Endocannabinoids regulate microglia in developing brain

内源性大麻素（Endocannabinoids，EDCs）和先天免疫系统是两个独立和共同影响大脑发育的早期信号系统。发展的关键窗口期往往也是易受外来刺激干扰的敏感时期。我们在新生大鼠杏仁核中发现了一个独特的关键时期，在此期间，大脑的先天免疫细胞，小胶质细胞，积极吞噬和杀死新生细胞，如果不受干扰，这些细胞将继续成为星形胶质细胞。通过这种机制，杏仁核中星形胶质细胞的未来密度被确定。最值得注意的是，男性的最佳密度比女性低，因为它是导致青少年社交游戏时期数周后神经元活动增加的原因。同样值得注意的是，男性杏仁核中小胶质细胞吞噬活性的增加是EDC张力较高的直接结果，这反过来又是新生男性雄激素升高的发育程序。在关键期内，雄激素水平的变化，或者更重要的是EDC的变化，包括暴露于THC，永久改变了青春期的神经元/星形胶质细胞群体和玩耍性。由于合法化、非刑事化和医疗化，怀孕和哺乳期间使用避孕药的情况普遍存在，而且越来越多，但我们在很大程度上不知道对胎儿和新生儿大脑的潜在后果。转录组学和腺相关病毒（AAV）技术的进步为大鼠提供了以前无法获得的探索和精确性的新工具。一个主要目标是深入了解内源性大麻素诱导的小胶质细胞吞噬星形胶质细胞祖细胞的机制，以前所未有的特异性阐明免疫，神经元，星形胶质细胞和祖细胞之间的相互作用。另一个目标是建立一个更大的框架来理解THC暴露的发展，该框架集中在调节性型社会行为的大脑区域中的抑制性神经元上。我们将通过以下具体目标实现这两个目标。目标1将充分表征内源性大麻素和先天免疫系统的发展杏仁核的高精度细胞表型，吞噬作用所需的补体蛋白的来源的鉴定，和转录组学，以确定基因介导的细胞存活。在目标2中，我们将一次选择性地减少一种细胞类型中的雄激素受体，以确定内源性大麻素音调的性别差异的来源，目标3将询问THC，EDCs和雄激素作用的影响之间的关系，重点是跨大脑区域的抑制性神经元。结合起来，这些研究将提供前所未有的清晰度，在细胞参与者和机制中建立社会回路中的性别差异，以及该过程如何由于发育THC暴露而出错。

Endocannabinoids (EDCs) and the innate immune system are two early signaling systems which independently and together profoundly influence brain development. Critical windows of development are also often sensitive periods for disruption by exogenous stimuli. We have discovered a unique critical period in the neonatal amygdala of the rat during which the innate immune cells of the brain, microglia, actively engulf and kill newborn cells that if left unmolested would have gone on to become astrocytes. Through this mechanism the future density of astrocytes in the amygdala is determined. Most remarkably, the optimal density is lower for males than females in that it is causally responsible for increased neuronal activity many weeks later during epochs of adolescent social play. Equally remarkable, the increased phagocytic activity of microglia in the male amygdala is a direct consequence of a higher EDC tone, which is in turn developmentally programmed by elevated androgens in neonatal males. Changes to either androgen levels or, more importantly, EDC tone, including by exposure to THC, during the critical period permanently alters the neuronal/astrocytic population and playfulness during adolescence. Marijuana use during pregnancy and breastfeeding is prevalent and increasing due to legalization, decriminalization and medicalization but we are largely ignorant of the potential consequences to the fetal and newborn brain. Advances in transcriptomics and adeno-associated virus (AAV) techniques provide new tools for exploration and precision not previously accessible for the rat. A primary goal is a deep mechanistic understanding of endocannabinoid-induced microglia phagocytosis of astrocytic progenitors to illuminate with unprecedented specificity the interaction between immune, neuronal, astrocytic and progenitor cells. An additional goal is to establish a larger framework for understanding development THC exposure that converges on inhibitory neurons in brain regions regulating sex-typic social behaviors. We will achieve these dual ends via the following Specific Aims. Aim 1 will fully characterize the endocannabinoid and innate immune systems of the developing amygdala by high precision cell phenotyping, identification of the source of complement proteins needed for phagocytosis, and transcriptomics to identify genes mediating cell survival. In Aim 2 we will selectively reduce androgen receptor in one cell type at a time to determine the source of the sex difference in endocannabinoid tone and Aim 3 will interrogate the relationship between the effects of THC, EDCs and androgen action with an emphasis on inhibitory neurons across brain regions. Combined, these studies will provide unprecedented clarity in the cellular participants and mechanisms establishing a sex difference in a social circuit and how that process can go awry as a consequence of developmental THC exposure.