mouse brain was analysed in hemibrains cut along the midline, starting from E14 to young (P60) and middle adulthood (6 months of age). Notably, all Kv3 transcripts were significantly expressed in embryonic age and showed a progressive increase of expression, reaching an asymptote within the last time point of 6 months (Fig. 1) with a constant expression in adulthood between 2 and 6 months (P>0.05). Most of the increase developed between P0 and P14, except for Kv3.4, whose increase in expression was mainly confined to foetal development, so that the asymptote was attained already around birth (Fig. 1D). To confirm these data, Kv3 protein content was evaluated in the contralateral hemibrains at E14, P7 and P60 (Fig. 2). Western blot analysis showed that only Kv3.1, Kv3.2 and Kv3.4 subunit proteins were present at E14, while Kv3.3 subunit was detected only by P7 (Fig. 2A). The developmental time course of Kv3 proteins was similar to that of Kv3 mRNAs: Kv3.1, Kv3.2 and Kv3.3 protein levels significantly increased between E14 and P7 and then, more markedly, between P7 and P60 (P<0.05; Fig. 2B, C and D). Conversely, Kv3.4 subunit reached a relatively high level of expression already at E14 and slightly increased during ontogenesis (Fig. 2E).
Time course analysis of Kv3 mRNAs and proteins in selected brain regions
The expression level in the whole brain is the average of different cell types in different regions of the central nervous system. The latter aspect, of regional pattern of expression, was directly assessed by dissecting and separately analysing selected brain regions (olfactory bulb, septum, neocortex, hippocampus, brainstem, cerebellum) starting from P7 (Fig. 3). For Kv3.1, Kv3.2 and Kv3.3 genes, the time course of the expression during postnatal development was well described by a single exponential function (Fig. 3A-C). In contrast, Kv3.4 in the forebrain regions showed no consistent variation during postnatal development, while, from P7 and P14, it increased in the cerebellum and it declined in the brainstem (Fig. 3D). Since P14, Kv3.4 remained stable also in these regions (Fig. 3D).
The data in Fig. 3 and Table I allow a direct comparison, for each subunit, of the expression levels between the different regions. The region with the highest levels of expression of Kv3 channels was the cerebellum, where Kv3.1 and Kv3.3 were at least twofold represented relative to the other regions. The cerebellum is also the only region in which one of the subunits (Kv3.2) is nearly absent. In the other regions analysed there was a clearly detectable expression of the whole set of the four Kv3 subunits.
To confirm these data, Kv3 protein content was evaluated in the forebrain and hindbrain of adult wild-type mice (Fig. 2F). Consistent with mRNA quantifications, Western blot experiments showed that Kv3.1, Kv3.3 and Kv3.4 subunits were significantly more expressed in the hindbrain regions (Kv3.1 and Kv3.4: P<0.05; Kv3.3: P<0.001; Fig. 2G, I and J), while Kv3.2 protein displayed a non-significant tendency to prevail in the forebrain (P>0.05; Fig. 2H).
Kv3 expression during aging
The expression of Kv3 subunits during aging was measured in the same selected regions (olfactory bulb, septum, neocortex, hippocampus, brainstem, cerebellum) analysed during postnatal development and in the adult. Similarly to the stability of expression throughout adulthood, the
levels of the transcripts at 18 months of age were comparable to the adult values derived from 6 month old mice (Fig. 4; see Table I for comparison with neonate and young adult ages). The only notable exceptions were an increase of expression of Kv3.1 and Kv3.2 in olfactory bulb (P<0.05) and a significant downregulation of Kv3.4 in septum and neocortex (P<0.05). No other significant variation was found for any other region or subunit.
DISCUSSION
Time course analysis of Kv3 subunit expression in mouse brain
Our time course analysis is the first complete measurement of the developmental profile of the four Kv3 subunits. In the whole brain, we found a low but significant expression of all Kv3 mRNAs even in embryonic age (E14). However, protein analysis showed that only Kv3.1, Kv3.2 and Kv3.4 subunit proteins were present at this early developmental stage. Previous studies showed that Kv3.1 subunit is expressed at a detectable level before birth in the murine and avian brain (Perney et al., 1992; Kuenzel et al., 2009; Feng and Morest, 2006). However, the expression of Kv3 subunits in the embryonic central nervous system was by large not investigated and the majority of the studies focused on Kv3 expression in the postnatal period and in single brain regions (Goldberg et al., 2011; Liu and Kaczmarek, 1998; Goldman-Wohl et al., 1994; Prüss et al., 20010). Kv3.1 and Kv3.2 were shown to be present already at the birth day in the cerebral cortex (Grabert and Wahle, 2008), while they were not detected in the hippocampus at the same time point (Tansey et al., 2002; Du et al., 1996). Since at the embryonic stage of life the FS phenotype is not yet present, the finding of such early expression of Kv3 suggests a novel role for these subunits before the appearance of mature firing competence. Very few studies concerning non-conventional functions of Kv3 channels have been published to date and were all focused on Kv3 involvement in cell proliferation (Chang et al., 2003; Liebau et al., 2006; Spitzner et al., 2007; Miguel-Velado et al, 2010;), migration (Hendriks et al., 1999) and neuronal growth cone extension (Pollock et al., 2002). Our
results underscore the need of further studies regarding the role of Kv3 currents in regulating neuronal and glial properties during the earliest phases of brain development.
After the embryonic age, all subunits increased in expression, with a stable level reached in the adulthood. Notably, Kv3.4 mRNA showed changes only in utero, with a constant level already since birth. Consistently, Kv3.4 subunit protein reached a relatively high level of expression already at E14 and slightly increased during the postnatal period. On the contrary, in the whole brain tissue, the other subunits showed a significant increase between the P7 and P14, which parallels brain electrophysiological maturation with acquisition of a FS phenotype by several neuronal populations. With the exception of Kv3.4, the postnatal changes in expression of Kv3 transcripts were all similar, although not identical. Since Kv3 subunits can form heteromultimeric channels, it might be possible that a coordinated regulation of expression could occur to reach the appropriate stoichiometry. The different time course of Kv3.4 expression in the brain tissue suggests a separate role for this rapidly inactivating subunit in neuronal function.
Kv3 expression changes were not synchronous in all regions, suggesting differences in the timing of maturation of these structures: in cerebellum, brain stem and olfactory bulb, Kv3 transcripts displayed the main increase during the second week of life, while in hippocampus and neocortex they peaked later, between P14 and P60. Interestingly, the Kv3.4 level increased only in the cerebellum between P7 and P14, while, in the same period it exhibited a tendency to decrease in the brain stem. In the cerebellum, Kv3 upregulation parallels the period in which granule neurons begin to receive synapses from the mossy fibres (Ito, 1984) and is likely triggered by the initiation of granule cell electrical activity elicited by afferent terminals (Liu and Kaczmarek, 1998; Grabert and Wahle, 2008). In the forebrain, Kv3.1, Kv3.2 and Kv3.3 proteins are remarkably expressed in subpopulations of cortical GABAergic (gamma-aminobutyric acid) interneurons in the neocortex (Goldberg et al., 2005; Chang et al., 2007) and hippocampus (Tansey et al., 2002; Chang et al., 2007). Cortical GABAergic interneurons are involved in the processing of information, in the regulation of plasticity, in the generation of cortical rhythms and the limiting of seizures (McBain
and Fisahn, 2001). The increase of Kv3 expression between the P14 and P60 is likely related to the maturation of the GABAergic circuitry, in agreement with the susceptibility to seizures of Kv3.2-deficient mice (Lau et al., 2000) and with the alterations in the gamma-frequency oscillations in Kv3.1 knockout mice, (Joho et al., 1999).
Few significant changes in Kv3 mRNAs expression were found between 6 and 18 months of age in the single brain regions, with the exception of a considerable upregulation of Kv3.1 and Kv3.2 in the olfactory bulb and a significant downregulation of Kv3.4 in septum and neocortex. Consistently, the age-related enhancement of post-burst AHP, associated with the impairment of both hippocampus- and cerebellum-dependent learning in normal subjects, was attributed to the alteration of potassium currents other than Kv3 (Disterhoft and Oh, 2007).
ACKNOWLEDGEMENTS
We wish to thank Dr. Annarita De Luca, Dr. Carlo Giachello and Dr. Federica Premoselli for precious help with Real Time RT-PCR and Western blot experimental setting. The technical support of Mr. Matteo Novello is gratefully acknowledged.
REFERENCES
Baranauskas G, Tkatch T, Nagata K, Yeh JZ, Surmeier DJ. 2003. Kv3.4 subunits enhance the repolarizing efficiency of Kv3.1 channels in fast-spiking neurons. Nat Neurosci. 6:258-266.
Boda E, Pini A, Hoxha E, Parolisi R, Tempia F. 2009. Selection of reference genes for quantitative Real-Time RT-PCR studies in mouse brain. J Mol Neurosci 33:170-179.
Bortone DS, Mitchell K, Manis PB. 2006. Developmental time course of potassium channel expression in the rat cochlear nucleus. Hear Res. 211:114-125.
Cartwright TA, Corey MJ, Schwalbe RA. 2007. Complex oligosaccharides are N-linked to Kv3 voltage-gated K+ channels in rat brain. Biochim Biophys Acta 1770 :666-71
Chang KW, Yuan TC, Fang KP, Yang FS, Liu CJ, Chang CS, Lin SC. 2003. The increase of voltage-gated potassium channel Kv3.4 mRNA expression in oral squamous cell carcinoma. Oral Pathol Med. 32:606-611.
Chang SY, Zagha E, Kwon ES, Ozaita A, Bobik M, Martone ME, Ellisman MH, Heintz N, Rudy B. 2007. Distribution of Kv3.3 potassium channel subunits in distinct neuronal populations of mouse brain. J Comp Neurol. 502:953-972.
Disterhoft JF, Oh MM 2007. Alterations in intrinsic neuronal excitability during normal aging. Aging Cell. 6:327-336.
Du J, Zhang L, Weiser M, Rudy B, McBain CJ. 1996. Developmental expression and functional characterization of the potassium-channel subunit Kv3.1b in parvalbumin-containing interneurons of the rat hippocampus. J Neurosci. 16:506-18.
Espinosa F, McMahon A, Chan E, Wang S, Ho CS, Heintz N, Joho RH. 2001. Alcohol hypersensitivity, increased locomotion, and spontaneous myoclonus in mice lacking the potassium channels Kv3.1 and Kv3.3. J Neurosci. 21:6657-6665.
Feng JJ, Morest DK. 2006. Development of synapses and expression of a voltage-gated potassium channel in chick embryonic auditory nuclei. Hear Res. 216-217:116-126.
Figueroa KP, Waters MF, Garibyan V, Bird TD, Gomez CM, Ranum LP, Minassian NA, Papazian DM, Pulst SM. 2011. Frequency of KCNC3 DNA Variants as Causes of Spinocerebellar Ataxia 13 (SCA13). PLoS One. 6:e17811.
Goldberg EM, Watanabe S, Chang SY, Joho RH, Huang ZJ, Leonard CS, Rudy B. 2005. Specific functions of synaptically localized potassium channels in synaptic transmission at the neocortical GABAergic fast-spiking cell synapse. J Neurosci. 25:5230-5235.
Goldberg EM, Jeong HY, Kruglikov I, Tremblay R, Lazarenko RM, Rudy B. 2011. Rapid Developmental Maturation of Neocortical FS Cell Intrinsic Excitability. Cereb Cortex. 21:666-82. Goldman-Wohl DS, Chan E, Baird D, Heintz N. 1994. Kv3.3b: a novel Shaw type potassium channel expressed in terminally differentiated cerebellar Purkinje cells and deep cerebellar nuclei. J Neurosci. 14:511-522.
Grabert J, Wahle P. 2008. Neuronal activity and TrkB ligands influence Kv3.1b and Kv3.2 expression in developing cortical interneurons. Neuroscience 156:618-629.
Hendriks R, Morest DK, Kaczmarek LK. 1999. Role in neuronal cell migration for high-threshold potassium currents in the chicken hindbrain. J Neurosci Res. 58:805-814.
Hernández-Pineda R, Chow A, Amarillo Y, Moreno H, Saganich M, Vega-Saenz de Miera EC, Hernández-Cruz A, Rudy B. 1999. Kv3.1-Kv3.2 channels underlie a high-voltage-activating component of the delayed rectifier K+ current in projecting neurons from the globus pallidus. J
Neurophysiol. 82:1512-1528.
Ho CS, Grange RW, Joho RH. 1997. Pleiotropic effects of a disrupted K+ channel gene: reduced body weight, impaired motor skill and muscle contraction, but no seizures. Proc Natl Acad Sci USA 94:1533-1538.
Ito M. 1984. The cerebellum and neural control. New York: Raven Press.
Joho RH, Ho CS, Marks GA. 1999. Increased gamma- and decreased delta-oscillations in a mouse deficient for a potassium channel expressed in fast-spiking interneurons. J Neurophysiol. 82:1855-1864.
Kuenzel T, Wirth MJ, Luksch H, Wagner H, Mey J. 2009. Increase of Kv3.1b expression in avian auditory brainstem neurons correlates with synaptogenesis in vivo and in vitro. Brain Res. 1302:64-75.
Lau D, Vega-Saenz de Miera EC, Contreras D, Ozaita A, Harvey M, Chow A, Noebels JL, Paylor R, Morgan JI, Leonard CS, Rudy B. 2000. Impaired fast-spiking, suppressed cortical inhibition, and increased susceptibility to seizures in mice lacking Kv3.2 K+ channel proteins. J Neurosci. 20:9071-9085.
Liebau S, Pröpper C, Böckers T, Lehmann-Horn F, Storch A, Grissmer S, Wittekindt OH. 2006. Selective blockage of Kv1.3 and Kv3.1 channels increases neural progenitor cell proliferation. J Neurochem. 99:426-37.
Liu SJ, Kaczmarek LK. 1998. The expression of two splice variants of the Kv3.1 potassium channel gene is regulated by different signaling pathways. J Neurosci. 18:2881-90.
Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402-408.
McBain CJ, Fisahn A. 2001. Interneurons unbound. Nat Rev Neurosci. 2:11-23.
McMahon A, Fowler SC, Perney TM, Akemann W, Knopfel T, Joho RH. 2004. Allele-dependent changes of olivocerebellar circuit properties in the absence of the voltage-gated potassium channels Kv3.1 and Kv3.3. Eur J Neurosci. 19:3317-3327.
Miguel-Velado E, Carretero FD, Colinas O, Cidad P, Heras M, López-López JR, Pérez-García MT. 2010. Cell cycle-dependent expression of Kv3.4 channels modulates proliferation of human uterine artery smooth muscle cells. Cardiovasc Res. 86:383-391.
Parameshwaran S, Carr CE, Perney TM. 2001. Expression of the Kv3.1 potassium channel in the avian auditory brainstem. J. Neurosci. 21: 485-494.
Parameshwaran-Iyer S, Carr CE, Perney TM. 2003. Localization of KCNC1 (Kv3.1) potassium channel subunits in the avian auditory nucleus magnocellularis and nucleus laminaris during development. J Neurobiol. 55:165-178.
Paxinos G, Franklin KBJ. 2001. The mouse brain in stereotaxic coordinates. San Diego, CA, USA: Academic Press.
Perney TM, Marshall J, Martin KA, Hockfield S, Kaczmarek LK. 1992. Expression of the mRNAs for the Kv3.1 potassium channel gene in the adult and developing rat brain. J Neurophysiol. 68:756-766.
Pollock NS, Ferguson SC, McFarlane S. 2002. Expression of voltage-dependent potassium channels in the developing visual system of Xenopus laevis. J Comp Neurol 452:381-391.
Prüss H, Grosse G, Brunk I, Veh RW, Ahnert-Hilger G. 2010. Age-dependent axonal expression of potassium channel proteins during development in mouse hippocampus. Histochem Cell Biol. 133:301-12.
Rudy B, McBain CJ. 2001. Kv3 channels: voltage-gated K+ channels designed for high-frequency repetitive firing. Trends Neurosci 24:517-526.
Spitzner M, Ousingsawat J, Scheidt K, Kunzelmann K, Schreiber R. 2007. Voltage-gated K+
channels support proliferation of colonic carcinoma cells. FASEB J 21:35-44.
Tansey EP, Chow A, Rudy B, McBain CJ. 2002. Developmental expression of potassium-channel subunit Kv3.2 within subpopulations of mouse hippocampal inhibitory interneurons. Hippocampus 12:137-148.
Waters MF, Minassian NA, Stevanin G, Figueroa KP, Bannister JP, Nolte D, Mock AF, Evidente VG, Fee DB, Muller U, Durr A, Brice A, Papazian DM, Pulst SM. 2006. Mutations in voltage-gated potassium channel KCNC3 cause degenerative and developmental central nervous system phenotypes. Nat Genet 38:447-451.
Weiser M, Vega-Saenz de Miera E, Kentros C, Moreno H, Franzen L, Hillman D, Baker H, Rudy B. 1994. Differential expression of Shaw-related K+ channels in the rat central nervous system. J Neurosci 14:949-972.
TABLE I. Kv3 mRNA expression in brain regions during mouse lifespan Kv3.1 Kv3.2 Kv3.3 Kv3.4 P7 P60 18M P7 P60 18M P7 P60 18M P7 P60 18M Olfactory bulb 2.33(±0,19) 4.19(±0.26) 9.01(±0.4) 0.99(±0.11) 2.5(±0.19) 3.7(±0.04) 2.15(±0.1) 2.95(±0.19) 3.68(±0.12) 0.9(±0.17) 0.51(±0.02) 0.48(±0.01) Septum 2.16(±0,18) 2.58(±0.12) 3.07(±0.35) 1.24(±0.12) 2.48(±0.25) 2.43(±0.4) 1.74(±0.16) 3.03(±0.37) 2.05(±0.09) 1.38(±0,11) 1.17(±0.11) 0.43(±0.07) Neocortex 1.9(±0,1) 3.36(±0.34) 3.32(±0.23) 2.32(±0.19) 3.95(±0.28) 3.64(±0.16) 1.84(±0.15) 4.13(±0.5) 3.7(±0.29) 1.45(±0.13) 1.54(±0.13) 1.2(±0.03) Hippocampus 1.7(±0.16) 3.08(±0.21) 2.53(±0.28) 2.13(±0.23) 4.37(±0.63) 3.88(±0.23) 5.2(±0.42) 11.3(±1.8) 4.54(±0.36) 1.03(±0.09) 1.29(±0.15) 1.14(±0.13) Brain stem 2.01(±0.23) 2.9(±0.23) 2.65(±0.29) 1.02(±0.13) 1.66(±0.11) 1.5(±0.15) 1.7(±0.15) 6.48(±0.59) 6.15(±0.63) 2.92(±0.08) 1.33(±0.11) 1.04(±0.1) Cerebellum 2.24(±0,24) 10.57(±1.3) 12.07(±1.5) 0.09(±0.02) 0.26(±0.05) 0.34(±0.08) 5.72(±1.28) 25.26(±3.7) 24.03(±2.5) 0.47(±0.10) 1.39(±0.10) 1.42(±0.16)
Data are expressed as mean 2-ddCT (± SE) after the normalization to the level of P7 hemibrain. Only three ages are included (the pup (P7), the adult
(P60) and the aged (18M) mouse) in order to highlight the main changes in Kv3 transcript expression during mouse lifespan in each selected brain region.
FIGURE LEGENDS
Fig 1. Time course of the expression of Kv3 mRNAs in mouse whole brain.
A-D: expression levels of Kv3.1, Kv3.2, Kv3.3 and Kv3.4 transcripts at different times after conception, normalized to the housekeeping gene Pgk1 and to the value at E14. The birth day is marked by an arrowhead. Time points are the embryonic day, the birth day, the postnatal days 7 and 14, 2 months, 6 months. Each circle corresponds to a single hemibrain. Note the early asymptote of Kv3.4. In all panels, solid lines represent the best fitting exponential functions.
Fig. 2. Time course of Kv3 subunit proteins in mouse brain.
A: Representative Western blots of Kv3 subunits in mouse brain at E14, P7 and P60. B, C, D, E: Quantification of the mean Kv3 subunit expression (optical density of reactive bands at 110kDa, reflecting the estimated size of glycosylated channel monomers) in three Western blot experiments on mouse brain extracts. Data are normalized to the housekeeping gene Act and to the values obtained at E14 for Kv3.1, Kv3.2 and Kv3.4. Data are normalized to the value at P7 for Kv3.3. F: Representative Western blots of Kv3 subunits in adult mouse forebrain and hindbrain. G, H, I, J: Quantification of the mean Kv3 subunit expression in three Western blot experiments in mouse forebrain and hindbrain. Data are normalized to the housekeeping gene Act and to the values obtained in the forebrain. Histograms represent mean ± standard error (SE). Asterisks indicate significant differences (* P<0.05; ** P<0.001).
Fig. 3. Time course of Kv3 mRNAs expression in selected brain regions.
A: Kv3.1; B: Kv3.2; C: Kv3.3; D: Kv3.4. Data are normalized to the housekeeping gene Pgk1 and to the value of hemibrains at P7. Symbols represent mean ± SE. The symbol legend in D
applies to all panels. The best fitting exponential curve (solid lines) is drawn in all cases in which the fitting converged properly.
Fig. 4. Variations of Kv3 subunits expression in aged relative to adult mice.
A: Kv3.1; B: Kv3.2; C: Kv3.3; D: Kv3.4. Data are normalized to the housekeeping gene Pgk1 and to the value of hemibrains at P7. Symbols represent mean ± SE. Points are connected by solid lines to better illustrate the tendency to increase or decrease in aging. The symbol legend in D applies to all panels. Asterisks indicate significant differences (* P<0.05).
SUPPLEMENTARY MATERIAL
Control of the specificity of Kv3 antibodies
To test the specificity of the anti-Kv3 antibodies, 20 g of protein extracted from whole brains were loaded on 12% SDS-PAGE and then blotted onto PVDF membranes. Different lanes of PVDF membrane were cut and probed in parallel with Kv3 antibodies alone or pre-absorbed with specific peptide antigens in two different ratios (1:1 and 1:5 ratio), according to the manufacturer’s instructions.
FIGURE LEGEND
Suppl. Fig. 1. Specificity of anti-Kv3 antibodies. A, B, C, Anti-Kv3.1, -Kv3.2, -Kv3.3
antibodies labelled two bands at 110kDa and 260kDa (first lanes), as already shown in (McMahon et al., 2004; Parameshwaran et al., 2001). After antibody pre-adsorption with specific antigen peptides, the intensity of the 110kDa band decreased in a dose dependent manner (second and third lanes).
D, Anti-Kv3.4 antibody labelled a 110kDa band (first lane), that decreased in a dose
dependent manner (second and third lanes) after pre-adsorption with different concentrations of its specific antigen peptide. CPA: control peptide antigen.
