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

<ol>
	<li>Buzs&#225;ki, G., Anastassiou, C. A., Koch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+extracellular+fields+and+currents--EEG,+ECoG,+LFP+and+spikes.">The origin of extracellular fields and currents--EEG, ECoG, LFP and spikes.</a> Nature Reviews Neuroscience. 13 (6), 407-420 (2012).</li><li>Singer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronization+of+cortical+activity+and+its+putative+role+in+information+processing+and+learning.">Synchronization of cortical activity and its putative role in information processing and learning.</a> Annual Review of Physiology. 55, 349-374 (1993).</li><li>Arroyo-Garc&#237;a, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+spike-gamma+coupling+of+area+CA3+fast-spiking+interneurons+as+the+earliest+functional+impairment+in+the+App(NL-G-F)+mouse+model+of+Alzheimer's+disease.">Impaired spike-gamma coupling of area CA3 fast-spiking interneurons as the earliest functional impairment in the App(NL-G-F) mouse model of Alzheimer's disease.</a> Molecular Psychiatry. 26 (10), 5557-5567 (2021).</li><li>Ozawa, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience-dependent+resonance+in+amygdalo-cortical+circuits+supports+fear+memory+retrieval+following+extinction.">Experience-dependent resonance in amygdalo-cortical circuits supports fear memory retrieval following extinction.</a> Nature Communications. 11 (1), 4358 (2020).</li><li>Vinck, M., Batista-Brito, R., Knoblich, U., Cardin, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arousal+and+locomotion+make+distinct+contributions+to+cortical+activity+patterns+and+visual+encoding.">Arousal and locomotion make distinct contributions to cortical activity patterns and visual encoding.</a> Neuron. 86 (3), 740-754 (2015).</li><li>Beck, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=long-term+dopamine+depletion+causes+enhanced+beta+oscillations+in+the+cortico-basal+ganglia+loop+of+parkinsonian+rats.">long-term dopamine depletion causes enhanced beta oscillations in the cortico-basal ganglia loop of parkinsonian rats.</a> Experimental Neurology. 286, 124-136 (2016).</li><li>Magill, P. J., Bolam, J. P., Bevan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+of+activity+in+the+subthalamic+nucleus-globus+pallidus+network+to+cortical+electroencephalogram.">Relationship of activity in the subthalamic nucleus-globus pallidus network to cortical electroencephalogram.</a> Journal of Neuroscience. 20 (2), 820-833 (2000).</li><li>Magill, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+functional+connectivity+within+the+rat+striatopallidal+axis+during+global+brain+activation+in+vivo.">Changes in functional connectivity within the rat striatopallidal axis during global brain activation in vivo.</a> Journal of Neuroscience. 26 (23), 6318-6329 (2006).</li><li>Rapeaux, A. B., Constandinou, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implantable+brain+machine+interfaces:+First-in-human+studies,+technology+challenges+and+trends.">Implantable brain machine interfaces: First-in-human studies, technology challenges and trends.</a> Current Opinion in Biotechnology. 72, 102-111 (2021).</li><li>Tort, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+cross-frequency+couplings+of+local+field+potential+oscillations+in+rat+striatum+and+hippocampus+during+performance+of+a+T-maze+task.">Dynamic cross-frequency couplings of local field potential oscillations in rat striatum and hippocampus during performance of a T-maze task.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (51), 20517-20522 (2008).</li><li>Yamamoto, J., Wilson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+chronically+implantable+precision+motorized+microdrive+array+for+freely+behaving+animals.">Large-scale chronically implantable precision motorized microdrive array for freely behaving animals.</a> Journal of Neurophysiology. 100 (4), 2430-2440 (2008).</li><li>Chang, E. H., Frattini, S. A., Robbiati, S., Huerta, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+microdrive+arrays+for+chronic+neural+recordings+in+awake+behaving+mice.">Construction of microdrive arrays for chronic neural recordings in awake behaving mice.</a> Journal of Visualized Experiments. (77), e50470 (2013).</li><li>Vandecasteele, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+recording+of+neurons+by+movable+silicon+probes+in+behaving+rodents.">Large-scale recording of neurons by movable silicon probes in behaving rodents.</a> Journal of Visualized Experiments. (61), e3568 (2012).</li><li>Lansink, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+split+microdrive+for+simultaneous+multi-electrode+recordings+from+two+brain+areas+in+awake+small+animals.">A split microdrive for simultaneous multi-electrode recordings from two brain areas in awake small animals.</a> Journal of Neuroscience Methods. 162 (1-2), 129-138 (2007).</li><li>Sato, T., Suzuki, T., Mabuchi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+multi-electrode+array+design+for+chronic+neural+recording,+with+independent+and+automatic+hydraulic+positioning.">A new multi-electrode array design for chronic neural recording, with independent and automatic hydraulic positioning.</a> Journal of Neuroscience Methods. 160 (1), 45-51 (2007).</li><li>van Daal, R. J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implantation+of+Neuropixels+probes+for+chronic+recording+of+neuronal+activity+in+freely+behaving+mice+and+rats.">Implantation of Neuropixels probes for chronic recording of neuronal activity in freely behaving mice and rats.</a> Nature Protocols. 16 (7), 3322-3347 (2021).</li><li>Unakafova, V. A., Gail, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+open-source+toolboxes+for+processing+and+analysis+of+spike+and+local+field+potentials+data.">Comparing open-source toolboxes for processing and analysis of spike and local field potentials data.</a> Frontiers in Neuroinformatics. 13, 57 (2019).</li><li>Mao, L., Wang, H., Qiao, L., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+Nrf2+enhances+the+upregulation+of+nuclear+factor-kappaB+activity,+tumor+necrosis+factor-alpha,+and+matrix+metalloproteinase-9+after+spinal+cord+injury+in+mice.">Disruption of Nrf2 enhances the upregulation of nuclear factor-kappaB activity, tumor necrosis factor-alpha, and matrix metalloproteinase-9 after spinal cord injury in mice.</a> Mediators of Inflammation. 2010, 238321 (2010).</li><li>Jin, Z., Zhang, Z., Ke, J., Wang, Y., Wu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise-linked+irisin+prevents+mortality+and+enhances+cognition+in+a+mice+model+of+cerebral+ischemia+by+regulating+Klotho+expression.">Exercise-linked irisin prevents mortality and enhances cognition in a mice model of cerebral ischemia by regulating Klotho expression.</a> Oxidative Medicine and Cellular Longevity. 2021, 1697070 (2021).</li><li>Ding, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spreading+of+TDP-43+pathology+via+pyramidal+tract+induces+ALS-like+phenotypes+in+TDP-43+transgenic+mice.">Spreading of TDP-43 pathology via pyramidal tract induces ALS-like phenotypes in TDP-43 transgenic mice.</a> Acta Neuropathologica Communications. 9 (1), 15 (2021).</li><li>Cao, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gamma+oscillation+dysfunction+in+mPFC+leads+to+social+deficits+in+neuroligin+3+R451C+knockin+mice.">Gamma oscillation dysfunction in mPFC leads to social deficits in neuroligin 3 R451C knockin mice.</a> Neuron. 97 (6), 1253-1260 (2018).</li></ol>

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.

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

Multichannel Extracellular Recording in Freely Moving Mice

We aim to uncover the properties of neuronal firing and network local field potentials in behaving mice carrying out specific tasks by correlating the electrophysiological signal with behaviors. Multichannel extracellular recording by the micro-drive system has been shown to be a suitable and a efficient technology for neural activity during behavioral tests. Multichannel recording in free-moving mice has been deemed to be a useful technology in neuroscience studies, but it's still quite challenging for beginners to acquire and analyze the signals.We introduced how to perform the multichannel extracellular recording in freely-moving mice with a more steady and lightweight micro-drive system, and optimize the processes of recording and data analysis for the beginners. We are going to increase the number of channels and reduce the volume of micro-driver system in a new version.

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

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.

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

1.9K Views.  South China Normal University. We aim to uncover the properties of neuronal firing and network local field potentials in behaving mice carrying out specific tasks by correlating the electrophysiological signal with behaviors. Multichannel extracellular recording by the micro-drive system has been shown to be a suitable and a efficient technology for neural activity during behavioral tests. Multichannel recording in free-moving mice has been deemed to be a useful technology in neuroscience studies, but it's still quite chal...

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

Micro-Drive System Assembly and Electrode Array Implantation for Multichannel Recording in Mice

Begin by assembling the Microdrive system. Connect two computer-designed boards using two stulls and a screw that holds the movable Microdrive, and attach the connector to one board. Ensure the Microdrive can carry two sets of eight guide tubes for each side of the motor cortex or MC region.Cut the guide tubes to the same length. Cut 16 nichrome wires, each measuring approximately five centimeters long and 35 micrometers in diameter. Load the wires into the guide tubes and apply glue to fix them.Strip the wire insulation and twine each exposed wire to each pin from the connector following the channel map and the reference and ground electrodes, then slowly coat conducting paint onto each pin. Cover the pins using epoxy resin, then perform gold plating via an impedance tester to reduce the impedance of the electrode tips to approximately 350 kiloohms. For electrode array implantation, fix the anesthetized mouse in a stereotaxic apparatus and use a temperature controller to maintain its rectal temperature at 37 degrees Celsius.Then make a small midline incision to expose the skull. Remove the residual tissue using scissors and clean the skull with sterile saline-soaked cotton buds. Using a glass microelectrode filled with ink, mark the desired locations of the bilateral MC for implantation.Next, using a skull drill, carefully drill two small holes on both the left and right sides of the coordinated skull in the MC regions. Gently remove the dura mater from the holes with fine forceps. Then insert the Microdrive system into the center of the holes, using a micromanipulator at 10 micrometers per second.After finishing the insertion, fill the petroleum jelly into the dental cement walls and join the bottom plate of the Microdrive system and the dental cement walls with the mixed dental cement.

Multichannel Recording in the Bilateral MC in Free-Moving Mice and Neurophysiological Data Analysis

For the multi-channel recording, move down the electrode arrays by twisting the screw on the movable part of the micro-drive system one day in advance. Hold the head of an awake mouse lightly and carefully. Then, link the center of the head stage and a helium balloon with a thread to offset the weight of the head stage and the micro-drive system.Capture raw signals using the recording electrodes and multi-channel systems by sampling at 30 kilohertz in the recording software. Then digitize using a digital analog converter from the multi-channel systems. Extract the local field potential, or LFP signals, from the raw data by re-sampling at 10 kilohertz.Then, using a notch filter, remove the 50-hertz line noise. For spike sorting and analysis, in the spike sorting software, click on File, then Open, and nev files to open the spike data sampled at 30 kilohertz. Click on Info to select the unsorted channel.Then choose Sort, Change Sort Method, and Use K-Means. Press the button Valley Seeking Sort, then K-Means Sorting to obtain the sorted units. Next, in the neurophysiological data analysis software, open the sorted spike file by clicking on File, Import Data, and BlackRock File.To obtain the autocorrelogram for the selected unit, click on Analysis, then Autocorrelograms, and set the parameters. Load the sorted spike data. Then, click on Analysis, and Interspike Interval Histograms to obtain the interspike interval histogram, and then set the desired parameters.Click on Analysis, then Crosscorrelograms to obtain the crosscorrelogram between two sorted unit events. And then set the reference events and parameters. For LFP analysis, click on File, Import Data, and BlackRock File, to open the continuous signal data sampled at 10 kilohertz.Then, by clicking Analysis and Spectrum for Continuous, analyze the power spectrum for the LFP from the selected channel. Next, click on Analysis, then Coherence for Continuous to analyze the coherence for two LFPs from the left and right sides of the MC.Then, click on Analysis, followed by Correlation with Continuous Variables to analyze the correlation between two LFPs from the left and right sides of the MC.Once done, click on Results, then Numerical Results to save the Power Spectrum Density, Coherence, and Correlation with a xls file name extension. To analyze correlations between the spike and LFP, click on File, then Import Data and BlackRock File to open the continuous signal data and spike data.Then, click on Analysis and Coherence Analysis to analyze the coherence between the spikes and LFP from the selected channel. Finally, click on Results, then Numerical Results to save the results of the spike field coherence with a xls file name extension. The valley width and waveform duration of the units in the MC of the mouse showed that both the valley width and waveform duration of the MC putative pyramidal neurons in mice are higher than those of the putative interneurons.The crosscorrelogram between putative pyramidal neurons and interneurons indicated that the putative pyramidal neurons spiking occurs before the putative interneurons with a window of 18 milliseconds. In the LFP analysis, the LFPs of the left and right MC in normal mice were similar in the power spectrum, suggesting synchronized activities between the left and right MC.Further, the coherence and correlation between the left and right MC were calculated. The curve of spike field coherence in the MC of a normal mouse showed a stronger low gamma coherence for the putative interneurons compared to the pyramidal neurons.