Ti 1200

Mice were anesthetized with 200 mg/kg pentobarbital and perfused transcardially with 4% paraformaldehyde (PFA) in PBS. Brains were isolated and fixed overnight in 4% PFA before storage in 1 X PBS. Brains were sectioned with a vibratome between 60–100 µm or a cryostat at 30 µm and mounted on slides (Fisher Permafrost). Vibratome sections were permeabilized with 1 X PBS with 0.2% Triton-X100, mounted on slides, and coverslipped with mounting media containing DAPI. All injection sites were aligned back to the Allen Brain Atlas, injection sites with substantial off-target infection were excluded. In the cannula experiments, signal from the AAV2/9-CAG-DIO-DREADD(h4Dmi) was amplified using anti-mCherry (Abcam: ab167453) at 1:500 by first blocking with 1 X PBS with 10% goat serum and 0.3% Triton X-100 for one hour at room temperature, incubated with anti-mCherry overnight at 4 °C, followed by incubation with goat anti-rabbit 568 (Thermo Scientific: A-110011) for one hour at room temperature, and cover slipped with DAPI mounting media. For identification of excitatory cortical neurons in layer II/III, anti-Foxp1 (Abcam: ab16645 1:500) was used with goat anti-rabbit 488 (Thermo Scientific: A-11008). Slides were visualized and imaged with a Nikon Ti-1200.

Mice were perfused transcardially with 4% paraformaldehyde (PFA) in PBS. Brains were isolated and fixed overnight in 4% PFA before storage in 1 X PBS. Brains were sectioned with a vibratome between 60–100μm or a cryostat at 30μm and mounted on slides (Fisher Permafrost). Vibratome sections were permeabilized with 1 X PBS with 0.2% Triton-X100, mounted on slides, and coverslipped with mounting media containing DAPI. All injection sites were aligned back to the Allen Brain Atlas, injection sites with substantial off-target infection were excluded. In the cannula experiments, signal from the AAV2/9-CAG-DIO-DREADD(h4Dmi) was amplified using anti-mCherry (Abcam: ab167453) at 1:500 by first blocking with 1 X PBS with 10% goat serum and 0.3% Triton X-100 for one hour at room temperature, incubated with anti-mCherry overnight at 4°C, followed by incubation with goat anti-rabbit 568 (Thermo Scientific: A-110011) for one hour at room temperature, and cover slipped with DAPI mounting media. For identification of excitatory cortical neurons in layer II/III, anti-Foxp1 (Abcam: ab16645 1:500) was used with goat anti-rabbit 488 (Thermo Scientific: A-11008). Slides were visualized and imaged with a Nikon Ti-1200.