Name: using baseplating and a miniscope preanchored with an objective lens for calcium transient research in mice
Uploaded: 2021-06-05T15:00:00
Description: Watch this Scientific Journal Video about Using Baseplating and a Miniscope Preanchored with an Objective Lens for Calcium Transient Research in Mice at JoVE.com

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

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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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...

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 activity1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16. The first miniaturized one-photon fluorescence microscope (miniscope) was designed in 2011 by Mark Schnitzer et al.5. This miniscope enables researchers to monitor the fluorescence dynamics of cerebellar cells in freely behaving animals5 (i.e., without any physical restraint, head restraint, sedation, or anesthesia to the animals). Currently, the technique can be applied to monitor superficial brain areas such as the cortex6,8,15,16; subcortical areas such as the dorsal hippocampus8,11,13,14 and striatum6,17; and deep brain areas such as the ventral hippocampus14, amygdala10,18, and hypothalamus8,12.
In recent years, several open-source miniscopes have been developed4,5,6,7,11,13,17,19. The miniscope can be economically assembled by researchers if they follow the step-by-step guidelines provided by the University of California, Los Angeles (UCLA) Miniscope team4,7,11,13. Because optical monitoring of neural activity is restricted by the limitations of light transmission7 to and from the neuronal population of interest, a miniscope was designed that requires an objective gradient refractive index (GRIN) lens (or objective lens) to be preanchored at the bottom of the miniscope to magnify the field of view that is relayed from a relay GRIN lens (or relay lens)6,7,8,10,16,17. This relay lens is implanted into the target brain region such that the fluorescence activity of the target brain region is relayed onto the surface of relay lens6,7,8,10,16,17. Approximately 1/4 of a full sinusoidal period of light travels through the objective GRIN lens (~ 0.25 pitch) (Figure 1A1), resulting in a magnified fluorescence image6,7. The objective lens is not always fixed at the bottom of the miniscope nor is the implantation of the relay lens necessary6,7,11,13,15. Specifically, there are two configurations: one with a fixed objective lens in the miniscope and a relay lens implanted in the brain8,10,12,14,16 (Figure 1B1) and another with just a removable objective lens6,7,11,13,15 (Figure 1B2). In the design based on the fixed objective and implanted relay lens combination, the fluorescence signals from the brain are brought to the top surface of the relay lens (Figure 1A1)7,8,10,12,14,16. Subsequently, the objective lens can magnify and transmit the visual field from the top surface of the relay lens (Figure 1A2). On the other hand, the removable objective GRIN lens design is more flexible, which means preimplantation of a relay lens into the brain is not mandatory (Figure 1B2)6,7,11,13,15. When using a miniscope based on a removable objective lens design, researchers still need to implant a lens into the target brain region but they can either implant an objective lens6,7,11,13,15 or a relay lens in the brain6,7. The choice of an objective or a relay lens for implantation determines the miniscope configuration that the researcher must use. For instance, the V3 UCLA Miniscope is based on a removable objective GRIN lens design. Researchers can choose either to directly implant an objective lens in the brain region of interest and mount the &#34;empty&#34; miniscope onto the objective lens6,7,11,13,15 (a one-lens system; Figure 1B2) or to implant a relay lens in the brain and mount a miniscope that is preanchored with an objective lens6,7 (a two-lens system; Figure 1B1). The miniscope then works as a fluorescence camera to capture livestream images of neuronal fluorescence produced by a genetically encoded calcium indicator1,2,3. After the miniscope is connected to a computer, these fluorescence images can be transferred to the computer and saved as video clips. Researchers can study neuronal activity by analyzing the relative changes in fluorescence with some analysis packages20,21 or write their codes for future analysis.
The V3 UCLA Miniscope provides flexibility for users to determine whether to image neuronal activity with a one- or two-lens system7. The choice of the recording system is based on the depth and size of the target brain area. In brief, a one-lens system can only image an area that is superficial (less than approximately 2.5 mm deep) and relatively large (larger than approximately 1.8 x 1.8 mm2) because the manufacturers only produce a certain size of objective lens. In contrast, a two-lens system can be applied to any target brain area. However, the dental cement for gluing the baseplate tends to cause misalignment with an altered distance between the objective and relay lenses, resulting in a poor image quality. If the two-lens system is being used, two working distances need to be precisely targeted to achieve the optimal imaging quality (Figure 1A). These two critical working distances are between the neurons and bottom surface of the relay lens, and between the top surface of the relay lens and the bottom surface of the objective lens (Figure 1A1). Any misalignment or misplacement of the lens outside of the working distance results in imaging failure (Figure 1C2). In contrast, the one-lens system requires only one precise working distance. However, objective lens size limits its application for monitoring of deep brain regions (the objective lens that fits the miniscope is approximately 1.8 ~ 2.0 mm6,11,13,15). Therefore, implantation of an objective lens is limited for the observation of the surface and relatively large brain regions, such as the cortex6,15 and dorsal cornu ammonis 1 (CA1) in mice11,13 . In addition, a large area of the cortex must be aspirated to target the dorsal CA111,13. Because of the limitation of the one-lens configuration that prevents imaging of deep brain regions, commercial miniscope systems offer only a combined objective lens/relay lens (two-lens) design. On the other hand, the V3 UCLA miniscope can be modified into either a one-lens or two-lens system because its objective lens is removable6,11,13,15. In other words, V3 UCLA miniscope users can take advantage of the removable lens by implanting it in the brain (creating a one-lens system), when performing experiments involving superficial brain observations (less than 2.5 mm in depth), or by preanchoring it in the miniscope and implanting a relay lens in the brain (creating a two-lens system), when performing experiments involving deep brain observations. The two-lens system can also be applied for superficially observing the brain, but the researcher must know the accurate working distances between the objective lens and relay lens. The main advantage of the one-lens system is that there is a decreased chance of missing the working distances than with a two-lens system, given that there are two working distances that need to be precisely targeted to achieve optimal imaging quality in the two-lens system (Figure 1A). Therefore, we recommend using a one-lens system for superficial brain observations. However, if the experiment requires imaging in the deep brain area, the researcher must learn to avoid misalignment of the two lenses.
The basic protocol for two-lens configuration of miniscopes for experiments includes lens implantation and baseplating8,10,16,17. Baseplating is the gluing of a baseplate onto an animal&#39;s head so that the miniscope can be eventually mounted on top of the animal and videotape the fluorescence signals of neurons (Figure 1B). This procedure involves using dental cement to glue the baseplate onto the skull (Figure 1C), but the shrinkage of dental cement can cause unacceptable changes in the distance between the implanted relay lens and the objective lens8,17. If the shifted distance between the two lenses is too large, cells cannot be brought into focus.
Detailed protocols for deep brain calcium imaging experiments using miniscopes have already been published8,10,16,17. The authors of these protocols have used the Inscopix system8,10,16 or other customized designs17 and have described the experimental procedures for viral selection, surgery, and baseplate attachment. However, their protocols cannot be precisely applied to other open-source systems, such as the V3 UCLA Miniscope system, NINscope6, and Finchscope19. Misalignment of the two lenses can occur during the recording in a two-lens configuration with a UCLA Miniscope due to the type of dental cement that is used to cement the baseplate to the skull8,17 (Figure 1C). The present protocol is needed because the distance between the implanted relay lens and the objective lens is prone to shift due to the undesirable shrinkage of dental cement during the baseplating procedure. During baseplating, the optimal working distance between the implanted relay lens and the objective lens must be found by adjusting the distance between the miniscope and the top of the relay lens, and the baseplate should then be glued at this ideal location. After the correct distance between the objective lens and the implanted relay lens is set, longitudinal measurements can be obtained at cellular resolution (Figure 1B; in vivo recording). Since the optimal range of working distances of a relay lens is small (50 - 350 &#181;m)4,8, excessive cement shrinkage during curing can make it difficult to keep the objective lens and the implanted relay lens within the appropriate range. The overall goal of this report is to provide a protocol to reduce the shrinkage problems8,17 that occur during the baseplating procedure and to increase the success rate of miniscope recordings of fluorescence signals in a two-lens configuration. Successful miniscope recording is defined as recording of a livestream of noticeable relative changes in the fluorescence of individual neurons in a freely behaving animal. Although different brands of dental cement have different shrinkage rates, researchers can select a brand that has been previously tested6,7,8,10,11,12,13,14,15,16,22. However, not every brand is easy to obtain in some countries/regions due to the import regulations for medical materials. Therefore, we have developed methods to test the shrinkage rates of available dental cements and, importantly provide an alternative protocol that minimizes the shrinkage problem. The advantage over the present baseplating protocol is an increase in the success rate of calcium imaging with tools and cement that can be easily obtained in laboratories. The UCLA miniscope is used as an example, but the protocol is also applicable to other miniscopes. In this report, we describe an optimized baseplating procedure and also recommend some strategies for fitting the UCLA miniscope two-lens system (Figure 2A). Both examples of successful implantation (n= 3 mice) and examples of failed implantation (n=2 mice) for the two-lens configuration with the UCLA miniscope are presented along with the discussions for the reasons of the successes and failures.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.7-mm drill bit</td>
			<td>&#160;#19008-07</td>
			<td>Fine Science Tools; USA</td>
			<td>for surgery</td>
		</tr>
		<tr>
			<td>0.1&#8211;10 &#956;l pipette tips</td>
			<td>104-Q; QSP</td>
			<td>Fisher Scientific; Singapore</td>
			<td>for testing dental cement</td>
		</tr>
		<tr>
			<td>20 G IV cathater</td>
			<td>#SR-OX2032CA</td>
			<td>Terumo Corporation; Tokyo, Japan</td>
			<td>for surgery</td>
		</tr>
		<tr>
			<td>27 G needle</td>
			<td>AGANI, AN*2713R</td>
			<td>Terumo Corporation; Tokyo, Japan</td>
			<td>for surgery</td>
		</tr>
		<tr>
			<td>AAV9-syn-jGCaMP7s-WPRE</td>
			<td>#104487-AAV9; 1.5*10^13</td>
			<td>Addgene viral prep; MA, USA</td>
			<td>for viral injection</td>
		</tr>
		<tr>
			<td>Atropine sulfate</td>
			<td></td>
			<td>Astart; Hsinchu, Taiwan</td>
			<td>for surgery/dummy baseplating/baseplating</td>
		</tr>
		<tr>
			<td>Baseplate</td>
			<td>V3</td>
			<td>http://miniscope.org</td>
			<td>for dummy baseplating/baseplating</td>
		</tr>
		<tr>
			<td>BLU TACK</td>
			<td>#30840350</td>
			<td>Bostik; Chelsea, Massachusetts, USA</td>
			<td>Reusable adhesive clay; for surgery/dummy baseplating/baseplating</td>
		</tr>
		<tr>
			<td>Bone Rongeur Friedman</td>
			<td>13 cm</td>
			<td>Diener; Tuttlingen, Germany</td>
			<td>for baseplating</td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td></td>
			<td>INDIVIOR; UK</td>
			<td>for surgery</td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Rimadyl</td>
			<td>Zoetis; Exton, PA</td>
			<td>analgesia</td>
		</tr>
		<tr>
			<td>Ceftazidime</td>
			<td></td>
			<td>Taiwan Biotech; Taiwan</td>
			<td>prevent infection</td>
		</tr>
		<tr>
			<td>Data Acquisition PCB for UCLA Miniscope</td>
			<td>purchased on https://www.labmaker.org/collections/neuroscience/products/data-aquistion-system-daq</td>
			<td></td>
			<td>for baseplating</td>
		</tr>
		<tr>
			<td>Dental cement set</td>
			<td>Tempron</td>
			<td>GC Corp; Tokyo, Japan</td>
			<td>for testing dental cement</td>
		</tr>
		<tr>
			<td>Dental cement set</td>
			<td>Tokuso Curefast</td>
			<td>Tokuyama Dental Corp.; Tokyo, Japan</td>
			<td>for testing dental cement/surgery/dummy baseplating/baseplating</td>
		</tr>
		<tr>
			<td>Dual Lab Standard with Mouse and Rat Adaptors</td>
			<td>#51673</td>
			<td>Stoelting Co; Illinois, USA</td>
			<td>for surgery/dummy baseplating/baseplating</td>
		</tr>
		<tr>
			<td>Duratear ointment</td>
			<td></td>
			<td>Alcon; Geneva, Switzerland</td>
			<td>for surgery/dummy baseplating/baseplating</td>
		</tr>
		<tr>
			<td>Ibuprofen</td>
			<td></td>
			<td>YungShin; Taiwan</td>
			<td>analgesia</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td></td>
			<td>Panion &#38; BF Biotech INC.; Taoyuan, Taiwan</td>
			<td>for surgery/dummy baseplating/baseplating</td>
		</tr>
		<tr>
			<td>Inscopix</td>
			<td>nVista System</td>
			<td>Inscopix; Palo Alto, CA</td>
			<td>for comparison with V3 UCLA Miniscope</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td></td>
			<td>Pfizer; NY, NY</td>
			<td>for euthanasia</td>
		</tr>
		<tr>
			<td>Normal saline</td>
			<td></td>
			<td></td>
			<td>for surgery</td>
		</tr>
		<tr>
			<td>Micro bulldog clamps</td>
			<td>#12.102.04</td>
			<td>Dimedo; Tuttlingen, Germany</td>
			<td>for lens implantation</td>
		</tr>
		<tr>
			<td>Microliter Microsyringes, 2.0 &#181;L, 25 gauge</td>
			<td>#88400</td>
			<td>Hamilton; Bonaduz, Switzerland</td>
			<td>for viral injection</td>
		</tr>
		<tr>
			<td>Molding silicone rubber</td>
			<td>ZA22 Thixo</td>
			<td>Zhermack; Badia Polesine, Italy</td>
			<td>for dummy baseplating</td>
		</tr>
		<tr>
			<td>Objective Gradient index (GRIN) lens</td>
			<td>#64519</td>
			<td>Edmund Optics; NJ, USA</td>
			<td>for dummy baseplating/baseplating</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>#PM996</td>
			<td>Bemis; Neenah, USA</td>
			<td>for dummy baseplating</td>
		</tr>
		<tr>
			<td>Portable Suction</td>
			<td>#DF-750</td>
			<td>Doctor&#39;s Friend Medical Instrument Co., Inc., Taichung, Taiwan</td>
			<td>for surgery</td>
		</tr>
		<tr>
			<td>Relay GRIN lens</td>
			<td>#1050-002177</td>
			<td>Inscopix; Palo Alto, CA, USA</td>
			<td>for dummy baseplating/baseplating</td>
		</tr>
		<tr>
			<td>Stainless steel anchor screws</td>
			<td>1.00 mm diameter, total length 3.00 mm</td>
			<td></td>
			<td>for surgery</td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>#SL720</td>
			<td>Sage Vison; New Taipei City, Taiwan</td>
			<td>for surgery/dummy baseplating/baseplating</td>
		</tr>
		<tr>
			<td>Stereotaxic apparatus</td>
			<td>#51673</td>
			<td>Stoelting; IL, USA</td>
			<td>for surgery/dummy baseplating/baseplating</td>
		</tr>
		<tr>
			<td>UV Cure Adhesive</td>
			<td>#3321</td>
			<td>Loctite; D&#252;sseldorf, Germany</td>
			<td>for testing dental cement</td>
		</tr>
		<tr>
			<td>V3 UCLA Miniscope</td>
			<td>purchased on https://www.labmaker.org/products/miniscope-complete-set-of-components</td>
			<td></td>
			<td>for surgery/dummy baseplating/baseplating</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>X1126</td>
			<td>Sigma-Aldrich; St. Louis, MO</td>
			<td>for euthanasia</td>
		</tr>
		<tr>
			<td>Xylocaine pump spray 10%</td>
			<td></td>
			<td>AstraZeneca; S&#246;dert&#228;lje, Sweden</td>
			<td>for surgery</td>
		</tr>
	</tbody>
</table>

using baseplating and a miniscope preanchored with an objective lens for calcium transient research in mice

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.

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 &#181;m4,8. Therefore, two commercially available dental cements were tested in this case, Tempron and Tokuso, before the implantation and baseplating procedure (Figure 5)....

Watch this Scientific Journal Video about Using Baseplating and a Miniscope Preanchored with an Objective Lens for Calcium Transient Research in Mice at JoVE.com

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

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 ...

This miniscope can reduce the shrinkage problem with dental cement during the baseplate procedure and increase the success rate of miniscope recordings of fluorescence signals in two length configuration. If the fluorescence transient cannot be observed during baseplating, check the expression of calcium indicator and location of a relay lens on the brain slice. Start with mounting the inner needle of a 20 gauge intravenous catheter with a diameter of one millimeter onto the stereotaxic arm.Lower the catheter into the ventral corn ammonis 1 into the target region. Then, lower the microinjection needle at a speed of 100 to 200 micrometers per minute into the ventral cornu ammonis 1, and infuse 200 nanoliters of the viral vector into the target region at a speed of 25 nanoliters per minute. After allowing the virus to diffuse for 10 minutes, withdraw the microinjection needle at a speed of 100 to 200 micrometers per minute.After disinfecting the relay lens, soak the lens in cold pyrogen-free saline until implantation. Using a micro bulldog clamp with heat shrink tubing, hold the relay lens and place it on top of the target region at a speed of 100 to 200 micrometers per minute. Then, stabilize the lens with dental cement.To set a miniscope, fasten the objective lens on the bottom and assemble the base plate onto the miniscope. Wrap 10 centimeters of paraffin film around the outside of the baseplate. Using reusable adhesive clay, hold the miniscope with the stereotaxic arm probe.Align the objective lens on top of the relay lens with as little space between the lenses as possible. Use dental cement to secure the positioning of the baseplate, such that the cement touches only the paraffin film. When the dental cement has dried, remove the paraffin film, the base plate, and the miniscope, keeping the dental cement base hollow.Seal the relay lens with molding silicone rubber and cover the silicone rubber with a thin layer of dental cement. Disengage the mouse from the stereotaxic instruments and place the mouse into a recovery chamber. For baseplating, cut the thin layer of the dental cement roof carefully with a bone rongeur and remove the silicone rubber.Clean the surface of the relay lens with 75%alcohol. Fasten the set screw beside the baseplate to fix it to the bottom of the miniscope. Adjust the focus slide to be approximately 2.7 to 3 millimeters from the main housing.When the setting is done, connect the miniscope to the data acquisition board and plug the miniscope into a USB 3.0 port on a computer. Run the data acquisition software developed by the UCLA team. Adjust the exposure to 255, the gain to 64x and the excitation LED to 5%To connect the miniscope to the data acquisition software, click the connect button and watch the livestream of the data acquisition software considering the margin of the relay lens as a landmark.Once the relay lens is found, align the miniscope objective lens to the relay lens by hand. Adjusting the various angles and distances of the miniscope, begin searching for the fluorescence signals. Holding the miniscope at the optimal position, move the stereotaxic arm towards the mini scope and adhere the miniscope to the stereotaxic arm with reusable adhesive clay.Search for the best view by adjusting the X, Y, and Z arms slightly. Adjust the angle between the surface of the objective lens and relay lens by turning the ear bar, tooth bar, or reusable adhesive clay on the Z axis. Viral expression and lens implantation are successful when at least one cell displaced fluorescence transients during baseplating.Fix the base plate firmly with the smallest possible amount of dental cement while monitoring the region of interest to ensure that the optimal position does not change. In the representative study, the procedure of dummy baseplating proved advantageous as it resulted in a smaller shift in the baseplate location than the original procedure. The shift of the baseplate was also influenced by the height of the dental cement.The baseplate that was anchored 0.5 centimeters above the skull shifted significantly less than the baseplate that was fixed one centimeter above the skull. In an example of successful imaging during the base plating procedure fluorescence transients along with blood vessels were noticed in the anesthetized animal. Fluorescence transients were observed five days after baseplating even when the mouse was freely behaving in its home cage with only a slight shift in a position.Subsequent calcium imaging of mice was carried out. In a few instances, problems occurred during the baseplating procedure, such as the absence of anything except a uniform background or visibility of white cell margins. without fluorescence transients.It is important to fix the baseplate firmly on the target region with the smallest possible amount of dental cement. With slate technique, researchers can longitudinally observe neural activity across this. With the proper license, this procedure can also apply to observe the neural network in the field of view.

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