5 mg/mL, 15 min), then hydrogen peroxide (0.01%, 3–5 min). Sections were mounted on gelatinized microscope slides, air dried, then Nissl stained (neutral red), dehydrated in alcohol, cleared (X-3B), and coverslipped. Stereology The numbers of tyrosine hydroxylase immunopositive (TH+) and tyrosine hydroxylase immunonegative (TH−) cells in the left SNc, ventral tegmental area (VTA), and

locus coeruleus (LC) were estimated using unbiased stereological methods (Stereo Investigator, MicroBrightField, VT). TH+ cells were immunoreactive for TH whereas TH− cells were not immunoreactive for TH but were Nissl stained. The stereologist was blind to the treatment received. The SNc, VTA, and LC were Inhibitors,research,lifescience,medical identified by the spatial distribution

of TH+ cells and anatomical landmarks/boundaries according to the atlas of Paxinos and Watson (Paxinos and Franklin 2004). Counts of TH+ and TH− cells within a counting frame (55 × 55 μm = 3025 μm2) were made at regular predetermined intervals (x = 140 μm, y = 140 μm for SNc; x = 100 μm, y = 100 μm for VTA and LC) throughout each nucleus Inhibitors,research,lifescience,medical in every fourth section. Only those cells with a visible nucleus were counted and glia were Inhibitors,research,lifescience,medical excluded on the basis of soma diameter <5 μm. Drug infusions Some mice had an osmotic minipump implanted to infuse a GABAA receptor antagonist (or vehicle) locally into the left SNc and surrounds via a cannula implanted in the midbrain. This surgery was performed immediately prior to the beginning of EE (or SH) and the infusion lasted throughout the 2-week period of EE (or SH). On the day before surgery, ALZET® Inhibitors,research,lifescience,medical micro-osmotic pumps (model 1002; DURECT Corporation, Cupertino, CA) attached to ALZET® brain infusion cannulae (brain infusion kit 1) were prefilled with drug or vehicle and immersed in 37°C sterile saline overnight to prime

the Inhibitors,research,lifescience,medical pumps. Mice were anesthetized with isofluorane (1–1.5%) in air and placed in a stereotaxic headframe. The infusion cannula was introduced through a ~1.5 mm diameter craniotomy and the cannula tip was placed in the left midbrain at coordinates: 3.0 mm posterior to Bregma, 1.5 mm lateral to the BLZ945 mw midline, and 4.0 before mm below the surface of the brain. The cannula was glued to the skull with dental cement and the attached pump was placed in a subcutaneous “pocket” created in the interscapular region. The location of the tip of the cannula was verified histologically to be in or just dorsal to SNc in each mouse (e.g., asterisks in Fig. ​Fig.33A). Figure 3 GABAA receptor blockade completely abolishes the environment enrichment-induced increase in number of tyrosine hydroxylase immunoreactive (TH+) neurons in the substantia nigra pars compacta (SNc). (A) Photomicrographs through SNc showing TH immunoreactive … Results Effects of mating Males paired with females for 7 days (mated males) had significantly more TH+ SNc neurons than males paired with males (control males) (Fig. ​(Fig.1A;1A; Table ​Table1).1).