Protocol for sleep analysis in the brain of genetically modified adult mice

Summary Elucidating the molecular pathways that regulate animal behavior such as sleep is essential for understanding how the brain works. However, to examine how a certain functional domain of protein is involved in animal behavior is challenging. Here, we present a protocol for inducing endogenous protein that lacks a specific functional domain using Cre-mediated allele modification in neurons followed by electroencephalogram/electromyogram (EEG/EMG) recording to study the role of kinases in sleep. This strategy is applicable to other gene targets or behaviors. For complete details on the use and execution of this protocol, please refer to Iwasaki et al. (2021).


SUMMARY
Elucidating the molecular pathways that regulate animal behavior such as sleep is essential for understanding how the brain works. However, to examine how a certain functional domain of protein is involved in animal behavior is challenging. Here, we present a protocol for inducing endogenous protein that lacks a specific functional domain using Cre-mediated allele modification in neurons followed by electroencephalogram/electromyogram (EEG/EMG) recording to study the role of kinases in sleep. This strategy is applicable to other gene targets or behaviors. For complete details on the use and execution of this protocol, please refer to Iwasaki et al. (2021).

BEFORE YOU BEGIN
Note: All animal work presented here was approved (Protocol#180094) and conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of the University of Tsukuba.
Here, we describe materials and methods for the induction of SIK3 lacking a protein kinase A (PKA)phosphorylation site in neurons using Sik3 ex13flox and synapsin1 CreERT2 mice (Funato et al., 2016;Honda et al., 2018;Iwasaki et al., 2021). Intraperitoneal injection of tamoxifen in late infancy leads to the expected recombination and increases non-rapid eye movement sleep (NREM sleep) in syn-apsin1 CreERT2 ; Sik3 ex13flox mice. Using other flox mice, the researchers are able to induce or delete the target domain or the entire protein in adult brains. To confirm the successful allele induction, it is desirable to obtain or create a variety of antibodies that detect allele-specific and/or general form of the target protein. When there are no available antibodies, RT-PCR can be used to confirm allele induction. To visualize Cre-mediated recombination induced by tamoxifen, crosses with reporter mouse lines such as ROSA26 LacZ mouse are useful to observe detailed spatial recombination.

Tamoxifen dosage
In general, higher doses of tamoxifen administration guarantee higher recombination rates, but at the same time lower survival rates (Table 1). Donocoff and colleagues showed that almost all C57BL/6 mouse given five daily 6 mg tamoxifen (equivalent to 200 mg/kg body weight (BW) in a 30 g mouse) were dead in 2 weeks whereas 70% of those given 3 mg tamoxifen survived (Donocoff  OPEN ACCESS et al., 2020). According to Valny and colleagues, peak concentrations of tamoxifen metabolite 4-hydroxytamoxifen in adult brains, which presumably be a major CreERT2 inducer, were unchanged between 2 daily 200 mg/kg BW and 100 mg/kg BW administration, but faster degradation was seen at the lower dose (Valny et al., 2016). After five daily intraperitoneal injection of 100 mg/kg BW tamoxifen from P28 to sodium-dependent L-glutamate/L-aspartate transporter (GLAST)-CreERT2 mice results in nearly 100% recombination in the cerebral cortex (Jahn et al., 2018). Five times 100 mg/kg BW tamoxifen injection were performed every alternative day for 10 days from 3 weeks old, which provided high efficient recombination (Huang et al., 2018). These reports suggest that repeated injections of 100 mg/kg BW tamoxifen are reasonable to achieve high recombination efficiency while maintaining low lethality.
Age at which tamoxifen is administered may affect recombination efficiency (Table 1). A single administration of 100 mg/kg BW of tamoxifen to GLAST-CreERT2 mice at P1 led to an almost complete deletion of the target protein, glutamate transporter 1. However, five daily administrations to P19 and adult mice led to a moderate or mild reduction, respectively (Aida et al., 2015). For neonatal mice, Pitulescu et al. intraperitoneally injected 50 mg tamoxifen for 3 consecutive days from P1 (equivalent to 25 mg/kg BW in a 2 g mouse), and 100 mg tamoxifen for 4 consecutive days from P5 (equivalent to 33 mg/kg BW in a 3 g mouse) (Pitulescu et al., 2010(Pitulescu et al., , 2017.
After preliminary experiments, we decided to administer 100 mg/kg BW tamoxifen once a day for five days from P28. For infants, we repeatedly injected 500 mg tamoxifen at P14, 17, 20 to minimum intervention of pup rearing. Given that pups weigh 6g, 500ug is equivalent to 83 mg/kg BW.

Animals
All animal experiments must be approved by relevant institutional review board. The animal experiments described here were approved and conducted according to the guidelines established by the Institutional Animal Care and Use Committee of the University of Tsukuba.
We use synapsin1 CreERT2 mouse line and Sik3 ex13flox mice in step-by-step protocol which were established in (Iwasaki et al., 2021). In synapsin1 CreERT2 ; Sik3 ex13flox mice, tamoxifen administration induces exon 13 skipping allele of Sik3, which produces Sleepy (Slp) mutant SIK3. Male mice were used in this protocol. EEG/EMG recording were performed between 10-to 12-week-old.
To breed mouse lines, genotyping is required. The reagents and primer information used in (Iwasaki et al., 2021) are shown in key resources table. PCR cycling conditions are shown below. In genotyping of synapsin1 CreERT2 , the size of PCR products of wild-type allele amplified with synapsin1-CreERT2_C1 and synapsin1-CreERT2_W1 is 396 bp, and knock-in allele amplified with synapsin1-CreERT2_C1 and synapsin1-CreERT2_M1 is 443 bp. In genotyping of Sik3 ex13flox , the size of PCR products of wild-type allele is 191 bp, and knock-in allele is 293 bp.

PCR cycling conditions for genotyping synapsin1 CreERT2
Steps Temperature Time Cycles Alternatives: This protocol can be applied to other CreERT2 and flox mouse lines. We recommend that you observe the spatial and temporal expressions of the target gene using Allen Brain Atlas and in situ hybridization before generating new CreERT2 mouse lines. It is recommended to verify enough recombination efficiency is achieved and consider dose/timing of tamoxifen administration as needed. Please refer prior part ''Tamoxifen dosage'' for protocol modification. To show spatial recombination pattern in expected outcomes, we used RO-SA26 LacZ mouse line in (Iwasaki et al., 2021).
Alternatives: Adeno-associated virus (AAV)-based approaches are also possible to induce a mutant allele in the adult brain. For example, the local administration of Cre-expressing AAV in Sik3 ex13flox mouse brains can induce the exon 13-skipping allele. The local administration of a double-inverted orientation (DIO) of Sik3 Slp (ex13-skipped)-expressing AAV in an appropriate Cre mouse brains enables to express the mutant protein at a specific cell type.

Preparation of materials required for EEG/EMG recording
We examined sleep/wake behavior using electroencephalogram/electromyogram (EEG/EMG) recording after CreERT2 induction. Specialized equipment is required such as recording chambers, stereotaxic apparatus, EEG/EMG electrodes, amplifiers and analog-to-digital converters, EEG/EMG analysis software. Protocol to make tether cable is shown below. Please refer to key resources table for equipment information.

Timing: 1 day
Preparation of tether cable 1. Cable preparation a. Prepare tools and materials shown in Figures 1A and 1B. b. Cut the four-core cable into 17 cm pieces. c. Strip 1-1.2 cm of the outer sheath from both ends of the four-core cable with a stripper. d. Cut the metallic shields and intervention of the four-core cable from each end with small scissors e. Strip 1-1.2 mm of the inner sheath from each end of the four wires with a wire stripper (Figure 1C). f. Prepare eight thin heat-shrinkable tubes of 1 cm in length and two thick heat-shrinkable tubes of 2 cm in length ( Figure 1B). g. Pass each wire through the thin heat-shrinkable tube ( Figure 1D). h. Pass the cable through the two thick heat-shrinkable tubes ( Figure 1E). 2. Detangler preparation a. Cut the detangler cable. b. Strip the outer sheath of the detangler cable from the end with small scissors. c. Strip 1-1.2 mm of the inner sheath from the four wires of the detangler cable using a wire stripper ( Figure 1F). a. Bend the pin headers with pliers and cut it so that each piece has three pairs of electrode pins ( Figure 1B).
4. Attach the four-core cable to the detangler cable a. Insert each detangler wire into the thin heat-shrinkable tube covering a specific wire ( Figure 1G).
CRITICAL: The conductors of the four-core cable and the detangler cable need to be in contact with each other.
b. Apply hot air to the heat-shrinkable tubes with a heat gun to shrink them ( Figure 1H).
Note: Do not expose the thick heat-shrinkable tube to hot air.
5. Connect the four-core cable to the electrode pins to make a tether cable a. Insert each pin into the thin heat-shrinkable tube covering a specific wire ( Figure 1I).
CRITICAL: The conductors of the four-core cable and the electrode pins need to be in contact with each other.
b. Apply hot air to the thin heat-shrinkable tubes with a heat gun to shrink them ( Figure 1J).
Note: Do not expose the thick heat-shrinkable tube to hot air.
6. Check energization a. Connect the tether cable to the multimeter tester with electrode pins and detangler. b. Make sure that the electrical resistance of all conductors are less than 20U and stable even when the detangler is twisted. 7. Fix connections with epoxy and attach hanger wire a. Fix the pins and cable connection with epoxy. i. Mix the two components of the epoxy resin.
ii. Apply the mixed epoxy resin from the outer sheath to the base of the electrode pins and leave it until it hardens ( Figure 1K). iii. Cover the border between sheath and wires with thick heat-shrinkable tube and seal it ( Figure 1L). iv. Apply the mixed epoxy resin to the connections between heat-shrinkable tube and fourcore cable, and heat-shrinkable tube to the base of the electrode pins. Leave it until it hardens ( Figure 1M). b. Fix the detangler and cable connection with epoxy.
i. Cover the connection from the four-core cable to the detangler cable with a thick heatshrinkable tube and shrink it with a heat gun. ii. Apply the mixed epoxy resin to the connections between the detangler cable and heat-shrinkable tube, and heat-shrinkable tube and four-core cable. Leave it until it hardens ( Figure 1N). (J) Shorter pins and four-core cable are connected after hot air is applied.
(K) Epoxy resin is applied from the outer sheath to the base of the electrode pins.
(L) Thick heat-shrinkable tube covering the border between sheath and wires after sealing.
(M) Epoxy resin is applied to the connections between heat-shrinkable tube and four-core cable, and heat-shrinkable tube to the base of the electrode pins.
(N) Epoxy resin is applied to the connections between the detangler cable and heat-shrinkable tube, and heat-shrinkable tube and four-core cable.
(O) A hanger wire is attached to the detangler. Finished tether cable.

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c. Attach a hanger wire to the detangler ( Figure 1O).
Alternatives: There are several ways to assess sleep/wakefulness without EEG/EMG recording. However, EEG/EMG-based sleep analysis is the most reliable, especially for rapid eye movement sleep (REM sleep) and enables spectrum analysis that is required for the deeper analysis of sleep.

MATERIALS AND EQUIPMENT
Note: Prepare on ice before use.
Note: Add MilliQ after the other reagents are dissolved by heating at 70 C for 15 min; Store the aliquot at À20 C without 2-mercaptoethanol and can be store for at least 6 months; Add 2mercaptoethanol immediately before use and store at 4 C.
Note: Store at 4 C protected from light, and do not store more than 6 months.
Note: Add TEMED and mix immediately before pouring the gel into the mold.

Reagents Final concentration Amount
Tris-HCl pH7.6 (1 M) 50 mM 50 mL NaCl (1 M Note: Add TEMED and mix immediately before pouring the gel into the mold.
Note: Store at 18 C-24 C, and do not store more than 1 year. Dilute before use.
Note: Store at 18 C-24 C, and do not store more than 1 year. Dilute before use.
Note: Store at 18 C-24 C, and do not store more than 1 year.

ll OPEN ACCESS
Note: Store at 18 C-24 C, and do not store more than 2 weeks.

Materials to confirm CreERT2 dependent recombination
Western blots using allele specific antibodies In general, useful antibodies for detecting allele induction or deletion are as follows: 1) antibodies specific for sequences deleted by Cre-dependent allele induction/deletion; 2) antibodies specific for sequences encompassing adjacent exons after Cre-dependent allele induction/deletion. 3) antibodies specific for functional features such as amino acid sequence containing phosphorylated amino acids. Standard equipment for western blots is required. Please refer key resources table for product information that we used in the protocol.
Anti-SIK3 ex13 antibody This rabbit polyclonal antibody was raised against LHAQQLLKRPRGPS using custom antibody production (Eurofins). The amino acid sequence is encoded by exon 13 and is expected to be less susceptible to phosphorylation because it is 8 amino acids away from 551 serine residue.

Anti-Slp-specific SIK3 antibody
This rabbit polyclonal antibody was raised against QLEYKAVPA spanning the sequence encoded by exon12 and exon 14 using custom antibody production (Eurofins). Resultant anti-serum was absorbed with QLEYKEQS (synthesized by Eurofins) spanning the sequence encoded by exon12 and exon 13 to eliminate the affinity for wild-type SIK3.
SIK3 sequence a part of exon12, 13, 14-encoded regions. QVAPNMNFTHNLLPMQSLQPTGQ LEYKEQSLLQPPTLQLLNGMGPLGRRASDGGANIQLHAQQLLKRPRGPSPLVTMTPAVPAVTPVDEES SDGEPDQEA Bold letters indicate amino acids encoded by exon 13 and RRAD (underlined) is the consensus sequence for PKA phosphorylation.
Alternatives: Alternative antibody is required depending on the flox mouse line. Consider RT-PCR if there is no available antibody.

STEP-BY-STEP METHOD DETAILS
Obtaining synapsin1 CreERT2 ; Sik3 ex13flox mice Timing: More than 4 weeks Synapsin1 CreERT2 and Sik3 ex13flox mice are crossed to obtain synapsin1 CreERT2 ; Sik3 ex13flox mice. Since the synapsin1 gene is located on the X chromosome, the synapsin1 CreERT2 of males is passed from their mothers. In order to increase efficiency, it is necessary to increase the number of mice with the appropriate sex and genotype. Young adult mice (2-4 months) are recommended when breeding starts. Littermates are recommended as control group because the genetic and environmental conditions can be identical except for the Sik3 ex13flox allele.
Note: Please refer to the key resources table for reagents used in the genotype, and PCR conditions 8. Wean at around 4 weeks of age.

Timing: 2 days
Tamoxifen is diluted to 20 mg/mL in corn oil. Dilution should be started the day before the first IP injection. Dissolved tamoxifen should be used within 1 month in 4 C.
CRITICAL: Tamoxifen needs to be handled carefully according to safety data sheet because of the toxicity.
9. Cover a 15 mL-tube with aluminum foil to protect it from light (Figures 2A and 2B). 10. Bring tamoxifen to room temperature in a dark room and heat 5 mL of corn oil to 42 C. 11. Weigh 80 mg of tamoxifen in the tube prepared in step 10 ( Figure 2C). 12. Add warmed 4 mL of corn oil to the tube and vortex (Figures 2D and 2E).
Note: Change the amount of tamoxifen dilution as needed.
CRITICAL: Repeat vortex until no more tamoxifen particle are visible.

Tamoxifen injection during late infancy
Timing: 7 days ll

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Tamoxifen is delivered with IP injection 17. Dilute tamoxifen stock (20 mg/mL) 2 times with corn oil to 10 mg/mL.
18. Intraperitoneally inject 50 mL tamoxifen in corn oil (10 mg/mL) to pups on postnatal days 14, 17, and 20. a. Fill a syringe with the tamoxifen solution and remove air in syringe ( Figures 3A and 3B).  Note: Because of the high viscosity of corn oil, this step takes several minutes.
b. Separate pups to a new cage. c. Gently stretch the pup along your hand by holding the back neck skin and tail ( Figure 3C). d. Insert the needle to the lower abdomen towards the head at a 30 to horizontal. The needle was inserted only 5 mm ( Figure 3D). e. Inject 50 mL of tamoxifen in corn oil intraperitoneally. f. Put the pups back to breeding cage.
Note: This injection procedure is the same as giving an IP injection to an adult mouse.
Note: It is recommended to fix injection time to avoid varying injection interval which may affect tamoxifen accumulation.
Note: For histological examination, we sacrificed the mice 7-8 days after the last injection.

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our laboratory, EEG signals are recorded from ipsilateral metal pins on left hemisphere, and EMG signals are obtained from neck extensor muscle.
19. EEG/EMG electrode implantation a. Prepare tools for electrode implantation (Figure 4 and its figure legend). b. Place an 8-9-week-old mouse in an anesthesia box and anesthetize for 3 min with 4% isoflurane. c. Fix the mouse head in a stereotaxic instrument with two ear bars and a nose clamp. (Figure 5A).
Note: Stable fixation capable of avoiding movement by drilling is required.
Note: Observe respiratory rate and decrease isoflurane concentration if it is unstable or too slow.
e. Apply Vaseline to the eyes, shave the fur from the top of the head, and disinfect the head skin with 70% ethanol. f. Make a longitudinal incision (2 cm long) along the midline of the scalp. g. Open the skin with four tissue clamps and clean the cranium surface with a cotton swab ( Figure 5B). h. Adjust the head holder to set bregma and lambda at the same dorsoventral and mediolateral level ( Figure 5B). i. Drill four holes in the skull for EEG electrodes and clean the skull surface with a cotton swab ( Figures 5C and 5D).
CRITICAL: Do not drill too deep so as not to damage the cerebral cortex. We use the tip of the drill reaching the outer surface of the skull as seen by the surgeon as a guide. But this varies depending on the drill used and needs to be adjusted for each researcher.
j. Attach the electrode to the implant holder with long pins (made of a pin header, Figure 1) of the 6-pin header ( Figure 5E). k. Lower the electrode pins into the holes under stereotaxic control until reach the four stoppers (small protrusion at the bottom of the implant to contact the skull) of the implant attach on the skull ( Figure 5F).
CRITICAL: Do not push the skull with the stopper.
CRITICAL: Of the four stoppers, at least two on the left side should be attached to the skull for EEG recording.
CRITICAL: When two stoppers on the left side is not attached to the skull because of displacement in horizontal direction of skull, correct the tilt with screws on the stereotaxic instrument.
l. Set the Z axis to zero and heighten the Z axis of the implant to 1.0 mm. m. Apply dental cement between the skull and the electrode base ( Figure 5G).
CRITICAL: Turn off the surgical light as the dental cement is cured by strong light.
CRITICAL: Make sure that the cement does not contain air when it is filled.
n. Lower the Z axis of the implant to zero and cure the dental cement with LED light. o. Cross the two EMG wires and insert them into neck extensor muscles under the fascia (Figure 5H). p. Apply dental cement from the base of the wires to the intersection and cure the dental cement with LED light ( Figure 5I). q. Apply two-four stitches using silk thread ( Figure 5J).

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Note: Make sure there is no gap between the implant and the skin to prevent suture from being broken by the mouse.
r. Stop the anesthesia and remove the mouse from the stereotaxic instrument. s. Remove the 6-pin header from the implant and then insert a one side 6-pin header (Figure 6A).
Note: Hold the implant, not the mouse head, when removing the 6-pin header and inserting the one side 6-pin header.
t. Carefully wipe Vaseline from eyes. u. Place the mouse in the cage on the heating pad to keep it warm until the mouse starts normal behavior.
20. EEG/EMG recording a. Allow the mouse to recover for at least 5 days in a home cage. b. Put the mouse in an anesthesia box (4% isoflurane) and wait until the mouse loses righting reflex. Take the mice from the anesthesia box and quickly remove the cap and attach a tether cable to the mouse. c. Acclimate the mouse to the recording environment for at least 7 days ( Figures 7B and 7C).
Note: Left side electrodes are used for EEG recording, so the electrode pins with three wires need to be left side ( Figure 6B).

Western blotting for verification of recombination
Timing: 3 days Sik3 recombination was verified with western blotting. Mice were sacrificed after EEG/EMG recording for brain sampling.
21. Brain sampling for western blotting a. Prepare liquid nitrogen (LN2), tubes and dissection tools. b. Quickly dissect the brain after cervical dislocation and freeze it in LN2. c. Store at À80 C until homogenization.
Pause Point: frozen brain can be stored at À80 C for at least 3 months. ; Sik3 ex13flox/+ mice that were administered tamoxifen or vehicle at P14, P17, and P20. One-way ANOVA followed by Tukey's test; 13 mice per group. Data are mean G SEM. ***p<0.001. For more information, please refer to the original paper (Iwasaki et al., 2021). (E) Western blotting of brain homogenates. An antibody specific to ex13 skipping SIK3 detected the protein (asterisks) in tamoxifen administrated synapsin1 CreERT2 ; Sik3 ex13flox/+ mice and Sik3 Slp/Slp mice (top row). An antibody specific to the exon 13-encoded region detected wild-type SIK3 protein in tamoxifen-or vehicle-administered synapsin1 CreERT2 ; Sik3 ex13flox/+ mouse brains, but not in Sik3 Slp/Slp mouse brains (middle row). An antibody specific to SIK3 c-terminus detected long isoform of SIK3 (third row). In wild-type brains, there are two major isoforms of SIK3: short isoform (50-75 kDa, top and second row) and long isoform (150-250 kDa, third row) (Park et al., 2020). b-tubulin was used as a loading control (bottom row).

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22. Western blotting a. Homogenize the brains using a rotor-stator homogenizer in ice-cold Lysis Buffer (100 mL Lysis Buffer for 1 mg brain). b. Rotate 15 min at 4 C for 30 min. c. Centrifuge at 178003g at 4 C for 12 min. d. Immediately aliquot supernatant. e. Determine protein concentration with micro BCA assay. f. Dilute a part of supernatant with 63 Sample Buffer for SDS-PAGE and store the diluted sample and remaining sample at À80 C.
Pause Point: frozen supernatants can be stored at À80 C.
g. Perform SDS-PAGE with 7% Running Gel (under the 3% Stacking Gel layer) with 50 mg of protein in 13 Running Buffer. h. Activate PVDF membrane with methanol for 10 min at room temperature (18 C-24 C). i. Transfer the protein to PVDF membranes in 13 Transfer Buffer.
Note: The concentration of acrylamide gel and protein concentration can be modified depending on your purpose.
j. Wash the membranes in TBST and incubate overnight (12-18 h) at 4 C with a primary antibody in TBST with 5% BSA. k. Wash in TBST and incubate with HRP-conjugated for 2 h at room temperature (18 C-24 C), donkey anti-rabbit IgG (1:2000 dilution in TBST with 5% BSA; Jackson ImmunoResearch Laboratories) l. Wash in TBST and expose the blots to Clarity Western ECL Substrate. m. Detect chemiluminescence signaling with FUSION Solo 6S.EDGE.

EXPECTED OUTCOMES
By following the protocol described here, we analyzed sleep/wake behavior of mice expressing SIK3 lacking exon 13 encoded region in neurons after late infancy ( Figure 7A). Total wake time was decreased and NREM sleep time was increased in tamoxifen administered synapsin1 CreERT2 ; Si-k3 ex13flox/+ mice ( Figures 7B and 7C), but REM sleep time remained unchanged ( Figure 7D). These result shows that CreERT2 knock-in to synapsin1 locus did not affect total time spent in each stage. Western blotting showed that SIK3 lacking exon 13 expressed in synapsin1 CreERT2 ; Sik3 ex13flox/+ mice only with tamoxifen administration ( Figure 7E, Iwasaki et al., 2021).

LIMITATIONS
The target exon that encodes functionally relevant amino acid sequence such as protein kinase recognition sequences is in-frame. Since the expression of synapsin 1 is not evenly expressed in all neurons in the brain and tend to be more broadly expressed in earlier stage as shown (Iwasaki et al., 2021), the injection schedule of tamoxifen needs to be optimized.

TROUBLESHOOTING Problem 1
Tamoxifen does not dissolve in corn oil (step 15).

Potential solution
Increase the frequency of vortex during overnight incubation. Many protocols state that tamoxifen is dissolved at 37 C, but in our experience, that doesn't dissolve very well. Then, we set the temperature to 42 C to make it easier to dissolve. Please see Figures 2E and 2F.

Problem 2
Mice are dead after tamoxifen administration (step 18).

Potential solution
We have rarely experienced death of mice after tamoxifen administration in the current protocol. However, if mice frequently die after tamoxifen administration, the route and timing of administration should be changed appropriately to maintain a balance between the efficiency of Cre-dependent recombination and the survival rate. Please refer ''Tamoxifen doseage'' in this protocol for more information.

Problem 3
Bleeding from the skull hole during the implantation surgery (step 19. i).

Potential solution
Apply a clean twisted paper (kimwipes) into the skull hole and absorb the blood. In case of a lot of bleeding, apply pressure with a cotton swab against the skull hole to stop the bleeding.

Problem 4
The implant dislodged from the skull after the implantation surgery (step 19. k).

Potential solution
Before applying dental cement, carefully remove the blood and tissues on the skull with cotton swab.

Problem 5
The tether cord restricts mouse's movement since the mouse is small or weak (step 20. c).

Potential solution
Using a thinner and softer tether will make it easier for the mouse to move. However, the tethers are weak, so always have spare tethers available, or change to a regular tether when the mouse grows.

Problem 6
The clean EEG/EMG signals are not obtained (step 20. d).

Potential solution
Replace the tether cable with a new one. During the implantation procedure, make sure that the two stoppers on the left side of the EEG are lightly touching the skull, the EMG wires are inserted into the neck extensors and are not touching each other, and that the electrode pins are clean.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, [Masashi Yanagisawa] (yanagisawa.masa.fu@u.tsukuba.ac.jp).

Materials availability
This study did not generate new reagents.

Data and code availability
This study did not generate any data, a sophisticated custom computer code, or an algorithm.