Synaptic alpha-dystrobrevin: Localization of a short alpha-dystrobrevin isoform in melanin-concentrating hormone neurons of the hypothalamus

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Synaptic alpha-dystrobrevin: Localization of a short alpha-dystrobrevin isoform in melanin-concentrating hormone neurons of the hypothalamus Diana Hazaia , Chun-Fu Lienb , Ferenc Hajósa , Katalin Halasya , Dariusz C. Góreckib , Veronika Jancsika,⁎ a

Faculty of Veterinary Science, Department of Anatomy and Histology, Szent István University, Budapest, Hungary Molecular Medicine, Institute of Biomedical and Biomolecular Sciences, School of Pharmacy and Biomedical Sciences, University of Portsmouth, Portsmouth, UK b

A R T I C LE I N FO

AB S T R A C T

Article history:

The expression of the two members of the dystrobrevin (DB) family in the adult brain was

Accepted 5 January 2008

thought to be highly specific for the two main cell types: alpha-dystrobrevin (α-DB) and beta-

Available online 26 January 2008

dystrobrevin (β-DB) has been identified as glial and neuronal proteins, respectively. In the present work we show that a subset of neurons in the hypothalamus contains α-DB.

Keywords:

Comparative immunohistochemical studies with two α-DB antibodies of different specificity

Immunohistochemistry

indicate that the neurons contain short α-DB isoform(s) α-DB-2 and/or α-DB-4. Immunoreactive

Dystrophin-associated protein

multipolar or spindle-shaped neurons form clusters with bilateral symmetry, localized

complex

predominantly in the lateral hypothalamic area, with extensions into the zona incerta and

Hypothalamus

the dorso-medial and ventro-medial hypothalamic region. α-DB immunoreactivity was

Melanin-concentrating hormone

localized in cell processes and at postsynaptic densities, furthermore in the endoplasmic reticulum within the perikarya. α-DB-positive neurons are β-dystrobrevin immunoreactive, but α- and β-DB do not co-localize with their usual molecular anchors like dystrophins or high molecular weight forms of utrophin. Colocalization with nNOS was also not observed. The pattern of α-DB immunoreactive neurons gave a perfect colocalization with melaninconcentrating hormone (MCH) neurons throughout the whole region studied. We propose that α-DB plays a role in a structure or regulation mechanism unique to MCHexpressing neurons. © 2008 Published by Elsevier B.V.

1.

Introduction

The dystrophin-associated protein complex (DAPC) is expressed in a wide variety of tissues. Protein components of the DAPC are traditionally subdivided into three sub-complexes: the

membrane-spanning dystroglycan complex, the membraneembedded sarcoglycan and sarcospan complex, and the cytoplasmic complex (Yoshida et al., 1994; Culligan and Ohlendieck, 2002). A remarkable attribute of DAPC is its tissue- and developmental-state specific molecular composition. These

⁎ Corresponding author. Faculty of Veterinary Science, Department of Anatomy and Histology, Szent István University, 1078 Budapest István u 2, Hungary. Fax: +36 1 478 42 24. E-mail address: [email protected] (V. Jancsik). 0006-8993/$ – see front matter © 2008 Published by Elsevier B.V. doi:10.1016/j.brainres.2008.01.046

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complexes are suggested to exert or be involved in a plethora of tissue-specific processes, yet in a number of tissues their biological functions are still largely unknown. The best understood is the DAPC in the skeletal muscle, where it provides a transmembrane link between the subplasmalemmal actin cytoskeleton and the extracellular matrix and thereby ensures the structural integrity of myofibers. Recently, the role of the DAPC in signal transduction from the cell surface towards its interior (in a manner similar to integrins) has been suggested by different lines of investigations (Ehmsen et al., 2002; Oak et al., 2001; Zhou et al., 2006). Noticeably, the signalling enzyme neuronal nitric oxide synthase (nNOS) was shown to coexist with DAPCs in specific tissues (Bredt, 1996; Brenman et al., 1996). The impact of the DAPC dysfunction is best understood in the skeletal muscle. Several muscular dystrophies of genetic origin are known to arise due to the lack of functional impairment of individual DAPC members (for a recent review see Kanagawa and Toda, 2006). Dystrophin is a key component of the cytoplasmic sub-complex and it anchors the entire DAPC in the membrane. Its mutations cause Duchenne muscular dystrophy (DMD), the most common and severe form of dystrophy also associated with other abnormalities, including mental retardation (Anderson et al., 2002; Montanaro and Carbonetto, 2003; Moore et al., 2002). Further elements of the cytoplasmic sub-complex are the dystrobrevins. These proteins provide a link between the members of the dystrophin and syntrophin protein families (Blake et al., 1996; Peters et al., 1997). Dystrobrevins, encoded by two different genes are designated as α-dystrobrevins (α-DB) and β-dystrobrevins (β-DB). Multiple transcripts arise from both genes by alternative promoter usage and alternative splicing, giving rise to multiple protein isoforms in different cell types and at specific developmental stages (Blake et al., 1996; Ambrose et al., 1997). In skeletal and cardiac muscle alternative splicing of α-DB mRNA generates at least five isoforms: α-DB-1 to α-DB-5 that differ both in primary sequence and tissue distribution (Rees et al., 2007). Several lines of evidence point to the key role of dystrobrevins in the formation of the multitude of distinct DAPCs in the adult as well as in the developing tissues, including the nervous system (Blake et al., 1999; Lien et al., 2004; Lien et al., 2007), by recruiting distinct syntrophin isoforms into the complexes (Peters et al., 1997). The α-dystrobrevin knockout mice show myopathic phenotype without alteration of the structural integrity of the muscle fibre (Grady et al., 1999) indicating an essential role for this protein in muscle. Furthermore, developmental studies demonstrated a specific spatio-temporal expression pattern of α-dystrobrevin in a number of other tissues, including the central nervous system. In the light of our findings of abundant α-dystrobrevin immunoreactivity in the developing CNS, including in the developing neurons (Lien et al., 2004) we have reevaluated the localization of this protein in the adult CNS. In accordance with previous findings, we established that α-DB was predominantly present in glia. However, with a combination of antibodies differentiating between α-DB-1 and α-DB-2/4 isoforms, we have identified a specific subset of immunopositive neurons in the hypothalamus. We performed analysis of the cellular and subcellular localization of α-DB in these neurons by combined light and

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electron-microscopic investigations. Immunoreactive neurons were also studied by double labelling immunofluorescence for the presence of the functionally important DAPC members: utrophins, β-DB and nNOS. The significance of the interactions with dystrophins was assessed by studying α-DB immunoreactivity in the hypothalamus of mdxβgeo mice, lacking all dystrophin isoforms (Wertz and Fuchtbauer, 1998). To identify and further characterize the α-DB immunopositive neurons, we considered their localization in the context of the physiological role of the lateral hypothalamus, an area classically implicated in the regulation of a multitude of physiological processes including feeding behavior and energy homeostasis (Bernardis and Bellinger 1993, 1996). On the grounds of their anatomical position and morphology (cf. later) it was feasible to suppose that α-DB immunopositivity occurs in neurons producing either melanin-concentrating hormone (MCH) or orexins (hypocretins). These neuropeptides, with feeding-stimulatory (orexigenic) activities (Qu et al., 1996) are expressed by two discrete but spatially overlapping neuron populations located predominantly in the lateral hypothalamic area (LHA) (Zamir et al., 1986; Bittencourt et al., 1992). Both populations are targets for excitatory and inhibitory signals from other brain regions, including the arcuate nucleus (Broberger et al., 1998) and send axonal projections to the cortex, thalamus, hypothalamus, brainstem and spinal cord (Bittencourt and Elias, 1998; Peyron et al., 1998). In addition, the MCH neurons can be divided into at least two subpopulations on the basis of their neuronal phenotypes and connections (Cvetkovic et al., 2004). Axons of one of the subpopulations, which express neurokinin 3 receptor and – paradoxically – the anorectic peptide cocaine and amphetamine regulated transcript (CART), can be traced throughout the telencephalon. The axons belonging to the second MCH neuron subpopulation, expressing neither of the above proteins, project mostly toward the brainstem. The intrinsic characteristics and the molecular composition of MCH and orexin/hypocretin neurons have been recently characterized further by multi-transcriptional profiling (Harthoorn et al., 2005). These results are suggesting that both GABA and glutamate are involved in their functioning. To assess the significance of the neuronal population that we found to be α-DB immunopositive, it was essential to establish their identity. Colocalization studies presented here

Fig. 1 – Expression of α-DB isoforms in the hypothalamus of mouse brain. A: V-19 (recognizing α-dystrobrevin-1, -2 and -4) staining B: α1-CT-FP (α-DB-1 specific) staining. Neurons are stained exclusively with V-19, while both antibodies gave perivascular immunoreactivity. Scale bar: 500 μm.

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show that all hypothalamic neurons identified as α-DB positive were simultaneously MCH positive and vice versa.

2.

Results

The glial endfeet supporting the microvasculature of the adult mouse brain is strongly immunopositive for α-DB-1, while during the mouse development no staining was observed in both glial and neuronal populations (Lien et al., 2004, 2007). Moreover, western blot analyses of brain protein extracts by using V-19 anti-dystrobrevin antibody detected the full-length α-DB-1 as well as shorter α-DB-2 and α-DB-4 (Lien et al., 2007). In order to check for the presence of a neuronal population expressing one of these α-DB isoforms we surveyed the adult mouse brain with two α-DB antibodies. The results of this study indicate that the neurons are devoid of V-19 immunoreactivity, with the exception of the hypothalamus, where a neuronal population with highly specific immunoreactivity was detected. These neurons were not stained with α1-CT-FP antibody, which exclusively recognizes α-DB-1 (Fig. 1). This indicates that V-19 immunopositive neurons contain α-DB-2 and/or 4 isoforms.

Fig. 3 – Cellular and subcellular localization of the neuronal α-dystrobrevin. A and B: Representative images showing the morphology of immunolabelled neuronal perikarya and processes. A: DAB technique. Arrows: immunopositive perikarya, arrowheads: immunopositive blood vessels. B: Confocal laser scanning microscopy. Arrows: punctate staining on the neuronal processes. White: nuclear counterstaining with ToPro-3. Scale bar: 40 μm. C–F: Transmission electron-microscopic images of immunolabelled structures. C and D: Within the perikarya, immunolabelling occurs exclusively on the rough endoplasmic reticulum membrane. Note the uneven distribution of the labelling on the membranes. D and E: Labelled neuronal processes of different caliber ( ), immunolabelled (→) and nonlabelled postsynaptic densities (1).

Fig. 2 – Distribution of α-dystrobrevin immunoreactive neurons within the hypothalamus. Representative low magnification images showing the distribution of α-DB-2/4 immunoreactive neurons. For better discerning immunopositive neurons on the low magnification overview images shown above, black dots were superposed on each labelled neuron.

Finally, the specificity of the anti-alpha-DB antibody used in this study has been verified on a selection of tissues from the α-DB knockout mice, including the brain (not shown here). For more accurate anatomical localization of α-DB-2/4 immunoreactive (α-DB-2/4 IR) neurons within the hypothalamus, rostro-caudal series of coronal brain sections were analysed. The first group of α-DB-2/4 IR neurons appeared at the level where the optic chiasma continue into the two optic tracts (approx. at Bregma −1.22 mm). The most rostral immunopositive neurons appeared in the region of the zona incerta. In subsequent sections, through approx. 0.7 mm, the immunopositive neurons constitute clusters of various form, dimension and localization with bilateral symmetry. The α-DB immunoreactive neurons were localized predominantly in the lateral hypothalamic area (LHA), with occasional extensions into the dorsomedial region. At the caudal limit of their occurrence α-DB-2/4 IR neurons extend towards the ventro-medial area of the hypothalamus. It is worth noting that α-DB-2/4 IR neurons were not

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Fig. 4 – Colocalization of α-DB-2/4 staining with β-DB and utrophin in the hypothalamus. A: β-DB IR. Note immunolabelling in the nucleus. B: α-DB-2/4 IR. Stars: β-DB immunopositive but α-DB-2/4 immunonegative neuron subpopulation. C: Merged image. D: Utrophin IR. Arrowheads: microvasculature. E: α-DB-2/4 IR. Arrowheads: microvasculature, arrows: neurons. F: Merged image. α-DB-2/4 immunoreactive neurons do not contain utrophin. Colocalization of α-DB-2/4 and utrophin occurs exclusively around brain microvasculature. Scale bar: 50 μm.

present in the dorso-medial and ventro-medial hypothalamic nuclei. Representative sections displaying typical clusters are shown in Fig. 2. Individual immunopositive neurons (Figs. 3A and B) are multipolar or spindle-shaped. The immunoreaction within the perikarya has a patchy appearance. Immunostained cell processes were frequently observed. The labelling along these processes was characteristically punctate, indicative of local accumulations of α-DB-2/4, very probably at synapses. Using electron microscope, immunoreaction end product was localized on the membranes of the rough endoplasmic reticulum within the cell body (Figs. 3C and D). A remarkable feature of these sacculi was their broad lumen as compared to sacculi in adjacent neurons. Immunopositivity was also present in dendrites and at postsynaptic densities, in clear agreement with the light microscopic observations. Immunolabelling of these structures was heterogeneous: immunopositive and unlabelled cell processes and synapses occurred intermingled. (Figs. 3E and F). α-DB-2/4 immunoreactivity was compared to β-DB immunoreactivity. Immunofluorescence studies, shown in Fig. 4, demonstrate that β-DB immunoreactive neurons, similar to α-DB-2/4 immunoreactive ones, are spindle-shaped or multipolar. Immunoreaction occurs both in the perikarya and in processes. Remarkably, within the perikarya, the immunoreaction is not restricted to the cytoplasm; nuclei are also immunopositive (Fig. 4A). The co-distribution pattern (Fig. 4C) reveals that β-DB immunoreactive neurons are more widely distributed within the hypothalamus than α-DB-2/4 immunoreactive ones, and that all α-DB-2/4 IR neurons show β-DB immunoreactivity as well. Thus, α-DB-2/4 positive neurons constitute a specific subset of the β-DB immunopositive ones. In order to ascertain the type of DAP complexes involving neuronal α-DB or β-DB, we have analysed the neurons described

above for utrophin. By using the Mupa-2 antibody (which recognizes all utrophin forms except the shortest Up71) no significant signal has been observed in neurons, while brain capillaries showed positive immunostaining, as expected (Fig. 4D). Next, the significance of dystrophin, another key component of the DAPC, was investigated in mdxβgeo mice — this knockout lacking all dystrophin isoforms (Wertz and Fuchtbauer, 1998). In mdxβgeo brains the perivascular dystrobrevin staining was strongly reduced, but the localization and immunostaining intensity of neuronal α-DB were similar to those observed in the wild type mice (Fig. 5). Immunoreactivity of nNOS, known to be a functionally-significant constituent of DAPC in several tissues, has also been analysed. However, no colocalization of α-DB with nNOS has been detected in LHA neurons (results not shown).

Fig. 5 – α-dystrobrevin immunoreactive neurons in the hypothalamus of mdxbgeo mice. V-19 immunoreactivity. Arrows: neurons, arrowheads: microvasculature. Note the extremely faint perivascular staining, typical in the absence of dystrophin. Scale bar: 50 μm.

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Fig. 6 – Colocalization of the neuronal α-dystrobrevin-2/4 with melanin-concentrating hormone. Double-label immunofluorescence images showing V-19 (A, D) and MCH immunoreactivities (B, E) and their co-occurrence (C, F) in hypothalamic neurons. Scale bar: 50 μm (A–C), 25 μm (D–F).

To find the possible correlation to individual neuronal populations of the highly specific α-DB immunoreactivity demonstrated above, we considered their distribution which suggested that this neuron population could represent the MCH or orexin expressing cells. Double labelling immunofluorescence studies for the colocalization of α-DB with MCH revealed completely matching patterns throughout the whole region studied (Fig. 6). High magnification images show overlapping subcellular distributions of these two proteins, mostly concentrated within the perikarya and in the proximal axon.

3.

Discussion

Until now the expression of the two members of the dystrobrevin family in the adult brain was believed to be highly segregated between neuronal and glial cells. The β-DB localizes to neurons, where it associates with specific dystrophin isoforms (Blake et al., 1998; Blake et al., 1999). In contrast, α-DB has been identified as a glial protein within the mature brain. However, during embryonic development α-DB-1 was found to be present in neuronal cells, exhibiting a complex spatio-temporal distribution pattern (Lien et al., 2004). Expression of α-DB isoforms in the early phase of the neuronal differentiation has been substantiated by the studies on the retinoic acid-triggered neuronal differentiation of cultured P19 cells (Ceccarini et al., 2002). Immunohistochemical studies presented here reveal the presence of short α-DB isoforms, α-DB isoforms, α-DB-2 and/or -4 in a specific neuronal subpopulation within the hypothalamus of the mature mouse brain. The protein localizes in neuronal perikarya, processes and postsynaptic densities. Within the perikarya, α-DB-2/4 is prominently present on the membranes of the ER sacculi. This subcellular localization is unusual for a DAPC

component protein. Up to now, only γ-syntrophin isoforms were localized to the endoplasmic reticulum in neurons, where they were suggested to form a scaffold for signalling and trafficking (Alessi et al., 2006). Other aspects of the subcellular localization of α-DB-2/4 namely its presence in neuronal processes and postsynaptic densities are more reminiscent of the localization of other DAPC components in neurons. It is however the first indication of the synaptic localization of α-DB. It expands the repertoire of the possible roles for this protein in brain functions. In search for interacting partners of α-DB-2/4, we checked the neurons for the presence of utrophins, dystrophins and nNOS. Utrophin and nNOS immunoreactivity were both absent. Taken into account the specificity of the utrophin antibody used, the conclusion can be drawn that the full-length utrophin, Up140 and G-utrophin are not participating in α-DB containing neuronal complexes. While these utrophin isoforms predominate in the brain (Blake et al., 1995) a selective interaction with Up71 cannot be excluded at this stage. The potential significance of dystrophins for the formation of the DAPC in the α-DB immunopositive LHA neurons was evaluated by comparing neuronal α-DB immunostaining in the wild type and in the mdxβgeo mice. Neuronal α-DB immunoreactivity – in contrast to the perivascular one – was not significantly affected in dystrophin-deficient mice. Taken together with the result on the utrophin colocalization, this finding can be regarded as an indication of the neuronal α-DB being a component of a complex highly diverse from the known DAPC or not being a part of any dystrophin-associated protein complex. We presumed that the high specificity of the neuronal α-DB-2/ 4 immunoreactivity within the hypothalamus was due to its presence in a subset of neurons of distinct physiological function (s). Amongst the several neuron subpopulations within the

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hypothalamic region in question, we considered MCH and/or orexin expressing neurons as good candidates for being the α-DB-2/4 immunoreactive neurons. Indeed, the colocalization studies showed that all neurons identified as α-DB positive were also MCH positive and vice versa. As MCH and orexin expression in the same cell is mutually exclusive (Elias et al., 1998 and own observations), it is safe to assume that orexin neurons are negative for α-DB. The question arises, whether α-DB-2/4 immunoreactive neurons also contain β-DB. β-DB is more widely expressed in the hypothalamus than α-DB. Colocalization studies presented here showed that all α-DB expressing neurons were immunopositive for β-DB as well. Noticeably, beside the perikaryon and cell processes, β-DB has also been detected in the nuclei. This finding is in agreement with the recent discovery of DAPC (including dystrobrevins) within the nuclear matrix of specific cells (Fuentes-Mera et al., 2006) and suggests that β-DB is likely to play a role as a component of the nucleoskeleton or as a transcriptional regulator in these cells. Taken together, a neuronal population has been defined in the lateral hypothalamic area, characterized by the presence of both α- and β-DB. The coexistence of α-DB and β-DB within the perikaryon of these neurons suggests the presence of two distinct dystrobrevincontaining DAPCs, with presumably distinct physiological roles. We suppose that the exclusive colocalization of α-DB with MCH at the synapses reflects a specific functional requirement and/or regulatory phenomenon. This could be associated with the role of α-DB as a multifunctional scaffold for signalling proteins (Blake et al., 1996; Peters et al., 1997). Alternatively, α-DB could interact with MCH and regulate its intracellular targeting into specific subcellular compartments or membrane sites by acting as kinesin motor protein receptor (Ceccarini et al., 2005) and/or by interacting with dynamin (Zhan et al., 2005). In conclusion, we suggest that the presence of α-DB and β-DB, either as DAPC components or as participants of hitherto unidentified macromolecular assemblies, may be important for cell-specific function of MCH-expressing neurons.

4.

Experimental procedures

4.1.

Animals

Adult C57BL10 control, the dystrophin-negative DMDmdxβgeo (Wertz and Fuchtbauer, 1998) and the alpha-dystrobrevin knockout mice (Grady et al., 1999) were used. Animals were maintained in a 12 hour light/dark cycle and fed normal diet and water ad libitum. All procedures were performed with permission of the local Animal Health and Welfare Committees and in accordance with the Hungarian and UK Home Office guidelines.

4.1.1.

Antibodies

• α-dystrobrevin: 1. V-19 (Santa Cruz Biotechnology, Inc.): polyclonal antibody, raised in goat, against a peptide near the carboxy terminus of human α-dystrobrevin. In rat and mouse tissues this antibody recognizes α-dystrobrevin-1, -2 and -4. 2. α1-CT-FP: rabbit polyclonal antibody recognizing exclusively α-dystrobrevin-1 (Blake et al., 1998; Nawrotzki et al., 1998).

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• β-dystrobrevin: beta-521 (Blake et al., 1998). • MCH: rabbit polyclonal antibody (H 070-47, Phoenix Pharmaceuticals, Inc.). • ncNOS (NOS1): mouse monoclonal antibody raised against amino acids 2–300 of NOS1 of human origin (sc5302, Santa Cruz Biotechnology, Inc). • Utrophin: Mupa-2, a rabbit polyclonal antibody raised against peptides corresponding to amino acids 2543–2738 next to the cysteine-rich domain of utrophin, recognizes all utrophin isoforms except Up71 (Jimenez-Mallebrera et al., 2003).

4.1.2.

Correlated light- and electron-microscopy studies

Mice weighing 25–35 g were transcardially perfused under deep Euthanyl anaesthesia with heparinized saline, followed by glutaraldehyde-supplemented Zamboni's fixative (4% paraformaldehyde, 16% saturated picric acid and 0.25% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4). Brains were removed from the skull and postfixed in buffered 4% paraformaldehyde for further 2 h. 60 μm thick coronal sections were cut with vibratome. Slices containing the hypothalamus were cryoprotected in an antifreeze mixture containing 25% sucrose and 8.7% glycerol. After repeated freezing and thawing (in liquid nitrogen) the slices were processed for immunohistochemistry. Sections were washed in 0.1 M phosphate buffer, pH 7.4 and then incubated in 1% sodium borohydride for 30 min. After washing, the sections were treated with 2% hydrogen-peroxide for further 15 min. Non-specific binding was blocked by incubation in 5% bovine serum albumin (Sigma) overnight at 4 °C. Blocking was followed by the incubation of the sections for 48 h at 4 °C with the V-19 antibody, diluted 1:1000. Sections were rinsed, then biotinylated anti-goat IgG (Amersham) secondary antibody and avidin–biotin–horseradish peroxidase (Vectastain ABC kit, Vector Laboratories) were used, (for 2 h each) according to the manufacturer's instructions. Bound peroxidase was visualized with 3,3′-diaminobenzidine tetrahydrochloride. For light microscopy examination the sections were dehydrated and mounted in DPX on gelatine-coated slides. Sections for electron microscopy were treated with 1% OsO4 for 30 min, then dehydrated with graded ethanol and propylene oxide, and then embedded flat in Durcupan ACM epoxy resin (Fluka). Uranyl acetate contrast staining was applied during dehydration. Tangential ultrathin sections were cut from the superficial zone of the vibratome slices with a Reichert Ultracut 2 and picked up on Formvar coated single-slot grids. A third contrast staining was performed with lead citrate, and then sections were viewed and photographed in a JEOL 100 B electron microscope. Control specimens were prepared by the same procedure described above but omitting the primary antibody from the incubation solution. No immunostaining was observed in controls. Several approaches were applied to confirm specificity of the dystrobrevin antibodies used in this study; variations in antibody dilutions (from 1:250 to 1:2000) and high salt concentrations (2.5% NaCl) in the antibody solution had no effect on the staining pattern. Finally, the specificity of the anti-alpha-DB antibodies has been verified on a selection of tissues from the α-DB knockout mice (Grady et al., 1999), where no staining was observed:

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(results not shown here) and substantiated in immunoblot analyses in various tissue extracts, as described before (Lien et al., 2007).

4.1.3.

Fluorescence double labelling

Mice were perfused as described above. After post-fixation of the brain, 40 μm coronal sections were cut by vibratome. Sections were washed thoroughly with 0.1 M PBS, pH 7.4, then incubated overnight with 20% normal donkey serum (Jackson Immunoresearch Laboratories) in 0.1 M PBS containing 0.5% Triton X-100 at 4 °C, in order to block the nonspecific binding. After blocking, sections were incubated for 40 h at 4 °C with the primary antibodies.V-19 α-DB antibody (dilution: 1:250) was combined with either anti-MCH (dilution: 1:100) or anti-NOS1 (dilution: 1:50). Dilutions were made with 0.1 M PBS containing 0.5% Triton X-100. After washing with PBS, the appropriate mixtures of fluorescent secondary antibodies were applied. Fluorescein (FITC)conjugated AffiniPure donkey anti-goat IgG, Cy3-conjugated AffiniPure donkey anti-rabbit IgG (both from Jackson Immunoresearch Laboratories) and Texas Red conjugated anti-mouse IgG (Vector Laboratories) were diluted 1:50, 1:100 and 1:50, respectively. Incubation was performed at room temperature for 3 h. Sections were finally washed with 0.1 M PBS at room temperature, then mounted using Vectashield medium (Vector Laboratories), and analysed/photographed under a Zeiss Axiophot epifluorescence microscope. Control specimens were prepared by omitting the primary antibodies from the incubation solution. No immunostaining was observed in controls.

4.1.4.

Confocal laser scanning microscopy analyses

For double labelling tissue sections were fixed as described before and blocked in 10% normal chicken serum (NCS) in 1× PBS for 30 min. Sections were then incubated overnight at 4 °C with combinations of two primary antibodies, in the same 10% blocking buffer. This was followed by 30 min incubation with a 2% (v/v) NCS–PBS-buffered cocktail mixture containing Alexa 488-conjugated chicken anti-rabbit IgG (1:200 dilution), Alexa 546-conjugated donkey anti-goat IgG (1:200 dilution), and TOPRO-3 nucleic acid stain (1:2000 dilution; Molecular Probes, Invitrogen Ltd., Paisley, UK). Specimens were mounted in nonfluorescing Vectashield® medium (Vector Laboratories Ltd., Peterborough, UK). Sections were examined using LSM 510 Meta confocal laser scanning microscope (Carl Zeiss, Jena, Germany). Parameters, including pinhole size, photomultiplier tube settings and laser intensities were kept constant for all the imaging analyses conducted so that the relative immunofluorescence intensity levels could be compared between samples.

Acknowledgments The authors thank Dr D. Blake, Department of Pharmacology, University of Oxford for α1-CT-FP antibody sample and Dr. R.M. Grady, Washington University School of Medicine, St. Louis, Missouri, for alpha-dystrobrevin knockout mice. The excellent technical assistance of J. Beveridge is greatly appreciated. This

work was supported by the Hungarian Scientific Research Fund (OTKA, T 037597) for V. Jancsik.

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