Dm6000 b widefield light microscope

Whole brains were extracted, fixed, cryoprotected, and sectioned into 40 μm coronal sections on a cryostat (Leica CM 3050 S, Leica Microsystems, Buffalo Grove, IL, USA). Sections were mounted on charged glass slides (FisherBrand Superfrost, ThermoFisher Scientific, Waltham, MA, USA) and coverslipped using HardSet VECTASHIELD mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA) for microscopy. Fluorescence microscopy (Leica DM6000 B widefield light microscope, Leica Microsystems, Buffalo Grove, IL, USA) was used to verify mCherry expression (proxy for DREADD expression) in the hippocampal target cell bodies and terminal regions. The data of rats without bilateral mCherry expression in the subiculum were eliminated from analysis.

At the end of the experiments, mice were anesthetized with an overdose of kethamine/xylazine (100/10 mg/kg) and transcardially perfused with phosphate-buffered saline (PBS) for 4 minutes followed immediately by 10% formalin. Brains were then collected and postfixed in 10% formalin for 24 h, followed by cryoprotection in 0.2 M Phosphate buffer (PB) at pH 7.4. Brains were sectioned using a vibratome into 40 μm slices and every other section was mounted on charged glass slides (FisherBrand Superfrost, Thermo Fisher Scientific, Waltham, MA, USA). After allowed to air dry under dark conditions, the mounted tissue was coverslip using HardSet VECTASHIELD mounting medium with DAPI (Vector Laboratories Inc., Burlingame, CA, USA). Tissue that was not mounted was placed in cryopreserve solution and stored in a −20 °C freezer. The location of viral vector expression in the LC were evaluated through of the mCherry fluorescent tag and for cannulas placements in the RMTg were both verified using fluorescent microscopy (Leica DM6000 B widefield light microscope, Leica Microsystems, Buffalo Grove, IL, USA), as presented in Fig. 1. Only animals with bilateral mCherry expression in LC and correct bilateral cannula positions were included in statistical analyses.