A total of 100 110 sections of 70 nm electron microscope images were reconstructed to visualize astrocyte shapes (see three-dimensional reconstruction of electron microscope images in Supplemental movies; non-treated male M5R+/+ mice, Movie S1; non-treated male M5R−/− mice, Movie S2; E2-treated male M5R+/+ mice, Movie S3; E2-treated male M5R−/− mice Movie S4).
The sections were observed under a light microscope, and the recording sites were reconstructed according to the lesions made by current injection and traces of electrode tracks.
Material was then stained with aniline blue, mounted on microscope slides, and imaged using a compound fluorescence microscope to obtain optical sections that were reconstructed into a 3D image.
Images magnified by factors of 12.5 and 40 were obtained using an optical microscope (Axioscop, Carl Zeiss, Jena, Germany) and the images were reconstructed to photos of the entire tissues using the Photoshop program.
3D neuron models were reconstructed using the Neurolucida system (MBF Bioscience, USA) and a bright-field light microscope (Olympus, BX51, Japan).
Axons, dendrites, and somata of stained neurons were reconstructed using the Neurolucida software (MBF Bioscience, Williston, VT) attached to a NIKON ECLIPSE microscope equipped with a 60× objective lens (NA 1.4, NIKON, Tokyo, Japan).
Representative examples were reconstructed using the Neurolucida software (MicroBrightfield Europe, Magdeburg, Germany) equipped to an Olympus BX61 microscope (Olympus, Hamburg, Germany).
All cells matching the selective criteria were reconstructed using the NEUROLUCIDA software (MicroBrightfield Europe; Magdeburg, Germany) equipped to an Olympus BX61 microscope.
VG3-ACs, W3-RGCs, and patterns of connections between them were reconstructed from confocal and 2-photon imaging stacks acquired on Fv1000 laser scanning microscopes (Olympus, Tokyo, Japan) using 60× 1.35 NA oil immersion or 20× 0.9 NA water immersion objectives.
Neurons were reconstructed with the aid of Neurolucida software (MicroBrightField, Colchester, VT, USA) using an Olympus Optical (Hamburg, Germany) BX50 microscope or a Zeiss Axioplan (Götttingen, Germany) microscope equipped with ×100 oil immersion objective.
