Dominance, Ocular

Cats (postnatal days 36–49) were anesthetized with isoflurane (1–2% in surgery, 0.5–1.0% during imaging)22 (link) and paralyzed with vecuronium bromide22 (link). A craniotomy was performed over area 18 of the visual cortex, the dura reflected, and the underlying cortex covered with agarose. Movement of the brain from respiratory and heart beat pulsations were negligible (Supplementary Fig. S8). The cell-permeant calcium indicator Oregon Green 488 Bapta-1 AM (1 mM) was prepared22 (link),30 (link) and co-loaded with 40 μM Alexa Fluor 594 into a glass patch pipette (2.5 μm diameter tip). Under continuous visual guidance, the pipette tip was advanced 200–250 μm below the cortical surface and the indicators were then pressure ejected (5–10 psi). This particular method of loading produces minimal staining of glial cells (see Ref 22 (link)) but it is possible that some of the stained cells in the present study were not neuronal. Fluorescence was monitored with a custom-built microscope (Prairie Technologies) coupled with a Mai Tai XF (Newport Spectra-Physics) mode-locked Ti:sapphire laser (850 or 920 nm). Drifting sine-wave gratings (2 Hz, 50% contrast) were presented on a CRT (100 Hz refresh rate) in a variety of configurations for orientation, direction of motion, spatial frequency, ocularity (left or right eye—for ocular dominance), and eight inter-ocular spatial phase disparities (0°, 45°, 90°, 135°, 180°, 225°, 270°, 315°). For ocular dominance and binocular disparity assays, animals viewed the monoptic and dichoptic visual stimuli through ultra-fast ferroelectric liquid crystal shutters (7 KHz switching time, 1,000:1 extinction contrast ratio, DisplayTech). Each stimulus period (8 s) was preceded by an equal blank period, repeated 3–8 times. Coarse retinotopic positions of monocular receptive fields were determined by using 5° wide flashing bars of light or strips of gratings at ten retinotopic positions. Two-photon images were analyzed inMatlab (Mathworks)—see Full Methods.
Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature.

Experimental protocols were carried out in strict accordance with the University of Rochester Committee on Animal Resources (UCAR) and conformed to the National Institute of Health’s “Guide for the Care and Use of Laboratory Animals, 8^th Edition, 2011.” Experiments characterizing baseline microglial function were carried out at ages representing early development (postnatal day (p) 15), adolescence (p28), or early adulthood (p60). Experiments examining visual cortical plasticity were carried out during the visual critical period for ocular dominance plasticity, between p26 and p34. Monocular deprivations were performed between p26 and p30 by removing the right eye lid margins and suturing the lid shut. Examination of retino-geniculate projections in the lateral geniculate nucleus were carried out during the same time period, as reorganization of these projections is complete by this time and the final organization of eye-specific layers can be assessed. Experiments examining microglial infiltration into thalamocortical axon (TCA) clusters were carried out at p7 and p10 to replicate previously published methods (Hoshiko et al. 2012 (link)). Both female and male mice were included in all experiments and all mouse lines were generated on a C57Bl/6 background. C57Bl/6 (Jackson Labs), Cx3cr1-EGFP (Jung et al. 2000 (link)), Cx3cr1-knockout (Taconic Biosciences), and thy1-YFP line H (Feng et al. 2000 (link)) mouse lines were used and bred together as follows: The Cx3cr1-EGFP mouse line was used both to visualize microglia and to achieve manipulation of CX₃CR1. For experiments involving in vivo imaging of microglia, because visualization of microglia requires at least one copy of GFP, Cx3cr1-EGFP heterozygous mice (Cx3cr1^G/+) were used as controls. It is important to note that some studies have observed gene dosage effects in Cx3cr1-EGFP (Jung et al. 2000 (link); Lee et al. 2010 (link); Rogers et al. 2011 (link)). While this finding comes from a small subset of studies conducted under mostly pathological conditions, it is therefore possible that heterozygous mice might not behave the same as wild-type mice. However, given that these experiments cannot be carried out without a fluorescent label, this question will need to be explored using a different approach in the future. Similarly, to assure similar levels of GFP expression and therefore similar visualization of microglia in Cx3cr1-null mice as in control mice, Cx3cr1-EGFP homozygous mice (Cx3cr1^G/G) were crossed to Cx3cr1-knockout mice (Cx3cr1^−/−) to generate Cx3cr1-null mice with a single copy of GFP (Cx3cr1^G/−). Cx3cr1^G/G mice were included in imaging experiments to assay the potential impact of additional GFP expression on visualization and/or GFP toxicity. We did not observe any differences in the dynamics of microglia expressing different levels of GFP, but we cannot rule out the possibility that GFP overexpression alters microglial behavior. For experiments examining in vivo interactions between neurons and microglia, thy1-YFP mice were crossed to generate Cx3cr1^G/+/YFP control mice, as well as both Cx3cr1^G/−/YFP and Cx3cr1^G/G/YFP Cx3cr1-null mice, again to assay the impact of additional GFP expression. For experiments examining in vivo dendritic spine turnover, YFP, Cx3cr1^G/+/YFP, and Cx3cr1^G/G/YFP mice were used, as microglia were not studied in these experiments.