slice cultures with the ABD of spectrin or closely related proteins, following the approach previously used to study a-actinin [30]. We suggest that bI-spectrin may play both a structural and functional role in dendritic spines through interaction with the actin cytoskeleton and Rac3. Methods Ethical approval All experimental protocols that involve tissue collected from animals in this study have been approved by The University of Maryland School of Medicine Institutional Animal Care and Use Committee (Protocol #0609015). Neuronal cultures Organotypic hippocampal slice cultures were prepared with the roller tube method [31]. Briefly, hippocampi were dissected from 5–7 day-old rat pups and cut into 400 mm-thick slices with a chopper, under sterile conditions. Slices were attached to cleaned, poly-lysine–coated coverslips in a mixture of lyophilized chicken plasma (Cocalico, Reamstown, PA) and concentrated fibrin solution (Tisseel, Baxter Health Care) that was then clotted with thrombin. Coverslips containing slices were placed in culture tubes on a slowly rotating roller drum in an incubator. After 4 days in vitro, cultures were treated with antimitotics overnight to reduce glial proliferation. Cultures remained in the incubator for 12–16 days before transfection. Cortical neuronal cultures were prepared and cultured from E18 rat neocortex, as described [12]. ABD and Rac constructs Plasmids were created using PCR to amplify the cDNAs of the actin-binding domains (ABDs) of dystrophin, utrophin, actinin-2, and bI-spectrin [detailed methods: 32]. The first 942 base pairs of the coding sequence of the ABD of bI-spectrin (NM_000347) was amplified from plasmid B259 [32]. With primers A, 59 CGTAGAAT TCCTATG-ACATCGGCCACAG 39 (sense), and B, 59 TCCCC- GCGGCTAAGGTGAG CAGGTCCGA 39 (antisense). cDNA was cloned into the DsRed2-c1 plasmid vector (BD Biosciences Clontech) via a EcoRI sense site and a SacII antisense site. For dystrophin, a plasmid (pDys246) [33] was used with primers A, 59 GCGAATTC- TATGTTGTTGTGGGAAGAAGTA 39 (sense), and B, 59 GCAGGTACCCTATTCAATGCTCACTTGTTG 39 (antisense), to amplify and clone the first 738 bases of the dystrophin gene (accession no. M18533), containing its ABD. The dystrophin ABD was cloned into the DsRed2-c1 vector via an EcoRI site on the sense primer and a KpnI site on the antisense primer. An utrophin plasmid (pet23Utr261) [34] served as a template to amplify the ABD. The primers A, 59 TATAAGCTTC-TATGGCCAAGTATGGGAC 39 (sense) and B, 59 TGTGGATCCCTAATCTATCGTGACTT- GCTG 39 (antisense) were used to amplify the first 783 bases of the utrophin gene (accession no. Y12229), which was then cloned into the DsRed2-c1 vector via a HinDIII sense site and a BamHI antisense site. The ABD of a-actinin-2 (CAA32078.1) was cloned into the DsRed2-c1 vector similarly, with the primers. A, 59 GTA- GAATTCTATGAACAGCATGAACCAG 39 (sense) and B, 59 TGTGGATCCCTATTTCTCAGCAATTTCCATG 39 (anti- sense) were used to amplify the first 671 bases of the a-actinin-2 gene, via an EcoRI sense site and a BamHI antisense site. GFP-actin was from Clontech (Mountain View, CA). cDNAs encoding wild type and dominant negative Rac 1 and 3 were myc- and HA-tagged constructs from the Guthrie cDNA Resource Center (Rollo, MO). Plasmids were introduced into bacteria and amplified using standard protocols. Amplified cDNA was isolated and purified using a HiSpeed Plasmid Maxi Kit (Qiagen; Valencia, CA) following the The Role of bI-Spectrin in Dendritic Spines manufacturer’s instructions and prepared for the biolistic transfection of organotypic hippocampal slice cultures. Biolistic transfection Cultures were transfected biolistically with a gene gun (Helios; Bio-Rad, Hercules, CA) as described [35]. Gold pellets (1.0 mm diameter) were coated with spermidine and then placed in a solution of 25 mg/ml DNA, which was precipitated onto the particles by the addition of CaCl2 to a final concentration of 500 mM. The coated pellets were attached to the walls of the cartridges with polyvinylpyrrolidone, as described. Prior to transfection, each slice was placed in a small volume of solution containing 40 mM amino-5-phosphonovaleric acid (AP5), 40 mM 6,7-dinitroquinoxaline-2,3-dione (DNQX), and 10 mM Mg2+ to block synaptic transmission and reduce excitotoxicity. Cultures were transfected by shooting the gold particles out of the gene gun through a nylon mesh (90 mm2 pore size) at a distance of 2 cm from the culture surface at a pressure of 200 psi. After transfection, slices were returned to roller tubes and placed in the incubator for 24–48 hrs before being used for electrophysiology or imaging. Electrophysiology Cultures were placed in a recording chamber and perfused with extracellular saline containing (in mM): 145 NaCl, 10 NaHCO3, 2 CaCl2, 2 MgCl2, and Phenol Red (10 mg/l). Transfected neurons were identified by fluorescence microscopy before recordings were performed. Patch pipettes were filled with the following (in mM): 130 gluconic acid, 10 KCl, 1 MgCl2, 2 ATP, 10 HEPES, and 0.1 EGTA, titrated to pH 7.4 with KOH. To ensure that transfected neurons were those being recorded from, the pipette solution also contained Alexa 488 (100 mM). After breakthrough to whole-cell configuration, transfected neurons contained both green Alexa 488 and red DsRed fluorescence, whereas untransfected cells, recorded within the same slice culture, displayed only green fluorescence. Miniature excitatory postsynaptic currents (mEPSCs) were recorded at a holding potential of 270 mV in the presence of tetrodotoxin (1 mm) and bicuculline (40 mm) at room temperature. Recording pipette resistances were between 6.5 and 8 MV. Recordings in which the access resistance exceeded 30 MV were discarded. Spontaneous mEPSCs were amplified using an Axopatch 200B amplifier and Clampex 9 software (Molecular Devices, Sunnyvale, CA), low-pass filtered at 2 kHz, and digitized at 10 kHz. mEPSCs were identified and characterized using a template-matching algorithm created from the average of 50 events. To be included for analysis, mEPSCs had to have a uniphasic rise time of ,6 ms, a decay time constant of ,25 ms and a peak amplitude .4 pA. Decay time constants were calculated by fitting each event with a single exponential. Microscopy and data analysis In experiments involving static imaging, spine head width and spine length were measured from confocal micrographs of all morphological spine categories. Confocal images were taken with an upright Zeiss LSM-510 Meta Confocal Microscope System using a water immersion objective (Zeiss; 60X/NA 1.2, C- 2 Apochromat, effective pixel size = 0.017 mm ). Fluorescence exci- tation was achieved using a 25 mW Argon ion laser (Spectra- physics, Mountain View, CA) and illuminating the specimen using the 488 nm and 514 nm lines. 3D image stacks were collected from living cells in unfixed cultures, background fluorescence was subtracted, and images were collapsed into maximal fluorescence 2D projections. Spines were then traced manually and the spine head width or the spine length was computed using the SimplePCI software with the PLoS ONE | www.plosone.org 2 January 2011 | Volume 6 | Issue 1 | e16197