Nonlinear analysis of intracellular recording of rostral ventrolateral medulla neurons using whole-cell patch-clamp technique

为了确定血压升高的机制，我们比较了新生儿Wistar-Kyoto（WKY）和自发性高血压大鼠（SHR）延髓头端腹外侧（RVLM）神经元的电生理特征。利用全细胞膜片钳技术，我们研究了保留交感神经元网络的脑干-脊髓标本中RVLM神经元的特性。RVLM神经元的基线膜电位较浅（-49 mV vs.- SHR（n=56）的放电频率（5.1± 0.3Hz）明显快于WKY（n=33）的（4.1 ± 0.5Hz）。血管紧张素Ⅱ（6 μ mol/L）灌流使SHR RVLM神经元去极化（8.9±1.9 mV），其程度大于WKY（1.8±0.8 mV）。血管紧张素Ⅱ 1型（AT_1）受体拮抗剂坎地沙坦（0.12 μ mol/L）可引起SHR RVLM神经元的膜超极化（4.9±1.1 mV）和放电频率降低，但对WKY无此作用。 关于我们 Ist，PD 123319（60 μ mol/L）可使心肌超极化消失，提示血管紧张素II通过AT_2受体参与了心肌超极化。结果表明，高血压大鼠RVLM神经元的电生理特性及其对血管紧张素Ⅱ的反应存在明显差异，坎地沙坦引起RVLM神经元的超极化和放电频率的降低，提示内源性血管紧张素Ⅱ确实与RVLM神经元上的AT_1受体结合，从而导致高血压大鼠RVLM神经元膜电位升高和放电频率加快。此外，通过记录清醒WKY和SHR的血压（BP）、心率、肾交感神经活动（RSNA）和同侧肾血流量（RBF），我们发现RSNA与BP、RSNA与RBF之间的传递函数在0.05和0.80 Hz处有相干峰，低于心脏和心脏相关的波动。在0 ~ 0.1Hz频率范围内，RSNA与BP和RBF的互信息值均呈现较强的线性和非线性关系，且两品系间的相关性相似。高血压大鼠的血压和RBF与RSNA的线性相关性更强，而正常血压大鼠的非线性相关性更强。时延值表明，RSNA在闭环条件下优于BP和RBF。由中枢神经系统引起的不同低频RSNA振荡在清醒大鼠中产生血流动力学波动，独立于压力反射。少

To determine mechanisms underlying blood pressure elevation, we compared electrophysiological characteristics of neurons in the rostral ventrolateral medulla (RVLM) of neonatal Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Using the whole-cell patch-clamp technique, we examined the properties of RVLM neurons in brainstem-spinal cord preparations with a preserved sympathetic neuronal network. The baseline membrane potential of RVLM neurons was shallower (-49±1 mV vs.-53±2 mV) and the firing rate was faster (5.1±0.3 Hz vs.4.1±0.5 Hz) in SHR (n=56) than in WKY (n=33). Superfusion with angiotensin II (6 micromol/L) depolarized the RVLM neurons in SHR (8.9±1.9 mV) to a greater extent than in WKY (1.8±0.8 mV). Angiotensin II type 1 (AT_1) receptor antagonist, candesartan (0.12 micromol/L), induced significant membrane hyperpolarization (4.9±1.1 mV) and a decrease in the firing rate in RVLM neurons of SHR but not in those of WKY.After superfusion with the AT_2 receptor antagon … More ist, PD123319 (60 micromol/L), the hyperpolarization was abolished, suggesting angiotensin II action through AT_2 receptors to be involved in this hyperpolarization. Electrophysiological properties of RVLM neurons and their response to angiotensin II differ between WKY and SHR.The significant hyperpolarization and decreased firing rate caused by candesartan suggest that endogenously generated angiotensin II actually binds to AT_1 receptors on RVLM neurons, thus tonically contributing to a higher membrane potential and a faster firing rate in hypertensive rats. These differences in RVLM neurons suggest a mechanism possibly leading to elevation in blood pressure.Additionally, by recording blood pressure (BP), heart rate, renal sympathetic nerve activity (RSNA), and ipsilateral renal blood flow (RBF) in conscious WKY and SHR, we found coherence peaks of the transfer function between RSNA and BP and between RSNA and RBF at 0.05 and 0.80 Hz, below the respiratory-and cardiac-related fluctuations. The coherence was significantly higher and gain smaller in SHR than in WKY.Mutual information values showed strong linear and nonlinear relationships between RSNA and both BP and RBF at 0-0.1 Hz, and the relationships were similar in the two strains. The higher coherence implies that BP and RBF were more linearly correlated with RSNA in hypertensive rats, whereas the nonlinear correlation was greater in normotensive rats. Time delay values demonstrated that RSNA preceded BP and RBF under closed-loop conditions. Distinct low-frequency RSNA oscillations arising from the central nervous system generated hemodynamic fluctuations in conscious rats, independently of the baroreflex. Less