All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Preparation of hardware and software for multi-channel recordings
- Set up a 16-channel data acquisition system according to manufacturer instructions.
NOTE: Several commercially available amplifiers and data acquisition systems can be used to collect multi-channel recordings. In the experiments described here, analog signals are delivered via an electrode reference panel to two amplifiers, where they are amplified (2000x) and filtered (0.1-10kHz). Analog inputs to the data acquisition system are digitized at 40kHz.
- Fasten the appropriate 16-channel head stage adaptor to a microscope micromanipulator. Orient the adaptor so that the female connector ports face downward.
- Adjust the angle of operation of this micromanipulator such that it is oriented downward toward the recording chamber at an angle of approximately 70° relative to horizontal.
- Connect the headstage input to a 16 x 1 probe for in vitro electrophysiology via the head stage adaptor anchored to the micromanipulator.
- Connect the head stage output connector to the data acquisition system.
- Install appropriate software for data acquisition. Configure 15 input channels to correspond to input signals from the first 15 multi-channel probe contacts. Configure the remaining channel to receive input from the intracellular electrode.
NOTE: When collecting and analyzing data, take care to consider electrode and adaptor maps to ensure the appropriate signal corresponds to the electrode contact from which it was collected.
2. Configuration of light stimulation protocols
- Set up a light delivery system and install the accompanying software.
- Open the software. Choose a hardware wiring configuration in which a Trigger Source (Digital/TTL(Transistor–transistor logic) Out) provides a Trigger In signal to the light delivery system, and the light delivery system provides a Trigger Out signal to a 470 nm LED (light-emitting diode).
- Mount high-power objective lens. Using a digital camera, calibrate high-power objectives with the light delivery system.
- Create a new profile sequence of light stimulation profiles.
- Create a pattern of choice. In the experiments described here, a circle of diameter 150 µm allows layer-specific activation of axon terminals.
- To construct a profile sequence, copy and paste this profile for each of any number of trials.
- Create a waveform list that contains waveforms of any light intensity, pulse duration, or pulse number.
- Randomly assign waveforms to each profile. Each profile with its assigned waveform corresponds to one trigger pulse from a Digital TTL input or one trial.
- Save the profile sequence.
- In the data acquisition software, create a new protocol.
- Set the number of trials to equal the number of profiles in the created profile sequence.
- Choose signal inputs to match those configured in Step 1.6. Configure a protocol that provides a single digital TTL output, recording from these 16 input channels for an appropriate amount of time before and after the digital trigger.
3. Placing multi-channel probe in ex vivo brain tissue slice
- Perfuse bubbled eACSF(Experimental Artificial cerebrospinal fluid) (not in sealed bags) at 3-6 mL/min.
- Transfer the brain slice containing an area of interest onto the mesh grid in a microscope perfusion chamber. Anchor with platinum harp
- Rotate the mesh grid such that the line of electrode contacts on the distal end of the multi-channel probe is approximately perpendicular to the pial surface.
- Lower the multi-channel probe toward the surface of the slice under broadfield illumination and fine control of the micromanipulator.
- Rotate the filter cube turret to engage the appropriate filter cube to visualize the fluorescent reporter protein expressed in axon terminals of cortical afferents. If necessary, rotate the slice to more precisely align the probe with the pial surface.
- Position the probe just above the plane of the slice, ~200 µm short of the final target position along the x-axis, leaving at least one channel outside the boundary of the area of tissue being recorded.
- Slowly insert the probe into the slice by moving the manipulator along its longitudinal axis. To minimize damage to the tissue, only advance the probe to the extent that the sharp tips are just visible below the tissue surface. This will minimize damage to the tissue while still ensuring the electrode contacts are in contact with the tissue.
4. Patch clamping targeted neurons and obtaining whole-cell configuration
- Switch eACSF source to the bagged Control solution.
- Identify fluorescently labeled cells for targeted patch clamp recording.
- Restrict the aperture iris diaphragm to the smallest diameter. Engage a low-power objective lens and bring the tissue into focus.
- Center the light over an area of tissue adjacent to (but not overlapping) the multi-channel probe.
- Engage the high-power (40x or 60x) water immersion objective, using caution to avoid contact between the multi-channel probe and the objective lens.
- Rotate the filter cube turret to engage the appropriate filter set to allow imaging of cells expressing Cre-dependent fluorescent marker.
- Identify a fluorescently labeled cell as a target for patch clamp recording. Raise the objective lens to create ample space to lower a patch pipette.
- Load a patch pipette (see Table 1) with internal solution (Table 2) and mount pipette into electrode holder. Using 1 mL syringe, apply positive pressure corresponding to ~0.1mL air.
- Lower the patch pipette into the solution. Bring the pipette tip into focus under visual guidance.
- Obtain whole-cell recording from the targeted cell using the steps previously demonstrated33.
- If planning to assess changes to intrinsic properties of the cell (e.g., input resistance, action potential firing rate in response to current steps), conduct these recordings. Otherwise, move to the axon stimulation protocol below.
Table 1: Composition of artificial cerebral spinal fluid and intracellular solution. Reagents and concentrations for eACSF, and intracellular pipette solution for patch clamp recordings are listed.
| Experiment ACSF, eACSF (in mM) |
| NaCl | 111 |
| NaHCO3 | 35 |
| HEPES | 20 |
| KCl | 1.8 |
| CaCl2 | 2.1 |
| MgSO4 | 1.4 |
| KH2PO4 | 1.2 |
| glucose | 10 |
| Internal Solution |
| K-gluconate | 140 |
| NaCl | 10 |
| HEPES | 10 |
| EGTA | 0.1 |
| MgATP | 4 |
| NaGTP | 0.3 |
| pH = 7.2 |
