Cx9000

Brains were sectioned at 40 μm using a cryostat, and subjected to immunohistochemistry for Fos as described previously [14 (link)]. Sections were incubated with a rabbit primary antiserum raised against the Fos protein (1:1000, sc-52, Santa Cruz Biotechnology, Inc., CA, USA) and processed with avidin-biotin-immunoperoxidase technique using DAB enhanced with 4% nickel sulfate as the chromogen. Thereafter, sections were mounted on slides, dehydrated with alcohol rinses, cleared with xylene, and coverslipped with Permount (Fisher Scientific, NJ, USA). For quantification, images were captured using a CCD video camera (CX9000, MBF bioscience, Williston, VT, USA) attached to a light microscope (Zeiss, Gottingen, Germany) with a 10× objective lens. Numbers of Fos-immunoreactive (Fos-ir) nuclei were counted in a standardized area within the PVN and subfields of the CA1, CA3, and dentate gyrus in the hippocampus using NIH Image J. The average of the counts from the two sections of the PVN and three sections of the hippocampus were used to represent the value for each animal. For the hippocampus, Fos-ir cells were counted along the molecular cell layer in the CA1 and CA3 and the granule cell layer in the dentate gyrus and calculated as cell counts per 10³ μm², and the values from left and right hemispheres were averaged.

Behaviorally naïve animals were used in this study. Grass rats were housed in either brLD or dimLD (n=7/condition) for 4 weeks prior to transcardial perfusion (at ZT 5–7) with a phosphate buffer followed with a Rapid-Golgi fixative solution (modified from (Patro et al., 2013 (link))). Brains were post-fixed in the same solution for 24 hours, then transferred to 3% potassium dichromate for three days before immersion in 1% AgNO₃ for eight days. Brains were placed in 20% sucrose for 48 hours prior to sectioning at 100 μm using a cryostat. Sections were processed through an ethanol dehydration series and were clarified with xylene. Sections were mounted onto gelatin-coated slides and coverslipped with Permount (Fisher Scientific, NJ, USA). For quantification, images of dendritic spines were captured using a CCD video camera (CX9000, MBF bioscience, VM, USA) attached to a light microscope using an oil immersion lens (Nikon Instruments Inc., NY, USA) and spines were quantified using ImageJ with the AnalyzeSkeleton plug-in (Ignacio Arganda-Carreras, http://fiji.sc/wiki/index.php/). CA1 apical dendritic spines were analyzed from 20μm segments of four distinct dendritic branches per neuron, a total of six neurons were analyzed per brain (Pyter et al., 2005 (link)).