Biology subjects

Potter, G. J.

Publications and source records attributed to Potter, G. J..

3 recordsLinked to original sources

bioRxiv2025

A validated method for banking mixed glial cultures that yield responsive microglia

Microglia, the primary immune cells of the brain, orchestrate immune responses to both external and internal stimuli in health and disease. Although several cell culture methods exist to model microglia in vitro, isolating loosely adherent microglia from primary murine mixed glial cultures remains valuable for recapitulating in vivo cell states from complex transgenic lines at relatively low cost. However, these methods are constrained by limited temporal control and scalability. To address these challenges, we established a protocol for generating microglia from frozen mixed glial cultures. While freezing introduced measurable differences in microglial morphology and lipid droplet content, microglia derived from frozen cultures responded to environmental stimuli (high glucose and LPS) in the same way as those from fresh cultures. Thus, while frozen primary microglia exhibit similar functional responses, maintaining consistency in using fresh or frozen cells within a given experiment is strongly recommended. MotivationAlthough our understanding of microglias role in brain development and disease continues to grow, a standardized in vitro model of primary mouse microglia remains absent in the field. Primary cell culture systems are essential for studying microglia, enabling the empirical assessment of gene function in transgenic models and testing novel interventions before committing to time- and cost-intensive in vivo studies. However, mixed glial culture-derived primary microglia (pMG) systems are constrained by the timing of animal availability and have limited scalability. In this study, we sought to establish a protocol for freezing and subsequently reviving primary mixed glial cultures from which responsive pMG could be isolated for downstream analysis. We validated this approach by quantifying microglia responses to distinct environmental stimuli before and after a freeze-thaw cycle. Research topicsCP: Cell biology Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/693524v2_ufig1.gif" ALT="Figure 1000"> View larger version (54K): org.highwire.dtl.DTLVardef@1b0e291org.highwire.dtl.DTLVardef@1eacfb2org.highwire.dtl.DTLVardef@e9c18aorg.highwire.dtl.DTLVardef@a151a5_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

Altered Metabolism and DAM-signatures in Female Brains and Microglia with Aging

Despite Alzheimers disease (AD) disproportionately affecting women, the mechanisms remain elusive. In AD, microglia undergo metabolic reprogramming, which contributes to microglial dysfunction and AD pathology. However, how sex and age contribute to metabolic reprogramming in microglia is understudied. Here, we use metabolic imaging, transcriptomics, and metabolic assays to probe age-and sex-associated changes in brain and microglial metabolism. Glycolytic and oxidative metabolism in the whole brain was determined using Fluorescence Lifetime Imaging Microscopy (FLIM). Young female brains appeared less glycolytic than male brains, but with aging, the female brain became male-like. Transcriptomic analysis revealed increased expression of disease-associated microglia (DAM) genes (e.g., ApoE, Trem2, LPL), and genes involved in glycolysis and oxidative metabolism in microglia from aged females compared to males. To determine whether estrogen can alter the expression of these genes, BV-2 microglia-like cell lines, which abundantly express DAM genes, were supplemented with 17{beta}-estradiol (E2). E2 supplementation resulted in reduced expression of DAM genes, reduced lipid and cholesterol transport, and substrate-dependent changes in glycolysis and oxidative metabolism. Consistent with the notion that E2 may suppress DAM-associated factors, LPL activity was elevated in the brains of aged female mice. Similarly, DAM gene and protein expression was higher in monocyte-derived microglia-like (MDMi) cells derived from middle-aged females compared to age-matched males and was responsive to E2 supplementation. FLIM analysis of MDMi from young and middle-aged females revealed reduced oxidative metabolism and FAD+ with age. Overall, our findings show that altered metabolism defines age-associated changes in female microglia and suggest that estrogen may inhibit the expression and activity of DAM-associated factors, which may contribute to increased AD risk, especially in post-menopausal women.

neuroscience↗

bioRxiv2023

Prior experience with behavioral control over stress facilitates social dominance

Dominance status has extensive effects on physical and mental health, and an individuals relative position can be shaped by experiential factors. A variety of considerations suggest that the experience of behavioral control over stressors should produce winning in dominance tests and that winning should blunt the impact of later stressors, as does prior control. To investigate the interplay between competitive success and stressor control, we first examined the impact of stressor controllability on subsequent performance in a warm spot competition test modified for rats. Prior experience of controllable, but not physically identical uncontrollable, stress increased later effortful behavior and occupation of the warm spot. Controllable stress subjects consistently ranked higher than did uncontrollable stress subjects. Pharmacological inactivation of the prelimbic (PL) cortex during behavioral control prevented later facilitation of dominance. Next, we explored whether repeated winning experiences produced later resistance against the typical sequelae of uncontrollable stress. To establish dominance status, triads of rats were given five sessions of warm spot competition. Reversible inactivation of the PL or NMDA receptor blockade in the dorsomedial striatum led to a long-term reduction in social rank. Stable dominance blunted the later stress-induced increase in dorsal raphe nucleus serotonergic activity, as well as prevented stress-induced social avoidance. In contrast, endocrine and neuroimmune responses to uncontrollable stress were unaffected, indicating a selective impact of prior dominance. Together, these data demonstrate that instrumental control over stress promotes later dominance, but also reveal that winning experiences buffer against the neural and behavioral outcomes of future adversity.

neuroscience↗