Donkey anti mouse alexa fluor 405

After patch-clamp recordings, the recording electrode was carefully withdrawn, and the slices were fixed overnight at 4 ºC in 4% paraformaldehyde in phosphate buffer solution (PBS). After thorough rinsing with PBS and permeabilization with 0.3% Triton X-100 (Sigma-Aldrich), the slices were incubated overnight with streptavidin-CY3 (1:50, 438,315, Invitrogen, USA) or streptavidin-CY5 (1:50, 438,315, Invitrogen, USA). Slices were then rinsed with PBS and mounted onto Fisher SuperFrost slides for visualization in an Olympus FV1000 confocal microscope. Low magnification (10×) was used to localize the recorded neurons, confirming that they were within the boundaries of the VTA. Slices that contained a labeled neuron were transferred back to a PBS solution and after additional permeabilization with 0.3% Triton X-100, they were incubated overnight with a mouse anti-TH antibody (1:100, MAB318, Millipore, Burlington, MA). Sections were rinsed with PBS and then incubated with secondary Donkey anti-Mouse Alexa Fluor 405 (1:100, ab175658, abcam, Cambridge, MA) for 1 hr at 30°C. Sections were rinsed with PBS and then mounted onto Fisher SuperFrost slides and visualized using higher magnification (40×) on an Olympus FV1000 confocal microscope.

Methylmercury chloride (MeHg) was purchased from ICN Biomedical Inc. (Costa Mesa, CA). Calcein acetoxymethylester (calcein AM) and ethidium homodimer-1 (EthD-1) were purchased as a cell viability kit from Molecular Probes (Invitrogen, Eugene, OR). Poly-L-ornithine, laminin, CNQX (cyanquixaline, 6-cyano-7-nitroquinoxaline-2,3-dione), NAS (1-naphthylacethyl spermine), AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), paraformaldehyde, tritonX-100, fura-2AM and ethylene glycol-bis (β-aminoethylether) N,N,N’,N’-tetra acetic acid (EGTA) were purchased from Sigma-Aldrich (St. Louis, MO). Normal goat serum and mounting medium Vecta shield with nuclear DNA stain 4, 6-diamino-2-phenylinndole (DAPI) were purchased from Vector Labs (Burlingame, CA).
Antibodies used were: rabbit anti-glutamate receptor subunit 1 (GluA1), rabbit anti-GluA2, rabbit anti-adenosine deaminase acting on RNA2 (ADAR2) conjugated with Alexa Fluor 488, and chicken anti-neurofilament heavy-200 (NFH) obtained from Abcam (Cambridge, UK). Mouse anti-neuronal specific nuclear protein NeuN (NeuN) were purchased from Millipore; secondary antibodies used were donkey anti-mouse Alexa Fluor 405, goat anti-rabbit Alexa Fluor 488 and goat anti-chicken Alexa Fluor 594 obtained from Abcam.