This work was supported by the National Institutes of Health (NIH) grant R01 EY030998 (J.F.M., A.B., and S.C.). This method is based on methods developed in Coop et al. (under review, 2022; https://www.biorxiv.org/content/10.1101/2022.10.11.511827v2.abstract). We would like to thank Dina Graf and members of the Mitchell lab for help with the marmoset care and handling.

Development of an X-Y Electrode Stage for Precise Neural Recording in Marmosets

<ol>
	<li>Mansfield, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marmoset+models+commonly+used+in+biomedical+research.">Marmoset models commonly used in biomedical research.</a> Comparative Medicine. 53 (4), 383-392 (2003).</li><li>Solomon, S. G., Rosa, M. G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simpler+primate+brain:+the+visual+system+of+the+marmoset+monkey.">A simpler primate brain: the visual system of the marmoset monkey.</a> Frontiers in Neural Circuits. 8, 96 (2014).</li><li>Remington, E. D., Osmanski, M. S., 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=An+operant+conditioning+method+for+studying+auditory+behaviors+in+marmoset+monkeys.">An operant conditioning method for studying auditory behaviors in marmoset monkeys.</a> PLoS One. 7 (10), e47895 (2012).</li><li>Walker, J. D., 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=Chronic+wireless+neural+population+recordings+with+common+marmosets.">Chronic wireless neural population recordings with common marmosets.</a> Cell Reports. 36 (2), 109379 (2021).</li><li>Jendritza, P., Klein, F. J., Fries, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-area+recordings+and+optogenetics+in+the+awake,+behaving+marmoset.">Multi-area recordings and optogenetics in the awake, behaving marmoset.</a> Nature Communications. 14 (1), 577 (2023).</li><li>Pinotsis, D. A., 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=Linking+canonical+microcircuits+and+neuronal+activity:+Dynamic+causal+modelling+of+laminar+recordings.">Linking canonical microcircuits and neuronal activity: Dynamic causal modelling of laminar recordings.</a> Neuroimage. 146, 355-366 (2017).</li><li>Ludwig, K. A., 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=Poly+(3,+4-ethylenedioxythiophene)(PEDOT)+polymer+coatings+facilitate+smaller+neural+recording+electrodes.">Poly (3, 4-ethylenedioxythiophene)(PEDOT) polymer coatings facilitate smaller neural recording electrodes.</a> Journal of Neural Engineering. 8 (1), 014001 (2011).</li><li>Ludwig, K. A., Uram, J. D., Yang, J., Martin, D. C., Kipke, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+neural+recordings+using+silicon+microelectrode+arrays+electrochemically+deposited+with+a+poly+(3,+4-ethylenedioxythiophene)(PEDOT)+film.">Chronic neural recordings using silicon microelectrode arrays electrochemically deposited with a poly (3, 4-ethylenedioxythiophene)(PEDOT) film.</a> Journal of Neural Engineering. 3 (1), 59 (2006).</li><li>Lu, T., Liang, 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=Neural+representations+of+temporally+asymmetric+stimuli+in+the+auditory+cortex+of+awake+primates.">Neural representations of temporally asymmetric stimuli in the auditory cortex of awake primates.</a> Journal of Neurophysiology. 85 (6), 2364-2380 (2001).</li><li>Osmanski, M. S., Song, X., 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=The+role+of+harmonic+resolvability+in+pitch+perception+in+a+vocal+nonhuman+primate,+the+common+marmoset+(Callithrix+jacchus).">The role of harmonic resolvability in pitch perception in a vocal nonhuman primate, the common marmoset (Callithrix jacchus).</a> Journal of Neuroscience. 33 (21), 9161-9168 (2013).</li><li>Nummela, S. U., 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=Psychophysical+measurement+of+marmoset+acuity+and+myopia.">Psychophysical measurement of marmoset acuity and myopia.</a> Developmental Neurobiology. 77 (3), 300-313 (2017).</li><li>Paxinos, G., Watson, C., Petrides, M., Rosa, M., Tokuno, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Marmoset Brain in Stereotaxic Coordinates. , (2012).</li><li>Mitchell, J. F., Reynolds, J. H., Miller, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+vision+in+marmosets:+A+model+system+for+visual+neuroscience.">Active vision in marmosets: A model system for visual neuroscience.</a> Journal of Neuroscience. 34 (4), 1183-1194 (2014).</li><li>Spitler, K. M., Gothard, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+removable+silicone+elastomer+seal+reduces+granulation+tissue+growth+and+maintains+the+sterility+of+recording+chambers+for+primate+neurophysiology.">A removable silicone elastomer seal reduces granulation tissue growth and maintains the sterility of recording chambers for primate neurophysiology.</a> Journal of Neuroscience Methods. 169 (1), 23-26 (2008).</li><li>Jun, 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=Fully+integrated+silicon+probes+for+high-density+recording+of+neural+activity.">Fully integrated silicon probes for high-density recording of neural activity.</a> Nature. 551 (7679), 232-236 (2017).</li><li>Mitzdorf, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+source-density+method+and+application+in+cat+cerebral+cortex:+investigation+of+evoked+potentials+and+EEG+phenomena.">Current source-density method and application in cat cerebral cortex: investigation of evoked potentials and EEG phenomena.</a> Physiological Reviews. 65 (1), 37-100 (1985).</li><li>Coop, S. H., Yates, J. L., Mitchell, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-saccadic+neural+enhancements+in+marmoset+area+MT.">Pre-saccadic neural enhancements in marmoset area MT.</a> bioRxiv. , (2022).</li><li>Okun, M., Lak, A., Carandini, M., Harris, K. D. <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+recordings+with+immobile+silicon+probes+in+the+mouse+cortex.">Long term recordings with immobile silicon probes in the mouse cortex.</a> PloS One. 11 (3), e0151180 (2016).</li></ol>

The authors have nothing to disclose.

Several methods (e.g., chronic, semi-chronic, acute) are currently available for performing neurophysiology experiments in non-human primates. The common marmoset poses unique challenges for neurophysiology experiments due to its small size and lack of gyri as anatomical landmarks. This requires researchers to use neurophysiological landmarks such as the retinotopy and tuning properties of areas of interest to identify the recording targets. Therefore, when initially mapping out a cortical area, daily adjustments to the electrode positioning may be needed. Current preparations for marmoset neurophysiology often use semi-chronic probe positioning, which does not allow for easy-access positioning mechanisms5. This protocol demonstrates a lightweight micro-drive for stable linear array recordings that enables flexible positioning with precise sub-millimeter movements across sessions to map out the retinotopy within a chamber.
The current approach, which involves acute daily electrode penetrations for precise electrode targeting and movement across retinotopic maps, differs from chronic electrode array implants in marmosets5. However, acute preparations intrinsically have drawbacks, such as an increased experimental preparation time and an increased risk of infection, as well as an increased risk of cortical tissue damage due to repeated penetrations. While the use of silastic has been optimized in this preparation to limit the risk of infection, applying the silastic does require some troubleshooting to ensure it is done correctly and to obtain a dry seal. This preparation also demands care to avoid electrode damage during the drive mounting and requires care in tightening the drive screws to ensure reliable targeting across sessions.
Long-term cortical damage can be avoided in this preparation when applied correctly with a secure drive, slow penetration of the tissue, and the avoidance of major blood vessels. Additionally, the use of silastic in the craniotomy to prevent infections has been optimized in this technique. The brain is covered with a thin layer of silastic after the craniotomy is performed to prevent infection and keep the interior of the chamber dry14. When the dura is clear from blood and granulation tissue (which is possible with a tight silastic seal), this enables the visualization of the major blood vessels in the underlying tissue and for targeting the silicon probes to avoid hitting the blood vessels during recordings. This technique requires some troubleshooting during learning, since it is critical that the silastic seal is thin (&#60;1 mm) in the center to allow electrode penetration without excessive dimpling while also being thick at the edges to provide mechanical purchase. These methods will be valuable in investigating the circuitry role of the layers across the marmoset cortex and in preventing tissue damage during multiple penetrations.
Using custom-printed plastic components along with commercially available supplies, this micro-drive design is easy to use once fabricated and can be personalized for different recording probes. While this method has been shown to be reliable, the initial fabrication can be difficult and costly to learn. However, the components of the probe are relatively economical to purchase, and, once constructed, the probes have been shown to last up to 1 year with continual use.
One of the key advantages of this preparation is the precise and repeatable targeting of areas of interest due to the X-Y stage. With this technology, researchers are able to map the retinotopic locations within the recording chambers and record from different areas across days using a coordinate system. To achieve this, it is critical to ensure the correct placement of the probe daily and to tighten all the exterior screws in a repeatable manner. Changes to the exterior screw tightness can lead to small shifts in the electrode placement across days.
Acute recordings offer the benefit of repeated independent measurements and can result in a higher yield of single-unit data. However, semi-chronic or chronic recordings may also offer advantages. In small animals such as mice, chronic recordings can be made with high-density silicon probes to obtain stable, long-term population recordings over multiple days18. Eliminating the repeated insertions of electrodes into the brain for each recording session can shorten the overall experimental preparation time and reduce the risk of infection or cortical tissue damage. Therefore, when evaluating the use of chronic or acute recording preparations, it is essential to consider the specific experimental requirements.
The proposed micro-drive design allows the flexibility of different recording techniques and array usage, meaning it could be easily adopted into existing experimental setups and pipelines. Furthermore, the flexibility of the array mounting on the micro-drive may lead to possible future applications, such as controlling laser stimulation for optogenetic purposes. This method may enable future studies to improve on targeting for laser stimulation in the marmoset.

The common marmoset (Callithrix jacchus) has quickly grown in popularity as a model to study brain function in recent years. This growing popularity is due to the accessibility of the marmoset&#39;s smooth cortex, the similarities in neural functional organization with humans and other primates, and the small size and fast breeding rate1. As this model organism has grown in popularity, there has been rapid development in the neurophysiological techniques suited for use in the marmoset brain. Electrophysiology methods are widely used in neuroscience to study the activity of single neurons in the cortex of both rodents and primates, resulting in unparalleled temporal resolution and location access. Due to the relative novelty of the marmoset monkey as a model of visual neuroscience, the optimization of awake-behaving electrophysiology techniques is still evolving. Previous studies have shown the establishment of robust protocols for electrophysiology in anesthetized preparations2, and early awake-behaving neurophysiology studies have shown the reliability of single-channel tungsten electodes3. In recent years, researchers have established the use of silicon-based microelectrode arrays for awake-behaving neurophysiology4. However, the marmoset poses unique targeting challenges due to its small brain size and lack of anatomical landmarks. This protocol outlines how to construct and use a micro-drive recording system suitable for the marmoset that allows for the recording of large populations of neurons with silicon linear arrays while producing minimal tissue damage.
Working with the marmoset poses a challenge due to the smaller scale of the retinotopic maps in the visual cortex as compared to larger primates. A slight shift of the electrodes by just 1 mm can result in significant changes in the maps. Moreover, researchers often need to alter the placement of the electrodes between the recording sessions to obtain a broader range of retinotopic positions in the visual cortex. Current semi-chronic preparations do not allow for the adjustment of the electrode positioning daily or with enough precision to target specific locations at sub-millimeter scales5. With this in mind, the proposed micro-drive system utilizes an X-Y electrode stage that mounts a lightweight micro-drive to a recording chamber and allows for the sub-millimeter targeting of cortical sites. The moveable X-Y stage components allow for vertical and horizontal movement of the linear array in order to traverse the cortical areas systematically, which is required to identify areas of interest (via retinotopy and tuning properties). Across recording sessions, researchers can also manually adjust the X-Y stage to shift the targeted sites within the area. This is a key advantage over alternative techniques using semi-chronic recording preparations, which do not have easy electrode targeting mechanisms.
The micro-drive is a versatile tool that enables the attachment of various silicon arrays to be mounted for lowering into the cortex. In this protocol, a custom probe with two 32-channel linear arrays spaced 200 &#181;m apart was used for the investigation of laminar circuits spanning the cortical depth. Most methods for probing the neural circuitry typically sample the electrical potentials or single units averaged across all the layers of the cerebral cortex. However, recent research has revealed intriguing findings about cortical laminar microcircuits6. By utilizing the micro-drive, researchers can use laminar probes and make fine adjustments to the recording depth to ensure comprehensive sampling across all the layers.
This system can be constructed with commercially available components and is easily modified for different experimental techniques or probes. The key advantages of this preparation are the ability to change the X-Y recording position with sub-millimeter precision and to control the depth of the recording within the cortex. This protocol presents step-by-step instructions for constructing the X-Y stage micro-drive and neurophysiology recording techniques.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1/4 Hp burr drill bit</td>
			<td>McMaster &#38; Carr</td>
			<td>Cat# 43035A32</td>
			<td>Carbide Bur with 1/4&#34; Shank Diameter, Rounded Cylinder Head, trade Number SC-1, single Cut(https://www.mcmaster.com/products/bur-bits/burs-7/?s=1%2F4%22+bur+bits)</td>
		</tr>
		<tr>
			<td>1x1mm Crist Grid</td>
			<td>Crist Instruments</td>
			<td>1 mm x 1 mm Grid</td>
			<td>https://www.cristinstrument.com/products/implant-intro/grids</td>
		</tr>
		<tr>
			<td>91% isopropyl alcohol</td>
			<td>Medline</td>
			<td>N/A</td>
			<td>https://www.medline.com/product/Medline-Isopropyl-Rubbing-Alcohol/Bulk-Alcohol/Z05-PF03807?question=91%25%20isopropyl%20alcohol</td>
		</tr>
		<tr>
			<td>Acquisition Board</td>
			<td>Open-Ephys</td>
			<td>N/A</td>
			<td>https://open-ephys.org/acquisition-system/eux9baf6a5s8tid06hk1mw5aafjdz1</td>
		</tr>
		<tr>
			<td>Bacitracin Ointment</td>
			<td>Medline: Cosette Pharmaceuticals Inc</td>
			<td>N/A</td>
			<td>https://www.medline.com/product/Bacitracin-Ointment/Antibiotics/Z05-PF86957?question=bacitr</td>
		</tr>
		<tr>
			<td>Blunt straight Forceps</td>
			<td>Medline</td>
			<td>N/A</td>
			<td>https://www.medline.com/category/Central-Sterile/Surgical-Instruments/Forceps/Z05-CA16_02_20/products</td>
		</tr>
		<tr>
			<td>Bone wax</td>
			<td>Medline</td>
			<td>ETHW31G</td>
			<td>https://www.medline.com/product/Ethicon-Bone-Wax/Bone-Wax/Z05-PF61528?question=bonewax</td>
		</tr>
		<tr>
			<td>C&#38;B Metabond Quick Adhesive Cement System</td>
			<td>Parkell, Inc.</td>
			<td>SKU: S380</td>
			<td>https://www.parkell.com/C-B-Metabond-Quick-Adhesive-Cement-System</td>
		</tr>
		<tr>
			<td>Clavamox</td>
			<td>MWI Animal Health</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Contact lens solution</td>
			<td>Bausch and lomb</td>
			<td></td>
			<td>Various sources available</td>
		</tr>
		<tr>
			<td>Custom Printed 3D printed parts</td>
			<td>ProtoLab</td>
			<td></td>
			<td>https://marmolab.bcs.rochester.edu/resources.html</td>
		</tr>
		<tr>
			<td>DB25-G2 25 Pin Male Plug Port Signal Connector</td>
			<td>Various Sources</td>
			<td>DB25-G2 25</td>
			<td>DB25-G2 25 Pin Male Plug Port Signal 2 Row Terminal Breakout Board Screw Nut Connector</td>
		</tr>
		<tr>
			<td>diamond saw attachement for dremmel</td>
			<td>Dremmel</td>
			<td>545 Diamond Wheel</td>
			<td>https://www.dremel.com/us/en/p/545-26150545ab</td>
		</tr>
		<tr>
			<td>Digitizing Head-stages</td>
			<td>Intan</td>
			<td>RHD 32channel (Part #C3314)</td>
			<td>https://intantech.com/RHD_headstages.html?tabSelect=RHD32ch&#38;yPos=120.80 
			000305175781</td>
		</tr>
		<tr>
			<td>EDOT</td>
			<td>Sigma Aldrich</td>
			<td>Product # 483028</td>
			<td>https://www.sigmaaldrich.com/US/en/product/aldrich/483028</td>
		</tr>
		<tr>
			<td>Helping Hands</td>
			<td>Harbor Freight</td>
			<td>N/A</td>
			<td>https://www.harborfreight.com/helping-hands-60501.html</td>
		</tr>
		<tr>
			<td>Hook Electrical Clips</td>
			<td>Various Sources</td>
			<td>N/A</td>
			<td>Hook test Cable wires</td>
		</tr>
		<tr>
			<td>Interface Cables (RHD 3-ft (0.9 m) ultra thin SPI cable)</td>
			<td>Intan</td>
			<td>&#160;Part #C3213</td>
			<td>https://intantech.com/RHD_SPI_cables.html</td>
		</tr>
		<tr>
			<td>Lab jack</td>
			<td>Various Sources</td>
			<td>N/A</td>
			<td>https://www.amazon.com/Stainless-Steel-Scissor-Stand-Platform/dp/B07T8FM85H/ref=asc_df_B07T8FM85H/?tag=&#38;linkCode=df0&#38;hvadid=366343 
			827267&#38;hvpos=&#38;hvnetw=g&#38;hvrand 
			=2036619536500717246&#38;hvpone 
			=&#38;hvptwo=&#38;hvqmt=&#38;hvdev=c&#38;hv 
			dvcmdl=&#38;hvlocint=&#38;hvlocphy=900 
			5674&#38;hvtargid=pla-795933567991&#38; 
			ref=&#38;adgrpid=71496544770&#38;th=1</td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td>MWI Animal Health</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-drive</td>
			<td>Crist Instrument</td>
			<td>3-NRMD</td>
			<td>https://www.cristinstrument.com/products/microdrives/miniature-microdrive-3-nrmd</td>
		</tr>
		<tr>
			<td>Multi-channel linear silicon arrays with 64 channel connector</td>
			<td>NeuroNexus</td>
			<td>A1x32-5mm-25-177</td>
			<td>https://www.neuronexus.com/products/electrode-arrays/up-to-10-mm-depth/</td>
		</tr>
		<tr>
			<td>NanoZ Omentics Adapter- 32 Channel</td>
			<td>NeuraLynx</td>
			<td>ADPT-NZ-N2T-32</td>
			<td>https://neuralynx.com/hardware/adpt-nz-n2t-32</td>
		</tr>
		<tr>
			<td>NanoZ System</td>
			<td>Plexon</td>
			<td>NanoZ Impedence Tester</td>
			<td>https://plexon.com/products/nanoz-impedance-tester/</td>
		</tr>
		<tr>
			<td>Narishige Micromanipulator</td>
			<td>Narishige</td>
			<td>Stereotaxic Micromanipulator</td>
			<td>https://usa.narishige-group.com/</td>
		</tr>
		<tr>
			<td>Open-Ephys GUI</td>
			<td>Open-Ephys</td>
			<td></td>
			<td>https://open-ephys.org/</td>
		</tr>
		<tr>
			<td>Polyimide Tubing (OD(in): 0.021 / ID(in) 0.018 )</td>
			<td>Various Sources (Chamfr)</td>
			<td>Chamfr Cat#HPC01895</td>
			<td>https://chamfr.com/sellers/teleflex-medical-oem-llc/</td>
		</tr>
		<tr>
			<td>Primate Chair</td>
			<td>Custom made by University of Rochester Machine Shop</td>
			<td>Designs online</td>
			<td>https://marmolab.bcs.rochester.edu/resources.html</td>
		</tr>
		<tr>
			<td>Poly(sodium 4-styrenesulfonate) (PSS)</td>
			<td>Sigma Aldrich</td>
			<td>Product # 243051</td>
			<td>https://www.sigmaaldrich.com/US/en/product/aldrich/243051</td>
		</tr>
		<tr>
			<td>RHD USB Interface board</td>
			<td>Intan</td>
			<td>RHD2000 Evaluation Board Version 1.0</td>
			<td>https://intantech.com/RHD_USB_interface_board.html</td>
		</tr>
		<tr>
			<td>Silastic gel</td>
			<td>World Precision Instuments</td>
			<td># KWIK-SIL</td>
			<td>Low Toxicity Silicone Adhesive ((https://www.wpiinc.com/kwik-sil-low-toxicity-silicone-adhesive)</td>
		</tr>
		<tr>
			<td>Slow release buprenorphine</td>
			<td>Compounding Pharmacy</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel wire 36 gauge</td>
			<td>McMaster &#38; Carr</td>
			<td>Cat# 6517K11</td>
			<td>Round Bend-and-Stay Multipurpose 304 Stainless Steel Wire, Matte Finish, 1-Foot Long, 0.008&#34; Diameter</td>
		</tr>
		<tr>
			<td>Stanley 6-Piece Precision Screwdriver Set</td>
			<td>Stanley</td>
			<td>1.4mm flathead screwdriver</td>
			<td>https://www.amazon.com/Stanley-Tools-6-Piece-Precision-Screwdriver/dp/B076621ZGC/ref=sr_1_3?crid=237VSK5FNFP9N&#38;keywords= 
			stanley+66-052&#38;qid=1672764369&#38;sprefix= 
			stanley+66-052%2Caps%2C90&#38;sr=8-3</td>
		</tr>
		<tr>
			<td>Steel Screws</td>
			<td>McMaster &#38; Carr</td>
			<td>type 00 stainless steel hex screws and 1/8&#8221; in length</td>
			<td>https://www.mcmaster.com/</td>
		</tr>
		<tr>
			<td>Steel Tube</td>
			<td>McMaster &#38; Carr</td>
			<td>28 gauge stainless steel tubing</td>
			<td>https://www.mcmaster.com/tubing/multipurpose-304-stainless-steel-6/id~0-055/</td>
		</tr>
		<tr>
			<td>Superglue</td>
			<td>Loctite</td>
			<td>SuperGlue Gel Control</td>
			<td>https://www.loctiteproducts.com/en/products/fix/super-glue/loctite_super_gluegelcontrol.html</td>
		</tr>
	</tbody>
</table>

electrophysiology of laminar cortical activity in the common marmoset

The marmoset monkey provides an ideal model for examining laminar cortical circuits due to its smooth cortical surface, which facilitates recordings with linear arrays. The marmoset has recently grown in popularity due to its similar neural functional organization to other primates and its technical advantages for recording and imaging. However, neurophysiology in this model poses some unique challenges due to the small size and lack of gyri as anatomical landmarks. Using custom-built micro-drives, researchers can manipulate linear array placement to sub-millimeter precision and reliably record at the same retinotopically targeted location across recording days. This protocol describes the step-by-step construction of the micro-drive positioning system and the neurophysiological recording technique with silicon linear electrode arrays. With precise control of electrode placement across recording sessions, researchers can easily traverse the cortex to identify areas of interest based on their retinotopic organization and the tuning properties of the recorded neurons. Further, using this laminar array electrode system, it is possible to apply a current source density analysis (CSD) to determine the recording depth of individual neurons. This protocol also demonstrates examples of laminar recordings, including spike waveforms isolated in Kilosort, which span multiple channels on the arrays.

This protocol describes how to build an X-Y electrode stage (Figure 1) that allows for the sub-millimeter targeting of sites and maintains reliable positioning across separate recording sessions. The reliability of the X-Y positioning is illustrated in Figure 6, which demonstrates that two recording sessions conducted a week apart showed a 70.8% overlap in their mean RF locations (Figure 6A). Furthermore, minor adjustments to the micro-drive positioning can support precise movement in the retinotopic space. Small X and Y stage movements up to 0.2 mm can be made by referencing the predrilled holes in each stage, which are 2 mm apart. In a series of recording sessions that transversed the MT/MTC border using minor positioning adjustments (Figure 6B), precise movements in retinotopic space could be seen (Figure 6C). The recording sites for the MT and MTC aligned with electrophysiology maps previously depicted in Solomon and Rosa2. Similarly, with this protocol, the Z-positioning can be tracked by the number of turns to the preferred electrode depth. While the Z-positioning of the drive to place electrodes in cortical depth must be adjusted daily and includes some variation, it is possible to obtain estimates of depth in early visual areas offline by using current source density analysis (CSD), as described previously16.
Using this X-Y stage technology, researchers can perform neurophysiological recording with linear arrays while the subjects perform a variety of foraging tasks for visual receptive field mapping and simple saccade tasks17. Electrode placement techniques are shown in Figure 4, which highlights the importance of retraction to alleviate pressure on the tissue and prevent long-term cortical damage. Using two shank linear arrays allows for sampling a large number of neurons across cortical depths during each recording. In this work, the spike-sorted array data obtained using Kilosort showed that the principle component features of a selection of spikes in a cluster were distinct from background spikes and that the recordings were stable over time (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65397/f65397ig01v2.jpg" /> 
Figure 1: Construction of the electrode micro-drive. The construction of an electrode micro-drive is possible after obtaining all the necessary components outlaid in section 1, step 1. Each successive step (section 1, steps 2-10)&#160;shows a before and an after image to aid in the visualization of the actions required. This construction is explained fully in protocol section 1. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65397/65397fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65397/65397fig02.jpg" /> 
Figure 2: Constructed micro-drive and electroplating setup. Overview image of the completed micro-drive from the (A) top and (B) bottom. (C) The micro-drive is attached to a protective sleeve (same dimensions as the chambers on the animal&#39;s implant, with an extended length for protection) and then attached to the animal&#39;s chamber by tightening three side screws. (D) Configuration of the micro-drive on the impedance tester for electroplating. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65397/65397fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65397/65397fig03.jpg" /> 
Figure 3: Schematic of the head cap and chamber placement (A) Drawing representation of an animal with a head cap, head post, and MT chamber. (B) Image of an MT chamber 6 months after craniotomy and sealed with a thin layer of silastic. (C) Schematic of the chamber and micro-drive preparation. The illustration shows the chamber preparation and the thin layer of silastic for use in the recordings. The micro-drive is secured to the recording chamber by tension from the side screws. (D) One should move parallel to the chamber to attach the micro-drive to the recording chamber. Coming from an angle will likely result in hitting the side of the chamber or the cement sides of the craniotomy. (E) The micro-drive secured on an example head cap. (F) Depiction of the three screws on the micro-drive to be tightened for secure placement. Note: One screw sits behind the connector and is usually tightened from above. The screws are tightened in a repeatable order to ensure drive location reliability. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65397/65397fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65397/65397fig04.jpg" /> 
Figure 4: Neurophysiology recordings (A) Depiction of the electrode before and after it touches the dura (top). High-pass-filtered recording signal from a single probe in a neural recording before and after the probe touches the dura (bottom). (B) Depiction of an electrode causing dimpling after initially popping through the silastic (top). High-pass-filtered recording signal from one probe in a neural recording after the probe has popped through the silastic (bottom). Channels with neurons are labeled as filled. (C) Depiction of the electrode in the cortex before and after retraction (top). High-pass-filtered recording signal from one probe in a neural recording before and after a 500 &#181;m retraction; the units have shifted one channel (bottom). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65397/65397fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65397/65397fig05.jpg" /> 
Figure 5: Post-processing of neurophysiology data. (A) Depiction of a laminar probe (left) and a selection of spike waveforms viewed across each channel (right). (B) Example Kilosort output. (Left) Example waveform across several channels with a peak waveform on channel 46. (Top right) The principle component features of a selection of spikes in the selected cluster (blue) against a set of background spikes (uniformly spaced in time across the entire recording, grey). Spiking clusters are easily discriminated from background spikes. (Bottom right) The amplitude of a selection of spikes belonging to the selected cluster over time. The recordings show stability over time for this example neuron. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65397/65397fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65397/65397fig06.jpg" /> 
Figure 6: Reliability of the X-Y stage positioning (A) Mean receptive field (RF) contours for two recording sessions 1 week apart with the same micro-drive coordinates, showing 70.8% overlap. (B) An MT/MTC map showing the successive targeting locations across recording sessions. The solid lines and numbers indicate eccentricity from the fovea in degrees. The dashed lines indicate the horizontal meridian (HM). The bolded lines indicate the vertical meridian (VM). (C) The corresponding RF locations for each shank outlined with a circle marking the center location show that minor adjustments to the micro-drive positioning can support precise movement in the retinotopic space. Panel B has been adapted with permission from Solomon and Rosa2. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65397/65397fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Electrophysiology of Laminar Cortical Activity in the Common Marmoset at JoVE.com

Author Spotlight: Optimizing Micro-Drive Systems for Precise Electrode Positioning in Marmoset

Custom-built micro-drives enable the sub-millimeter targeting of cortical recording sites with linear silicon arrays.

Electrophysiology of Laminar Cortical Activity in the Common Marmoset

The marmoset monkey provides an ideal model for examining laminar cortical circuits due to its smooth cortical surface, which facilitates recordings with ...

electrophysiology-of-laminar-cortical-activity-in-the-common-marmoset

Nanyang Technological University

Research

JoVE Journal

Neuroscience

805 Views.  University of Rochester. Custom-built micro-drives enable the sub-millimeter targeting of cortical recording sites with linear silicon arrays.

Video: Author Spotlight: Optimizing Micro-Drive Systems for Precise Electrode Positioning in Marmoset

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Electrophysiology of Scorpion Peg Sensilla

We describe a modification of an existing tip-recording technique1,2 for electrophysiologically investigating short, peg-like sensory sensilla3,4. On the mid-ventral surface of all scorpions are two appendages called pectines, which have dense fields of mechano- and chemosensory peg sensilla5,6. One method for assessing chemoresponsiveness of these sensilla uses a tungsten electrode for extracellularly recording neural activity within a sensillum as a volatile odorant is introduced to the sensory field5,7. The limitations of this method include slow data collection and uncontrolled stimulant introduction to, and removal from, the peg field. To overcome these limitations, we developed a new tip-recording technique that uses nonpolar mineral oil as a medium through which to deliver water-based tastants to individual peg sensilla8,9. We have successfully applied this method to obtain sensillar chemoresponses to citric acid, ethanol, and salt. Here we describe the experimental protocol for such a study9. We think this new method may be useful for studying the response properties of other arthropod chemosensory systems, including those of insects10, 11 and crustaceans12.

This article describes an electrophysiological method for isolating chemical stimulation to individual sensilla via extracellular, tip-recordings under mineral oil.

We describe a modification of an existing tip-recording technique1,2 for electrophysiologically investigating short, peg-like sensory sensilla3,4. On the ...

Examining Local Network Processing using Multi-contact Laminar Electrode Recording

Cortical layers are ubiquitous structures throughout neocortex1-4 that consist of highly recurrent local networks. In recent years, significant progress has been made in our understanding of differences in response properties of neurons in different cortical layers5-8, yet there is still a great deal left to learn about whether and how neuronal populations encode information in a laminar-specific manner.
Existing multi-electrode array techniques, although informative for measuring responses across many millimeters of cortical space along the cortical surface, are unsuitable to approach the issue of laminar cortical circuits. Here, we present our method for setting up and recording individual neurons and local field potentials (LFPs) across cortical layers of primary visual cortex (V1) utilizing multi-contact laminar electrodes (Figure 1; Plextrode U-Probe, Plexon Inc).
The methods included are recording device construction, identification of cortical layers, and identification of receptive fields of individual neurons. To identify cortical layers, we measure the evoked response potentials (ERPs) of the LFP time-series using full-field flashed stimuli. We then perform current-source density (CSD) analysis to identify the polarity inversion accompanied by the sink-source configuration at the base of layer 4 (the sink is inside layer 4, subsequently referred to as granular layer9-12). Current-source density is useful because it provides an index of the location, direction, and density of transmembrane current flow, allowing us to accurately position electrodes to record from all layers in a single penetration6, 11, 12.

A fundamental issue in our understanding of cortical circuitry is how networks in different cortical layers encode sensory information. Here we describe electrophysiological techniques utilizing multi-contact laminar electrodes to record single-units and local field potentials and present analyses to identify cortical layers.

Cortical layers are ubiquitous structures throughout neocortex1-4 that consist of highly recurrent local networks. In recent years, significant progress ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the underlying neuronal components in the circuits1-6. Application of optogenetics7,8 in the larval neural circuit allows us to manipulate neuronal activity in spatially and temporally patterned ways9-13. Typically, specimens are broadly illuminated with a mercury lamp or LED, so specificity of the target neurons is controlled by binary gene expression systems such as the Gal4-UAS system14,15. In this work, to improve the spatial resolution to "sub-genetic resolution", we locally illuminated a subset of neurons in the ventral nerve cord using lasers implemented in a conventional confocal microscope. While monitoring the motion of the body wall of the semi-intact larvae, we interactively activated or inhibited neural activity with channelrhodopsin16,17 or halorhodopsin18-20, respectively. By spatially and temporally restricted illumination of the neural tissue, we can manipulate the activity of specific neurons in the circuit at a specific phase of behavior. This method is useful for studying the relationship between the activities of a local neural assembly in the ventral nerve cord and the spatiotemporal pattern of motor output.

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Here we describe a protocol for optogenetic manipulation of motoneuronal activity while monitoring changes in motor output (muscle contraction) in semi-intact Drosophila larvae using lasers within a conventional confocal microscope. This technique enables researchers to achieve local perturbation of neural activity in a few neuromeres to elucidate the dynamics of motor circuits.

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...