, 2005; Fig. 1). The VTA was further subdivided along its rostrocaudal

extent because of previous reports of functional specificity in rats and mice (Olson et al., 2005; Ikemoto, 2007) and a relative lack of region-specific analysis in the hamster. Rostral sections were defined as having TH cells adjacent to the fasciculus retroflexus prior to the onset of the interpeduncular nucleus; caudal sections were defined as having interpeduncular nucleus present prior to the medial lemniscus merging with the cerebral peduncle; tail sections were defined as having a rounded interpeduncular nucleus prior to the oral part of the pontine nuclei (Fig. 1). Upon completion of microscopic inspection and analysis, similar effects of age and swab exposure

were found in Decitabine mouse the rostral and caudal portions of each VTA subregion; therefore, data from rostral and caudal IF, PN and PBP sections were combined within subregion for statistical analysis and presentation here. Anatomically matched tissue sections throughout the extent of each region of interest (2–5 sections per subregion, depending on size) were selected at INK128 4× magnification. In the Acb, Me and VMH, subregion contours were manually traced bilaterally according to the atlas and cytoarchitecture in Nissl-stained sections and then overlaid 4-Aminobutyrate aminotransferase onto corresponding immunohistochemically treated tissue sections for cell counting. In the mPFC, 600 × 600 μm boxes were placed in the mPFC relative to the medial brain edge and corpus callosum. In the hypothalamus, boxes were drawn to surround all orexin-ir cells medial or lateral to the lateral edge of the fornix in immunohistochemically treated tissue sections. In the VTA, contours were drawn unilaterally in immunohistochemically treated tissue sections. Cell counts were made within a contour by a single experimenter blind to hamster treatment with an UPlanSApo 40 ×  (0.9NA)

objective on an Olympus BX51 microscope under brightfield illumination using Neurolucida (version 7; Microbrightfield, Williston, VT, USA). All quantification was performed on double-labeled immunohistochemically treated tissue; cells were considered Fos-ir if they had a distinct nucleus with visible puncta stained dark red-brown and TH- or orexin-ir if the cytoplasm was stained gray-blue. In all regions, single-labeled Fos-ir cells were counted; the number of Fos-ir cells within each subregion contour was divided by the area of that contour to create a measure of cell density within a section. These density data control for any change in subregion area with age, and generally detect similar effects of treatment as do cell count data.