Introduction: The preoptic area of the hypothalamus (POA) is a functionally heterogeneous region known to contribute primarily to thermoregulation and energy expenditure. Studies have suggested that cells in the POA that regulate response to hyperthermic environmental conditions and those that regulate food intake and energy expenditure may indeed be the same cell population.

Methods: Leptin receptor-Cre (LepRCre) knock-in mice at full maturity underwent either a warm stress challenge at 37℃ for three hours or were kept at room temperature for the same amount of time. Brains were collected immediately following temperature challenging, resulting in one successful sample from a warm-stressed mouse and two samples from room-temperature mice. The brains were fixed in paraformaldehyde and then 10% sucrose for approximately 48 hours before being frozen in optimal cutting temperature compound. The frozen brains were cryosectioned coronally at 15μm, resulting in approximately 10 slices that spanned the POA. These slices were incubated in cFos, a neuronal activation marker used here to determine temperature regulatory function, and Hoechst antibodies. Confocal microscopy (Zeiss) and subsequent image processing (Imaris, FIJI) were used to localize POA cells that were LepR and cFos positive respectively. Individual cells were located via Hoechst immunofluorescence, LepR positive cells via endogenous Cre-GFP fluorescence, and cFos positive cells via cFos immunofluorescence. Data analysis was performed on individual signal intensity amplitudes by observing cFos intensity within LepR positive cells as well as comparing calculated z-scores of cFos and LepR signal intensities in individual cells.

Results: cFos and LepR signal intensities were measured from individual cells determined via Hoechst immunofluorescence in the POA. Z-scores were found for the respective intensities. Using a Z-score cutoff of 2, all defined cells were determined to be either be cFos positive or negative and LepR positive or negative. In the room-temperature mice, using the above listed conditions, 2.0% of total cells were determined to be both cFos and LepR positive. In the warm-stressed mouse, 9.0% of total cells were both cFos and LepR positive.

Conclusion: Due to the small sample size, we cannot confidently conclude that cells in the POA that regulate response to hyperthermic environmental conditions, as represented by cFos and cells that regulate energy intake, as represented by LepR, are the same cell population. However, the preliminary data as well as existing studies indicate that there may be a correlation between the two cell populations and that more studies should be performed in order to fully characterize the relationship between the functional cell groups.

Email: nohmm@mail.uc.edu

Key Words: ophthalmology, neurology, immunofluorescence, confocal imaging