Fluoro-Gold

Experimental procedures for tracer injections have been described previously^{52 (link)}. Briefly, double coinjections of anterograde and retrograde tracers were delivered to virtually all anatomically delineated regions of the cortex and into select regions of the amygdala and thalamus. Phaseolus vulgaris leucoagglutinin (PHAL; 2.5%; Vector Laboratories) and cholera toxin subunit b (AlexaFluor 647 conjugate, 0.25%; Invitrogen) were coinjected, while biotinylated dextran amine (BDA; FluoroRuby, 5%; Invitrogen) was injected in combination with Fluorogold (FG; 1%; Fluorochrome, LLC). Small localized injections (~200–500 μm) were confined within domains of cortical areas and produced consistent, specific, and highly topographic patterns across the rostral-caudal extent of the CP (Supplementary Fig. 1a). The labeling from PHAL injections was primarily used for automated quantification (see below). Multiple retrograde tracers were injected into different CP domains within a single animal to validate the anterograde tracing data (Supplementary Fig. 1b). Retrograde tracers included FG and CTb 647, 488, and 549 conjugates (0.25%; Invitrogen). Adeno-associated viruses encoding enhanced green fluorescent protein (AAV-GFP; AAV2/1.hSynapsin.EGFP.WPRE.bGH; Penn Vector Core) and tdTomato (AAV1.CAG.tdtomato.WPRE.SV40; Penn Vector Core) were used in cases in which multiple anterograde tracer injections were used to reveal direct spatial correlations of axonal terminals arising from different cortical areas (i.e., topography or interdigitation) (Supplementary Fig. 2a). Although the images in the paper are unique exemplars, the majority of injections were successfully repeated anywhere from 1–17 times (see Supplementary Table 1). For zQ175 and MAO A/B knockout mice, only PHAL tracer injections and labeling were used for quantification. Either one (PHAL) or three weeks (for AAV-GFP) was allowed for tracer transport after which animals were perfused and their brains were extracted.
Surgeries for tracer infusions were performed under isoflurane anesthesia (Hospira, Inc.). Mice were initially anesthetized in an induction chamber primed with isoflurane and were subsequently mounted to the stereotaxic apparatus where they were maintained under anesthetic state via a vaporizer (Datex-Ohmeda). The isoflurane was vaporized and mixed with oxygen (0.5 L/min) and nitrogen (1 L/min). The percent of isoflurane in the gas mixture was maintained between 2 and 2.5 throughout the surgery. Tracers were delivered iontophoretically using glass micropipettes whose outside tip diameters measured approximately 10–30 μm. A positive 5 μAmp, 7-second alternating injection current was delivered for 10 minutes (Stoelting Co.).