Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

All experiments were performed in accordance with the animal welfare guidelines of the Animal Protection Committee of Université Laval and the Canadian Council on Animal Care.

1. Preliminary Preparation (Optional: Prepare 1 Day in Advance)

1. Prepare three types of artificial cerebrospinal fluid (ACSF) solution (Normal, Sucrose and Recovery Solutions, 1 L each; see Table 1). Adjust the osmolality of the solutions to 300 ± 10 mOsm²⁵ and cool the ACSF-Sucrose down to a near-freezing point (0-4 °C).
   NOTE: The use of a low-sodium cutting solution (ACSF-Sucrose) helps preserve the viability of neurons in the superficial layers of acute slices²⁶.
2. Prepare 0.75 mL of patch-solution containing a red fluorophore (e.g., Alexa-594) and a green synthetic calcium indicator (e.g., Oregon Green BAPTA-1; see Table 1). Include biocytin (see Table 1) in the patch-solution if post hoc morphological identification of recorded cells is required.
3. Adjust the osmolality of the solution to 280 ± 5 mOsm and pH to 7.35 ± 0.5. Keep the solution in the refrigerator or on ice at all times.
   NOTE: Choice of the calcium indicator should be predicated on its dynamic range and on the nature of the investigated Ca²⁺ events.
4. Using a micropipette puller, prepare patch pipettes from borosilicate glass capillaries (see Table of Materials) with a ≈ 2 µm tip (pipette resistance of 3-5 MΩ) and stimulation pipettes from borosilicate theta-glass capillaries (see Table of Materials) with a 2-5 µm tip.
   NOTE: The puller settings to make pipettes of a given diameter and shape will be determined by the puller filament type and the ramp test results for a given type of glass.

2. Hippocampal Slice Preparation

1. Anesthetize the mouse (P13-20; the strain and sex of the mouse is determined by the experimental question being addressed) with 1 mL of isoflurane placed onto a paper towel within an inhalation narcosis chamber. When the animal is deeply anesthetized, as evidenced by a lack of movement and slow respiration, remove it from the narcosis chamber and decapitate using a guillotine.
2. Expose the skull by cutting the skin with a small pair of scissors from the back of the skull to the nose. Use a small pair of mini bone rongeurs to make a hole at the bregma level. Then use the scissors to make a caudal-to-rostral cut along the sagittal suture. With the rongeurs grasp the back of each skull flap and lift upward and outward to remove the skull covering each hemisphere.
3. After the brain is fully exposed, undercut the optic nerves with a small spatula. Remove the brain from the skull and put it into a Petri dish containing continuously oxygenated, cold ACSF-Sucrose (see Table 1 for sucrose concentration).
   1. If tissue from older mice is required, perform an intracardiac perfusion using a syringe (25G) filled with 20 mL of ice-cold ACSF-Sucrose prior to the brain extraction to obtain good quality slices.
4. Prepare hippocampal slices from the extracted animal brain.
   1. Let the brain chill for around 2 min. Place a piece of filter paper on an ice-packed Petri dish, transfer the brain onto the filter paper. Dissect the two hemispheres.
   2. Glue the dissected hemispheres (the orientation is determined by the experimental goal to obtain coronal, transversal, or horizontal slices) onto a vibratome table and immerse it into the vibratome tray filled with ice-cold oxygenated ACSF-Sucrose. Make 300-µm thick slices at a temperature of about 1 °C by using a vibratome cooler or placing the ice around the vibratome tray.
   3. Using a flat paint brush, accumulate the slices containing the hippocampus (up to 6 slices per hemisphere) in a bath containing oxygenated ACSF-Recovery heated to 37 °C.
   4. Leave the slices in the ACSF-Recovery bath for at least 45 min.

3. Whole-cell Patch-clamp Recordings

1. Set a hippocampal slice in place in a bath positioned under the objective (magnification: 40x, numerical aperture: 0.8) of a laser-scanning two-photon microscope. Continually perfuse the bath with oxygenated ACSF-Normal heated at 30-32 °C at a rate of 2.5-3 mL/min.
2. Use infrared differential interference contrast (IR-DIC) or Dodt-SGC microscopy to locate a cell of interest using its size, shape, and position.
   NOTE: Depending on the targeted neuron population, a transgenic mouse model can be used to easily locate cells of interest. In this case, this step can be substituted with use of a fluorescent light source.
3. Fill a stimulation pipette with an ACSF-Normal solution containing the red fluorophore (e.g., Alexa-594).
4. Set the stimulation pipette (connected to an electrical stimulation unit) on top of the slice so that the tip is in the same region as the cell of interest.
5. After filling the patch pipette with patch solution and attaching it to a head stage, set it over the slice so that its tip is directly above the cell of interest.
6. Fit a syringe into a three-way stopcock and connect it to the patch-pipette through the plastic tube. Inject constant positive pressure into the patch pipette (≈ 0.1 mL) using the syringe. Keep the stopcock in "close" position.
7. Activate the amplifier control software module and switch to voltage-clamp mode by clicking on the "VC" button. Open the electrophysiology data acquisition software (e.g., clampex) for acquisition of electrophysiological signals and click on the "Membrane Test" icon to have it continually send out a square voltage pulse (5 mV, 10 ms), which is necessary to monitor changes in the pipette resistance.
8. Lower the patch pipette until it is right on top of the targeted cell. Upon making contact with the cell membrane, remove the pressure by turning the stopcock into the "open" position.
   NOTE: Contact with the cell membrane is detected when a small increase in the pipette resistance (≈ 0.2 MΩ) is seen and a small indentation on the cell is caused by positive pressure.
9. Apply a slight negative pressure to the patch pipette using an empty syringe fitted into the stopcock until the pipette resistance provided by the software reaches 1 GΩ. In the 'Membrane Test' window of the data acquisition software, clamp the cell at -60 mV.
10. Continue applying negative pressure until the cell is opened and the whole-cell configuration is achieved. Detection of this cell state is based upon the sudden change in the pipette resistance (from 1 GΩ to 70-500 MΩ depending on the interneuron type) and the appearance of large capacitive transients in the 'Membrane Test' window.

4. Two-photon Ca²⁺ Imaging

1. If interested in the excitatory postsynaptic responses, add the GABA_A receptor blocker gabazine (10 µM) and the GABA_B receptor blocker CGP55845 (2 µM) in the ACSF bath.
2. In amplifier control software module, set the patch configuration to current-clamp by clicking on the "CC" button and record the cell's firing pattern in response to somatic injections of depolarizing current (0.8-1.0 nA, 1 s). Wait for at least 30 min for the cell to be filled with the indicators present in the patch solution.
   NOTE: The cell's firing pattern and active properties can be used to identify the cell's subtype; e.g., fast-spiking cells. It is important for the indicator concentration to stabilize through diffusion before starting to record CaTs (see Table 2 for troubleshooting).
3. AP-evoked CaTs
   1. Using the image acquisition software, start acquiring images. Locate a dendrite of interest using the red fluorescence signal. To ensure a visible response, first, choose a proximal dendrite (≤ 50 µm) as the efficiency of AP backpropagation may significantly decline with distance in GABAergic interneurons²².
   2. Using the 'rectangular tool' in the image acquisition software, zoom in on the targeted region and switch to the "xt" (linescan) mode. Position the scan across the dendritic branch of interest. With the laser intensity controllers in the acquisition software, set the two-photon laser at a minimal power level where baseline green fluorescence is just slightly visible, to avoid phototoxicity.
   3. In the electrophysiology data acquisition software 'Protocol' window and image acquisition software, create a recording trial of the desired duration containing a somatic current injection of the desired amplitude.
      1. Using the image acquisition software, click the "Start record" button and acquire the fluorescence continuously for 1-2 s. Repeat the image acquisition 3-10 times. Wait at least 30 s between single scans to avoid photodamage. If acquiring linescans at multiple points along a dendrite, take them in random order to avoid order effects.
4. Synaptically-evoked CaTs
   1. Locate a dendrite of interest using the red fluorescence signal.
   2. Set the stimulation pipette on the surface of the slice above the dendrite of interest. Slowly lower the stimulation pipette into place, minimizing movement to avoid disturbing the whole-cell configuration. Position the pipette at 10-15 µm from the dendrite.
   3. To visualize the location of synaptic microdomains in aspiny dendrites, in the image acquisition software switch to the 'xt' (linescan) mode and position the line along the dendritic branch of interest.
   4. In the 'Protocol' Window (of electrophysiology data acquisition software) and image acquisition software, create a recording trial that triggers the stimulation unit after the trial start.
   5. Using the acquisition software, click the "Start record" button to scan along the dendrite continuously for 1-2 s. Repeat acquisition 3-5 times, waiting 30 s between scans to prevent photodamage.
      NOTE: The length of scan can be adjusted depending on the evoked event's kinetics, but should be minimized to prevent photodamage (see Table 2 for troubleshooting).
   6. Repeat step 4.5.5 in different conditions (e.g., different intensity/length of stimulation, introduction of a pharmacological blocker, etc.) depending on the specific question being addressed.
   7. Stop the acquisition when the cell shows signs of deteriorating health: depolarization below -45 mV, increase in the baseline Ca²⁺ level, morphological deterioration of dendrites (e.g., blebbing, fragmentation; see Table 2 for troubleshooting).
   8. To obtain preliminary information on the cell's morphology and keep a record of the location of the stimulation pipette, acquire a Z-stack of the cell in the red channel. Using the acquisition software in 'xyz' mode, set the upper and lower stack limits to image the entire cell with all processes included. Set the 'step size' at 1 µm and initiate the stack acquisition using the "Start record" button.
   9. When the Z-stack acquisition is complete, use the "Maximum projection" option in the software to superimpose all focal plans of the stack and verify the quality of acquisition. Then, slowly retract the patch pipette out of the slice.
   10. To fix the slice for post hoc morphological identification, quickly remove the slice from the bath using a flat paint brush, and place it between two filter papers, in an ACSF-filled Petri dish. Replace the ACSF with a 4% paraformaldehyde (PFA) solution and leave the dish in a 4 °C room or refrigerator overnight.

5. Immunohistochemistry for Post Hoc Morphological Identification of Recorded Cells

NOTE: After 1 night of fixation in PFA, the slice can be stored in phosphate buffer (PB) with sodium azide (0.5%) for up to 1 month.

1. Put the slice in Tris-buffered saline solution (TBS) with Triton X-100 (0.3%) and perform 4 washes (5-10 min each).
2. Put the slice in TBS-Triton 0.3% with 10% normal goat serum (NGS) for 1 h.
3. Put the slice in TBS-Triton 0.3% with 1% NGS and a Streptavidin-conjugated fluorophore (e.g., Alexa Fluor 546-conjugated Streptavidin, 1:200) for overnight incubation.
4. Wash 4 times (5-10 min) in TBS.
5. Mount slices on microscope slides and image using a confocal microscope. To have a complete cell reconstruction, acquire a Z-stack (step size: 1 µm) using a 20x objective. For imaging an Alexa-546-conjugated label, use a 543 nm laser and a 515-560 nm detection filter set.

6. Analysis of CaTs

1. Extract the fluorescence values from the regions of interest (ROIs) in the linescan images from the red and green channels and average the values of the multiple repeats for each condition to reduce noise.
   NOTE: When linescan acquisition is performed along the dendrite, it is possible to extract data from multiple ROIs, which may correspond to dendritic microdomains (2-5 µm), and thus allowing the study of the Ca²⁺ signal compartmentalization.
2. For each ROI, express the changes in Ca²⁺ amplitude as:
   
   NOTE: Here G is the fluorescence value from the green channel; G₀ is the baseline fluorescence value from the green channel during the pre-stimulation period; R is the fluorescence value from the red channel¹⁸. As two-photon excitation is highly localized and does not generate a significant background, subtraction of background fluorescence is not required.