This work was supported by grants from the National Natural Science Foundation of China (31871170, 32170950, and 31970915), the Natural Science Foundation of Guangdong Province (2021A1515010804 and 2023A1515010899), the Guangdong Natural Science Foundation for Major Cultivation Project (2018B030336001), and the Guangdong Grant: Key Technologies for Treatment of Brain Disorders(2018B030332001).

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 &#176;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
<ol>
	<li>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).</li>
	<li>Ensure the micro-drive can carry two sets of eight guide tubes (~3 cm long, ~50 &#181;m internal diameter, ~125 &#181;m external diameter) for each side of the MC region, and then cut it to the same length (at least 15 mm; Figure 1Biv, v).</li>
	<li>Cut 16 Ni-chrome wires (~5 cm long) with a 35 &#181;m diameter, and then load them successively into the guide tubes, followed by applying the glue to fix them (Figure 1Bi, vi, vii).</li>
	<li>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).</li>
	<li>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: &#8722;10.08 &#181;A direct current for 1 s with gold plating solution, including 5 mM PtCl4.</li>
	<li>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.</li>
</ol>
2. Electrode array implantation
<ol>
	<li>Sterilize the surgery kits, wear sterile gloves and put on a doctor&#39;s sterile white coat before the operation starts.</li>
	<li>To manage the pain, use subcutaneous (s.c.) injection of meloxicam&#160;injectable&#160;(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 chamber18,19. Apply a supplemental dose of pentobarbital (20 mg/kg/h) if the toe pinch reflex is still present.</li>
	<li>Fix the mouse in a stereotaxic apparatus and maintain its rectal temperature at 37 &#176;C by using a temperature controller.</li>
	<li>Apply tetracycline eye ointment to both eyes of the mouse and change sterile gloves&#160;again before surgery.</li>
	<li>Shave the mouse&#39;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&#160;(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&#160;(Figure 2iii).</li>
	<li>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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>Remove the dura mater from the holes carefully with fine forceps (Figure 2xiii).</li>
	<li>Insert the micro-drive system into the center of the holes using a micromanipulator at 10 &#181;m/s (Figure 2xiv-xvii).</li>
	<li>Fill the petroleum jelly into the dental cement walls after finishing the insertion of the micro-drive system (Figure 2xviii).</li>
	<li>Join the bottom plate of the micro-drive system and the dental cement walls with the mixed dental cement (Figure 2xix)</li>
	<li>Wash the incision with saline followed by local treatment with a gel containing lincomycin hydrochloride and lidocaine hydrochloride to relieve post-surgical pain.</li>
	<li>Wind the conductive copper foil tape around the implanted micro-drive system (Figure 2xx).</li>
	<li>Move the mouse into a cage kept at 31-33 &#176;C and monitor the mouse for recovery from the anesthesia.</li>
	<li>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.</li>
</ol>
3. Multichannel recording in the bilateral MC in free-moving mice
<ol>
	<li>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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Store the recorded data in the computer and analyze it offline (Figure 4 and Figure 5).</li>
	<li>After finishing the experiment, perform euthanasia as per the institute&#39;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&#39;s brain into 30 &#181;m slices using a freezing microtome, collect the MC sections, and then capture the images with a microscope (Figure 3C, D).</li>
</ol>
4. Spike sorting and analysis
<ol>
	<li>Click on File &#62; Open &#62; Nev files in the spike sorting software to open the spike data sampled at 30 kHz (Figure 4Ai).</li>
	<li>Click on Info to select the unsorted channel, and then select Sort &#62; Change Sort Method &#62; Use K-Means. Press the button Valley-Seeking Sort &#62; K-Means Sorting to obtain the sorted units (Figure 4Aii, iii).</li>
	<li>Click on File &#62; Save as, save the sorted spike data with a .nev filename extension, and select File &#62; Export Per-Waveform Data to export the PCA results with a .txt filename extension (Figure 4Aiv).</li>
	<li>Click on File &#62; Import Data &#62; Blackrock File in the software for the neurophysiological data analysis to open the sorted spike file (Figure 4Bi).</li>
	<li>Click on Analysis &#62; Autocorrelograms to obtain the autocorrelogram for the selected unit, and then set the parameters as follows: the X Minimum value at &#8722;0.05 s, the X Maximum value at 0.05 s, and the Bin value at 0.001 (Figure 4Bii, iii).</li>
	<li>Load the sorted spike data, click on Analysis &#62; 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).</li>
	<li>Click on Analysis &#62; 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 &#8722;0.1 s, the X Maximum value at 0.1 s, and the Bin value at 0.001 (Figure 4Bvi, vii).</li>
	<li>Click on Results &#62; 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.</li>
</ol>
5. LFP analysis
<ol>
	<li>Click on File Import Data &#62; Blackrock File in the software for the neurophysiological data analysis to open the continuous signal data sampled at 10 kHz (Figure 5Ai).</li>
	<li>Click on Analysis &#62; 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).</li>
	<li>Click on Analysis &#62; 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).</li>
	<li>Click on Analysis &#62; 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 &#8722;0.1 s, the X Maximum value at 0.1 s, and the Bin value at 0.001 (Figure 5Avi, vii).</li>
	<li>Click on Results &#62; Numerical Results to save the results of the PSD, coherence, and correlation with an .xls filename extension (Figure 5Aviii, ix).</li>
	<li>Select the channel for which the representative traces need to be extracted for each frequency band, click on Edit &#62; 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 (&#948;, 1-4 Hz), theta (&#952;, 5-12 Hz), beta (&#946;, 13-30 Hz), low gamma (low &#947;, 30-70 Hz), and high gamma (high &#947;, 70-100 Hz) oscillations.</li>
	<li>Analyze the data and draw the graph.</li>
</ol>
6. Correlations between the spike and LFP
<ol>
	<li>Click on File &#62; Import Data &#62; Blackrock File in the software for the neurophysiological data analysisto open the continuous signal data and spike data.</li>
	<li>Click on Analysis &#62; 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).</li>
	<li>Click on Results &#62; Numerical Results to save the result of the spike field coherence with an .xls filename extension (Figure 5Ciii, iv).</li>
	<li>Analyze the data and draw the graph.</li>
</ol>

Extracellular Electrophysiological Recording in the Motor Cortex of Mice

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The authors have nothing to disclose.

Multichannel recording in free-moving mice has been deemed to be a useful technology in neuroscience studies, but it is still quite challenging for beginners to record and analyze the signals. In the present study, we provide simplified guidelines for making micro-drive systems and performing electrode implantation, as well as simplified procedures for capturing and analyzing the electrical signals via spike sorting software and software for neurophysiological data analysis.
Given tha...

The local field potential (LFP), an important component of extracellular signals, reflects the synaptic activity of large populations of neurons, which form the neural code for multiple behaviors1. Spikes generated by neuronal activity are considered to contribute to the LFP and are important for neural coding2. Alterations in spikes and LFPs have been proven to mediate several brain diseases, such as Alzheimer&#39;s disease, as well as emotions such as fear, etc.3,4. It is worth noting that many studies have highlighted that spike activity significantly differs between awake and anesthetized states in animals5. Although recordings in anesthetized animals offer an opportunity to assess LFPs with minimal artifacts in highly defined cortical synchronization states, the results differ to some extent from what can be found in awake subjects6,7,8. Thus, it is more meaningful to detect neural activity over long time scales and large spatial scales in various diseases in an awake brain state using electrodes implanted in the brain. This manuscript provides information for beginners on how to make the micro-drive system and set the parameters using common software for computing the spike and LFP signals in a fast and straightforward way in order to get the recording and analysis started.
Although the non-invasive recording of brain functions, such as by using electroencephalograms (EEGs) and event-related potentials (ERPs) recorded from the scalp, has been used extensively in human and rodent studies, EEG and ERP data have low spatial and temporal properties and, thus, cannot detect the precise signals produced by nearby dendritic synaptic activity within a specific brain area1. Currently, by taking advantage of multichannel recording in conscious animals, neural activity in the deeper layers of the brain can be recorded chronically and progressively by implanting a micro-drive system into the brains of primates or rodents during multiple behavioral tests1,2,3,4,5,6,7,8,9. Briefly, researchers can construct a micro-drive system that can be used for the independent positioning of the electrodes or tetrodes to target different parts of the brain10,11. For example, Chang et al. described techniques to record spikes and LFPs in mice by assembling a light and compact micro-drive12. In addition, micro-machined silicon probes with custom-made accessory components are commercially available for recording multiple single neurons and LFPs in rodents during behavioral tasks13. Although various designs have been used for assembling micro-drive systems, these still have limited success in terms of the complexity and weight of the whole micro-drive system. For example, Lansink et al. showed a multichannel micro-drive system with a complex structure for recording from a single brain region14. Sato et al. reported a multichannel micro-drive system displaying an automatic hydraulic positioning function15. The main disadvantages of these micro-drive systems are that they are too heavy for mice to move freely and are difficult to assemble for beginners. Although multichannel extracellular recording has been shown to be a suitable and efficient technology for measuring neural activity during behavioral tests, it is not easy for beginners to record and analyze the signals acquired by the complex micro-drive system. Given that it is difficult to get the entire operation process of the multichannel extracellular recording and data analysis started in freely moving mice16,17, this present article presents simplified guidelines to introduce the detailed process of making the micro-drive system using commonly available components and settings; the parameters in the common software for computing the spike and LFP signals in a fast and straightforward way are also provided. Additionally, in this protocol, the mouse can move freely due to the use of a helium balloon, which contributes to offsetting the weight of headstage and micro-drive system. Generally, in the present study, we describe how to easily build a micro-drive system and optimize the processes of recording and data analysis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2.54 mm pin header</td>
			<td>YOUXIN Electronic Co., Ltd.</td>
			<td>1 x 5</td>
			<td>Applying for the movable micro-drive which can slide on its stulls.</td>
		</tr>
		<tr>
			<td>Adobe Illustrator CC 2017</td>
			<td>Adobe</td>
			<td>N/A</td>
			<td>To optimize images from GraphPad.</td>
		</tr>
		<tr>
			<td>BlackRock Microsystems</td>
			<td>Blackrock Neurotech</td>
			<td>Cerebus</td>
			<td>This systems includes headsatge, DA convert, amplifier and computer.</td>
		</tr>
		<tr>
			<td>Brass nut</td>
			<td>Dongguan Gaosi Technology Co., Ltd.</td>
			<td>M0.8 brass nut</td>
			<td>The nut fixes the position of screw.</td>
		</tr>
		<tr>
			<td>Brass screw</td>
			<td>Dongguan Gaosi Technology Co., Ltd.</td>
			<td>M0.8 x 11 mm brass screw</td>
			<td>A screw that hold the movable micro-drive.</td>
		</tr>
		<tr>
			<td>C57BL/6J</td>
			<td>Guangdong Zhiyuan Biomedical Technology Co., LTD.</td>
			<td>N/A</td>
			<td>12 weeks of age.</td>
		</tr>
		<tr>
			<td>Centrifuge tube</td>
			<td>Biosharp</td>
			<td>15 mL; BS-150-M</td>
			<td>To store mice brain with sucrose sulutions.</td>
		</tr>
		<tr>
			<td>Conducting paint</td>
			<td>Structure Probe, Inc.</td>
			<td>7440-22-4</td>
			<td>To improve the lead-connecting quality between connector pins and Ni-wires.</td>
		</tr>
		<tr>
			<td>Conductive copper foil tape</td>
			<td>3M</td>
			<td>1181</td>
			<td>To reduce interferenc.</td>
		</tr>
		<tr>
			<td>Connector</td>
			<td>YOUXIN Electronic Co., Ltd.</td>
			<td>2 x 10P</td>
			<td>To connect the headtage to micro-drive system.</td>
		</tr>
		<tr>
			<td>DC Power supply</td>
			<td>Maisheng</td>
			<td>MS-305D</td>
			<td>A power device for&#160; electrolytic lesion.</td>
		</tr>
		<tr>
			<td>Dental cement</td>
			<td>Shanghai New Century Dental Materials Co., Ltd.</td>
			<td>N/A</td>
			<td>To fix the electrode arrays on mouse&#39;s skull after finishing the implantation.</td>
		</tr>
		<tr>
			<td>Digital analog converter</td>
			<td>Blackrock</td>
			<td>128-Channel</td>
			<td>A device that converts digital data into analog signals.</td>
		</tr>
		<tr>
			<td>Epoxy resin</td>
			<td>Alteco</td>
			<td>N/A</td>
			<td>To cover pins.</td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td>N/A</td>
			<td>To summarize data after analysis.</td>
		</tr>
		<tr>
			<td>Eye scissors</td>
			<td>JiangXi YuYuan Medical Equipment Co.,Ltd.</td>
			<td>N/A</td>
			<td>For surgery or cutting the Ni-chrome wire.</td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>JiangXi YuYuan Medical Equipment Co.,Ltd.</td>
			<td>N/A</td>
			<td>For surgery.</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>JiangXi YuYuan Medical Equipment Co.,Ltd.</td>
			<td>N/A</td>
			<td>For surgery or assembling the mirco-drive system.</td>
		</tr>
		<tr>
			<td>Freezing microtome</td>
			<td>Leica</td>
			<td>CM3050 S</td>
			<td>&#160;Cut the mouse&#8217;s brain into slices</td>
		</tr>
		<tr>
			<td>Fused silica capillary tubing</td>
			<td>Zhengzhou INNOSEP Scientific Co., Ltd.</td>
			<td>TSP050125</td>
			<td>To&#160; serve as the guide tubes for Ni-chrome wires.</td>
		</tr>
		<tr>
			<td>Glass microelectrode</td>
			<td>Sutter Instrument Company</td>
			<td>BF100-50-10</td>
			<td>To mark the desired locations for implantation using the filled ink.</td>
		</tr>
		<tr>
			<td>GraphPad Prism 7</td>
			<td>GraphPad Software</td>
			<td>N/A</td>
			<td>To analyze and visualize the results.</td>
		</tr>
		<tr>
			<td>Guide-tube</td>
			<td>Polymicro technologies</td>
			<td>1068150020</td>
			<td>To load Ni-chrome wires.</td>
		</tr>
		<tr>
			<td>Headstage</td>
			<td>Blackrock</td>
			<td>N/A</td>
			<td>A tool of transmitting signals.</td>
		</tr>
		<tr>
			<td>Helium balloon</td>
			<td>Yili Festive products Co., Ltd.</td>
			<td>24 inch</td>
			<td>To offset the weight of headstage and micro-drive system.</td>
		</tr>
		<tr>
			<td>Ink</td>
			<td>Sailor Pen Co.,LTD.</td>
			<td>13-2001</td>
			<td>To mark the desired locations for implantation.</td>
		</tr>
		<tr>
			<td>Iodine tincture</td>
			<td>Guangdong Hengjian Pharmaceutical Co., Ltd.</td>
			<td>N/A</td>
			<td>To disinfect mouse&#39;s scalp.</td>
		</tr>
		<tr>
			<td>Lincomycin in Hydrochloride and Lidocaine&#160; hydrochloride gel</td>
			<td>Hubei kangzheng pharmaceutical co., ltd.</td>
			<td>10g</td>
			<td>A drug used to reduce inflammation.</td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td>Vicki Biotechnology Co., Ltd.</td>
			<td>71125-38-7</td>
			<td>To reduce postoperative pain in mice.</td>
		</tr>
		<tr>
			<td>Micromanipulators</td>
			<td>Scientifica</td>
			<td>Scientifica IVM Triple</td>
			<td>For electrode arrays implantation.</td>
		</tr>
		<tr>
			<td>Microscope&#160;</td>
			<td>Nikon</td>
			<td>ECLIPSE Ni-E</td>
			<td>&#160;Capture the images of brain sections</td>
		</tr>
		<tr>
			<td>nanoZ impedance tester</td>
			<td>Plexon</td>
			<td>nanoZ</td>
			<td>To measure impedance or performing electrode impedance spectroscopy (EIS) for multichannel microelectrode arrays.</td>
		</tr>
		<tr>
			<td>NeuroExplorer</td>
			<td>Plexon</td>
			<td>NeuroExplorer</td>
			<td>A tool for analyzing the electrophysiological data.</td>
		</tr>
		<tr>
			<td>NeuroExplorer&#160;</td>
			<td>Plexon, USA</td>
			<td>N/A</td>
			<td>A software.</td>
		</tr>
		<tr>
			<td>Ni-chrome wire</td>
			<td>California Fine Wire Co.</td>
			<td>M472490</td>
			<td>35 &#956;m Ni-chrome wire.</td>
		</tr>
		<tr>
			<td>Offline Sorter</td>
			<td>Plexon</td>
			<td>Offline Sorter</td>
			<td>A tool for sorting the recorded multi-units.</td>
		</tr>
		<tr>
			<td>PCB board</td>
			<td>Hangzhou Jiepei Information Technology Co., Ltd.</td>
			<td>N/A</td>
			<td>Computer designed board.</td>
		</tr>
		<tr>
			<td>Pentobarbital</td>
			<td>Sigma</td>
			<td>P3761</td>
			<td>To anesthetize mice.</td>
		</tr>
		<tr>
			<td>Pentobarbital sodium</td>
			<td>Sigma</td>
			<td>57-33-0</td>
			<td>To anesthetize the mouse.</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Longer</td>
			<td>BT100-1F</td>
			<td>A device used for perfusion</td>
		</tr>
		<tr>
			<td>Polyformaldehyde&#160;</td>
			<td>Sangon Biotech</td>
			<td>A500684-0500</td>
			<td>The main component of fixative solution for fixation of mouse brains&#160;</td>
		</tr>
		<tr>
			<td>PtCl4</td>
			<td>Tianjin Jinbolan Fine Chemical Co., Ltd.</td>
			<td>13454-96-1</td>
			<td>Preparation for gold plating liquid.</td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>Guangdong Hengjian Pharmaceutical Co., Ltd.</td>
			<td>N/A</td>
			<td>To clean the mouse&#39;s skull.</td>
		</tr>
		<tr>
			<td>Silver wire</td>
			<td>Suzhou Xinye Electronics Co., Ltd.</td>
			<td>2 mm diameter</td>
			<td>Applying for ground and reference electrodes.</td>
		</tr>
		<tr>
			<td>Skull drill</td>
			<td>RWD Life Science</td>
			<td>78001</td>
			<td>To drill carefully two small holes on mouse&#39;s skull.</td>
		</tr>
		<tr>
			<td>Stainless steel screws</td>
			<td>YOUXIN Electronic Co., Ltd.</td>
			<td>M0.8 x 2</td>
			<td>To protect the micro-drive system and link the ground and reference electrodes.</td>
		</tr>
		<tr>
			<td>Stereotaxic apparatus</td>
			<td>RWD Life Science</td>
			<td>68513</td>
			<td>To perform the stereotaxic coordinates of bilateral motor cortex.</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Damao</td>
			<td>57-50-1</td>
			<td>To dehydrate the mouse brains&#160; after perfusion.</td>
		</tr>
		<tr>
			<td>Super glue</td>
			<td>Henkel AG &#38; Co.</td>
			<td>PSK5C</td>
			<td>To fix the guide tube and Ni-chrome wire.</td>
		</tr>
		<tr>
			<td>Temperature controller</td>
			<td>Harvard Apparatus</td>
			<td>TCAT-2</td>
			<td>To maintain mouse&#39;s rectal temperature at 37&#176;C</td>
		</tr>
		<tr>
			<td>Tetracycline eye ointment</td>
			<td>Guangdong Hengjian Pharmaceutical Co., Ltd.</td>
			<td>N/A</td>
			<td>To protect the mouse&#39;s eyes during surgery.</td>
		</tr>
		<tr>
			<td>Thread</td>
			<td>Rapala</td>
			<td>N/A</td>
			<td>To link ballon and headstage.</td>
		</tr>
		<tr>
			<td>Vaseline</td>
			<td>Unilever plc</td>
			<td>N/A</td>
			<td>To cover the gap between electrode arrays and mouse&#39;s skull.</td>
		</tr>
	</tbody>
</table>

multichannel extracellular recording in freely moving mice

The protocol aims to uncover the properties of neuronal firing and network local field potentials (LFPs) in behaving mice carrying out specific tasks by correlating the electrophysiological signals with spontaneous and/or specific behavior. This technique represents a valuable tool in studying the neuronal network activity underlying these behaviors. The article provides a detailed and complete procedure for electrode implantation and consequent extracellular recording in free-moving conscious mice. The study includes a detailed method for implanting the microelectrode arrays, capturing the LFP and neuronal spiking signals in the motor cortex (MC) using a multichannel system, and the subsequent offline data analysis. The advantage of multichannel recording in conscious animals is that a greater number of spiking neurons and neuronal subtypes can be obtained and compared, which allows the evaluation of the relationship between a specific behavior and the associated electrophysiological signals. Notably, the multichannel extracellular recording technique and the data analysis procedure described in the present study can be applied to other brain areas when conducting experiments in behaving mice.

A high-pass (250 Hz) filter was applied to extract the multi-unit spikes from the raw signals (Figure 6A). Further, the recorded units from the MC of a normal mouse sorted by PCA were verified (Figure 7A-D), and the valley width and waveform duration of the units in the MC of the mouse were recorded. The results showed that both the valley width and waveform duration of the MC putative pyramidal neurons (Pyn) in mice are higher ...

Watch this Scientific Journal Video about Advancements in Multichannel Extracellular Recording for Studying Neuronal Activity in Freely Moving Mice at JoVE.com

Author Spotlight: Advancements in Multichannel Extracellular Recording for Studying Neuronal Activity in Freely Moving Mice 

The protocol describes the methodology of extracellular recording in the motor cortex (MC) to reveal extracellular electrophysiological properties in freely moving conscious mice, as well as the data analysis of local field potentials (LFPs) and spikes, which is useful for evaluating the network neural activity underlying behaviors of interest.

Multichannel Extracellular Recording in Freely Moving Mice

The protocol aims to uncover the properties of neuronal firing and network local field potentials (LFPs) in behaving mice carrying out specific tasks by ...

multichannel-extracellular-recording-in-freely-moving-mice

Research

JoVE Journal

Neuroscience

2.5K Views.  South China Normal University. The protocol describes the methodology of extracellular recording in the motor cortex (MC) to reveal extracellular electrophysiological properties in freely moving conscious mice, as well as the data analysis of local field potentials (LFPs) and spikes, which is useful for evaluating the network neural activity underlying behaviors of interest. 

Video: Author Spotlight: Advancements in Multichannel Extracellular Recording for Studying Neuronal Activity in Freely Moving Mice 

High-density EEG Recordings of the Freely Moving Mice using Polyimide-based Microelectrode

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a noninvasive brain monitoring tool in cognitive neuroscience as well as a diagnostic tool for epilepsy and sleep disorders in neurology. However, the underlying mechanism of EEG rhythm generation is still under the veil. Recently introduced polyimide-based microelectrode (PBM-array) for high resolution mouse EEG1 is one of the trials to answer the neurophysiological questions on EEG signals based on a rich genetic resource that the mouse model contains for the analysis of complex EEG generation process. This application of nanofabricated PBM-array to mouse skull is an efficient tool for collecting large-scale brain activity of transgenic mice and accommodates to identify the neural correlates to certain EEG rhythms in conjunction with behavior. However its ultra-thin thickness and bifurcated structure cause a trouble in handling and implantation of PBM-array. In the presented video, the preparation and surgery steps for the implantation of PBM-array on a mouse skull are described step by step. Handling and surgery tips to help researchers succeed in implantation are also provided.

In this article, we described the surgery procedure and handling tips for implantation of ultra-thin polyimide-based microelectrode array (PBM-array) on the mouse skull for acquisition of high-density encephalography (EEG) in a mouse model.

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Recording Single Neurons' Action Potentials from Freely Moving Pigeons Across Three Stages of Learning

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed single-neuron activity while animals are acquiring an operantly conditioned response, or when that response is extinguished. But even in these cases, observation periods usually encompass only a single stage of learning, i.e. acquisition or extinction, but not both (exceptions include protocols employing reversal learning; see Bingman&#160;et al.1 for an example). However, acquisition and extinction entail different learning mechanisms and are therefore expected to be accompanied by different types and/or loci of neural plasticity.
Accordingly, we developed a behavioral paradigm which institutes three stages of learning in a single behavioral session and which is well suited for the simultaneous recording of single neurons&#39; action potentials. Animals are trained on a single-interval forced choice task which requires mapping each of two possible choice responses to the presentation of different novel visual stimuli (acquisition). After having reached a predefined performance criterion, one of the two choice responses is no longer reinforced (extinction). Following a certain decrement in performance level, correct responses are reinforced again (reacquisition). By using a new set of stimuli in every session, animals can undergo the acquisition-extinction-reacquisition process repeatedly. Because all three stages of learning occur in a single behavioral session, the paradigm is ideal for the simultaneous observation of the spiking output of multiple single neurons. We use pigeons as model systems, but the task can easily be adapted to any other species capable of conditioned discrimination learning.

Learning new stimulus-response associations engages a wide range of neural processes which are ultimately reflected in changing spike output of individual neurons. Here we describe a behavioral protocol allowing for the continuous registration of single-neuron activity while animals acquire, extinguish, and reacquire a conditioned response within a single experimental session.

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed ...

Extracellular Wire Tetrode Recording in Brain of Freely Walking Insects

Increasing interest in the role of brain activity in insect motor control requires that we be able to monitor neural activity while insects perform natural behavior. We previously developed a technique for implanting tetrode wires into the central complex of cockroach brains that allowed us to record activity from multiple neurons simultaneously while a tethered cockroach turned or altered walking speed. While a major advance, tethered preparations provide access to limited behaviors and often lack feedback processes that occur in freely moving animals. We now present a modified version of that technique that allows us to record from the central complex of freely moving cockroaches as they walk in an arena and deal with barriers by turning, climbing or tunneling. Coupled with high speed video and cluster cutting, we can now relate brain activity to various parameters of the movement of freely behaving insects.

We previously developed a technique for implanting tetrode wires into the central complex of cockroach brains that allows us to monitor activity in individual units of tethered cockroaches. Here we present a modified version of that technique that allows us to also record brain activity in freely moving insects.

Increasing interest in the role of brain activity in insect motor control requires that we be able to monitor neural activity while insects perform ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Noninvasive EEG Recordings from Freely Moving Piglets

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel telemetric electroencephalography system in combination with standard self-adhesive hydrogel electrodes. The piglets are calmed down without the use of sedatives. After their release into the pigpen, the piglets behave normally&#8212;they drink and sleep in the same cycle as their siblings. Their sleep phases are used for the EEG recordings.

Here, we present a protocol to record telemetric electroencephalograms (EEGs) from freely moving piglets directly in the pigpen without the use of a sedative, making it possible to record typical EEG patterns during non-REM sleep, like spindle bursts.

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel ...

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.

Here, we describe a method for in vivo microdialysis to analyze aspartate and glutamate release in the ventral hippocampus of epileptic and non-epileptic rats, in combination with EEG recordings. Extracellular concentrations of aspartate and glutamate may be correlated with the different phases of the disease.

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain ...

Long-term Sensory Conflict in Freely Behaving Mice

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning in mice. By permanently wearing a device fixed on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages. Therefore, this protocol readily enables the study of the visual system and multisensory interactions over an extended timeframe that would not be accessible otherwise. In addition to lowering the experimental costs of long-term sensory learning in naturally behaving mice, this approach accommodates the combination of in vivo and in vitro experiments. In the reported example, video-oculography is performed to quantify the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) before and after learning. Mice exposed to this long-term sensory conflict between visual and vestibular inputs presented a strong VOR gain decrease but exhibited few OKR changes. Detailed steps of device assembly, animal care, and reflex measurements are hereby reported.

The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages.

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for ...

Hybrid Microdrive System with Recoverable Opto-Silicon Probe and Tetrode for Dual-Site High Density Recording in Freely Moving Mice

Multi-regional neural recordings can provide crucial information to understanding fine-timescale interactions between multiple brain regions. However, conventional microdrive designs often only allow use of one type of electrode to record from single or multiple regions, limiting the yield of single-unit or depth profile recordings. It also often limits the ability to combine electrode recordings with optogenetic tools to target pathway and/or cell type specific activity. Presented here is a hybrid microdrive array for freely moving mice to optimize yield and a description of its fabrication and reuse of the microdrive array. The current design employs nine tetrodes and one opto-silicon probe implanted in two different brain areas simultaneously in freely moving mice. The tetrodes and the opto-silicon probe are independently adjustable along the dorsoventral axis in the brain to maximize the yield of unit and oscillatory activities. This microdrive array also incorporates a set-up for light, mediating optogenetic manipulation to investigate the regional- or cell type-specific responses and functions of long-range neural circuits. In addition, the opto-silicon probe can be safely recovered and reused after each experiment. Because the microdrive array consists of 3D-printed parts, the design of microdrives can be easily modified to accommodate various settings. First described is the design of the microdrive array and how to attach the optical fiber to a silicon probe for optogenetics experiments, followed by fabrication of the tetrode bundle and implantation of the array into a mouse brain. The recording of local field potentials and unit spiking combined with optogenetic stimulation also demonstrate feasibility of the microdrive array system in freely moving mice.

This protocol describes the construction of a hybrid microdrive array that allows implantation of nine independently adjustable tetrodes and one adjustable opto-silicon probe in two brain regions in freely moving mice. Also demonstrated is a method for safely recovering and reusing the opto-silicon probe for multiple purposes.

Multi-regional neural recordings can provide crucial information to understanding fine-timescale interactions between multiple brain regions. However, ...