Fluorescent labeling of specific neuron populations

Mice were anesthetized with 2% isoflurane and mounted in a stereotactic frame. Craniotomies were made according to stereotaxic coordinates relative to Bregma. To label interneurons in the mPFC, we injected AAV encoding the Dlxi1/2b enhancer driving mCherry into the ipsilateral mPFC as previously reported ¹⁷. To selectively label subcortical projection (SC) or intratelencephalic (IT) neurons, we injected fluorescently-labeled latex microspheres (Retrobeads, Lumafluor) or fluorescently-labeled cholera toxin subunit B (CTB, Molecular Probes) into contralateral mPFC or ipsilateral MD thalamus. Coordinates for injection into contralateral mPFC were (in millimeters relative to Bregma): +1.7 anterior-posterior (AP), –0.3 mediolateral (ML), and –2.75 dorsoventral (DV). Coordinates for injection into ipsilateral MD thalamus were –1.7 AP, +0.3 ML, and –3.5 DV. We injected 500 nL at 150 nL/min for mPFC and 400 nL at 100 nL/min for MD thalamus. We waited 5 minutes after the end of the injection before slowly withdrawing the syringe. We waited 3–5 days following retrograde tracer injections before performing experiments. At the time of the experiments, we visually verified that retrograde tracer injections were targeted appropriately and that tracer was not present in nearby structures.