Bergamo 2

Seven to fourteen days after the craniotomy surgery, we started monitoring hippocampal activity. The same procedure was followed for all mice before, during, and after the cuprizone diet period. Mice were anesthetized (3% isoflurane) and injected with Chlorprothixene Hydrochloride (IM, 30 μl of a 0.33 mg/ml solution, Santa Cruz). Recordings started at least 30 min after injection. Mice were put on a 37°C heating pad in the dark, and isoflurane levels were reduced to 0.5–0.75% prior to recording to bring the mice to a lightly anesthetized state, where they do not move but are sensitive to pain. Spontaneous activity was recorded using 20–120 mW of 1100 nm excitation light (Insight X3, Spectra-Physics) and a resonant-scanner two-photon microscope (512 × 512 pixels, 30 Hz acquisition rate, Bergamo II, Thorlabs). Light intensity was controlled using a Pockels Cell (model 350-105, Conoptics) to maintain similar signal-to-noise ratios across different animals. Data were recorded on a weekly basis from all animals, and each recording session included acquisition of 200 s for each field of view (FOV), 3–5 FOVs from CA1 and 2–4 FOVs from dentate gyrus (DG) for each mouse.

Imaging experiments were carried out as previously described^{28 (link)} using a two-photon microscope with a movable stage (Thorlabs Bergamo II) and a fast piezoelectric objective scanner (Physik Instrumente P725) for volumetric imaging. We used a Chameleon Vision-S Ti-sapphire femtosecond laser tuned to 940 nm for two-photon excitation. Images were collected using a 20× 0.95-NA objective (Olympus). Emission fluorescence was filtered with a 525-nm bandpass filter (Thorlabs) and collected using a GaAsP photomultiplier tube (Hamamatsu).
For EPG neurons, the imaging region was centred on the protocerebral bridge, where EPG axons terminate. The imaging view was 256 × 128 pixels, and 8–12 slices deep in the z axis (4-6 µm per slice), resulting in a 6–9 Hz volumetric scanning rate. For ExR2 neurons, the imaging region was centred on the bulb and ellipsoid body. The imaging view was 256 × 128 pixels, and 12 slices deep in the z axis (6-8 µm per slice), resulting in a 6–7 Hz volumetric scanning rate.
Volumetric z-scanning signals from the piezoelectric objective scanner were acquired simultaneously with analog output signals from the visual panorama and/or analog outputs from FicTrac 2.1 through a NiDAQ PCI-6341 at 40 kHz. Data were acquired using ScanImage 2018 (Vidrio Technologies) with National Instruments hardware from Vidrio (NI PXIe-6341).

It is taken with a commercial multiphoton microscope with both 2P and 3P light path (Bergamo II, Thorlabs). A high numerical aperture (NA) water immersion microscope objective (Olympus XLPLN25XWMP2, 25 X, NA 1.05) is used. For GFP and THG imaging, fluorescence and THG signals are separated and directed to the detector by a 488 nm dichronic mirror (Di02-R488, Semrock) and 562 nm dichronic mirror (FF562-Di03). Then the GFP and THG signals are further filtered by a 525/50 nm band-pass filter (FF03-525/50, Semrock) and 447/60 nm (FF02-447/60, Semrock) band-pass filter, respectively. The signals are finally detected by GaAsP photomultiplier tubes (PMTs) (PMT2101, Thorlabs).

Fluorescence images were obtained using a custom-built in vivo two-photon microscope (Bergamo II, Thorlabs), equipped with a Nikon ×16 (0.8NA, 3 mm WD) or an Olympus ×20 objective lens (1.0NA, 2.5 mm WD) and GaAsP PMTs (Hamamatsu) and controlled by ThorImage. GCaMP6f and Rhodamine B dextran or Texas Red dextran were excited at 920 nm using a tunable Ti:sapphire laser (Tiberius, Thorlabs). Green fluorescence signals were obtained using a 525/50-nm band-pass filter, and orange/red signals were obtained using a 605/70-nm band-pass filter. Imaging was performed at a rate of 3.2 or 3.8 Hz. Animal behaviors were captured using a near-infrared LED (780 nm) and a camera. A 5-s air puff that deflected all whiskers on the contralateral side without impacting the face was applied using a Picospritzer, and vessel surface area and astrocyte Ca²⁺ responses were monitored in the barrel cortex (layers I–III).