Neuroscience 292 (2015) 101–111

DEVELOPMENTAL CHANGES IN THE FLOTILLIN-1 EXPRESSION PATTERN OF THE RAT VISUAL CORTEX K. NAKADATE *

where it exerts effects at the presynaptic sites of excitatory and inhibitory neurons. Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Department of Basic Biology, Educational and Research Center for Pharmacy, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan

Abstract—Ocular dominance plasticity is activity dependent, changes in response to eye competition, and is transitory during developmental stages. Lipid rafts have modulatory functions in cellular, physiological, and behavioral processes. Although many of these modulatory roles are mediated by flotillin-1, a lipid raft-associated protein, the ontogenetic changes in the cellular and subcellular distribution patterns of flotillin-1 are unclear. I investigated the developmental pattern of the distribution of flotillin-1 in the rat visual cortex with immunohistochemistry at both light and electron microscopic levels. An affinity-purified antiflotillin-1 antibody reacted with a single band of about 40– 50 kDa in total proteins prepared from the rat visual cortex. Flotillin-1 levels transiently increased on postnatal days 21–35. Flotillin-1 immunoreactivity at 3 weeks of age was broadly distributed though all visual cortical layers, but it exhibited a relatively higher density in layers II/III and V/VI. Flotillin-1 immunoreactivity at 3 months of age was significantly decreased compared with that at 3 weeks of age. Strong flotillin-1 immunoreactivity was observed in both neuronal perikarya and processes at 3 weeks of age. Double-labeling experiments with anti-microtubule-associated protein 2, anti-neurofilament, anti-synaptophysin, anti-vesicular glutamate transporter 1, anti-vesicular glutamate transporter 2, anti-glial fibrillary acidic protein, and flotillin-1 mainly labeled the somata of excitatory neurons and corticocortical synapses. Some flotillin-1 was distributed in excitatory neuron axons, thalamocortical synapses, astrocytes, oligodendrocytes, and microglial cells. Immunoelectron microscopy revealed numerous regions of flotillin-1 immunoreactivity near the rough endoplasmic reticulum in neurons and presynaptic regions at 3 weeks of age. These findings illustrate early developmental changes in the cellular and subcellular localization of flotillin-1 protein in the rat visual cortex. Moreover, the ultrastructural distribution of flotillin-1 immunoreactivity suggested that flotillin-1 was transported mainly into presynaptic terminals

Key words: flotillin-1, lipid raft, immunohistochemistry, immunoelectron microscopy, synaptic plasticity.

INTRODUCTION Neuronal development is important for learning and memory. After neurite outgrowth, neurites begin to form synapses with other neurons to promote the formation of the neuronal circuitry. Synapses, which are necessary for interactions with environmental stimuli, are added and removed before they mature. Activitydependent changes in synaptic plasticity play an important role in the central nervous system during the development (Kaminska et al., 1995; Hensch et al., 1998; Lamsa and Taira, 2003; Nudo, 2003; Weber et al., 2003; Thakur et al., 2004). Ocular dominance (OD) plasticity has been examined in developmental animal models following monocular deprivation in the visual cortex (Wiesel et al., 1974; Hubel et al., 1977; Issa et al., 1999; Levelt and Hubener, 2012). OD plasticity is activity dependent, changes in response to competition in both eyes, and is transitory during developmental stages. Monocular deprivation normally alters OD in the rat visual cortex during the postnatal critical period (postnatal days 17–45) (Maffei et al., 1992; Fagiolini et al., 1994). The noradrenergic (NA) system plays important roles in OD plasticity through beta-1 adrenoceptors (Kasamatsu and Pettigrew, 1976, 1979; Kasamatsu et al., 1979; Kasamatsu and Shirokawa, 1985; Shirokawa and Kasamatsu, 1986; Imamura and Kasamatsu, 1989, 1991; Shirokawa et al., 1989). Furthermore, in the developing visual cortex, the NA system, which acts through alpha-1 adrenoceptors, plays important roles in the synapse formation (Blue and Parnavelas, 1982), synaptic plasticity (Kirkwood et al., 1999; Inaba et al., 2009), and maintenance of excitatory synapses (Nakadate et al., 2006). The activation of alpha-1 adrenoceptors selectively suppresses the horizontal propagation of excitation in the supragranular layers of the rat visual cortex (Kobayashi et al., 2000). These receptors that are related to the modulation of synaptic plasticity have also been shown to be distributed on lipid rafts (Chini and Parenti, 2004).

*Tel/fax: +81-42-495-8634. E-mail address: [email protected] Abbreviations: CNPase, 2-3-cyclic nucleotide 3-phosphodiesterase; GFAP, glial fibrillary acidic protein; MAP2, microtubule-associated protein 2; OD, ocular dominance; PB, phosphate buffer; PBS, phosphate-buffered saline; PBS-T, PBS containing 0.3% Triton X-100; RT, room temperature; SDS, sodium dodecyl sulfate; TPBS, PBS containing 0.1% Tween 20. http://dx.doi.org/10.1016/j.neuroscience.2015.02.035 0306-4522/Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved. 101

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In addition to the regulation of synaptic formation with receptors on lipid rafts, it is possible that lipid rafts themselves influence the synapse formation. Lipid rafts are microdomains of the plasma membrane that are rich in glycosphingolipids, cholesterol, and acylated proteins and act as signaling platforms (Pike, 2006). Many receptors that are expressed on lipid rafts (Chini and Parenti, 2004) are involved in various cellular functions, such as signal transduction, molecular sorting, and membrane trafficking, through each of these expressed receptors (Simons and Ikonen, 1997; Lafont et al., 1999). The cholesterol that is derived from glial cells induces the formation of synapses (Mauch et al., 2001). By increasing the number of lipid rafts, the cholesterol synthesis within neurons can enhance synaptogenesis (Suzuki et al., 2007). In addition, intact lipid rafts (Hering et al., 2003) and cholesterol homeostasis (Goritz et al., 2005) are necessary for synapse stability. Flotillin-1, which is a lipid raft-associated protein, has been shown to be important in the early stages of neuronal development (Carcea et al., 2010; Swanwick et al., 2010a,b). Flotillin-1 was identified as a caveolaeassociated integral membrane protein in the brain and the lung (Bickel et al., 1997). Although neurons do not express caveolin or possess caveolae, flotillin-1 is abundant in the brain. It is expressed in pyramidal neurons and astrocytes in the human brain tissue (Kokubo et al., 2000). Flotillin-1 is a 428-amino-acid protein that is associated with the cytoplasmic side of lipid raft membranes (Solis et al., 2007). It is ubiquitously expressed in all cell types in adulthood (Morrow and Parton, 2005), and the flotillin-1 expression increases during developmental stages (Volonte et al., 1999). Flotillin-1 induces filopodia formation in neurons (Hazarika et al., 1999; NeumannGiesen et al., 2004) and neuritic outgrowth (Swanwick et al., 2010a) as well as promotes hippocampal neuronal differentiation (Munderloh et al., 2009; Swanwick et al., 2010b). Previous studies of the distribution of flotillin-1 have mainly been conducted with biochemical or immunofluorescence microscopy methods in cultured cells. Moreover, the subcellular localization of flotillin-1 in tissues has not been demonstrated. Therefore, I investigated the localization of flotillin-1, the lipid raftassociated protein, to clarify developmental changes. In the present study, I performed an immunoelectron microscopic analysis of flotillin-1 and double immunostaining of flotillin-1 and various markers to investigate developmental changes in the subcellular localization of flotillin-1 in the rat visual cortex around the time of the critical period of OD plasticity.

EXPERIMENTAL PROCEDURES Animals In this study, 30 Sprague–Dawley male rats (Charles River Laboratories Japan, Inc., Yokohama, Japan) were used. The animals were housed under temperature- and humidity-controlled conditions with a 12-h light/dark cycle and ad libitum access to food and water. All experiments were performed in accordance with the

National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the animal research committee of the Meiji Pharmaceutical University. In the present study, all efforts were made to minimize the suffering of animals and to reduce the number of animals used. Antibody for flotillin-1 A polyclonal antibody to flotillin-1 was purchased from Immuno-Biological Laboratories Co., Ltd. (Gunma, Japan). It had been raised against the C-terminal part of the common site of the rat and mouse flotillin-1 sequence (SQVNHNKPLRTA). This sequence is 100% conserved in the rat and mouse flotillin-1. To examine the specificity, antibodies were preadsorbed with synthetic peptides that were based on the findings of the epitope mapping. In brief, 100 lg of the synthetic peptide (SQVNHNKPLRTA) was incubated with 10 lg of the anti-flotillin-1 polyclonal antibody at 4 °C for 24 h on a rotation vortex. The supernatant solution containing the blocked antibody was then tested with immunoblotting and immunohistochemical analyses, as described below. Protein preparation for western blotting Proteins were prepared for western blotting in accordance with methods described previously (Nakadate et al., 2006; Amaddii et al., 2012). Rats of each age were perfused through the left ventricle with ice-cold saline. The brains were rapidly removed and homogenized in 10 volumes of ice-cold homogenate buffer (20 mM Tris–HCl at pH 7.5, 1 mM ethylenediaminetetraacetic acid, 1 mM dithiothreitol, and 150 mM NaCl) containing protease inhibitors (one tablet/10 mL homogenate buffer, Completeä Mini, Roche Diagnostics, Basel, Switzerland). Homogenates were then centrifuged at 500g for 5 min at 4 °C, and supernatants were dissolved in sodium dodecyl sulfate (SDS) sample buffer (62.5 mM Tris–HCl, pH 6.8, containing 3% SDS, 5% glycerol, and 2% 2-mercaptoethanol) and then boiled for 5 min. According to the Bradford method, protein concentrations were measured with a protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and determined with bovine serum albumin, as previously described (Nakadate et al., 2006). Immunoblotting SDS–polyacrylamide gel electrophoresis and western blotting were performed with the ECL-Plus immunoblotting detection system (GE Healthcare Life Sciences, Buckinghamshire, UK), as previously described (Nakadate et al., 2006). The proteins were separated by SDS–polyacrylamide gel electrophoresis (12% gels) and electrophoretically transferred at 50 V for 60 min onto a polyvinylidene difluoride membrane (Immobilonä-P, EMD Millipore, Billerica, MA, USA). After blocking with 5% (w/v) skim milk (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and Block-Ace

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(Yukijirushi Nyugyo Co., Ltd., Sapporo, Japan) in phosphate-buffered saline (PBS) containing 0.1% Tween 20 (TPBS, pH 7.5) for 1 h at room temperature (RT), the membrane was washed and incubated with the primary anti-flotillin-1 antibody (1:4000 dilution in TPBS) for 1 h at RT. After incubation with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:2000 dilution in TPBS) for 45 min at RT, immunoreactive bands were detected with an ECL-Plus kit (GE Healthcare Life Sciences). The densities of bands were quantified with an ImageJ software (http://imagej.nih.gov/ij/). Each value from each group was statistically compared with an analysis of variance (ANOVA). Tissue preparation for immunohistochemistry and immunoelectron microscopy Tissue preparation for immunohistochemistry and immunoelectron microscopy was performed according to the protocol described previously (Nakadate et al., 2006). The animals were deeply anesthetized with an overdose of sodium pentobarbiturate (50 mg/kg, intraperitoneal; NembutalÒ; Abbott Laboratories, Abbott Park, IL, USA). The animals were then perfused through the left ventricle with saline. For the immunohistochemical and immunofluorescence analyses, animals were perfused with a fixative containing 4% paraformaldehyde and 15% saturated picric acid in 0.1 M phosphate buffer (PB, pH 7.4). The brains were then removed and postfixed in the same fixative overnight at 4 °C. The brains were cryoprotected in graded concentrations of sucrose (final: 30%) in 0.1 M PB, quickly frozen on dry ice, and then cut into 50-lm coronal sections with a freezing microtome (REM-710, Yamato Co., Ltd., Tokyo, Japan). For the immunoelectron microscopic study, animals were perfused with saline, which was followed by perfusion with a fixative containing 4% paraformaldehyde, 15% saturated picric acid, and 0.05% glutaraldehyde in 0.1 M PB (pH 7.4). The brains were quickly removed and postfixed in the same fixative for 24 h at 4 °C. The brains were cut into 50-lm-thick sections with a microslicer (DTK-1000, Dosaka EM Co., Ltd., Kyoto, Japan). These sections were processed for electron microscopy as described below. Filipin staining To label the free cholesterol in the cell membrane, 50-lmthick sections from rats of different developmental stages were stained with the filipin solution according to methods described in previous reports (Hering et al., 2003; Liao et al., 2007; Suzuki et al., 2007; Nicholson and Ferreira, 2009). The sections were rinsed with PBS and incubated with 0.15% glycine in PBS for 10 min at RT to quench the paraformaldehyde. The sections were stained with 0.005% filipin (Sigma–Aldrich Co. LLC, St. Louis, MO, USA) in PBS for 2 h at RT. The sections were then rinsed with PBS, mounted on gelatin-coated slides, and coverslipped. All sections were viewed with fluorescence microscopy (BX51 fluorescence system, Olympus Corporation, Tokyo, Japan) with a UV filter set and captured with a CCD camera system (DP-50, Olympus Corporation).

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Immunohistochemistry for light microscopy Immunohistochemistry was performed with the freefloating method. The sections were washed in PBS containing 0.3% Triton X-100 (PBS-T) and incubated at RT for 60 min in PBS-T containing 1% hydrogen peroxide. After several washes, sections were incubated with a blocking solution containing 5% normal goat serum (Vector Laboratories, Inc., Burlingame, CA, USA) in PBS-T at RT for 2 h and then incubated for 2 days at 4 °C with anti-flotillin-1 antibody (diluted 1:300 in PBST). After washing with PBS-T, sections were incubated with a biotinylated anti-rabbit antibody (Vector Laboratories, Inc.) at RT for 2 h. The sections were then washed and reacted with the avidin–biotin peroxidase complex (ABC kit, Vector Laboratories, Inc.) at RT for 2 h. The sections were subsequently incubated in 50 mM Tris–HCl (pH 7.3) containing 0.05% 3,30 diaminobenzidine tetrahydrochloride (DAB; Dojindo Laboratories, Kumamoto, Japan) and 0.003% hydrogen peroxide. All sections were mounted on gelatin-coated slides, dehydrated through graded concentrations of ethanol and then xylene, and then mounted with MountQuick (Daido Sangyo Co., Ltd., Tokyo, Japan). In addition, control sections were incubated in a solution that did not contain the primary antibody or that contained a primary antibody that was preadsorbed with the synthetic peptide. Photomicrographs of horseradish peroxidase-DABreacted sections and Nissl-stained sections were captured with a CCD camera system (FUJIX DIGITAL CAMERA HC-2500 3CCDÒ, Fujifilm Corporation, Tokyo, Japan). The anatomical structures were identified by direct observation of Nissl-stained sections. Double-immunofluorescence staining The sections were incubated with a blocking solution and then with a cocktail of primary antibodies overnight at 4 °C. The primary and secondary antibodies that were used in the present study are listed in Table 1. One primary antibody was an anti-rabbit anti-flotillin-1 antibody (1:300 in PBS-T) and the other was one of the following mouse monoclonal antibodies: anti-Hu C/D (1:2000, Life Technologies, Grand Island, NY, USA), anti-neurofilament (1:1000, BioScience Products AG, Emmenbruecke, Switzerland), anti-synaptophysin (1:1000, Dako North America, Inc., Carpinteria, CA, USA), anti-vesicular glutamate transporter 1 (vGlut1; 1:5000, EMD Millipore), anti-vesicular glutamate transporter 2 (vGlut2; 1:5000, EMD Millipore), antimicrotubule-associated protein 2 (MAP2; 1:1000, Sigma–Aldrich Co. LLC), anti-glial fibrillary acidic protein (GFAP; 1:2000, Sigma–Aldrich Co. LLC), anti-OX-42 (1:300, Bio-Rad Laboratories, Inc.), or anti-2-3-cyclic nucleotide 3-phosphodiesterase (CNPase; 1:1000, Sigma–Aldrich Co. LLC). The sections were rinsed with PBS-T and incubated with conjugated secondary antibodies, including Alexa 488-conjugated goat antirabbit IgG (1:1,000 in PBS-T, Life Technologies) and Alexa 568-conjugated goat anti-mouse IgG (1:1000 in PBS-T, Life Technologies). All immunofluorescence

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Table 1. Primary and secondary antibodies used in this study Antibodies

Serum code and immunogen of primary antibodies or conjugation of secondary antibodies

Primary antibody Flotillin-1 18951: Peptide sequence (SQVNHNKPLRTA) corresponding to amino acid sequence conserved in rat and mouse Flotillin-1 proteins Hu C/D A21271: Peptide sequence (QAQRFRLDNLLN) corresponding to amino acid sequence conserved in human Hu proteins Neurofilament M53: Neurofilament from human brain Synaptophysin M0776: Synaptophysin from bovine brain vGlut1 AB5905: Synthetic peptide from rat vGlut1 protein

Host species

Dilution

Source

Rabbit

1:300

Mouse

1:2000

Immuno-Biological Laboratories Invitrogen

1:1000 1:1000 1:5000

Bio-Science Dako Cytomation Chemicon

1:5000

Chemicon

1:1000 1:2000 1:300

vGlut2

AB5907: Synthetic peptide from rat vGlut2 protein

MAP2 GFAP OX-42

M4403: Rat brain microtubules G3893: GFAP from pig spinal cord MCA275GA: Rat peritoneal macrophages

Mouse Mouse Guinea pig Guinea pig Mouse Mouse Mouse

CNPase

C5922: Human 20 , 30 -cyclic nucleotide-30 -phosphohydrolase (CNPase)

Mouse

1:1000

Sigma–Aldrich Sigma–Aldrich SEROTEC Immunological Excellence Sigma–Aldrich

Goat Goat Goat

1:250 1:1000 1:1000

Vector Laboratories Invitrogen Invitrogen

Goat

1:1000

Invitrogen

Secondary antibody Anti-rabbit IgG Biotin Anti-rabbit IgG Alexa 568 Anti-mouse Alexa 488 IgG Anti-guinea Alexa 488 pig IgG

images were obtained under a LSM510 confocal laser scanning microscope (Carl Zeiss AG, Jena, Germany). Immunoelectron microscopy For the immunoelectron microscopic observations, all sections were cryoprotected in sequential sucrose solutions (final: 30%) in 0.1 M PB. The sections were then freeze–thawed with liquid nitrogen. The sections were incubated in a blocking solution containing 10% normal goat serum in 0.1 M PBS for 2 h. After blocking, sections were incubated with primary antibodies (diluted 1:300) in PBS containing 3% normal goat serum overnight at 4 °C. The sections were incubated with biotinylated secondary antibody (diluted 1:250 in PBS) and then with the ABC kit (Vector Laboratories, Inc.), and finally, they were reacted with the DAB solution (Dojindo Laboratories). After incubation with OsO4 for 2 h, sections were stained with uranyl acetate, dehydrated with ethanol, and embedded in Epon-812 resin (TAAB Laboratories Equipment, Ltd., Aldermaston, UK). Ultrathin sections (70-nm thickness) were cut on an ultramicrotome (Ultracut S; Reichert-Nissei, Tokyo, Japan) and examined with an H-7100 electron microscope (Hitachi, Ltd., Tokyo, Japan).

RESULTS Developmental changes in the free cholesterol in the visual cortex Sections from 7-, 14-, 21-, and 35-day-old and 3-monthold rats were stained with the filipin solution to determine developmental changes in the unesterified

cholesterol (free cholesterol) in the visual cortex. In layers II/III of the primary visual cortex (area 17, Oc1B) on postnatal day 7, the fluorescence of filipin was weakly detected (Fig. 1). Filipin-positive free cholesterol was detected in neuronal cell somata without cell nuclei throughout all sections from rats of all developmental stages (Arrows in Fig. 1). Moreover, the fluorescence of filipin was observed in the perineural space and neuropil. The intensity of the filipin fluorescence in the perineural space and neuropil transiently increased with age, and the accumulation of filipin in neuronal cell somata slightly changed with age (Fig. 1).

The detection of flotillin-1 protein in the visual cortex Flotillin-1, which is a lipid raft-associated protein, is important in the early stages of neuronal development. However, the developmental changes of flotillin-1 and the subcellular localization of flotillin-1 were unknown. Therefore, I first examined developmental changes in flotillin-1 in the visual cortex. The specificity of the affinity-purified antibody to flotillin-1 protein was examined by the immunoblotting of membrane fractions that were obtained from 56-day-old rat brains (Fig. 2A). The flotillin-1 antibody labeled a single band with a molecular weight of about 44–48 kDa (Flot1 lane in Fig. 2A). This molecular weight was expected from the amino-acid-sequence database (NCBI reference sequence: NP 073192.2). The immunoreactivity was completely adsorbed by the preincubation of primary antibodies with excess amounts of respective epitope peptides (P.A. lane in Fig. 2A).

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Fig. 1. Postnatal developmental changes in lipid accumulation in the rat visual cortex. Photomicrographs of the layer II/III of the primary visual cortex (area 17, Oc1B). Fluorescent filipin staining for the unesterified cholesterol (free cholesterol) was examined in 7-, 14-, 21-, and 35-day-old and 3-month-old rats. Filipin-stained free cholesterol is localized to cell somata (arrows) and perineural space and neuropil. The accumulation of cholesterol in cell somata exhibited little change (constantly higher expression), but the cholesterol in the perineural space and neuropil increased transiently. The scale bars=20 lm.

filipin staining observations, western blots showed that flotillin-1 protein immunoreactivity transiently increased, and the peak in immunoreactivity was from postnatal days 21–35 (Fig. 2B). The optical densities of bands were significantly higher on postnatal days 21–35 than those on other days (ANOVA, Fisher posthoc test, P < 0.05; Fig. 2B,C). To determine developmental changes in flotillin-1, I also examined developmental changes in actin, which is a housekeeping protein. These results indicated that the flotillin-1 expression transiently increased with age. Next, I performed an immunohistochemical analysis in the visual cortex (Fig. 3). The flotillin-1 immunoreactivity on postnatal day 21 was stronger than that in the 3month-old rat (Fig. 3C vs. D, respectively). Fig. 2. Specificity of the polyclonal anti-flotillin-1 antibody and postnatal developmental changes in flotillin-1 expression. (A) The anti-flotillin-1 antibody recognized a single band with a molecular weight of 44–48 kDa within the rat visual cortical membrane fraction (Flot1 lane). No immunoreactivity was observed in western blots that were conducted with serum that was preadsorbed with the synthetic peptide of the epitope (P.A. lane). (B) The brain homogenates (20 lg of protein/lane) that were prepared from rats that were 1-, 7-, 14-, 21-, and 35-postnatal (P)-days old and 3- and 6-months (M) old were electrophoresed on a sodium dodecyl sulfate–polyacrylamide gel, blotted onto a membrane, and reacted with an antibody against flotillin-1 or actin. (C) The histogram shows the relative amount of protein as a percentage of the P21 expression level. Each point represents the mean ± standard deviation (n = 3). Each level was measured by densitometric scanning of western blots.

Moreover, lack of immunohistochemical crossreactivity was confirmed by observations that flotillin-1 immunoreactivity was completely absent after the omission of the primary antibody (data not shown) and that the immunoreactivity was completely adsorbed after the preincubation of primary antibodies with an excess amount of the synthetic peptide of the antigen (Fig. 3B). Thus, this antibody appeared to specifically bind to the flotillin-1 protein. Postnatal changes in flotillin-1 expression in the rat visual cortex Because the intensity of filipin transiently increased with age, I next analyzed developmental changes in flotillin1, the lipid raft-associated protein. In accordance with

Laminar distribution of flotillin-1 in the rat visual cortex The laminar pattern of flotillin-1 immunolabeling was examined (Fig. 3). Although the immunoreactivity on postnatal day 21 was intense in all layers of the visual cortex, it was relatively low in the layer IV (Fig. 3C). Flotillin-1 immunoreactivity was localized to cell somata, and it exhibited a small dot-like pattern of immunoreactivity in the perineural space. In the layer II, large numbers of immunoreactive somata and fibers were present, and no density was observed in nuclei (Fig. 3E). There were less flotillin-1-immunoreactive somata in the layer IV compared with those in the layer II, but large numbers of immunoreactive fibers were detected (Fig. 3F). In the layer V, moderate numbers of immunoreactive somata and fibers that appeared as dot-like structures were present (Fig. 3G). In 3-monthold rats, flotillin-1 immunoreactivity was also detected, but the intensity was lower than that on postnatal day 21 (Fig. 3D). Moderate flotillin-1 immunoreactivity was present in the layer II/III, and low levels of reactivity were distributed in all layers (Fig. 3D). In the layer II, moderate levels of immunoreactive somata and fibers, which appeared as dot-like structures, were present, and a relatively low density was observed in nuclei (Fig. 3H). No staining was detected in the tissue that was either incubated in antisera that were preadsorbed with the synthetic peptide (Fig. 3B) or after the omission of primary antisera (data not shown).

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Fig. 3. Distribution of flotillin-1 in the visual cortex of postnatal 3-week-old and 3-month-old rats. These photomicrographs of immunostaining were taken from the primary visual cortex (area 17, Oc1B). (A) A Nissl-stained section showing the anatomic appearance of the primary visual cortex. Layers I–VI of the visual cortex and white matter are represented in Fig. 3A as I, II/III, IV, V/VI, and WM. (B) Incubation with the antiserum that was preadsorbed with immunizing peptide. The laminar pattern of flotillin-1 staining in 3-week-old (C) and 3-month-old (D) rats is shown. The black rectangular areas in Fig. 3C, D are enlarged in Fig. 3E–H. Fig. 3E–H show flotillin-1 immunoreactivity in layers II/III, IV, and V/VI in 3-week-old rats and in layers II/III in 3-month-old rats, respectively. The scale bars=100 lm in D (applies to A–D) and 50 lm in H (applies to E–H).

The cellular localization of flotillin-1 in the rat visual cortex Double immunostaining with fluorescently labeled antibodies to flotillin-1 (red color in Fig. 4) and many neuronal subcellular markers (green color in Fig. 4) was used to determine cell types and cellular compartments that contained flotillin-1. The images in Fig. 4F,P were obtained from the layer IV of the primary visual cortex, and other images were obtained from the layer II/III of the primary visual cortex. To analyze changes in flotillin-1 localization by ontogeny, the immunoreactivities of flotillin1 and subcellular markers were examined on postnatal day 21 (Fig. 4A–F) and at 3 months (Fig. 4G–L). On postnatal day 21, strong flotillin-1 immunoreactivity was colocalized with Hu, which is a neuronal somata marker (Fig. 4A–A00 ). Moreover, strong flotillin-1 immunoreactivity was observed along the plasma membrane (strong yellow color in Fig. 4A00 ). However, the colocalization of flotillin-1 with Hu in the 3-month-old rats was significantly reduced (Fig. 4G–G00 ). Immunohistochemistry with an antibody to neurofilament, which is an axonal marker, showed that flotillin-1 protein was localized in the axons of young rats, but in 3-month-old rats, the percentage of axons showing the colocalization of flotillin-1 with the axonal marker was significantly decreased (Fig. 4B00 vs. H00 ). The coexpression of flotillin-1 and synaptophysin (Fig. 4C00 , I00 ) and vGlut1 (Fig. 4D00 , J00 ) at each age showed a trend that was similar to that of the axonal marker. The coexpression of flotillin-1 and vGlut2 was detected in the layer II/III of the young primary visual cortex (Fig. 4E00 ), and in the adult visual cortex, flotillin-1 was not coexpressed with vGlut2 (Fig. 4K00 ). mRNA for vGlut2 is expressed in the lateral geniculate nucleus of the thalamus (Barroso-Chinea et al., 2007), and vGlut2 proteins are expressed in thalamocortical axon terminals (Fujiyama et al., 2003, 2004; Nakamura et al., 2005; Barroso-Chinea et al., 2008). Therefore, the coexpression of both flotillin-1 and vGlut2 in the layer IV of

the visual cortex was examined. These results were similar to those in the layer II/III in that coexpression was only detected in young rats (Fig. 4F00 ,L00 ). To determine if flotillin-1 was expressed in dendrites and spines, double staining with flotillin-1 and MAP2 was examined. These results revealed that a fraction of flotillin-1 was observed in dendrites and spines in both young rats and older rats (data not shown). Next, to determine whether flotillin-1 was expressed in glial cells, double immunostaining with fluorescently labeled antibodies to flotillin-1 (red color in Fig. 5) and many glial cell markers (green color in Fig. 5) was examined. The images were obtained from the layer II/III of the primary visual cortex. In young rats, double staining with anti-flotillin-1 (red color), anti-GFAP antibody (green color, Fig. 5A0 ,A00 ), anti-CNPase antibody (green color, Fig. 5B0 ,B00 ), and anti-OX42 antibody (green color, Fig. 5C’,C00 ) demonstrated that large amounts of flotillin-1 were localized in astrocytes, oligodendrocytes, and microglia in the young rat visual cortex. However, in older rats, only a small amount of flotillin-1 was detected in glial cells (Fig. 5D00 –F00 ).

Observations of flotillin-1 in presynaptic terminals To determine subcellular components that contained flotillin-1 in the rat visual cortex, an immunoelectron microscopic analysis was performed with the antiflotillin-1 antibody (Fig. 6). The immunoperoxidase products within the neuronal perikarya were associated with the endoplasmic reticulum in the rat visual cortex on postnatal day 21 and at 3 months (arrows in Fig. 6A,C, respectively). According to the type of synaptic contact (gray type I, excitatory synapse; gray type II, inhibitory synapse) (Gray, 1959), strong flotillin-1 immunoreactivity was observed in the presynaptic terminals of both excitatory and inhibitory synaptic sites

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Fig. 4. Laser scanning microscopic images of the double immunofluorescence of flotillin-1 (red fluorescence) with various neuronal markers (green fluorescence). Fig. 4A–F show flotillin-1 immunoreactivity in 3-week-old rats, and Fig. 4G–L show immunoreactivity in 3-month-old rats. All images, except for Fig. 4F, L, are from layers II/III. The images in Fig. 4F, L are from the layer IV. The red fluorescence color in each panel indicates the flotillin-1 expression patterns (A–L). The green fluorescence colors in each panel indicate neuronal markers (A0 –L0 ). Fig. 4A00 –L00 show the merge. The neuronal markers (green fluorescence) include Hu (A0 , G0 ), neurofilament (B0 , H0 ), synaptophysin (C0 , I0 ), vesicular glutamate transporter 1 (vGlut1; D0 , J0 ), and vesicular glutamate transporter 2 (vGlut2; E0 , F0 , K0 , and L0 ). All scale bars=10 lm.

(excitatory synapse: arrowheads in Fig. 6B; inhibitory synapse: double arrowheads in Fig. 6B) in the young visual cortex. Moreover, flotillin-1 was localized to the presynaptic region both near and far from the site of synaptic contact in young rats (Fig. 6B). In the adult rat visual cortex, flotillin-1 immunoreactivity was detected in presynaptic terminals and exhibited a punctate-like distribution (arrowheads in Fig. 6D). However, flotillin-1 expression in older rats was distributed only in presynaptic regions far from the site of synaptic contact (Fig. 6D). In the double immunohistochemical analysis, a very small amount of weak flotillin-1 immunoreactivity was observed in postsynaptic regions in rats of both ages (3 months; double arrow in Fig. 6E).

DISCUSSION Antibody specificity In this study, the distribution of flotillin-1 was demonstrated in the rat visual cortex. Because the specificity of the anti-flotillin-1 antibody in the rat brain was unclear, I first evaluated its specificity with immunoblotting and immunohistochemical analyses. In immunoblotting experiments, the anti-flotillin-1 antibody labeled a single band with a molecular weight of 44– 48 kDa. The molecular weight was the same as that expected by the amino-acid sequence of rat flotillin-1 (NCBI reference sequence: NP 073192.2). In the immunohistochemical analysis, a specific pattern of

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Fig. 5. Laser scanning microscopic images of the double immunofluorescence of flotillin-1 (red fluorescence) with various glial markers (green fluorescence). Fig. 5A–C show flotillin-1 immunoreactivity in 3-week-old rats, and Fig. 5D–F show immunoreactivity in 3-month-old rats. All images are from layers II/III. The red fluorescence color in each panel indicates flotillin-1 expression patterns (A–F). The green fluorescence colors in each panel indicate the glial markers (A0 –F0 ). Fig. 5A00 –F00 show the merge. The glial cell markers (green fluorescence) include GFAP (A0 , D0 ), CNPase (B0 , E0 ), and OX42 (C0 , F0 ). All scale bars=10 lm.

immunoreactivity for flotillin-1 was observed. Because the immunoreactivity was completely adsorbed after the preincubation of primary antibodies with excess amounts of respective epitope peptides, the antibody appeared to specifically bind to the flotillin-1 protein.

Developmental changes in lipid rafts First, I demonstrated the developmental changes of the free cholesterol in the rat visual cortex. Fluorescent filipin staining for the unesterified cholesterol transiently increased with age in the rat visual cortex. These results may reflect developmental changes in neuronal network formation that underlie OD plasticity in the rat visual cortex. Cholesterol synthesis within neurons can enhance synaptogenesis (Suzuki et al., 2007), and intact lipid rafts are necessary for synapse stability (Hering et al., 2003). In addition to this regulation of synaptic formation and/or regulation through receptors on lipid rafts, lipid rafts themselves might influence synapse formation. However, it was not possible with this technique to detect in detail whether the subcellular localization of filipin was changed during development. Therefore, I next examined the developmental changes of flotillin-1, which is a lipid raft-associated protein, in the rat visual cortex. Similar to filipin staining results, western blots showed that the contents of flotillin-1 increased transiently. Because flotillin-1 is expressed on lipid rafts, changes in flotillin-1 contents have been related to various cellular changes. Flotillin-1 is important in the early stages of neuronal development (Carcea et al., 2010; Swanwick et al., 2010a,b), filopodia formation (Hazarika et al., 1999; Neumann-Giesen et al.,

2004), hippocampal neuronal differentiation (Munderloh et al., 2009; Swanwick et al., 2010b), and neurite outgrowth (Swanwick et al., 2010a). Therefore, these findings in the present study of transient increases in both lipid rafts (filipin staining) and flotillin-1 protein might reflect synaptic formation or maintenance in the visual cortex. Furthermore, glia-derived cholesterol induces the formation of synapses (Mauch et al., 2001). In the present study, I demonstrated transient changes in the expression of glial cells in the visual cortex. Thus, developmental changes in flotillin-1 in glial cells might also reflect synaptic formation and maintenance. In the present study, it was not possible to elucidate whether the functions of lipid rafts were related to flotillin-1. However, their similar developmental expression pattern suggested that the developmental changes in flotillin-1 expression were closely related to the functions of lipid rafts.

Functional correlation with OD plasticity The well-studied developmental changes in synaptic plasticity in the visual cortex involve OD plasticity (Wiesel et al., 1974; Hubel et al., 1977). In the present study, an immunoelectron microscopic and double immunostaining analysis of flotillin-1 was performed to investigate developmental changes in the subcellular localization of flotillin-1 in the rat visual cortex around the time of the critical period for OD plasticity. OD plasticity is activity dependent, changes in response to competition in both eyes, and is transitory during cortical development. Monocular deprivation normally alters OD in the rat visual cortex during the postnatal critical period

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Fig. 6. Electron microscopy reveals flotillin-1 within a perikaryon and presynaptic terminal in the rat visual cortex. The flotillin-1 immunoreactivity in the visual cortex of 3-week-old (A, B) and 3-month-old (C–E) rats is shown. These photomicrographs were taken from ultrathin sections in which the lead citrate counterstain was omitted to facilitate the visualization of the immunolabeling. The arrows in Fig. 6A, C point to immunoreactivity near the rough endoplasmic reticulum in the neuronal cell body. The arrowhead and double arrowhead in Fig. 6B point to strong immunoreactivity in excitatory and inhibitory presynaptic terminals, respectively. Flotillin-1 immunoreactivity is shown in presynaptic terminals as a punctate-like distribution in Fig. 6D. A small amount of flotillin-1 immunoreactivity is present in postsynaptic regions (double arrowhead in Fig. 6E). All scale bars=500 nm.

(postnatal days 17–45) (Maffei et al., 1992; Fagiolini et al., 1994). Many key factors, such as the GABAergic system (Iwai et al., 2003), NMDA receptors (Sawtell et al., 2003), serotonergic system (Gu and Singer, 1995), cholinergic system (Bear and Singer, 1986), and NAergic system (Kasamatsu and Pettigrew, 1976, 1979; Kasamatsu et al., 1979) regulate OD plasticity. The multitude of physiological effects resulting from NA activation is likely to be a result of different types of postsynaptic responses to NA that is released at different loci. It is possible that various functional effects that are attributed to biogenic amines, including serotonin, acetylcholine, and NA, are the result of differential patterns of activation of each receptor subtype. In the NAergic system, the NA projections in the visual cortex play important roles in the formation of synapses (Blue and Parnavelas, 1982) and in synaptic plasticity during development (Kirkwood et al., 1999; Inaba et al., 2009). Many receptors, including adrenoceptors, are distributed on lipid rafts (Chini and Parenti, 2004). The density of beta-1 adrenoceptors gradually increases from postnatal days 4 to 24 and decreases from days 40 to 160 (McDonald et al., 1982). Therefore, it is possible that changes in flotillin-1 in lipid rafts may influence the stability of the distribution and function of receptors on lipid rafts.

These findings provide the first evidence of developmental changes in flotillin-1, a lipid raftassociated protein, in the rat visual cortex. The transient increase in flotillin-1 in the rat visual cortex may influence receptor stability in both neurons and glial cells and several functions, including OD plasticity. Moreover, the ultrastructural distributions of flotillin-1 in neurons indicated that flotillin-1 acts at presynaptic sites. These new findings suggested that flotillin-1 and receptors on lipid rafts have modulatory effects on many neuronal developmental functions, including OD plasticity.

CONCLUSIONS I demonstrated developmental changes in lipid raft and flotillin-1 in the rat visual cortex. These findings provide the first evidence of developmental changes in the cellular and subcellular localization of flotillin-1 immunoreactivity in rat visual cortex. Furthermore, the ultrastructural analysis of flotillin-1 immunoreactivity showed that flotillin-1 was localized to the presynaptic terminal. These results suggested that flotillin-1 becomes localized to presynaptic terminals with development and that it exerts effects at the presynaptic sites of excitatory and inhibitory neurons.

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Acknowledgments—This work was supported in part by a Grantin-Aid for Scientific Research (C) to K.N.

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(Accepted 19 February 2015) (Available online 27 February 2015)

Developmental changes in the flotillin-1 expression pattern of the rat visual cortex.

Ocular dominance plasticity is activity dependent, changes in response to eye competition, and is transitory during developmental stages. Lipid rafts ...
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