Neurolucida

Sections were imaged on an inverted confocal microscope (Fluoview FV3000, Olympus) and projection images with maximum intensity were acquired. Images were analysed using Imaris and adjusted for contrast and brightness in Adobe Photoshop. Data were collected from at least three brains per timepoint and presented as mean ± SEM. For 3D reconstruction of lineages, whole brain sections photographed by Olympus VS120 were reconstructed for clones spanning several 80‐μm adjacent sections using Neurolucida. Neurons and glia were distinguished based on their morphology, and cortical areas were identified using the Allen Brain Atlas. The intensities of CFSE in determinate lineage were captured using Live Cell Imaging System (UltraVIEW VOX, Andor and SpinsR, Olympus).

In vivo images were acquired under MMF-anesthesia, using a custom- built 2PLSM (Janelia Farm Research Campus, model Non-MIMMS in vivo microscope^{77 (link)}. To record the 2PLSM images, we used custom software written in MATLAB (Scanimage, Vidriotech and Janelia Farm Research Campus). A pulsed Ti:sapphire laser (Chameleon ultra II, Coherent) was tuned to 910 nm to excite the GFP. The green channel was equipped with a photomultiplier tube (Hamamatsu H10770PA-40SEL). We used a 20x water immersion objective (NA 0.95, XLUMPlanFI, Olympus, Japan), and images were acquired at 2 ms/line (image size 1024 × 1024 pixels, pixel size 0.87 μm², z-plane steps: 2 μm for large z-stacks used for exploration of the preparation and the Neurolucida reconstructions, and 1024 × 1024 pixels z-stacks with a pixel size of 0.0049 μm² and z-steps of 1 μm, for the acquisition of images that were used for spine tracking). Neurolucida (MBF Bioscience) software was used to trace the 3D morphology in the 2PLSM image stacks (pixel size 0.87 μm²) 2PLSM z-stacks.

We used exhaustive plotting to map the distribution of calretinin-positive (CR+) neurons in representative coronal sections of the hypothalamus (Case AX) as described previously (Joyce and Barbas 2018 (link)). Briefly, we used a semi-automated commercial system (Neurolucida, Version 2018.1.1; Olympus BX60 microscope) to plot the architectonic landmarks of the limits of the hypothalamus, including the third ventricle, the optic tract, the optic chiasm, the infundibulum, and the cerebral peduncle, at 40× magnification under brightfield illumination. We then plotted each CR+ labeled neuron in the hypothalamus at 400× magnification using a marker (red) to represent each CR+ neuron. We then compared each map to matched sections of the hypothalamus stained for AChE to trace the limits of hypothalamic nuclei for qualitative analysis of the distribution of CR+ neurons. No other adjustments were made.