C57Bl6 mice were obtained from Charles River Laboratories (Wilmington, MA). Post-natal day (P)35-P49 and P120 mice were used to compare dsAAV serotypes. P12, P21, and P84–P98 mice were used to determine the timeline of dsAAV-mediated expression. The animal and experimental protocols were approved by the University of Rochester University Committee on Animal Resources (UCAR) in accordance with the PHS Policy on Humane Care and Use of Laboratory Animals and conformed to the National Institute of Health guidelines.
Packaging, Purification and Titering of AAV vectors
The construction of dsAAV-GFP packaging plasmid is based on AAV serotype 2 genome and has been described previously (Wang et al., 2003
). To generate pssAAV-GFP, the 5’ ITR of dsAAV-GFP was replaced with the 5’ITR of pAAV-LacZ (Stratagene, La Jolla, CA). Both plasmids contain the cytomegalovirus immediate-early enhancer/promoter to drive expression of GFP. Viral stocks were prepared using the triple-transfection method (Xiao et al., 1998
; Howard et al., 2008
). Briefly, twenty 15 cm dishes containing HEK293 cells at 85–95% confluency were transfected by CaCl2 method with pHelper (Stratagene, La Jolla, CA), pdsAAV-GFP (Wang et al., 2003
) or pssAAV-GFP (Xiao et al., 1998
) and a plasmid containing rep/cap genes for serotype1 pXR1 (aka pXX12; (Rabinowitz, 2002
#170)), pXX2 (Xiao et al., 1998
), pXR5 (Rabinowitz et al., 2002
), pAAV2/6 (Rutledge et al., 1998
), pAAV7 (Gao et al., 2002
), pAAV8 (Gao et al., 2002
), and pAAV9 (Gao et al., 2004
). Plasmids used for packaging AAV were generously provided by Dr. Xiao Xiao (UNC, Chapel Hill, NC). Approximately 48 hours post-transfection, cells were harvested, lysed by freeze/thaw, and purified by centrifugation on CsCl gradient. Final samples were dialyzed in PBS to 1013
vg/ml, aliquoted and stored at −80°C until use. All vectors were titered by quantitative PCR using GFP as the target sequence. Viral titers are recorded as viral genome/ml.
Mice were anesthetized with Avertin (200mg/kg, IP, Sigma), administered Bupronex (0.1 mg/kg, SC, Bedford Labs, Bedford, OH), secured in a stereotaxic frame with ear cups, and their skulls exposed. For mice P21 and older, the skull was thinned over visual cortex in both hemispheres and a small portion of skull removed. Injections were made bilaterally. For mice P12 and younger, a micropipette was inserted directly through the skull. A glass micropipette with a diameter of 20 µm was used to inject dsAAV-GFP or ssAAV-GFP 500 µm below the surface of the cortex in P21 and older mice, or 400 um below the surface of the cortex in P12 and younger mice. One microliter of virus (titer 1013 vg/ml) was delivered over a 10 minute period via a mechanical plunger controlled by a micropump injector (World Precision Instruments, Sarasota, FL). Following virus injection the pipette was withdrawn and the scalp sutured. Animals were allowed to recover under a heat lamp before being returned to the animal facility. Seven days after injection, mice were perfused transcardially with 0.1M phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in 0.1M PBS and brains were harvested. In order to determine the timeline of dsAAV expression, mice were also perfused 2 days or 4 weeks following dsAAV injection. In the text, n denotes the number of individual injections for each serotype.
After overnight fixation in 4% PFA at 4°C, brains were cryoprotected for 24-hours each in 10, 20, 30% sucrose at 4°C. Brains were sectioned coronally on a freezing, sliding microtome (Microme; Global Medical Instrumentation, Inc., Ramsey, MN) at 50 µm thickness into 0.1M PBS. For immunofluorescent labeling, sections were washed several times in 0.1M PBS. To block endogenous peroxidase activity, sections were placed in a solution containing 3% H2O2 in 0.1M PBS for 20 minutes at room temperature. Sections were washed briefly in 0.1M PBS and blocked for 1 hour at room temperature in a solution containing 0.3% Triton-X, 5% normal donkey serum (NDS) in 0.1M PBS. Sections were washed in 0.1M PBS and transferred into an antibody dilution buffer containing 0.3% Triton-X, 3% NDS in 0.1M PBS along with one of the following primary antibodies: mouse anti-neuronal nuclei (NeuN, 1:250, IgG, Millipore), rabbit anti-Iba1 (1:500, IgG, Wako Pure Chemical Industries, Ltd., Richmond, VA). rabbit anti-glial fibrillary acidic protein (GFAP,1:1,500,IgG, DakoCytomation, Glostrup, Denmark) or mouse anti-GAD67 (1:1000, Millipore) at 4°C in a humidified chamber for 48-hours. Tissue was washed with 0.1M PBS and incubated for 4-hours at room temperature with Alexa Fluor 594 donkey anti-rabbit IgG or Alexa Fluor 594 donkey anti-mouse IgG (1:250, Molecular Probes, Carlsbad, CA) in a solution containing 0.5% bovine serum albumin (BSA), 3% NDS in 0.1M PBS. Sections were washed in 0.1M PBS. Tissue sections were mounted in 0.1M PBS and cover-slipped with Prolong Gold anti-fade reagent (Molecular Probes, Carlsbad, CA), slide edges sealed and protected from light until analysis.
Image acquisition and analysis
Injection sites were viewed on an AX70 Microscope (Olympus, Center Valley, PA) using epifluorescence. Digital images were obtained using a MicroFire camera (Optronics, Muskogee, OK) and Image Pro software (Media Cybernetics, Bethesda, MD). Images were analyzed off-line using free-ware NIH Image-J. Labeled neurons were manually counted in all sections taken from each injection site and divided into one of three categories (see ). Type 1 cells were defined as thoroughly-labeled neurons where proximal and distal dendrites and dendritic spines were clearly visible. Type 2 cells were defined by having clearly labeled cell bodies and proximal dendrites, while in Type 3 cells only the cell bodies were apparent. Injections were typically characterized by very strong, dense labeling at the center where individual cells were difficult to identify surrounded by a less dense “halo” of individual cells. Neuronal counts were made in the less dense “halo” region surrounding the bright injection core. While this suggests that our numbers underestimate the number of neurons labeled by the dsAAV, the counts are relevant for imaging purposes as the dense core labeling precludes the visualization of individual cells. Additionally, using immunohistochemistry we determined that the majority of cells in the dense core were GFAP+ suggesting that glial labeling is most pronounced in this region. Indeed while individual labeled glia were observed, they were generally very near the edges of the dense core unlike labeled neurons which could be found quite far from the site of injection. To compare the amount of glial labeling in injections of different serotypes, we identified the section in which the area of the core was largest, outlined the bright core labeling and measured its area in that section ().
Serotype comparison of dsAAV-GFP transduction of cells in mouse visual cortex
To verify cell identification, a subset of injections was labeled immunocytochemically for either astrocytes (GFAP), microglia (Iba1), or neurons (NeuN) and imaged on a confocal microscope (Zeiss, Thornwood, NY). To determine whether inhibitory cells were labeled in addition to morphologically identifiable pyramidal cells, a subset of injections was labeled immunocytochemically with the inhibitory cell marker GAD67. Sections were imaged on an AX70 microscope (Olympus) using epifluorescence and images were analyzed using ImageJ freeware to determine the extent of colocalization of marker label and GFP. Statistical comparisons of different serotypes and timeline of dsAAV1 expression were made using a one-way ANOVA analysis with Tukey's post-hoc test. Statistical comparisons of dsAAV and ssAAV expression were made using a paired t-test.
A custom-made two-photon laser scanning microscope (Majewska et al., 2000
) was used for in vivo
imaging. The microscope consists of a modified Fluoview confocal scanhead (Olympus) and a Ti:S laser providing 100 fs pulses at 80 MHz at a wavelength of 920 nm (Mai-Tai, Spectra-physics). Fluorescence was detected using photomultiplier tubes (HC125-02, Hamamatsu, Bridgewater, NJ) in whole field detection mode. For in vivo
imaging, mice were anesthetized with Avertin (200mg/kg, IP). The skull over the injection site was thinned or removed. The craniotomy was initially identified under whole field fluorescence illumination and areas with superficial dendrites and glia were identified using a 20×, 0.9 5 NA lens (IR2, Olympus). Image acquisition was accomplished using Fluoview software. In cases of repeat imaging the animal’s scalp was sutured between imaging sessions. Axon terminals were identified based on their morphology (Majewska et al., 2006
) and their locations were marked on different days. Stable, lost and new terminals are expressed as a percentage of total terminals observed on the first day of imaging. For acute slice imaging, two photomultiplier tubes were used for the GFP and Alexa-594 channels. Slices were placed in a recording chamber under the two-photon microscope and were imaged after a whole cell recording was obtained. Fluorescence from the slices was directed through a dichroic (Chroma, Rockingham, VT) to the photomultipler detectors. Analysis of images was carried out offline in ImageJ.
Mice (P16–18) were decapitated 1 week after dsAAV1-GFP was injected bilaterally in visual cortex. The brains were rapidly removed and immersed in ice-cold artificial cerebral spinal fluid (ACSF) saturated with 95%O2 and 5%CO2. The ACSF contained (mM): 126 NaCl, 3 KCl, 2.5 CaCl2.2H2O, 1.3 MgCl2.6H2O, 1.1 NaH 2PO4, 10 Glucose, 26 NaHCO3. Coronal slices (400 µm) were cut on a Vibratome (1000 plus, TPI, St. Louis, MO) and incubated for at least 1 hour in a recovery chamber prior to recording. Slices were transferred to a recording chamber on the two-photon microscope, and perfused continuously with ACSF saturated with 95% O2 and 5%CO2 at a flow of 2–3 ml/min. Injection sites in visual cortex were identified under epifluorescence. Recording patch pipettes (5–8 MΩ) were filled with (mM) 135 k-gluconate, 10 KCl, 10 HEPES, 8 NaCl, 4 MgATP, 0.3Na-ATP, 0.1 Alexa594 (Invitrogen, Carlsbad, CA), (pH7.25, 290mOsm). Whole-cell current clamp recordings were made using a patch clamp amplifier (MultiClamp 700A, Axon Instruments, Toronto, Canada). Data acquisition and analysis were performed using a digitizer (DigiData 1322A, Axon Instruments) and pClamp 9.2 (Axon Instruments). Signals were filtered at 2 kHz, and sampled at 10 kHz. The membrane potential was determined from the value recorded immediately after achieving the whole-cell configuration. All chemicals for electrophysiology were purchased from Sigma Aldrich unless otherwise stated. Statistical comparisons were made using a one-tail Student’s t-test.