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All experiments and procedures were performed in accordance with protocols approved by the Allen Institute Animal Care and Use Committee. Mice were maintained on a reverse 12-hour light-cycle (off at 9am, on at 9pm) and all experiments were performed during the dark cycle. Experiments were performed on male and female C57 mice (wild-type mice) or on transgenic mice expressing channelrhodopsin-2 (ChR2) or archaearhodopsin (Arch) in cholinergic neurons. Transgenic mice were produced by crossing Chat-IRES-Cre mice (Jax 006410, B6;129S6-Chattm2(cre)Lowl/J) with Ai32D (Jax 012569, 129S-Gt(ROSA)26Sortm32(CAG-COP4*H134R/EYFP)Hze/J) or Ai35D (Jax 012735, B6;129S-Gt(ROSA)26Sortm35.1(CAG-AOP3/GFP)Hze/J). Where transgenic mice were employed, the optogenetic illumination experiments used a wavelength that does not activate the relevant opsin: 473 nm illumination for Arch-expressing mice and 589 or 640 nm illumination for ChR2-expressing mice. For head restraint, a custom-made titanium plate was attached to the skull with C&B Metabond (Parkell S380), under isoflurane anesthesia (1–2.5%, inhaled). A guide cannula (C300GS-5/SPC, Plastics One) was implanted in the left hemisphere using stereotaxic coordinates (from bregma): -2.0 mm lateral, -0.5 mm posterior, -4.0 mm ventral. Surgery was performed at postnatal day 40–60. Before recovery from surgery, topical antibiotic (bacitracin-neomycin-polymyxin ointment) was applied to wound margins to minimize risk of infection. For alleviation of pain, mice were given ketoprofen (2-5mg/kg sub-cutaneous) immediately before recovery from anesthesia and twice daily for two days after surgery. Mice recovered for 7–10 days before starting water scheduling. Once experiments were complete, mice were euthanized by cervical dislocation under anesthesia (2–4% isoflurane, inhaled) or, usually, by transcardial perfusion under anesthesia (2–4% isoflurane, inhaled).
The start of water scheduling (1–1.5 ml per day) coincided with 5–7 days of habituation, during which mice were handled, allowed to explore the behavioral apparatus (unrestrained) for ≤10 minutes per day and conditioned to head restraint in the behavioral box for ≤10 minutes per day. After habituation mice began training. Behavioral training and testing was performed in a custom-made light- and sound-attenuating chamber during the dark-cycle. Training and testing occurred once per day, 5 days per week, with no training (1.5 ml water per day) 2 days per week. Mice were supplemented with additional water or high-calorie food, as necessary, to maintain >80% of initial body weight. During behavioral sessions the mouse was head-restrained and allowed to run on a 16.5 cm diameter disk while visual objects were presented on an LED monitor (Asus PA248Q) centered 15 cm from the right eye. The medial edge of the monitor was positioned 30° from the midline and the temporal edge at 150°. Hence the center of the monitor was at 90° from the midline. The luminance of the monitor ranged from 0 (black) to 90 (white) cd/m2. The visual object was a 20° diameter circle containing a stationary grating, 0.15 cycles per degree with a half-cosine mask, generated by the PsychoPy package. The behavioral session was conducted with custom-written software in Python. Movement of the object was yoked to running speed via the rate of rotation of the running disk (0.17 degrees visual space/degree rotation of the disk), and the mouse collected a reward by slowing its running to select the visual object. A region subtending 40° in the center of the monitor was designated as the reward window. In order to select the object, the mouse held the object in the window for a minimum of 0.75–1.2 seconds. The reward for successful target selection was 5–10 μL of water, with mice performing 150–1000 trials in a single session. A behavioral session continued until the mouse received its daily allotment of water or until 60 minutes had passed, whichever was less. Where the mouse failed to collect all its water during behavioral training or testing, supplementary water was provided to bring the final volume to 1–1.5 ml. Mice were eliminated from further study if, after 6 weeks of training, they routinely failed to run on the disk or stop to select the object (30% behavioral attrition rate). After mice learned to select the rewarded object (3–6 weeks), an unrewarded object (a novel orientation of the grating) was introduced randomly at a 1:1 ratio with the rewarded object. Mice learned to discriminate between the two objects within several sessions. Once a mouse learned to discriminate two high-contrast objects (criterion: d’>1.0 in 60% of sessions) additional low-contrast (0%, 25%, 38%) objects were added, but 0% contrast objects were not rewarded.
Data analysis was performed using custom routines written in Python. Psychometric functions were fit to a Weibull distribution: y=γ+(1−γ−λ)*(1−e−(x/α)β) Where α is the midpoint of the curve; β is slope at the midpoint; γ and λ are the lower and upper limits, respectively. Error bars for single-session behavioral data denote the 95% confidence-interval [16] implemented in the Python SciPy package. The discriminability index (d’) was calculated as d’= z(stop probability for rewarded objects) − z(stop probability for unrewarded objects) where z is the inverse of the cumulative Gaussian distribution.
Optogenetic illumination was provided via diode-pumped solid-state (DPSS) lasers (Opto-Engine: MGL-III-589 yellow-orange laser, MRL-III-640 red laser, MBL-III-473 blue laser). Illumination was delivered with a 300 μm diameter optical fiber (ThorLabs FT300UMT), inserted into the guide cannula so that the tip of the fiber extended just beyond the end of the cannula. Illumination was provided on 50% of trials and began as the leading edge of the object first appeared on the left side of the monitor and lasted until the object completely exited the screen (1–5 seconds depending on running speed). The inter-trial interval (time between the start of consecutive trials) was 4.6 ± 1.4 seconds (42 behavioral sessions, 11 mice). Hence fiber illumination, which occurred on 1 of every 2 trials on average, occurred for approximately 3 in every 9 seconds.
ERG recordings were performed under anesthesia (1–2.5% isoflurane) with periorbital silver wire electrodes placed in contact with the cornea. Two reference electrodes were inserted beneath the skin near the eyes. Amplifier headstages were positioned ~4 cm from each eye. Voltage signals were acquired (model 1800, AM systems) and digitized at 25 kHz with the Ecube acquisition system (White Matter LLC, Mercer Island, WA). Optogenetic illumination was presented for 200 milliseconds, with ≥1 second between stimuli. When pooling results across mice, to correct for differences in recordings conditions we normalized ERG voltage amplitudes to those evoked by blue illumination through the fiber tip (20 mW, 200 ms) with the fiber tip positioned outside the brain, in front of the head.
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