Model s48 stimulator

Stereotaxic injections were performed as previously described
(Livneh et al., 2017 (link)). Mice
were anesthetized with isoflurane in 100% O₂ (induction, 3%;
maintenance, 1–2%), and placed into a stereotaxic apparatus (Kopf
model 963 or Stoelting). After exposing the skull via a small incision,
a small hole was drilled for injection. A pulled-glass pipette with
20–40 μm tip diameter was inserted into the brain, and
virus was injected using an air pressure system (Picospritzer). A
micromanipulator (Grass Technologies, model S48 stimulator) was used to
deliver the injection at 25 nl/min and the pipette was withdrawn 5 min
after injection. For postoperative care, mice were injected
intraperitoneally with meloxicam (0.5 mg/kg). Mice were 8–14
weeks old at the time of injection, except for CRACM experiments, for
which mice were 7–10 weeks old.
We used the following volumes of virus and injection
coordinates: InsCtx (100–200 nl, Bregma: AP: 0.0, 0.4 mm, DV:
−4.1, −4.3 mm, ML: ~4.0 mm), SFO (50 nl per DV
depth, Bregma: AP: −0.65 mm, DV:
−2.3/−2.45/−2.6 mm, ML: 0 mm), MnPO (50 nl, Bregma:
AP: +0.5 mm, DV: −5.2 mm, ML: 0 mm), ARC (200 nl, Bregma: AP:
−1.45 mm, DV: −5.85 mm, ML: ±0.25 mm), PVT
(25–50 nl, Bregma: AP: −1.0, −1.3 mm, DV:
−3.0, −3.0 mm, ML: 0.0, 0.0 mm), BLA (100 nl, Bregma: AP:
−1.6 mm, DV: −4.5, −4.76 mm, ML: ±3.3
mm).

Rats were anesthetized using vaporized isoflurane (2–3%) in supplemental oxygen sufficient to achieve surgical anesthetic depth. Twenty minutes after anesthetization, the sciatic nerve of the left hindlimb was isolated just proximal to the point of trifurcation. Contraction of the hindlimb musculature was elicited by stimulating the sciatic nerve at 100 Hz for 10 minutes at one 10 millisecond pulse per second and 15 volts (Grass Model S48 Stimulator, Quincy, MA). During the contraction bout, the foot was held at approximately 90°. Sciatic nerve stimulation activates both the chronically weight-bearing plantarflexors as well as the dorsiflexors of the hindlimb, including the gastrocnemius (GAST) and tibialis anterior (TA) muscles. GAST and TA were removed immediately after contraction and frozen at the temperature of liquid nitrogen. Both muscles were analyzed to allow assessment of the AMPK system in two distinct muscles with different ambulatory functions, with the gastrocnemius being a weight-bearing muscle, while the TA only stabilizes and dorsiflexes the ankle during ambulation. The right hindlimb was not subjected to electrical stimulation and was removed prior to stimulation of the left hindlimb and served as a resting control. All tissue samples were frozen between metal tongs cooled to the temperature of liquid nitrogen and then frozen at −95°C until further analysis.

Stereotaxic injections were performed as previously described ^{7 (link)}. Briefly, mice were anesthetized with xylazine (5mg per kg) and ketamine (75mg per kg) diluted in saline and placed into a stereotaxic apparatus (model 963, David Kopf Instruments, Tujunga, CA). For postoperative care, mice were injected intraperitoneally with meloxicam (0.5mg per kg). After exposing the skull via a small incision, a small hole was drilled for injection. A pulled-glass pipette was inserted into the brain and virus was injected by an air pressure system. A micromanipulator (model S48 stimulator, Grass Technologies, Rockland, MA) was used deliver the injection at 5nl per min and the pipette was withdrawn 5 min after injection. For electrophysiology and tracing, AAV1-CBA-Flex-ChR2(H134R)-mCherry (University of Pennsylvania School of Medicine, Philadephia, PA) was injected unilaterally into the arcuate (2–5nL; from Bregma, AP: −1.35mm, DV: −6.00mm, ML: ±0.2mm). For in vivo chemogenetic experiments, AAV8—hSyn-DIO-hM3Dq-mCherry (University of North Carolina Vector Core, Chapel Hill, NC) was bilaterally injected into the arcuate (2–5nL, coordinates as above). For profiling of Arc-ME RIP-Cre neurons, AAV8-EF1a-DIO-eYFP (University of North Carolina Vector Core, Chapel Hill, NC) was injected into arcuate (100nL, coordinates as above).