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, ...

Our hybrid microdrive design allows experimenters to not only record from multiple brain regions, but also to choose different types of electrodes to optimize yield and stability. We combined nine tetrodes, a silicon linear probe, and fiber optics that are adjustable and reusable in a microdrive array for in vivo experiments with freely moving mice. This technique can easily be adapted to other larger animals such as rats or marmosets.Silicon probes are extremely fragile. We need to be careful when dealing with the probes, for example, when adapting the optical fiber and when rotating the shuttle assembly into the microdrive. The microdrive array consists of five parts.To prepare the microdrive screw for the silicon probe first 3D print a plastic pattern of the microdrive and place tape around the pattern to make a temporary wall around the pattern. Pour liquid silicon gel into the pattern and remove air bubbles with gentle shaking. When the silicon has cured, carefully remove the silicon gel mold from the pattern.Next use a rotary grinder to cut 18 and 9.5mm lengths of 23 gauge stainless steel wire tube and use the grinder to roughen the top 2 to 3mm of the tubes. Apply a small volume of silicon oil to a custom screw and set the tubes and screw into the mold. Pour dental acrylic into the mold and use a syringe to eliminate any air bubbles around the tubes and the screw.When the dental acrylic has fully cured remove the microdrive screw from the mold and use pliers to bend 6mm from one end of the 18mm wire tip to a 60 degree angle. Using a turning tool rotate the microdrive screw to check the quality then install the microdrive screw into the microdrive array body and turn the screw to check whether it is able to move up and down smoothly. To prepare the shuttle for the silicon probe use sharp scissors to cut two 5mm lengths of PEEK and align the tubes at both sides of the shuttle.Glue the tubes to the shuttle with epoxy and apply a small volume of silicon oil to the guide posts. When the epoxy has dried, place the shuttle over the guide posts of the microdrive array body to check the quality. The shuttle should move smoothly without excessive friction.To prepare the optorode use a Rubi cutter to cleave a piece of optical fiber to 21mm in length and grind the fiber tip until it is flat and shiny. When the tip is ready, gently place the optical fiber on the front side of the silicon probe 200 to 300 micrometers above the top of the electrode sites and temporarily secure the fiber with transparent tape then glue the optical fiber to the base of the silicon probe with a small volume of epoxy and allow the epoxy to cure for five hours. To attach the shuttle to the silicon probe apply a small volume of epoxy at the back of the silicon probe base and gently hold the bottom part of the shuttle against the silicon probe base for two to three minutes to avoid the formation of a gap between the shuttle and the silicon probe base.When the epoxy has completely cured hold the groove of the shuttle with fine tweezers and use a microscope to carefully place the shuttle tubes on to the guide posts of the main body. Turn the screw to insert the microdrive screw into the screw hole and insert the tip of the L shaped tube into the groove of the shuttle head to engage the silicon probe and microdrive screw. Next cut two number 0 screws to a 3.5mm thread length and grind the tips to remove burrs.Place the probe connector holder on the array body and insert the silicon probe electrical connector into the holder. Insert the number 0 screws to hold the probe connector holder. Use epoxy to secure the silicon probe connector in the holder taking care not to glue the probe to the microdrive array body.To attach the ferrule holder to the opto-silicon probe and the microdrive array body, cut two number 0 screws to a 6mm thread length and grind the tips. Grind the outside of two number 0 machine screw nuts to make small hex nuts with 2.5 to 3mm outer diameters and insert the number 0 screw into component A of the holder. Glue the screw heads with epoxy.Apply a small volume of silicon grease to components A and B to reduce the friction with the body. Use inverse tweezers to temporarily insert component A into the body and place component B on to the screws of component A.Thread the customized nuts on to the screws and use pliers to tighten the nuts to secure the ferrule holder on to the body. Insert the fiber ferrule into the groove of the fiber ferrule holder taking care that the fiber ferrule is sticking 4 to 5mm out from the holder.Apply a small amount of epoxy between the ferrule and the holder groove. When the epoxy is cured, loosen the nuts and turn the microdrive screw to check the shuttle and ferrule holder for smooth motion then confirm that the probe tip completely retracts into the body when the ferrule holder is at the top position and the shuttle tubes are still associated with the guide posts. To attach the shield cone, fasten two 3.5mm number 0 screws from the outside of the cone to hold the microdrive in place.The microdrive array can be set up within five days if the construction is completed according to the timeline outlined in the table. After the tetrode adjustment behavioral performance can be tested on a linear track and in an open field. In both types of experiments the mouse is allowed to explore freely for approximately 30 minutes while the electrophysiological signals are recorded without severe motion related noise throughout the recording session.Light stimulation can then be performed at the medial entorhinal cortex to stimulate the medial entorhinal cortex layer 3 neurons that project to the CA1 subfield of the hippocampus. Spontaneous spiking activities and local field potentials are then recorded from the tetrodes and the silicon probe when the mouse is sleeping. In this representative experiment the left field potentials recorded in the tetrodes demonstrated large ripple activities suggesting that all of the tetrodes were positioned in the vicinity of the CA1 pyramidal cell layer.Light induced responsive activities in this assay were first observed in the medial entorhinal cortex followed by activities within CA1 with 13 to 18 milliseconds of latency. Take care to insert the opto-silicon probe into the microdrive body in a electrostatic free environment. The shielding Cone can be replaced with materials such as a sheet of paper and tape.This will decrease the rate of the microdrive by up to 20%Our microdrive design provides flexible choices for effective combination of recording electrodes to measure and manipulate multiple brain regions and to investigate the dynamics and interactions of different brain structures.

Research

JoVE Journal

Neuroscience

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

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

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

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

Extracellular Wire Tetrode Recording in Brain of Freely Walking Insects

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

Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle (Mercynorrhina torquata)

Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle Mercynorrhina torquata

The rise of radio-enabled digital electronic devices has prompted the use of small wireless neuromuscular recorders and stimulators for studying in-flight ...

Fabrication of High Contact-Density, Flat-Interface Nerve Electrodes for Recording and Stimulation Applications

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

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

Noninvasive EEG Recordings from Freely Moving Piglets

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

Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and ...

Intracerebroventricular Delivery of Gut-Derived Microbial Metabolites in Freely Moving Mice

The impact of gut microbiota and their metabolites on host physiology and behavior has been extensively investigated in this decade. Numerous studies have ...

Extracellular Electrophysiological Recording in the Motor Cortex of Mice

Multichannel Extracellular Recording in Freely Moving Mice

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

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