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and was then slowly withdrawn.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.