Introduction

The rostral ventrolateral medulla (RVLM) is a vital vasomotor nucleus involved in hypertension control. However, the molecular intricacies within RVLM cells, particularly in non-neuronal cell populations such as astrocytes, under hypertensive conditions remain to be systematically investigated.

Objectives

We aimed to elucidate cellular heterogeneity in the RVLM at single-cell resolution, with a focus on astrocytes, to further characterize its role in hypertension.

Methods

Transcriptional changes in cells in the RVLM after hypertension were examined by using single-nucleus RNA sequencing (snRNA-seq). Further experiments, like radiotelemetry and immunofluorescence assays, were conducted to support the findings of snRNA-seq.

Results

In total, 36 982 high‐quality nuclei were obtained, with identification of nine cell types. Three transcriptomically distinct subtypes of astrocytes were defined within the astrocyte cell population: Cntnap2⁺, Pld5⁺, and Ppp1r16b⁺. The expression of Cntnap2 in RVLM astrocytes was notably increased to approximately 2.0-fold (P < 0.001) upon hypertension. Astrocyte-specific Cntnap2 overexpression in RVLM of WKY rats significantly enhanced neuronal excitability (∼21.5-fold, P < 0.001), sympathetic outflow (∼4.2-fold, P < 0.001), and blood pressure (BP, ∼1.5-fold, P < 0.001). Astrocyte-specific Cntnap2 knockdown in RVLM of SHRs resulted in 71 % reduction in neuronal excitability (P < 0.001), 51.6 % decrease in sympathetic outflow (P < 0.01), and 12.6 % drop in BP (P < 0.01). Elevated levels of Cntnap2 in primary astrocytes markedly increased primary neuron excitability to approximately 8.2-fold (P < 0.001). Mechanistically, Cntnap2 upregulation decreased Eaat2 expression by 26.5 % (P < 0.001), impairing astrocytic glutamate uptake. Importantly, reintroducing Eaat2 into astrocytes blocked 90.5 % (P < 0.001) of Cntnap2-induced neuronal excitability.

Conclusion

Our findings suggest that Cntnap2 may impede glutamate homeostasis at least partially through regulating Eaat2 in astrocytes, which could consequently enhance RVLM neuronal excitability, increase sympathetic tone, and contribute to elevated BP.

中文翻译：

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单核 RNA 测序显示 Cntnap2+ 星形胶质细胞通过破坏谷氨酸摄取在喙腹外侧延髓中触发高血压作用

  介绍

喙腹外侧延髓 （RVLM） 是参与高血压控制的重要血管舒缩核。然而，在高血压条件下，RVLM 细胞内的分子复杂性，特别是在非神经元细胞群（如星形胶质细胞）中的分子复杂性仍有待系统研究。

  目标

我们旨在以单细胞分辨率阐明 RVLM 中的细胞异质性，重点是星形胶质细胞，以进一步表征其在高血压中的作用。

  方法

使用单核 RNA 测序 （snRNA-seq） 检查高血压后 RVLM 细胞的转录变化。进行了进一步的实验，如放射遥测和免疫荧光测定，以支持 snRNA-seq 的研究结果。

  结果

总共获得了 36 982 个高质量细胞核，鉴定了 9 种细胞类型。在星形胶质细胞群中定义了三种转录组不同的星形胶质细胞亚型： Cntnap2 ⁺ 、 Pld5 ⁺ 和 Ppp1r16b ⁺ 。高血压时，RVLM 星形胶质细胞中 Cntnap2 的表达显着增加至约 2.0 倍 （P < 0.001）。星形胶质细胞特异性 Cntnap2 在 WKY 大鼠 RVLM 中的过表达显著增强了神经元兴奋性 （∼21.5 倍，P < 0.001）、交感神经流出 （∼4.2 倍，P < 0.001） 和血压 （BP，∼1.5 倍，P < 0.001）。在 SHR 的 RVLM 中，星形胶质细胞特异性 Cntnap2 敲低导致神经元兴奋性降低 71% （P < 0.001），交感神经流出减少 51.6% （P < 0.01），血压降低 12.6% （P < 0.01）。原代星形胶质细胞中 Cntnap2 水平升高显着使原代神经元兴奋性增加至约 8.2 倍 （P < 0.001）。从机制上讲，Cntnap2 上调使 Eaat2 表达降低了 26.5 % （P < 0.001），损害了星形胶质细胞谷氨酸的摄取。重要的是，将 Eaat2 重新引入星形胶质细胞阻断了 90.5% （P < 0.001） 的 Cntnap2 诱导的神经元兴奋性。

  结论

我们的研究结果表明，Cntnap2 可能至少部分地通过调节星形胶质细胞中的 Eaat2 来阻碍谷氨酸稳态，从而增强 RVLM 神经元的兴奋性，增加交感神经张力，并导致血压升高。