The Stanford Administrative Panel on Laboratory Animal Care approved all procedures. We used male CD-1 wild type mice 7-14 weeks old and performed surgery using isoflurane (1.5–2.5%; mixed with 1–2 l/min O2) anesthesia as previously described6 (link),9 (link). In brief, 1–6 days prior to imaging we exposed and cleaned the skull above cerebellum. (See Supplementary Note 1 regarding the hippocampal preparation). Using dental acrylic (Coltene/Whaledent, H00335) we fixed to the skull a custom metal plate allowing cranial access. On the imaging day, we opened a craniotomy (1.5–2.5 mm diameter; 6.5 mm posterior to bregma; 0.5 mm lateral) and irrigated the exposed tissue with warm artificial cerebral spinal fluid (ACSF; 125 mM NaCl, 5 mM KCl, 10 mM D-Glucose, 10 mM HEPES, 2 mM MgSO4, 2 mM CaCl2, pH adjusted to 7.4 with NaOH). To dampen heartbeat- and breathing-induced brain motion, we filled the craniotomy with agarose (2%; Type III-A, high EEO; Sigma) in ACSF and covered it with a coverslip fixed to the head plate, creating an optical window. We sealed the coverslip edges with dental acrylic. In Ca2+ imaging experiments, we left the dura intact for Ca2+ indicator injections but then removed it prior to agarose application.
In Ca2+ imaging studies, we labeled cerebellar cortex by multi-cell bolus-loading using the Ca2+-indicator Oregon-Green-488-BAPTA-1-acetoxymethyl (OGB-1-AM)6 (link),9 (link). Concentrations of OGB-1-AM and DMSO in our pipette solution were 500 μM and 5%, respectively. In microcirculatory studies, we labeled the blood plasma by injection into a tail vein of 0.15–0.25 ml fluorescein-dextran (Sigma, FD2000S, 2000 kDa, 10 mg/ml)6 (link).
We positioned the miniature microscope above the optical window and lowered it towards the brain using a translation stage, until fluorescent surface structures were visible under weak illumination (90–200 μW). After locating a suitable recording site and focal depth, we turned the illumination off and fixed the microscope to the metal head plate using Cerebond™ adhesive (myNeuroLab.com, 39465030) and dental acrylic (Henry Schein, 5478203EZ). We allowed the mouse to recover from anesthesia before placing it into the behavioral arena. Imaging began once the mouse exhibited vigorous locomotor activity, typically 15-60 min after removal from isoflurane. To minimize the possibility of photo-induced alterations in physiology, the duration and mean power of continuous illumination were typically < 5 min and ∼170-600 μW at the specimen plane for each recording. There were at least 2 min between recordings, and the total recording duration was typically < 45 min. In Ca2+ imaging experiments, we assessed tissue health before and after imaging using an upright two-photon microscope equipped with a 20× water-immersion objective (Olympus, 0.95 NA, XLUMPlanFl) and an ultra-short pulsed Ti:sapphire laser (Tsunami, Spectra-Physics) tuned to 800 nm. The frame acquisition rates of the integrated microscope were 100 Hz for studies of microcirculation and 30–46 Hz for Ca2+ imaging studies.
For studies of freely moving mice, we placed the mouse into a 45 cm × 45 cm × 15 cm arena made of transparent acrylic. A thin layer of bedding, a few food pellets, and an exercise wheel (Bio-Serv, K3250 and K3328) were inside the arena to provide a comfortable environment for the mouse. To record mouse behavior, we used either a video rate monochrome CMOS camera (Prosilica, EC640) with a high-resolution lens (Computar, M0814-MP2) situated above the arena or a video rate color CCD camera (Sony, DCR-VX2000NTSC) placed adjacent to the arena. We used two sets of infrared LED arrays (Lorex, VQ2120) for illumination with the overhead camera, but simply dim room lighting with the color camera.
In Ca2+ imaging studies, we labeled cerebellar cortex by multi-cell bolus-loading using the Ca2+-indicator Oregon-Green-488-BAPTA-1-acetoxymethyl (OGB-1-AM)6 (link),9 (link). Concentrations of OGB-1-AM and DMSO in our pipette solution were 500 μM and 5%, respectively. In microcirculatory studies, we labeled the blood plasma by injection into a tail vein of 0.15–0.25 ml fluorescein-dextran (Sigma, FD2000S, 2000 kDa, 10 mg/ml)6 (link).
We positioned the miniature microscope above the optical window and lowered it towards the brain using a translation stage, until fluorescent surface structures were visible under weak illumination (90–200 μW). After locating a suitable recording site and focal depth, we turned the illumination off and fixed the microscope to the metal head plate using Cerebond™ adhesive (myNeuroLab.com, 39465030) and dental acrylic (Henry Schein, 5478203EZ). We allowed the mouse to recover from anesthesia before placing it into the behavioral arena. Imaging began once the mouse exhibited vigorous locomotor activity, typically 15-60 min after removal from isoflurane. To minimize the possibility of photo-induced alterations in physiology, the duration and mean power of continuous illumination were typically < 5 min and ∼170-600 μW at the specimen plane for each recording. There were at least 2 min between recordings, and the total recording duration was typically < 45 min. In Ca2+ imaging experiments, we assessed tissue health before and after imaging using an upright two-photon microscope equipped with a 20× water-immersion objective (Olympus, 0.95 NA, XLUMPlanFl) and an ultra-short pulsed Ti:sapphire laser (Tsunami, Spectra-Physics) tuned to 800 nm. The frame acquisition rates of the integrated microscope were 100 Hz for studies of microcirculation and 30–46 Hz for Ca2+ imaging studies.
For studies of freely moving mice, we placed the mouse into a 45 cm × 45 cm × 15 cm arena made of transparent acrylic. A thin layer of bedding, a few food pellets, and an exercise wheel (Bio-Serv, K3250 and K3328) were inside the arena to provide a comfortable environment for the mouse. To record mouse behavior, we used either a video rate monochrome CMOS camera (Prosilica, EC640) with a high-resolution lens (Computar, M0814-MP2) situated above the arena or a video rate color CCD camera (Sony, DCR-VX2000NTSC) placed adjacent to the arena. We used two sets of infrared LED arrays (Lorex, VQ2120) for illumination with the overhead camera, but simply dim room lighting with the color camera.
