Grin lens

At two weeks after AAV1-CaMKII-GCaMP6f injection, a gradient refractive index (GRIN) lens was implanted at the injection site in CA1. A 1.8 mm-diameter circular craniotomy was centered at the coordinates (AP −2.30 mm and ML +1.75 mm relative to bregma). ACSF was repeatedly applied to the exposed tissue; the cortex directly below the craniotomy was aspirated with a 27-gauge blunt syringe needle attached to a vacuum pump. The unilateral cortical aspiration might affect part of the anteromedial visual area determined using Allen Brain Atlas (www.brain-map.org/), but the procedure left the primary visual area intact. The GRIN lens (0.25 pitch, 0.55 NA, 1.8 mm diameter and 4.31 mm in length, Edmund Optics) was slowly lowered with a stereotaxic arm to CA1 with a depth of −1.60 mm relative to the bregma. Next, a skull screw was used to anchor the GRIN lens to the skull. Both the GRIN lens and skull screw were fixed with cyanoacrylate and dental cement. Kwik-Sil (World Precision Instruments) was used to cover the lens. Two weeks later, a small aluminum baseplate was cemented onto the animal’s head atop the existing dental cement. A miniscope was fitted into the baseplate and locked in a position so that the field of view was in focus to visualize GCaMP6f expressing neurons and visible landmarks, such as blood vessels.
In order to motivate animals to run on a linear track, access to water was regulated between every Sunday afternoon and every Friday afternoon. Mice were provided water starting at 1ml per day and with adjustments up or down until weights stabilized around 82–85% of their original body weight. In the meantime, mice were handled 5 min per day for 3 days. As illustrated in Supplementary Fig. 7, mice were trained to run on a 1-meter linear track with water rewards placed at both ends of the track for 60 laps per session for 5 days. Mice were then trained with a head-mounted miniscope (without recording) to run on the linear track with water reward for 60 laps per session for another 5 days. After all training was completed, in vivo GCaMP6f-based calcium imaging of population CA1 neurons in an awake behaving mouse was performed for the linear track experiments with miniscope recordings for 2 sessions of baseline control, 2 sessions of CNO inactivation, and 2 sessions of post-baseline control. In order to avoid GCaMP6f fluorescence bleaching, mice usually ran 1 session every other day, with 15 min recording. CNO was administered 45 min through intraperitoneal injection prior to imaging, saline was administered during control and post-control sessions in the same way. Intervals between the last CNO session and first post-control session were longer than 48 hours to ensure clearing of CNO.
For open field experiments, mice were handled for at least one week and habituated for i.p. injections and head-mounted miniscopes. Mice were then habituated for 3 days in an open field arena with miniscopes for 15 min on each day. The open field arena is a circular environment (36 cm in diameter) with fixed local and distal cues. Mice were randomly divided into two groups, with one group receiving CNO injection for DREADD-based inactivation of CA1-projecting subiculum neurons. On day 1, mice from both groups run in the open field arena for 15 min each while calcium signals from CA1 neurons are recorded. These recordings act as baseline controls. On day 2, mice from the saline control group received a single dose of saline injections 45min prior to the open field recording, while mice from the CNO treated group received a single dose of CNO injections (1.4 mg/kg) 45 min prior to the open field recording. Day 3 was off to ensure clearance of CNO. Experiments on day 4 were similar to day 1; mice from both groups explored in the open field arena for 15 min each with miniscope imaging. These recordings were used as post-controls.
For miniscope imaging during object location memory (OLM) tasks, mice were handled for at least one week and habituated for i.p. injections and head-mounted miniscopes. Mice were then habituated for 3 days in the OLM behavior box with miniscopes for 10 min on each day. For baseline recordings, animals were put in the OLM behavior box without objects and released for 10 min. At 45 min prior to training, mice were randomly divided into two groups, and one group received saline injections while the other group received CNO (1.4 mg/kg) injections. During recordings in training, two identical objects were presented to the animal during the 10 min training session. For the retention test, one of the two objects were moved to a new location and animals were allowed to explore for 10 min in order to get sufficient coverage for calcium imaging analysis. With respect to behavior, we found that the 10 min retention test gave rise to a more robust measurement with less variability in the discrimination index compared to the data analyzed based on the 1^st 5 min of recording.
The linear track, open field, as well as the OLM experiments were placed in a dedicated animal behavior testing room. Two distinct shelving racks (195-cm height × 122-cm width versus 178-cm height × 152-cm width) with different objects and visual cue decorations were placed against the north and east room walls. Two identical fear-conditioning boxes (60 cm tall) were placed against the west room wall. The linear track was placed in parallel to the north rack with distinct visual cues at each end of the track. The open field enclosure was placed against the southwest edge of the east rack with cue cards inside to serve as local cues. The OLM testing arena was placed against the northwest edge of the east rack with 5 cm wide cue tape on the east wall of the enclosure. All the environments/cues remain constant during the entire series of the experiments.