Cf175

We performed calcium imaging of the OB using an Ultima two-photon microscope from Prairie Technologies, equipped with a × 16 water-immersion objective lens (0.8 NA; CF175, Nikon). We delivered two-photon excitation at the 920nm using a DeepSee femtosecond laser (Spectraphysics). Acquisition rate was 7Hz. Before awake imaging and ∼3 weeks after implanting the window, we habituated the mice under the microscope in the head-fixed configuration.
In order to recover the same neurons on multiple sessions, we followed the same procedures that we recently described (e.g., Vinograd et al., 2017b (link); Shani-Narkiss et al., 2020 (link); Kudryavitskaya et al., 2021 (link)). In short, in each mouse we found a clear anatomical mark on the surface of the OB (a blood vessel pattern), which was directly above the imaging field of view. This anatomical mark was used as an anchor and documented for future use. In a following session, the microscope was targeted to this anchor, and focusing down to the mitral cell layer revealed a region roughly including the previously imaged MCs. This region was compared to the previously stored micrograph of the field of view and manually aligned in the x, y, and z coordinates.

Images were acquired using a resonant scanning two-photon microscope (Ultima Investigator) at a 30 Hz frame rate and 512 × 512 pixel resolution through a 16× water-immersion lens (16×/0.8 numerical aperture; model CF175, Nikon). On separate days, either AC or PPC was imaged at a depth between 150 and 300 μm, corresponding to layers 2/3 of cortex. For AC imaging, the objective was rotated 35–45° from vertical, and for PPC imaging, it was rotated to 5–15° from vertical, matching the angle of the cranial window implant. Fields of view were 500 μm² and contained 187 ± 95 neurons, 20 ± 10 (mean ± SD) of which were classified as SOM neurons. Excitation light was provided by a femtosecond infrared (IR) laser (Insight X3, Spectra-Physics) tuned to 920 nm. Green and red wavelengths were separated through a 565 nm low-pass filter before passing through bandpass filters (catalog #ET525/70 and #ET595/50, Chroma). PrairieView software (version 5.5; Bruker) was used to control the microscope.

We performed calcium imaging of the OB using an Ultima two-photon microscope from Prairie Technologies, equipped with a ×16 water-immersion objective lens (0.8 NA; CF175, Nikon). We delivered two-photon excitation at 920 nm using a DeepSee femtosecond laser (Spectraphysics). Acquisition rate was 7 Hz. Before awake imaging and ~3 weeks after implanting the window, we habituated the mice under the microscope in the head-fixed position. Two-photon microscopy was operated with PrairieView software (version 5.5).

Cell-attached recordings were obtained using targeted patch-clamp recording by a previously described procedure (Margrie et al., 2003 (link); Cohen and Mizrahi, 2015 (link); Maor et al., 2016 (link)). For visualization, the electrode was filled with a green fluorescent dye (Alexa Flour-488; 50 μM). Imaging of A1 was performed using an Ultima two-photon microscope from Prairie Technologies equipped with a 16 × water-immersion objective lens (0.8 numerical aperture; CF175; Nikon). Two-photon excitation at wavelength of 930 nm was used in order to visualize both the electrode, filled with Alexa Flour-488, and PV⁺ somata, labeled with tdTomato (DeepSee femtosecond laser; Spectraphysics). The recording depths of cell somata were restricted to subpial depths of 180–420 μm, documented by the multiphoton imaging. Spike waveform analysis was performed on all recorded cells, verifying that tdTomato+ cells in L2/3 had faster/narrower spikes relative to tdTomato-negative (tdTomato-) cells (see also Cohen and Mizrahi, 2015 (link)).

Time-lapse imaging of new-born GCs started 27 days post-injection (d.p.i), and was performed again at 28 d.p.i. (i.e., a 24 hr interval). Mice were anesthetized (using ketamine (50 mg per kg)/medetomidine (0.42 mg per kg)) and placed under the microscope in a custom-made stereotaxic device via the metal bar glued to the skull in a fixed orientation relative to the objective lens. We performed the imaging of the OB using an Ultima two-photon microscope from Prairie Technologies (Middleton, WI), equipped with a 16X water-immersion objective lens (0.8 NA; CF175, Nikon). We delivered two-photon excitation (1000 nm) with a DeepSee femtosecond laser (Spectraphysics), and expanded the laser beam to fill the large back aperture of the 16X objective. We acquired images of dendritic spines (512 × 512 pixels) at 0.23 μm/pixel resolution in the xy dimension and at 0.9 μm/frame in the z dimension. Each dendritic tree was identified in the additional imaging sessions by its location in 3D relative to the blood vessel map (Mizrahi, 2007 (link); Kopel et al., 2012 (link)).