Este trabalho foi apoiado pelo National Institutes of Health (NIH) grant R01 EY030998 (J.F.M., A.B., e S.C.). Esse método é baseado em métodos desenvolvidos em Coop et al (em revisão, 2022; https://www.biorxiv.org/content/10.1101/2022.10.11.511827v2.abstract). Gostaríamos de agradecer a Dina Graf e aos membros do laboratório Mitchell pela ajuda com o cuidado e manuseio do sagui.

Desenvolvimento de um estágio de eletrodo X-Y para registro neural preciso em saguis

<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>

Os autores não têm nada a revelar.

Vários métodos (por exemplo, crônico, semicrônico, agudo) estão atualmente disponíveis para a realização de experimentos de neurofisiologia em primatas não humanos. O sagui-comum apresenta desafios únicos para experimentos de neurofisiologia devido ao seu pequeno tamanho e à falta de giros como marcos anatômicos. Isso requer que os pesquisadores usem pontos de referência neurofisiológicos, como a retinotopia e as propriedades de sintonia de áreas de interesse, para identificar os alvos de registro. Portant...

O sagui-comum (Callithrix jacchus) cresceu rapidamente em popularidade como um modelo para estudar a função cerebral nos últimos anos. Essa crescente popularidade deve-se à acessibilidade do córtex liso do sagui, às semelhanças na organização funcional neural com humanos e outros primatas, e ao pequeno tamanho e à rápida taxa de reprodução1. Como este organismo modelo tem crescido em popularidade, tem havido um rápido desenvolvimento nas técnicas neurofisiológicas adequadas para uso no cérebro de sagui. Métodos eletrofisiológicos são amplamente utilizados em neurociência para estudar a atividade de neurônios únicos no córtex de roedores e primatas, resultando em resolução temporal e acesso de localização incomparáveis. Devido à relativa novidade do macaco sagui como modelo da neurociência visual, a otimização das técnicas de eletrofisiologia do comportamento acordado ainda está em evolução. Estudos prévios mostraram o estabelecimento de protocolos robustos para eletrofisiologia em preparações anestesiadas2, e estudos neurofisiológicos com comportamento precoce em vigília mostraram a confiabilidade de electos de tungstênio monocanal3. Nos últimos anos, pesquisadores estabeleceram o uso de arranjos de microeletrodos à base de silício para neurofisiologia do comportamento acordado4. No entanto, o sagui apresenta desafios de mira únicos devido ao seu pequeno tamanho cerebral e à falta de marcos anatômicos. Este protocolo descreve como construir e usar um sistema de gravação de micro-drive adequado para o sagui que permite o registro de grandes populações de neurônios com matrizes lineares de silício enquanto produz dano tecidual mínimo.
Trabalhar com o sagui representa um desafio devido à menor escala dos mapas retinotópicos no córtex visual em comparação com primatas maiores. Um ligeiro deslocamento dos eletrodos em apenas 1 mm pode resultar em mudanças significativas nos mapas. Além disso, os pesquisadores muitas vezes precisam alterar o posicionamento dos eletrodos entre as sessões de gravação para obter uma gama mais ampla de posições retinotópicas no córtex visual. As preparações semicrônicas atuais não permitem o ajuste do posicionamento do eletrodo diariamente ou com precisão suficiente para atingir locais específicos em escalas submilimetradas5. Com isso em mente, o sistema de micro-acionamento proposto utiliza um estágio de eletrodo X-Y que monta um micro-drive leve em uma câmara de gravação e permite o direcionamento submilimétrico de locais corticais. Os componentes móveis do estágio X-Y permitem o movimento vertical e horizontal da matriz linear para atravessar as áreas corticais sistematicamente, o que é necessário para identificar áreas de interesse (via retinotopia e propriedades de sintonia). Nas sessões de gravação, os pesquisadores também podem ajustar manualmente o estágio X-Y para mudar os locais de destino dentro da área. Esta é uma vantagem fundamental em relação a técnicas alternativas que usam preparações de gravação semi-crônicas, que não têm mecanismos fáceis de direcionamento de eletrodos.
O micro-drive é uma ferramenta versátil que permite a fixação de várias matrizes de silício a serem montadas para abaixar no córtex. Neste protocolo, uma sonda personalizada com duas matrizes lineares de 32 canais espaçadas de 200 μm foi usada para a investigação de circuitos laminares abrangendo a profundidade cortical. A maioria dos métodos para sondar os circuitos neurais tipicamente amostram os potenciais elétricos ou unidades únicas médias em todas as camadas do córtex cerebral. No entanto, pesquisas recentes têm revelado achados intrigantes sobre microcircuitos laminares corticais6. Ao utilizar o micro-drive, os pesquisadores podem usar sondas laminares e fazer ajustes finos na profundidade de gravação para garantir uma amostragem abrangente em todas as camadas.
Este sistema pode ser construído com componentes comercialmente disponíveis e é facilmente modificado para diferentes técnicas experimentais ou sondas. As principais vantagens desta preparação são a capacidade de alterar a posição de gravação X-Y com precisão submilimétrica e controlar a profundidade da gravação dentro do córtex. Este protocolo apresenta instruções passo a passo para a construção do microdrive de estágio X-Y e técnicas de registro da neurofisiologia.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<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>
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			<td>91% isopropyl alcohol</td>
			<td>Medline</td>
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			<td>Acquisition Board</td>
			<td>Open-Ephys</td>
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			<td>Medline: Cosette Pharmaceuticals Inc</td>
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			<td>Medline</td>
			<td>N/A</td>
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			<td>Medline</td>
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			<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>
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			<td>MWI Animal Health</td>
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			<td>Contact lens solution</td>
			<td>Bausch and lomb</td>
			<td></td>
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			<td>Custom Printed 3D printed parts</td>
			<td>ProtoLab</td>
			<td></td>
			<td>https://marmolab.bcs.rochester.edu/resources.html</td>
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			<td>DB25-G2 25 Pin Male Plug Port Signal Connector</td>
			<td>Various Sources</td>
			<td>DB25-G2 25</td>
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			<td>diamond saw attachement for dremmel</td>
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			<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 
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			<td>EDOT</td>
			<td>Sigma Aldrich</td>
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			<td>Helping Hands</td>
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			<td>Hook Electrical Clips</td>
			<td>Various Sources</td>
			<td>N/A</td>
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			<td>Interface Cables (RHD 3-ft (0.9 m) ultra thin SPI cable)</td>
			<td>Intan</td>
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			<td>Lab jack</td>
			<td>Various Sources</td>
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			<td>Meloxicam</td>
			<td>MWI Animal Health</td>
			<td>N/A</td>
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			<td>Micro-drive</td>
			<td>Crist Instrument</td>
			<td>3-NRMD</td>
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			<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>
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			<td>NanoZ Omentics Adapter- 32 Channel</td>
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			<td>ADPT-NZ-N2T-32</td>
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			<td>NanoZ System</td>
			<td>Plexon</td>
			<td>NanoZ Impedence Tester</td>
			<td>https://plexon.com/products/nanoz-impedance-tester/</td>
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			<td>Narishige Micromanipulator</td>
			<td>Narishige</td>
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			<td>Open-Ephys GUI</td>
			<td>Open-Ephys</td>
			<td></td>
			<td>https://open-ephys.org/</td>
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			<td>Polyimide Tubing (OD(in): 0.021 / ID(in) 0.018 )</td>
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			<td>Chamfr Cat#HPC01895</td>
			<td>https://chamfr.com/sellers/teleflex-medical-oem-llc/</td>
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			<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>
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			<td>Poly(sodium 4-styrenesulfonate) (PSS)</td>
			<td>Sigma Aldrich</td>
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			<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>
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			<td>World Precision Instuments</td>
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			<td>Slow release buprenorphine</td>
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			<td>Stainless steel wire 36 gauge</td>
			<td>McMaster &#38; Carr</td>
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			<td>Stanley 6-Piece Precision Screwdriver Set</td>
			<td>Stanley</td>
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			<td>Steel Screws</td>
			<td>McMaster &#38; Carr</td>
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			<td>https://www.mcmaster.com/</td>
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</table>

electrophysiology of laminar cortical activity in the common marmoset

O macaco sagui fornece um modelo ideal para examinar circuitos corticais laminares devido à sua superfície cortical lisa, o que facilita gravações com matrizes lineares. O sagui tem crescido recentemente em popularidade devido à sua organização funcional neural semelhante a outros primatas e suas vantagens técnicas para registro e imagem. No entanto, a neurofisiologia neste modelo apresenta alguns desafios únicos devido ao pequeno tamanho e à falta de giros como pontos de referência anatômicos. Usando microdrives personalizados, os pesquisadores podem manipular a colocação de matriz linear para precisão submilimétrica e gravar de forma confiável no mesmo local alvo retinotopicamente ao longo dos dias de gravação. Este protocolo descreve o passo-a-passo da construção do sistema de posicionamento de micro-drive e a técnica de registro neurofisiológico com arranjos lineares de eletrodos de silício. Com o controle preciso da colocação do eletrodo nas sessões de gravação, os pesquisadores podem facilmente atravessar o córtex para identificar áreas de interesse com base em sua organização retinotópica e nas propriedades de sintonia dos neurônios gravados. Além disso, usando este sistema de eletrodos de arranjo laminar, é possível aplicar uma análise de densidade de fonte de corrente (CSD) para determinar a profundidade de registro de neurônios individuais. Este protocolo também demonstra exemplos de gravações laminares, incluindo formas de onda de pico isoladas em Kilosort, que abrangem vários canais nas matrizes.

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.

Autor em destaque: Otimizando sistemas de microacionamento para posicionamento preciso de eletrodos no sagui

Eletrofisiologia da Atividade Cortical Laminar no Sagui-comum

The common marmoset poses unique challenges for neurophysiology due to its small size and lack of gyre as anatomical landmarks. A slight shift of the electrode by just a millimeter can result in significant changes in the retinotopy map. The proposed micro drive system utilizes an XY electrode stage that allows for vertical and horizontal movement on a sub millimeter scale.Due to the relative novelty of the marmoset monkey as a model for visual neuron sign, awake behaving electrophysiology techniques are still evolving. Current preparations often use semi chronic probes that do not allow access for the positioning mechanisms. This protocol demonstrates a lightweight micro drive for useful linear array recordings, which allow flexible positioning across sessions to map out retinotopy within the chamber.Long-term cortical damage can be avoided in the preparation when used correctly with the secure drive, slow penetration of tissue, and avoidance of major blood vessels. Additionally, the use of silastic in the craniotomies to avoid infections has been optimized in this technique.

Este protocolo descreve como construir um estágio de eletrodo X-Y (Figura 1) que permite a segmentação submilimétrica de locais e mantém o posicionamento confiável em sessões de gravação separadas. A confiabilidade do posicionamento X-Y é ilustrada na Figura 6, que demonstra que duas sessões de registro realizadas com uma semana de intervalo mostraram sobreposição de 70,8% em suas localizações médias de FR (Figura 6A...

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

Microdrives personalizados permitem a segmentação submilimétrica de locais de gravação corticais com matrizes lineares de silício.

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

O sagui-comum apresenta desafios únicos para a neurofisiologia devido ao seu pequeno tamanho e à falta de giro como marcos anatômicos. Um ligeiro deslocamento do eletrodo em apenas um milímetro pode resultar em mudanças significativas no mapa de retinotopia. O sistema de microacionamento proposto utiliza um estágio de eletrodo XY que permite o movimento vertical e horizontal em uma escala submilimétrica.Devido à relativa novidade do macaco sagui como modelo para o sinal visual do neurônio, as técnicas de eletrofisiologia do comportamento acordado ainda estão evoluindo. As preparações atuais frequentemente utilizam sondas semicrônicas que não permitem o acesso aos mecanismos de posicionamento. Este protocolo demonstra um micro drive leve para gravações de matriz linear úteis, que permitem o posicionamento flexível entre sessões para mapear a retinotopia dentro da câmara.Danos corticais a longo prazo podem ser evitados na preparação quando usados corretamente com a unidade segura, penetração lenta do tecido e evitar os principais vasos sanguíneos. Além disso, o uso de silastic nas craniotomias para evitar infecções tem sido otimizado nessa técnica.

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

Research

JoVE Journal

Neuroscience

Electrophysiology of Laminar Cortical Activity in the Common Marmoset

682 visualizações.  University of Rochester. O sagui-comum apresenta desafios únicos para a neurofisiologia devido ao seu pequeno tamanho e à falta de giro como marcos anatômicos. Um ligeiro deslocamento do eletrodo em apenas um milímetro pode resultar em mudanças significativas no mapa de retinotopia. O sistema de microacionamento proposto utiliza um estágio de eletrodo XY que permite o movimento vertical e horizontal em uma escala submilimétrica.Devido à relativa novidade do macaco sagui como modelo para o sinal visual ...

Vídeo: Autor em destaque: Otimizando sistemas de microacionamento para posicionamento preciso de eletrodos no sagui

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.

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

Neurociência

Construction of the Electrode Micro-Drive and Polytrode Plating of Electrodes for Neurophysiology Recordings

To begin, collect the parts required for the construction of electrode micro-drive. Place a nut in the slot of the base and add super glue for stability. Then insert a screw through the nut in the base.Using a rotary tool with a diamond wheel cutting attachment, cut a four by three by three millimeter section of the plastic grid with one by one millimeter hole spacing. Place the grid section onto the top of the Y stage. Apply epoxy to fix the grid section onto the top of the Y stage.Then fit the micro-drive over the grid using a screw. Add epoxy at the base of the micro-drive for stability. Attach a plastic rectangular platform to a 28 gauge steel tube using epoxy, thread a 23 gauge guide tube through the grid.Then thread the steel tube attached to the plastic platform through the 23 gauge guide tube. Place the 28 gauge steel tube into one of the micro-drive slots. Secure the steel tube to the micro-drive with epoxy.Using a hand tapping tool, tap the pre-made screw holes in the X stage and base to build the base for the drive. Then insert screws through the long slots in the X stage. Secure them in the corresponding screw holes of the base.Insert two screws through the long slot in the Y stage and secure them to the pre-made screw holes in the X stage. After ensuring the micro manipulator drive is outfitted with a plastic platform, use tape to attach the 64 pin connector to the platform. Then carefully place a dab of bacitracin onto the plastic tube and attach the electrode connected to the 64 pin connector using a flexible ribbon cable.Using the micro manipulator drive, hold the silicon electrode and its connector while threading the plastic tube through the hole in the Y stage. Detach the 64 pin connector from the micro manipulator drive and use glue gel to secure the connector to the platform on the X stage. The next day, carefully detach the electrode from the plastic tube and remove the micro manipulator drive from the assembly.Gently move the alligator clip to flip the drive over to see the unsecured electrode. Place the electrode onto the plastic holder below the Y stage and secure it with silicone elastomer. After five minutes, using the screw control on the micro-drive, retract the electrode and place the protective sleeve over the electrode.Tighten the three side located screws on the base component of the micro-drive. Connect the head stage for recordings onto the 64 pin connector. To prepare the PDOT solution, combine EDOT and PSS in distilled water a day before the use.Open the impedance tester software and in the upper dropdown menu at the top left, select the adapter. In the second dropdown menu, select the electrode. Ensure that the number of channels is correct.Using a connector, connect the probe to the impedance tester system and lower the probe into grounded saline to obtain a baseline reading. Press the test impedances button and verify the correct setting before pressing the test probe button. Record and save the baseline readings for future reference for both shanks of the electrode.Next, lower the lab jack to remove the probe from the saline. Replace the saline in the beaker with distilled water. Raise the lab jack to submerge the electrode and rinse the remaining saline solution before lowering the lab jack again.Replace the distilled water with PDOT solution and raise the lab jack until the electrode is submerged. Select the DC ELECTROPLATE button and verify the correct settings. Then press the Autoplate button and save the results.After testing, first, rinse the probe with distilled water, then fill the beaker with saline, and raise the lab jack until the electrode is submerged. Press the test impedances button and check for the correct settings. Then press the test pro button.

Construção do Micro-Drive de Eletrodos e Revestimento de Politrodos para Registros de Neurofisiologia 

Neurophysiology Recordings in Cortical Tissue of Marmoset

To begin, secure the micro-drive on a head cap. Secure all three screws on the micro-drive system. Using the screw control on the micro-drive, lower the silicon probe, making one turn every 1 to 2 seconds until units are observed at the array tip.Once the neurons are observed, retract the one turn of the micro-drive to decrease the electrode's speed of entry as it moves through the Silastic and dura into the cortical tissue. Slowly continue to drive the array into the cortex, advancing 4 to 6 turns over 20 to 30 minutes until neurons are evenly distributed across the length of the probe. Then slowly retract the array by 1 to 2 turns to reduce the pressure on the tissue.After the recording is complete, slowly retract the array from the cortex at a speed similar to the initial insertion. Once no more neurons are visible on the probe, retract the array more quickly until it returns to the fully retracted starting position. After removing the probe from the recording chamber, soak it in contact lens solution for 20 minutes to clean off any tissue or blood.Place the probe in alcohol for one minute to remove the contact lens solution and allow the electrode to dry. The spike sorted array data obtained through Kilosort revealed that the principle component features of selected spikes in a cluster were distinct from background spikes, confirming the recording's stability over time. The reliability of the X-Y positioning demonstrated a 70.8%overlap in mean RF locations between two sessions a week apart.Precise retina topic space movements were observed in a series of recording sessions crossing the MT and MTC borders using minor positioning changes.