Fluorescent Calcium Indicator Protein Expression in the Mouse Brain Using Recombinant Adeno-Associated Viruses.

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Protocol

Fluorescent Calcium Indicator Protein Expression in the Mouse Brain Using Recombinant Adeno-Associated Viruses Matthias Heindorf and Mazahir T. Hasan

One method for gene delivery and long-term fluorescent calcium indicator protein (FCIP) expression in mammalian neurons in vivo involves the introduction of FCIPs via recombinant adeno-associated virus (rAAV) vectors using constitutive and cell type-specific promoters. This protocol describes the use of rAAVs to express FCIPs in the brain for imaging. Human embryonic kidney 293 cells are first transfected using calcium phosphate. rAAV is then prepared using either an iodixanol gradient or a heparin column. After the virus is purified, its quality is assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, estimation of genomic and functional virus titers by quantitative polymerase chain reaction, and expression in dissociated neurons. Mice are injected with rAAV using a stereotactic instrument and can be imaged 3 wk later.

MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.

Reagents

Adeno-associated viruses (AAV) plasmids (see Discussion) AAV packaging plasmids Set 1: pDP1 and pDP2 (Jürgen A. Kleinschmidt, German Cancer Research Center, Heidelberg, Germany) Set 2: pFdelta6, pNLrep, and pH21 (Matthias J. During, Auckland, New Zealand) Choose one of the sets of packaging plasmids for use in the experiment.

AAV plasmids pAAV-hSYN-eGFP (pAAV-6P-SEWB) (equipped with a human synapsin promoter to drive gene expression [Sebastian Kügler and Mazahir T. Hasan]) pAM-CAG-pL-WPRE-βGH-pA (equipped with a CMV enhancer and a chicken β-Actin promoter to drive gene expression [Matthias J. During]) pAAV-Ptetbi (for simultaneous expression of two different genes [Mazahir T. Hasan and Rolf Sprengel]) Adapted from Imaging in Neuroscience: A Laboratory Manual (ed. Helmchen and Konnerth). CSHL Press, Cold Spring Harbor, NY, USA, 2011. © 2015 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot087635

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M. Heindorf and M.T. Hasan

pAAV-hSYN-tTA and pAAV-hSYN-rtTA (for expressing tTA and rtTA, respectively, under control of a human synapsin promoter [Mazahir T. Hasan and Rolf Sprengel] Benzonase, 300 U/μL stock solution (25,000 U; Sigma-Aldrich E1014) BES-buffered saline (BBS) for transfection, 2× CaCl2, 2 M stock solution (filter-sterilized) Coomassie Brilliant Blue gel staining solution Destaining solution For destaining solution, prepare 50% methanol, 7.5% acetic acid in H2O; mix well.

Dissociated cultured neurons, for estimation of functional titer (Step 31) Fetal calf serum (FCS), heat-inactivated (Gibco/Life Technologies) Human embryonic kidney 293 cells (HEK 293 cells; American Tissue Culture, ATCC no. CRL-1573) For transfection and virus production, HEK 293 cells of 80%. If this is the case, proceed to the next step. 13. Scrape transfected cells from the plate without removing the medium. Transfer the cell suspension to 50 mL conical tubes. For 10 × 15 cm plates, use 4 × 50 mL conical tubes. Retaining the medium is important because transfected cells tend to detach and start floating in the medium.

14. Pellet cells at 1000g for 5 min at 4˚C.

15. Discard supernatant and resuspend cell pellets in 10 mL of 1× PBS (filter-sterilized) and transfer into a single 50 mL conical tube. Centrifuge again at 2000g for 10 min to pellet. 16. Proceed to virus purification using either an iodixanol gradient (Step 17) or a heparin column (Step 32) as described below. Purification of rAAV Purify the rAAV using either an iodixanol gradient (Steps 17–31) or a heparin column (Steps 32–41). After purification, continue with Step 42. Commercial services for rAAV purification are also available (see http://www.vectorbiolabs .com/vbs/index.html and http://www.PlasmidFactory.com).

Purification of rAAV by an Iodixanol Gradient

17. Resuspend the cell pellet (0.5 mL) from Step 15 above in 9 mL of lysis buffer 1, add 500 µL of NaDOC (10%) to yield a final concentration of 0.5%, and mix well by pipetting up and down several times. 18. Add Benzonase to a final concentration of 50 U/mL, and mix well by pipetting up and down several times to ensure that Benzonase has thoroughly mixed with the lysate. Incubate the lysate for 1 h at 37˚C. Optional: The crude lysate can be frozen at −70˚C at this step.

19. To thaw, incubate the lysate for 20 min at 37˚C. Remove cell debris by centrifugation at 4000g for 15 min at 4˚C. Optional: The crude lysate can be frozen at −70˚C at this step.

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Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot087635


FCIP Expression in the Mouse Brain Using rAAVs

20. To thaw, incubate the lysate for 20 min at 37˚C. Add 584 mg of NaCl to the lysate and incubate for 10 min at room temperature. Perform three freeze–thaw cycles in liquid nitrogen and 37˚C, respectively. (It takes 15 min for each freeze–thaw cycle.) Centrifuge at 3000g for 15 min, and transfer the clear crude lysate into a fresh 50 mL conical tube. If the lysate is still not clear, follow the recentrifugation step once or twice more. 21. Prepare 15%, 25%, 40%, and 54% iodixanol solutions with 1× PBS-MK. 22. Load 6 mL of 15% iodixanol in Beckman centrifuge tubes. Underlay the 15% iodixanol with 4 mL of 25% iodixanol, followed by 3 mL of 40% iodixanol, and finally 3 mL of 54% iodixanol. (Four discernible phases should be visible.) With a permanent pen marker, label the boundary between the phases of 40% and 54% iodixanol. 23. Gently load the cell lysate (9 mL) on top of the gradient. Set aside 100 µL of the unpurified lysate for analysis on SDS-PAGE. 24. Centrifuge in an ultracentrifuge at 60,000 rpm for 1.5 h at 18˚C.

25. Extract the virus from the 40% to 54% iodixanol interphase as well as the majority of the 40% iodixanol phase using a 5 mL syringe attached to a 20-G × 2¾-inch needle bent at a 90˚ angle. Make sure that the beveled side of the needle is facing downward. Avoid removing the “white film” above and below the 40% iodixanol layer.

26. Put the collected fraction in an Amicon concentrator (100 kDa), and fill the remaining volume with 1× PBS-MK, mix well, and centrifuge at 3000g for 5 min. Discard the flowthrough, refill the reservoir with 1× PBS-MK again, and repeat the whole procedure twice more. 27. After the last spin, transfer the dialyzed and concentrated virus suspension (250–300 µL) into a microcentrifuge tube, and filter-sterilize it using an Acrodisc filter (0.22 µm) that is 13 mm in diameter. Do not use larger filter devices! 28. Store viruses at −70˚C as 10 μL aliquots in small microcentrifuge tubes. 29. Analyze unpurified and purified viruses on SDS-PAGE. i. Prepare an SDS-PAGE gel. ii. Take 10 µL of virus mix and add 2.5 µL of 5× Laemmli buffer. iii. Heat samples for 5 min at 99˚C, then centrifuge briefly at 1000g. Load virus samples and molecular mass markers in gel slots. Run samples in the stacking gel at 80 V and in the separation gel at 150 V. iv. Disassemble the gel, stain in Coomassie Brilliant Blue solution for 1 h, and destain the gel with the destaining solution once for 10 min and one more time for 45 min. If gel has destained well, wash twice in water for 10 min. To further destain the background, leave the gel in water overnight at room temperature on a tray set on a shaker. Gel bands can be quantified with a luminescent image analyzer. The purified virus should show three predominant bands that correspond to the capsid proteins (VP1, VP2, and VP3) (Fig. 1C).

30. Perform a quantitative polymerase chain reaction for estimating viral genome particles per milliliter. We use the Applied Biosystems 7500 Fast Real-Time PCR System, following the manufacturer’s instructions.

31. To estimate functional titer, infect dissociated cultured neurons with serially diluted purified rAAVs. Two weeks after infection, calculate the fraction of GFP-positive neurons. Purification of rAAV by a Heparin Column The heparin column purification method is suitable for serotype-2-containing viruses. Serotype-2 viruses use the heparan sulfate proteoglycan as its primary cell surface attachment receptor for entering cells.


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100 m

FIGURE 1. Recombinant adeno-associated virus (rAAV)-mediated FCIP expression in vitro and in vivo. (A) Inducible and constitutive expression systems. (Top panel) In the inducible expression system, two rAAVs are required: one to express tTA under control of the human synapsin promoter (virus 1) and the other to inducibly express FCIP and Cre genes under control of a Ptetbi (virus 2) in a tTA-dependent manner. (Bottom panel) In the constitutive expression system, the human synapsin promoter drives constitutive expression of FCIP. (TB) Synthetic transcription blocker; (WPRE) a woodchuck hepatitis virus posttranscriptional control element; (pA) polyadenylation signal; (ITR) inverted terminal repeats of AAV2. (B) Coomassie-stained gel (SDS-PAGE) with capsid proteins (VP1, VP2, VP3) from a purified virus (lane 3), unpurified virus in cell lysate (lane 2), and protein molecular mass markers (lane 1). (C ) Electron microscopic images of virus particles visualized by negative staining with uranyl acetate. Empty virus particle (black arrow) and DNA-filled virus particle (white arrow) are indicated in example 1. Bars, 50 nm. (D) FCIP expression in dissociated cultured neurons. (E,F) A tetracycline-inducible promoter expresses an FCIP (D3cpv) in hippocampal organotypic slices acquired at low and high zoom (both images are collapsed two-photon image stacks). (G) Human synapsin promoter (PhSYN)driven FCIP (D3cpv) expression in vitro. Fluorescence in a fixed brain slice was detected by wide-field imaging with AAV injections at depths of 250 µm and 1.5 mm. (CA3) Hippocampal layer CA3; (L2/3) layer 2/3; (L5) layer 5. (H ) A versatile combinatorial genetic approach for rAAVmediated Ptetbi responder gene expression in tTA/rtTA-expressing cells delivered via three different approaches: (1) rAAV, (2) lentivirus, and (3) transgenic mice. (I ) Example of an rAAV equipped with a Ptetbi responder. When injected into a tTA or rtTA transgenic mouse (left panel), this allows for forebrain-specific, neuron-specific gene expression in the presence of doxycycline (right panel).



32. Resuspend the cell pellet from Step 15 above in 50 mL of lysis buffer 2, and split into two 50 mL conical tubes to help handle the volume adequately. To each tube, add 1.25 mL of NaDOC (10%) to give a final concentration of 0.5% NaDOC and mix well by pipetting up and down several times. 33. Add Benzonase to a final concentration of 50 U/mL, and mix well by pipetting up and down several times to ensure that Benzonase has thoroughly mixed with the lysate. Incubate the lysate for 1 h at 37˚C. Optional: The crude lysate can be frozen at −70˚C at this step.

34. To thaw, incubate lysate for 20 min at 37˚C. Remove cell debris by centrifugation at 4000g for 15 min at 4˚C. Optional: The crude lysate can be frozen at −70˚C at this step.

35. To thaw, incubate lysate for 20 min at 37˚C. Do three freeze–thaw cycles in liquid nitrogen and 37˚C, respectively. (It takes 15 min for each freeze–thaw cycle.) Centrifuge at 3000g for 15 min, and transfer the clear crude lysate into a fresh 50 mL conical tube. If the lysate is still not clear, follow the recentrifugation step once or twice more. Freeze the crude lysate at −70˚C and prepare for heparin column purification steps. 36. Equilibrate a heparin column with 10 mL of cell lysis buffer 2 (150 mM NaCl, 20 mM Tris–HCl at pH 8). Use a 60 mL syringe pump with the attachment provided and pump solution at a flow rate of 1 mL/min. 37. Disconnect the heparin column, and fill the 60 mL syringe with 50 mL of crude clear lysate. Reconnect the heparin column to the syringe, and pump lysate through it at a flow rate of 1 mL/min. Set aside 100 µL of unpurified lysate for analysis on SDS-PAGE. 38. Again disconnect the heparin column, and fill the 60 mL syringe with 20 mL of Solution A for a first wash. Reconnect the heparin column to the syringe, and pump Solution A through it at a flow rate of 1 mL/min. The next steps (Steps 39–40) can be done manually (without using the pump).

39. Disassemble the heparin column, connect it to a 1 mL syringe, and gently and slowly wash the heparin column first with 1 mL of Solution B and finally with 1 mL of Solution C. 40. For the elution steps, use a 3 mL syringe. Elute successively with 1.5 mL of Solution D, 3 mL of Solution E, and 1.5 mL of Solution F. 41. Dialyze and concentrate the 6 mL of eluted volume, and analyze as described above for the iodixanol gradient purification method (Steps 26–31). Stereotactic Injection of rAAV For fluorescent calcium indicator protein (FCIP) expression in vivo, we have successfully used 50–250 nL of high-titer rAAV to label neurons in selective brain regions. Virus suspension with 10% D-mannitol solution for in vivo injection (Mastakov et al. 2001) increased the infection diameter and even expression levels. Systematic control of virus concentration, amounts, and the use of D-mannitol should allow one to label neurons both sparsely and densely and with small and large expression diameters in the brain. We have been able to label neurons in vivo with expression diameters of 0.3–2 mm.

42. Anesthetize a mouse with ketamine plus xylazine by i.p. injection (ketamine, 100 mg/kg body weight; xylazine, 10 mg/kg body weight), and fix it on a stereotactic instrument. 43. Load 150–300 nL of purified virus suspension into a glass capillary (tip size 8–12 µm), and gently lower the glass pipette into the brain to a depth of 250 µm, over which a small craniotomy has been performed, 1.5 mm posterior to the bregma and 3 mm lateral to the midline for the whiskerrelated somatosensory cortex. 44. Follow the stereotactic procedure for virus injection as described previously (Cetin et al. 2006). The mice can be used for functional analyses 3 wk after the rAAV injection. Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot087635

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In Vivo Imaging The methods below provide examples of two different imaging experiments.

In Vivo Imaging in the Olfactory Bulb

45. Anesthetize mice with pentobarbital, and perform surgical procedures as described previously (Wachowiak and Cohen 2001). 46. Deliver odors through a flow dilution olfactometer. 47. Collect images at rates of 5–15 Hz using the appropriate imaging setup. In Vivo Two-Photon Imaging

48. Anesthetize mice with urethane (1–2 g/kg), and monitor anesthesia by pinching one of the paws or the tail. Monitor core body temperature by a rectal probe and maintain at 37˚C by a heating blanket. Apply local anesthesia (lidocaine, as recommended by the vendor) underneath the head skin before beginning surgical procedures (Cetin et al. 2006). 49. Glue a removable head plate with a cranial window of 3 mm in diameter to the top of the skull using cyano-acrylate, and attach it to a fixed bar before thinning the skull. The combination of a rigid head plate and a thinned skull reduces respiration motion as well as cardiacpulsation-induced brain motion.

50. Thin the exposed skull through the cranial window (50 µm in thickness) using a dental drill. After the head-plate chamber is filled with Ringer’s buffer, blood vessels become clearly visible under the binocular.

51. Position the two-photon microscope objective so that the optical axis is perpendicular to the surface of the cortex. 52. Perform two-photon imaging using the appropriate imaging setup. Although we image mice for hours, each field of view is usually imaged in short time blocks. In whiskerstimulation experiments, we imaged mice through the thinned skull at cortical depths of 50 and 250 µm with laser powers of 50 and 150 mW, respectively, after the objective.

DISCUSSION

The AAV genome is 4.8 kb long and is composed of single-stranded DNA, either positive or negative sense. The genome comprises inverted terminal repeat (ITR) sequences of 145 bases each at both ends of the DNA strand. ITRs are required in cis to the gene of interest, which replaces structural (cap) and packaging (rep) genes. The Rep proteins are required for the AAV life cycle, and the capsid proteins (VP1, VP2, and VP3) self-organize to form a capsid of an icosahedral symmetry. After infection of host cells, AAVs form episomal concatamers in the nucleus. In nondividing cells, these concatamers remain intact throughout the life of the cell. Random integration of AAV DNA into the host genome is quite rare. This is advantageous because it reduces the chances of mutagenesis due to random DNA insertion. In dividing cells, AAV DNA is diluted every cell division because the episomal DNA is unable to replicate in host cells. The other major advantage of the rAAVs is that they can be targeted to selected cell types at any time in the animal’s life, thereby allowing detailed investigation of gene function without interference during development as is the case with traditional transgenic mice derived by pronuclei DNA microinjections. This not only obviates the use of conditional transgenics, but also easily expands the use of transgenics to species other than rodents and allows the use of multiple viruses to express multiple genes simultaneously, even in different locations. Maximum transgene expression can be observed in a matter of weeks and typically persists throughout life. We have found that the rAAV expression systems are highly suitable for imaging neuronal activity both in vitro and in vivo. Purified rAAVs can be used to express an FCIP in dissociated neurons, in cultured brain slices, and in vivo. FCIPs can be expressed under control of either constitutive or Tet-inducible promoters (Fig. 1A). 704




Both expression systems allow for fast and efficient gene delivery in different cell types and long-term gene expression. Moreover, they can also be applied to different animal species, from rats (Xu et al. 2001) to monkeys (Stettler et al. 2006). This makes rAAV vectors and expression systems valuable tools for creating animal models of diseases, for gene therapy applications, and for functional genomics. A variety of DNA expression vectors are available to generate rAAVs for cell type-specific, constitutive (Xu et al. 2001, 2005; Lu 2004; Shevtsova et al. 2005) and inducible (Zhu et al. 2007) gene expression both in vitro and in vivo. These viruses are powerful tools for investigating the role of a particular gene product in cellular signaling and animal behavior. For functional brain imaging, hightiter, high-quality viruses are required for recording functional Ca2+ signals without tissue damage. The first critical step to produce high-quality viruses is to ensure that the AAV-plasmid transfection efficiency in HEK 293 cells is >80%. Once this is accomplished, a suitable virus purification procedure should be selected. Although there are different methods for virus purification (Hermens et al. 1999; Auricchio et al. 2001; Zolotukhin et al. 2002; Hauck et al. 2003; Zeltner et al. 2010), here we recommend virus purification by either iodixanol gradient or heparin column (Wallace et al. 2008). After the virus is purified, it is analyzed for purity (Fig. 1B,C) and infectivity both in vitro and in vivo (Fig. 1D–G). It is important to determine that the purified rAAVs show distinct capsid proteins on a gel (Fig. 1B, lane 3). Electron microscopic images of different virus preparations help to determine the fraction of empty and DNA-filled virus particles (Fig. 1C). It is highly recommended that a cloned DNA fragment between the two ITRs not be >4.6 kb in length. With larger insert sizes, the packaging efficiency drops sharply; thus empty virus particles dominate the fraction (Fig. 1C). We recommend that virus titers be determined in dissociated neurons and later functionally tested for Ca2+ transients in cultured hippocampal organotypic slices. This saves both time and resources. Depending on the quality of the DNA transfection, packaging efficiency, and virus purification steps, DNA-containing AAV titers are usually 1012–1013 particles/mL with particle-to-infectivity ratios of 2–5 × 108 transducing units per microliter, injection of 50–200 nL of virus in 10% D-mannitol can provide high expression levels in neurons, which is suitable for longterm functional in vivo two-photon imaging. In our experience, 10–60 µM fluorescein equivalent units of FCIP in neurons should be sufficient to detect single action potentials (APs) in vivo (Wallace et al. 2008). With both constitutive and inducible systems, we have observed robust FCIP expression 2 wk after applying viruses to dissociated cultured neurons, rat hippocampal organotypic slices, and in the living animal (Fig. 1D–G; Wallace et al. 2008). To achieve cell type-specific FCIP expression amplification, rAAVs equipped with a Tet-responsive promoter (Ptetbi) can be combined with transactivators provided via rAAVs, lentiviruses, and transgenic mice (Fig. 1H; Zhu et al. 2007). (See Fig. 1I for an example of a combination of rAAV-Ptetbi and rtTA-expressing transgenic mice for cell type-specific gene expression on treatment with doxycycline.) In CA3 neurons of cultured rat hippocampal slices, it is possible to detect fluorescence changes reliably with D3cpv in response to single APs (Wallace et al. 2008). During whole-cell recordings, current was injected in D3cpv-expressing neurons (Fig. 2A), and simultaneous optical two-photon recording allowed detection of fluorescence changes, a decrease in CFP and an increase in YFP signal, respectively (Fig. 2B). For one, two, and three APs, the average peak amplitudes of Ca2+ transients (%ΔR/R) were 8.3 ± 0.9, 15.0 ± 2.4, and 20.0 ± 4.3, respectively (Fig. 2C). In the superficial layer of the somatosensory cortex (Fig. 2D,E), D3cpv detected spontaneous (%ΔR/R = 3.6 ± 0.16) and whiskerevoked (%ΔR/R = 3.2 ± 0.16) Ca2+ transients (Fig. 2F; Wallace et al. 2008). In layer 2/3 cortical neurons, whisker stimulation evoked Ca2+ transients both in cell somata and in the adjacent neuropil. Simultaneous recording of the local field potential (LFP) and fluorescence changes in the neuropil showed that LFP and fluorescence changes correlated well. Moreover, in vivo targeted cell-attached recording of D3cpv-expressing neurons in layer 2/3 showed good correlation between single AP and Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot087635

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A

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FIGURE 2. In vitro and in vivo functional responses with D3cpv. (A) Whole-cell recordings in organotypic slices of CA3 pyramidal cells shown as YFP, CFP, and merged images. The recording pipette is indicated in the merged image. (B) Time courses for YFP (green trace) and CFP (blue trace) channels show that there are increases in fluorescence changes (both in CFP and YFP raw traces) with increasing number of action potentials (APs); (a.u.) arbitrary units. (C ) Single-trial and averaged ratios of CFP and YFP fluorescence in response to one, two, and three APs. (D) In vivo two-photon imaging experimental setup for recording neuronal activity in response to whisker stimulation by air puffs. (E) Mouse somatosensory cortex layer 1 neuropil (CFP and YFP channels). (F ) Spontaneous and whisker-stimulation-induced fluorescence transients (examples of single-trial raw traces for CFP [cyan], YFP [yellow], and YFP/CFP [black]). An average of 15 trials across the whole frame without and in response to air-puff whisker stimulation; (black) averages over trials; (gray) single trials. (Adapted from Wallace et al. 2008.)

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FIGURE 3. Fiber-optic recording of neuronal activity in freely moving mice. (A) The tip of an optical fiber is placed on the cortical surface with fluorescence excitation and emission collected from the same fiber. (B) Bulk Ca2+ signals were recorded in somatosensory cortex through a single-core optical fiber with fluorescence changes in the YFP-channel over a period of 25 sec. The fluorescence changes are shown here together with the position of the mouse in an open field box. Animal behavior (sitting still, moving, touches, or having contact with the wall) is indicated for each time period. (Adapted from Lütcke et al. 2010.)

Ca2+ transients. Recordings from seven cells (four mice) showed an average Ca2+ transient %ΔR/R = 3.5 ± 0.2 in response to a single AP (Wallace et al. 2008). Similar results were obtained with YC3.60 (Lütcke et al. 2010), which could also detect single AP responses both in vitro and in vivo. Moreover, with a fiber-optic telescope implanted on top of a thinned skull (Fig. 3A), it was possible to detect spontaneous and behaviorally related changes in YFP fluorescence (YC3.60) even in freely moving mice (Fig. 3B; Lütcke et al. 2010). RECIPES AAV Plasmids

1. Add ampicillin (Sigma-Aldrich A0166) to 1 L of Terrific Broth (TB) (final concentration 100 µg/mL). 2. Inoculate with 1–3 mL of bacterial culture (AAV plasmid-transformed SURE 2 supercompetent cells [Stratagene 200152]). 3. Grow 1 L of bacterial culture for 24 h in a 4 L autoclaved flask with constant shaking and aeration at 37˚C. 4. Isolate plasmids using the HiSpeed Plasmid Maxi Kit (QIAGEN 12662) according to the manufacturer’s instructions. BES-Buffered Saline for Transfection, 2×

50 mM N,N-bis(2-hydroxyethyl)-2-amino-ethanesulfonic acid (BES), pH 6.95 280 mM NaCl 1.5 mM Na2HPO4 With 2 M NaOH and 2 M HCl, adjust the pH to 6.95. Filter-sterilize by passing through 0.2-μm nitrocellulose filters (Nalge), and store aliquots at −20˚C. Coomassie Brilliant Blue Gel Staining Solution

1.06% Coomassie Brilliant Blue 50% methanol 10% acetic acid in H2O Prepare 1 L. Mix well, and then filter through Whatman #1 paper. Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot087635

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Phosphate-Buffered Saline for Cells

1. To make 1 L of 10× phosphate-buffered saline (PBS), combine the following and bring to a final volume of 1000 mL with distilled H2O (pH 7.2). 10.9 g anhydrous Na2HPO4 3.2 g anhydrous NaH2PO4 9 g NaCl 2. To make 1 L of 1× PBS, thoroughly mix 100 mL of 10× PBS with 900 mL of distilled H2O.

SDS-PAGE Gel

1. Prepare the separation gel (10%). Mix in the following order: H2O Acrylamide/bis (30% 37.5:1; Bio-Rad) Tris–HCl (1.5 M, pH 8.8) SDS, 10% N,N,N′ ,N′ -tetramethylethylene-diamine (TEMED) (Bio-Rad) Ammonium persulfate (APS), 10%

4.1 mL 3.3 mL 2.5 mL 100 µL 10 µL 32 µL

After adding TEMED and APS to the SDS-PAGE separation gel solution, the gel will polymerize quickly, so add these two reagents when ready to pour. 2. Pour gel, leaving 2 cm below the bottom of the comb for the stacking gel. Make sure to remove bubbles. 3. Layer the top of the gel with isopropanol. This will help to remove bubbles at the top of the gel and will also keep the polymerized gel from drying out. In 30 min, the gel should be completely polymerized. 4. Remove the isopropanol and wash out the remaining traces of isopropanol with distilled water. 5. Prepare the stacking gel (4%). Mix in the following order: H2O Acrylamide/bis (30%, 37.5:1) Tris–HCl (0.5 M, pH 6.8) SDS, 10% TEMED Ammonium persulfate (APS), 10%

6.1 mL 1.3 mL 2.5 mL 100 µL 10 µL 100 µL

6. Pour stacking gel on top of the separation gel. 7. Add combs to make wells. In 30 min, the stacking gel should become completely polymerized. 8. Clamp gel into apparatus, and fill both buffer chambers with gel running buffer according to the instructions for the specific apparatus. 9. Load samples and molecular mass protein markers into wells for separation by electrophoresis.

SDS-PAGE Laemmli Buffer, 5×

Tris–HCl (pH 6.8) SDS Glycerol Bromophenol blue

312.5 mM 10% 25% 0.5%

Add 10% β-Mercaptoethanol before use. 708




SDS-PAGE 10× SDS Running Buffer

Tris base Glycine SDS

30.3 g 144.4 g 10 g

Dissolve in 1 L of MilliQ-filtered H2O. Terrific Broth

Terrific broth (TB)-A: Yeast extract Tryptone Glycerol TB-B: KH2PO4 K2HPO4

24 g 12 g 4g 2.3 g 16.4 g

Prepare TB-A by combining the ingredients in a final volume to 900 mL with MilliQ water, and then autoclave. Prepare TB-B by combining the ingredients in a final volume to 100 mL with MilliQ water, and then autoclave. To prepare TB, mix 900 mL of TB-A with 100 mL of TB-B.

ACKNOWLEDGMENTS

This work was supported by the Max Planck Society, the Schloessmann Foundation, Collaborative Research Center (SFB 488), Fritz Thyssen Stiftung, and Deutsche Forschungsgemeinschaft. REFERENCES Auricchio A, Hildinger M, O’Connor E, Gao GP, Wilson JM. 2001. Isolation of highly infectious and pure adeno-associated virus type 2 vectors with a single-step gravity-flow column. Hum Gene Ther 12: 71–76. Cetin A, Komai S, Eliava M, Seeburg PH, Osten P. 2006. Stereotaxic gene delivery in the rodent brain. Nat Protoc 1: 3166–3173. Hauck B, Chen L, Xiao W. 2003. Generation and characterization of chimeric recombinant AAV vectors. Mol Ther 7: 419–425. Hermens WT, ter Brake O, Dijkhuizen PA, Sonnemans MA, Grimm D, Kleinschmidt JA, Verhaagen J. 1999. Purification of recombinant adeno-associated virus by iodixanol gradient ultracentrifugation allows rapid and reproducible preparation of vector stocks for gene transfer in the nervous system. Hum Gene Ther 10: 1885–1891. Lu Y. 2004. Recombinant adeno-associated virus as delivery vector for gene therapy—A review. Stem Cells Dev 13: 133–145. Lütcke H, Murayama M, Hahn T, Margolis DJ, Astori S, Zum Alten Borgloh SM, Gobel W, Yang Y, Tang W, Kugler S, et al. 2010. Optical recording of neuronal activity with a genetically-encoded calcium indicator in anesthetized and freely moving mice. Front Neural Circuits 4: 9. doi: 10.3389/fncir.2010.00009. Mastakov MY, Baer K, Xu R, Fitzsimons H, During MJ. 2001. Combined injection of rAAV with mannitol enhances gene expression in the rat brain. Mol Ther 3: 225–232. Rohr UP, Heyd F, Neukirchen J, Wulf MA, Queitsch I, Kroener-Lux G, Steidl U, Fenk R, Haas R, Kronenwett R. 2005. Quantitative real-time PCR for titration of infectious recombinant AAV-2 particles. J Virol Methods 127: 40–45. Shevtsova Z, Malik JM, Michel U, Bahr M, Kugler S. 2005. Promoters and serotypes: Targeting of adeno-associated virus vectors for gene transfer in the rat central nervous system in vitro and in vivo. Exp Physiol 90: 53–59.


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Fluorescent Calcium Indicator Protein Expression in the Mouse Brain Using Recombinant Adeno-Associated Viruses Matthias Heindorf and Mazahir T. Hasan Cold Spring Harb Protoc; doi: 10.1101/pdb.prot087635 Email Alerting Service Subject Categories

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