Hormones heavily influence feeding behavior and body weight. Estrogen depletion in post-menopause women leads to high risk of obesity, while estrogen replacement therapy suppresses body weight. While previous studies have started to reveal the neural substrate of feeding behavior control, how they are modulated by estrogen action is not well understood. The paraventricular hypothalamus (PVH) is known to control satiety in animals. Thus, in this study, we hypothesized that neurons responding to estrogen in the PVH is related to hormone-dependent modulation of feeding. To evaluate the causal relationship between the activity of PVH estrogen responsive cells and feeding behavior, we virally targeted channel rhodopsin (ChR2) to PVH expressing estrogen receptor beta (Esr2) using Esr2-Cre transgenic mice and Cre-dependent virus. The virus delivers ChR2 mRNA nonspecifically to cells in the PVH. In Esr2-Cre mice, only cells expressing Esr2 will also express the Cre recombinase enzyme to allow successful expression of the virally delivered ChR2. ChR2 is activated by light delivered into the PVH via fiber optics and the feeding behavior of the animal is observed and quantified under on or off lighting conditions. Preliminary results show that the light stimulation of ChR2 expressing neurons decreased feeding behavior, suggesting that the activation of PVHEsr2+ cells are sufficient to suppress feeding. Further studies to reveal the neural basis of hormone-dependent regulation of satiety can shed light on eating disorders in humans, potentially leading to the discovery of new drug targets to treat these disorders.

In opioid addiction, withdrawal presents a major obstacle to recovery after the development of dependence. Microglia, immune cells of the central nervous system, have been shown to play a role in mediating symptoms of opioid withdrawal. When activated, microglia undergo morphological changes and release inflammatory factors which can contribute to hyperalgesia and other effects associated with withdrawal. The purinergic Gi-protein coupled receptor P2Y12 mediates microglial activation. In this study, we will inhibit P2RY12 in the microglia of fentanyl-conditioned mice using the irreversible antagonist clopidogrel prior to inducing withdrawal. We will assess the effects of P2RY12 inhibition on hyperalgesia associated with opioid withdrawal using the tail-flick assay. Additionally, we will use immunohistochemistry to analyze the morphological effects of withdrawal on microglia in clopidogrel-treated and untreated mice. The results of this study will allow us to study the role of microglia in producing the physiological symptoms of opioid withdrawal, and assess the viability of inhibiting this pathway to attenuate opioid withdrawal. Our findings could potentially aid the search for new drugs to assist in the treatment of opioid addiction by treating withdrawal.

Dandy-Walker Malformation (DWM) is a common malformation of the cerebellum in humans. While it can be diagnosed by prenatal magnetic resonance imaging (MRI) using radiological criteria, almost nothing is known about the developmental mechanisms which elicit Dandy-Walker Malformation. Therefore, we sought to use a wide spectrum of techniques to understand the underlying cause of this malformation. We obtained 27 cases diagnosed as either DWM or Cerebellar Vermis Hypoplasia (CVH), another common cerebellar malformation. Using a combination of radiological, histopathological, and transcriptomic analyses we were able to determine the developmental pathogenesis of DWM. Our analysis included atypical measurements of different aspects of the cerebellar development, including growth and foliation of the cerebellar midline called vermis, proliferation in various germinal zones, and morphobiometric changes. Furthermore, we found that an important stem cell zone called the rhombic lip, which produces nearly 80% all neurons of the adult human brain, is specifically disrupted in DWM. Finally, transcriptomic analysis revealed specific genes related to normal growth and cell division were downregulated in the DWM cerebellum. Using this information, we propose a model for how DWM arises during development, and hypothesize that a disruption to the germinal rhombic lip by vascular insults causes a reduction in cell proliferation, resulting in the characteristic presentation of DWM that can be seen on MRI. We acknowledge that although mouse models have provided us many clues on how the cerebellum develops, they have many limitations since they lack critical germinal zones present during human development. Our study underlines the urgency for further human-based analysis of brain birth defects.

Chemokines are important regulators of neuroinflammation and interact with brain endothelial cells that comprise the blood-brain barrier (BBB) to modulate their function and promote their interactions with leukocytes. Chemokines can also cross the intact BBB, which may contribute to their functions in the brain, but interactions of chemokines with the BBB in vivo are understudied. Here, we show that the chemokines C-C motif ligand 2 (CCL2) and C-C motif ligand 5 (CCL5), which have been well-studied in context of their neuroinflammatory functions, are amenable to labeling with iodine-125. This affords highly sensitive and quantitative detection of CCL2 and CCL5 in vivo, and allows for rapid assessment of the distribution of circulating chemokines. Multiple-time regression analysis was used to characterize chemokine transport across the mouse BBB. In untreated mice, we evaluated the brain uptake of CCL2 and CCL5 and characterized saturability of uptake and endothelial binding vs. transport. We then determined whether CCL2 and CCL5 interactions with the BBB are altered in mice treated with three doses of 3mg/kg lipopolysaccharide (LPS). Our results indicate that in addition to endothelial binding at the glycocalyx, there is uptake of both CCL5 and CCL2 into the mouse brain parenchyma. We found no significant difference in the rate of chemokine uptake in mice treated with LPS versus untreated mice. However, the iodine-125 counts per minute (CPM) was significantly higher in the brains of LPS mice versus untreated mice brains, indicating that more radio-labelled chemokine had entered the brain after LPS-induced inflammation. In conclusion, we have found that chemokines interact with the intact BBB, and alterations in these interactions with inflammation can be detected in vivo. As a future direction, this method can be utilized to evaluate pharmacological approaches to inhibiting BBB/chemokine interactions, which may protect against harmful neuroinflammation during neurological disease.

Ischemic preconditioning (IPC) is an experimental phenomenon in which a brief ischemic event confers neuronal and axonal protection against subsequent ischemic exposure. The cell types responsible for IPC in the brain are unknown. In a novel model of white matter (WM) IPC and ischemic injury, we identified specific innate immune signaling pathways in microglia as required for IPC-mediated axonal protection, leading us to suspect that microglia are required for IPC. The model of WM IPC involves exposing the mouse optic nerve (MON) to a brief ischemic event 72 hours before the MONs are isolated and exposed to oxygen-glucose deprivation. Animals were treated with PLX5622 - a colony stimulating factor-1 receptor (CSF1R) pharmacologic antagonist – to deplete microglia in the central nervous system, including the MON, to test our hypothesis. By recording axonal function, we determined that microglial depletion eliminated IPC-mediated axonal protection in the WM. This study examines the impacts of IPC and ischemia on the nodes of Ranvier, which we hypothesize are protected by preconditioned microglia. The length of the nodes of Ranvier affects conductance and action potential propagation through an axon, with recent publications suggesting elongation of the notes in disease or injury states. We hypothesize the nodes of Ranvier will be shorter in preconditioned MONs than non-preconditioned MONs, which would support IPC-mediated protection. Additionally, we expect the nodes of Ranvier in PLX5622-treated MONS will be of similar length as non-preconditioned control MONs, indicating loss of IPC-mediated protection in animals without microglia. We will use immunofluorescence and confocal microscopy to measure the nodes of Ranvier and correlate these anatomical findings to prior electrophysiology experiments. The results of this research would contextualize a novel understanding of how microglia and ischemia affect the WM in specific regions of the axon, which is vital for advancing the development of neurotherapeutics for stroke.

The rhombic lip is a dorsal germinal zone that produces nearly 80% of all neurons in the human brain and assists in the production of all glutamatergic neurons in the cerebellum. In the mouse which has traditionally been used as a model organism, the rhombic lip is a transient structure and a dorsal stem cell zone that appears between embryonic days 12.5 and 17.5. While the mouse rhombic lip is molecularly compartmentalized, it lacks structural compartmentalization. The human rhombic lip on the other hand is long-lived and seen throughout gestation. It also exhibits substructure and is compartmentalized into a ventricular and subventricular zone. The extended duration of rhombic lip is a unique feature of human development and not seen in other model organisms including non-human primates like the macaque. This indicates that model organisms have limitations as they cannot fully recapitulate human development. Histological and immunohistochemical analyses of over 27 Dandy Walker Malformation (DWM) and Cerebellar Vermis Hypoplasia (CVH) samples suggest that rhombic lip disruption leads to cerebellar hypoplasia that disproportionately affects the posterior vermis. DWM and CVH are birth defects of the cerebellum that are diagnosed using ultrasound and MRI. Our histopathological analysis suggests that reduced proliferation and self-renewal of the rhombic lip progenitor pool leads to underdevelopment of the posterior cerebellum. These findings add to the understanding of the developmental pathogenesis of DWM, and underlines the need for further research of the human rhombic lip which can further improve our understanding of cerebellar brain defects.