Fig 1: Improper functioning of Cre system in the BNST. (A) VGLUT3-Cre mouse acquired from the Jacksons Laboratory (USA; stock No.: #028534) and bred locally at the Institute of Experimental Medicine was injected with AAV containing the sequence of EYFP in a DIO construct (Addgene, catalog No.: #20298-AAV5) (see Figure 2B). Brain slices of 30 µm thickness were stained with a-VGLUT3 (rabbit, Synaptic Systems, stock No.: #135203) and a-EYFP (chicken, Life Technologies, stock No.: #A10262), then with Alexa-594 conjugated a-rabbit (goat host, Life Technologies, stock No.: #A11012) and Alexa-488 conjugated a-chicken (goat host, Life Technologies, stock No.: #A11039) antibodies, respectively. EYFP-positive (green) cells represent Cre activity. (B) Example of a proper Cre system functioning: VGLUT3+ (red) neuron was able to inverse and express the gene sequence of EYFP that was coded in the injected AAV in the BNST. (C) Example of an improper Cre system functioning: numerous other cells in the BNST that are VGLUT3+ (red) failed to express the EYFP, while VGLUT3 negative neurons were able. Thus, this VGLUT3-Cre mouse could not be used for the specific manipulation of BNST VGLUT3+ neurons. Double fluorescent immunohistochemistry was done by László Bíró, and the pictures were taken by him at the Nikon Centre at the Institute of Experimental Medicine with C2 confocal laser-scanning microscope at 20× and 60× magnification. AAV: adeno-associated viral vectors; BNST: bed nucleus of stria terminalis; DIO: double-floxed inverted open reading frame; EYFP: enhanced yellow fluorescent protein; VGLUT3: vesicular glutamate transporter type 3.