Regional expression and subcellular ... - Wiley Online Library

JOURNAL OF NEUROCHEMISTRY

| 2012 | 122 | 1095–1107

doi: 10.1111/j.1471-4159.2012.07853.x

Dept. Ciencias Me´dicas, Instituto de Investigacioń en Discapacidades Neurolo´gicas (IDINE), Facultad de Medicina, Universidad Castilla-La Mancha, Albacete, Spain

Abstract Ca2+ channel b subunits determine the maturation, biophysical properties and cell surface expression of high voltage-activated channels. Thus, we have analysed the expression, regional distribution and subcellular localization of the Cavb subunit family in mice from birth to adulthood. In the hippocampus and cerebellum, Cavb1, Cavb3 and Cavb4 protein levels increased with age, although there were marked region- and developmental stage-specific differences in their expression. Cavb1 was predominantly expressed in the strata oriens and radiatum of the hippocampus, and only weakly in the cerebellum. The Cavb3 subunit was mainly expressed in the strata radiatum and lucidum of the hippocampus and in the molecular layer of the cerebellum. During development, Cavb3 protein expression in the cerebellum peaked at postnatal days (P) 15 and 21, and had diminished drastically by P60, and in the

hippocampus increased with age throughout all subfields. Cavb4 protein was detected throughout the cerebellum, particularly in the molecular layer, and in contrast to the other subunits, Cavb4 was mainly detected in the molecular layer and the hilus of the hippocampus. At the subcellular level, Cavb1 and Cavb3 were predominantly located post-synaptically in hippocampal pyramidal cells and cerebellar Purkinje cells. Cavb4 subunits were detected in the pre-synaptic and postsynaptic compartments of both regions, albeit more strongly at post-synaptic sites. These results shed new light on the developmental regulation and subcellular localization of Cavb subunits, and their possible role in pre- and post-synaptic transmission. Keywords: calcium channel, cerebellum, electron microscopy, hippocampus, immunohistochemistry. J. Neurochem. (2012) 122, 1095–1107.

Voltage-gated calcium channels (Cav) play a key role in mediating calcium entry into nerve termini. Calcium influx is regulated strictly to control calcium-dependent processes, such as neurotransmitter release, neuronal excitation and the regulation of gene expression. Biophysical and pharmacological studies have identified distinct channel types activated by high voltage- (HVA: CaV1 and CaV2) or low voltage-activated (LVA: CaV3) channels. In neurons, CaV1 (L-type) and CaV2 (N-, P/Q- and R-type) channels in neurons are composed of several subunits, including a pore-forming a1 subunit, and several auxiliary subunits, such as the b, a2d and c subunits (Buraei and Yang 2010). Four Cavb isoforms (b1–b4) have been identified, encoded by four distinct genes (Cacnb1–4), and each with several splice variants. The association between CaV1 and CaV2 channels and the cytoplasmic b subunits influence the biophysical properties of the channels, hyperpolarizing the voltage dependence of activation and increasing their open probability (reviewed by Buraei and Yang 2010). In addition,

Cavb subunits bind with high affinity to an intracellular motif between the I-II linker of CaV1 and CaV2 channels known as the a interaction domain (AID; De Waard et al. 1996; Pragnell et al. 1994), and mutations within this motif eliminated its cell surface expression and biophysical modulation (Leroy et al., 2005; Butcher et al. 2006). This molecular interaction between Cavb subunits and the I-II linker of Cav1 and Cav2 channels results in the protection

Received February 23, 2012; revised manuscript received June 17, 2012; accepted June 18, 2012. Address correspondence and reprint requests to Dr Rafael Lujań, Department of Ciencias Me´dicas, Instituto de Investigacioń en Discapacidades Neurolo´gicas (IDINE), Facultad de Medicina, Universidad Castilla-La Mancha, Campus Biosanitario, C/Almansa 14, 02006 Albacete, Spain. E-mail: [email protected] Abbreviations used: AP, alkaline phosphatase; IN, intracellular sites; PB, phosphate buffer; PM, plasma membrane; SDS, sodium dodecyl sulphate; TBS, tris-buffered saline.

2012 The Authors Journal of Neurochemistry 2012 International Society for Neurochemistry, J. Neurochem. (2012) 122, 1095–1107

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from proteasomal degradation, thus promoting expression in the cell surface (Altier et al. 2011; Waithe et al. 2011). As a mechanism for the effect on expression, mutagenesis approaches identified an acidic-residue putative ER export motif responsible for the I-II loop-mediated increase in channel surface density (Fang and Colecraft 2011). All Cavb subunits are expressed in the brain, particularly in the cerebellum and hippocampus (Ludwig et al. 1997). However, the membrane association and subcellular localization of Cavb subunits has yet to be described in detail. Although cytosolic, Cavb subunits localize to the plasma membrane (PM) through their association with Cav2.1 and they may be targeted to different subcellular compartments depending on the Cav2.1 subunit type with which they associate (Ruth et al. 1989; Perez-Reyes et al. 1992). For example, Cavb3 and Cavb4 associate predominantly with pre-synaptic Cav2 channels, and they are found in the axons of cultured neurons, whereas Cavb1 appears to associate with somato-dendritic Cav1.2 channels (Obermair et al. 2010). A deeper understanding of the ontogeny and distribution of Cavb subunits is crucial to define the role of CaV channels in the developing and adult brain (Pravettoni et al. 2000; Splawski et al. 2004). The goal of this study was to analyse the expression and regional distribution of endogenous Cavb1, Cavb3 and Cavb4 subunits in the mouse brain, specifically in the hippocampus and cerebellum. In addition, we analysed the subcellular localization of Cavbs in the adult mouse brain using electron microscopy. Our results demonstrate that the expression of Cavb subunits is developmentally regulated, and that these subunits have a characteristic region-specific distribution. Moreover, the subcellular localization of Cavbs reveals key differences between individual subunits and their effects on synaptic plasticity.

Materials and methods Tissue preparation Swiss mice (P0 to P60) obtained from the Animal House Facility at the School of Medicine of the University of Castilla-La Mancha were analysed in immunoblots, immunohistoblots and by light microscopy immunohistochemistry. The mice were housed on a 12-h light/dark cycle with ad libitum access to food and water. The care and handling of animals prior to and during the experimental procedures was in accordance with Spanish (RD 1201/2005) and European Union regulations (86/609/EC), and the protocols were approved by the University’s Animal Care and Use Committee. For each developmental stage, animals from different litters were used. For immunoblot and immunohistoblot experiments, animals were deeply anaesthetized by hypothermia (P0–P5) or by intraperitoneal injection of ketamine–xylazine (1 : 1, 0.1 mL/kg), and the hippocampus and cerebellum were extracted and frozen rapidly at )80C. For immunohistochemistry, animals were anaesthetized and transcardially perfused with ice-cold fixative containing 4% paraformaldehyde and 15% (v/v) saturated picric acid in 0.1 M phosphate buffer (PB, pH 7.4). After perfusion, the animal’s brain was

removed and immersed in the same fixative for 2 h or overnight at 4C. Tissue blocks were washed thoroughly in 0.1 M PB and coronal Vibratome sections were obtained [60 lm thick: Leica V1000 (Leica, Wetzlar, Germany)]. Antibodies The following primary antibodies were used: mouse monoclonal anti-a-tubulin (1 : 5000; Calbiochem, Merk4Biosciences, Darmstadt, Germany); rabbit polyclonal anti-Cavb3 (1 : 1000 for immunoblotting, 1 : 400 for immunohistoblotting and 1 : 250 for immunohistochemistry; Alomone Labs, Jerusalem, Israel); mouse monoclonal anti-Cavb1 and anti-Cavb4 (1 : 1000 for immunoblotting and immunohistoblotting, and 1 : 250 for immunohistochemistry; both from Neuromab, Davis, CA, USA). The specificity of the antibodies against Cavb1 and Cavb4 has been described previously (Obermair et al. 2010). The secondary antibodies used were as follows: goat anti-mouse IgG-horseradish peroxidase (1 : 2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat anti-rabbit IgG-horseradish peroxidase (1 : 15 000; Pierce, Rockford, USA), alkaline phosphatase (AP)goat anti-mouse IgG (H+L) and AP-goat anti-rabbit IgG (H+L) (1 : 5000; Invitrogen, Paisley, UK), goat anti-rabbit and anti-mouse IgG coupled to 1.4 nm gold (1 : 100; Nanoprobes Inc., Stony Brook, NY, USA). Immunoblotting The hippocampus and cerebellum from mice at different postnatal stages were homogenized using a motor and pestle in homogenization buffer [320 mM sucrose, 2 mM EDTA, 10 mM HEPES (pH 7.4)] containing a Protease Inhibitor Cocktail (Sigma-Aldrich, St Louis, MO, USA). The homogenized tissue was centrifuged for 15 min at 16 000 g and 4C in a microcentrifuge, and the pellet resuspended in ristocetin-induced platelet agglutination buffer [140 mM NaCl, 10 mM Tris, 1 mM EDTA, 1% Triton X-100, 0.1% sodium dodecyl sulphate (SDS), 0.1% deoxycholic acid (pH 8)] containing a Protease Inhibitor Cocktail. This solution was then incubated on ice for 15 min and centrifuged for 30 min at 16 000 g and 4C in a microcentrifuge. The supernatant (protein extract) was quantified and 50 lg of total protein were analysed in western blots using the ECL blotting detection kit (SuperSignal West Dura, Pierce, Rockford, USA). Blots were quantified by densitometry using a LAS4000 MINI (Fujifilm, Tokyo, Japan) and the band densities as load control normalized against those of a-tubulin. Developmental differences between normalized band densities were assessed using a two-way analysis of variance (ANOVA), and further compared using a Bonferroni post hoc test where necessary, with a minimum confidence level of p < 0.05. Immunohistoblotting The regional distribution of Cavb subunits was analysed in the mouse brain using an in situ blotting technique (To¨nnes et al. 1999; Fernandez-Alacid et al. 2011). Briefly, sagittal cryostat sections (10 lm) were placed on nitrocellulose membranes moistened with 48 mM Tris-base, 39 mM glycine, 2% (w/v) SDS and 20% (v/v) methanol for 15 min at room temperature (20C). After blocking in 5% (w/v) non-fat dry milk in phosphate-buffered saline, the nitrocellulose membranes were treated with DNase I (5 U/mL), washed and incubated in 2% (w/v) SDS, 100 mM b-mercaptoethanol in 100 mM Tris–HCl (pH 7.0) for 60 min at 45C to remove


Localization of Cavb subunits in the mouse brain | 1097

adhering tissue residues. After extensive washing, the blots were incubated overnight at 4C with Cavb antibodies (0.5 lg/mL) in blocking solution. The bound primary antibodies were detected with AP-conjugated secondary antibodies. To compare expression levels of each protein during development, all nitrocellulose membranes were processed in parallel, using the same incubation times and antibody/reagent concentrations. Indeed, for this reason, some regions with very low levels of expression may have been considered as negative and thus, we only performed quantitative analysis of expression from P5 onwards. Only labelling patterns obtained with the same antibody were compared. Digital images were acquired by scanning the nitrocellulose membranes using a desktop scanner (HP Scanjet 8300, HewlettPackard, Palo Alto, CA, USA), processing and analysing the Images using Adobe Photoshop software (Adobe Systems, San Jose´, CA, USA), as described previously (Kopniczky et al. 2005; FernandezAlacid et al. 2011). All the images were processed with the same equipment in the same way to allow comparison of the intensity of greyscale images at different postnatal ages and in different brain regions on different days. The pixel density (arbitrary units) of immunoreactivity was measured using open circular cursors with a diameter of 0.10 mm. We used background correction to eliminate potential differences in optical densities across different sections in different experiments. The average of eight background determinations carried out near the brain protein-containing areas of the immunostained nitrocellulose membranes was subtracted from the average pixel densities measured within brain regions. Following background corrections, the average pixel density for the whole region from one animal counted as one ‘n’. Under these conditions, labelling performed on different days produced very consistent results. Data were analysed and plotted using the software Analysis (Soft Imaging Systems, Munster, Germany). The differences between brain regions over time were assessed using one-way analysis of variance (ANOVA) and further compared with the Bonferroni post hoc test, with a minimum confidence level of p < 0.05. Immunohistochemistry for electron microscopy Immunohistochemical reactions for electron microscopy were carried out using the pre-embedding immunogold method, as described previously (Lujan et al. 1996). Briefly, free-floating sections were incubated in 10% (v/v) normal goat serum diluted in tris-buffered saline (TBS). Sections were then incubated with Cavb antibodies at a final protein concentration of 3–5 lg/mL diluted in TBS containing 1% (v/v) normal goat serum. After several washes in TBS, sections were incubated in goat anti-mouse IgG coupled to 1.4 nm gold (Nanoprobes Inc., Stony Brook, NY, USA). The sections were post-fixed in 1% (v/v) glutaraldehyde and washed in double-distilled water, and an HQ Silver kit (Nanoprobes Inc.) was used for silver enhancement of the gold particles. Sections were then treated with osmium tetraoxide (1% in 0.1 M PB), block-stained with uranyl acetate, dehydrated in a graded series of ethanol dilutions and flat-embedded on glass slides in Durcupan resin (Fluka). Regions of interest were cut at a thickness of 70–90 nm on an ultramicrotome (Reichert Ultracut E, Leica, Austria) and collected on single-slot pioloform-coated copper grids. Ultrastructural analyses were performed on a Jeol-1010 electron microscope (Jeol, Tokyo, Japan). As Ca2+ channels are synthesized in the rough endoplasmic reticulum and then are targeted to their final destination to specific subcellular compartments, the expected labelling pattern should be

detected along the plasma membrane and/or associated with membranes at the cytoplasmic site. We considered non-specific or background labelling when immunoparticles for Cavb subunits were observed in the lumen of capillary, over myelin sheath of myelinated axons or over cristae of mitochondria. Regarding subcellular structures containing Cavb subunits, labelled structures were classified as dendritic shafts, spines and axon terminals based on established ultrastructural criteria. Dendritic shafts of CA1 pyramidal cells or Purkinje cells were identified by the presence of dendritic spines, lack of small vesicles, diffuse filaments and numerous mitochondria. Dendritic spines were identified as small cell protrusion from dendritic shafts that typically contain a postsynaptic density forming the post-synaptic component of most excitatory synapses. Axon terminals were identified by the presence of synaptic contacts and small round and/or large granular vesicles. Synapses were identified as parallel membranes separated by widened clefts that are associated with membrane specializations. Synapses displaying a prominent density on the post-synaptic side were characterized as asymmetrical (putative excitatory), whereas synapses showing equivalent densities on both sides were characterized as symmetrical (putative inhibitory). Semi-quantitative analysis of Cavb subunits in the adult To determine the relative abundance of Cavb subunits in the hippocampus and cerebellum, immunolabelling was quantified in coronal slices (60 lm) processed for pre-embedding immunogold immunohistochemistry, as described previously (Lujan et al. 1996; Lujan and Shigemoto 2006). Briefly, for each of three animals, three tissue samples were obtained and mounted in embedding blocks. To minimize the false-negative results, ultrathin sections were cut close to the surface of each block. We estimated the quality of immunolabelling by selecting areas with optimal gold labelling at approximately the same distance from the cut surface. Randomly selected areas from the chosen ultrathin sections were then photographed at a final magnification of 45 000x. Immunogold labelling was quantified in reference areas of each brain region totalling 2000 lm2. Immunoparticles were identified for individual Cavb subunits in each reference area and counted along the PM and at intracellular sites in dendrites, spines and axon terminals. The percentage of immunoparticles in each subcellular compartment for each Cavb subunit type was then calculated. Controls To determine the specificity of the immunoblot, histoblot and electron microscopy procedures, primary antibodies were either omitted or replaced with 5% (v/v) normal serum from the species of the primary antibody. Under these conditions, no selective labelling was observed (Figure S1). Similarly, labelling patterns observed by electron microscopy were compared with that of Calbindin (Figure S1); only antibodies against Cavb subunits consistently labelled the plasma membrane.

Results Cavb subunits display different expression profiles during postnatal development To determine the developmental profile of Cavb1, Cavb3 and Cavb4 protein expression, total extracts of protein obtained



P10–P21, subsequently declining dramatically (Fig. 1e). The expression observed during cerebellar maturation corroborated that of a previous study (Vance et al. 1998).

from the hippocampus and cerebellum of individual mice at different developmental stages were analysed using Western blots. In accordance with the size of the three Cavb subunits (Ludwig et al. 1997), anti-Cavb1 recognized a band of approximately 80 kDa, anti-Cavb3 recognized a band of approximately 60 kDa and anti-Cavb4 recognized a band of 55 kDa in both brain regions. In the hippocampus, the expression of Cavb1 protein increased with age until P60, when the protein levels remained constant (Fig. 1a). Similarly, low-level Cavb3 expression was detected at earlier developmental stages, which progressively increased until they stabilized at P21 (Fig. 1b). Notably, the amount of both proteins detected increased fourfold between P0 and P60. Similarly, Cavb4 expression increased significantly during development until adulthood, resulting in a 25-fold increase between P0 and P60 (Fig. 1c). The expression profile of Cavb4 in the cerebellum was similar to that observed in the hippocampus, although Cavb4 levels increased 100-fold between p5 and p60 (Fig. 1f). Cavb1 protein accumulated progressively between birth and P60, although this effect was not significant (Fig. 1d). The cerebellar expression of Cavb3 differed from that observed in the hippocampus, as peak expression was reached at

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Differential regional expression of Cavb proteins during postnatal development To determine the regional expression of Cavb1, Cavb3 and Cavb4 subunits in the mouse brain, b subunit-specific antibodies were used in conventional immunohistoblotting. This method is a reliable and convenient way to compare the regional distribution of different proteins in brain samples without compromising the integrity of antibody-binding sites by tissue fixation, which is required for conventional immunocytochemistry (To¨nnes et al. 1999). Fixation introduces covalent modifications and cross-linking of proteins. These chemical modifications may alter the antibody-binding sites and cross-linked molecules may hinder the access of antibody to epitopes. The direct transfer of native proteins from unfixed frozen tissue sections to an immobilizing matrix offers much improved accessibility of the transferred proteins for immunochemical analysis. Two different anti-Cavb2 antibodies were also tested, but failed to provide an acceptable signal, possibly because of

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Fig. 1 Cavb subunit expression in the developing mouse hippocampus and cerebellum. Cavb subunit expression was determined at various time points during postnatal development. (a and d) Cavb1 protein levels increased significantly from P0 to P60 in the hippocampus and the cerebellum. (b and e) In the hippocampus, Cavb3 levels increased progressively between P0 and P60, whereas cerebellar Cavb3 levels increased between P5 and P10, remained similar

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between P10 and P21 and decreasing until P60. (c and f) Cavb4 levels increased significantly during development in both regions. The expression of these subunits was normalized to that of a-tubulin, and the densitometric measurements from four independent experiments were averaged for each developmental stage and normalized to those detected at P60. *p < 0.05; **p < 0.01; ***p < 0.001; two-way ANOVA with Bonferroni post hoc test; error bars represent the SEM.



Postnatal development The Cavb1, Cavb3 and Cavb4 proteins were expressed in the developing brain from P0 in a region-specific manner

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Adult brain (P60) Cavb1, Cavb3 and Cavb4 were widely distributed in the adult brain. Very strong Cavb1 immunoreactivity was observed in the hippocampus, caudate putamen and septum, with high levels in the neocortex and superior colliculus (Fig. 2a). Faint staining was observed in the thalamus and very weak staining in the cerebellum (Fig. 2a). Strong Cavb3 immunoreactivity was observed in the hippocampus, caudate putamen and neocortex (Fig. 3a), with moderate staining in the thalamus and cerebellum, and weak staining in the midbrain nuclei, including the inferior and superior colliculi (Fig. 3a). Cavb4 subunit immunoreactivity was very strong in the cerebellum, and strong immunoreactivity in the neocortex, caudate putamen and thalamus (Fig. 4a). Moderate staining was observed in the hippocampus and septum, and weak staining in the midbrain nuclei, including the inferior and superior colliculi (Fig. 4a). The hippocampus and cerebellum, in which the most intense Cavb1, Cavb3 and Cavb4 immunoreactivity was detected, were further examined in region- and layer-specific analyses. Densitometric measurements from six different experiments were averaged to compare the protein expression at each developmental stage and dendritic subfield. This analysis revealed an overlapping distribution of Cavb1 and Cavb3 in the hippocampus. Immunoreactivity for both subunits was strong in the strata oriens and radiatum of the CA1 and CA3, the molecular layer of the dentate gyrus and the stratum lucidum of CA3 (Figs 2b and 3b). However, weaker immunoreactivity was evident in the stratum lacunosum-moleculare of CA1 and CA3, and in the hilus of the dentate gyrus (Figs 2b and 3b). By contrast, immunoreactivity for Cavb4 was strong in the molecular layer of the dentate gyrus, and moderate in the strata oriens, radiatum and lacunosum-moleculare of the CA1 and CA3 and in the stratum lucidum of CA3 and the hilus of the dentate gyrus (Fig. 4b). In the cerebellum, all three subunits were distributed in a similar pattern, although the expression levels differed considerably: high for Cavb4, moderate for Cavb3 and weak for Cavb1. Thus, immunoreactivity for Cavb1, Cavb3 (Fig. 3a and c) and Cavb4 (Fig. 4a and c) was stronger in the molecular layer than the granule cell layer, in which moderate to weak staining was consistently detected.

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the limited accessibility of the Cavb2 subunit epitope in the native channel complex, or more likely because of its weak expression in the brain (Ludwig et al. 1997). In both the developing and adult brain, the overall expression of Cavb1, Cavb3 and Cavb4 subunits revealed marked regional- and developmental stage-specific differences.

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Fig. 2 Regional distribution of the Cavb1 subunit in the developing mouse brain. (a) The distribution of the Cavb1 protein was visualized in histoblots of saggital brain sections at various time points during postnatal development. The strongest expression was detected in the hippocampus and cortex, with weaker expression in the cerebellum. Scale bar = 0.4 cm. (b) Cavb1 expression in the different layers of the hippocampus was determined by densitometric analysis of the scanned histoblots. The expression of this subunit increased until P21 before it stabilized, and it was detected mainly in the stratum oriens and stratum radiatum of CA1 and the stratum moleculare of the dentate gyrus. The layers of the hippocampus are represented by their initials: stratum oriens (so), stratum radiatum (sr), stratum lacunosummoleculare (slm), stratum moleculare of dentate gyrus (sm), hilum of dentate gyrus (h) and stratum lucidum of CA3 (sl). Nine independent experiments were averaged to determine the protein density. In each layer of the hippocampus, the quantified density at different developmental time points was compared with that at P60. One-way ANOVA followed by Bonferroni post hoc test; error bars represent the SEM, df = 5, *p < 0.05; **p < 0.01; ***p < 0.001.

(Figs 2a, 3a and 4a). In the hippocampus, weak Cavb1 and Cavb3 expression was detected at P0, which increased after P5 to reach a peak at P21–P60 (Figs 2a and 3a). Weak Cavb4 expression was detected at P5 that increased progressively from P10 to P60, both in the CA1 and dentate gyrus (Fig. 4a). At all developmental stages, similar levels of Cavb1 and Cavb3 immunoreactivity were observed in CA1 and the dentate gyrus (Figs 2b and 3b), whereas Cavb4 immunoreactivity was generally highest in



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Fig. 3 Regional distribution of Cavb3 in the developing mouse brain. (a) Histoblots revealed Cavb3 expression to be strongest in the hippocampus, followed by the cortex and the cerebellum. Scale bar = 0.4 cm. (b and c) Hippocampal Cavb3 levels increased continuously during development, while they peaked at P15–P21 in the cerebellum. Cavb3 was predominantly expressed in the stratum oriens of CA1 The layers of the hippocampus are represented by their initials: stratum oriens (so), stratum radiatum (sr), stratum lacunosum-moleculare (slm), stratum moleculare of dentate gyrus (sm), hilum of dentate gyrus (h) and stratum lucidum of CA3 (sl). The cerebellar layers are denoted as molecular layer (ml), granule cells (gc) and white matter (wm). Nine independent experiments were averaged to determine the protein density. In each layer of the hippocampus and cerebellum, the quantified density at different developmental time points was compared with that at P60. One-way ANOVA followed by Bonferroni post hoc test; error bars represent the SEM, df = 5, *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 4 Regional distribution of the Cavb4 subunit in the developing mouse brain. (a) In histoblots, prominent Cavb4 expression was evident in the cerebellum, with weaker levels in the cortex and hippocampus. Scale bar = 0.4 cm. (b and c) Densitometric analysis demonstrated an increase in Cavb4 expression with age in both the cerebellum and hippocampus. Cavb4 protein was mainly expressed in the stratum radiatum of the dentate gyrus and the molecular layer of the cerebellum. The layers of the hippocampus are represented by their initials: stratum oriens (so), stratum radiatum (sr), stratum lacunosum-moleculare (slm), stratum moleculare of dentate gyrus (sm), hilum of dentate gyrus (h) and stratum lucidum of CA3 (sl). Cerebellar layers are denoted as molecular layer (ml), granule cells (gc) and white matter (wm). Nine independent experiments were averaged to compare protein density. In each layer of the hippocampus and cerebellum, the quantified density at different developmental time points was compared with that at P60. One-way ANOVA followed by Bonferroni post hoc test; error bars represent the SEM, df hippocampus = 4 and df cerebellum = 5; *p < 0.05; **p < 0.01; ***p < 0.001.

the dentate gyrus and lowest in CA1 (Fig. 4b). Moreover, in all the subfields analysed, Cavb1, Cavb3 and Cavb4 expression increased from the lowest levels at P5–P10 to

reach peak levels at P21–P60 (Figs 2b, 3b and 2b). Similar expression patterns were observed in immunoblots (Fig. 1a–c).



In the cerebellum, weak Cavb3 expression was observed in the molecular and granule cell layers at P5, which increased at P10 and subsequently decreased between P15 and P60 (Fig. 3a and c). By contrast, Cavb4 levels increased steadily in the molecular and granule cell layers over this period (Fig. 4a and c). In the white matter of the cerebellum, Cavb3 and Cavb4 expression was consistently lower than that detected in the molecular and granule cell layers, with the strongest expression observed during the second postnatal week, that then gradually decreased until adulthood (Figs 3c and 4c). Similar expression patterns were observed in immunoblot experiments (Fig. 1d–f), although Cavb1 expression was not analysed quantitatively in histoblots because of its weak expression during postnatal development. Although the expression of the three protein subunits increased progressively up to P60 in the hippocampus and cerebellum, some remarkable differences were detected in other brain regions. For instance, Cavb1 expression in the thalamus was stronger during the first postnatal week than at later stages, and it remained relatively consistent in the cortex, with only minor changes (Fig. 2a). Subcellular localization of Cavb subunits in the adult brain Given the role of Cavb subunits in scaffold functions and the targeting of Ca2+ channel complexes to specific subcellular compartments (Bichet et al. 2000; Sokolov et al. 2000; Hibino et al. 2003), we studied the subcellular localization of Cavb1, Cavb3 and Cavb4 in the brain, focusing on the hippocampus and cerebellum. In the hippocampus, Cavb1 and Cavb3 were localized to the strata oriens and radiatum of the CA1, and Cavb4 was found in the molecular layer of the dentate gyrus, areas in which the most prominent expression of the respective subunits was observed in immunohistoblot analyses. Moreover, these regions receive different glutamatergic inputs (the stratum oriens receives input from other pyramidal cells, septal fibres and commissural fibres from the contralateral hippocampus whereas Schaffer collaterals represent the main input to the stratum radiatum), allowing us to investigate the subcellular localization of Cavb subunits in an inputdependent manner. In the cerebellum, the distribution of Cavb1, Cavb3 and Cavb4 subunits was analysed in the molecular layer of the cerebellar cortex. We did not include the pyramidal cell layer of the Purkinje cell layer in the present quantitative analysis. However, we detected a very low immunoreactivity for all CaVb subunits in the nucleus of CA1 pyramidal cells and cerebellar Purkinje cells, with an immunoparticle density similar to background levels (but see Hibino et al. 2003). Hippocampus The pre-embedding immunogold method revealed Cavb1 immunoreactivity (Fig. 5a–c) along the PM and at intracellular sites (IN) in pyramidal cells, with a similar abundance in both

compartments in the two subfields analysed (50% PM vs. 50% IN, n = 790 immunoparticles in so, n = 560 immunoparticles in stratum radiatum). By contrast, Cavb3 (Fig. 5d–g) and Cavb4 (Fig. 5h and i) immunoreactivity was mainly detected at intracellular sites, with a small proportion observed at the PM (28% PM vs. 72% IN for Cavb3 in the strata oriens (n = 958 immunoparticles) and radiatum (n = 566 immunoparticles); 28% PM vs. 72% IN for Cavb4 in the molecular layer of the dentate gyrus, n = 893 immunoparticles). At the PM, Cavb1 (Fig. 5a–c), Cavb3 (Fig. 5d–g) and Cavb1 (Fig. 5h and i) immunoreactivity was mainly detected post-synaptically in dendritic spines and shafts (62%, 62%, 60% of all particles examined for Cavb1, Cavb3 and Cavb4 respectively), and it was less abundant at pre-synaptic sites in axon terminals that established asymmetrical synapses with dendritic spines (38%, 38%, 40% of all particles examined for Cavb1, Cavb3 and Cavb4 respectively). Cerebellum Using the pre-embedding immunogold method, Cavb1 (Fig. 6a–c), Cavb3 (Fig. 6d–f) and Cavb4 (Fig. 6g and h) immunoreactivity was predominantly detected at intracellular sites in the molecular layer of the cerebellar cortex, with a smaller proportion observed at the PM (23% PM vs. 77% IN for Cavb1, n = 835 immunoparticles; 30% PM vs. 70% IN for Cavb3, n = 977 immunoparticles; 27% PM vs. 73% IN for Cavb4, n = 1081 immunoparticles). At the PM, Cavb1 (Fig. 6a–c), Cavb3 (Fig. 6d–f) and Cavb1 (Fig. 6g and h) immunoreactivity was mainly detected post-synaptically in dendritic spines and on the shafts of Purkinje cells (60%, 60%, 55% of all particles examined for Cavb1, Cavb3 and Cavb4 respectively). Less abundant immunoreactivity for all three subunits was detected at pre-synaptic sites in axon terminals that established asymmetrical synapses with the dendritic spines of Purkinje cells (40%, 40%, 45% of all particles examined for Cavb1, Cavb3 and Cavb4 respectively).

Discussion We have investigated the spatiotemporal expression and the subcellular localization of Cavb subunits in the mouse brain during postnatal development. Cavb1, Cavb3 and Cavb4 expression increased throughout development, with all three subunits distributed throughout the adult and developing brain, consistent with previous studies (Ludwig et al. 1997; McEnery et al. 1998; Vance et al. 1998). The subunits displayed partially overlapping expression patterns with several notable regional differences. Immunoelectron microscopy combined with quantitative analysis revealed that Cavb subunits were predominantly localized in the post-synaptic compartment, although pre-synaptic expression was also detected, both in hippocampal pyramidal cells and cerebellar Purkinje cells. These findings demonstrate that the three Cavb subunits are differentially regulated, suggesting their



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Fig. 5 Subcellular localization of Cavb subunits in the adult mouse hippocampus. Electron micrographs of the stratum oriens (so) and stratum radiatum (sr) of the CA1 region of the hippocampus and the stratum moleculare (ml) of the dentate gyrus showing immunogold particles for Cavb proteins in the adult mouse brain, detected using a pre-embedding method. (a–c) Cavb1 immunoparticles were mainly detected post-synaptically in pyramidal cells, both in dendritic shafts (Den) and spines (s). Quantitative analysis (c) revealed a similar distribution in the plasma membrane (arrows) and the intracellular membranes (crossed arrows). (d–g) Cavb3 immunoparticles were mainly localized intracellularly, and they were detected in both the pre(arrowheads) and post-synaptic (crossed arrows) compartments in the

so of the CA1 region (d and e). In the sr, major post-synaptic Cavb3 immunoreactivity was observed in pyramidal cells, both intracellularly and along the plasma membrane of dendrites (Den). (h and i) Cavb4 was predominantly localized intracellularly (crossed arrows), and it was detected pre- and post-synaptically in the hippocampus. (c, g and i) Immunoparticles from each reference area were counted along the plasma membrane and at the intracellular sites in dendrites, spines and axon terminals, and represented as a percentage of the total number of immunoparticles per region from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; unpaired Student¢s t-test; error bars represent the SEM. axon terminal (at). Scale bar = 0.5 lm.

involvement in pre- and post-synaptic aspects of neurotransmission.

spatiotemporal profile of subunit expression to fully understand the molecular basis of Cav signalling. Native CaV channels are heteromultimeric complexes composed of one a subunit, which forms the pore, and other auxiliary subunits: one b, one a2d and one c (Buraei and Yang 2010). However, it is generally agreed that the b subunits mediate the biophysical modulation, and the maturation and cell surface expression, of CaV channels.

Differential expression of Cavb subunits The dramatic changes in calcium conductances that occur during neuronal maturation suggest that underlying and equally dramatic changes occur in the expression of voltagegated Ca2+ channels. Thus, it is important to determine the


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Fig. 6 Subcellular localization of Cavb subunits in the adult mouse cerebellum. Electron micrographs of the molecular layer (ml) of the cerebellum showing immunogold particles for Cavb proteins detected using a pre-embedding method. (a–c) Cavb1 immunoparticles were mainly located intracellularly (crossed arrows) in the spines (s) of Purkinje cells and in parallel fibre axon terminals (at), and a few were observed along the plasma membrane (arrows and c). (d–f) Cavb3 immunoparticles were mainly located intracellularly (crossed arrows) in the spines (s) of Purkinje cells and in parallel fibre axon terminals (at), and they were detected in both pre- and post-synaptic compart-

ments in the molecular layer (c). (g–h) Cavb4 immunoparticles were predominantly localized intracellularly (crossed arrows), and they were detected both in pre-synaptic axon terminals (at) and post-synaptically in the spines (s) and dendrites of Purkinje cells. (c, f and h) Immunoparticles from each reference area were counted along the plasma membrane and at the intracellular sites in dendrites, spines and axon terminals, and represented as a percentage of the total number of immunoparticles per region from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; unpaired Student¢s t-test; error bars represent the SEM. Scale bar = 0.2 lm.

If we consider the expression of the distinct b subunits, Cavb4 mRNA is prominently expressed in the cerebellum and up-regulated during development (Ludwig et al. 1997; Vance et al. 1998; Schlick et al. 2010). Indeed, we observed pronounced differences in Cavb4 protein levels between P0 and P60, with expression increasing in parallel with axon outgrowth and synapse formation in the cerebellum (P7), as reported previously (Vance et al. 1998). This expression profile was mirrored in the molecular layer of the cerebellum, where the most prominent staining was detected. These results suggest that the Cavb4 subunit plays a more important role in the mature than in the developing cerebellum. While stable Cavb3 mRNA expression has been described in the cerebellum (Vance et al. 1998), we primarily detected Cavb3 protein in the molecular layer of the cerebellum where low levels were observed from P0–P5,

increasing at P15 and subsequently decreasing drastically until P60. The distribution of Cavb3 mRNA transcripts overlaps with that of Cav2.2, suggesting that these subunits can associate to form N-type channels in the cerebellum (Ludwig et al. 1997). As Cavb4 can also associate with Cav2.2 in the cerebellum (Scott et al. 1996), the loss of Cavb3 in this structure at P60 may be linked with the increase in Cavb4, reflecting an adaptive developmental response. We observed low levels of Cavb1 protein in the cerebellum when compared with the hippocampus and cortex, consistent with the distribution of Cavb1 mRNA described previously (Schlick et al. 2010). One possibility is that the low levels of Cavb1 expression may explain why the loss of Cavb4 could produce an aberrant targeting of Cav2.1, giving rise to major defects in cerebellar development and function (McEnery et al. 1998).



In contrast to the patterns of mRNA expression described (Schlick et al. 2010), high levels of Cavb1 and Cavb3 protein were detected in the hippocampus, increasing progressively with age. Both subunits were predominantly expressed in the strata oriens, radiatum and lucidum of the CA1 and CA3. Both Cavb1 and Cavb3 can associate with the a1 subunit of L-type channels in the hippocampus (Pichler et al. 1997; Obermair et al. 2010) and significantly, Cavb3 KO mice exhibit no changes in L-type Ca2+ currents in hippocampal CA1 pyramidal neurons, or in the expression of Cav1.2 and Cav1.3 (Jeon et al. 2008). Although Cavb3 is known to associate with Cav2.2, which plays a crucial role in neurotransmitter release at hippocampal CA3–CA1 synapses (Dunlap et al. 1995; Scott et al. 1996; Vance et al. 1998; Jeon et al. 2007), basal synaptic transmission remains unchanged at hippocampal CA3–CA1 synapses in Cavb3 KO mice (Jeon et al. 2008). Our results suggest a compensatory increase in Cavb1 expression in Cavb3 KO mice, as both proteins exhibit overlapping expression patterns in the hippocampus. Furthermore, as the expression levels of Cavb2 and Cavb3 seem to be similar (Vance et al. 1998), another possibility is that the Cavb2 subunit compensates for Cavb3. On the other hand, unlike Cavb1 and Cavb3, the co-localization of Cavb4 with Cav1.2 channels in hippocampal neurons is significantly diminished (Obermair et al. 2010). However, Cavb4 co-localizes with the a1 subunits of the P/Q channel in hippocampal neurons (Wittemann et al. 2000), the main Ca2+ channel triggering neurotransmitter release in the perforant pathway from the enthorinal cortex to the dentate gyrus (Qian and Noebels 2001). We found Cavb4 immunoreactivity to be prominent in the molecular layer of the dentate gyrus, corroborating previous findings (Ludwig et al. 1997) and implicating Cavb4 in the formation of P/Q channels in this structure. Taken together, these findings indicate that the highly differentiated regional expression of Cavb subunits is important for correct neuronal function and synaptic plasticity in the hippocampus. Cellular and subcellular localization of Cavb subunits Because of the promiscuous nature of the a1-b pairings, the subunit composition of Ca2+ HVA channels depends on the relative concentrations of the Cavb subunits (Obermair et al. 2008). To determine the role of Ca2+ channels in hippocampal and cerebellar circuits, we analysed the subcellular localization of Cavb proteins in the adult mouse brain. At the ultrastructural level, we detected strong Cavb1 and Cavb3 immunoreactivity in the CA1 region of the hippocampus, and Cavb4 labelling in the molecular layer of the dentate gyrus. These patterns were attributed to the presence of ion channel subunits in pyramidal and granule cells respectively. This finding is in agreement with previous in situ hybridization studies demonstrating abundant Cavb1, Cavb3 and Cavb4 expression in hippocampal principal cells (Ludwig et al. 1997).

Immunogold labelling for the three Cavb subunits was evident along the surface of hippocampal pyramidal cells and of Purkinje cells in the cerebellum. However, while Cavb1 was distributed equally between the intracellular compartment and the plasma membrane of dendritic shafts and spines, Cavb3 and Cavb4 were predominantly localized intracellularly. Although not directly comparable to the in vivo situation where multiple a1 subunits are expressed, those findings agree with previous in vitro studies reporting the membrane association of Cavb1, but not for Cavb3 or Cavb4 (Brice et al. 1997; Bogdanov et al. 2000). A similar intracellular localization of different Cav channel subunits has been described using immunohistochemistry to detect Cav1.2 and other Cav channels in cultured hippocampal neurons (Obermair et al. 2004) and in CA1 dendrites in vivo (Vinet and Sik 2006). In addition, electron microscopy to analyse immunogold labelling of Cav1.2 channels in the globus pallidus revealed that these channels predominantly remain at intracellular sites rather than inserting into the plasma membrane of dendrites (Hanson and Smith 2002). Intracellular Cavbs may represent newly synthesized ion channel subunits in transit to their sites of action at the plasma membrane. Another possibility is that there is an excess of Cavb subunits, and the interaction with the channels is reversible. However, neuronal CaV channels are also internalized as a consequence of G-protein-coupled receptor activation (Altier et al. 2006; Tombler et al. 2006). Thus, intracellular Cavbs may also correspond to CaV channels that have undergone endocytosis and entered into recycling or degradation pathways. In accordance with the histoblot analyses that indicated a similar intensity of Cavb1 and Cavb3 in CA1 dendrites, electron microscopy revealed a comparable subcellular distribution of these two subunits in certain regions. Cavb1 and Cavb3 immunoreactivity was observed in both pre- and post-synaptic compartments, although post-synaptic staining was most frequently detected in dendrites and spines (55– 70%) and in axon terminals (30–45%). By contrast, Cavb4 was distributed similarly at pre-synaptic and postsynaptic sites in the molecular layer of the dentate gyrus, suggesting that this subunit associates with pre-synaptic Ca2+ channels in the hippocampus. These findings are consistent with previous studies describing a pre-synaptic role for the Cavb4 subunit in synaptic transmission (Wittemann et al. 2000). Interestingly, we observed a similar distribution for the three subunits studied, which were located subcellularly in Purkinje cells of the cerebellum, suggesting that different cell types in the brain may use similar mechanisms to target and traffic CaVb subunits. The only significant difference in expression observed was that the Cavb1 subunit was more frequently detected at intracellular sites in Purkinje cells than in CA1 pyramidal cells. Overall, our findings suggest that CaVb subunits exert their effects on CaV channels, Ca2+



signalling and excitability in the hippocampus and cerebellum, in a diverse range of locations. Immunogold labelling for Cavb1, Cavb3 and Cavb4 in the spines of CA1 pyramidal cells and cerebellar Purkinje cells was largely extrasynaptic, which may reflect the limited access of the antibodies to post-synaptic compartments, as previously reported for pre-embedding immunogold methods (Lujan et al. 1996). However, the frequent exclusion of immunogold staining for all three subunits from the postsynaptic density supports the extrasynaptic localization of these channel subunits, as previously described for subunits such as Cav1.2 (Tippens et al. 2008) and Cav2.1 (Kulik et al. 2004), and not overlapping with the synaptic localization observed in cultured hippocampal pyramidal neurons (Obermair et al. 2004; Di Biase et al. 2008). Physiological consequences of the localization of Cavb subunits in the brain Neuronal excitability is thought to depend on the precise spatial distribution of voltage-gated ion channels. Given their scaffold function and their regulation of Ca2+ channel trafficking (Buraei and Yang 2010), Cavb subunits exert specific effects on neuronal excitation and neurotransmitter release when expressed in particular synapses, neuronal circuits, brain regions or at certain developmental stages. For example, our results provide evidence that functional Cav channels are present in the dendritic spines of hippocampal pyramidal neurons (Davare et al. 2001; Hoogland and Saggau 2004). Spines receive the excitatory input to hippocampal pyramidal neurons and they experience depolarization-induced increases in Ca2+ that are mediated by different Cav channel types (Segal 1995). Spines are particularly abundant in the stratum radiatum and they serve as the primary targets for the excitatory synapses formed by the Schaffer collaterals (Bannister and Larkman 1995; Megias et al. 2001). The prevalence of Cavb1 and Cavb3 in spines and dendrites of CA1 pyramidal cells is consistent with a role for post-synaptic Cavb subunits in the formation of the Cav channels that promote LTP and LTD at the Schaffer collateral-CA1 synapse (Moosmang et al. 2005). Accordingly, some forms of hippocampal-dependent learning and memory appear to be enhanced in Cavb3 KO mice, although working memory is impaired (Murakami et al. 2007; Jeon et al. 2008). In the cerebellum, we detected high post-synaptic levels of all three subunits in the spines and dendrites of Purkinje cells. Cavb4 null mice exhibit cerebellar ataxia (Burgess et al. 1997), which is likely to be correlated with impaired synaptic transmission in Purkinje cells. Therefore, localization of specific Cavb subunits both within and beyond synaptic terminals may represent an important mechanism for the regulation of synaptic plasticity. In conclusion, the results presented here demonstrate that the Cavb1, Cavb3 and Cavb4 subunits of Cav channels are

distributed in the somato-dendritic and axonal plasma membrane compartments of CA1 pyramidal neurons and cerebellar Purkinje cells. Correlating their spatial distribution with that of other Cav channel subunits, mainly the poreforming a subunits, may further advance our understanding of Ca2+ signalling, and of the excitability of brain neurons.

Acknowledgements We thank Mr. Mark Sefton for the English revision of this manuscript. This work was supported by grants from the Spanish Ministry of Education and Science (BFU-2009-08404/BFI) and the CONSOLIDER programme (CSD2008-00005) to R.L. The monoclonal antibodies Cavbeta1 (Clone N7/18) and Cavbeta4 (Clone N10/7) were developed by and/or obtained from the UC Davis/NIH NeuroMab Facility (Department of Neurobiology, Physiology and Behaviour, College of Biological Sciences, University of California, Davis, CA). All authors have no conflict of interest to declare.

Supporting information Additional supporting information may be found in the online version of this article. Figure S1. Controls used for immunoelectron microscopic techniques. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

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