Stereotactic surgery for retrograde tracing and fibre photometry
Stereotactic surgery was performed using an Angle Two™ system (Leica) according to our previously published protocol (e.g. ). Both mice per littermate pair were operated consecutively on the same day. Analgesia was conducted with buprenorphine and anaesthesia with isoflurane in pure oxygen. Cranial burr holes (Ø = 300 µm) were drilled for bilateral injection of retrograde tracer (ex vivo assessment of neuronal activation) or unilateral injection of viral vectors and placement of an optic fibre (in vivo GCaMP6 fibre photometry). Injections were conducted at a rate of 50 nl/min using 10 µl NanoFil™ microsyringes fitted with a 33G beveled stainless-steel needle and connected to an ultra-micro pump (UMP3, Micro4; World Precision Instruments). After injection completion, the microsyringe remained in position for 5 min and was then slowly withdrawn. Concerning stereotactic injection coordinates, based on a mouse brain atlas , for nucleus accumbens (NAc) these were bregma anterior-posterior (AP) +1.1 mm or +1.6 mm, medial-lateral (ML) ±1.1 mm, dorsal-ventral (DV) -4.8 mm. Coordinates selection was based on previous studies and to target BA projectors to both core and shell NAc sub-regions. For fibre photometry, coordinates for AAV vector injection in basal amygdala (BA) were bregma AP -1.8 mm, ML ±3.2 mm, DV -5.1 ; these were based on current ex vivofindings and published evidence that the absolute number of BA neurons projecting to NAc at bregma +1.1 is highest at bregma -1.6 to -2.0. Mice were weighed and wound healing controlled for 10 days post-surgery.