Protocol to study inter-tissue communication between the hypothalamus and white adipose tissue and lifespan using a chemogenetic approach in aged mice

Summary

Here, we present a protocol for assessing the impact of a chemogenetic manipulation in a subpopulation of the hypothalamic neurons on aging and lifespan control using a mouse model developed specifically for this purpose. We describe steps for stereotaxic viral injection and assess inter-tissue communication between protein phosphatase 1 regulatory subunit 17 (Ppp1r17)-expressing neurons in the dorsomedial hypothalamus and white adipose tissue. We then detail procedures for lifespan measurements following chemogenetic manipulation in aged mice.

For complete details on the use and execution of this protocol, please refer to Tokizane et al.¹

Graphical abstract

Highlights

• •Stereotactic surgery for targeted infusion of virus into the aged mouse hypothalamus
• •Manipulation of neuronal activity to assess inter-tissue communication in aged mice
• •The longitudinal assessment for lifespan in aged mice under chemogenetic manipulation

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Here, we present a protocol for assessing the impact of a chemogenetic manipulation in a subpopulation of the hypothalamic neurons on aging and lifespan control using a mouse model developed specifically for this purpose. We describe steps for stereotaxic viral injection and assess inter-tissue communication between protein phosphatase 1 regulatory subunit 17 (Ppp1r17)-expressing neurons in the dorsomedial hypothalamus and white adipose tissue. We then detail procedures for lifespan measurements following chemogenetic manipulation in aged mice.

Before you begin

The protocol in this article describes the specific steps to use the chemogenetic approach to analyze aging and lifespan in mice. We have also used this protocol in other mouse models for lifespan studies. In our recent study, we investigated the impact of neuronal activation of a specific neuronal subpopulation localized in the dorsomedial hypothalamus (DMH), marked by protein phosphatase 1 regulatory subunit 17 (Ppp1r17) expression (DMH^Ppp1r17 neurons), on mouse aging and lifespan, monitoring the mice for >400 days (over 13 months) until their endpoint in comparison to control mice. We have demonstrated that the activation of DMH^Ppp1r17 neurons delays aging and extends lifespan.¹

In this protocol, we describe our chemogenetic approach in aged mice and how to assess the impact of this approach on their lifespan. Our protocol will be helpful for other mouse models that can evaluate aging and lifespan because there are no straightforward guidelines or protocols for long-term chemogenetic experiments using aged mice. Our study used a chemogenetic approach, the Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) system, to longitudinally activate a specific subset of hypothalamic neurons. Our protocol for the DREADD system enables activation of DMH^Ppp1r17 neurons only during the night, the active time for mice, and allows activation throughout their life. We also describe the assessments for inter-tissue communication between the hypothalamus and white adipose tissue (WAT) after chemogenetic manipulation in aged mice. DMH^Ppp1r17 neurons specifically regulate WAT function, including lipolysis and extracellular nicotinamide phosphoribosyltransferase (eNAMPT) secretion, by stimulating the sympathetic nervous system (SNS) directed to WAT.¹ Thus, the concept of this protocol will also help assess inter-tissue communication between the central nervous system and peripheral tissues in aged mice. The time estimates in this protocol may vary depending on unpredictable factors such as differences in mouse husbandry conditions.

Institutional permissions

Animal care and experimental procedures were performed according to the recommendations of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocol was approved by the Washington University School of Medicine in St. Louis Institutional Animal Care and Use Committee (IACUC). Prior approval from the relevant institutional animal ethical committee must be obtained to perform the protocol described in this article.

Breeding and husbandry of aged Ppp1r17-Cre mice

• 1.Obtain Ppp1r17-Cre [MMRRC:036188; Tg (Ppp1r17-Cre) NL163Gsat/Mmucd] mice for having Cre-recombinase expression in Ppp1r17-positive DMH neurons.
• 2.Backcross to wild-type C57BL/6J mice (Jackson Laboratories) for at least 6–7 generations before starting to make breeding units for the experiment.

Note: Since heterozygous mice have a wild-type chromosome present, using heterozygous instead of homozygous Cre genotypes might minimize unintended consequences of random transgene insertion. Cre toxicity can also occur when Cre expression is high enough to affect cell physiology. Nonetheless, expression levels of a heterozygous transgene should be sufficient to detect recombination, particularly in this specific Cre mouse line.

• 3.Generate heterozygous Ppp1r17-Cre mice by crossing heterozygous Ppp1r17-Cre and wild-type C57BL/6J mice.
• 4.To minimize uneven housing conditions, avoid single housing and combine different littermates into the same cage before starting the experiment, considering that mice are social animals.
• 5.Keep the heterozygous Ppp1r17-Cre mice for the lifespan cohort until they become 22-month old.

Note: In this protocol, we initiated viral injections using 22-month-old male mice. It would be ideal to obtain and assess each sex in lifespan studies. Female C57BL/6J mice live shorter than male mice in our facility, as reported in other facilities.² Therefore, starting the experiment earlier would be a better option if you plan to use female mice in this study.

CRITICAL: It will be crucial to make enough breeding units and keep larger cohorts of heterozygous Ppp1r17-Cre mice than you need for the lifespan study because some may die unexpectedly or need to be removed from the study for various reasons before the experiment is complete. If 60 heterozygous Ppp1r17-Cre mice are required at 22 months of age, it would be recommended to obtain 90 mice and euthanize the ones with visible lesions. Troubleshooting 1.

Obtaining the adeno-associated virus

Muscarinic receptor-based DREADD receptors are delivered to the DMH via viral injections. Adeno-associated virus (AAV) is a widely used tool for achieving the in vivo expression of chemogenetic receptors, and various AAV-encoding chemogenetic plasmids are available. Based on our pilot experiments, AAV5 would give us the best result for this experiment because it spreads less in the DMH compared to other serotypes.

• 6.Obtain AAV5-hSyn-DIO-mCherry (#50459-AAV5: control virus) and AAV5-hSyn-DIO-hM3D(Gq)-mCherry (#44361-AAV5: activation) from Addgene.
• 7.Make 5 μL aliquots of the virus in each tube, and snap-freeze them in liquid nitrogen or powdered dry ice before storing at −80°C.

Note: AAV stocks should be thawed immediately before use on ice and then kept on ice.

DREADD agonist 21 (compound 21) dihydrochloride solution

A combination of DREADDs and corresponding designer drugs, such as clozapine N-oxide (CNO), allows the modification of neuronal activity non-invasively in animal models. DREADD agonist 21 (Compound 21) hydrochloride is a water-soluble salt of Compound 21, which represents an alternative chemogenetic actuator for muscarinic-based DREADDs to CNO for in vivo studies in which the metabolic conversion of CNO to clozapine could be an issue.³ It has been reported that this compound crosses the blood-brain barrier to activate DREADD-expressing neurons and does not undergo metabolism back to clozapine.⁴ It is beneficial to use this compound for longitudinal administration because converted clozapine may affect animal behaviors, and effects on lifespan are unknown.

• 8.Dissolve 2.5 mg of DREADD agonist 21 in 10 mL of PBS (0.25 mg/mL).
• 9.Aliquot the solution into tightly sealed vials and store them at −20°C for up to one month.
• 10.Allow the product to equilibrate to 20°C for at least 1 h before opening and using it.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Rabbit anti-HSL (1:2,000)	Cell Signaling Technology	4107 RRID:AB_2296900
Rabbit anti-p-HSL (1:2,000)	Cell Signaling Technology	4126S RRID:AB_490997
Rabbit anti-NAMPT (1:1,000)	Bethyl	A700-058 RRID:AB_2891856
Rabbit anti-transferrin (1:1,000)	Abcam	ab82411 RRID:AB_1659060
HRP-conjugated goat anti-rabbit (1:10,000)	Vector Laboratories	PI-1000-1 RRID:AB_2916034
Bacterial and virus strains
AAV5-hSyn-DIO-mCherry	Addgene	50459-AAV5
AAV5-hSyn-DIO-hM3D(Gq)-mCherry	Addgene	44361-AAV5
Chemicals, peptides, and recombinant proteins
DREADD agonist 21 (Compound 21)	HelloBio	HB6124
Phosphate-buffered saline (PBS)	Gibco	C10010500BT
Isoflurane	Covetrus	11695-6777-2
Eye ointment	Stye	63736-0238-24
Triple antibiotic ointment	Taro	51672-2120-2
Betadine	Avrio Health L.P.	67618-154-16
Webcol alcohol prep	McKesson	191320
Ethanol 200 Proof	Decon	2701
Buprenorphine ER	Wedgewood Connect	79926-058-17
Hydrogen peroxide solution	Sigma-Aldrich	H1009
Tween 20	Thermo Fisher Scientific	P7949
Trizma base	Sigma-Aldrich	RDD008
Methanol	Thermo Fisher Scientific	A412P-4
HEPES	Sigma-Aldrich	H3375
NaCl	Sigma-Aldrich	746398
SDS	Thermo Fisher Scientific	BP166
EDTA	Sigma-Aldrich	E5134
DTT	Sigma-Aldrich	D9779
Phenylmethanesulfonyl fluoride (PMSF)	Sigma-Aldrich	P7626
Sodium fluoride (NaF)	Sigma-Aldrich	S6521
Sodium orthovanadate (Na3Vo4)	Sigma-Aldrich	S6508
cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail	Roche	04693159001
Critical commercial assays
HR series NEFA-HR kit	Wako Pure Chemical Industries	No. 999-34691
Experimental models: organisms/strains
Tg (Ppp1r17-Cre) NL163Gsat/Mmucd (C57BL/6 background, heterozygous, 22-month-old, male)	MMRRC	036188
Software and algorithms
Prism 9	GraphPad	https://www.graphpad.com/
Other
Standard chow diet	LabDiet	5053
Hamilton Neuros syringe	Hamilton	65457-02
3 ½ʹʹ iris scissors	Miltex	18-1396
Cotton tip wood shaft	McKesson	24-106-2S
Ethilon nylon suture	Ethicon	668G
Incubator	–	–
Standard insulin syringe with needle	McKesson	102-SN1C2805P
Purifier class I safety enclosures	Labconco	3730001
Model 940 small animal stereotaxic instrument	KOPF	940223A
Hand drill	Osada	CS132
Stereo microscope	Olympus	SZ61
Heating pad	Cell MicroControls	TCII
Intensive care unit dome cover small	Braintree Scientific, Inc.	DW 1N
Nanoliter syringe pump	KDScience	KDS 310 plus
Pestle	Argos	7339-901
Capillary tube	Fisher	22-260950
PVDF membrane	Millipore	IPVH00010
Mini Trans-Blot electrophoretic transfer cell	Bio-Rad	1703930
PowerPac Basic power supply	Bio-Rad	1645050
Carnation dry milk non-fat instant	Nestle	N/A
Pierce BCA Protein Assay Kit	Thermo Fisher Scientific	23227
NanoDrop OneC UV-vis spectrophotometer	Thermo Fisher Scientific	13-400-519
4%–15% SDS-PAGE	Bio-Rad	4561084 or 4561086
Pierce ECL western blotting substrate	Thermo Fisher Scientific	32209
Restore western blot stripping buffer	Thermo Fisher Scientific	21059

Materials and equipment

TBS-T

Reagent	Final concentration	Amount
Tris pH 7.4–7.6 (1 M)	10 mM	10 mL
NaCl (5 M)	150 mM	30 mL
Tween 20	0.1%	1 mL
ddH2O	N/A	959 mL
Total	N/A	1000 mL

Store at 20°C.

Homogenization buffer

Reagent	Final concentration	Amount
HEPES (1 M)	50 mM	500 μL
NaCl (5 M)	100 mM	200 μL
SDS (20%)	5%	2500 μL
EDTA (0.5 M)	2 mM	40 μL
DTT (0.5 M)	0.5 mM	10 μL
PmSF (0.1 M)	0.1 mM	10 μL
NaF (0.5 M)	0.1 mM	2 μL
NaVo4 (0.2 M)	0.1 mM	5 μL
HCl (2 N)	8 mN	40 μL
cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail (10x stock)	N/A	1000 μL
ddH2O	N/A	5693 μL
Total	N/A	10 mL

Do not store. Make this solution right before using.

Step-by-step method details

Part 1: Viral injection

DURATION: Approximately 1 h.

Here, we describe stereotaxic injection of AAV solution (30 nL) into the DMH of Ppp1r17-Cre mice to generate hM3D (Gq)-mCherry-expressing experimental (DREADD) and mCherry-expressing control (control) mice.

CRITICAL: Aged mice usually show physiological and pathophysiological variability, even among littermates (Figure 1A). With limited sample sizes, assigning subjects to groups semi-randomly can minimize confounding variables. The mice are evenly divided into two groups by counterbalancing their body weight and cage distributions. Then, the two groups are randomly assigned to be either the control or the DREADD group. Additionally, the mice should be kept in their original home cages after this procedure to avoid fighting and stress caused by changing the environment. Troubleshooting 2.

• 1.
  Preparing and setting up before viral injection.

  • a.
    Disinfect the instruments used in the injection experiment with 70% ethanol (Figure 1B). For specific instructions on proper aseptic techniques, please refer to intuitional guidelines.
  • b.
    Dilute AAV solution with PBS to 5.0 × 10¹² GC/mL.
  • c.
    Place the diluted solution on ice.

    Note: The solution may be preserved at 4°C for up to three weeks.

    Anesthetize a Ppp1r17-Cre mouse (22-month-old) with 1.5%–1.8% isoflurane gas and shave the fur off around the top of the skull.

    CRITICAL: Because aged mice are vulnerable to anesthesia, decrease isoflurane concentration compared to a regular concentration (2.0%) to avoid the risk of death during this procedure. Monitor the breathing of mice carefully. Depending on the breathing condition, the isoflurane concentration should be adjusted. Under optimal anesthesia, the breathing rate should be 55–65 breaths per minute. Ideally, this survival surgery can be performed within 50 min to avoid the risk of death from a long period of anesthesia. Troubleshooting 3.

  • d.
    Mount the anesthetized mouse by placing the ear bars and nose fixing clamp until the Lambda and Bregma are equal in height (flat-skull position) (Figure 1C).

    Note: At this moment, it is acceptable even if the flatness adjustment is not completed because the later step will precisely adjust the flatness. Troubleshooting 4.

  • e.
    Cover the anesthetized mouse’s eyes with sterile ocular lubricant to keep them moist during the surgery.
  • f.
    Clean the skin’s surface with alcohol and Iodine prep wipes, then use small scissors to make an incision along the midline to expose the skull.
  • g.
    Apply 30% hydrogen peroxide to a cotton swab to visualize the sagittal and coronal sutures and define the Bregma and Lambda (Figure 1D).

    CRITICAL: The different suture lines of the four bone plates of the skull surface must be visible to correctly identify the stereotaxic landmarks Bregma and Lambda. The quality of the approach to the target structure depends on the precise determination of these two reference points because there are individual variations in the sagittal and coronal sutures. Bregma corresponds to the intersection of the sagittal and coronal sutures at the rostral level of the mouse head but not the actual crossing point of the sagittal and coronal sutures (Figure 1E). Troubleshooting 5.

• 2.
  Making holes.

  • a.
    Aspirate approximately 100 nL of an AAV solution into a Hamilton Neuros Syringe (7803–03) and place the syringe in the injector.
  • b.
    Adjust the syringe needle tip to Bregma, then set the position as anteroposterior (AP): 0.00 mm, medial-lateral (ML): 0.00 mm, and dorsal-ventral (DV): 0.00 mm.
  • c.
    Rotate the syringe to ensure the needle is straight. If the needle tip is going to be off from the Bregma, adjust the straightness of the needle. Troubleshooting 6.

    Note: This step is required when the syringe is placed for the first time on the day of the procedure.

  • d.
    Adjust the needle tip to Lambda and record the coordination.
  • e.
    Adjust the mouth fixture to make DV coordinates equal between Bregma and Lambda.

    Note: The gap between Bregma and Lambda should be less than ±0.05 mm, so the mouse head is flat.

  • f.
    Reposition the needle tip to Bregma and set the position as AP: 0.00 mm, ML: 0.00 mm, and DV: 0.00 mm.
  • g.
    Move the needle tip to the DMH.
  • h.
    Mark the skull.
  • i.
    Make a 2 mm hole on the skull above the marked site using a hand drill with a steel hole cutter.

    Note: The injection coordinates for the DMH are as follows: AP -1.80 mm, ML ±0.35 mm from Bregma.

    CRITICAL: Mislocations and smaller holes can cause the needle to bend when inserted. Troubleshooting 7.

• 3.
  Viral injection.

  • a.
    Set the injector to 50 nL/min speed and 30 nL volume.

    CRITICAL: Eject a drop of viral solution from the tip of the needle to ensure that it is not clogged before inserting it into the brain. Troubleshooting 8.

  • b.
    Move the needle to the brain surface and slowly insert it into the target. Pull the needle upward approximately 0.05 mm to allow the viral solution to diffuse and wait for 3 min.
  • c.
    Inject the viral solution into the DMH.
  • d.
    Leave the needle in place for 5 min, then remove it slowly.
  • e.
    Repeat steps 2-i and 3-a to d on the other side of the brain.
• 4.
  After viral injection.

  • a.
    Administer antibiotics locally after closing the incision with a 4–0 nylon suture and sterilizing the wound.
  • b.
    Recover the mouse in a temperature-regulated incubator (32°C) until fully awake.
  • c.
    Monitor the mouse at least once a day for a week.

Note: If significant impairment or distress is observed, such as persistent abnormal movements, vocalization, seizure, and animals losing significant weight (>20%), they should be euthanized.

CRITICAL: The ideal injection is that the hM3D expression, labeled by mCherry in the hypothalamus, is restricted within DMH^Ppp1r17 neurons bilaterally (Figure 1F). For the lifespan study, it is almost impossible to check all the brains in the cohort after their endpoint because there is severe post-mortem degradation. Therefore, practicing the viral injection as much as possible before starting the actual experiment and confirming the successful rate of the injection should be close to 100% is required in this experiment.

Figure 1.

Surgical instruments and critical procedures of stereotactic injection into the dorsomedial hypothalamus (DMH) of aged mice

(A) A representative photograph of aged male littermates.

(B) Main components of the stereotactic system.

(C) Head fixation of an anesthetized mouse using a stereotactic instrument.

(D) Stereotaxic markers on the skull before (left) and after (right) 30% hydrogen peroxide application.

(E) A photograph of a mouse skull’s surface shows the Bregma and Lambda reference points. Diagram showing different bone suture lines in an animal with an asymmetric junction of bone plates. In this example, the Lambda and Bregma points are not aligned with the mediator (line “a”). Curve “b” represents the frontal sutures. The centerline “a” is equidistant from the lateral bone edges. This distance is designated by the segment “c”.

(F) Representative immunostaining image of Ppp1r17 (green) and mCherry (red) in hM3D (Gq)-mCherry-expressing Ppp1r17-Cre mice. Scale bars are 200 μm.

Part 2: Checking the effects of the DREADD in the DMH

Timing: at least 2 weeks after DREADD AAV injections

Timing: before collection of tissues from the mice, Agonist 21 injection is performed for 4 days (for step8)

DURATION: 9 days for habituation, 8 days for agonist injection, 1 day for free fatty acid (FFA) measurement, and 2 days for eNAMPT and the phosphorylated hormone-sensitive lipase (pHSL) in each.

This step ensures the impact of the activation of the DMH^Ppp1r17 neurons by measuring WAT functions since DMH^Ppp1r17 neurons mediate WAT functions through the SNS. In this step, plasma levels of free fatty acid (FFA) and eNAMPT are compared between PBS and Agonist 21 injections. Troubleshooting 9.

Then, the phosphorylated hormone-sensitive lipase (p-HSL) level in WAT is assessed after tissue collection.

• 5.
  Injections (PBS and Agonist 21) and sample collections.

  Note: For FFA and eNAMPT measurement, it would be ideal to compare before and after the DREADD activation in each individual mouse between mCherry control and hM3D groups. For p-HSL measurement, the hM3D groups will be compared with the mCherry control group.

  CRITICAL: The purpose of this administration method is that neuronal activation by DREADDs should be induced right after the light turns off when mice start waking up and show the highest locomotor activity (ZT12-15).^1,5,6 The activation of DMH^Ppp1r17 neurons during daytime is inappropriate for this experiment because DMH^Ppp1r17 neurons induce locomotor activity and could mess up their circadian rhythm. The effects of DREADDs on neuronal activity begin approximately 30 min after ligand administration and last for several hours.^7,8,9

  • a.
    Inject PBS for habituation intraperitoneally (i.p.) once daily at ZT 11.5–12 for 9 days.
  • b.
    Collect blood from their tail vein into capillary tubes coated with heparin sulfate on the last day of PBS injection around ZT 15. Approximately 40 μL of whole blood would be enough for FFA and eNAMPT measurements.

    Note: Because aged mice have slower blood flow than young mice, the blood may not come out from their tails as efficiently as young mice.

  • c.
    Spin down blood at 3000 × g for 10 min at 4°C and collect only plasma supernatant. Troubleshooting 10.
  • d.
    For FFA measurement, snap-freeze at least 10 μL of plasma using dry ice and store it at −80°C until use.
  • e.
    For eNAMPT measurement, incubate 1 μL of freshly collected plasma with 100 μL of 1x SDS Sample Buffer at 95°C for 10 min and snap-freeze them using dry ice before storing it at −30°C until use.
  • f.
    Inject Agonist 21 by i.p. at a 0.5 mg/kg dose at ZT 11.5–12 for 8 days.
  • g.
    Repeat steps 5-b to e for the Agonist 21 injection.
• 6.
  FFA measurement.

  • a.
    Measure FFA using the HR series NEFA-HR kit No. 999–34691. Aliquot 2 volumes (50 μL) of reagent A to wells, add 1 μL of plasma or standard and incubate the plate for 15 min at 20°C.
  • b.
    Add 1 volume (25 μL) of reagent B to all wells to develop the color. Incubate the plate for an additional 15 min at 20°C.
  • c.
    Read at 540 nm and correct for blanks and a secondary wavelength at 660 nm.
• 7.
  eNAMPT measurement by Western blotting (WB).

  • a.
    Right before the analysis, add 5 μL of each sample to 35 μL of 1x SDS Sample Buffer and incubate at 95°C for 30 min.

    CRITICAL: This 30 min boiling is necessary to make eNAMPT bands discrete and quantifiable.

  • b.
    Run 10–30 μL of the final mixture on a 4%–15% SDS-PAGE gel (#4561084 or 4561086, Bio-Rad) and transfer proteins to the PVDF membrane (IPVH00010).
  • c.
    Incubate the membrane with 5% non-fat dried milk in TBS-T (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) for blocking at 20°C for 1 h. Incubate the membrane with rabbit anti-NAMPT antibody (1:1000; A700–058, Bethyl) diluted in the blocking solution with gentle agitation at 4°C for 16 h.
  • d.
    Wash three times for 10 min each with TBS-T. Incubate with HRP-conjugated secondary antibody (PI-1000-1) at 1:10,000 in TBST-5% non-fat dried milk for 1 h at 20°C .
  • e.
    Wash three times again for 10 min each with TBS-T. For detection, use Pierce ECL WB Substrate (32209, Thermo) and prepare according to instructions.
  • f.
    For loading control, use Rabbit anti-transferrin antibody (1:1000; ab82411, Abcam) as the primary antibody.
• 8.
  p-HSL measurement in WAT by WB.

  • a.
    Perfuse the mice with PBS and collect WATs. After the PBS perfusion, freeze the WATs in liquid nitrogen and store them at −80°C.
  • b.
    Homogenize the WATs to extract protein using pestles (7339–901) in homogenization buffer (50 mM HEPES, 100 mM NaCl, 5% SDS, 2 mM EDTA, 0.5 mM DTT, 0.1 mM PMSF, 0.1 mM NaF, 0.1 mM NaVO₄, with cOmplete Protease Inhibitor Cocktail (#04693159001, Roche), see materials and equipment section).

    Note: This homogenization buffer should be made freshly right before the homogenization.

  • c.
    Spin down homogenized samples at 8000 × g for 5 min at 4°C and collect only protein-containing clear supernatant.
  • d.
    Repeat step 8-c one more time.

    Note: Do not take lipids (white layer) and remaining tissues.

  • e.
    Quantify the protein concentration using the Pierce BCA Protein Assay Kit (#23227, Thermo Fisher) and Add 8 μg of the above-prepared sample to the SDS Sample Buffer in a total volume of 15 μL.
  • f.
    After incubating the SDS samples at 95°C for 10 min, apply them to a 4%–15% SDS-PAGE gel and run the gel until the dye reaches the bottom.
  • g.
    Transfer proteins from the gel to the PVDF membrane and incubate the membrane with 5% non-fat dried milk in TBS-T for blocking at 20°C for 1 h. Incubate the membrane with rabbit anti-p-HSL (1:2000; 4126S, Cell Signaling) diluted in the blocking solution with gentle agitation at 4°C for 16 h.
  • h.
    The secondary antibody and detection steps are the same as eNAMPT WB (Step 6-d, e). Troubleshooting 11.
  • i.
    After detection, incubate the membrane with Restore Western Blot Stripping Buffer (21059, Thermo) for 15 min at 20°C with gentle shaking to reblot rabbit anti-HSL (1:2000; 4107S, Cell Signaling) at 4°C for 16 h.
  • j.
    The secondary antibody and detection steps are the same as eNAMPT WB (Step 6-d, e ).

Part 3: Longitudinal DREADD agonist 21 administration for lifespan analysis

DURATION: Approximately 0.5 h, four consecutive days per week, throughout the lifespan analysis.

Here, we describe the administration of DREADD agonist 21 to activate DMH^Ppp1r17 neurons using chemogenetics.

• 9.Administer DREADD agonist 21 by i.p. injection at a dose of 0.25 mg/kg at Zeitgeber Time (ZT) 11.5–12 every day, Monday through Thursday (four consecutive days in each week) until their endpoint.

CRITICAL: The timing of the injection is essential to this experiment, too, as described in Part 2 (Injections and sample collections). Although the exact mechanisms underlying the diverse physiological effects of chronic DREADD activation remain unclear, more evidence for receptor desensitization can be found with the employment of DREADDs.¹⁰ Therefore, pausing the administration of DREADD agonist 21 for three days each week is required in this experiment to avoid receptor desensitization after four days of consecutive administration.

• 10.Longitudinal mouse monitoring.

  DURATION: Approximately 400 days.

  To assess whether the activation of DMH^Ppp1r17 neurons extends the healthspan and lifespan, mice in both control and DREADD groups should be monitored until their endpoints.

  • a.
    Inspect all mice in the aging cohorts at least once daily and measure their body weights every Wednesday (once a week).

    Note: The endpoint of life is when each mouse is found dead during the daily inspection. Moribund mice are euthanized according to our institutional animal care guidelines, and the time at euthanasia is its endpoint. Do not combine animals in a cage from different cages at this moment, even if they become singly housed, because they will fight in the cage and have injuries that would affect their lifespan, especially males.

  • b.
    Perform a necropsy immediately after the death or euthanasia.

    Note: While examining an animal yourself is preferable to not inspecting it at all, the best approach is to consult a veterinary pathologist or technician for a professional assessment. Tumors are common in aged rodents, and best of all is a histopathological autopsy. Quick microscopic observations of the obvious lesions are required to obtain cancer-dependent death incidence.

  • c.
    Record the date of death and any details available, including the possible cause and appearance of age-related conditions.
• 11.
  Survival analysis.

  • a.
    Generate a Kaplan-Meier curve to assess differences in survival between control and DREADD mice.
  • b.
    Use log-rank tests (e.g., Mantel-Cox) to assess whether differences between experimental groups are statistically significant using GraphPad Prism 9.

    Note: The log-rank test (Mantel-Cox) is a nonparametric hypothesis test and the most commonly used test to compare the survival trends of two or more groups. It is most likely to detect a difference between groups when the risk of an event is consistently greater for one group than another.

  • c.
    Estimate the age-associated mortality rate (qx) as the number of animals alive at the end of the interval over the number of animals at the start of the interval.

    Note: The hazard rate (hz) is estimated by hz = 2qx/ (2-qx),¹¹ the natural logarithm of hz is plotted, and statistical analysis is performed using ANCOVA.

Expected outcomes

A successful stereotaxic injection procedure in aged mice results in no deaths and allows us to deliver the transgene to the restricted area of the mouse’s DMH. As outcomes of inter-tissue communication between DMH^Ppp1r17 neurons and WAT through the SNS, circulating eNAMPT levels, plasma levels of free fatty acids, and phosphorylated hormone-sensitive lipase (p-HSL) levels in WAT are expected to increase after agonist 21 treatment in DREADD mice, but not in control mice. The continuous activation of DMH^Ppp1r17 neurons will display a significant delay in age-associated mortality and eventually extend the lifespan compared to control mice (Figure 2).¹

Figure 2.

DREADD-mediated activation of DMH^Ppp1r17 neurons increases wheel-running activity and lipolysis and extends lifespan in aged mice

Schematic representation of the DREADD-mediated activation of DMH^Ppp1r17 neurons in aged mice. Expressing hM3D (Gq)-mCherry selectively in DMH^Ppp1r17 neurons increases wheel-running activity, lipolysis, eNAMPT secretion, and lifespan in aged mice. Plasma-free fatty acid (FFA) levels in 20-month-old control and DREADD male mice before (PBS) and after (agonist 21) activation (∗∗p < 0.01, two-way ANOVA with Sidá k’s multiple comparisons test, n = 5). Phosphorylated hormone-sensitive lipase (p-HSL) and total HSL levels in inguinal white adipose tissue from 20-month-old control and DREADD male mice after treatment with agonist 21 were analyzed by western blotting. p- HSL levels were normalized to total HSL levels (∗p < 0.05, Student’s t-test, n = 5). Plasma eNAMPT levels of 20-month-old control and DREADD male mice before (PBS) and after (agonist 21) activation. eNAMPT levels were normalized to transferrin levels (∗p < 0.05, two-way ANOVA with Šidák’s multiple comparisons test, n = 5). Kaplan-Meier curves of control and DREADD male mice (n = 13 for each group). Age-associated mortality rates of control and DREADD male mice (ANCOVA, p = 0.0004).

Limitations

This protocol is optimized to combine chemogenetics and lifespan analysis using aged mice. In theory, it should be applied to any other research goal using aged mice, but the protocol must be validated for such use outside the scope of this protocol. Because of the nature of lifespan analysis, which takes years to finish, conducting even one experiment comes with a significant cost. Whereas it is necessary to design and conduct the experiment carefully and accurately, finding the optimal conditions and obtaining a sufficient number of DREADD virus-injected aged animals in both males and females is extremely challenging. Additionally, DREADD agonist 21 needs to be administered intraperitoneally (i.p.) at ZT 11.5–12 four consecutive days per week until the endpoint to keep activating specific neurons throughout the experiment. Thus, managing the entire administrative procedure for a large mouse cohort could be a technical limitation of this protocol.

Troubleshooting

Problem 1

The number of heterozygous Cre mice cannot be predicted (before you begin step 5).

Potential solution

Following Mendel’s laws of inheritance, the expected number of heterozygous Cre mice, crossing heterozygous Cre and wild-type C57BL/6J mice, is expected to be half of the total number of newborn pups. Nonetheless, given sex and unexpected unevenness, it is recommended to set up as many breeding units as possible, probably more than 15 cages, simultaneously. Wild-type mice and surplus heterozygous Cre mice can then be eliminated from the lifespan cohort or used to optimize viral injections. Although it is not ideal, preparing other cohorts is an alternative option if enough Cre mice for one lifespan experiment are not obtained at the same period.

Problem 2

The control group unexpectedly shows a shorter lifespan (Viral injection before step 1).

Potential solution

As described in the step-by-step method details, unhealthy-aged mice could be assigned to one group of the lifespan cohort unevenly because of the limited sample sizes. Assigning subjects to groups using a semi-random method can minimize confounding variables. The easiest and most reliable way to divide them into two groups is to counterbalance their body weights and cage distributions. Then, the two groups are randomly assigned to be either the control or the DREADD group.

Problem 3

Aged mice die during or after viral injection.

Potential solution

Decreasing isoflurane concentration and surgical time can prevent the death of aged mice during or after the surgical procedure. Although it may depend on the isoflurane vaporizer, isoflurane concentrations of 1.5%–1.8% and less than 50 min for the procedure are recommended for aged mice. If the above solutions do not resolve the problem, using mice that are a few months younger would be considered. Inserting too deep ear bars, subdural bleeding, and infection would be the next considerations for causes of death and should be prevented the same as using young mice.

Problem 4

The mouse skull is unstable in the stereotaxic stage (Viral injection, step 1-d).

Potential solution

Change the positions of the inserted ear bars if the animal’s head is not entirely fixed. The ear bars might have been inserted incorrectly. Initially, one of the two bars must be securely fixed to the instrument. Place the ear bar just behind the ear bone spur. Proper insertion is confirmed by a gentle, distinctive click, which should not be mistaken for the louder popping sound that occurs when the eardrum is perforated due to excessive insertion of the bar. When the ear bars are correctly positioned, the animal’s head should be immobilized laterally. The only possible movements at this stage are forward and backward tilting of the head.

Problem 5

The location of the viral injection in the brain is inconsistent every time (Viral injection, step 1-g).

Potential solution

Define Bregma correctly and make the flat skull position as consistent as possible, as described in the main step-by-step method. The combination of these two factors is the primary cause of injection failure. The ideal injection is that the hM3D expression, which is labeled by mCherry in the hypothalamus, is restricted within DMH^Ppp1r17 neurons bilaterally (Figure 1F).

Problem 6

The injection sites on both sides are slightly off the target region, or the needle tracts are tilted (Viral injection, step 2-c).

Potential solution

The needle can be easily bent, and the Neuros Syringes can be separated into two parts so that it can be inclined when assembled. Rotate the syringe to ensure that the needle is straight. If the needle tip is off from the Bregma, ensure the needle is straight properly.

Problem 7

Damaging cortical brain regions and heavy bleeding occur by drilling holes (Viral injection, step 2-i).

Potential solution

Wet the surface of the skull with PBS appropriately when making holes with a hand drill to avoid damage to the cortices by frictional heat. Do not drill too deep, and avoid contacting the brain region. Carefully remove the remaining bone using fine-tip tweezers to expose the brain’s surface.

Problem 8

The amount of virus in the target region varies every time (Viral injection, step 3-a).

Potential solution

Brain tissue or tiny debris in the viral solution can easily clog the tip of the needle. Before inserting the needle into the brain, it is crucial to check whether the solution can be slowly ejected without clogging. A beveled needle would be better than a blunt needle if clogging occurs frequently.

Problem 9

FFA and eNAMPT plasma levels are highly variable, even in the PBS control (Part 2).

Potential solution

Each aged mouse has developed its age-associated physiologies, even if they are littermates. Because of the variability, it would not be easy to see significant differences. A paired test would be recommended between PBS (pre-treatment) and Agonist 21 (post-treatment) to avoid unnecessarily increasing the number of aged animals.

Problem 10

The collected plasma is pinkish (Part 2, step 5-c).

Potential solution

Gently squeeze their tails from the base of the tail to the tip, but not strongly. It may cause hemolysis. After collection, the blood should be on the ice before centrifugation to obtain plasma.

Problem 11

The phosphorylation of HSL is not detected (Part 2, step 8-h).

Potential solution

Do not thaw WAT samples before homogenization; do not repeat freeze-thaw cycles for the homogenized WAT extracts before use. Multiple freeze-thaw cycles will cause dephosphorylation of HSL.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Shin-ichiro Imai (imaishin@wustl.edu).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by technical contact Kyohei Tokizane (k.tokizane@wustl.edu).

Materials availability

No new reagents were generated in this study.

Data and code availability

This study did not generate any new data or codes.

Acknowledgments

We particularly thank Hanyue Cecilia Lei, Cynthia S. Brace, Kathryn F. Mills, and Kentaro Mori for their technical assistance; Ernesto Gonzales for his support of stereotactic injection in the Animal Surgery Core; Leslie Wilson for necropsy in the lifespan study in the DCM Research Animal Diagnostic Laboratory; and members of the Imai lab for critical comments and suggestions on this study. This work was mainly supported by grants to S.-i.I. from the National Institute on Aging (AG037457 and AG047902), the American Federation for Aging Research, and the Tanaka Fund at the Washington University School of Medicine. K.T. was supported as a Glenn Foundation for Medical Research Postdoctoral Fellow and Tanaka Scholar for this study.

Author contributions

K.T. performed experiments and wrote the original manuscript. S.-i.I. supervised and supported the project and revised the manuscript with K.T.

Declaration of interests

S.-i.I. declares the following competing financial interests: S.-i.I. receives a part of patent-licensing fees from MetroBiotech (USA) and the Institute for Research on Productive Aging (Japan) through Washington University. S.-i.I. also serves as the Chairman of the Institute for Research on Productive Aging (Japan) and a Co-CEO of LongGen Bioscience (Japan). S.-i.I.’s external professional activities have already been reported to and a potential conflict of interest has been properly resolved through the Washington University Conflict of Interest Committee.