Three types of male (PND 14–21) mice (wild-type C57BL/6 mice, nAChR β2 subunit knockout mice on a C57BL/6 background and the glutamate decarboxylase-67 (GAD67)-green fluorescent protein (GFP) knock-in mice on a CD-1 background  were used in this study. Experiments were approved by the Institutional Animal Care and Use Committee at the Barrow Neurological Institute, St. Joseph's Hospital and Medical Center. Mice were group-housed in plexiglas shoebox-style cages with ad libitum access to food and water. PCR genotyping was performed to confirm the genetic status of these mice. Genomic DNA from mice newly born to heterozygotic, nAChR β2 subunit knock-out parents was extracted from mouse tail tips by using the QIAgen DNeasy Blood & Tissue Kit following the manufacture's protocol. PCR amplification of the nAChR β2 subunit or lac-Z (an indicator for the knock-out) was performed and PCR products were then resolved on 1% agarose gels and stained for visualization as described previously . Phenotyping of GAD67-GFP knock-in mice was achieved by examining the heads of the mice during postnatal 1–5 days, and these GAD67-GFP knock-in mice exhibited a striking green fluorescence in the brain that can be visualized through the skull at this age, as described previously .
Cells were injected with biocytin (5 mg/ml included in the intracellular solution) during patch-clamp recordings for immunostaining in 35 mm culture dishes. After recordings, cells were fixed in 4% paraformaldehyde for 10 min and washed with PBS 3 times at room temperature. Then a PBS-based blocking solution containing 5% normal goat serum and 0.3% Triton X-100 was then applied for 1 hr. After incubations at 4°C overnight with the primary GAD 67 antibody (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), the cultures were then washed with PBS three times. Thereafter, Avidin (AF488) and GAD 67 secondary antibody (Alexa 555-conjugated, anti-goat) were applied in the blocking solution for 2 hr at room temperature (all used at 1:1000 dilutions; all from Invitrogen, Carlsbad, CA). Cells were then finally washed three times for 5 min with PBS.
Acutely dissociated neurons from hippocampus and patch-clamp whole-cell current recordings
Neuron dissociation and patch-clamp recordings were performed as described by Wu et al. [17, 30, 67]. Briefly, postnatal 2 to 4-week-old mice were anesthetized using isoflurane, and the brain was rapidly removed. Several 400 μm coronal slices, which contained the dorsal CA1 region of the hippocampus were cut using a vibratome (Vibratome 1000 plus; Jed Pella Inc., Redding, CA) in cold (2–4°C) artificial cerebrospinal fluid (ACSF) containing (in mM): NaCl, 119; KCl, 2.5; NaHCO3, 26; MgSO2, 1.3; NaH2PO4, 1.0; CaCl2, 2.5 and glucose, 11, pH = 7.4. The ACSF was continuously bubbled with 95% O2 - 5% CO2. The slices were then incubated in a chamber (Warner Instruments, Hamden, CT) and allowed to recover for 2 hr at room temperature in oxygenated ACSF. Thereafter, the slices were treated with pronase (1 mg/ml) at 31°C for 30 min and subsequently treated with protease (1 mg/ml) for another 30 min. The ventral CA1 region was extracted by punching slices using a well-polished needle. The punched tissue was then dissociated mechanically by using several fire-polished micro-Pasteur pipettes in a 35 mm culture dish filled with oxygenated standard external solution [in mm: 150 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 10 HEPES; pH 7.4 (with Tris-base)]. Perforated-patch whole-cell recordings coupled with a three-barrel drug application system were used (Warner Instruments, Hamden, CT). To prepare for perforated-patch whole-cell recording, glass microelectrodes (GC-1.5; Narishige) were fashioned on a two-stage vertical pipette puller (P-830; Narishige, NY, USA), and the resistance of the electrode was 4–6 MΩ when filled with the internal solution. A tight seal (>2 GΩ) was formed between the electrode tip and the cell surface, which was followed by a transition from on-cell to whole-cell recording mode due to the partitioning of amphotericin B (200 μg/ml, Sigma, St. Louis, MO) into the membrane underlying the patch. After whole-cell, an access resistance lower than 60 MΩ was acceptable for perforated-patch recordings under voltage-clamp mode. The series resistance was not compensated in the experiments using dissociated neurons. Data were acquired by Axopatch 200B amplifier at 5 kHz with pClamp 9.2 software (Molecular Devices, Sunnyvale, CA) and analyzed with Clampfit 9.2 software (Molecular Devices, Sunnyvale, CA).
Drugs and Aβ preparation
Drugs used in this study were choline, methyllycaconitine (MLA), dihydro-β-erythroidine (DHβE) (Sigma, St. Louis, MO), brefeldin A (Calbiochem, San Diego, CA), scramble Aβ1-42, and Aβ1-42 (rPeptide, Athens, GA). Aβ1–42 was reconstituted in distilled water to a concentration of 100 μM and stored at −80°C as previously described . Aβ was used within 7 days after reconstitution. Aliquots diluted in standard extracellular solution yielded a predominantly oligomeric form. AFM was used to monitor aggregation forms of Aβ. For each use, Aβ stock (100 μM) was then diluted into desired concentrations. In this study, 1 nM Aβ within 4 hr after preparation mostly forms smaller oligomers. In all experiments within this study, Aβ was used within 4 hr before discarded each time.
Atomic force microscope (AFM) imaging
AFM was used to monitor the morphology of the Aβ aggregates before experiments. Aliquots were removed from Aβ samples, and then immediately spotted on freshly cleaved mica. After 2 min the mica was washed with 1 ml of de-ionized water, and then dried with compressed nitrogen. Topographic AFM images were obtained in air at room temperature using a Tapping Mode AFM with a Nanoscope IIIa controller (Veeco, Santa Barbara, CA). Images were acquired using oxide sharpened Si3N4 AFM tips (k = 40 N/m, fo ~ 300kHz) (Model: OTESPA, Veeco, Santa Barbara, CA) at scan rates of 2–3 Hz and at scan resolution of 512 samples per line. Images were subjected to 2nd order polynomial flattening as needed to reduce the effects of image bowing and tilt. AFM images were analyzed with the Scanning Probe Imaging Processor (SPIP) software (Image Metrology, http://www.imagemet.com) to generate height distribution histograms for each sample.
Immunoprecipitation and electrophoresis
Tissues were Dounce homogenized (10 strokes) in ice-cold lysis buffer [1% (v/v) Triton X-100, 150 mm EDTA, 10% (v/v) glycerol, 50 mm Tris–HCl, pH 8.0] containing 1× general protease inhibitor cocktails (Sigma-Aldrich, St. Louis, MO). The lysates were transferred to microcentrifuge tubes and further solubilized for 30 min at 4°C. The detergent extracts (supernatants) were collected by centrifugation at 15,000 × g for 15 min at 4°C, and protein concentration was determined for sample aliquots using bicinchoninic acid (BCA) protein assay reagents (Pierce Chemical, Rockford, IL). The detergent extracts were then precleared with 50 μl of mixed slurry of protein A-Sepharose and protein G-Sepharose (1:1) (Amersham Biosciences, NJ) twice, each for 30 min at 4°C. Detergent extracts were mixed with 1 μg of rabbit anti-α7 antiserum (H302, Santa Cruz Biotechnology, Santa Cruz, CA) and incubated at 4°C overnight with continuous agitation. Protein A-Sepharose and protein G-Sepharose mixtures (50 μl) were added and incubated at 4°C for 1 hr. The beads were washed four times with ice-cold lysis buffer containing protease inhibitors. Laemmli sample buffer eluates were resolved by SDS-PAGE. Proteins were transferred onto Hybond ECL nitrocellular membranes (Amersham Biosciences, NJ). The membranes were blocked with TBST buffer [20 mm Tris–HCl, pH 7.6, 150 mm NaCl, and 0.1% (v/v) Tween 20 containing 2% (w/v) nonfat dry milk for at least 2 hr and incubated with rat monoclonal anti-β2 antibody (mAb270; Santa Cruz) or rabbit anti-α7 antiserum (H302), respectively, at 4°C overnight. After three washes in TBST, the membranes were incubated with goat anti-rat or goat anti-rabbit secondary antibodies (1:10,000) (Pierce Chemical, Rockford, IL) for 1 hr and washed. The bound antibodies were detected with SuperSignal chemiluminescent substrate (Pierce Chemical, Rockford, IL).
Aβ 1–42 peptides were analyzed with electrophoresis to test the exact form of oligomer during aggregation state. Pre-cast 10-20% SDS-polyacrylamide Tris-Tricine gels (Bio-Rad, Hercules, CA) or 16% Tris-Tricine gels in the presence or absence of SDS or Urea 8M were used. 100 μg of Aβ1-42 per sample was resuspended with 4X Tricine loading buffer. Aβ1-42 samples dissolved with water or DMSO were aggregated for 2 hr before loaded.
All data were presented as mean ± standard error (SE). Statistical comparisons using Student’s t-test (independent or paired) were performed with Origin 5.0 (Microcal Software, Inc., Northampton, MA). p values less than 0.05 were considered statistically significant.