Thesis Seminars

Gregory Reilly, MS - PhD Candidate, Neuroscience Graduate Program

Biological sex is a fundamental dimension of internal state that can have deep influences on behavior. Understanding the mechanisms behind these influences can provide insight into how shared neural circuits are tuned to produce sex-specific behavioral variation. Biological sex can influence both short-term behaviors and longer, more persistent forms of behavior known as behavioral states. In C. elegans, persistent motor behavior, called locomotor states, is well-studied in hermaphrodites. On a patch of food, hermaphrodites will switch between two states of foraging and feeding, called roaming and dwelling respectively. However, while some work has examined motor states in males, these remain poorly characterized. Previous work from our lab has demonstrated that male locomotion is sex-specific; the sexual state of muscle tissue and the nervous system is essential for sex differences in speed and body posture. Therefore, biological sex may also similarly influence locomotor states. We trained a supervised machine learning Random Forest model to detect three locomotor states: roaming, dwelling, and tail chase. In addition, we used a dimensionality reduction analysis, Linear Discrimination Analysis (LDA), to compare the overall characteristics of these states. Furthermore, to measure the transition probability between states, we used a Markov model. While both males and hermaphrodites share the locomotor states of roaming and dwelling, the characteristics of these differ by sex- the amount of time spent in each state, state durations, and transitions between states (temporal differences), as well as the linear speed, curvature, and other characteristics (feature differences), have sexual dimorphism. To understand how sex tunes these locomotor states, we manipulated the sex determination pathway to sex reverse the nervous system in both males and hermaphrodites. Interestingly, we found that pan-neuronally feminized males had similar locomotor state characteristics to hermaphrodites; both temporal and state feature sex differences were eliminated in the feminized males. Yet, masculinized hermaphrodites were indistinguishable from their wildtype counterparts indicating that either male-specific neurons or other tissue played a role in mediating these sex differences. To uncover the mechanisms that biological sex leverages to achieve this sex-specific variance in locomotor states, various neuromodulator knockout mutants known to affect locomotor states were tested. PDFR-1 emerged as a strong candidate as it removed differences in both temporal and features of locomotor states. Preliminary data suggests that PDFR-1 signaling may regulate the temporal sex differences through daf-7, a TGF-ß signal. daf-7 knockout mutants appear to maintain differences in state features but both spend similar amounts of time roaming and dwelling. Given that PDFR-1 signaling has been implicated as the mechanism that regulates sexual dimorphism in daf-7 expression in the ASJ neuron, these results remain promising. Together, our results provide a mechanistic framework for understanding how sex-specific neuronal tuning influences behavioral states.

 Jun 14, 2024 @ 10:00 a.m.

 Medical Center | SMD Lg. Aud. (2-6424)

Host: Advisor: Doug Portman, PhD

Mark Stoessel, MS - PhD Candidate, Neuroscience Graduate Program

Synaptic plasticity allows the central nervous system to incorporate new sensory experiences and information, and its disruption is associated with many neurological and psychiatric disorders. Much recent work has focused on the contribution of non-neuronal central nervous system cells, especially microglia, to synaptic plasticity. Though classically defined by their immune capacities, microglia are vital to many homeostatic processes, including synaptic plasticity of nascent and adult neuronal networks. Despite the emerging consensus that microglial dynamics are critical to brain function during physiological as well as pathological conditions, it is unclear whether these microglial roles and their underlying mechanisms are universal or differ between brain regions. There is a growing body evidence to suggest microglia exhibit a high degree of regional specialization. Cerebellar microglia in particular exhibit unique transcriptional and epigenetic profiles, and distinct functional properties, such as being morphologically less ramified, and less densely distributed than cortical microglia. As a consequence, cerebellar microglia survey less of the parenchyma than cortical microglia but compensate for this by undergoing frequent somatic translocations under homeostatic conditions, a phenomenon not observed in cortex. Despite such differences, cerebellar microglia maintain common microglial functions. Two pathways of interest to cortical microglial mediated synaptic plasticity are purinergic signaling through the P2Y12 receptor and noradrenergic signaling through the β2 adrenergic receptor (β2-AR), both of which have been shown to be critically involved in microglial roles in synaptic remodeling and rapid chemotaxis to sites of injury.

To address this question of regional heterogeneity in microglial signaling we investigated the roles of P2Y12 and β2-AR in cerebellar microglial with a comparison to the known roles of these signaling pathways in cerebral cortex. We desired to understand the contribution of these pathways to the many aspects of microglial function in the adult brain and therefore characterized cerebellar microglial morphology, surveillance, injury response dynamics, gene expression patterns, and contributions to cerebellar learning and plasticity, while manipulating either microglial purinergic or adrenergic signaling. On the whole, our findings suggest that signaling pathways that are present in both cortical and cerebellar microglia may play differential roles in microglial function depending on brain area.

 Jun 12, 2024 @ 1:00 p.m.

 Medical Center | K-207 (2-6408)

Host: Advisor: Ania Majewska, PhD

MaKenna Cealie - PhD Candidate, Neuroscience Graduate Program

Fetal alcohol spectrum disorders (FASD), caused by prenatal alcohol exposure, are the most common cause of non-heritable, preventable mental disability and have no known cure. Physical, cognitive, and behavioral deficits have been reported in FASD, including impairments related to the cerebellum. To elucidate the mechanisms of FASD, we examined microglia, the immune cells of the central nervous system, as well as Purkinje cells, the sole output of the cerebellar cortex, which are both impacted by developmental ethanol exposure. Microglia are dynamic cells and shape neuronal circuit development and connectivity in the cerebellum. However, how cerebellar microglia dynamics and their interactions with neurons are affected by early life exposure to ethanol is unknown. We explored the impact of a third-trimester equivalent binge-level ethanol exposure on cerebellar microglia and microglia-Purkinje cell interactions in adolescent and young adult mice.

We subcutaneously injected Ai9+/-/C3xcr1G/+/L7cre mice with 5.0 g/kg/day of either ethanol or saline from postnatal (P) days 4-9. Mice were then aged to adolescence (P28) and cranial windows were implanted above the cerebellum to allow for two-photon in vivo imaging in both adolescence and young adulthood (P60). We found that in vivo cerebellar microglia dynamics, microglia morphology, and microglia-Purkinje cell interactions were largely unaffected by developmental ethanol exposure in both adolescence and young adulthood. We also examined if a “second-hit” laser ablation injury in young adulthood would uncover differences, but found no changes in cerebellar microglia injury response between ethanol- and saline-dosed animals. We collected the young adults’ brains for confocal imaging to examine a larger number of microglia and Purkinje cells. Microglia density, morphology, and interactions with Purkinje cells were largely unaltered by developmental ethanol exposure. However, Purkinje cell linear frequency was significantly decreased in ethanol-dosed mice.

Overall, we found that cerebellar microglia in adolescent and young adult mice were largely unaffected by developmental ethanol exposure, but Purkinje cells appeared to be more susceptible to its effects. Our work suggests that microglia may return to homeostasis later in life after an early life insult. This work is important to narrow down the mechanisms leading to FASD so future therapies can be developed.

 May 13, 2024 @ 11:00 a.m.

 Medical Center | Lower Adolph Aud. (1-7619)

Host: Advisor: Ania Majewska, PhD

Cody McKee - PhD Candidate, Neuroscience Graduate Program

Protein Phosphatase 1 (PP1) is a major Serine (Ser)/Threonine (Thr) phosphatase responsible for more than half of all Ser/Thr dephosphorylation events in eukaryotic cells. Three genes encode the three major isoforms of PP1 (α, β, and γ). While PP1α and PP1γ are considered major players in synaptic physiology, the neuronal function of PP1β is unknown. Recently, de novo mutations in PP1β have been linked to intellectual developmental disabilities in children, suggesting a critical role for PP1β in the central nervous system. While correlations between PP1 and various other neurodevelopmental/neurodegenerative diseases have been suggested, a causative role for PP1 in many of these contexts has yet to be established. The current study seeks to investigate the neuronal role of PP1β in vivo, and to uncover potential mechanisms by which PP1β influences neuronal function.

A Thy1-Cre mouse line was used to generate neuron specific PP1β conditional KO (PP1β cKO) mice. These mice exhibit a failure to thrive and typically die by 2-3 postnatal weeks. Hippocampal slice recordings demonstrated increased paired-pulse facilitation, suggesting impaired neurotransmitter release. In agreement with studies suggesting activity influences myelination within specific brain regions, we found significantly lower levels of myelin basic protein in the cortex of PP1β cKO mice. Furthermore, to assess the influence of PP1β on myelin function in a predominately activity-independent context, we measured compound action potentials (CAPs) along the optic nerve. Deficits in CAP recordings suggested impaired optic nerve myelination. However, analysis of the electron micrographs failed to detect a significant difference in myelinated axons. Using immunofluorescence, we then uncovered significantly fewer nodes of Ranvier in PP1β cKO mice that could potentially explain the CAP recordings. This deficit in nodes coincided with an increase in phosphorylation of PP1β-specific substrate, myosin light chain, which localizes to nodes of Ranvier. These data suggest a potential role for PP1β in nodal structure that could influence action potential propagation.

To then study the role of PP1β in adolescent mice, we generated a neuron specific inducible PP1β KO mouse line (iKO). These iKO mice exhibit progressive deterioration of hind limb functionality and premature demise at ~4 weeks post recombination. We then uncovered significant changes in various respiratory parameters suggesting a potential mechanism to explain the premature demise. However, while no morphological changes were observed within neuromuscular junctions in the diaphragm, it is possible that neurotransmitter release at these synapses is abrogated, and this will be investigated in the future.

These data support the hypothesis that PP1β alters action potential propagation in a way that disrupts downstream functionality. These results shed light on the role of PP1β and potential mechanisms that could be disrupted by PP1β in pathological states. Future studies will seek to uncover the molecular substrates underlying these effects and provide potential therapeutic targets for diseases in which PP1β functionality may be altered.

 May 10, 2024 @ 2:00 p.m.

 Medical Center | K-207 (2-6408)

Host: Advisor: Houhui (Hugh) Xia