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.
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.