Achroplan

Electrophysiological protocols have been previously described57 and details are provided as Supplementary Information. Briefly, at CFC day 14^th, the brains of 15 mice (5 mice/phenotype) were cut in 250 μm coronal sections, incubated in oxygenated ACSF, transferred to a recording chamber and submerged in continuously flowing oxygenated ACSF for electrophysiological recordings.
To study the sEPSCs using a Cs-methanesulfonate internal solution, whole-cell patch-clamp recordings in voltage clamp mode (holding potential −70 mV) were performed from mPFC pyramidal neurons, visually identified in slices using an upright infrared microscope (Axioskop 2 FS, Zeiss, Germany), a 40× water-immersion objective (Achroplan, Zeiss, Germany), and a CCD camera (Cool Snap, Photometrics, AZ, USA).
In some experiments the AMPA/Kainate receptor antagonist CNQX (10 μM) and the NMDA receptor antagonist AP-5 (50 μM) were added to the ACSF. In this condition all synaptic events were blocked, indicating that they were due to glutamatergic receptor activation57 .
Spontaneous synaptic events were analyzed off-line using the Mini Analysis Program (Synaptosoft Inc., USA). sEPSCs were manually detected using a 10 pA threshold crossing algorithm. The inter-event interval, event amplitude and kinetic parameters (rise and decay times) were compared among phenotypes.

NIH 3T3 (ATCC CRL1658) and NIH 3T3 X2 (ref. 54 (link)) fibroblasts were grown under standard conditions in Dulbecco's MEM containing 3.7 NaHCO₃, 4.5 D-Glucose (Biochrom), 100 Penicillin/Streptomycin (PAA), and 10% fetal bovine serum (Biochrom). Cells were mycoplasm free.
Live cell imaging was carried out on a Zeiss Axio Oberver.Z1 equipped with an incubation system at 37 °C and 5% CO₂. A Zeiss Achro Plan 10 × (NA 0.25) and Zeiss Plan Apochromat 40 × (NA 0.95) were used for phase contrast and fluorescence imaging respectively in conjunction with a Zeiss AxioCam MRm camera. No PDGF was added to the cell medium in experiments with live cell imaging (except for Supplementary Fig. 5). Actin was visualized using pLifeAct-TagGFP2 (Ibidi) and Lipofectamin2000 (Invitrogen) transfection.
Confocal imaging of fixed cells was done using a Zeiss LSM 780 equipped with a Plan-Apochromat 63 × (NA 1.4) objective. Actin was stained via Rhodamin/Phalloidin (Biotium) and the nucleus with DAPI (Roche). CDRs initiated via 30 hPDGF-BB (Cell Signaling Technology) in serum-free DMEM. Cells were then fixed 20 min after stimulation using methanol/acetate (1:1).

Cerebellar Purkinje cells were lesioned at 6 dpf specifically using transgenic expression of the photosensitizer, KillerRed. Larvae were mounted dorsal-up in agarose and anesthetized in MESAB. Control, transgenic larvae were anesthetized in MESAB in parallel. Larvae were randomly allocated into groups without blinding. Illumination conditions on a widefield microscope (Axio Imager M1, Zeiss, Oberkochen, Germany) were set under blue light (480/30 excitation filter from filter set 19002, Chroma Technology, VT) to visualize but not activate KillerRed. Light was focused through a 40x, 0.75NA water immersion objective (Zeiss Achroplan), stopped down to fill a 0.3 mm diameter region, and focused on the Purkinje cell somata. Green light (540/25 excitation filter from filter set 19004, Chroma Technology, VT) was then applied for 15 min, quenching KillerRed fluorescence. The power at the sample plane, measured at 540 nm with a 9.5 mm aperture silicon photodiode (PM100D power meter, S130C sensor, Thorlabs, NJ) was 14 mW. Fish were allowed to recover for 16–24 hr before behavioral measurements.

Multiphotonic microscopy was performed using an LSM 710NLO laser-scanning microscope (Zeiss) equipped with a 20 × dry objective with numerical aperture of 0.8 (Achroplan; Zeiss). The whole tissue sections were entirely scanned by tiling (with an overlap of 10%) multiple 512 × 512 pixel frames. Each tissue section was also scanned for a Z-stack of 3 µm steps with no image average. The Chameleon Ultra Ti:Sapphire laser (Coherent) was tuned at 810 nm. Signals were collected using non-descanned detectors (Zeiss). The second harmonic generation (SHG) signal was detected through a bandwidth filter 395 to 425 nm of a non-descanned detector in a backscattering geometry. Simultaneously, the retinoid fluorescence (RF) signal was detected through a bandwidth filter 500 to 550 nm of a non-descanned detector in a backscattering geometry. The quantitative analysis of multiphoton microscopy images was made through a home-made automated image analysis workflow implemented in Fiji (NIH, Bethesda, MD, USA) environment as a macro.

Confocal laser scanning microscopy (CLSM) observations were carried out on a Zeiss LSM 510 microscope using a 63× (Zeiss Achroplan) objective and with 0.4 μm z-section intervals. FITC fluorescence was detected after excitation at λ = 488 nm using a cut-off dichroic mirror of 488 nm and an emission band-pass filter of 505–530 nm (green emission). Alizarin complexone fluorescence was detected after excitation at λ = 543 nm using a dichroic mirror of 543 nm, and an emission long pass filter of 585 nm (red emission).