Endocannabinoids regulate microglia in developing brain

内源性大麻素(EDCs)和先天免疫系统是两个早期的信号系统，它们独立而共同地深刻地影响着大脑的发育。发展的关键时期也往往是受到外部刺激干扰的敏感期。我们在大鼠新生的杏仁核中发现了一个独特的关键时期，在此期间，大脑的固有免疫细胞-小胶质细胞-活跃地吞噬并杀死新生的细胞，如果不受干扰，这些细胞将继续成为星形胶质细胞。通过这一机制，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，男性的最佳密度低于女性，因为它是青少年社交游戏时代几周后神经元活动增加的因果关系。同样值得注意的是，雄性杏仁核小胶质细胞吞噬活性的增加是EDC张力升高的直接结果，而EDC张力的升高反过来又是新生儿雄性雄激素升高的发育编程。在关键时期，雄激素水平或更重要的是EDC音调的变化，包括暴露于THC，都会永久性地改变青春期神经元/星形胶质细胞的数量和玩耍。由于合法化、非刑事化和医学化，怀孕和哺乳期间使用大麻的现象很普遍，而且还在增加，但我们在很大程度上忽视了对胎儿和新生儿大脑的潜在后果。转录学和腺相关病毒(AAV)技术的进步为探索和精确提供了新的工具，这是以前大鼠无法获得的。一个主要的目标是深入了解内源性大麻素诱导星形胶质细胞前体细胞吞噬小胶质细胞的机制，以前所未有的特异性阐明免疫细胞、神经元细胞、星形胶质细胞和星形胶质细胞之间的相互作用。另一个目标是建立一个更大的框架，用于理解发育过程中的暴露，集中在大脑区域调节性社交行为的抑制性神经元上。我们将通过以下具体目标实现这两个目标。目的1通过高精度的细胞表型鉴定、吞噬所需的补体蛋白来源的鉴定和鉴定细胞存活调节基因的转录，全面描述发育中的杏仁核的内源性大麻素和先天免疫系统。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张力性别差异的来源，目标3将询问THC、EDCs和雄激素作用之间的关系，重点放在大脑区域的抑制神经元上。总而言之，这些研究将为细胞参与者和机制提供前所未有的澄清，以建立社会关系中的性别差异，以及这一过程如何可能因发育的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.