Kisspeptin (encoded by Kiss1) is a hypothalamic neuropeptide that is directly regulated by sex steroids and directly stimulates gonadotropin-releasing hormone (GnRH) neurons. Kisspeptin cell models were established in order to facilitate future molecular analysis of kisspeptin. mHypoA-51 and mHypoA-63 cell lines were found to express kisspeptin, estrogen receptor α and β, substance P, but not tyrosine hydroxyase. Furthermore, estrogen decreased Kiss1 expression in both cell lines. Based on these results, it was concluded that mHypoA-51 and mHypoA-63 are representative of arcuate kisspeptin neurons. Accumulating evidence also indicates that kisspeptin indirectly stimulates GnRH neurons through afferent neurons. Kisspeptin receptor expression was detected in native neuropeptide Y (NPY) neurons. Using the mHypoE-38 cell line, kisspeptin was found to directly regulate NPY mRNA expression and secretion via the ERK1/2 and p38 MAPK pathways. This is the first evidence that kisspeptin directly stimulates NPY neurons to potentially exert indirect effects on GnRH neurons.

Kisspeptin (encoded by Kiss1) is a hypothalamic neuropeptide that is directly regulated by sex steroids and directly stimulates gonadotropin-releasing hormone (GnRH) neurons. Kisspeptin cell models were established in order to facilitate future molecular analysis of kisspeptin. mHypoA-51 and mHypoA-63 cell lines were found to express kisspeptin, estrogen receptor α and β, substance P, but not tyrosine hydroxyase. Furthermore, estrogen decreased Kiss1 expression in both cell lines. Based on these results, it was concluded that mHypoA-51 and mHypoA-63 are representative of arcuate kisspeptin neurons. Accumulating evidence also indicates that kisspeptin indirectly stimulates GnRH neurons through afferent neurons. Kisspeptin receptor expression was detected in native neuropeptide Y (NPY) neurons. Using the mHypoE-38 cell line, kisspeptin was found to directly regulate NPY mRNA expression and secretion via the ERK1/2 and p38 MAPK pathways. This is the first evidence that kisspeptin directly stimulates NPY neurons to potentially exert indirect effects on GnRH neurons.

This dissertation evaluated the feasibility of using commercially available immortalized cell lines in building a tissue engineered in vitro blood-brain barrier (BBB) co-culture model for preliminary drug development studies. Mouse endothelial cell line and rat astrocyte cell lines purchased from American Type Culture Collections (ATCC) were the building blocks of the co-culture model. An astrocyte derived acellular extracellular matrix (aECM) was introduced in the co-culture model to provide a novel in vitro biomimetic basement membrane for the endothelial cells to form endothelial tight junctions. Trans-endothelial electrical resistance (TEER) and solute mass transport studies were engaged to quantitatively evaluate the tight junction formation on the in-vitro BBB models. Immuno-fluorescence microscopy and Western Blot analysis were used to qualitatively verify the in vitro expression of occludin, one of the earliest discovered tight junction proteins. Experimental data from a total of 12 experiments conclusively showed that the novel BBB in vitro co-culture model with the astrocyte derived aECM (CO+aECM) was promising in terms of establishing tight junction formation represented by TEER values, transport profiles and tight junction protein expression when compared with traditional co-culture (CO) model setups and endothelial cells cultured alone. Experimental data were also found to be comparable with several existing in vitro BBB models built from various methods. In vitro colorimetric sulforhodamine B (SRB) assay revealed that the co-cultured samples with aECM resulted in less cell loss on the basal sides of the insert membranes than that from traditional co-culture samples. The novel tissue engineering approach using immortalized cell lines with the addition of aECM was proven to be a relevant alternative to the traditional BBB in vitro modeling.

Serotonin (5-HT) has been implicated in energy homeostasis. There is growing evidence that 5-HT, acting through the 5-HT1BR in the paraventricular nucleus of the hypothalamus (PVN), is important to this regulation. To investigate the cellular events underlying 5-HT1BR action, a PVN neuronal cell model was established. The mHypoA-2/30 cell line expresses a complement of markers and neuropeptides specifically localized to the PVN. 5-HT induces neuronal activation in a dose-dependent manner as determined by an elevation in cFos mRNA levels. As 5-HT exerted limited transcriptional control, the integrity of 5-HT signaling machinery was assessed. 5-HT signals through cAMP and calcium secondary messenger systems by suppressing cAMP and elevating intracellular calcium, effects that are mimicked by activating the 5-HT1BR and that are attenuated in the presence of inhibitors. These findings support the use of this novel PVN cell model for delineating components involved in direct 5-HT action in PVN neurons.

Serotonin (5-HT) has been implicated in energy homeostasis. There is growing evidence that 5-HT, acting through the 5-HT1BR in the paraventricular nucleus of the hypothalamus (PVN), is important to this regulation. To investigate the cellular events underlying 5-HT1BR action, a PVN neuronal cell model was established. The mHypoA-2/30 cell line expresses a complement of markers and neuropeptides specifically localized to the PVN. 5-HT induces neuronal activation in a dose-dependent manner as determined by an elevation in cFos mRNA levels. As 5-HT exerted limited transcriptional control, the integrity of 5-HT signaling machinery was assessed. 5-HT signals through cAMP and calcium secondary messenger systems by suppressing cAMP and elevating intracellular calcium, effects that are mimicked by activating the 5-HT1BR and that are attenuated in the presence of inhibitors. These findings support the use of this novel PVN cell model for delineating components involved in direct 5-HT action in PVN neurons.