All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review.
1. Preliminary Preparation (Optional: Prepare 1 Day in Advance)
- 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 mOsm 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 slices.
- 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.
- 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: The choice of the calcium indicator should be predicated on its dynamic range and on the nature of the investigated Ca2+ events.
- 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. Whole-cell Patch-clamp Recordings
- 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.
- 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 locate cells of interest easily. In this case, this step can be substituted with the use of a fluorescent light source.
- Fill a stimulation pipette with an ACSF-Normal solution containing the red fluorophore (e.g., Alexa-594).
- 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.
- 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.
- 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 a "close" position.
- 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 the 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.
- 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.
- 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.
- 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.
3. Two-photon calcium (Ca2+)Imaging
- If interested in the excitatory postsynaptic responses, add the GABAA receptor blocker gabazine (10 µM) and the GABAB receptor blocker CGP55845 (2 µM) to the ACSF bath.
- In the 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 its subtype, e.g., fast-spiking cells. It is important for the indicator concentration to stabilize through diffusion before starting to record calcium transients (CaTs) (see Table 2 for troubleshooting).
- AP-evoked CaTs
- 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 interneurons.
- 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.
- 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.
- 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.
- Synaptically-evoked CaTs
- Locate a dendrite of interest using the red fluorescence signal.
- 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.
- 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.
- 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.
- 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 the scan can be adjusted depending on the evoked event's kinetics but should be minimized to prevent photodamage (see Table 2 for troubleshooting).
- Repeat step 3.4.5 in different conditions (e.g., different intensity/length of stimulation, introduction of a pharmacological blocker, etc.) depending on the specific question being addressed.
- 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).
- 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.
- 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.
- 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.
Table 1: Solution recipes. Compounds and concentrations for solutions used during the protocol.
| Solution | Component | Concentration (mM) |
| ACSF-Normal | NaCl | 124 |
| KCl | 2.5 |
| NaH2PO4 | 1.25 |
| MgSO4 | 2 |
| NaHCO3 | 26 |
| Glucose | 10 |
| CaCl2 | 2 |
| ACSF-Recovery | NaCl | 124 |
| KCl | 2.5 |
| NaH2PO4 | 1.25 |
| MgSO4 | 3 |
| NaHCO3 | 26 |
| Glucose | 10 |
| CaCl2 | 1 |
| ACSF-Sucrose | KCl | 2 |
| NaH2PO4 | 1.25 |
| MgSO4 | 7 |
| NaHCO3 | 26 |
| Glucose | 10 |
| Sucrose | 219 |
| CaCl2 | 1 |
| K+-based Patch solution | K+-gluconate | 130 |
| HEPES | 10 |
| MgCl2 | 2 |
| Phosphocreatin di(tris)salt | 10 |
| ATP-Tris | 2 |
| GTP-Tris | 0.2 |
| Biocytin | 72 |
| Alexa-594 | 0.02 |
| Oregon Green-BAPTA-1 | 0.2 |
Table 2: Troubleshooting table. Solutions to common problems that may arise during a Ca2+ imaging experiment.
| Problem | Solution |
| Unable to patch healthy neuron | Check for signs that the slices are unhealthy: shrunken or swollen cells, visible nuclei, etc. If so, discard the slices. Also verify the patch-pipette resistance and the patch-solution's osmolality; replace them if the values are not in the appropriate range. |
| The fluorescence signal from dendrites is low | Wait longer for the cell to fill. If an obstruction is keeping the patch-solution from diffusing into the cell, try to apply a small amount of negative pressure in the patch-pipette. |
| Electrical stimulation does not evoking a Ca2+ response | Check for the presence of an artifact in the electrophysiological recording. If absent, check for a short-circuit/stimulating unit malfunction. If present, raise the stimulation intensity or move the stimulation pipette closer to the dendrite. It is to be noted that the distance between the stimulation electrode and the dendrite should not exceed 8 um to prevent direct depolarization of dendrites when studying synaptic responses. |
| Evoked Ca2+ signal is too high/saturates the Ca2+ indicator | Reduce laser power. If the problem persists in multiple cells, use a different Ca2+ indicator with a lower Ca2+ affinity. |
| The baseline Ca2+ signal is gradually increasing during the experiment | Wait for a longer period between individual scans. If the baseline is still increasing, stop acquisition; it indicates that the cell's health is likely declining. |
| The amplitude of the Ca2+ signal decreases during scans | Reduce laser power or zoom out (if applicable). |
| Dendrite is fragmenting ("blebbing") after scanning | Reduce the laser power or zoom out. If blebbing is limited to the targeted dendrite, choose another dendrite and reduce laser power/ zoom out. If multiple dendrites are blebbing, stop the acquisition. |
| Fluorescence signals are "drifting" out of the scan line after sweeps | Reduce movement in the slice by reducing the speed of ACSF perfusion. Before patching, make sure that the slice is strongly fixed in place by a net. |