Development of neuronal subtypes and local circuits in the hippocampus

项目概要/摘要 在大脑发育过程中，神经元必须正确分化成不同的亚型才能组装健康的细胞 电路。因此，这个过程的破坏会影响神经结构和接线，并导致疾病 例如自闭症、精神分裂症和癫痫症。海马体是对学习和学习至关重要的大脑结构 记忆及其功能在这些疾病中受到损害。 CA1 区的兴奋性锥体细胞提供 海马计算的主要输出到其他大脑区域。这些单元格可以根据它们的 CA1 内的物理位置为“深层”或“浅层”。海马深部和浅部锥体细胞是 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电路连接。确定 其分化背后的机制对于理解海马的发育和功能至关重要 在健康和疾病方面。 CA1 中的浅表锥体细胞优先表达转录 调节因子 Satb2，通过修饰染色质结构来控制基因表达。在人类中，突变 Satb2 会导致发育迟缓、智力障碍、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 Satb2 会破坏浅表细胞的分化 CA1 中的锥体细胞。此外，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。在本提案中，三项具体内容 目的将检验 Satb2 的早期表达对于海马锥体细胞是必需的这一假设 CA1 中的分化和电路发育，而后期表达对于促进体验是必要的- 依赖的突触可塑性。这些实验将在小鼠身上使用分子遗传工具来有条件地 在早期和晚期发育阶段从锥体细胞中敲除 Satb2。目标 1 将使用 电生理学和电刺激研究不同传入源的强度和可塑性 输入到急性切片中深层和浅层 CA1 锥体细胞。这个目标将检验早期的假设 Satb2 表达对于建立传入输入强度的差异是必要的，而后来的表达是 这些输入的活动驱动的突触可塑性是必需的。目标 2 将使用配对的全细胞记录 锥体细胞（深层和浅层）之间和已识别的中间神经元亚型之间，以绘制电路图和 研究其突触生理学的细节。该目标将检验早期 Satb2 表达是的假设 对于在局部抑制性中间神经元和浅表锥体细胞之间建立电路基序是必要的，同时 后期的表达对于招募新的抑制性突触以响应环境富集是必要的。目标 3 将使用单细胞 RNA-seq 和 ATAC-seq 来确定 Satb2 敲除如何改变基因表达和 CA1 中多个发育时间点的染色质可及性。这一目标将提供分子洞察 Satb2 如何在发育过程中控制 CA1 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.