Vetalar

To allow optogenetic activation of LC neurons, a Canine-Adenoviral vector (CAV) was used (CAV-PRS-ChR2-mCherry) (Li et al., 2016 (link)). The prolyl tRNA synthetase (PRS) promoter in this vector has been previously demonstrated to achieve LC-specific expression by harnessing the Phox2B transcription factor, which drives endogenous dopamine-β-hydroxylase (the final enzyme in the synthesis pathway for NA).
Stereotaxic injection of the CAV vector was made into the dorsal pons in the region of the LC of PD 19 Wistar rats using an established protocol (Li et al., 2016 (link)). Rats were anesthetized (intraperitoneally) with ketamine (5 mg/100 g, Vetalar; Pfizer, New York, NY, United States) and medetomidine (30 μg/100 g, Domitor; Pfizer) until loss of paw withdrawal reflex. The animal was placed in a stereotaxic frame and core temperature was maintained at 37°C using a homeothermic blanket (Harvard Apparatus, Holliston, MA, United States). Following a craniotomy (from bregma: –1 mm AP, +1.1 mm ML), a microinjection pipette was advanced into the brain with a 10° rostral tilt. Two hundred nanoliters of vector was injected at 4.6, 4.9. 5.2, and 5.5 mm depth. The titre of vector used was 2.15 × 10¹¹ physical particles/ml. Aseptic surgical techniques were used throughout.

Surgical procedures for stereotaxic injection of stimulatory Cre‐dependent DREADD viral construct (AAV‐hSyn‐DIO‐hM₃D(Gq)‐mCherry, Serotype:5; University of North Carolina at Chapel Hill Vector Core, NC, USA) to express the hM₃Dq‐DREADD in MePD Kiss1 neurones were performed under aseptic conditions with general anaesthesia induced by ketamine (Vetalar, 100 mg kg^‐1, i.p.; Pfizer, Sandwich, UK) and xylazine (Rompun, 10 mg kg^‐1, i.p.; Bayer, Leverkusen, Germany). Kiss‐Cre male mice (age 7‐8 weeks, n = 12) were secured in a David Kopf stereotaxic frame and small holes were drilled into the skull at a location above the MePD after a midline incision of the scalp. The stereotaxic injection coordinates used to target the MePD were obtained from the mouse brain atlas of Paxinos and Franklin27 (2.1 mm lateral, 1.64 mm posterior to bregma and 5.1 mm below the surface of the dura). Using a 2‐μL Hamilton micro syringe (Esslab, Essex, UK), 1 μL of AAV‐hSyn‐DIO‐hM₃D(Gq)‐mCherry was injected bilaterally into the MePD over 10 minutes. The needle was left in position for a further 5 minutes and then removed slowly over 1 minute. After recovery from surgery, mice were left undisturbed for 4 weeks to allow the time‐course for effective and stable receptor expression.28