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Neuroscience 167 (2010) 786 –798

Department of Physiology and Medical Physics, Innsbruck Medical University, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria

Abstract—The importance and diversity of calcium signaling in the brain is mirrored by the expression of a multitude of voltage-activated calcium channel (CaV) isoforms. Whereas the overall distributions of 1 subunits are well established, the expression patterns of distinct channel isoforms in specific brain regions and neurons, as well as those of the auxiliary and 2 subunits are still incompletely characterized. Further it is unknown whether neuronal differentiation and activity induce changes of CaV subunit composition. Here we combined absolute and relative quantitative TaqMan reverse transcription PCR (RT-PCR) to analyze mRNA expression of all high voltage-activated CaV 1 subunits and all and 2 subunits. This allowed for the first time the direct comparison of complete CaV expression profiles of mouse cortex, hippocampus, cerebellum, and cultured hippocampal neurons. All brain regions expressed characteristic profiles of the full set of isoforms, except CaV1.1 and CaV1.4. In cortex development was accompanied by a general down regulation of 1 and 2 subunits and a shift from 1/ 3 to 2/ 4. The most abundant CaV isoforms in cerebellum were CaV2.1, 4, and 2 -2, and in hippocampus CaV2.3, 2, and 2 -1. Interestingly, cultured hippocampal neurons also expressed the same CaV complement as adult hippocampus. During differentiation specific CaV isoforms experienced up- or downregulation; however blocking electrical activity did not affect CaV expression patterns. Correlation analysis of 1, and subunit expression throughout all examined prepara2 tions revealed a strong preference of CaV2.1 for 4 and 2 -2 and vice versa, whereas the other 1 isoforms were nonselectively expressed together with each of the other and isoforms. Together our results revealed a remarkably 2 stable overall Ca2 channel complement as well as tissue specific differences in expression levels. Developmental changes are likely determined by an intrinsic program and not regulated by changes in neuronal activity. © 2010 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: VGCC, Ca2 , realtime RT-PCR, beta, alpha(2)delta, mRNA distribution.

*Corresponding author. Tel: 43-512-9003-70841 or 43-512-900370836; fax: 43-512-9003-73800 or 43-512-9003-73836. E-mail addresses: (G. J. Obermair) or (B. E. Flucher). Abbreviations: CaV, voltage-activated calcium channel; cDNA, complementary DNA; Ct, cycle threshold; DIV, days in vitro; DL-AP5, DL-2-amino-5-phosphonopentanoic acid; E16, embryonic day 16; HPRT1, hypoxanthine phosphoribosyl-transferase 1; LOD, limit of detection; LOQ, limit of quantification; mRNA, messenger RNA; NMDA, N-methyl-D-aspartate; PD1, postnatal day 1; qRT-PCR, quantitative reverse transcription PCR; TTX, tetrodotoxin.

Voltage-activated Ca2 channels (CaV) control multiple neuronal functions including transmitter release, gene transcription, and synaptic plasticity (Catterall, 2000). High voltage-activated CaVs are heteromultimers consisting of a pore-forming 1 and the auxiliary 2 and subunits (Catterall et al., 2005). Seven genes encode for 1 subunits of L-type (CaV1.1 to CaV1.4) and non L-type (CaV2.1 to CaV2.3) channels, and four genes for each of the auxiliary and 2 subunits (Dolphin, 2003; Davies et al., 2007). Most of the subunit isoforms are expressed in the central nervous system (Ludwig et al., 1997; Dolphin, 2003; Davies et al., 2007). The L-type channels CaV1.2 and CaV1.3 perform primarily postsynaptic functions in transcriptional regulation and synaptic plasticity, whereas the non-L-type channels (CaV2.1, CaV2.2, CaV2.3) are responsible for neurotransmitter release. While some peripheral neurons, like superior cervical ganglion cells, are known to express only one presynaptic channel (Mochida et al., 2003), it is evident that the majority of brain regions and neurons express the whole plethora of CaVs (Vacher et al., 2008). Considering the additional diversity of the auxiliary subunits and the fact that all 1 subunits seem to be capable of assembling with all and 2 isoforms, the complexity of possible subunit compositions becomes enormous. For example three distinct presynaptic CaV2 1 isoforms plus three 2 and four subunits already give 36 possible channel compositions; and that is without including the splice variants existing for all of the isoforms. In light of this subunit diversity specific mechanisms must exist to assemble distinct 1/ / 2 complexes in neurons. The simplest possible mechanism is to limit the number of isoforms expressed in a single cell at a given time. This is the case in skeletal muscle (CaV1.1/ 1a/ 2 1), cardiac myocytes (CaV1.2/ 2/ 2 -1), and retina photoreceptor cells (CaV1.4/ 2/ 2 -4) (Mori et al., 1991; Ball et al., 2002; Barnes and Kelly, 2002; Arikkath and Campbell, 2003; Wycisk et al., 2006). Similarly, the cerebellum shows a strong preference towards one subunit combination (CaV2.1/ 4/ 2 -2) (Ludwig et al., 1997; Brodbeck et al., 2002). In contrast, the cerebral cortex shows a more heterogenous expression of CaV isoforms (Ludwig et al., 1997; Klugbauer et al., 1999). Existing evidence from electrophysiological, pharmacological, and immunostaining experiments indicates that these narrow and broad expression patterns in cerebellum and cortex respectively, are also reflected in the 1 subunit expression of specific types of neurons, like cerebellar granule cells and hippocampal pyramidal cells (Hell et al., 1993; Lorenzon and Foehring, 1995; Randall and Tsien, 1995; Westenbroek et al., 1995).

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TX. From each mouse total RNA was isolated from the entire cerebral hemispheres (referred to as cortex). TaqMan gene expression assays for all high-voltage activated Ca2 channel 1. IKA. . 2 and 8 weeks) were euthanized by CO2 exposure and decapitated. USA) and random hexamer primers (Promega. Madison WI.878 2583 25. USA).769 44. and 2 subunits Subunit GenBank accession NM_014193 NM_009781 NM_028981 NM_019582 NM_007578 NM_007579 NM_009782 NM_031173 NM_023116 NM_007581 NM_146123 NM_009784 NM_020263 NM_009785 NM_001033382 Assay ID Exon boundary 9–10 8a–9 29–30 18–19 40–41 38–39 43–44 1–2 13–14 1–2 11–12 33–34 1–2 5–6 8–9 Intron length (bp) 1768 7241 5412 541 3281 2530 4240 2575 1148 4274 12. Germany) and sequenced (Eurofins MWG Operon Sequencing Department. Germany) were designed to amplify templates for the standard curves using cDNA from mouse whole brain (Table 2). The hippocampus was removed from one hemisphere using fine scissors. Carlsbad CA. For each assay. Standard curve dilution series ranging from 101 to 107 DNA molecules were generated in water containing 1 g/ml of poly-dC-DNA (Midland. and each of the four and 2 subunit genes. Reverse transcription was performed on 1 g of RNA using Superscript II reverse transcriptase (Invitrogen.2 CaV2. Martinsried. Hilden. GmbH. see above) over the course of 2 years. PCR products were separated on 1. Hilden. Briefly. and information on expression patterns of auxiliary subunits is sparse. Germany). USA). Germany) supplemented with Glutamax and B27 supplements (Invitrogen GmbH). harvested by trypsin treatment. The entire cerebral hemispheres were isolated by separation from the diencephalon. DNA was extracted using Nucleospin Extract II columns (Macherey-Nagel. dissected hippocampi were dissociated by trypsin treatment and trituration. 2006). Schlick et al. Carlsbad. the hippocampus. GmbH.477 2407 EXPERIMENTAL PROCEDURES RNA isolation from cultured hippocampal neurons Low-density cultures of hippocampal neurons were prepared from 16. and homogenized using QiaShredder columns (Qiagen. Total RNA was extracted from homogenized brain tissue using the RNeasy Protect Mini Kit (Qiagen.2 CaV1. Tissue samples were disrupted by using a rotor-stator homogenizer (Ultraturrax T8.4. In order to determine standard reliability all standard curves were repeated two to three times (including all steps. PD1. USA). Germany). Düren. Germany).4 CaV2.5-day-old embryonic BALB/c mice as described (Obermair et al. All examined tissues and cells expressed the full complement of subunit isoforms. / Neuroscience 167 (2010) 786 –798 787 However. qRT-PCRs of the standard curve samples were performed in triplicates and samples without template (background) served as negative controls. Neurons were plated on poly-L-lysine-coated glass coverslips at a density of 7000 cells/cm2. RNA isolation from tissue BALB/c mice of different age (E16.B. alteration of the electrical activity of cultured hippocampal neurons did not affect the CaV expression patterns.1 and CaV1. The limits of detection (LOD) and quantification (LOQ) of each TaqMan assay were obtained by subtracting 3 and 10 times. RT mix was incubated for 60 min at 37 °C. Kaech and Banker. were purchased from Applied Biosystems (Foster City. Five or 24 days after plating coverglasses with neurons were removed from the dishes with glia cells. flanking primer pairs (Eurofins MWG Operon. GmbH. cerebellum. and cultured hippocampal neurons. Animal handling was in accordance with national and international standards of animal welfare. Together these data emphasize the great complexity of CaV expression in brain as well as in hippocampal pyramidal cells. average linear regressions were calculated for the combined results of all standard curve replicates. Hilden.1 CaV1. and indicate a limited role of differential expression in controlling the subunit and isoform composition in neurons.1 CaV2. Hilden. Karlsruhe. little to no quantitative comparable data exist on the expression of CaV isoforms in different brain tissues and cells. After plating. cells were allowed to attach for 3– 4 h before transferring the coverslips neuron-side-down into 60-mm culture dishes with a glial feeder layer. RNA concentrations were determined photometrically. Finally. hippocampus. USA). USA). and a rigorous normalization to endogenous reference genes allowed the direct comparison of the expression levels in mouse cortex. and the cerebellum. Characteristic developmental changes in the CaV subunit expression were evident in brain regions and cultured neurons. To bring more clarity into this complex situation we employed TaqMan quantitative RT-PCR (qRT-PCR) to measure the mRNA expression of all seven high voltageactivated CaV 1. Table 1. GmbH. OH. 2003. TaqMan gene expression assays (Table 1). Staufen. However.. further cut in four to six pieces and immediately transferred into RNAlater RNA Stabilization Reagent (Qiagen. CA.5% low melting point agarose gels (Amresco. designed to span exon– exon boundaries. Total RNA was extracted using the RNeasy Protect Mini Kit (Qiagen. Only data points in the logarithmic amplification range of the respective standard curve were included for regression analysis.3 CaV1. Germany). Germany) and QiaShredder columns. The respective tissues were CaV1. To this end the skull was opened from caudal to rostral and the brain was carefully removed and placed in ice cold Hank=s buffered salt solution. USA) and random hexamer primers (Promega. Solon. with the exception of CaV1. Reverse transcription was performed on 5 l of RNA using Superscript II reverse transcriptase (Invitrogen. Next the entire cerebellum was cut from the brainstem. Bands were excised. Concentrations of PCR products were determined using Quant-IT PicoGreen dsDNA Assay Kit (Invitrogen). the RT mix was incubated at 37 °C for 60 min.3 1 2 3 4 2 2 2 2 -1 -2 -3 -4 Mm00489257_m1 Mm00437917_m1 Mm01209919_m1 Mm00490443_m1 Mm00432190_m1 Mm00432226_m1 Mm00494444_m1 Mm00518940_m1 Mm00659092_m1 Mm00432233_m1 Mm00521623_m1 Mm00486607_m1 Mm00457825_m1 Mm00486613_m1 Mm01190105_m1 . Quantitative real time PCR The relative abundance of different CaV subunit transcripts was assessed by TaqMan qRT-PCR using a standard curve method based on PCR products of known concentration. Neurons and glial feeder layer were cultured in serum-free neurobasal medium (Invitrogen GmbH. Ebersberg. Germany) to confirm the integrity of the obtained fragments. Madison WI. The generation of standard curves enabled the quantitative comparison of the transcript levels between the individual genes.

01 36.506 3.520 3.0 software. To correct for multiplicity in analyses involving many pairwise comparisons the Holm procedure (Bonferroni step-down correction) was applied (Bender and Lange.048 0. beta-2-microglobulin (B2M).22 0. data were normalized based on the most Table 3.043 0.992 0.989 0.028 0.995 0. Schlick et al.029 0.043 0.1 CaV1. Properties of standard curves and limits of detection (LOD) and quantification (LOQ) B CaV1.48 37.036 0.995 0. IL. HPRT1 and SDHA were determined to be the most stably expressed reference genes when comparing all preparations (Suppl.25 0. Statistical significance was determined by Student’s t-test and ANOVA. Mm00437762_m1. Endogenous controls and data normalization To compare the relative expression of distinct CaV subunits in different preparations. / Neuroscience 167 (2010) 786 –798 Table 2.997 0.13 0.2 CaV1. normalized molecule numbers were calculated for each CaV subunit from their respective standard curve. -1 -2 -3 -4 SE-B.535 3.533 3. Measurements were performed on at least three independent RNA preparations from each tissue and developmental stage.035 0. Mm99999915_g1.050 0.788 B.05 were considered as statistically significant. Mm00441941_m1.053 0.11 0. d LOQ.556 3. 2001).3 1 2 3 4 2 2 2 2 Reverse primer 5=-atgagcatttcgatggtgaag-3= 5=-caggtagcctttgagatcttcttc-3= 5=-aggacttgatgaaggtccacag-3= 5=-gtaccaccttctccttgggtacta-3= 5=-ccagtcttctggaacatctcttg-3= 5=-tatcatgagagcagcatagacctt-3= 5=-gtctgttaccaccagagattgttg-3= 5=-ctgcctccttccttaaggcttc-3= 5=-ctctcttgggtttcagagtcaaa-3= 5=-acagtagctgacattggtcctcac-3= 5=-tgtctcattcgctgactctgtaat-3= 5=-acagtccagtaaaccactgaatga-3= 5=-cttcctgtccagcaggctct-3= 5=-atttaatccctgggtactgtctga-3= 5=-caaggaagtctctgcaaccag-3= Fragment size (bp) 349 201 327 324 306 408 267 266 317 216 288 347 273 305 337 -1 -2 -3 -4 5=-gttacatgagctggatcacacag-3= 5=-atgcaagacgctatgggctat-3= 5=-acattctgaacatggtcttcacag-3= 5=-ctcttcatctgtggcaactacatc-3= 5=-ggtcacacctcacaagtccac-3= 5=-cacttagacgaattcattcgagtct-3= 5=-aaggtaaagaaacagagacagcag-3= 5=-gatcctctccatggtccagaa-3= 5=-gactatctggaggcatactggaag-3= 5=-cccatgtatgacgactcctacg-3= 5=-gctgattaagtccagaggaaagtc-3= 5=-gcatgatgagacacctggttaata-3= 5=-ccgctcttgctcttgctg-3= 5=-gtatgaatacttcaatgctgtgctg-3= 5=-cacatctcccaaagacatcgt-3= respectively. To allow a direct comparison between the expression levels in different tissues. LOQ was determined by subtracting 10 times the root mean squares of the residuals.3 CaV1. qRT-PCR (50 cycles) was performed in duplicates using 20 ng total RNA equivalents of cDNA and the specific TaqMan gene expression assay for each 20 l reaction in TaqMan Universal PCR Master Mix (Applied Biosystems).18 0. limit of detection (number of transcripts). Austria) using a 10 objective.3 CaV1. we normalized all experiments to the Ct value of HPRT1 in the RNA preparation yielding the highest HPRT1 Ct value. Mm00446968_m1. molecule numbers were calculated from the raw Ct values. The number of neurons per coverslip was extrapolated to the total number of neurons per RNA preparation and RT reaction and. Mm00607939_s1.474 3. transferrin receptor (TFRC).13 0.1 CaV1. Subsequently.23 0. finally.993 0. the root mean squares of the residuals. Wien.037 0.449 3.b 38.80 38.040 0. 1).998 0.996 LODc 3 37 60 5 4 5 4 3 6 12 5 5 22 4 3 LOQd 15 142 78 34 102 155 59 34 190 114 137 127 117 59 27 Data analysis and statistics Data were organized and analyzed using MS Excel and SPSS statistical software (SPSS Inc. including background values.998 0.14 37. SE-Ba 3..412 0. -Y.17 0.692 3.19 0.86 37. For determining the number of molecules per neuron (see below)..23 36.2 CaV1. Y-intercept (Ct value).995 0.17 0. .54 41. cDNA specific primer sequences for standard template amplification Forward primer CaV1. succinate dehydrogenase complex.028 Y-int. 2002). limit of quantification (number of transcripts).993 0. Mm01352363_m1. standard error of B and Y intercept.4 CaV2.039 0.2 CaV2.1 CaV2.4 CaV2. tata box binding protein (TBP). Mm00446973_m1.436 3. from the Ct value of the Y-intercept (Table 3).. The endogenous control genes included were [name (gene symbol).491 3.995 0. subunit A (SDHA). Analyses were performed using the 7500 Fast System (Applied Biosystems). c LOD.572 3. Graphs and figures were generated using MS Excel. respectively.420 3. After Holm correction P-values 0.058 0. to the amount of RNA equivalents used for the qRT-PCR reaction. USA) as indicated.997 0.02 38.88 37. In cases where assays did not show background Ct values or the spread of the background Ct values was large. Origin 7.07 37.990 0. glyceraldehyde-3-phosphate dehydrogenase (GAPD).497 3. Coverslips of four 24 DIV hippocampal cultures were stained with Hoechst dye to distinguish nuclei and analyzed on an Axiovert 200M microscope (Carl Zeiss GmbH.648 3.20 0.023 0.41 38. assay ID (Applied Biosystems)]: -cytoplasmic actin (ACTB).18 0.58 38.13 R2 0. 2003). excluding background.1 CaV2. All standard curve data were included for this analysis.36 37.3 1 2 3 4 2 2 2 2 a b stable control gene determined as previously described (Vandesompele et al. The Ct values for each CaV gene expression assay were recorded for each individual preparation. Estimating the number of transcript molecules per cultured neuron To estimate the number of transcript molecules of each CaV subunit we first determined the number of neurons per coverslip. Y-int. and Adobe Photoshop 8.994 0. from the cycle threshold (Ct) value of the Y-intercept (Table 3) (Corley.27 0.34 39.16 0.70 SE-Ya 0. hypoxanthine phosphoribosyl-transferase 1 (HPRT1). All data are presented as mean SE for the indicated number of experiments. Chicago.2 CaV2.

Nevertheless. CaV2. except the skeletal muscle CaV1.. One reason being the inherent difficulty to quantitatively compare the outcomes of methods based on different antibodies. 2003). Expression profile of the high voltage-activated Ca2 channel . or PCR primers. 1. Quantitative TaqMan RT-PCR analysis is the state-of-the art approach to analyze relative mRNA amounts in distinct probes. Arikkath and Campbell. lum. respectively. Overall.4. Cerebellar expression levels of L-type channels also differed from those in the other two brain regions in that the ratio of CaV1. provided a suitable and stably expressed reference gene has been identified. 2005) and thus strengthens the confidence in our new quantification method. and CaV2. when compared to the other auxiliary subunits its expression levels were negligible. the L-type channels (CaV1. and CaV2. 1A–C). Levels of 2 -3 are uniform throughout the brain regions tested. * P 0. cortex.2 is higher and CaV1.1 and CaV1.1. Cole et al. However. which is highest expressed in hippocampus. which were expressed at 1000-fold lower levels. (A) Hippocampus (blue). 2000.B. around the limit of quantification (Fig. and cerebel1.01. Schlick et al.4. 1A).1 is the most abundant isoform.3. and to get an accurate estimate on the quantities of each transcript relative to each other. whereas it was 1:1 in Fig. C). 2 -3). quantitatively comparable expression levels of the distinct CaV subunit isoforms in different brain regions of a single mammalian species were missing.1. 1997. and 2 subunits in mouse hippocampus.2. CaV1. Barclay et al. riboprobes.. and cerebellum (yellow) express all CaV 1 subunits except CaV1. cortex (green). cerebellum showed some striking differences. 2 and 4 are the dominant isoforms in hippocampus and cerebellum. cortex. 2 -2.3. Subunit expression levels in hippocampus and cortex are similar with the exception of CaV2. Hobom et al.3 lower than in hippocampus and cortex. (C) 2 -1 is the major 2 isoform in hippocampus and cortex.3 was 4:1 in cerebellum.1 and the retinal CaV1.2 is uniformly expressed in all three brain regions. and of all four subunits in the mammalian brain (for review see Catterall. 2-way ANOVA plus post hoc ANOVA with Holm correction. The high expression of these three CaV isoforms in the cerebellum confirms previous findings (Ludwig et al.2 to CaV1. CaV2. although above the limit of quantification in all qRT-PCR runs. and cerebellum Previous studies demonstrated the existence of mRNA and protein of CaV 1 (CaV1. In cerebellum CaV2. error bars: SEM.. Compared to the other auxiliary subunits 2 -4 expression levels were negligible. Moreover.3).. cortex. the quantitative comparison of the expression level of different genes is hampered by variations in the sensitivities and amplification kinetics of the various qRT-PCR assays. and 2 -2 were the dominating isoforms in cerebellum and were expressed two to threefold higher compared to cortex and hippocampus (Fig.2 and CaV1.2. Hippocampus. Whereas the expression profile was fairly similar between cortex and hippocampus. Therefore we combined relative quantification based on endogenous control genes with absolute quantification based on individual standard curves. In cortex subunits are expressed at similar levels. 2 ( 2 -1.1.3). 2001. / Neuroscience 167 (2010) 786 –798 789 RESULTS CaV expression profiles of mouse hippocampus.05. . all four subunit genes and three of the four 2 subunit genes were robustly expressed in these brain tissues (Fig. 4. 2000. CaV2. 1B. CaV2.2. and cerebellum of adult (8 weeks old) mice expressed all high voltage-activated CaV 1 subunits. ** P 0. CaV2. and cerebellum. CaV1. This allowed us to determine the amount of each CaV isoform transcript in different mouse brain regions and cultured hippocampal neurons. Furthermore. cortex. 2 -4 molecule numbers were above the limit of quantification in all qRT-PCR runs. (B) mRNA of all four subunits is present in hippocampus.3) were expressed at lower levels than each of the non-L-type channels (CaV2. whereas in cerebellum it is 2 -2.

2. (B) In hippocampus the overall developmental changes are less striking than in cortex. but not for total subunits. Moreover. 2A. / Neuroscience 167 (2010) 786 –798 Fig. In contrast to whole cortex.3. four isoforms showed stable expression levels (CaV1. 2 -2). 1).790 B. the increase in 2 and the significant drop of 3 levels between E16 and PD1 are more pronounced. and 2 -2 significantly decline. Individually.. 2 -1 and 2 -3). B). 2 and 4 expression levels significantly increased. A similar trend was observed for the 2 subunits. 2007.2. whereas levels of 2 and 4 increase. the total number of CaV 1 transcripts was about twofold larger in developing than in mature brain. Sinnegger-Brauns et al. 1. CaV2. we next sought to investigate whether and how their expression patterns change from late embryonic until adult stages. and at 2 weeks and 8 weeks of age (Fig. CaV2. However. this isoform was markedly reduced in the cerebellum (Fig. To this end we analyzed the expression profiles on embryonic day 16 (E16). Finally. and 2 and 8 weeks old BALB/c mice.. In cortex three different patterns of developmental changes could be observed (Fig. Our observation that CaV1. error bars: SEM. postnatal day 1 (PD1). the expression of CaV2.3. and 2 -1 and 2 -3 remain stable. 1C). 2004).2. 2000. Thus.2. CaV2. CaV2. 3. Developmental changes of CaV subunit mRNA expression in cortex and hippocampus.1 and CaV1. 2. Unexpectedly the CaV2. Interestingly. CaV subunit expression profiles were determined in embryonic (E16). cortex and hippocampus. * P 0. CaV2. CaV1. Actually they were the highest expressed 1 and isoforms in the hippocampus. in contrast to the 1 subunits. the total number of subunit transcripts remained fairly . Levels of CaV1. 2009).. levels of CaV2. CaV2.01.3. Whereas 1 and 3 levels declined as the majority of the 1 subunits. expression of 2 -4 increases 20-fold during development. Although total mRNA levels are negligible in comparison with the other 2 subunits. 1.3.4 were absent from both tissues at all time points (Fig. (A) During development cortical mRNA levels of CaV1. whereas 2 -1 was the dominating 2 subunit isoform in cortex and hippocampus.2 and 2 -3 was remarkably uniform throughout the three brain regions. 2-way ANOVA plus post hoc ANOVA with Holm correction. 2A): the majority of isoforms showed decreasing expression levels (CaV1. and the levels of two isoforms ( 2 and 4) showed a significant increase with development. CaV expression patterns change during development Because CaVs have been previously demonstrated to be important for neuronal maturation and development (Pravettoni et al.3 did not decline.3 transcripts accounted for only 20% of cerebellar L-type channels is again consistent with previous observations (Koschak et al.05. ** P 0..1. 2). postnatal (PD1). subunits showed very pronounced developmental the changes. 3. Splawski et al. Schlick et al.3 and 2 mRNAs were twofold higher in the hippocampus than in cortex and cerebellum.1. In line with the expression profiles of 8 weeks old mice (Fig.

2 -4 levels were analyzed separately in five DIV and 24 DIV old neurons (cf. First. Second.1 was the most abundant 1 subunit. Cultured hippocampal neurons express the same subunit isoforms as adult hippocampus The overall CaV expression profile in hippocampus. Therefore. with one exception (CaV2. (B) In cultured neurons all isoforms are expressed at equal amounts. On the other hand. to mostly 2 and 4 in the mature cortex. Interestingly. ** P 0. However. The most striking difference was the fact that CaV2. Specific developmental upregulation of CaV2. In light of this it is remarkable that qRT-PCR analysis revealed the same set of CaV subunit isoforms in differentiated cultured hippocampal neurons (24 DIV) as in adult hippocampus expressed (Fig. As a consequence. 2B) expression levels of many subunits showed the same tendency as in whole cortex. 3B. Low density cultured hippocampal neurons serve as an ideal model to address this question. 2-way ANOVA plus post hoc t-test with Holm correction. 1994.. 3. 3). / Neuroscience 167 (2010) 786 –798 791 constant during development. However. This drop was also observed in cortex (Fig. 2003). Schlick et al. In the developing hippocampus (Fig.. and cerebellum represents the sum of RNAs derived from a large number of different neuronal and non-neuronal cell types. their ratio shifted from predominantly 1 and 3 in embryonic cortex. 3A).3) the expression levels of the 1 isoforms were similar to those in hippocampus (Fig.3 and 1 levels did not show the developmental decline observed in cortex. Similar to CaV2. and 2 subunits in differentiated cultured hippocampal neurons (24 DIV). Obermair et al. 2A). Consequently the hippocampus-specific expression profile of the auxiliary subunits was not observed in the cultured neurons. In the culture CaV2. but always below detectability. this cell culture system is a pure neuronal culture without glia. 4 and Suppl. a decline of a specific transcript in one cell type might coincide with an increase of the same transcript in another cell type and thus mask the developmental change. C). 2).3. in addition to understanding the overall CaV channel expression patterns in brain and distinct brain regions. 2 and 2 -1 (and to a lesser degree also 1 and 4) showed substantially lower expression levels in hippocampal neurons than in whole hippocampus.01. Fig. although still at a very low level.3 in the culture were much lower than in hippocampus. but at significantly lower levels than in hippocampus. cortex. The developmental increase of 2 was even more pronounced whereas 3 showed a dramatic drop within the short period between E16 and PD1. Fig. Fig. error bars: SEM. Transcript levels of CaV2. developmental changes may indicate an overall change in all cells. it represents a highly homogenous culture with 90% glutamatergic pyramidal cells (Benson et al. expression of CaV2. (A) The majority of 1 subunits show similar expression levels in the cultured neurons as in 8 weeks old hippocampus. it is vitally important to understand the subunit expression in single types of neurons. but 1 to 4 and 2 -1 to 2 -3 were all expressed at approximately equal amounts (Fig.B. (C) 2 subunits are expressed at equal amounts and generally lower levels than in hippocampal tissue. in hippocampus expression of 2 -4 increased 20 fold during development. . or it may result from manifold greater changes in subpopulations of cells.1 and 2 -2 in cultured hippocampal neurons Cultured hippocampal neurons undergo dramatic morphological changes during growth and differentiation in vitro. This indicates that the complexity of CaV expression pattern exists in individual neuron types also and does not only arise from a mix of different cell types with distinct sets of calcium channels. Expression profile of high voltage-activated Ca2 channel 1. Surprisingly. .3 was 5-fold lower in cultured neurons.

amounts of CaV2. Obermair et al. 2006. 2003) and likely also directly (Gomez-Ospina et al. (A) The majority of CaV isoforms shows a slight increase in transcript levels between 5 and 24 DIV. Schlick et al. error bars: SEM. and 4. These homeostatic alterations are caused by both presynaptic and postsynaptic adaptations also involving gene transcription.and postsynaptic compartments and are indirectly (Deisseroth et al. 2 -2. and blockage by DL-2-amino-5-phosphonopen- Fig. Blocking neuronal activity does not alter the CaV expression pattern in cultured hippocampal neurons Neurons adapt to alterations in the activity status and synaptic transmission by homeostatic plasticity (Turrigiano. In contrast. Subramanyam et al. overnight application of TTX did not affect the expression level of any CaV subunit isoforms (Fig. This increase is most obvious for CaV2. 2003). 2003. whereas increased activity leads to a decrease therein.. (B) Blocking electrical activity by TTX for 24 h did not alter the expression level of any CaV subunit isoform.g. Nmethyl-D-aspartate (NMDA) receptors are critically involved in the control of plasticity-related gene expression and long-term memory formation (e. Well differentiated cultured hippocampal neurons display robust spontaneous activity. Among the subunits 4 showed the strongest increase. 4B) although the same experimental paradigm induced the nuclear translocation of the CaV 4b subunit (Subramanyam et al.1.05. which can be suppressed by tetrodotoxin (TTX) or further increased by blocking inhibitory input with bicuculline (Harms and Craig.3 and 2 -1 transcripts decrease during in vitro development.3 and 2 -1 decreased during the same time frame. Decreasing overall activity generally leads to an increase in the cellular excitability.. In contrast to the overall trend. 2-way ANOVA plus post hoc t-test with Holm correction. Surprisingly. 4A).1 and the 2 -2 subunit. 2009) involved in the regulation of transcription. Interestingly. In the following weeks the neurons develop a large and elaborate dendritic tree with numerous dendritic spines and an increasing number of synapses (Fletcher et al. 2008).. / Neuroscience 167 (2010) 786 –798 The first week is characterized by massive neurite outgrowth. Dolmetsch. the maturation of the dendritic tree starts around 5–7 DIV. 4. 2009).. 1994. Because CaVs are major constituents of both pre.792 B.. we tested whether their expression patterns change upon blocking neuronal activity. While first synapses appear around 3 DIV. expression levels of CaV2. Effects of development and neuronal activity on CaV subunit expression in cultured hippocampal neurons. 2004).. both of which experienced a significant up-regulation. during this period of continuous differentiation from 5 DIV to 24 DIV most CaV subunit isoforms showed only a slight increase or remained expressed at constant levels (Fig. 2009). The notable exceptions were the P/Q-type channel CaV2. Jordan and Kreutz. Turrigiano and Nelson. * P 0. . 2005.

CaV2.1 in cerebellum was paralleled by high expression of 4 and 2 -2.. 2009). Pichler et al. and it adds the new finding that.1 mRNA that has been reported in a study on human brain (Takahashi et al. Apparently specific properties of the 2 transcript. 1997. Doering et al. Fig. This is consistent with previous results of a Western blot and in situ hybridization study (Ludwig et al. This confirmed the high 2 expression levels (data not shown). this expression profile is characteristic for cultured hippocampal neurons and distinct from CaV expression patterns in other differentiated neurons.and postsynaptic compartments must be responsible for assembling channels with distinct subunit compositions (Obermair et al. However. like the secondary structure. Thus CaV expression of individual neuron types may differ greatly from the overall hippocampal profile. or transcriptional regulation in the hip- . Sinnegger-Brauns et al. DISCUSSION This is the first study providing a comprehensive calcium channel expression profile of mouse brain and cultured hippocampal neurons. 2008).. Nevertheless. Doyle et al.3 were the only L-type calcium channels expressed in mouse brain. In hippocampus 2 and 2 -1 were the highest expressed auxiliary subunits... In the family of the non-L-type calcium channels.3 were expressed at equal levels. Thus. a restricted expression of auxiliary subunit isoforms is not the strategy to achieve specific CaV subunit compositions..B. Postsynaptically. and 2 -1 levels in hippocampal neurons... and 2 -2 (ducky and entla) (Burgess et al. unpublished results). 1997. In cortex and hippocampus CaV1. Overall. The CaV expression profile in hippocampus reflects the sum of expression patterns in a variety of different neuronal and non-neuronal cells. Nevertheless.. CaV2. 4 (lethargic).. Interestingly. 2009).1 in the cerebellum. we reanalyzed selected samples with a second 2 assay. hippocampus. although the expression levels of all four subunits and 2 -1 through 2 -3 were fairly uniform. 1997. as well as with the similarity of phenotypes reported for mutant mice of CaV2.3 expression levels (cf. Each of the four subunits and three of the four 2 subunits were expressed in all examined brain regions.2 and CaV1. like dorsal root ganglion or cerebellar granule neurons (Obermair et al. 2005) and the involvement of CaV1.3 has been shown to be involved in presynaptic transmitter release in calyx-type terminals in the trapezoid body (Wu et al. 2003. 1998. expression of L-type calcium channels was lower than that of non-L-type channels. 1999) and in presynaptic LTP in mossy fiber synapses (Dietrich et al. Turrigiano. Therefore it was quite surprising to find that cultured hippocampal pyramidal cells express all the same CaV subunit isoforms as hippocampus. This study is unique in that it quantifies the expression of the full complement of high voltageactivated calcium channels 1 together with all 2 and subunits... and in that for the first time this quantification is thoroughly based on a quantitative assessment of the underlying assay variability. 2003). the previously reported low signals of in situ hybridization and the high raw Ct values observed in our own. 1998. Schlick et al. also chronic blockage of NMDA receptors did not change the CaV expression profile (Suppl.. which consist of mostly pyramidal cells.. 2002. The CaV transcriptome of mouse brain regions and cultured hippocampal neurons Comparing the calcium channel expression profiles of cortex.3). Generally this is in agreement with published data on the relative L-type CaV distribution in different rat and mouse brain regions (Qin et al. 2007. CaV2. This suggests that in cultured hippocampal pyramidal cells.3 is a major contributor to action potential evoked calcium transients in dendritic spines and is involved in postsynaptic plasticity (Yasuda et al..3 in depression (Busquet et al. 2. Either neurons other than pyramidal cells account for the prominent expression of these isoforms in the hippocampus. Consequently specific targeting properties and interactions with anchoring proteins in pre.1 (tottering. Because our 2 TaqMan assay showed a comparably low sensitivity (high Ct values and standard curves). may render it less accessible to riboprobes and PCR primers. One striking difference to hippocampus tissue was the reduction of CaV2.. 1997. Day et al. This would explain both. 2003).. However. levels of CaV1.. 1997) the overall high levels of 2 transcripts in all brain regions was not to be expected. we observed the expected high levels of CaV2. and thus was the dominant CaV isoform. In the cerebellum they were found at a ratio of 4:1 (CaV1.3 mRNA in the cortex and hippocampus can be as high as those of CaV1. our expression profile confirms previous data on the expression of L-type calcium channels in the brain. did not show similarly prominent CaV2.. CaV2.2: CaV1.. SinneggerBrauns et al. Based on experiments involving specific 2 riboprobes and antibodies (Ludwig et al. CaV1. The tissue-specific prevalence of CaV2..2. thus allowing a direct comparison of expression levels between all isoforms and across multiple preparations. / Neuroscience 167 (2010) 786 –798 793 tanoic acid (DL-AP5) leads to a local increase of synaptic NMDA receptors without inducing overall homeostatic changes (Rao and Craig. This is important in light of their distinct roles in the formation of long term memory (Moosmang et al. hippocampal neurons in culture. at least in some species or mouse strains. 2). 3A).. 2004). which is consistent with results from previous dihydropyridine binding studies and qRT-PCR analysis (Koschak et al.3.3 was expressed at twice the levels found in cortex and cerebellum. both individually and in sum. we found no evidence for the expression relatively high levels of Cav1. 1997. 1997). The high expression levels observed here may correspond to this twofold role in hippocampal neurons. 2010).. Brill et al. 2007. Barclay et al. In hippocampus however. and cerebellum revealed expression of the same complement of CaV isoforms. leaner). Obermair et al. 1997). 2001...1 was also the highest expressed 1 subunit in the cortex. 2009). 2003). previous Western Blot experiments performed on rat and rabbit tissues still indicate a low 2 subunit abundance in brain (Ludwig et al. Pichler et al.2 and CaV1.

CaV2. During the period when the cultured pyramidal cells differentiate into axons and dendrites and form numerous synaptic contacts..1. 2005.3 remains expressed at constantly high levels.3 in the cortex experiences a dramatic decline. even though transcription rates are reduced. 2008).3 and CaV2. and 2 -3 (Fig. the same four auxiliary subunit isoforms correlate negatively with CaV2. and activity-dependent regulation of membrane turnover of CaV1. Leroy et al. At the late embryonic stage total 1 transcripts are in excess of total transcripts and around birth total expression of 1 subunits declines. while transcript levels of CaV1. 2008. brane expression of CaVs in heterologous cells as well as in neurons (Dolphin. CaV2.2. reflecting their disproportional high expression in cerebellum and their concomitant up-regulation during development of hippocampal pyramidal cells. Global changes in activity quickly induce synaptic scaling mechanisms involving gene-expression (Ibata et al. The isoform shift during neuronal differentiation is expected to result in a functional switch of calcium channels without actually changing the subunits are essential for mem1 subunit. Correlated expression of specific subunit isoforms Recurring 1. or upon differentiation the stabilization of CaVs in pre... Schlick et al. Within one cell type the most likely interpretation of a correlated up-regulation of CaV2. developmental expression of the subunits shows a marked isoform switch.794 B.1. 2010). Furthermore. Thus. Developmental changes of selected isoforms in brain and hippocampal neurons In the weeks following birth expression of CaV1. Together these changes give rise to the predominant expression levels of CaV2. The preference for the same auxiliary subunits may lead to a functional redundancy of CaV2. at 8 weeks after birth total 1 and transcript levels are nearly balanced. 2003) and is consistent with a change in the channels coupled to glutamate release from mainly N-type channels to P/Qtype channels during in vitro development (Scholz and Miller. Our correlation analysis is based on the following assumptions: (1) if CaV 1 isoforms have a preference for specific auxiliary isoforms. and 2 -2) could also be observed during the differentiation of cultured neurons (Fig. . or (2) if the expression of certain isoforms is co-regulated. thus becoming the predominant 1 subunit isoform in mature hippocampus.and down-regulation of these subunits in different cells. Thus correlation analysis may be useful to suggest or exclude the existence of such preferential CaV complexes. 2007).2.2 and CaV2. in which electrical activity sets in and synaptic connections are established. Therefore it is remarkable that a correlated up-regulation of the same subunits (CaV2. then this should be revealed in a correlation analysis. and 4 in- creases. and (3) if this preference or co-regulation is constant in different brain regions. the overall decline of functionally expressed CaV proteins may be smaller than indicated by the numbers of 1 subunit transcripts. Contrary to our expectation treating cultured hippocampal neurons with TTX or DL-AP5 did not result in any changes in CaV isoform expression.. This observation is consistent with the dramatic increase in the synapse number (Obermair et al. 5A). In tissues such a correlation could also arise from parallel but independent up. Therefore we examined whether the specific changes in CaV isoform expression observed during differentiation are activity dependent. 4. 5A) clearly identified the preference of CaV2. During early development of the cortex all these channels and auxiliary subunits undergo a strong and concomitant down-regulation. Expression of these 1 subunits correlates positively with 1.2.. In fact. 5B).1. In vitro differentiating cultured hippocampal neurons do not reflect the developmental changes observed in whole hippocampus. CaV2. their differential expression in cortex and hippocampus suggests that CaV2.1 for 4 and 2 -2 subunits. 8 weeks after birth the quantity of total 1 subunit mRNA is reduced to about half of that expressed in late embryonic development.2 and CaV2. If the available amount of subunits is limiting membrane expression of CaVs. CaV2.and postsynaptic compartments reduces their turnover rate and thus high levels of protein expression may be maintained. However. 1A. while total expression of subunits remains constant. 4. and 2 -2 is indeed that these subunit isoforms actually form a channel together.3.and down-regulation can be indicative of the existence of preferential subunit combinations in neurons throughout the brain.2 expression has been suggested (Green et al. none of the 1 subunits is up-regulated indicates that the pore-forming subunits do not undergo a developmental isoform switch. Another striking upshot of the correlation analysis is the similarity of correlations between CaV1. 1995).1 remain constant. Whereas 1 and 3 decline in parallel to the subunits. Either CaVs play more important roles in developing neurons than so far anticipated.3 in supporting synaptic transmission.1.3 and 2 in the 8 weeks old hippocampus when compared to cortex (see Fig.3 declines. independent of electrical or synaptic activity. and CaV2. 2003. and 2 expression patterns in different samples or their coordinated up. In contrast. Considering the numerous important functions of voltage-gated calcium channels in mature neurons this developmental drop is remarkable. 2 -2. Obermair et al. / Neuroscience 167 (2010) 786 –798 pocampus is strongly dependent on cell-cell interactions or trophic factors missing in the culture system. The fact that during this critical period. expression of 2 and 4 experiences a significant increase. This indicates that the observed developmental changes in CaV expression in neurons may be intrinsically regulated. B). cells and conditions. In hippocampus developmental changes of CaV isoforms mirror those of cortex with one notable exception. Furthermore the developmental up-regulation of 2 is more pronounced than that of 4. 3. Interestingly. 2 -1. For example the correlation analysis (Fig. whereas expression of mainly presynaptic isoforms CaV2.3 may . suggesting that calcium channels change their subunit composition upon neuronal differentiation.2 and CaV2. The same is true for the 2 subunits.

.2 or 2. However. a The assay for 2 -4 was not included in the same qRT-PCR experiments.0 2. ** P 0. respectively. 4 and Suppl. 1975) a single ribosome yields approximately 10 proteins per hour. Fig. Interestingly. The total number of transcripts per cell was 26 1.3 CaV1.1 CaV2. Ino et al. each consisting of eight channels on average (Obermair et al.3 results in little to no neurological phenotype (Saegusa et al. 2005).1 in null-mutant mice causes severe neurological disease (Jun et al. Transcript numbers of individual neurons Estimated transcript numbers of the different CaV subunit isoforms per cultured hippocampal neuron ranged from 0 to 12 (Table 4).1 CaV1. in total 32.3 did not reveal a strong correlation with any auxiliary subunit. 2000.1 and CaV2. and 1200 2 molecules per hour and neuron. and 2 -3 on the other side may explain why loss of CaV2.0 5.. Asterisks mark significant correlations and the gray area indicates the cut-off (r 0.2. The abscissa represents the size and direction of the correlation coefficients whereby direct (positive) and indirect (negative) coefficients are indicated by green and red bars.3 by including only the measurements from cultured hippocampal neurons. Assuming a maximal translation rate of 5 amino acids per second (Boehlke and Friesen.8 2.2 4.2 CaV2. The specific subunit combination CaV2.3 1 2 3 4 2 2 2 2 Number of transcript molecules 0.1 0.4 CaV2. (B) Similar correlation coefficients were identified for CaV2. depending on the length of the reading frame a single transcript will yield different numbers of proteins per unit time. 2 -1.4 2. Number of mRNA molecules in a single cultured hippocampal neuron CaV subunit CaV1.4) for weak correlations.B.01. be dominating in distinct cell types.3 1.3 3. then our present data predict the translation of 2600 1.1 1 subunit with 4 and 2 -2. 2001). With the translation rates estimated Table 4. whereas loss of CaV2. 3.2 0.3 showed similar degrees of correlations with the same set of and 2 subunits.2 clusters.05. . However. Expression of CaV1.000 proteins. 20 2 . and 40 assume that every transcript is occupied by 10 ribosomes (Mata et al. 16 .0 -1 -2 -3 -4a Calculations are based on the qRT-PCR experiment on cultured hippocampal neurons (24 DIV) with highest RNA yield. and 6 2 s.4 3. 2004). / Neuroscience 167 (2010) 786 –798 795 Fig. in other qRT-PCR runs on cultured hippocampal neurons 2 -4 levels were always below detectability (cf.. If we further 1.. CaV2.2. 5. 2). * P 0.2 CaV1. Correlation analysis of CaV 1 subunit expression with individual and 2 isoforms. CaV1.1/ 4/ 2 -2 on one side and a promiscuous association of CaV2.6 4.2 5.0 11.8 2. Schlick et al. Pearson correlation. On the protein level our previous quantitative immunofluorescence analysis revealed that an average cultured hippocampal neuron may contain approximately 4000 CaV1. 6400 . and CaV2.2 and CaV2. Correlation coefficients were calculated between the transcript amounts of the individual 1 and the auxiliary and 2 subunits including measurements from all tissue and culture preparations.3 with any combination of 1. 1999).. Fig. (A) Correlation analysis clearly identified the previously demonstrated association of the CaV2.

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