Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908, USA.
J Neurosci. 2011 Feb 9;31(6):1981-90. doi: 10.1523/JNEUROSCI.4639-10.2011.
We used optogenetics to determine the global respiratory effects produced by selectively stimulating raphe obscurus (RO) serotonergic neurons in anesthetized mice and to test whether these neurons detect changes in the partial pressure of CO(2), and hence function as central respiratory chemoreceptors. Channelrhodopsin-2 (ChR2) was selectively (∼97%) incorporated into ∼50% of RO serotonergic neurons by injecting AAV2 DIO ChR2-mCherry (adeno-associated viral vector double-floxed inverse open reading frame of ChR2-mCherry) into the RO of ePet-Cre mice. The transfected neurons heavily innervated lower brainstem and spinal cord regions involved in autonomic and somatic motor control plus breathing but eschewed sensory related regions. Pulsed laser photostimulation of ChR2-transfected serotonergic neurons increased respiratory frequency (fR) and diaphragmatic EMG (dEMG) amplitude in relation to the duration and frequency of the light pulses (half saturation, 1 ms; 5-10 Hz). dEMG amplitude and fR increased slowly (half saturation after 10-15 s) and relaxed monoexponentially (tau, 13-15 s). The breathing stimulation was reduced ∼55% by methysergide (broad spectrum serotonin antagonist) and potentiated (∼16%) at elevated levels of inspired CO(2) (8%). RO serotonergic neurons, identified by their entrainment to short light pulses (threshold, 0.1-1 ms) were silent (nine cells) or had a low and regular level of activity (2.1 ± 0.4 Hz; 11 cells) that was not synchronized with respiration. These and nine surrounding neurons with similar characteristics were unaffected by adding up to 10% CO(2) to the breathing mixture. In conclusion, RO serotonergic neurons activate breathing frequency and amplitude and potentiate the central respiratory chemoreflex but do not appear to have a central respiratory chemoreceptor function.
我们使用光遗传学来确定选择性刺激麻醉小鼠中中缝背核（RO）5-羟色胺能神经元所产生的全局呼吸效应，并测试这些神经元是否能检测到二氧化碳分压的变化，从而作为中枢呼吸化学感受器发挥作用。通过将 AAV2 DIO ChR2-mCherry（腺相关病毒载体的 ChR2-mCherry 双缺失反向开放阅读框）注射到 ePet-Cre 小鼠的 RO 中，将通道视紫红质-2（ChR2）选择性地（∼97%）掺入到约 50%的 RO 5-羟色胺能神经元中。转染的神经元大量支配参与自主和躯体运动控制以及呼吸的脑桥下部和脊髓区域，但避开了与感觉相关的区域。ChR2 转染的 5-羟色胺能神经元的脉冲激光光刺激与光脉冲的持续时间和频率有关，增加了呼吸频率（fR）和膈神经肌电图（dEMG）振幅（半饱和值为 1 ms；5-10 Hz）。dEMG 振幅和 fR 缓慢增加（光刺激 10-15 s 后半饱和）并呈单指数松弛（τ，13-15 s）。麦角酰二乙胺（广泛的 5-羟色胺拮抗剂）将呼吸刺激减少了约 55%，并在高浓度吸入 CO2（8%）时增强了（∼16%）。RO 5-羟色胺能神经元通过对短光脉冲的节律性发放（阈值为 0.1-1 ms）来识别，这些神经元处于静息状态（9 个细胞）或具有低而规则的活动水平（2.1±0.4 Hz；11 个细胞），且与呼吸不同步。这些细胞和 9 个具有相似特征的周围神经元对呼吸混合物中添加高达 10%的 CO2 没有反应。总之，RO 5-羟色胺能神经元激活呼吸频率和幅度，并增强中枢呼吸化学反射，但似乎没有中枢呼吸化学感受器功能。
