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Jun 2020

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An Improved Method for Individual Tracking of Voluntary Wheel Running in Pair-housed Juvenile Mice
一种对饲养的幼鼠自主跑轮运动个体跟踪的改进方法   

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Abstract

Rodent cages equipped with access to a voluntary running wheel are commonly used to study the effects of aerobic physical activity on physiology and behavior. Notable discoveries in exercise neurobiology, including the key role of brain-derived neurotrophic factor (BDNF) in neural plasticity and cognition, have been made using rodents housed with voluntary running wheels. A major advantage of using home-cage running wheels over treadmills is the elimination of stress potentially associated with forced running. In addition, voluntary wheel running may simulate a more natural running pattern in laboratory mice. Singly housing mice with voluntary running wheels is traditionally employed to obtain exact quantitation of the distance ran; however, social isolation stress is often ignored to obtain precise running distances. Moreover, voluntary exercise studies in adolescent mice must consider the neurodevelopmental implications of isolation stress. In this protocol, we wean 21-day-old mouse pups directly into running wheel-equipped cages and pair-house them to reduce the impact of social isolation and other developmentally specific factors that could adversely affect their behavior or development. Individual running distances are obtained from each mouse in the cage using a radio-frequency identification (RFID) ear tag and a hidden antenna placed directly under the running wheel. We have demonstrated that voluntary running during a specific juvenile-adolescent developmental period can improve hippocampal memory when tested during adolescence (Ivy et al., 2020). Individual exercise tracking of group-housed mice can enable future studies to precisely correlate the amount of exercise with readouts such as cell-specific gene expression, epigenetic mechanisms, serum biomarkers, and behavior, in an intra-individual manner.


Graphic abstract:



Figure 1. Illustration of the dual RFID and Vital View system for individual mouse running in a pair-housed cage


Keywords: Group-housing (群居), Exercise (运动), Voluntary wheel running (自主跑轮运动), In-cage mouse tracking (笼子里的老鼠跟踪), Radio frequency identification (RFID) (射频识别(RFID)), Enrichment (富集), Juvenile mice (幼鼠)

Background

The use of voluntary running wheels in laboratory rodent cages is a common approach for investigating the impact of exercise on brain function and neurodegeneration (Liu et al., 2019). Employing a voluntary exercise study design eliminates the need for a stressful stimulus, such as a foot shock or investigator handling, to encourage the animal to exercise. Studies utilizing in-cage voluntary running wheels have traditionally housed rodents individually to generate accurate running distances and capture individual variations in exercise amounts (Goh and Ladiges, 2015). Individual running data can then be correlated with other intra-individual datasets. For example, total distance run over a 2-6 hour period positively correlates with brain-derived neurotrophic factor (BDNF) gene expression in the hippocampus (Oliff et al., 1998). However, a disadvantage of individual housing is the potential for stress induced by social isolation. Since mice are social animals, their innate social behaviors (whether in wild or captive environments) develop most naturally in social arrangements similar to those found in wild colonies (Reimer and Petras, 1967). The controlled laboratory environments required for accurate data collection frequently result in housing conditions that challenge the formation of natural social structures. The lack of a social environment when mice are individually housed must be considered not only for rodent welfare but also for its impact on the quality of data produced and the interpretation of findings (Kappel et al., 2017; Arakawa, 2018).


Prior research has demonstrated that individual housing of mice can impact brain function in a number of ways. It can alter hypothalamic-pituitary-adrenocortical (HPA) axis function, specifically glucocorticoid regulation and feedback (Hawkley et al., 2012). Individual housing can also confound performance on various behavioral tests, including those assessing anxiety (Koike et al., 2009) and learning and memory (Okada et al., 2015). Finally, single housing can lower the expression of neuroplasticity-related genes in the hippocampus and prefrontal cortex (Ieraci et al., 2016). Social isolation stress in singly housed rodents is therefore an important variable to consider when interpreting studies that assess the neurobiological effects of voluntary exercise.


On the other hand, group housing of mice can invoke male-on-male aggression. This usually emerges in rodents after the onset of puberty and tends not to be present during juvenile and adolescent developmental stages (Terranova et al., 1998). Female mice have a lower tendency to exhibit this aggression, even post-puberty (Hayes, 2000). Social isolation in juvenile rodents adversely impacts myelination in the medial prefrontal cortex (Makinodan et al., 2012), whereas social play in juveniles can enhance neural plasticity in this region (Himmler et al., 2013). Moreover, juvenile mice use the body temperature of their cage mates for thermoregulation (Batchelder et al., 1983). This underscores the importance of paired or grouped housing for maintaining basal body temperature, particularly in the setting of shifting metabolic demands with exercise. Therefore, in studies linking juvenile voluntary wheel running with neural function and behavior, a group housing-based approach may be preferred to eliminate isolation stress.

Individual home-cage rodent tracking has been accomplished through the use of video (Krynitsky et al., 2020) (Poffe et al., 2018) (Wang et al., 2018), subcutaneously implanted RFID microchips (Peleh et al., 2019) (Frahm et al., 2018), a combination of the two, or passive infrared sensors (Matikainen-Ankney, Garmendia-Cedillos et al., 2019). Current protocols utilizing subcutaneously implanted RFID microchips require prolonged restraint and anesthesia, which may produce undesired stress and pain. Unified Information Devices details a protocol that describes safely implanting a mouse RFID microchip (UID UC-1485) without anesthesia on postnatal day 12. This approach is used primarily for mouse identification at a single point in time, such as for taking rapid mouse inventory within a group-housed cage, but its use has not been demonstrated for live, continuous tracking. Another factor is the size of the RFID chip, which must be large enough to be detected by the antenna. For juvenile mice, typically weighing 6-9 grams, this size requirement prohibits administration without the use of anesthesia. In video tracking, wire cage tops obstruct continuous top-down video recording but are required for many home-cage running wheel systems. Finally, existing home-cage RFID activity-tracking systems currently on the market use a matrix of RFID antennae arranged throughout the base of the cage or in strategic locations to monitor baseline ambulatory activity (Voikar and Gaburro, 2020). However, if the objective of the experiment is to track voluntary wheel-running, only one antenna with a read range precisely limited to the area inside the wheel is required.


Our apparatus uses strong, low-profile RFID ear tags and one RFID antenna at the base of each cage to present a minimally invasive alternative to existing video-based and implantation-based tracking systems (Figure 1). The major advantage of our protocol is the ability to individually track running wheel activity of pair-housed juvenile mice starting at the age of weaning (postnatal day 21). We reduce isolation stress and issues with thermoregulation by pair-housing the mice and providing nesting material. Indeed, this model can be scaled up to >2 mice per cage if preferred. This procedure has been tested in cages containing mice of the same sex, but we did not investigate mixed-sex population effects on individual running activity. Our experimental design allows for the comparison of individual running distances between female and male pairs of mice within the same cage. Although there may be territorial issues once social hierarchy is established in cages housing two or more male mice, a precise quantity of exercise from each individual mouse in a group-housed cage can be correlated with any desired experimental readout.


A minor limitation of this apparatus is that in our hands, about 5-10% of running activity is unable to be reconciled; however, we believe that our 90-95% accuracy of allocating individual running data in group-housed environments is an acceptable yield. Although this method does not encounter the same issues with accuracy-diminishing antenna cross-talk as observed in RFID antenna matrices (Voikar and Gaburro, 2020), the metal wheel in very close proximity to the antenna may have the same interfering effect (thus necessitating a large ear tag). Our design effectively limits the read range of the antenna to the wheel area only.


Investigating the effects of voluntary physical activity has numerous implications for increasing our understanding of its benefits toward brain function and behavior. Determining individualized amounts of exercise for specific outcomes or targets requires accurate monitoring of running distances, which can now be performed as described in this protocol. Our approach can track individual mice by recognizing the unique RFID tags of pair-housed mice coupled with time-stamped data obtained from a magnetic sensor on a home-cage running wheel. Importantly, the procedure can be scaled up to tracking running distances of multiple mice in a cage sharing access to one running wheel. Our method brings RFID applications to juvenile mouse exercise studies, while minimizing stress from isolation and restraint, eliminating the need for anesthesia, and producing highly precise and individualized running data.

Materials and Reagents

  1. UHF RFID Tag for Metal Product Management (Murata Manufacturing, catalog number: LXTBKZMCMG-010)

  2. CommandTM Small Poster Strips (3M, catalog number: 17024ES)

  3. Pegboard, 5/32” thickness, holes spaced 1” apart

  4. LOCTITE® SUPER GLUE PRECISION PEN (Henkel, catalog number: 2066118)

  5. Netting material (Industrial Netting, catalog number: NG3060-164)

  6. Ear Tag, Mouse, Light Blue, 1-100 (Stoelting, catalog number: 56782)

  7. Ear Tag, Backing only, Pk/100 (Stoelting, catalog number: 56792)

  8. Etching Engraver Pen (Porsin, catalog number: LX1323)

  9. Teklad 1/8” Corncob Bedding (Envigo, catalog number: 7092A)

  10. Cotton Nestlets (Ancare nestlets)

  11. Animals: Jackson mice WT C57BL/6J (Jackson Laboratories, catalog number: 000664)

    Mice were progeny of C57BL/6J dams obtained from Jackson Laboratories, and were bred, born, and reared in our vivarium. Mice had free access to food and water, and the lights were maintained on a 12-hour light/dark cycle. Upon weaning on postnatal day (P) 21, mice were pair-housed in standard cages with free access to an in-cage stainless-steel running wheel equipped with a plastic net fitted around the rim for the safety of the juvenile mice. All mice are typically 6-9 g at the time of weaning. Tracking was conducted continuously 24 hours a day for three weeks from P21 to P41. All experiments were conducted according to U.S. National Institutes of Health guidelines for animal care and use and were approved by the Institutional Animal Care and Use Committee of the University of California, Irvine.

  12. Teklad Global Soy Protein-Free Extruded Rodent Diet (Envigo, catalog number: 2020X)

Equipment

  1. RFID Passive Antenna (Abracon LLC: ARRAN5-915.000MHz)

  2. L-Size Mouse Cage Body, 36.5 × 20.7 × 14.0 cm, Polycarbonate (Tecniplast Group: 1284L001; sourced from Starr Life Sciences)

  3. Autoclavable Filter Top (Tecniplast Group: 1284L-400SU; sourced from Starr Life Sciences)

  4. 4.5" Running wheel with reed switch (Bio-Lynx scientific equipment, Inc.: 610-0003-00; sourced from Starr Life Sciences)

  5. MMCX (J)-LL100HF-RPTNC (M) 60'' Cables (Federal Custom Cable, LLC: SCA1086-60)

  6. Ear Tag, Applicator (Stoelting Co: 56791)

  7. Keonn AdvanReader-160 UHF RFID Reader (4-Port) (Keonn Technologies: ADRD-M4-ESMA-160.01)

  8. AdvanMux-8 UHF RFID Multiplexer (8-Port) (Keonn Technologies: ADMX-8-e-110.04)

  9. CAT5 Ethernet Cable

  10. USB 3.0-to-Gigabit Ethernet Adapter (Best Buy Co., Inc., Insignia: NS-PU98635-C)

  11. Power Over Ethernet Injector (Phihong: PSA16U-480(POE); sourced from Keonn Technologies)

  12. SMA Male to SMA Male 24” Cable (Bracke Manufacturing, LLC: BM92046.24)

  13. Data Port 24 channel box (Starr Life Sciences)

  14. Data Port USB Cable (Starr Life Sciences)

  15. Electric Drill

  16. Table Saw

  17. 5/32”, 3/16” Drill Bits

  18. Two Laptops (one for each software program) with the following requirements:

    1. Minimum Requirements for VitalView®: Windows PC (Windows XP or newer), 2 GHz Processor, 2 GB Ram, 800 MG Free Hard Disk space, USB port

    2. Minimum Requirements AdvanNetTM Software: Firefox or Chrome browser, Internet connection, USB port

  19. Computer (for IndividualRFIDELEMus program)

    Minimum Requirements: Windows 10, 12 GB of RAM

Software

  1. AdvanNetTM Software (Keonn Technologies, S.L., https://keonn.com/software-product/advannet/)

  2. VitalView® Activity Data Acquisition Software (Starr Life Sciences Corp, https://www.starrlifesciences.com/product/activity-software/)

  3. IndividualRFIDELEMus program (https://github.com/anthonyraus/IndividualRFIDELEMus/releases)

Procedure

  1. RFID Antenna Setup (Figure 2)

    1. Cut 2” × 2” pegboard squares for each cage with two holes in each square: Take the pegboard and draw lines to make cuts orthogonally such that:

      1. One set of lines runs across every other hole.

      2. The other set of lines runs ½” between every other hole.

        Using a table saw, carefully cut through the lines (Figure 2A).
    2. Place the pegboard square white-side-up. Using a drill and a 3/16” drill bit, drill diagonally through one of the holes, such that the angled hole points away from the center of the square (distal end on the bottom). Repeat this process for every square.

      Note: The purpose of this step is to make the existing pegboard hole wider for the antenna head to lie flat on the pegboard once the antenna wire is threaded through.

    3. Thread the wire of an antenna through the angled hole of a pegboard square, such that the wire comes out the bottom (brown side). Have the base of the antenna cover both holes as much as possible.

    4. Place a double-sided CommandTM strip on the surface of the pegboard, covering the hole opposite to the hole that the antenna wire is threaded through. Affix the base of the antenna to the top (white side) of the pegboard square to the double-sided CommandTM strip (Figure 2B).



      Figure 2. Antenna setup. A. Proper dimensions of the pegboard square in relation to the antenna and CommandTM strip. B-C. Proper assembly of the antenna and pegboard square in top-down (B) and side-profile (C) views.


  2. Cage Setup (Figure 3)

    1. Pre-determine and assign two mice of the same sex to each running cage. Plan to construct RFID-compatible running cages based on the number of mouse pairs used in your study.

    2. Position the wheel housing on the underside of the wire lid adjacent to the food hopper of the Techniplast cage so that:

      1. The wheel is in front of and to the left of the food and water area.

      2. The left edge of the wheel housing is three slots away from the left edge of the cage.

      3. The screw of the housing is flush with the crossbar closest to the hopper.

      4. The slot for the reed switch faces toward the nearest wall of the cage.

        Screw the washer and wingnut onto the screw to affix the housing to the wire lid (Figure 3A).

    3. Using scissors, cut a strip of netting material the width of the running wheel, such that the mesh fits around the outer rim of the wheel. This is used so the limbs of the small juvenile mice do not fall through the rungs of the wheel.

      CAUTION: Ensure that this fit is precise. Improperly fitted netting (when cut too narrow) can slide on the wheel and potentially injure the mouse tail.

    4. Unscrew the axle of the wheel housing, place the wheel inside the housing with the wheel magnet facing outward, and thread the axle through the housing and the wheel, screwing the axle cap back on.

    5. Place the assembled RFID antenna and pegboard square directly underneath the wheel in its cage (aim the wire end of the antenna toward the back wall of the cage) and secure the antenna and pegboard square into place with a double-sided CommandTM strip. Mark a circle with the diamond-tipped pen next to where the hole with the wire is (Figure 3B). Take away the antenna-pegboard square by pulling out the CommandTM strip laterally.

    6. Flip over the cage such that the base is on top. Using the drill and a 5/32” drill bit, drill the spot that was marked. When a complete hole has been made, use the 3/16” drill bit to widen the hole. Drill diagonally through the hole, such that the hole exit on the bottom of the cage points toward the back wall.

      Note: Steps B1-B6 only need to be performed once for each cage.

    7. Flip over the cage to an upright position. Place the antenna and pegboard square back into the cage. Thread the antenna wire through the hole, such that the wire points out of the bottom of the back wall. Readjust the antenna and pegboard square until it is squarely under the wheel. Affix the bottom of the pegboard to the cage bottom using a double-sided CommandTM strip (Figure 3C).

      Note: In subsequent assemblies using pre-drilled cages, the orientation of the antenna can be modified to a non-orthogonal position with respect to the cage base. It is crucial to ensure that the center of the antenna is directly under the center of the wheel.

    8. Add a thin layer of bedding to the cage bottom until the bedding submerges the pegboard but not the antenna. Place two nesting squares into the bottom of the cage, away from the wheel. Place the wire lid with the wheel over the cage, such that the wheel lies above the antenna with adequate clearance (Figure 3D). Cover the cage with the filtered cage top.



      Figure 3. Cage setup. A. Proper assembly of the wheel housing in relation to the wire lid and cage layout. B. Bottom-up view of the Techniplast cage showing the area under the wheel. Proper placement of the antenna and pegboard square under the wheel. The drill site is adjacent to the wire exit hole of the pegboard and is indicated by the black circle drawn on the cage bottom. C. Threading of the antenna wire through the cage hole. D. Clearance between the antenna and the wheel.


  3. RFID + VV System Setup (Figure 4)

    1. Set up the VitalView® system and RFID reader system per the manufacturer’s instructions. Initialize as many VitalView® channels and RFID antennae as you anticipate possibly needing at one time. Keep the data recording configurations for each system as default, such that recording intervals for VitalView® are less than or equal to 1 minute, and the channel names remain numeric and sequential (1-24). Using one VitalView® system (24-port), one RFID reader (4-port), and three RFID multiplexers (8-port), a maximum of 24 cages can be equipped for individual tracking.

    2. Connect one antenna wire to one port on the RFID reader or multiplexer using the 60” LMR 100 cables (Figure 4).

    3. Thread one reed switch into the reed switch slot in each running cage. Plug the other end of the reed switch cable into the port on the VitalView® box (Figure 4). Test your wheel setup by spinning the running wheel, and adjust the depth of insertion of the reed switch until wheel revolutions are recorded by the VitalView® system.

      Note: The configuration with one RFID reader and one multiplexer can track 11 cages at a time, with two mice in each cage. Additional multiplexers can be added to increase the capacity of simultaneously recorded cages.



      Figure 4. RFID and VitalView® system setup. Schematic of data feeds from parallel RFID and VitalView® systems. RFID reader connections can be expanded to multiple cages using an RFID multiplexer. Each RFID antenna is connected to the reader or multiplexer via a 60” LMR 100 cable. We typically use separate computers for VitalView® and RFID reader software (AdvanNetTM Software) programs.


  4. Mouse Tagging (Figure 5)

    1. Etch each RFID tag based on the cage number (e.g., 1, 2, …) and animal ID within a cage (e.g., A, B) using the diamond-tipped pen.

    2. On the laptop using the RFID software (instructions in AdvanNetTM User Guide), change an RFID tag’s EPC code to e0000000000000000000001A, e0000000000000000000001B, e…2A, e…2B, etc. based on the tag number that was etched into it (Figure 5A). Assign these numbers, as well as the mice, so that they match the channel names 1-24 from the VitalView® channels; e.g., mice tagged 1A and 1B must be in the cage connected to VitalView® channel 1, and so forth.

    3. Scruff the P21 mouse. Using the Stoelting ear tags and ear tag applicator, tag each mouse on one ear (Figure 5B). Alternate ears (left vs. right), if possible, for easy visual identification of pair-housed mice.

    4. Place a small drop of super glue on the green base of an RFID tag (Figure 5C). Using the tweezers, carefully affix the glued side of the RFID tag onto the Stoelting tag on the mouse’s ear. Holding the RFID tag and ear tag in place between the tweezer tips, let the glue dry for approximately 30 seconds (Figure 5D).

    5. Place the mice in the cage. Ensure that mice 1A and 1B, indicated by the EPC code of the RFID tag affixed to them, are in the cage connected to VitalView® port 1. Repeat with the other cages.

      Note: It is recommended that daily in-person monitoring is performed to identify and prevent the possibility of jammed wheels or insecure or fallen tags. These are infrequent occurrences in our experience; however, they can introduce error into the data collected if not addressed quickly. These issues can be easily remedied with proper supervision of the apparatus.



      Figure 5. Mouse tagging. A. Example of RFID tags etched with their new EPC designation. B. The mouse is scruffed, and an ear tag is attached to mouse’s left ear. C. Super glue has been added to the base of the RFID tag, and the RFID tag can be picked up with the tweezers. Only a minimal amount of super glue is needed to affix the RFID tag to the ear tag. D. The RFID tag is applied to the ear tag and held in place for about 30 seconds to allow the glue to dry.


  5. RFID Cage and Antenna Reuse

    1. Once the experiment is completed, unplug the antenna from the LMR100 cable. Remove the antenna and pegboard square by pulling the CommandTM strips laterally. Dispose of the pegboard. Save the antenna and wash carefully with water and a sponge.

    2. Pre-drilled cages and washed antennae may be reused for future experiments. A new pegboard square must be used for each cage.

Data analysis

  1. Aligning VitalView® and RFID raw data to generate individual running distances (Figures 6, 7)

    Note: Wheel revolutions are determined minute-by-minute per cage by the VitalView® Activity Software and exported as a .csv file. For each RFID-tagged mouse, individual tag reads are logged by the RFID reader and exported as a .csv file via the AdvanNetTM Software.

    1. Per the manufacturers’ instructions, export the data from the VitalView® system and AdvanNetTM Software. The AdvanNetTM Software automatically names the file “data.csv.” The VitalView software is named according to user designation.

    2. Make six new directories (folders) to hold your files during processing. These directories will accept and store files for (suggested names): 1) VV Input 2) RFID Input, 3) VV Output, 4) RFID Output, 5) Distances, and 6) Stats.

    3. Place the Vital View export .csv file(s) into the “VV Input” directory.

    4. ”Preprocess” the RFID data file before proceeding: The RFID export .csv data file needs to be reformatted (preprocessed) in order for the program to align it with the VV data. Open the RFID export .csv file in Microsoft Excel (Figure 7A). Locate the time stamp column (in our case, column L). Convert the number format of the column to “MM/DD/YY HH:MM” (use a 24-hour clock). Select and cut the whole timestamp column and paste it into column B to the left of the EPC column (in our case, column C). Delete all columns except the timestamp and EPC columns, such that the timestamp column is column A and the EPC column is column B. Select the whole timestamp column, sort by oldest to newest, and expand the selection. Insert a new row 1. In cell 1A, write “TIME_STAMP” and in cell 1B, write “HEX_EPC” (Figure 7B). If these words appear anywhere else in the document, delete those rows and keep only the words written in row 1. Save the Excel sheet as a new “preprocessed” export .csv file and place it in the “RFID input” directory.



      Figure 6. Data processing pipeline in 3 steps. Illustration of the pipeline for processing RFID export and VV export files using the process_file_ui.exe program. A. Step 1: Processing of Vital View files from the input directory. B. Step 2: Processing of RFID export data. C. Step 3: Cross-referencing of RFID and Vital View files into individual daily distances and daily error rates.


    5. Open the project “Individual RFIDELEMus” on GitHub (downloadable link can be found in the above “Software” section). Use the RFID code program, process_file_ui.exe, to process the data in three steps:

      1. The “Vital View Processing” section of the process_file_ui.exe program collects one or multiple Vital View export files in the VV input directory and combines them into one processed file. The Program then resamples the data over 1-minute time intervals so that the data can be directly compared with the RFID data. Ensure that the VV input directory only contains Vital View export files. Select the “VV input” directory as your input and select the “VV output” directory as your output. Type in a name for your output VV file and run this section of the program (Figure 6A). The output file will be written to the “VV output” directory (Figure 7C).

      2. The “RFID Processing” section of the process_file_ui.exe program accepts a preprocessed RFID file .csv and bins its tag reads into rows of minutes for each mouse (each mouse has its own column) based on their unique EPC code. Select the preprocessed RFID data file in the “RFID Input” directory as your input and select the “RFID Output” directory as your output (Figure 6B). Type in a name for your output RFID file and run this section of the program. The output file will appear in the “RFID Output” directory (Figure 7D).

      3. The “Cross Reference Vital View Data with RFID Data” section of the process_file_ui.exe program assigns the sum of the number of wheel revolutions within each 1-minute interval to the mouse in the cage with the most tag reads during that minute. It then converts wheel revolutions to distance run (using our running wheel diameter of 4.5 inches) and outputs individual running distances as one .csv file per day (for example, if you recorded for seven days, there will be seven separate files in the output directory) (Figure 7E). Select the “VV Output” directory (containing the file from step 1) as your VV input to the program and select the “RFID Output” directory (containing the file from step 2) as your RFID input to the program. Select the “Distances” directory as your Distances output and select the “Stats” directory as your Stats output. Provide dates and times in “YYYY-MM-DD HH:MM:SS” (24-hour) format for the start and end of the desired data processing and run this section of the program (Figures 6C, 8A, 8B).



      Figure 7. RFID and VV data file reformatting examples. A. Excerpt of the raw RFID export file format before reformatting (preprocessing). B. Excerpt of the final reformatted (preprocessed) RFID export file, ready for use in RFID processing. C. Excerpt of the VV output file of the “Vital View Processing” step. D. Excerpt of the RFID output file of the “RFID Processing” step. E. Excerpt of the Distances output file for one day of the “Cross Reference Vital View Data with RFID Data” step. When cross-referencing multiple days, each day will produce one output file in the “Distances” directory. F. Excerpt of the Stats output file for one day of the “Cross Reference Vital View Data with RFID Data” step. When cross-referencing multiple days, each day will produce one output file in the “Stats” directory.


  2. Data Validation (Figure 8)

    Note: Running distances are assigned and compiled on a minute-by-minute basis. The process_file_ui.exe program provides separate .csv files logging a) the number of minutes with at least one wheel revolution, or “total vv counts”, and b) the number of minutes with a wheel revolution but without a tag read, or “vv counts without RFID.” These files are in the “Stats” folder. One Stats file is produced per day (Figure 7F).

    1. Divide b) by a) to yield the proportion (error rate) of running minutes that were not captured by the RFID system (Figure 8C).



      Figure 8. Representative data. A. Individual running distances of male mice per day, recorded for 13 consecutive days (between postnatal days 25 and 37) in two cages, Cage 1 and Cage 3. Mice 1A and 1B were pair-housed together, and mice 3A and 3B were pair-housed together. Mice were acclimated to the running wheel during P21-24 (data not shown). B. Average distance ran per day, demonstrating a significant intra-cage difference in the average running volume (unpaired t-test, *P < 0.05). C. Total number of minutes with wheel revolutions (as recorded by VitalView®) and total minutes of running without an accompanying RFID tag read (as recorded by the RFID reader) per day for each cage. .

Notes

  1. System Monitoring

    1. RFID-equipped cages must be routinely monitored for wheel obstructions by nesting material or excessive bedding under the wheel. This can be determined by daily visual inspection or viewing Vital View software recordings.

    2. Check the RFID export files regularly to make sure the tags are still being detected as expected. Check the Vital View data exports regularly to ensure that mouse running is being recorded as expected. We monitor these exports every other day. Data files can be exported to an external drive while the experiment is in progress.

    3. Monitor the mice daily for any disruptions in the RFID tag and/or ear tag attached to their ear. A separated ear tag and RFID tag from a mouse has only occurred twice in our experiments. If a mouse is missing an RFID tag or ear tag, give the mouse a new set, making sure the EPC of the new RFID tag is the same as that of the tag that has fallen off.


  2. Mouse Running

    1. Tracking is conducted continuously 24 h a day, through the light and dark cycle, and may start as soon as pups are weaned from their mother (postnatal day 21).

    2. Mice were not given a stationary wheel acclimation period. They were instead allowed to run on the running wheel from the time they were weaned. The plastic netting fitted around the rim of the wheel ensures that the wheel is safe for juvenile mice to run on, so an acclimation period was not deemed necessary to guarantee safety. Our data showed that juvenile mice gradually increase their running over time post-weaning. The first few days of acclimation may be removed during data analysis if needed.

Acknowledgments

All images were generated with Biorender. Special thanks to atlasRFIDstore, Keonn Technologies, and Luke Raus for their technical assistance and expertise.

Competing interests

There are no competing interests to disclose.

Ethics

All experiments using live animals described in this protocol have been approved for use by our Institutional Animal Care and Use Committee (IACUC) at the University of California – Irvine. All steps were taken to ensure minimal pain and distress to mice in this paradigm. The IACUC Approved Use Protocol for this experiment is 19-057.

References

  1. Arakawa, H. (2018). Ethological approach to social isolation effects in behavioral studies of laboratory rodents. Behav Brain Res 341: 98-108.
  2. Batchelder, P., R. O. Kinney, L. Demlow and C. B. Lynch. (1983). Effects of temperature and social interactions on huddling behavior in Mus musculus. Physiol Behav 31(1): 97-102.
  3. Frahm, S., V. Melis, D. Horsley, J. E. Rickard, G. Riedel, P. Fadda, M. Scherma, C. R. Harrington, C. M. Wischik, F. Theuring and K. Schwab. (2018). Alpha-Synuclein transgenic mice, h-α-SynL62, display α-Syn aggregation and a dopaminergic phenotype reminiscent of Parkinson’s disease. Behavioural Brain Research 339: 153-168.
  4. Goh, J. and W. Ladiges. (2015). Voluntary Wheel Running in Mice. Curr Protoc Mouse Biol 5(4): 283-290.
  5. Hawkley, L. C., S. W. Cole, J. P. Capitanio, G. J. Norman and J. T. Cacioppo. (2012). Effects of social isolation on glucocorticoid regulation in social mammals. Horm Behavior 62(3): 314-323.
  6. Hayes, L. D. (2000). To nest communally or not to nest communally: a review of rodent communal nesting and nursing. Anim Behav 59(4): 677-688.
  7. Himmler, B. T., S. M. Pellis and B. Kolb. (2013). Juvenile play experience primes neurons in the medial prefrontal cortex to be more responsive to later experiences. Neurosci Lett 556: 42-45.
  8. Ieraci, A., A. Mallei and M. Popoli. (2016). Social Isolation Stress Induces Anxious-Depressive-Like Behavior and Alterations of Neuroplasticity-Related Genes in Adult Male Mice. Neural Plast 2016: 6212983.
  9. Ivy, A. S., T. Yu, E. Kramar, S. Parievsky, F. Sohn and T. Vu. (2020). A Unique Mouse Model of Early Life Exercise Enables Hippocampal Memory and Synaptic Plasticity. Sci Rep 10(1): 9174.
  10. Kappel, S., P. Hawkins and M. T. Mendl. (2017). To Group or Not to Group? Good Practice for Housing Male Laboratory Mice. Animals (Basel) 7(12).
  11. Koike, H., D. Ibi, H. Mizoguchi, T. Nagai, A. Nitta, K. Takuma, T. Nabeshima, Y. Yoneda and K. Yamada. (2009). Behavioral abnormality and pharmacologic response in social isolation-reared mice. Behav Brain Res 202(1): 114-121.
  12. Krynitsky, J., A. A. Legaria, J. J. Pai, M. Garmendia-Cedillos, G. Salem, T. Pohida and A. V. Kravitz. (2020). Rodent Arena Tracker (RAT): A Machine Vision Rodent Tracking Camera and Closed Loop Control System. eNeuro 7(3).
  13. Liu, Y., T. Yan, J. M. Chu, Y. Chen, S. Dunnett, Y. S. Ho, G. T. Wong and R. C. Chang. (2019). The beneficial effects of physical exercise in the brain and related pathophysiological mechanisms in neurodegenerative diseases. Lab Invest 99(7): 943-957.
  14. Makinodan, M., K. M. Rosen, S. Ito and G. Corfas. (2012). A critical period for social experience-dependent oligodendrocyte maturation and myelination. Science 337(6100): 1357-1360.
  15. Matikainen-Ankney, B. A., M. Garmendia-Cedillos, M. Ali, J. Krynitsky, G. Salem, N. L. Miyazaki, T. Pohida and A. V. Kravitz. (2019). Rodent Activity Detector (RAD), an Open Source Device for Measuring Activity in Rodent Home Cages. eNeuro 6(4).
  16. Okada, R., H. Fujiwara, D. Mizuki, R. Araki, T. Yabe and K. Matsumoto. (2015). Involvement of dopaminergic and cholinergic systems in social isolation-induced deficits in social affiliation and conditional fear memory in mice. Neuroscience 299: 134-145.
  17. Oliff, H. S., N. C. Berchtold, P. Isackson and C. W. Cotman. (1998). Exercise-induced regulation of brain-derived neurotrophic factor (BDNF) transcripts in the rat hippocampus. Brain Res Mol Brain Res 61(1-2): 147-153.
  18. Peleh, T., X. Bai, M. J. H. Kas and B. Hengerer. (2019). RFID-supported video tracking for automated analysis of social behaviour in groups of mice. J Neurosci Methods 325: 108323.
  19. Poffe, C., S. Dalle, H. Kainz, E. Berardi and P. Hespel. (2018). A noninterfering system to measure in-cage spontaneous physical activity in mice. J Appl Physiol 125(2): 263-270.
  20. Reimer, J. D. and M. L. Petras. (1967). Breeding structure of the house mouse, Mus musculus, in a population cage. J Mammal 48(1): 88-99.
  21. Terranova, M. L., G. Laviola, L. de Acetis and E. Alleva. (1998). A description of the ontogeny of mouse agonistic behavior. J Comp Psychol 112(1): 3-12.
  22. Voikar, V. and S. Gaburro. (2020). Three Pillars of Automated Home-Cage Phenotyping of Mice: Novel Findings, Refinement, and Reproducibility Based on Literature and Experience. Front Behav Neurosci 14: 575434.
  23. Wang, Z., S. A. Mirbozorgi and M. Ghovanloo. (2018). An automated behavior analysis system for freely moving rodents using depth image. Med Biol Eng Comput 56(10): 1807-1821.

简介

[摘要]配备有自愿跑步轮的啮齿动物笼子通常用于研究有氧体育活动对生理和行为的影响。运动神经生物学方面的显着发现,包括脑源性神经营养因子 (BDNF) 在神经可塑性和认知中的关键作用,是使用装有自愿跑轮的啮齿动物进行的。A M ajor优点使用家庭笼的运行超过跑步机轮小号是应力消除潜在的具有强制相关奔跑克 此外,自愿轮跑可以模拟实验室小鼠更自然的跑步模式。SINGL ÿ自愿转轮壳体小鼠被traditionaly用来获得精确的孔定量吨的通货膨胀的距离RAN ; ^ h H但是,社会隔离压力常常被忽视,以获得精确的跑动距离。此外,青少年小鼠的自愿运动研究必须考虑隔离压力对神经发育的影响。在这个协议中,我们断奶21天-老的小鼠幼仔直接进入运行车轮配备笼,以及对-房子他们减少社会孤立的影响,和其他发育可能产生负面影响的具体因素的行为或发展。单独的R unning距离小号是从每只小鼠中获得的笼使用射频识别(RFID )耳标和一个隐藏的天线放置直接运行车轮下。我们已经证明,在青春期进行测试时,在特定的青少年-青少年发育时期自愿跑步可以改善海马记忆(Ivy等人,2020 年)。个人锻炼的跟踪分组饲养小鼠可以使将来的研究,以精确地相关联的量与读出,如运动的细胞特异性基因表达,表观遗传机制,血清生物标志物,和行为,在一个个体内的方式。



图文摘要:



图1 。图示的该双RFID和生命查看系统单个鼠标运行一对收纳笼


[背景]在U瑟自愿走行轮的实验室啮齿动物的笼子是研究运动对脑功能和神经退行性疾病的影响的常用方法(刘等人。,2019) 。采用自愿运动研究设计消除了对压力刺激的需要,例如足部电击或调查员处理,以鼓励动物运动。使用笼内自愿跑步轮的研究传统上将啮齿动物单独饲养,以产生准确的跑步距离并捕捉运动量的个体差异(Goh 和 Ladiges ,2015)。然后可以将个人跑步数据与其他个人内部数据集相关联。例如,在2-6小时的时间内奔跑总距离正相关与在脑源性神经营养因子(BDNF)的基因表达海马(Oliff等人,1998) 。然而,不利的个人住房是在由压力引起的潜在的社会隔离。由于老鼠是群居动物,它们天生的社会行为(无论是在野外还是圈养环境中)在与野生群体中发现的社会安排相似的社会安排中最自然地发展(Reimer 和 Petras ,1967)。准确数据收集所需的受控实验室环境经常导致住房条件挑战自然社会结构的形成。在缺乏一个社会环境,当小鼠单独饲养不仅要对啮齿动物的福利,但也被认为是其对生产数据的质量和影响的研究结果的解释(卡普尔等人,2017年,荒川,2018 )。

先前的研究表明,单独居住的小鼠可以通过多种方式影响大脑功能。它可以改变下丘脑-垂体-肾上腺皮质 (HPA) 轴功能,特别是糖皮质激素调节和反馈(Hawkley等,2012)。个人住房还会混淆各种行为测试的表现,包括评估焦虑(Koike等,2009)和学习和记忆(Okada等,2015)的测试。最后,单个壳体可以降低所述的在神经可塑性相关基因的表达的海马和前额叶皮质(Ieraci等人,2016) 。在单独社会孤立的应力因此安置啮齿动物是口译研究时要考虑的一个重要变量是评估自由运动的神经生物学效应。

另一方面,老鼠的群居会引起雄性对雄性的攻击。Ť他通常出现在啮齿类动物青春期开始后并趋向于没有少年和青少年发育阶段期间存在(诺瓦等人,1998) 。雌性小鼠表现出这种攻击性的倾向较低,甚至在青春期后(Hayes ,2000) 。幼年啮齿动物的社交隔离会对内侧前额叶皮层的髓鞘形成产生不利影响(Makinodan等,2012),而幼年的社交活动可以增强该区域的神经可塑性(Himmler等,2013)。此外,幼年小鼠使用体温的其笼配合用于温度调节(巴彻尔德等人,1983) 。这强调了成对或成组的住房对于维持基础体温的重要性,尤其是在随着运动而改变代谢需求的情况下。因此,在研究联少年自愿车轮运行与神经功能和行为,一组壳体-基于方法可以优选地消除隔离的应力。

个人家庭-笼啮齿动物跟踪已通过使用视频来完成(Krynitsky等人,2020年)(Poffe等,2018) (王等,2018) ,皮下植入RFID微芯片(Peleh等人,2019 )(弗拉汉姆等人,2018) ,二者的组合,或被动红外传感器(Matikainen-Ankney,迪亚-Cedillos等人。,2019) 。Ç urrent协议利用皮下植入RFID微芯片需要延长克制和麻醉,这可能产生不希望的应力和疼痛。统一的信息设备详细说明的协议,描述安全地植入小鼠RFID微(UID UC-1485)在无麻醉第12日龄该方法在主要用于鼠标识别一个时间单点,诸如用于拍摄内快速鼠标库存一组-装笼,但它的使用还没有被证实的现场,连续跟踪。另一个因素是RFID芯片的尺寸,它必须足够大才能被天线检测到。对于幼鼠,通常重 6-9 克,这个尺寸要求禁止在不使用麻醉的情况下给药。在视频跟踪,铁丝笼顶小号壅连续自上而下的录像,但所需要的许多家庭-笼跑轮系统。最后,现有的家庭-在市场上使用RFID天线的矩阵排列在整个笼子的底部或者到监视器基线活动能力的战略位置笼RFID活动跟踪系统目前(Voikar和Gaburro ,2020年)。然而,如果实验的目的是跟踪车轮的随意行驶,则只需要一根天线,其读取范围精确地限制在车轮内部的区域内。

我们的设备在每个笼子的底部使用坚固、低调的 RFID 耳标和一个 RFID 天线,为现有的基于视频和基于植入的跟踪系统提供一种微创替代方案(图 1)。我们协议的主要优点是能够从断奶年龄(产后第 21 天)开始单独跟踪成对饲养的幼鼠的跑轮活动。我们减少通过对隔离的压力和问题与体温调节-住房的小鼠,并提供筑巢材料。事实上,如果愿意,这个模型可以放大到每个笼子 > 2 只小鼠。此程序已在包含同性小鼠的笼子中进行了测试,但我们没有研究混合性别群体对个人跑步活动的影响。我们的实验设计允许比较同一笼子内雌性和雄性小鼠之间的个体跑步距离。虽然可能有领土问题一旦社会等级被建立在网箱壳体的两个或更多的雄性小鼠,运动的从每个单独小鼠一组中的精确量-容纳笼可以用任何所希望的读出实验相关联。

该设备的一个小限制是,在我们手中,大约有 5-10% 的跑步活动无法调和;^ h H但是,我们相信,我们在组分配各个运行数据的90-95%的精度-安置环境是一个ñ可接受的产率。尽管这种方法不会遇到与在 RFID 天线矩阵(Voikar 和 Gaburro,2020 年)中观察到的精度降低天线串扰相同的问题,但非常靠近天线的金属轮可能具有相同的干扰效应(因此需要一个大耳标)。我们的设计有效地将天线的读取范围限制在车轮区域。

调查自愿体育活动的影响对于增加我们对其对大脑功能和行为的益处的理解具有许多意义。确定个体化的量练习特定结果或目标的要求的准确的监测运行的距离,如在该协议描述的,现在可以进行。我们的方法可以通过识别独特的RFID标签跟踪个体小鼠š对收纳小鼠的耦合与从上运行车轮家用笼的磁传感器获得的时间标记的数据。重要的是,第È过程可以按比例扩大到跟踪的行驶距离在笼子共享访问一个运行车轮多鼠标。我们的方法将 RFID应用于幼鼠运动研究,同时最大限度地减少隔离和约束带来的压力,消除麻醉需求,并产生高度精确和个性化的跑步数据。

关键字:群居, 运动, 自主跑轮运动, 笼子里的老鼠跟踪, 射频识别(RFID), 富集, 幼鼠

材料和试剂


用于金属产品管理的UHF RFID 标签(村田制作所,目录号:LXTBKZMCMG-010)
Command TM小海报条(3M ,目录号:17024ES)
钉板,5/32” 厚,孔间距 1”
LOCTITE ® SUPER GLUE PRECISION PEN(汉高,目录号:2066118)
网材料(工业网,目录号:NG3060-164)
耳标,鼠标,浅蓝色,1-100(Stoelting ,目录号:56782)
耳标,仅靠背,Pk/100(Stoelting ,目录号:56792)
蚀刻雕刻笔(Porsin ,目录号:LX1323)
Teklad 1/8”玉米芯床上用品(Envigo,目录号:7092A)
棉花雀巢 (Ancare nestlets)
动物:Jackson 小鼠 WT C57B L / 6J(Jackson Laboratories ,目录号:000664)
小鼠C57B的后代L /大坝从杰克逊实验室获得6J ,并孕育,诞生,并在我们的生态饲养场饲养。小鼠对食物和水自由进入,并在灯都维持在12小时光照/黑暗周期。在断奶日龄(P)21,将小鼠一对收纳在标准笼子里,自由获取的在笼不锈钢-配备有塑料网钢运行车轮嵌合在轮缘为幼年小鼠的安全性。所有小鼠通常6-9在g的断奶时间。跟踪从 P21到 P 41每天 24 小时连续进行三周。所有实验均按照 U 进行。小号。美国国立卫生研究院动物护理和使用指南,并获得加州大学欧文分校动物护理和使用机构委员会的批准。


Teklad 全球无大豆蛋白挤压啮齿动物饮食(Envigo ,目录号:2020X)
设备


RFID 无源天线 (Abracon LLC: ARRAN5-915.000MHz)
L 尺寸鼠标笼体,36.5 × 20.7 × 14.0 厘米,聚碳酸酯(Tecniplast Group:1284L001;来自 Starr Life Sciences)
高压灭菌过滤器顶部(Tecniplast Group:1284L-400SU;来自 Starr Life Sciences)
4.5" 带簧片开关的跑轮(Bio-Lynx 科学设备公司:610-0003-00;来自 Starr Life Sciences)
MMCX (J)-LL100HF-RPTNC (M) 60'' 电缆(Federal Custom Cable, LLC:SCA1086-60)
耳标,涂抹器(Stoelting Co:56791)
Keonn AdvanReader-160 UHF RFID 阅读器(4 端口)(Keonn Technologies:ADRD-M4-ESMA-160.01)
AdvanMux-8 UHF RFID 多路复用器(8 端口)(Keonn Technologies:ADMX-8-e-110.04)
CAT5 以太网电缆
USB 3.0 转千兆以太网适配器(Best Buy Co., Inc.,标志:NS-PU98635-C)
以太网供电注入器(Phihong:PSA16U-480(POE);来自 Keonn Technologies)
SMA 公头到 SMA 公头 24” 电缆(Bracke Manufacturing, LLC:BM92046.24)
数据端口 24 通道盒(Starr Life Sciences)
数据端口 USB 电缆(Starr Life Sciences)
电钻
台锯
5/32”、3/16” 钻头
两台笔记本电脑(每个软件程序一台)具有以下要求:
VitalView ® 的最低要求:Windows PC(Windows XP 或更新版本)、2 GHz 处理器、2 GB 内存、800 MG可用硬盘空间、USB 端口
最低要求 AdvanNet TM软件:Firefox 或 Chrome 浏览器、互联网连接、USB 端口
计算机(用于个人RFIDELEMus程序)
最低要求:Windows 10、12 GB RAM


软件


AdvanNet TM软件(Keonn Technologies, SL, https://keonn.com/software-product/advannet/)
VitalView ®活动数据采集软件(Starr Life Sciences Corp,https: //www.starrlifesciences.com/product/activity-software/ )
个人RFIDELEMus计划(https://github.com/anthonyraus/IndividualRFIDELEMus/releases)
程序


RFID天线设置(图 2)
为每个笼子切割2” × 2”钉板方块,每个方块上有两个孔:取钉板并画线进行正交切割,使得:
一组线穿过每隔一个洞。
另一组线在每个其他孔之间延伸 ½”。
使用台锯,小心地切割线条(图 2A)。


将钉板方形白色面朝上放置。使用钻头和 3/16" 钻头,斜钻穿过其中一个孔,使成角度的孔远离正方形的中心(底部的远端)。对每个正方形重复此过程。
注意:该步骤的目的是使现有的栓板孔宽为天线头部到平躺在配挂板,一旦天线导线是线程编通过。


螺纹的天线的导线通过一个栓板正方形的成角度的孔,使得导线出来的底部(棕色侧)。让天线底座尽可能覆盖两个孔。
将双面 Command TM条放在钉板表面,覆盖与天线线穿过的孔相对的孔。将天线底座固定在钉板正方形的顶部(白色侧)到双面Command TM条(图 2B)。
图2.天线小号etup。一个。与天线和 Command TM条相关的钉板方块的正确尺寸。乙-丙。正确装配的天线的和在自顶向下(B)和侧栓板正方形-轮廓(C)的意见。


笼子设置(图 3)
预先确定并将两只相同性别的老鼠分配到每个运行的笼子里。计划到c onstruct RFID兼容运行笼基础上的鼠标对数中使用你的学习。
将轮罩放置在靠近 Techniplast 笼子食物漏斗的金属丝盖下侧,以便:
轮子位于食物和饮水区的前面和左侧。
轮罩的左边缘距离笼子的左边缘三个槽。
外壳的螺钉与最靠近料斗的横杆齐平。
为簧片开关槽面朝所述保持器的最近的壁。
将垫圈和蝶形螺母拧到螺钉上,将外壳固定到电线盖上(图 3A)。


使用剪刀,切割网材料的一个条带的运行车轮的宽度,以使得围绕所述轮的外轮缘的网格拟合。这是用来使四肢小幼年小鼠不通过掉落的的梯级的车轮。
注意:确保这种配合是精确的。不适当地适合泰德网(当切太窄)可以上滑动的轮子和潜在地伤害小鼠尾部。


拧下轮罩的轴,将轮放在外壳内,轮磁铁朝外,然后将轴穿过外壳和轮,然后重新拧上轴盖。
放置组装RFID天线和栓板平方直接在其笼中的轮下方(一个IM朝向天线的导线端部背面笼壁)和天线固定和栓板平方与到位双面命令TM条。用金刚石笔尖在带有电线的孔旁边标记一个圆圈(图 3B)。横向拉出 Command TM条带,取下天线钉板方块。
翻转笼子,使底座位于顶部。使用钻头和 5/32" 钻头,在标记的位置钻孔。打完一个完整的孔后,使用 3/16" 钻头加宽孔。通过孔斜钻,使得在朝着笼子点底部的孔出口回はLL。
注意:小号TEPS乙1-乙6只需要一次为每个笼子进行。


将笼子翻转到n 直立位置。将天线和钉板方块放回笼子里。通过该孔螺纹天线导线,使得导线分列的底部的背面壁。重新调整天线和钉板方块,直到它正好位于车轮下方。使用双面Command TM条(图 3C)将钉板的底部固定到笼子底部。
注意:在使用预先钻孔的笼子s 的后续组件中,天线的方向可以修改为相对于笼子底座的非正交位置。这是至关重要的,以确保该天线的中心是直接在轮的中心下。


在笼子底部添加一层薄薄的垫料,直到垫料浸入钉板而不是天线。将两个嵌套方块放入笼子底部,远离轮子。放置电线盖与所述轮在所述笼,使得轮在于用适当的间隙(图3D)天线的上方。用过滤后的笼顶盖住笼子。
图3.笼小号etup。一个。与金属丝盖和笼子布局相关的轮罩的正确组装。乙。Techniplast 笼子的自底向上视图,显示了车轮下方的区域。将天线和钉板方块正确放置在车轮下方。Ť他钻部位是相邻于栓板的金属丝出口孔,并通过在笼子底部取出的黑色圆圈表示。Ç 。Threadin克透笼孔的天线导线。d 。天线和车轮之间的间隙。


RFID + VV 系统设置(图 4)
按照制造商的说明设置 VitalView ®系统和 RFID 阅读器系统。一次初始化尽可能多的 VitalView ®通道和 RFID 天线e 。保持数据记录配置为每个系统作为默认,使得对于VitalView记录间隔®是小于或等于1分钟,和信道名称保持数字和顺序(1-24)。使用一个VitalView ®系统(24端口),一个RFID ř EADER(4端口),和三个RFID米ultiplexers(8端口),最大的24个笼可配备单个跟踪。
使用 60 英寸 LMR 100 电缆将一根天线线连接到 RFID 阅读器或多路复用器上的一个端口(图 4)。
将一个簧片开关拧入每个运行笼中的簧片开关槽。将簧片开关电缆的另一端插入 VitalView ®盒上的端口(图 4)。通过旋转运行轮来测试您的车轮设置,并调整簧片开关的插入深度,直到 VitalView ®系统记录车轮转数。
请注意:与一个RFID读取器和一个多路转换器的配置可以在一个时间跟踪11个笼,在每个笼两只小鼠。附加的多路复用器可以被加入以增加同时记录的容量编笼中。


图4. RFID和VitalView ®小号ystem小号etup。来自并行 RFID 和 VitalView ®系统的数据馈送示意图。RFID 阅读器连接可以使用 RFID 多路复用器扩展到多个笼子。每个 RFID 天线都通过 60 英寸 LMR 100 电缆连接到阅读器或多路复用器。我们通常为 VitalView ®和 RFID 阅读器软件(AdvanNet TM软件)程序使用单独的计算机。


鼠标标记(图 5)
蚀刻每个RFID标签根据所述(笼数例如,1,2,...)的保持架(内和动物ID例如,A,B)用金刚石尖笔。
在笔记本电脑ü唱RFID软件(指令AdvanNet在TM用户指南),改变RFID标签的EPC代码e0000000000000000000001A,e0000000000000000000001B,E ...... 2A,E ...... 2B,等基于所蚀刻到它的标签号(图5A)。分配这些编号以及鼠标,使它们与 VitalView ®通道中的通道名称 1-24 相匹配;例如,标记为 1A 和 1B 的小鼠必须在与 VitalView ®通道 1相连的笼子中,等等。
揉搓 P21 鼠标。使用 Stoelting 耳标和耳标涂抹器,在一只耳朵上标记每只老鼠(图 5B)。备用耳(左VS 。右),如果可能的话,用于对收纳小鼠的容易视觉识别。
在RFID 标签的绿色底座上放一小滴强力胶(图 5C)。使用镊子,小心翼翼地贴上RFID标签的粘胶面到鼠标上的Stoelting标签的耳朵。将RFID标签和耳标固定在镊子尖端之间,让胶水干燥约 30 秒(图 5D)。
把老鼠放在笼子里。确保小鼠 1A 和 1B(由贴在其上的 RFID 标签的 EPC 代码指示)在连接到 VitalView ®端口 1的笼子中。对其他笼子重复上述操作。
注意:建议每天进行现场监控,以识别和防止卡住车轮或不安全或掉落的标签的可能性。这些在我们的经验中很少发生;然而,他们可以在引入误差,以收集如果不能迅速解决的数据。这些问题可以很容易地纠正有个妥善监督ê设备。


图5.小鼠吨agging。一个。蚀刻有新 EPC 标识的 RFID 标签示例。乙。鼠标被套住,耳标贴在鼠标的左耳上。Ç 。RFID 标签底部添加了强力胶,可以用镊子夹起 RFID 标签。只需极少量的强力胶即可将 RFID 标签粘贴到耳标上。d 。RFID 标签贴在耳标上并固定约 30 秒,让胶水变干。


RFID 笼子和天线重复使用
一旦在实验完成后,拔掉从LMR100电缆的天线。横向拉动Command TM条带,卸下天线和钉板方块。处理钉板。保存天线和水并仔细冲洗一个海绵。
预钻孔的笼子和洗过的触角可重复用于未来的实验。每个笼子必须使用一个新的钉板方块。
数据一nalysis


对准VitalView ®和RFID原始数据,以产生单个行驶距离S(图小号6,7)
注意:车轮转数由 VitalView ®活动软件按每个笼子每分钟确定并导出为 .csv 文件。对于每只带有 RFID 标签的鼠标,RFID 阅读器都会记录单个标签读取,并通过AdvanNet TM软件导出为 .csv 文件。


每对制造商的说明,导出了从VitalView数据®系统和AdvanNet TM软件。该AdvanNet TM软件自动命名文件“data.csv”。VitalView软件根据用户指定命名。
在处理过程中创建六个新目录(文件夹)来保存您的文件。这些目录将接受和存储(建议名称)的文件:1) VV 输入 2) RFID 输入,3) VV 输出,4) RFID 输出,5) 距离,和 6) 统计。
将 Vital View 导出 .csv 文件放入“VV 输入”目录。
“预处理”继续之前的RFID数据文件:将RFID导出的.csv数据文件需要以重新格式化(预处理)计划与对齐的VV数据。在 Microsoft Excel 中打开RFID导出 .csv文件(图7 A)。找到时间戳列(在本例中为 L 列)。将列的数字格式转换为“MM/DD/YY HH:MM” (使用 24 小时制)。选择并剪切整个时间戳列,然后将其粘贴到EPC 列左侧的B列(在我们的示例中为 C 列)。删除除timestamp 和EPC 列以外的所有列,使timestamp 列为A 列,EPC 列为B 列。选择整个timestamp 列,按最旧到最新排序,然后展开选择。插入新行1.在电池1A,写入“TIME_STAMP”,并在电池1B,写入“HEX_EPC”(图7的B)。如果出现其他地方在文档中这些话,删除这些行,只保留写在第1行的话保存在é艾克赛尔板作为一个新的“预处理”出口.csv文件,并将其放置在“RFID输入”目录。
图6 。数据p rocessing p 3 ipeline小号依照步骤。管道的插图用于处理RFID出口和使用VV导出文件process_file_ui.exe程序。一个。第1步:从生命查看文件处理的输入目录。乙。第二步:RFID出口数据的处理。Ç 。第 3 步:将 RFID 和 Vital View 文件交叉引用为个人每日距离和每日错误率。


在 GitHub 上打开项目“Individual RFIDELEMus”(下载链接可以在上面的“软件”部分找到)。使用 RFID 编码程序process_file_ui.exe分三步处理数据:
process_file_ui.exe程序的“Vital View 处理”部分在 VV 输入目录中收集一个或多个 Vital View 导出文件,并将它们组合成一个处理文件。程序然后重新采样过1的数据-分钟尤特的时间间隔,从而使数据可被直接比较与RFID数据。保证是维维输入目录仅包含生命观导出文件。选择“VV input”目录作为输入,选择“VV output”目录作为输出。输入输出 VV 文件的名称并运行程序的这一部分(图6A )。输出文件将写入“VV 输出”目录(图 7C)。
process_file_ui.exe程序的“RFID 处理”部分接受预处理的 RFID 文件 .csv,并根据每个鼠标的唯一 EPC 代码将其标签读取分为每行分钟(每只鼠标都有自己的列)。选择在“RFID输入”的目录中输入的预处理后的RFID数据文件,并选择“RFID输出”目录作为输出(图6的B)。输入输出 RFID 文件的名称并运行程序的这一部分。输出文件将出现在“RFID 输出”目录中(图 7D)。
process_file_ui.exe 程序的“Cross Reference Vital View Data with RFID Data”部分将每 1 分钟间隔内的滚轮转数总和分配给该分钟内标签读取次数最多的笼子中的鼠标。然后将车轮转数转换为距离 r u n(使用我们的跑步轮直径 4.5 英寸),并将个人跑步距离作为每天一个 .csv 文件输出(例如,如果您记录了 7 天,则在其中将有七个单独的文件)输出目录)(图 7E)。选择“VV 输出”目录(包含步骤 1 中的文件)作为程序的 VV 输入,并选择“RFID 输出”目录(包含步骤 2 中的文件)作为程序的 RFID 输入。选择“Distances”目录作为距离输出,选择“Stats”目录作为统计输出。在“:MM:YYYY-MM-DD HH SS”提供的日期和时间所期望的数据处理的开始和结束(24小时)格式并运行该程序的该部分(图小号6C,8A,8B )。
图 7. RFID 和 VV 数据文件重新格式化示例。一个。重新格式化(预处理)之前的原始 RFID 导出文件格式的摘录。乙。节选的最终重新格式化(预处理)RFID导出文件,准备在RFID处理使用。Ç 。节选中的VV输出文件的“重要视图处理”的步骤。d 。节选中的RFID输出文件的“RFID处理”的步骤。乙。节选的为一天距离输出文件的“交叉引用命门查看数据与RFID数据”步骤。当交叉引用多天时,每天都会在“距离”目录中生成一个输出文件。˚F 。节选的为一天统计输出文件的“交叉引用命门查看数据与RFID数据”步骤。当交叉引用多天时,每天都会在“Stats”目录中生成一个输出文件。


数据验证(图 8)
注意:跑步距离是按分钟分配和编制的。该process_file_ui.exe程序提供单独的.csv文件小号日志记录)的分钟数与至少一个车轮转动,或“总VV计数”,以及b)分钟与车轮转动,但没有数的标签读取,或者“没有 RFID,vv 也算数。”这些文件位于“Stats”文件夹中。每天生成一个 Stats 文件(图 7F)。


b) 除以 a) 得出 RFID 系统未捕获的运行分钟数的比例(错误率)(图 8C)。
图8.代表性d ATA。一个。每天雄性小鼠的个别运行距离,记录连续13天(之间产后25天并在两个保持架,保持架1和保持架3.小鼠1A和1B 37)被成对圈养在一起和小鼠3A和3乙分别成对住在一起。小鼠被P21-24中(数据未显示)驯化的运行车轮。乙。每天平均距离跑,证明在一个显著帧内笼差的平均运行量(非配对吨-test,* P <0.05)。Ç 。每个笼子每天带轮转的总分钟数(由 VitalView ®记录)和在没有随附 RFID 标签读取的情况下运行的总分钟数(由 RFID 阅读器记录)。.


笔记


系统监控
必须定期监测配备 RFID 的笼子是否因车轮下的嵌套材料或过多的垫料而造成车轮障碍物。这可以通过日常目视检查或查看 Vital View 软件记录来确定。
定期检查RFID导出文件,以确保将仍然被检测到的代码如预期。定期检查的重要视图数据出口,以保证该预期鼠标运行正在记录。我们每隔一天监控这些出口。在实验过程中,数据文件可以导出到外部驱动器。
每天监测老鼠的 RFID 标签和/或贴在耳朵上的耳标是否有任何中断。在我们的实验中,从老鼠身上分离出的耳标和 RFID 标签只发生过两次。如果鼠标缺少 RFID 标签或耳标,请给鼠标一套新的,确保新 RFID 标签的 EPC 与脱落的标签相同。
鼠标运行
通过明暗循环,每天 24 小时连续进行跟踪,并且可能会在幼崽与母亲断奶后(产后第 21 天)开始。
没有给小鼠固定轮适应期。他们是不是允许从他们的时间运行轮运行,我们再断奶。塑料网套设于轮确保了轮辋是车轮是安全的幼年小鼠上运行,所以在适应期不被认为有必要保证安全。我们的数据显示,幼鼠在断奶后随着时间的推移逐渐增加它们的跑动。驯化的最初几天可能数据分析过程中,如果需要除去编。
致谢


所有图像均使用 Biorender 生成。特别感谢 atlasRFIDstore、Keonn Technologies 和 Luke Raus 的技术援助和专业知识。


利益争夺


没有要披露的竞争利益。


伦理


本协议中描述的所有使用活体动物的实验均已被我们在加州大学欧文分校的机构动物护理和使用委员会 (IACUC) 批准使用。采取所有步骤以确保在这种范例中小鼠的疼痛和痛苦最小。该实验的IACUC 批准使用协议是 19-057。


参考


荒川,H.(2018 年)。实验室啮齿动物行为研究中社会隔离效应的伦理学方法。行为大脑研究341:98-108。
Batchelder, P., RO Kinney, L. Demlow 和 CB Lynch 。(1983)。温度和社会互动对小家鼠蜷缩行为的影响。生理学行为31(1): 97-102。
Frahm, S.、V. Melis、D. Horsley、JE Rickard、G. Riedel、P. Fadda、M. Scherma、CR Harrington、CM Wischik、F. Theuring 和 K. Schwab 。(2018)。α-突触核蛋白转基因小鼠 h-α-SynL62 显示出 α-Syn 聚集和让人想起帕金森病的多巴胺能表型。行为大脑研究339:153-168。
Goh, J. 和 W. Ladiges 。(2015)。在小鼠中运行的自愿轮。Curr Protoc Mouse Biol 5(4): 283-290。
Hawkley, LC, SW Cole, JP Capitanio, GJ Norman 和 JT Cacioppo 。(2012)。社会隔离对社会哺乳动物糖皮质激素调节的影响。荷尔蒙行为62(3):314-323。
海斯,LD (2000)。共同筑巢或不共同筑巢:啮齿动物共同筑巢和护理的回顾。动画行为59(4):677-688。
Himmler, BT, SM Pellis 和 B. Kolb 。(2013)。青少年的游戏经历使内侧前额叶皮层的神经元对以后的经历更加敏感。Neurosci Lett 556:42-45。
Ieraci, A.、A. Mallei 和 M. Popoli 。(2016)。社会隔离压力会导致成年雄性小鼠的焦虑抑郁样行为和神经可塑性相关基因的改变。神经塑料 2016:6212983。
Ivy、AS、T. Yu、E. Kramar、S. Parievsky、F. Sohn 和 T. Vu 。(2020)。早期生活运动的独特小鼠模型使海马记忆和突触可塑性成为可能。科学报告10(1): 9174。
Kappel, S., P. Hawkins 和 MT Mendl 。(2017)。分组还是不分组?饲养雄性实验室小鼠的良好做法。动物(巴塞尔)7(12)。
Koike, H.、D. Ibi、H. Mizoguchi、T. Nagai、A. Nitta、K. Takuma、T. Nabeshima、Y. Yoneda 和 K. Yamada 。(2009)。社会隔离饲养小鼠的行为异常和药理反应。行为大脑研究202(1): 114-121。
Krynitsky, J., AA Legaria, JJ Pai, M. Garmendia-Cedillos, G. Salem, T. Pohida 和 AV Kravitz 。(2020)。Rodent Arena Tracker (RAT):机器视觉啮齿动物跟踪相机和闭环控制系统。电子神经网络7(3)。
Liu, Y.、T. Yan、JM Chu、Y. Chen、S. Dunnett、YS Ho、GT Wong 和 RC Chang 。(2019)。体育锻炼对大脑的有益作用和神经退行性疾病的相关病理生理机制。实验室投资99(7):943-957。
Makinodan, M., KM Rosen, S. Ito 和 G. Corfas 。(2012)。社会经验依赖性少突胶质细胞成熟和髓鞘形成的关键时期。科学337 (6100):1357-1360。
Matikainen-Ankney, BA, M. Garmendia-Cedillos, M. Ali, J. Krynitsky, G. Salem, NL Miyazaki, T. Pohida 和 AV Kravitz 。(2019)。啮齿动物活动检测器 (RAD),一种用于测量啮齿动物家庭笼中活动的开源设备。电子神经网络6(4)。
Okada, R.、H. Fujiwara、D. Mizuki、R. Araki、T. Yabe 和 K. Matsumoto 。(2015)。多巴胺能和胆碱能系统参与社会隔离引起的小鼠社会归属和条件恐惧记忆缺陷。神经科学299:134-145。
Oliff, HS, NC Berchtold, P. Isackson 和 CW Cotman 。(1998)。运动诱导的大鼠海马脑源性神经营养因子 (BDNF) 转录的调节。Brain Res Mol Brain Res 61(1-2):147-153。
Peleh, T.、X. Bai、MJH Kas 和 B. Hengerer 。(2019)。支持 RFID 的视频跟踪,用于自动分析小鼠群体的社会行为。J Neurosci 方法325:108323。
Poffe, C.、S. Dalle、H. Kainz、E. Berardi 和 P. Hespel 。(2018)。一种用于测量小鼠笼内自发身体活动的无干扰系统。J Appl Physiol 125(2): 263-270。
Reimer、JD 和 ML Petras 。(1967)。家鼠 Mus musculus 在种群笼中的繁殖结构。J 哺乳动物48(1):88-99。
Terranova, ML, G. Laviola, L. de Acetis 和 E. Alleva 。(1998)。小鼠激动行为个体发育的描述。J Comp Psychol 112(1):3-12。
Voikar, V. 和 S. Gaburro 。(2020)。小鼠自动化家笼表型分析的三大支柱:基于文献和经验的新发现、改进和再现性。前行为神经科学14:575434。
Wang, Z.、SA Mirbozorgi 和 M. Ghovanloo 。(2018)。一种使用深度图像自由移动啮齿动物的自动行为分析系统。Med Biol Eng Comput 56(10): 1807-1821。
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引用:Valientes, D. A., Raus, A. M. and Ivy, A. S. (2021). An Improved Method for Individual Tracking of Voluntary Wheel Running in Pair-housed Juvenile Mice. Bio-protocol 11(13): e4071. DOI: 10.21769/BioProtoc.4071.
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