Multichannel Extracellular Recording in Freely Moving Mice

All the mice were obtained commercially and maintained in a 12 h light/12 h dark cycle (light on at 08:00 A.M. local time) at a room temperature of 22-25 °C and a relative humidity of 50%-60%. The mice had access to a continuous supply of food and water. All the experiments were carried out in accordance with the Guidelines for Care and Use of Laboratory Animals of South China Normal University and approved by the Institutional Animal Ethics Committee. Male C57BL/6J mice aged 3-5 months old were used for the experiments.

1. Micro-drive system assembly

1. Connect two computer-designed boards using two stulls and a screw that holds the movable micro-drive and attach the connector to one board (Figure 1A, Bi-iii). Drive the micro-drive by twisting a screw (0.5 mm/circle).
2. Ensure the micro-drive can carry two sets of eight guide tubes (~3 cm long, ~50 µm internal diameter, ~125 µm external diameter) for each side of the MC region, and then cut it to the same length (at least 15 mm; Figure 1Biv, v).
3. Cut 16 Ni-chrome wires (~5 cm long) with a 35 µm diameter, and then load them successively into the guide tubes, followed by applying the glue to fix them (Figure 1Bi, vi, vii).
4. Strip the wire insulation, successively twine each exposed wire to each pin from the connector following the channel map, as well as the reference and ground electrodes, and then slowly coat conducting paint onto each pin (Figure 1Bviii-x).
5. Cover the pins using epoxy resin (Figure 1Axi, xii), and then perform gold plating via an impedance tester to reduce the impedance of the electrode tips to ~350 kOhm (Figure 1Bxiii, xiv). Set the parameters of the impedance tester as follows: −10.08 µA direct current for 1 s with gold plating solution, including 5 mM PtCl₄.
6. Finally, move the micro-drive to the top by twisting a screw. Check the overall size of the micro-drive system modified as shown in Figure 1A (approximately 15 mm long, 10 mm width, 20 mm height, ~1 g weight). Check the detailed specifications of the computer-designed board and movable component in Figure 1Ai, ii.

2. Electrode array implantation

1. Sterilize the surgery kits, wear sterile gloves and put on a doctor's sterile white coat before the operation starts.
2. To manage the pain, use subcutaneous (s.c.) injection of meloxicam injectable (5 mg/kg) for the mouse in an induction chamber. Then anesthetize the mouse by an intraperitoneal (i.p.) injection of pentobarbital (80 mg/kg) in an induction chamber^18,19. Apply a supplemental dose of pentobarbital (20 mg/kg/h) if the toe pinch reflex is still present.
3. Fix the mouse in a stereotaxic apparatus and maintain its rectal temperature at 37 °C by using a temperature controller.
4. Apply tetracycline eye ointment to both eyes of the mouse and change sterile gloves again before surgery.
5. Shave the mouse's fur and disinfect the surgical site with three alternating rounds of betadine scrub and alcohol using a sterile cotton-tipped applicator in concentric circles starting at the center and moving outward (Figure 2i, ii). Make a small midline incision (~15 mm) to expose its skull. Immediately, apply the 1% lidocaine locally to the neck muscles for pain relief. Then, remove the residual tissue using scissors, and clean the skull using saline-coated cotton buds (Figure 2iii).
6. Using a glass microelectrode filled with ink, mark the desired locations of the bilateral MC for implantation (Figure 2iv, v). Based on a previous study²⁰, the locations of the bilateral MC are as follows: 0.74 mm anterior to the bregma, and 1.25 mm lateral to the midline.
7. Implant four stainless steel screws (0.8 mm diameter) to protect the micro-drive system, and then link all the screws together with the reference and ground electrodes, followed by covering with mixed dental cement to form walls (Figure 2vi-xi).
8. Carefully drill two small holes (~1.5 mm²) with a skull drill on both the left and right sides of the coordinated skull in the MC regions (Figure 2xii). Use the stereotaxic coordinates of the bilateral MC: 0.74 mm anterior to the bregma, 1.25 mm lateral to the midline, and 0.5 mm ventral to the dura.
9. Remove the dura mater from the holes carefully with fine forceps (Figure 2xiii).
10. Insert the micro-drive system into the center of the holes using a micromanipulator at 10 µm/s (Figure 2xiv-xvii).
11. Fill the petroleum jelly into the dental cement walls after finishing the insertion of the micro-drive system (Figure 2xviii).
12. Join the bottom plate of the micro-drive system and the dental cement walls with the mixed dental cement (Figure 2xix)
13. Wash the incision with saline followed by local treatment with a gel containing lincomycin hydrochloride and lidocaine hydrochloride to relieve post-surgical pain.
14. Wind the conductive copper foil tape around the implanted micro-drive system (Figure 2xx).
15. Move the mouse into a cage kept at 31-33 °C and monitor the mouse for recovery from the anesthesia.
16. Allow the mice to recover for 1 week with separate feeding. Check and treat the incision with 3 days of continuous application of a gel containing lincomycin hydrochloride and lidocaine hydrochloride.

3. Multichannel recording in the bilateral MC in free-moving mice

1. Hold the head of an awake mouse lightly and carefully. Move down the electrode arrays (~0.1 mm depth) by twisting the screw on the movable part of the micro-drive system (Figure 1Aii) at least 1 day in advance.
2. Hold the head of the awake mouse lightly and carefully. Link the center of the headstage and a helium balloon (filled with approximately 0.02 L of helium) with a thread to offset the weight of the headstage and the micro-drive system (Figure 3A, B).
3. Capture raw signals using the recording electrodes and multichannel systems by sampling at 30 kHz in the recording software, and then digitize using a digital-analog (DA) converter from the multichannel systems.
4. Extract the LFP signals from the raw data by resampling at 10 kHz in the recording software, and then use a notch filter from the recording software to remove the 50 Hz line noise.
5. Record raw data in a stable state from a free-moving mouse for at least 60 s. After finishing the recording, slowly remove the connection between the headstage and the micro-drive system and return the mouse back to its home cage.
6. Store the recorded data in the computer and analyze it offline (Figure 4 and Figure 5).
7. After finishing the experiment, perform euthanasia as per the institute's guidelines and then confirm the locations of the electrodes by using the power supply at 3 V output to perform a 1 min electrolytic lesion, followed by performing histological analysis. Cut the mouse's brain into 30 µm slices using a freezing microtome, collect the MC sections, and then capture the images with a microscope (Figure 3C, D).

4. Spike sorting and analysis

1. Click on File > Open > Nev files in the spike sorting software to open the spike data sampled at 30 kHz (Figure 4Ai).
2. Click on Info to select the unsorted channel, and then select Sort > Change Sort Method > Use K-Means. Press the button Valley-Seeking Sort > K-Means Sorting to obtain the sorted units (Figure 4Aii, iii).
3. Click on File > Save as, save the sorted spike data with a .nev filename extension, and select File > Export Per-Waveform Data to export the PCA results with a .txt filename extension (Figure 4Aiv).
4. Click on File > Import Data > Blackrock File in the software for the neurophysiological data analysis to open the sorted spike file (Figure 4Bi).
5. Click on Analysis > Autocorrelograms to obtain the autocorrelogram for the selected unit, and then set the parameters as follows: the X Minimum value at −0.05 s, the X Maximum value at 0.05 s, and the Bin value at 0.001 (Figure 4Bii, iii).
6. Load the sorted spike data, click on Analysis > Interspike Interval Histograms to obtain the inter-spike interval histogram, and then set the parameters as follows: the Min. interval value at 0 s, the Max. interval value at 0.1 s, and the Bin value at 0.001 (Figure 4Biv, v).
7. Click on Analysis > Crosscorrelograms to obtain the cross-correlogram between two sorted unit events, and then set the reference events and parameters as follows: the X Minimum value at −0.1 s, the X Maximum value at 0.1 s, and the Bin value at 0.001 (Figure 4Bvi, vii).
8. Click on Results > Numerical Results to save the results of the autocorrelogram, inter-spike interval histogram, and cross-correlogram with .xls filename extensions (Figure 4Bviii, ix). Analyze the data, and draw the graph.

5. LFP analysis

1. Click on File Import Data > Blackrock File in the software for the neurophysiological data analysis to open the continuous signal data sampled at 10 kHz (Figure 5Ai).
2. Click on Analysis > Spectrum for Continuous to analyze the power spectrum for the LFP from the selected channel. Set the parameters as follows: the Number of Frequency Values at 8,192, the Multiple Tapers value at 3-5, the Normalization of the percentage of the total power spectral density (PSD), and the Frequency range from 1 Hz to 100 Hz (Figure 5Aii, iii).
3. Click on Analysis > Coherence for Continuous to analyze the coherence for two LFPs from the left and right sides of the MC. Set the reference channel and parameters as follows: Calculate at Coherence Values, the Number of Frequency Values at 8,192, the Multiple Tapers value at 3-5, and the frequency range from 1 Hz to 100 Hz (Figure 5Aiv, v).
4. Click on Analysis > Corr. with Cont. Variables to analyze the correlation between two LFPs from the left and right sides of the MC. Set the reference channel (LFP data) and parameters as follows: the X Minimum value at −0.1 s, the X Maximum value at 0.1 s, and the Bin value at 0.001 (Figure 5Avi, vii).
5. Click on Results > Numerical Results to save the results of the PSD, coherence, and correlation with an .xls filename extension (Figure 5Aviii, ix).
6. Select the channel for which the representative traces need to be extracted for each frequency band, click on Edit > Digital Filtering of Continuous Variables to obtain each frequency band, and then set the parameters as follows: the Filter Freq. Response as Bandpass, the Filter Implementation at infinite impulse response (IIR) Butterworth, and the Filter Order value at 2. Finally, set the frequency range of interest (Figure 5Bi-iv).
   NOTE: The frequency ranges used here were as follows: delta (δ, 1-4 Hz), theta (θ, 5-12 Hz), beta (β, 13-30 Hz), low gamma (low γ, 30-70 Hz), and high gamma (high γ, 70-100 Hz) oscillations.
7. Analyze the data and draw the graph.

6. Correlations between the spike and LFP

1. Click on File > Import Data > Blackrock File in the software for the neurophysiological data analysisto open the continuous signal data and spike data.
2. Click on Analysis > Coherence Analysis to analyze the coherence between the spikes and LFP from the selected channel. Set the reference variable (spike timing) and parameters as follows: Calculate at Coherence Values, the Number of Frequency Values at 512, the Multiple Tapers value at 3-5, and the frequency range from 1 Hz to 100 Hz (Figure 5Ci, ii).
3. Click on Results > Numerical Results to save the result of the spike field coherence with an .xls filename extension (Figure 5Ciii, iv).
4. Analyze the data and draw the graph.