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Neuroscience

Using Baseplating and a Miniscope Preanchored with an Objective Lens for Calcium Transient Research in Mice

Published: June 5th, 2021

DOI:

10.3791/62611

1Department of Veterinary Medicine, School of Veterinary Medicine, National Taiwan University

The shrinkage of dental cement during curing displaces the baseplate. This protocol minimizes the problem by creating an initial foundation of the dental cement that leaves space to cement the baseplate. Weeks later, the baseplate can be cemented in position on this scaffold using little new cement, therebyreducing shrinkage.

Neuroscientists use miniature microscopes (miniscopes) to observe neuronal activity in freely behaving animals. The University of California, Los Angeles (UCLA) Miniscope team provides open resources for researchers to build miniscopes themselves. The V3 UCLA Miniscope is one of the most popular open-source miniscopes currently in use. It permits imaging of the fluorescence transients emitted from genetically modified neurons through an objective lens implanted on the superficial cortex (a one-lens system), or in deep brain areas through a combination of a relay lens implanted in the deep brain and an objective lens that is preanchored in the miniscope to observe the relayed image (a two-lens system). Even under optimal conditions (when neurons express fluorescence indicators and the relay lens has been properly implanted), a volume change of the dental cement between the baseplate and its attachment to the skull upon cement curing can cause misalignment with an altered distance between the objective and relay lenses, resulting in the poor image quality. A baseplate is a plate that helps mount the miniscope onto the skull and fixes the working distance between the objective and relay lenses. Thus, changes in the volume of the dental cement around the baseplate alter the distance between the lenses. The present protocol aims to minimize the misalignment problem caused by volume changes in the dental cement. The protocol reduces the misalignment by building an initial foundation of dental cement during relay lens implantation. The convalescence time after implantation is sufficient for the foundation of dental cement to cure the baseplate completely, so the baseplate can be cemented on this scaffold using as little new cement as possible. In the present article, we describe strategies for baseplating in mice to enable imaging of neuronal activity with an objective lens anchored in the miniscope.

Fluorescent activity reporters are ideal for imaging of the neuronal activity because they are sensitive and have large dynamic ranges1,2,3. Therefore, an increasing number of experiments are using fluorescence microscopy to directly observe neuronal activity1,2,3,4,5,6,7,8,9

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All procedures performed in this study were approved by the National Taiwan University Animal Care and Use Committee (Approval No.: NTU-109-EL-00029 and NTU-108-EL-00158).

1. Assessment of the volume alteration of dental cement

NOTE: Changes in the volume of dental cement occur during the curing process. Test the volume changes of dental cement before implantation and baseplating. Researchers can test any brand of dental cement an.......

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Assessment of the dental cement volume alteration
Since the volume of dental cement changes during the curing process, it may significantly impact the imaging quality, given that the working distance of a GRIN lens is approximately 50 to 350 µm4,8. Therefore, two commercially available dental cements were tested in this case, Tempron and Tokuso, before the implantation and baseplating procedure (Figure 5)........

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The present report describes a detailed experimental protocol for researchers using the two-lens UCLA Miniscope system. The tools designed in our protocol are relatively affordable for any laboratory that wishes to try in vivo calcium imaging. Some protocols, such as viral injection, lens implantation, dummy baseplating, and baseplating, could also be used for other versions of the miniscope system to improve the success rate of calcium imaging. Other than general problems with viral injection, and lens implanta.......

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This work was supported by the Ministry of Science and Technology, Taiwan (108-2320-B-002 -074, 109-2320-B-002-023-MY2).

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Name Company Catalog Number Comments
0.7-mm drill bit  #19008-07 Fine Science Tools; USA for surgery
0.1–10 μl pipette tips 104-Q; QSP Fisher Scientific; Singapore for testing dental cement
20 G IV cathater #SR-OX2032CA Terumo Corporation; Tokyo, Japan for surgery
27 G needle AGANI, AN*2713R Terumo Corporation; Tokyo, Japan for surgery
AAV9-syn-jGCaMP7s-WPRE #104487-AAV9; 1.5*10^13 Addgene viral prep; MA, USA for viral injection
Atropine sulfate Astart; Hsinchu, Taiwan for surgery/dummy baseplating/baseplating
Baseplate V3 http://miniscope.org for dummy baseplating/baseplating
BLU TACK #30840350 Bostik; Chelsea, Massachusetts, USA Reusable adhesive clay; for surgery/dummy baseplating/baseplating
Bone Rongeur Friedman 13 cm Diener; Tuttlingen, Germany for baseplating
Buprenorphine INDIVIOR; UK for surgery
Carprofen Rimadyl Zoetis; Exton, PA analgesia
Ceftazidime Taiwan Biotech; Taiwan prevent infection
Data Acquisition PCB for UCLA Miniscope purchased on https://www.labmaker.org/collections/neuroscience/products/data-aquistion-system-daq for baseplating
Dental cement set Tempron GC Corp; Tokyo, Japan for testing dental cement
Dental cement set Tokuso Curefast Tokuyama Dental Corp.; Tokyo, Japan for testing dental cement/surgery/dummy baseplating/baseplating
Dual Lab Standard with Mouse and Rat Adaptors #51673 Stoelting Co; Illinois, USA for surgery/dummy baseplating/baseplating
Duratear ointment Alcon; Geneva, Switzerland for surgery/dummy baseplating/baseplating
Ibuprofen YungShin; Taiwan analgesia
Isoflurane Panion & BF Biotech INC.; Taoyuan, Taiwan for surgery/dummy baseplating/baseplating
Inscopix nVista System Inscopix; Palo Alto, CA for comparison with V3 UCLA Miniscope
Ketamine Pfizer; NY, NY for euthanasia
Normal saline for surgery
Micro bulldog clamps #12.102.04 Dimedo; Tuttlingen, Germany for lens implantation
Microliter Microsyringes, 2.0 µL, 25 gauge #88400 Hamilton; Bonaduz, Switzerland for viral injection
Molding silicone rubber ZA22 Thixo Zhermack; Badia Polesine, Italy for dummy baseplating
Objective Gradient index (GRIN) lens #64519 Edmund Optics; NJ, USA for dummy baseplating/baseplating
Parafilm #PM996 Bemis; Neenah, USA for dummy baseplating
Portable Suction #DF-750 Doctor's Friend Medical Instrument Co., Inc., Taichung, Taiwan for surgery
Relay GRIN lens #1050-002177 Inscopix; Palo Alto, CA, USA for dummy baseplating/baseplating
Stainless steel anchor screws 1.00 mm diameter, total length 3.00 mm for surgery
Stereo microscope #SL720 Sage Vison; New Taipei City, Taiwan for surgery/dummy baseplating/baseplating
Stereotaxic apparatus #51673 Stoelting; IL, USA for surgery/dummy baseplating/baseplating
UV Cure Adhesive #3321 Loctite; Düsseldorf, Germany for testing dental cement
V3 UCLA Miniscope purchased on https://www.labmaker.org/products/miniscope-complete-set-of-components for surgery/dummy baseplating/baseplating
Xylazine X1126 Sigma-Aldrich; St. Louis, MO for euthanasia
Xylocaine pump spray 10% AstraZeneca; Södertälje, Sweden for surgery

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