True confocal scanner spe

Rats (n = 4) were anesthetized with 1%–5% isoflurane, perfused with phosphate buffered saline for 5 min, followed by 4% paraformaldehyde (PFA) for 15 min. Brains were removed and sliced into 40 micronsections using a cryostat. NAc and VTA sections were stained as described in previous studies [33 (link),43 (link),46 (link)]. Briefly, sections were washed twice in 1× PBS, and then antigen-unmasked with 1% SDS for 5 min. Next, sections were blocked in donkey serum and incubated in 1:150 primary antibody rabbit αNMUR2 (NBP1-02351, Novus Biologicals, Littleton, CO, USA) overnight. The following day, sections were washed three times in 1X PBS, then incubated with 1:200 donkey αrabbit IgG Alexa Fluor 568 (A10042, Invitrogen, Carlsbad, CA, USA). Images were acquired using Leica True Confocal Scanner SPE in confocal mode and Leica Application Suite × software (Leica Microsystems, Wetzlar, Germany).

Accumbal NMUR2 was visualized in perfused brains using immunohistochemistry methods described previously (Benzon et al., 2014 (link); Kasper et al., 2016 (link); McCue et al., 2016 (link)). Briefly, 40 μm sections were taken from an anterior to posterior range of the NAc (three slices from each rat at 2.76, 1.92 and 1.32 mm from Bregma) to mimic the range included in the tissue dissections for RT-PCR. Slices were permeabilized, blocked with serum in phosphate buffered saline, and incubated with rabbit αNMUR2 (1:150; NBP-02351, Novus Biologicals). The sections were washed and incubated with donkey αrabbit AF-488 (1:100). Images were acquired using a Leica True Confocal Scanner SPE and Leica Application Suite Advanced Software (Leica Microsystems, Wetzlar, Germany) with 20× objective and tile scan mode.
Accumbal NMUR2 staining was quantified using FIJI (ImageJ). Image background was subtracted using rolling ball radius of 100 pixels. To quantify neuronal cell bodies and synapses, the range for desired size of events was set to >10 pixels. This quantifies the number of NMUR2 positive cell bodies and synapses as “events.” To analyze images, the NAc was defined from the rat brain atlas (Paxinos and Watson, 2007 ), and the number of NMUR2 positive neuronal events were quantified.

Rats were anesthetized with a combination of ketamine and xylazine, euthanized by cardiac puncture, and transcardially perfused for 5 min with 1XPBS (75 mL), followed by 15 min of 4% paraformaldehyde in 1XPBS (225 mL). Brains were then frozen with dry ice; using a microtome (Leica SM 2010R), 40 μm coronal slices containing the LH were collected and stored in vials of 0.01% sodium azide in 1XPBS at 4 °C until use. Immunohistochemistry staining for GFP was conducted as previously described [10 (link),12 (link)]. Slices were mounted onto Superfrost Plus microscope slides and allowed to dry overnight. Slides were rehydrated and cover-slipped according to previously published protocols [10 (link),12 (link)]. Targeting for each rat was assessed by examining immune-enhanced GFP expression under a confocal microscope. All images for targeting were captured using Leica True Confocal Scanner SPE and Leica Application Suite Advanced software (Leica Microsystems, Germany) [12 (link)].