3. Neuroanatomical Investigation of Daily and Estral Regulation of RFRP-3 Neurons
This protocol is extracted from research article:
Daily and Estral Regulation of RFRP-3 Neurons in the Female Mice
J Circadian Rhythms, Apr 15, 2021; DOI: 10.5334/jcr.212

Adult female mice at proestrus or diestrus stage were sacrificed by exposure to increasing concentration of CO2 at six different time points (ZT0, ZT4, ZT8, ZT12, ZT16 and ZT20; n = 5 per experimental point). After intracardiac blood puncture, mice were intracardially perfused with 10 mL phosphate buffer saline 0.1 M (PBS, pH 7.4) followed by 20 mL of periodate-lysine-paraformaldehyde fixative (formaldehyde 4%, NaIO4 10 mM and lysine 75 mM in 100 mM phosphate buffer, pH 7.3). The brains were collected, post-fixated in periodate-lysine-paraformaldehyde for 12 hours, washed out with PBS, dehydrated and embedded in polyethylene glycol as previously described [44].

Twelve series of 12 μm-thick coronal brain sections were cut using a microtome throughout the DMH as presented in the Paxinos mouse brain atlas. For each mouse, one section in every twelve (i.e. 1 section every 144 µm giving 6–7 DMH-containing brain sections) was rehydrated and mounted on a SuperFrost Plus (Menzel-Glaser, Braunschweig, Germany) slide. For each immunolabeling, DMH-containing slices of all mice of different time points and estrous stages were processed at the same time in order to limit variations in the labeling background.

The number of c-Fos expressing RFRP-3 neurons and the density of AVP fibers surrounding RFRP-3 neurons were assessed by dual immunohistochemistry. Brain sections were first incubated either with a rabbit polyclonal antiserum raised against c-Fos (1:2000; Santa Cruz Biotechnology; RRID: AB_627251) or a rabbit polyclonal antiserum raised against neurophysin II, a cleavage product of prepro-vasopressin (1:15000; Sigma-Aldrich; RRID: AB_260747) diluted in 154 mM PBS buffer containing 10% donkey serum and 0.3% Tween 20, for 24 hours at room temperature. Brain sections were washed with PBS, incubated with biotinylated donkey antirabbit (1:2000; Jackson Labs; in 154 mM PBS buffer containing 10% donkey serum and 0.3% Tween 20) for 1 hour and then washed again with PBS. Immunoreactive signal was amplified by a treatment with the avidin biotin complex coupled to peroxidase (1:250; Vector Laboratories) for 1 hour, then revealed using a solution of 0.5 mg/mL 3,3-diaminobenzidine (DAB; Sigma-Aldrich) diluted in water and 0.001% hydrogen peroxide urea (Sigma-Aldrich) for 30 minutes. Before performing the second immunolabelling, the antibodies were eluted with 2 x 15 minute washes in a solution of 100 mM glycine containing 0.3% Triton X-100 (pH 2.2). Brain sections were then incubated with a primary antibody directed against RFRP-3 (1:15000; rabbit anti-RFRP-3, University of Otago, NZ; RRID: AB_2877670; in a PBS buffer containing 10% donkey serum and 0.3% Tween 20) overnight at room temperature. The sections were then washed with PBS, incubated with biotinylated donkey anti-rabbit (1:2000; Jackson Labs; in 154 mM PBS buffer containing 10% donkey serum and 0.3% Tween 20) for 1 hour, and then washed again with PBS. The RFRP-3 signal was detected using streptavidin-peroxidase at 1/3000 (Roche) for 1 hour and revealed with Fast blue- BB (Sigma-Aldrich) for 7 minutes. Finally, sections were mounted with CC/mount (Sigma-Aldrich), dehydrated in toluene twice for 10 minutes, and mounted with Eukitt resin (Sigma-Aldrich).

Only five sections located at comparable levels of the DMH were taken into account for the analysis in order to limit differences stemming from rostrocaudal variations. The five sections were selected based on neuroanatomical markers such as the median eminence, the tuberoinfundibular sulcus and the pituitary stalk. Counting was done by the first author unaware of animal’s identity. For each mouse, the total number of RFRP-3-immunoreactive (ir) neurons, the number of RFRP-3 neurons containing nuclear c-Fos, and the number of RFRP-3 neurons receiving direct AVP-fiber projections were counted manually. AVP-ergic appositions were defined as terminal fibers directly contacting RFRP-3 cell somas. For each mouse, the number of RFRP-3 neurons is given as the total number counted in 5 sections. The number of c-Fos expressing RFRP-3 neurons is given as a percentage of this total number of RFRP-3 neurons. Also, the number of RFRP-3 neurons with close AVP-fiber appositions is given as a percentage of the total number of RFRP-3 neurons.

The density of AVP-ir fibers was quantified in selected regions of interest (ROI) bilaterally in the DMH defined by neuroanatomical landmarks (such as the median eminence, the tuberoinfundibular sulcus and the pituitary stalk), the DMH boundaries and the anatomical position of the RFRP-3 neurons. The density of AVP-fibers in the DMH was estimated by counting manually the number of points that a fiber crossed the intersections of a grid applied on the ROIs. For each mouse AVP-ir fiber density is given as total number of crossing points/total grid number of the ROIs.

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