The Impact of Visual Cues, Reward, and Motor Feedback on the Representation of Behaviorally Relevant Spatial Locations in Primary Visual Cortex.

Animals

All animal experiments were approved by the Animal Welfare and Ethical Review Board (AWERB) of the University of Edinburgh and were performed under a project license granted by the UK Home Office, and conformed with the UK Animals (Scientific Procedures) Act 1986 and the European Directive 86/609/EEC on the protection of animals used for experimental purposes. Seven male and female mice with a C57BL/6 background ( Sst tm2.1(cre)Zjh /J [RRID:IMSR_JAX:013044] cross-bred with B6.Cg- Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze /J [RRID:IMSR_JAX:007914]; Jackson Laboratory, ME, USA), aged 6–7 weeks, were used for the experiments. Animals were group housed in a reverse day/night cycle.

Surgical Procedures

For cranial window implantation and virus injection, mice were anaesthetized with isoflurane (4% for induction and 1%–2% maintenance during surgery) and mounted on a stereotaxic frame (David Kopf Instruments, CA, USA). Eye cream was applied to protect the eyes (Bepanthen; Bayer, Germany), and analgesics and anti-inflammatory drugs were injected subcutaneously (Vetergesic, buprenorphine, 0.1 mg/kg of body weight; carprofen, 0.15 mg; and dexamethasone, 2 μg). A section of scalp was removed and the underlying bone cleaned before a craniotomy (around 2 × 2 mm) was made over the left V1 (centered 2.5 mm lateral and 0.5 mm anterior to lambda). Then adeno-associated virus (AAV) (AAV1.Syn.GCaMP6f.WPRE.SV40; University of Pennsylvania Vector Core, PA, USA) was injected, to drive the expression of the fluorescent calcium indicator GCaMP6f in all neurons, using a pipette with 20-μm tip diameter (Nanoject; Drummond Scientific, PA, USA) at a speed of 10 nL min −1 at three different depths (around 250, 400, and 600 μm deep; 50 nL per site). The craniotomy was then sealed with a glass coverslip and fixed with cyanoacrylate glue. A custom-built head post was implanted on the exposed skull with glue and cemented with dental acrylic (Paladur; Heraeus Kulzer, Germany). Animals were returned to their home cage for 2–3 weeks to allow for virus expression and clearing of the imaging window ( Holtmaat et al., 2009 ) before habituation and imaging.

Virtual Reality System

Animals were trained on a virtual reality system consisting of two angled computer screens ( Figure 1 B), a cylindrical treadmill, head fixation system, and a reward spout. The computer screens (51 × 29 cm; Dell, UK) were placed at a 90° angle in front of the animal covering the majority of its field of view. A cylindrical polystyrene treadmill (20 cm diameter, 7.5 cm wide) was mounted on a central axle with an incremental rotary encoder (E6-2500-472-IE; Pewatron, Switzerland). A microcontroller (Arduino Uno) received rotational displacement information from the encoder and forwarded it the virtual reality software. The reward spout (59-8636; Harvard Apparatus, UK) was fitted with a capacitive touch sensor (SEN-12041; Sparkfun, CO, USA) to detect animal licking behavior and put into place at the beginning of each session, such that the animal was always able to reach it. Reward release was controlled by a pinch-valve (225PNC1-21; NResearch, NJ, USA) that dispensed 4- to 8-μL boluses per instance. The MATLAB-based package ViRMEn ( Aronov and Tank, 2014 ) combined with custom-written code was used to design and run the presentation of the virtual environment and collect related data (see Supplemental Experimental Procedures ). Visually Guided Rewarded Task Mice were water deprived (see Supplemental Experimental Procedures ) and water rewards could either be self-initiated by licking in the first half of the reward zone (early, successful trial), or were dispensed at a default location at the halfway point of the reward zone (late, missed trial). Behavioral training was divided into three phases. For each phase, the first day was taken as the “novice” day. The animals were considered “expert” and promoted to the next phase of training, when successful trials made up >75% of the total trials. For phase 1, the mice were exposed to a single virtual corridor condition with the reward zone visually cued by black corridor walls. Phase 2 introduced uncued trials on every fifth trial, where the rules for reward remained the same but the black corridor walls were removed. For three mice, an additional phase 3 was performed where in a single session the gain relating the rotation of the cylindrical treadmill to the progression in the virtual corridor was reduced from 1 to 0.75 (see Supplemental Experimental Procedures ).

Two-Photon Calcium Imaging

Two-photon calcium imaging was performed in head-fixed mice that ran freely on the cylindrical treadmill ( Figure 1 B) ( Dombeck et al., 2007 ) using a custom-built resonant scanning two-photon microscope with a Ti:sapphire pulsing laser (Chameleon Vision-S; Coherent, CA, USA; 1). In contrast, if the animal licks in a spatially indiscriminate pattern, the number will approach 1. If the animal licks often but keeps missing the reward zone, the SMI will be