Name: hybrid microdrive system with recoverable opto-silicon probe and tetrode for dual-site high density recording in freely moving mice
Uploaded: 2019-08-10T15:00:00
Description: Watch this Scientific Journal Video about Hybrid Microdrive System with Recoverable Opto-Silicon Probe and Tetrode for Dual-Site High Density Recording in Freely Moving Mice at JoVE.com

This work was supported in part by Japan Society for the Promotion of Science Overseas Research Fellowships (HO), Endowed Scholar Program (TK), Human Frontier Science Program (TK), Brain Research Foundation (TK), Faculty Science and Technology Acquisition and Retention Program (TK), Brain &#38; Behavior Research Foundation (TK), and by The Sumitomo Foundation Research Grant (JY), NARSAD Young Investigator Research Grant (JY). We thank W. Marks for valuable comments and suggestions during the preparation of the manuscript.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Texas Southwestern Medical Center.
1. Preparations of microdrive array parts
<ol>
	<li>Print the microdrive array parts using a 3D printer using dental model resin (Figure 1A,B). Ensure that the thickness of individual 3D printed layers is less than 50 &#181;m to keep the small holes on the printed parts clear and viable. 
	NOTE: The microdrive a...

The protocol demonstrates how to construct and implant a hybrid microdrive array that allows recording of neural activities from two brain areas using independent adjustable tetrodes and a silicon-probe in freely behaving mice. It also demonstrates optogenetic experiments and the recovery of the silicon probe after experiments. While adjustable silicon probe33 or opto-silicon probe36 implantation are previously demonstrated in mice, this protocol has clear advantages in the...

It is crucial to understand how neuronal activity supports cognitive process, such as learning and memory, by investigating how different brain regions dynamically interact with each other. To elucidate dynamics of the neural activity underlying cognitive tasks, large-scale extracellular electrophysiology has been conducted in freely moving animals with the aid of microdrive arrays1,2,3,4. In the past two decades, several types of microdrive array have been developed to implant electrodes into multiple brain regions for rats5,6,7,8 and mice9,10,11,12. Nonetheless, current microdrive designs generally do not allow for the use of multiple probe types, forcing researchers to choose a single electrode type with specific benefits and limitations. For example, tetrode arrays work well for densely populated brain regions such as the dorsal hippocampus CA11,13, while silicon probes give a better geometrical profile for studying anatomical connections14,15.
Tetrodes and silicon probes are often used for in vivo chronic recording, and each has its own advantages and disadvantages. Tetrodes have been proven to have significant advantages in better single unit isolation than single electrodes16,17, in addition to cost effectiveness and mechanical rigidity. They also provide higher yields of single unit activities when combined with microdrives8,18,19,20. It is essential to increase the number of simultaneously recorded neurons for understanding the function of neural circuits21. For example, large numbers of cells are needed to investigate small populations of functionally heterogeneous cell types such as time-related22 or reward coding23 cells. Much higher cell numbers are required to improve the decoding quality of spike sequences13,24,25.
Tetrodes, however, have a disadvantage in recording spatially distributed cells, such as in the cortex or thalamus. In contrast to tetrodes, silicon probes can provide spatial distribution and interaction of local field potentials (LFPs) and spiking activities within a local structure14,26. Multi-shank silicon probes further increase the number of recording sites and allow recording across single or neighboring structures27. However, such arrays are less flexible in the positioning of electrode sites compared to tetrodes. In addition, complex spike sorting algorithms are required in high-density probes to extract information about action potentials of neighboring channels to mirror the data acquired by tetrodes28,29,30. Hence, the overall yield of single units is often less than tetrodes. Moreover, silicon probes are disadvantageous due to their fragility and high cost. Thus, the choice of tetrodes vs. silicon probes depends on the aim of the recording, which is a question of whether obtaining a high yield of single-units or spatial profiling at the recording sites is prioritized.
In addition to recording neural activity, optogenetic manipulation has become one of the more powerful tools in neuroscience to examine how specific cell types and/or pathways contribute to neural circuit functions13,31,32,33. However, optogenetic experiments require additional consideration in microdrive array design to attach the fiber connector to stimulation light sources34,35,36. Often, connecting fiber-optics requires a relatively large force, which may lead to a mechanical shift of the probe in the brain. Therefore, it is not a trivial task to combine an implantable optical fiber to conventional microdrive arrays.
For the above reasons, researchers are required to optimize the selection of the type of electrode or to implant an optical fiber depending on the aim of the recording. For example tetrodes are used to achieve higher unit yield in hippocampus1,13, while silicon probes are used to investigate the laminar depth profile of cortical areas, such as the medial entorhinal cortex (MEC)37. Currently, microdrives for simultaneous implantation of tetrodes and silicon probes had been reported for rats5,11. However, it is extremely challenging to implant multiple tetrodes and silicon probes in mice because of the weight of the microdrives, limited space on the mouse head, and spatial requirements for designing the microdrive to employ different probes. Although it is possible to implant silicon probes without a microdrive, this procedure does not allow for adjustment of the probe and lowers the success rate of silicon-probe recovery12,38. Furthermore, optogenetic experiments require additional considerations in microdrive array design. This protocol demonstrates how to construct and implant a microdrive array for chronic recording in freely moving mice, which allows implantation of nine independently adjustable tetrodes and one adjustable opto-silicon probe. This microdrive array also facilitates optogenetic experiments and recovery of the silicon probe.

<table>
	<tbody>
		<tr>
			<td>#00-90 screw</td>
			<td>J.I. Morris</td>
			<td>#00-90-1/8</td>
			<td>EIB screws</td>
		</tr>
		<tr>
			<td>#0-80 nut</td>
			<td>Small Parts</td>
			<td>B00DGB7CT2</td>
			<td>brass nut for holding fiber ferrule holder</td>
		</tr>
		<tr>
			<td>#0-80 screw</td>
			<td>Small Parts</td>
			<td>B000FMZ57G</td>
			<td>brass machine screw for probe connector mount, fiber ferrule holder, and shielding cone</td>
		</tr>
		<tr>
			<td>22 Ga polyetheretherketone tubes</td>
			<td>Small Parts</td>
			<td>SLPT-22-24</td>
			<td>for attaching to the shuttle, 0.025 inches inner diameter</td>
		</tr>
		<tr>
			<td>23 Ga stainless tubing</td>
			<td>Small Parts</td>
			<td>HTX-23R</td>
			<td>for tetrode</td>
		</tr>
		<tr>
			<td>23 Ga stainless wire</td>
			<td>Small Parts</td>
			<td>HTX-23R-24-10</td>
			<td>for L-shape/support wire</td>
		</tr>
		<tr>
			<td>26 Ga stainless wire</td>
			<td>Small Parts</td>
			<td>GWX-0200</td>
			<td>for guide-posts</td>
		</tr>
		<tr>
			<td>30 Ga stainless wire</td>
			<td>Small Parts</td>
			<td>HTX-30R</td>
			<td>for tetrode</td>
		</tr>
		<tr>
			<td>3-D CAD software package</td>
			<td>Dassault Syst&#232;mes</td>
			<td>SolidWorks 2003</td>
			<td></td>
		</tr>
		<tr>
			<td>3D printer</td>
			<td>FormLab</td>
			<td>Form2</td>
			<td></td>
		</tr>
		<tr>
			<td>5.5mil polyimide insulating tubes</td>
			<td>HPC Medical</td>
			<td>72113900001-012</td>
			<td></td>
		</tr>
		<tr>
			<td>aluminum foil tape</td>
			<td>Tyco</td>
			<td>Tyco Adhesives 617022 Aluminum Foil Tape</td>
			<td>for the alternative shielding cone</td>
		</tr>
		<tr>
			<td>conductive paste</td>
			<td>YSHIELD</td>
			<td>HSF54</td>
			<td>for shielding cone</td>
		</tr>
		<tr>
			<td>customized screws for silicon-probe microdrive</td>
			<td>AMT</td>
			<td>UNM1.25-HalfMoon</td>
			<td>half-moon stainless screw, 1.5 mm diameter, 300 &#181;m thread pitch</td>
		</tr>
		<tr>
			<td>customized screws for tetrode microdrive</td>
			<td>AMT</td>
			<td>Yamamoto_0000-160_9mm</td>
			<td>slotted stainless screw, 0.5 mm diameter, 160 &#181;m thread pitch, custom-made to order for our design</td>
		</tr>
		<tr>
			<td>dental acrylic</td>
			<td>Stoelting</td>
			<td>51459</td>
			<td></td>
		</tr>
		<tr>
			<td>dental model resin</td>
			<td>FormLab</td>
			<td>RS-F2-DMBE-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Dremel rotary tool</td>
			<td>Dremel</td>
			<td>model 800</td>
			<td>a grinder</td>
		</tr>
		<tr>
			<td>drill bit</td>
			<td>Fine Science Tool</td>
			<td>19007-05</td>
			<td></td>
		</tr>
		<tr>
			<td>electric interface board</td>
			<td>Neuralynx</td>
			<td>EIB-36-Narrow</td>
			<td></td>
		</tr>
		<tr>
			<td>epoxy</td>
			<td>Devcon</td>
			<td>GLU-735.90</td>
			<td>5 minutes epoxy</td>
		</tr>
		<tr>
			<td>eye ointment</td>
			<td>Dechra</td>
			<td>Puralube Ophthalmic Ointment</td>
			<td>to prevent mice eyes from drying during surgery</td>
		</tr>
		<tr>
			<td>fiber polishing sheet</td>
			<td>Thorlabs</td>
			<td>LFG5P</td>
			<td>for polishing the optical fiber</td>
		</tr>
		<tr>
			<td>fine tweezers</td>
			<td>Protech International</td>
			<td>15-368</td>
			<td>for loading/recovering the silicon probe</td>
		</tr>
		<tr>
			<td>gold pins</td>
			<td>Neuralynx</td>
			<td>EIB Pins Small</td>
			<td></td>
		</tr>
		<tr>
			<td>ground wire</td>
			<td>A-M Systems</td>
			<td>781500</td>
			<td>0.010 inch bare silver wire</td>
		</tr>
		<tr>
			<td>headstage preamp</td>
			<td>Neuralynx</td>
			<td>HS-36</td>
			<td></td>
		</tr>
		<tr>
			<td>impedance meter</td>
			<td>BAK electronics</td>
			<td>Model IMP-2</td>
			<td>1 kHz testing frequency</td>
		</tr>
		<tr>
			<td>mineral oil</td>
			<td>ZONA</td>
			<td>36-105</td>
			<td>for lubricating screws and wires</td>
		</tr>
		<tr>
			<td>optical fiber</td>
			<td>Doric</td>
			<td>MFC_200/260-0.22_50mm_ZF1.25(G)_FLT</td>
			<td></td>
		</tr>
		<tr>
			<td>Recording system</td>
			<td>Neuralynx</td>
			<td>Digital Lynx 4SX</td>
			<td></td>
		</tr>
		<tr>
			<td>ruby fiber scribe</td>
			<td>Thorlabs</td>
			<td>S90R</td>
			<td>for cleaving the optical fiber</td>
		</tr>
		<tr>
			<td>silicon grease</td>
			<td>Fine Science Tool</td>
			<td>29051-45</td>
			<td></td>
		</tr>
		<tr>
			<td>silicon probe</td>
			<td>Neuronexus</td>
			<td>A1x32-Edge-5mm-20-177</td>
			<td>Fig. 3, 4A, 4B, 5</td>
		</tr>
		<tr>
			<td>silicon probe</td>
			<td>Neuronexus</td>
			<td>A1x32-6mm-50-177</td>
			<td>Fig. 4C</td>
		</tr>
		<tr>
			<td>silicon probe washing solution</td>
			<td>Alcon</td>
			<td>AL10078844</td>
			<td>contact lens cleaner</td>
		</tr>
		<tr>
			<td>silicone lubber</td>
			<td>Smooth-On</td>
			<td>Dragon Skin 10 FAST</td>
			<td>for preparation of microdrive mold</td>
		</tr>
		<tr>
			<td>silver paint</td>
			<td>GC electronic</td>
			<td>22-023</td>
			<td>silver print II coating, used for ground wires</td>
		</tr>
		<tr>
			<td>skull screw</td>
			<td>Otto Frei</td>
			<td>2647-10AC</td>
			<td>0.8 mm diameter, 0.200 mm thread pitch</td>
		</tr>
		<tr>
			<td>standard surgical scissors</td>
			<td>ROBOZ</td>
			<td>RS-5880</td>
			<td></td>
		</tr>
		<tr>
			<td>stereotaxic apparatus</td>
			<td>Kopf</td>
			<td>Model 942</td>
			<td></td>
		</tr>
		<tr>
			<td>super glue</td>
			<td>Loctite</td>
			<td>LOC230992</td>
			<td>for applying to guide-posts</td>
		</tr>
		<tr>
			<td>surgical tweezers</td>
			<td>ROBOZ</td>
			<td>RS-5135</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrode Twister</td>
			<td>Jun Yamamoto</td>
			<td>TT-01</td>
			<td></td>
		</tr>
		<tr>
			<td>tetrode wires</td>
			<td>Sandvik</td>
			<td>PX000004</td>
			<td></td>
		</tr>
	</tbody>
</table>

hybrid microdrive system with recoverable opto-silicon probe and tetrode for dual-site high density recording in freely moving mice

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

The microdrive array was constructed within 5 days. The timeline of microdrive preparation is described in Table 2. Using this microdrive, nine tetrodes and one silicon probe were implanted into the hippocampal CA1 and MEC of the mouse [21 week old/29 g body weight male pOxr1-Cre (C57BL/6 background)], respectively. This transgenic mouse expresses Cre in MEC layer III pyramidal neurons. The mouse was injected with 200 nL of AAV5-DIO-ChR2-YFP (titer: 7.7 x 1012 gc/mL) into the MEC 10 weeks befo...

Watch this Scientific Journal Video about Hybrid Microdrive System with Recoverable Opto-Silicon Probe and Tetrode for Dual-Site High Density Recording in Freely Moving Mice at JoVE.com

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

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

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

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