Development of neuronal subtypes and local circuits in the hippocampus

项目总结/摘要 在大脑发育过程中，神经元必须适当地分化成不同的亚型， 电路.因此，这一过程的中断会影响神经结构和布线，并导致疾病 例如自闭症、精神分裂症和癫痫。海马体是一种对学习至关重要的大脑结构， 记忆力和它的功能在这些疾病中受到损害。CA 1区的兴奋性锥体细胞提供了一个 海马计算向其他大脑区域的主要输出。这些单元格可以根据其 在CA 1内的物理位置为“深”或“浅”。海马深、浅锥体细胞 不同类别的神经元表现出不同的分子特征，电生理特性， 传入输入的来源，以及与局部抑制性中间神经元的回路连接。确定 它们分化的机制对于理解海马的发育和功能至关重要 无论是健康还是疾病。CA 1区的浅层锥体细胞优先表达转录因子 Satb 2，通过修饰染色质结构控制基因表达。在人类中， satb 2导致发育迟缓、智力残疾、癫痫和自闭症行为。我们的初步数据 表明在小鼠早期发育过程中敲除Satb 2会破坏表浅的 CA 1区锥体细胞。此外，对于细胞的迁移和存活，存在非细胞自主的变化。 与对照组相比，突变小鼠的中间神经元有不同的亚型。在本建议中，三个具体的 本研究旨在验证Satb 2的早期表达是海马锥体细胞发育所必需的假说， 分化和电路的发展，而后来的表达是必要的，以促进经验- 突触可塑性这些实验将在小鼠中使用分子遗传工具， 在早期和晚期发育阶段从锥体细胞中敲除Satb 2。目标1将使用 电生理学和电刺激来研究不同来源的传入的强度和可塑性 在急性切片中输入到深和浅CA 1锥体细胞。这一目标将检验这一假设， satb 2表达是必要的，以建立传入输入强度的差异，而后来的表达， 这些输入的活动驱动的突触可塑性所必需的。AIM 2将使用成对的全细胞记录 锥体细胞（深层和浅层）和已识别的中间神经元亚型之间的映射电路， 研究它们的突触生理学细节。这一目的将检验Satb 2早期表达是一种基因治疗的假设。 在局部抑制性中间神经元和浅层锥体细胞之间建立回路基序是必要的， 随后的表达对于响应环境富集而募集新的抑制性突触是必需的。目的 3将使用单细胞RNA-seq和ATAC-seq来确定Satb 2敲除如何改变基因表达， 在多个发育时间点的CA 1染色质可及性。这一目标将提供分子洞察力， Satb 2如何通过发育控制CA 1中的基因表达，以及其功能如何随时间变化。

PROJECT SUMMARY/ABSTRACT During brain development, neurons must properly differentiate into distinct subtypes to assemble healthy circuits. Thus, disruption of this process can impact neural architecture and wiring, and contribute to disorders such as autism, schizophrenia, and epilepsy. The hippocampus is a brain structure crucial for learning and memory, and its function is compromised in these disorders. Excitatory pyramidal cells in area CA1 provide a major output of hippocampal computations to other brain regions. These cells can be parsed based on their physical position within CA1 as “deep” or “superficial.” Deep and superficial hippocampal pyramidal cells are distinct classes of neurons that exhibit differential molecular signatures, electrophysiological properties, sources of afferent input, and circuit connectivity with local inhibitory interneurons. Determining the mechanisms underlying their differentiation is crucial for understanding hippocampal development and function in both health and disease. Superficial pyramidal cells in CA1 preferentially express the transcriptional regulator Satb2, which controls gene expression by modifying chromatin structure. In humans, mutations of Satb2 cause developmental delay, intellectual disability, epilepsy, and autistic behaviors. Our preliminary data show that knocking out Satb2 during early development in mice disrupts the differentiation of superficial pyramidal cells in CA1. Furthermore, there are non-cell-autonomous changes to the migration and survival of distinct subtypes of interneurons in mutant mice relative to controls. In the present proposal, three specific aims will test the hypothesis that early expression of Satb2 is necessary for hippocampal pyramidal cell differentiation and circuit development in CA1, while later expression is necessary to promote experience- dependent synaptic plasticity. These experiments will use molecular genetic tools in mice to conditionally knock out Satb2 from pyramidal cells during both early and late developmental stages. Aim 1 will use electrophysiology and electrical stimulation to study the strength and plasticity of different sources of afferent input to deep and superficial CA1 pyramidal cells in acute slices. This aim will test the hypothesis that early Satb2 expression is necessary to establish differences in afferent input strength, while later expression is necessary for activity-driven synaptic plasticity of these inputs. Aim 2 will use paired whole-cell recordings between pyramidal cells (deep and superficial) and identified subtypes of interneurons to map circuits and study details of their synaptic physiology. This aim will test the hypothesis that early Satb2 expression is necessary to establish circuit motifs between local inhibitory interneurons and superficial pyramidal cells, while later expression is necessary to recruit new inhibitory synapses in response to environmental enrichment. Aim 3 will use single-cell RNA-seq and ATAC-seq to determine how Satb2 knockout alters gene expression and chromatin accessibility in CA1 at multiple developmental timepoints. This aim will provide molecular insight into how Satb2 controls gene expression in CA1 through development, and how its function may change over time.