Edited by: Dirk Feldmeyer, Julich Research Centre, Germany
Reviewed by: Serena M. Dudek, National Institute of Environmental Health Sciences (NIEHS), United States; Michele H. Jacob, Tufts University, United States
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
The advent of optogenetic methods has made it possible to use endogeneously produced molecules to image and manipulate cellular, subcellular, and synaptic activity. It has also led to the development of photoactivatable calcium-dependent indicators that mark active synapses, neurons, and circuits. Furthermore, calcium-dependent photoactivation can be used to trigger gene expression in active neurons. Here we describe two sets of protocols, one using CaMPARI and a second one using Cal-Light. CaMPARI, a calcium-modulated photoactivatable ratiometric integrator, enables rapid network-wide, tunable, all-optical functional circuit mapping. Cal-Light, a photoactivatable calcium sensor, while slower to respond than CaMPARI, has the capacity to trigger the expression of genes, including effectors, activators, indicators, or other constructs. Here we describe the rationale and provide procedures for using these two calcium-dependent constructs (1)
A fundamental goal of neuroscience research is to understand what the activity of neurons represents: is the activity correlated with a particular sensory input or to a particular behavior? Do the neurons involved in learning, memory or behavior express specific markers or genetic programs that are activated during the learning or consolidation phase? To study why some neurons are active while neighboring neurons are inactive, or why some neurons show genetic changes during learning or consolidation or memory formation, requires a detailed understanding of their input and their genetic and physiological properties. To begin such analysis, we first need to identify a population of active neurons
Immediate early gene (IEG) expression has provided means to recover active neurons in experimental paradigms for decades. IEGs show low expression when cells are quiescent but stimulation can elicit transient high expression within minutes (Greenberg et al.,
CaMPARI (calcium-modulated photoactivatable ratiometric integrator) is a calcium indicator that can be rapidly photoconverted in active neurons to perform circuit mapping (Fosque et al.,
Cal-Light is another photoactivatable calcium-sensitive indicator that is able to trigger the expression of a variety of genes in active neurons (Lee et al.,
Both CaMPARI and Cal-Light require illumination, coincident to the cytosolic calcium increase, to trigger conversion or activation, respectively. However, CaMPARI and Cal-Light operate on different time scales: CaMPARI converts rapidly within seconds (Fosque et al.,
CaMPARI is a bright green fluorescent protein that—via allosteric modulation of the chromophore—converts to a bright red fluorescent species upon illumination with violet light during high calcium availability (Fosque et al.,
Schematic drawings of CaMPARI and Cal-Light.
Cal-Light is light-sensitive and calcium-dependent (Lee et al.,
The protocols presented here describe how we use these tools, and some of our modifications. The labs that developed these constructs have published papers demonstrating that they work
Flowchart of the four main procedures described. After each final process, confocal imaging and data analysis may be performed. Estimated amount of time needed for the main steps is included in the boxes in blue; necessary delays between major steps are given near the arrows. rec., recovery; hab., habituation.
The key requirement of photoconversion and photactivation is that calcium entry is coupled to exposure to light. However, the minimum duration of light exposure and the optimal timing of illumination and calcium entry are still not completely known. To measure the effectiveness of different light parameters, preparations such as dissociated neuronal culture combined with wide-field stimulation and live-cell imaging prove to be useful (
Procedure for transfection, conversion and imaging.
CaMPARI and Cal-Light expression and functionality in neuronal cell culture.
A fundamental problem of all calcium sensors is to identify the cause and source of calcium entry. Cytosolic calcium levels rise when neurons fire action potentials, when synaptic activity depolarizes neurons, or when calcium is released from internal stores (Sabatini et al.,
CaMPARI for all-optical functional connectivity mapping. CaMPARI was expressed in S1 and ChR2-EYFP was expressed in M1 cortex. In acute
The main purpose of these methods is to mark active neurons
Sparse Cal-Light expression triggered through a cranial window in an awake quietly sitting head-fixed mouse.
This method allows for recovery of CaMPARI-expressing neurons (including the photoconversion snapshot) in fixed tissue, opening up the possibility to mount sections and use them for confocal imaging at a later stage (
CaMPARI expression: Effects of fixation and use of immunostaining to recover converted neurons.
We split procedures into several modules (
Neural Basal A media (ThermoFisher Scientific, 10888022)
B27 (ThermoFisher Scientific, 17504001)
Glutamax (ThermoFisher Scientific, 35050-038)
Penicillin-Streptomycin (10,000 U/ml, ThermoFisher Scientific, 15140-122)
Poly-L-Lysine (Sigma, P1399 Coverslip coating)
Papain (Sigma, P4762)
Bovine Serum Albumin (Sigma, A3294)
Hibernate A low fluorescence media (Brain Bits Ltd, HALF)
NaCl (Carl Roth, HN00.2)
KCl (Carl Roth, HN02.2)
NaH2PO4 (monohydrate, Carl Roth, K300.1)
NaHCO3 (Carl Roth, HN01.2)
CaCl2 (dihydrate, Carl Roth, HN04.2)
MgCl2 (hexahydrate, Carl Roth, HN03.1)
D-glucose (Carl Roth, HN06.3)
Choline chloride (Sigma, C7527)
Na-L-ascorbate (Sigma, A4034)
Na-pyruvate (Sigma, P2256)
KH2PO4 (Carl Roth, 3904.1)
NaOH (Carl Roth, 9356.1)
HCl (37%, Carl Roth, 9277.2)
Paraformaldehyde (Merck, 1.04005)
Gabazine (SR-95531 hydrobromide, Tocris, 1262)
Carbachol (carbamoylcholine chloride, Tocris, 2810)
Ethanol (96%, Carl Roth, T171.4)
Normal goat serum (Gibco, 16210-072)
Triton X-100 (Sigma, T8787)
Glycerol (Sigma, G5516)
DAPCO (Carl Roth, 0718.1).
Ketamine (10%, Medistart)
Xylazine (Xylavet, 20 mg/ml, CP-Pharma)
Isoflurane (Forene, Abbvie)
Buprenorphine (Temgesic, Reckitt Benckiser)
Carprofen (Rimadyl, Zoetis)
Lidocaine (Sigma, L7757).
All viruses were made in house by the Charité Viral Core Facility and were aliquoted and stored at −80°C. Aliquots of viruses in use (5 μl aliquots are typical) can be stored in a standard refrigerator at 4°C for several months. Viruses we used are:
pAAV-TM-CaM-NES-TEV-N-AsLOV2-TEVseq-tTA
pAAV-M13-TEV-C-P2A-TdTomato
pAAV-TetO-GFP
pAAV-Syn-CaMPARI2
pAAV-ChR2-H134R-EYFP
1st antibody, CaMPARI 4F6, made at Janelia Farm Research Campus, Schreiter Lab, 1:1000
2nd antibody, Alexa 633, goat anti-mouse, Invitrogen A21050, 1:500
Wide-field epifluorescence (used for cell cultures; Nikon Ti2)
Epifluorescence (used to check
Confocal laser scanning (Nikon A1Rsi+)
Two-photon (Femto 2D two-photon laser scanning system, Femtonics Ltd, Budapest, Hungary).
Dumont no. 5/45 cover slip forceps (Fine Science Tools, 11251-33)
Dumont no. 3, 4, 5, 7 forceps, assorted styles, straight (Fine Science Tools, 11231-30, 11254-20, 11241-30, 11251-10, 11271-30)
Standard-pattern forceps (Fine Science Tools, 11000-12, various lengths & diameters)
Spatula
Fine scissors (Fine Science Tools, 14060-09, 14058-09, 14090-09).
Dental drill (Osada Success 40 or Foredom Micromotor, HPA917).
Drill bits (Fine Science Tools, 19007-05, 19007-07, 19007-09)
Sterile single-use syringe, 0.4 ml (Omnican, B. Braun, 9161627)
Sugi absorbant swabs (Kettenbach Medical, 31602)
Parafilm (Sigma, P7793)
PCR Micropipettes, 1–5 μl (Drummond, 5-000-1001-X)
Eye care cream (Bepanthen, Bayer)
Mineral oil (Sigma, M3516)
Heating pad (Temperature Regulation System, FHC)
Self-adhesive resin cement (RelyX Unicem, 3M, Applicaps, 56815)
Contemporary Ortho-jet powder (black, Lang Dental, 1520BLK)
Kwik-Cast sealant (World Precision Instruments)
Pressure injector for low rate and small volume (Stoelting Quintessential Pressure injector, 53311)
Micropipette puller for virus-injection pipettes (Sutter Instrument, P-97)
Stereotaxic apparatus for small animals (KOPF, 940)
Vibratome (Leica, VT1200S)
Ti:sapphire laser (MaiTai HP DeepSee; Spectra-Physics/Newport)
455 nm LED (for
405 nm LED (for
Optic fiber (for
Mercury lamp (X-cite 200 W, Excelitas Technologies)
Optical power meter (ThorLabs, PM200 & S120VC)
For two-photon image acquisition: Matlab-based MES software package (Femtonics)
Image-processing software (ImageJ,
Eppendorf tubes, 0.5 ml (Sigma, T891)
Falcon tubes, 50 ml (Corning, 430921)
Glass coverslips (12 mm round; Roth, P231.1)
Glass coverslips (3–5 mm round; Warner Instruments, CS-3R-0, CS-4R, CS-5R-1)
Glass-bottom dishes (Eppendorf, 0030740017)
0.2 μm filters (Carl Roth, P668.1)
Haemocytometer (A. Hartenstein, ZK06)
24-well cell culture plates (Corning, 353047)
Dissociate wild type cortico-hippocampal tissue from Wistar rat pups (post-natal days 0–2).
Estimate cell densities using a haemocytometer or automated cell counter.
Grow cells on 12 mm round-glass coverslips coated with Poly-L-Lysine (1 h coating; 20 μg/ml concentration).
Plate cells in 24-well cell culture plates, at a density of 400 cells per μl in a 500 μl droplet (total cells per well: 2 × 105).
Culture cells in Neural Basal A medium, supplemented with B27 (at 1 × concentration), GlutaMAX (at 1 × concentration) and Penicillin-Streptomycin (100 U/ml). The incubator temperature should be 37°C.
Feed cells weekly by removing 100 μl of conditioned cell culture medium and adding 200 μl of freshly made cell culture medium.
Prepare virus solutions in sterile aliquots. The total volume should be 1 μl per coverslip to be transfected.
Wait 5–20 days for cultures to grow before infecting.
Virus solutions:
For CaMPARI, prepare pAAV-Syn-CaMPARI2 with a titer of ~1011-1012 GC/ml.
For Cal-Light, mix three components:
° pAAV-TM-CaM-NES-TEV-N-AsLOV2-TEVseq-tTA
° pAAV-M13-TEV-C-P2A-TdTomato
° pAAV-TetO-GFP
The pAAV-TetO-GFP can be replaced by viruses linking other genetic constructs, e.g., ChR2, iChloC, or ArCHT to TetO. GFP is expressed when pAAV-TetO-GFP is used. The ratio of the TetO construct to the two other viruses can be varied, depending on the experimental requirements (see Lee et al.,
Dilute the virus solution in 20 μl sterile PBS per 1 μl of virus. Vortex thoroughly.
Take the well plate(s) out of the incubator and rapidly add 20 μl of the solution to each well in the plate containing coverslips to be transfected and then put them back into the incubator.
To test CaMPARI photoconversion and Cal-Light photoactivation, cells have to be active and cytosolic calcium increases have to be coupled to light exposure. To promote network activity in cell cultures, cells were incubated with the GABAA receptor antagonist gabazine blocking fast GABA-mediated synaptic inhibition. To further promote network activity, some cultures were also treated with the muscarinic receptor agonist carbachol.
Thaw frozen stock solutions of gabazine and carbachol at room temperature for 15 min. Incubate a conical tube containing 8 ml of hibernate A complete media in a water bath (37°C) for 15 min.
Following incubation, pipette 2 ml of hibernate A complete media to a glass-bottom plate. Transfer a 24-well plate from the incubator to a laminar flow hood. Use sharp forceps to transfer a coverslip containing cells from the 24-well plate to the glass-bottom plate. Use a platinum ring (or suitable alternative) to hold the coverslip in place. Quickly transfer the 24-well cell culture plate back to the incubator.
Place the glass-bottom dish inside the microscope sample holder and focus on the cell layer. Depending on the experiment, drugs may be carefully pipetted into the hibernate solution to increase network activity. To reduce inhibition and to generate repetitive bursts of action potentials and increased network activity, apply 10 μM gabazine (final concentration) to cell cultures. If after 10 min gabazine does not increase network activity (activity can be monitored if cells express CaMPARI, see below), apply 10 μM carbachol (final concentration) to promote a further increase in activity. Take pre-stimulation images in any fluorescence channels that are of interest (see next step).
Image acquisition:
In cultures expressing CaMPARI, network activity can be monitored before photoconversion. This is possible because CaMPARI is also a calcium indicator related to GCaMP3, which dims rapidly and reversibly upon calcium influx (Fosque et al.,
In cultures expressing Cal-Light, images are acquired at 555 nm (to check tdTomato expression) and at 470 nm (to check GFP expression). In the pre-stimulation period, when 470 nm light has not been applied, there should be no fluorescent neurons in the green (470 nm) channel.
Macros:
For CaMPARI, set up an automated macro script to acquire images in both 470 and 550 nm light channels, interspersed with 395 nm light stimulus (
For Cal-Light, set up an automated macro script that repetitively triggers a light stimulus at 470 nm (ON), followed by an interval of darkness (OFF). We suggest applying either 2 s ON/8 s OFF or 1 s ON/4 s OFF for 40–60 min, leading to 8–12 min of total light delivery. The light power of the stimulus should be ~4–10 mW·cm−2.
Once photoconversion occurs or light for photoactivation of Cal-Light expression has been delivered, transfer the coverslip from the hibernate solution to a well-filled with cell culture medium in a laminar flow hood. Return the cell culture plate to the incubator.
Further handling:
CaMPARI remains in its converted form for 2–3 days but will be progressively removed by protein turnover. In other words, the most reliable CaMPARI signal is detected right after conversion. Cells expressing CaMPARI can be used for additional experiments once protein turnover has removed the conversion, i.e., red CaMPARI has been fully replaced by newly produced green CaMPARI.
Cells expressing Cal-Light require at least 2–5 days to show reliable expression of the photoactivated construct. Make sure that incubator conditions (atmosphere and temperature) are optimal during that time and feed as required.
Virus solutions:
Cal-Light: Prepare Cal-Light as described above [in section
pAAV-TM-CaM-NES-TEV-N-AsLOV2-TEVseq-tTA,
pAAV-M13-TEV-C-P2A-TdTomato and
pAAV-TetO-GFP at a ratio of 1:1:2 (titers ~1012-1013 GC/ml)
CaMPARI: Prepare pAAV-Syn-CaMPARI2 with a final titer of ~1011-1012 GC/ml in a sterile 0.5 ml Eppendorf tube (total volume should be ~5 μl).
Pull glass pipettes for injections (5 or 10 μl) on a Sutter puller. Carefully cut the pulled pipettes back to ~10–20 μm with sharp scissors under a stereo-microscope. Before loading the virus, place a drop of mineral oil on a piece of sterile Parafilm.
To load the virus, insert a pulled glass-micropipette tip-first into a plastic-pipetting tip attached to an insulin syringe. Make sure that there is no air leaking when negative pressure is applied on the syringe. Carefully back-load 300–500 nl of virus from an Eppendorf tube into the open end of the micropipette. After loading the virus, release the pressure on the insulin syringe. Position the plastic pipette opening over the prepared oil drop on the piece of Parafilm. Apply negative pressure on the syringe and load ~500 nl of mineral oil. The boundary between virus solution and mineral oil should be visible as a clear contrast of phases. Carefully remove the filled glass micropipette from the plastic syringe and place it into the injector attached to a stereotaxic arm. Dispose the Parafilm in a biohazard waste bin and keep the remaining virus solution in the refrigerator at 4°C.
Before surgery, anesthetize mice deeply with an intraperitoneal injection of ketamine/xylazine (100/10 mg kg−1) solution. Once mice no longer react to tail or toe pinches, trim the fur on the head with sharp scissors or with an electric trimmer. Place mice into the stereotaxic apparatus (Kopf Instruments Inc., California, USA). Make sure that ear bars and the mouth piece are positioned correctly to hold mice in place for the duration of the surgery.
Provide local analgesia by injecting lidocaine (1–2%, 0.1–0.2 ml) locally under the scalp where the craniotomy is to be made.
When mice are fully sedated and positioned properly in the stereotaxic frame, ensure that the head is leveled and aligned. For injections deep into the brain it is necessary to ascertain that the
Once the fur is trimmed, disinfect the scalp using 70% ethanol. Carefully cut the scalp with a sterile scalpel and the splay the skin out with a forceps (Dumont, no 5). If needed, irrigate the wound edge with saline. Remove any excess liquid using absorbent swabs (Sugi, Kettenbach).
Define the stereotaxic coordinates by setting the reference point “0” at bregma. Mark the cortical area of interest (in our case, barrel cortex, medial-lateral 2.5 mm, anterior-posterior −2.0 mm) with a pen or carefully with a scalpel blade. Drill a circular craniotomy of 1 mm radius around the mark. Apply careful and slow drilling without fully perforating the bone. By constantly applying sterile PBS to the bone, heating, and damaging of the dura can be avoided. After thinning the bone by continuous drilling, it should be possible to remove the perforated piece of bone.
Apply sterile PBS onto the craniotomy and carefully clean the injection site using absorbent swabs. Keep the brain moist with PBS.
Put the virus-filled injection micropipette into the stereotaxic holder and place the pipette over the injection site in a 90° angle to the brain. Use the micro-injection controller to apply positive pressure and to generate a small drop of virus, visible at the tip of the pipette. This is to assure that the pipette is not clogged and virus solution can be injected smoothly.
Lower the injection micropipette and penetrate the dura to reach the desired depth (in our experiments, we injected at 0.6, 0.4, 0.2, and 0.1 mm) below the pial surface. Once the pipette is at its correct position, the virus can be injected with positive pressure (100–200 nl at 15–20 nl min−1).
After injection, wait for 5 min before removing the micropipette slowly from the cortex. Remove the pipette from the stereotaxic holder and dispose in a biohazard waste bin. Carefully clean the brain again with sterile PBS.
Inject the analgesics carprofen (5 mg/kg) and buprenorphine (0.05–0.1 mg/kg) intraperitoneally to ensure a pain-free recovery of the animal.
Remove the mouse from the stereotaxic frame by loosening the ear bars and the nose piece.
Suture the scalp with sterile suture sewing thread. Carefully put the mouse back into its home cage, which is put on a warming device. Monitor the mouse until it has woken up.
Allow CaMPARI to express for >14 days following viral injection(s)
Deeply anesthetize the mouse (postnatal age >P21) with isoflurane (1.5–3% in O2). Remove and section the brain into coronal, 300 μm thick slices with a vibratome under cold (~0°C) choline-based artificial cerebrospinal fluid (ACSF).
Transfer each slice after sectioning to an incubation chamber at 32°C for 5 min in a solution containing choline ACSF saturated with 95% O2/5% CO2.
Transfer the slices into an incubation chamber containing normal ACSF at 32°C for 25 min and then at room temperature for an additional 30 min before use in experiments.
CaMPARI can be used to map cortical circuit activity driven by optogenetically defined inputs in brain slices (Zolnik et al.,
Deliver violet light for optogenetic stimulation and CaMPARI photoconversion using an X-cite 200 W mercury lamp (Excelitas Technologies, Mississauga, Ontario, Canada) and light guided through a 405/10 nm bandpass filter (Semrock, FF01-405/10-25). In one-photon imaging experiments, photoconversion/stimulation light is delivered by a UPlanFL 4 × /NA 0.13 objective.
Measure the light stimulus intensity with a Thor Labs optical power meter (PM 200) and a photodiode sensor that works in the UV range (S120VC). For the experiments in
Photoconvert neurons with 10 pulses, 15 ms in duration, delivered at 10 Hz, followed by a 5 s-long light pulse. This protocol—especially the 5 s light pulse at the end of the stimulus train—ensures that the photoconversion light is delivered when calcium is elevated in the post-synaptic target neurons.
For quantification of the red/green ratios of each neuron, two-photon imaging is necessary. A standard brain slice immersion chamber is needed to maintain slice viability during live imaging for these experiments.
Use a two-photon laser scanning system equipped with a femtosecond pulsed Chameleon Ti:Sapphire laser controlled by the MES software package.
Tune the laser to λ = 820 nm for excitation of CaMPARI red and green fluorescence.
Detect fluorescence in epifluorescence mode with a water immersion objective (LUMPLFL 60 × /1.0 NA, Olympus, Hamburg, Germany), and trans-fluorescence and transmitted infrared light with an oil immersion condenser (Olympus; 1.4 NA). Emission light can be divided with a dichroic mirror at ~590–600 nm, and green and red signals filtered using 525/50 and 650/50 bandpass filters, respectively.
Allow >14 days for expression of constructs after surgery.
Follow steps (1)–(4) of module
Incise the scalp and scrape the skull. Carefully remove the fascia and let the skull air dry. When dry, place a metal head post (ours are custom-made, the shape varies) on the clean and dry skull. Next, use self-adhesive resin cement (RelyX Unicem, 3M ESPE) to glue the head post in place.
Once the cement has hardened, drill a circular craniotomy of 3 mm radius around the spot where virus had been injected previously. Apply careful and slow drilling without fully perforating the bone. By constantly applying sterile PBS to the bone, heating and damaging of the dura can be avoided. After thinning the bone by continuous drilling, it should be possible to remove the perforated piece of bone.
Prepare a 3 mm glass coverslip by cleaning in 70% ethanol. Carefully place the coverslip on the part of the mouse brain that is uncovered by the craniotomy. Use the arm on the stereotaxic apparatus to position a wooden tipped applicator or toothpick over the coverslip and apply slight pressure to push the coverslip into the craniotomy and hold in place.
Apply superglue at the edges of the coverslip and bone to fix the coverslip into the craniotomy, then wait for the glue to dry and carefully release the wooden tip off the window.
Cover the head post using Ortho-jet powder (Lang Dental, Black) to fill any gaps and to increase stability.
Cover the glass and well-around the craniotomy with Kwik-Cast Sealant silicone (WPI, World Precision Instruments).
Once the sealant firmed up, apply analgesics [see procedure
For the main photostimulation session, head-fix the mouse in the stimulation setup and remove the sealant. Place the tip of an optic fiber connected to the LED light source directly on top of the glass window at a 90° angle. Make sure that the light spot overlaps with the area of expression.
Photostimulation.
For Cal-Light-expressing animals, set up an automated macro script that repetitively triggers a light stimulus at 470 nm (ON), followed by an interval of darkness (OFF). We suggest applying either 2 s ON/8 s OFF or 1 s ON/4 s OFF for 40–60 min, leading to 8–12 min of total light delivery. The light power of the stimulus should be ~20–30 mW (measured at the tip of the optic fiber).
For CaMPARI, set up an automated macro that repetitively triggers a light stimulus at ~395–405 nm (ON), followed by an interval of darkness (OFF). We suggest applying either 2 s ON/8 s OFF or 1 s ON/4 s OFF for 40–60 min, leading to 8–12 min of total light delivery. The light power of the stimulus should be ~20–30 mW (measured at the tip of the optic fiber).
After imaging of the live brain slices, fix them in formaldehyde-based (4%) fixative at 4°C overnight.
Wash slices in phosphate buffered saline solution (PBS) and block in blocking solution for 2 h at room temperature.
Incubate brain slices in primary antibody containing solution (CaMPARI 4F6, at a dilution of 1:1,000 in blocking solution) at 4°C overnight.
Dispose the primary antibody, rinse slices in PBS before incubation in the secondary antibody solution (Alexa 633, goat anti mouse, Invitrogen A21050, at a dilution of 1:500 in blocking solution) for 2 h at room temperature.
Dispose the secondary antibody, rinse slices in PBS.
Mount slices in mounting solution (80% glycerol + 2.5% DAPCO in PBS) on a regular slide and coverslip.
After transfection, cultured neurons begin to express CaMPARI within ~10 days, this appears as a green fluorescent signal. Calcium transients in these neurons are noticeable as dimming in fluorescence and occur when network activity increases (e.g., via blocking inhibition and/or by application of carbachol; see
Cultured neurons express Cal-Light within ~10 days, indicated by a red fluorescent signal via tdTomato expression. When network activity increases (e.g., by blocking inhibition and/or by application of carbachol), active neurons expressing Cal-Light are photoactivated and express GFP within 2–5 days after exposure to 470 nm light (
Two to three weeks after virus injection, neurons begin to express CaMPARI. At this point,
Two to three weeks after virus injection, neurons begin to express Cal-light or CaMPARI. This expression (green CaMPARI or red Cal-Light) is visible through the cranial window using epifluorescence microscopy (
Immunostaining recovers the CaMPARI red signal in photoconverted neurons that is quenched after fixation (
All datasets generated for this study are included in the manuscript and/or the
We performed all procedures in accordance with protocols approved by the Charité—Universitätsmedizin Berlin and the Berlin Landesamt für Gesundheit und Soziales (LAGeSo) for the care and use of laboratory animals.
CE, SD, PT, PP, BE, IV, ML, and RS designed the study. CE, JL, TZ, SD, and AP did the experiments. CE, JL, PT, and RS wrote the paper. All authors read and edited text, contributed to manuscript revision and approved the submitted version.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We would like to thank Alexander Schill, Jan-Erik Ode, and Daniel Deblitz of the Charité Workshop for technical assistance; Thorsten Trimbuch, Anke Schönherr, and Bettina Brokowski of Viral Core Facility of the Charité (vcf.charite.de) for the production of viruses; Friedrich Johenning and the Schmitz lab for the use of a Femtonics two-photon system; Jan Schmoranzer and the Charité AMBIO facility for the training and use of the wide field imaging setup and Marti Ritter for assistance during cell culture photostimulation experiments. We would also like to thank Eric Schreiter and Hyung-Bae Kwon for commenting on an earlier version of this manuscript.
The Supplementary Material for this article can be found online at:
Photoconversion of CaMPARI-expressing cells in neuronal cell culture using wide-field epifluorescence imaging. Momentary dimming of fluorescence indicates calcium-intensive events. Videos contain 60 frames, each taken prior to a 5-s long pulse of 395 nm light. Green and red imaging channels are merged. Video 1, using 10 × objective and Video 2, using 20 × objective.