Quantification of immunolabeled RFRP-3 cells and AVP-ergic fibers
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

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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