Mice were anesthetized with an isoflurane/oxygen vapor selleck bio mixture (1%�C3%), and osmotic minipumps were inserted subcutaneously using aseptic surgery techniques. Minipumps were placed parallel to the spine at shoulder level with the flow moderator directed away from the wound. The wound was closed with 7-mm stainless steel wound clips (Reflex, Cellpoint Scientific, Gaithersburg, MD). Two weeks following minipump implantation, withdrawal was induced by surgical removal of the osmotic minipumps (saline, nicotine, and varenicline) at specific timepoints prior to testing (24 hr, 48 hr, 72 hr, or 7 days). Marble-Burying Test After 1-hr acclimation, mice (n = 6 per group) were placed individually in small cages (26 �� 20 �� 14 cm) in which 20 marbles had been equally distributed on top of mouse bedding (5-cm deep), and a lid was placed on top of the cage.

Mice were left undisturbed for 15 min, after which time a blind observer counted the number of buried marbles (i.e., those covered by bedding three quarters or more). Receptor Binding Brain regions examined were constrained by a minimal tissue amount required for homogenate-binding assays. Tissues were harvested from animals immediately following behavioral testing. The samples were homogenized in 50 mM Tris�CHCl (Sigma-Aldrich) buffer, pH 7.4 at 24��C, and centrifuged twice at 35,000 �� g for 10 min in fresh buffer. The membrane pellets were resuspended in fresh buffer and added to tubes containing a saturating concentration (2 nM) of [3H]epibatidine ([3H]EB; PerkinElmer, Boston, MA), an excellent ligand because of its extremely low nonspecific binding and its high-affinity binding to all heteromeric nAChRs.

Incubations were performed in Tris buffer at pH 7.4 for 2 hr at 24��C with [3H]EB. Bound receptors were separated from free ligand by vacuum filtration over GF/C glass fiber filters (Brandel, Gaithersburg, MD) that were pretreated with 0.5% polyethyleneimine (Sigma-Aldrich). The filters were then counted in a liquid scintillation counter. Nonspecific binding was determined in the presence of 300 ��M nicotine, and specific binding was defined as the difference between total binding and nonspecific binding. Data Analysis Using the GraphPad Prism 5.0 software package (GraphPad Software, San Diego, CA), statistical analyses of the differences between groups were assessed using two-way analysis of variance followed by Bonferroni’s multiple comparison tests.

To better assess the time course and significant correlations between nAChR-binding densities and the number of marbles buried in the marble-burying test, we used group data in the generation of plotted points for a Pearson correlation analysis with 95% CI. Results Both nicotine and varenicline treatment results in long-lasting upregulation of nicotinic receptors Batimastat in the cortex, striatum, hippocampus, and thalamus.