Viral construction and preparation
Viruses were constructed and rescued using standard methods (Whelan et al., 1995
). The A/RG chimeric glycoprotein was cloned from pCMMP EnvARGCD IRES GFP (gift from Ian Wickersham, MIT, Boston, MA) into the G locus of the VSV(ΔG) backbone using the MluI and NotI restriction sites (Chandran et al., 2005
In order to pseudotype VSV(A/RG) with RABV G, four 10-cm dishes of 90% confluent 293T TVA800 cells (gift from John Young, Salk Institute, La Jolla, CA ; Narayan et al., 2003
) were each transfected with 5 μg of pCAG RABV G (gift from Ed Callaway, Salk Institute, La Jolla, CA). The subsequent day, these plates were infected at an MOI of 0.01 with VSV(A/RG). Supernatants were collected every day for 4 days after infection, ultracentrifuged at 21,000 RPM for 90 minutes, resuspended in 30 μL DMEM, and frozen at -80°C. Viral stocks were titered on 293T cells. Viral focus-forming units (ffu) were calculated 2 dpi by identifying GFP-expressing cells, and the viral titer (ffu/mL) was determined by performing a dilution series.
VSV(A/RG) infection specificity for TVA-expressing cells
To test VSV(A/RG)infection specificity, relative viral infectivity of TVA-expressing and non-TVA-expressing tissue culture cells was quantified. A virus preparation of VSV(A/RG) was serially diluted onto five different cell lines: two that expressed the TVA receptor, including 293T TVA800 and mouse embryonic fibroblasts with both the cTVA and Cre alleles (MEF+/+) (Beier et al., 2011
), and three that did not express TVA, including NIH 3T3, 293T, and MEF+/- (with the cTVA but not the Cre allele) (Beier et al., 2011
). Fluorescent foci were quantified, and the ratio of infection relative to 293T TVA800 cells was quantified.
cTVA and Cre mice were genotyped using ubiquitin-C promoter and Cre coding sequence-specific primers, respectively, as described previously (Beier et al., 2011a
). The R26TdT (B6.Cg-Gt(ROSA)26Sor<tm9(CAG-tdTomato)Hze>/J; Madisen et al., 2010
) and ChAT-Cre (Jackson Laboratory strain B6;129S6-Chattm1(cre)Lowl
/J) lines were obtained from the Jackson Laboratory. The Parvalbumin-Cre (B6;129P2-Pvalb<tm1(cre)Arbr>/J) line was also obtained from the Jackson Laboratory (Hippenmeyer et al., 2005
). The VGlut3-Cre mouse line was a generous gift from Botond Roska (FMI, Basel CH).
DRD4-EGFP, CB2-GFP and Cdh3-GFP transgenic mouse lines were obtained from the MMRRC GENSAT collection (Gong et al., 2003
), crossed with C57/Bl6 mice or maintained on a mixed pigmented background. DRD4-GFP mice express GFP in one subtype of DSGCs (Huberman et al., 2009
), Cdh3-GFP mice express GFP in 2-3 subtypes of RGCs that project to non-image-forming visual nuclei (Osterhout et al., 2011
) and CB2-GFP mice express GFP in transient Off-alpha RGCs (Huberman et al., 2008b
For caudate putamen (CP) injections, 100 nL of 1×109 ffu/ml VSV(A/RG) pseudotyped with RABV G was injected into cTVA/R26TdT/PV-Cre triple transgenic mice between 5-8 weeks old, or cTVA/R26TdT/VGlut3-Cre transgenic mice. Mice were perfused 3 dpi. For animals injected with Adenovirus-Cre (Baylor College of Medicine Vector Core), VSV(A/RG) pseudotyped with RABV G was injected into the left hemisphere of the motor cortex of cTVA/R26TdT transgenic mice, while 100 nL of 1×1010 cfu/mL Adenovirus-Cre was injected into the equivalent coordinates of the opposite hemisphere at the same time. Mice were perfused 3 dpi.
For LGN injections, animals were injected with 250 nL of VSV(A/RG) pseudotyped with RABV G, and perfused between 2-7 dpi.
- Transgenic mice of either sex were used throughout.
- Injection coordinates used were:
- Lateral geniculate nucleus (LGN): A/P -2.46 from bregma, L/M 2, D/V -2.75
- CP: A/P +1 from bregma, L/M 1.8, D/V -2.5
- Motor cortex: A/P +1.34 from bregma, L/M 1.7, D/V -1.0.
For analyses of RGCs, 42 retinae were used from mice injected into the LGN. This resulted in sparse labeling of RGCs (i.e. ), permitting unambiguous analysis of single RGCs. Only labeled RGCs without overlapping dendritic arbors with other labeled RGCs were used for analysis. includes tables of these data.
Injection of VSV(A/RG) pseudotyped with RABV G into the LGN leads to infection of RGCs and transmission specifically to TVA-expressing SACs
Quantification of SAC labeling from LGN injection of VSV(A/RG) pseudotyped with RABV G
Mice were perfused with saline followed by 4% PFA in PBS, their eyes removed whole, and postfixed 4 hours in 4% PFA in PBS at 4°C. The cornea and lens were removed and the eyecup cryoprotected in 30% sucrose, before embedding in 2:1 sucrose: OCT for cryosectioning. Sections were cut at 14μm and thaw mounted onto slides. Primary antibodies used: rabbit anti-GFP and guinea pig anti-VAChT.
Brain tissue was perfused and then postfixed for 8 hours in 4% PFA in PBS, transferred to 30% sucrose, allowed to sink, and sectioned at 40 μm on a freezing sliding microtome. Anti-GFP staining was carried out as above with rabbit anti-GFP.
Polar plots to analyze the relative positions of DSGCs and SACs were created using Matlab software.
Targeted filling of genetically labeled RGCs and analysis of co-stratification with cholinergic laminae
Retinas from age P25-60 mice were dissected and kept in an oxygenated (95% O2/ 5% CO2) solution of Ames’ medium (Sigma) supplemented with 23 mM NaHCO3. RGCs were visualized with DIC optics and epifluorescence to target GFP+ somata and filled with borosilicate glass electrodes containing 10 mM solution of Alexa Fluor 555 hydrizide (Invitrogen) in 200 mM KCl. Hyperpolarizing current pulses ranging between 0.1-0.9 nA were applied for 5-20 minutes to obtain the complete cell fill (evidenced by filled axon and fine processes and comparison with GFP).
After filling, retinas were fixed for 1 hour with 4% PFA, washed with 1X PBS for 1 hour and incubated for 1 hour in a blocking solution (10% goat serum, 0.25 % Triton-X) at room temperature. The retinas were incubated overnight at 4°C with the following primary antibodies in blocking solution: rabbit anti-GFP (1:1000, Invitrogen) and guinea pig anti-VAChT (1:1000, Millipore). Sections were rinsed with PBS (3x, 30 minutes), and incubated for 2 hours at room temperatures with the following secondary antibodies: Alexa Fluor 488 goat anti-rabbit and Alexa Fluor 647 goat anti-guinea pig (1:1000, Invitrogen). Sections were rinsed with PBS and mounted with Prolong Gold with DAPI (Invitrogen).
Neurons were imaged using a 40x water immersion objective lens on a Zeiss LSM 710 laser scanning confocal microscope. Image stacks were collected at a scanning resolution of 1024×1024 pixels, and a step size through the Z- axis of 0.5 μm.
Recordings were made in whole-mount retinas from three- to six-week old C57/B6 mice of either sex. The mice studied were the offspring of two transgenic lines: the ChAT-Cre line and a second line, in which expression of a channelrhodopsin-2-YFP fusion protein is Cre-dependent (ChR2; Jackson Laboratory strain B6;129S-Gt(ROSA)26Sortm32(CAG-COP4*H134R/EYFP)Hze/J). The offspring (ChAT-CRE::ChR2) expressed ChR2 selectively in SACs throughout the retina.
Retinas were prepared in accordance with protocols approved by Yale University and using standard methods published previously (Borghuis et al., 2011
). Eyes were dissected in oxygenated Ames medium (95% O2
; Sigma-Aldrich). The retina was removed from the pigmented epithelium and placed in a perfusion chamber on an upright Olympus BX-51 microscope that was modified for two-photon fluorescence microscopy. Retinas were perfused with oxygenated Ames medium at physiological temperature (34 - 36°C) throughout the recordings. Two-photon fluorescence images were obtained with ScanImage software v3.6 (www.scanimage.org
) using an Olympus 60x, 0.9 NA, LUMPlanFl/IR objective and an ultra-fast pulsed laser (Chameleon Vision II; Coherent) tuned to 910 nm. ChR2 was activated with a high-power blue LED (λpeak
450 nm; 1W power corresponding to 3 · 1017
photons / cm2
/ s) focused on the retina through the condenser lens of the microscope.
Intracellular solution for whole-cell patch clamp recordings contained (in mM): 120 Cs-methanesulfonate, 5 TEA-Cl, 10 HEPES, 10 BAPTA, 3 NaCl, 2 QX-314-Cl, 4 ATP-Mg, 0.4 GTP-Na2
, and 10 Phosphocreatine (pH 7.3, 280 mOsm); 10 μM Alexa Fluor 568 (Invitrogen; Carlsbad, CA) was added to visualize the recorded cell. Currents were recorded with an Axon Multiclamp 700B amplifier controlled with PClamp 10 software (Molecular Devices; Sunnyvale, CA). Data were analyzed with custom algorithms in MATLAB (The MathWorks; Natick, MA). To isolate GABAergic currents evoked by ChR2 activation, the following drugs were added to Ames medium: 100 μm DNQX (AMPA/kainate receptor antagonist), 100 μM D-AP5 (NMDA receptor antagonist), 20 μm L-AP4 (group III mGluR agonist), 1 μM strychnine (glycine receptor antagonist) and 50 μM tubocurarine (nicotinic acetylcholine receptor antagonist). GABAergic currents were then blocked by adding 50 μm gabazine (SR-95531; GABA-A receptor antagonist). To isolate cholinergic currents, the following drugs were added to Ames medium: 100 μm DNQX, 100 μM D-AP5, 20 μm L-AP4, 1 μM strychnine and 50 μM gabazine. Cholinergic currents were then blocked by adding either 50 μM tubocurarine or 100 μM hexamethonium. All of the above synaptic blockers were purchased from Tocris Bioscience (www.tocris.com
). Responses evoked by ChR2 activation were quantified by averaging a leak-subtracted current over a 500-ms window. The leak current was measured over a time period just prior to the ChR2-activating stimulus. For ON-alpha cells, the response to the first light pulse on the first trial was excluded to avoid the melanopsin-mediated response (see Results). In some cells, light-evoked responses were measured with intact synaptic function using a modified Dell projector described previously (Estevez et al., in press
Series resistance during the recording was <30 MΩ and compensated by 50%. Excitatory currents were recorded with a holding potential (Vhold
) at the calculated chloride reversal potential (ECl
= -67 mV); inhibitory currents were recorded at Vhold
= 0 mV. Errors in the holding potential introduced by the uncompensated series resistance were corrected as described previously (Manookin et al., 2010
All mouse work involving viruses was conducted in biosafety containment level 2 conditions, and all procedures were approved by the Harvard University Committee on Microbiological Safety and Harvard University, Yale University, and University of California San Diego Institutional Animal Care and Use Committees.
Goat anti-Choline acetyltransferase, Millipore (AB144P), 1:30
Rabbit anti-tyrosine hydroxylase, Millipore (AB152), 1:500
Chicken anti-GFP, Abcam (ab13970), 1:2000
Rabbit anti-DsRed (tdTomato), Clontech (632496), 1:1000
Rabbit anti-GFP, Invitrogen (632375), 1:1000
Guinea pig anti-VAChT, Chemicon (AB1588), 1:1000.
Donkey anti-chicken Dylight 488, Jackson ImmunoResearch, 1:250
Donkey anti-rabbit Dylight 549, Jackson ImmunoResearch, 1:250
Donkey anti-rabbit Dylight 649, Jackson ImmunoResearch, 1:250
Donkey anti-goat Dylight 549, Jackson ImmunoResearch, 1:250
Donkey anti-goat Dylight 649, Jackson ImmunoResearch, 1:250
Goat anti-rabbit Alexa 488 highly cross-absorbed, Molecular Probes, 1:1000
Goat anti-guinea pig Alexa 594 highly cross absorbed, Molecular Probes, 1:1000