文獻光遺傳學(xué)激活膽堿能神經(jīng)元在PPT和LDT

1、Optogenetic activation of cholinergic neurons in thePPT or LDT induces REM sleep Christa J. Van DortProc Natl Acad Sci U S A. 2014 Dec 29. pii: 201423136. Epub ahead of print Department of Anesthesia, Critical Care, and Pain Medicine, Massachusetts General Hospital, Harvard Medical School, Boston一般認(rèn)

2、為廣義的基底前腦包括以下結(jié)構(gòu):(1)下丘腦視前區(qū)和前區(qū)，(2)隔核群，(3)終紋床核，(4)斜角帶核群, (5)Meynert基底核在內(nèi)的無名質(zhì)，(6)伏核(nucleus accumbens),(7)嗅結(jié)節(jié)，(8)嗅皮質(zhì)和(9)杏仁核群。狹義的基底前腦主要指半球前內(nèi)側(cè)面和基底面的一些靠近腦表面的灰質(zhì)，它們是(1)腹側(cè)紋狀體蒼白球(ventral striatopallidal system)，又稱腹側(cè)基底節(jié);(2)杏仁核的延伸部(bed nucleus-amygdalacontinuum, extended amygdala); (3)Meynert基底核(basalnucleus of M

3、eynert):(4)隔核及Broca氏斜角帶核。近年來文獻所指的基底前腦多指這些核群。而靈長類的無名質(zhì)(substantia innominata)也包括在這些灰質(zhì)群中。basal forebrainBackground Early pharmacological and unit recording studies Suggested that ACh was important for REM sleep regulation injection of cholinergic drugs into the dorsal mesopontine tegmentum reliably ind

4、uced a state very similar tonatural REM sleep in cats Electrical stimulationof the laterodorsal tegmentum (LDT) 被蓋背外側(cè)in cats increased the percentage of time spent in REM sleep (14), and activation of the pedunculopontine tegmentum (PPT)腳間腦橋被蓋 in rats induced wakefulness and REM sleep If cholinergic

5、 PPT and LDT neurons are necessary for REM sleep to occur, as the early studies suggest, then lesioning the PPT or LDT should decrease REM sleep. In cats, lesions of the PPT and LDT do disrupt REM sleep, but lesions in rodents have had little effect on REM sleep or increased REM sleep The PPT and LD

6、T are made up of heterogeneous populations of cells, including distinct populations of cholinergic, GABAergic, and glutamatergic neurons Rapid eye movement (REM) sleep is a critical component of restful sleep, yet the mechanisms that control REM sleep are incompletely understood. Brainstem cholinerg

7、ic neurons have been implicated in REM sleep regulation, but heterogeneous cell types in the area have made it difficult to determine the specific role of each population, leading to a debate about the importance of cholinergic neurons. Therefore, we selectively activated brainstem cholinergic neuro

8、ns to determine their role in REM sleep regulation. 2 光遺傳學(xué) 光遺傳學(xué)（optogenetics）是結(jié)合遺傳工程與光這一新的光控方法來選擇和打開某種生物的某一類細(xì)胞，并操作個別神經(jīng)細(xì)胞的活動，發(fā)現(xiàn)腦部如何產(chǎn)生波（gamma oscillations），并為它們在調(diào)控腦部功能中的角色提供新證據(jù)的嶄新學(xué)科。 該技術(shù)的關(guān)鍵是：先給動物的某種特定細(xì)胞轉(zhuǎn)入一種特殊基因，這種基因表達產(chǎn)物能夠?qū)Σ煌伾獾拇碳ぷ鞒雒舾械姆磻?yīng)，再利用不同顏色的激光照射來激活或者抑制細(xì)胞的功能。光遺傳學(xué)技術(shù)的運用包括四個步驟：第一、找尋合適的光敏蛋白。蛋白質(zhì)可以是具有天然

9、的光敏性，也可以是經(jīng)過化學(xué)修飾而具有光敏性。第二、遺傳信息傳遞。通過轉(zhuǎn)染、病毒轉(zhuǎn)導(dǎo)、轉(zhuǎn)基因動物系的建立等方式將光敏蛋白的遺傳信息傳遞給目標(biāo)細(xì)胞。第三、可控性演示。通過從時間和空間上控制演示光線的特定性，實現(xiàn)對細(xì)胞活動的精確演示。第四、讀取研究結(jié)果?？刹捎秒姌O通過檢測細(xì)胞膜內(nèi)外電壓來測量光敏蛋白的熒光效果變化，并可用熒光性生物傳感器來檢測不同細(xì)胞的讀出值。3 REM 快速動眼睡眠又稱異相睡眠或快波睡眠。此睡眠時相的腦電圖特征是呈現(xiàn)去同步化的快波。各種感覺和軀體運動功能進一步減退。 此外，還可有間斷性的陣發(fā)性表現(xiàn)：如出現(xiàn)眼球快速運動、部分肢體抽動、心率變快、血壓升高、呼吸加快等表現(xiàn)。此時易導(dǎo)致心絞

10、痛、哮喘、阻塞性肺氣腫缺氧的發(fā)作。快波睡眠期間，腦內(nèi)蛋白質(zhì)合成增加，新的突觸聯(lián)系建立，這有利于幼兒神經(jīng)系統(tǒng)的成熟、促進學(xué)習(xí)記憶活動和精力的恢復(fù)。 在快波睡眠時，將受試者喚醒，80%的人報告說正在做夢，所以做夢也是快波睡眠的一個特征。中腦橫斷面4 被蓋中腦借中腦水管分為背側(cè)的頂蓋和腹側(cè)的大腦腳，大腦腳又被黑質(zhì)分為腹側(cè)的大腦腳底和背側(cè)的被蓋。腦橋的內(nèi)部結(jié)構(gòu)以斜方體為界，分為腹側(cè)的基底部和背側(cè)的被蓋部。Materials and Methods Adult male mice (n = 23) expressing ChR2 under the ChAT and their WT littermat

11、es were implanted with EEG and EMG electrodes and bilateral fiber optics in the PPT or LDT. Blue light from a laser was used to stimulate the cholinergic PPT or LDT neurons optogenetically during NREM sleep. Stimulations were 60 s, 80 s, or 180 s long, separated by at least 1 min, Immunohistochemist

12、ry for ChAT confirmed that ChR2（通道視紫紅質(zhì) ）was expressed selectively in cholinergic neurons in the PPT and LDT Colocation of ChAT and ChR2-YFP in this range is consistent with previous reports demonstrating selective expression in the cortex (100%), striatum(100%), globus pallidus (100%), and medial ha

13、benula 中系帶(98.2%)for the same (31).No ChR2-YFPonly mouse strain neurons were found (Fig. 1)1 1 ChannelrhodopsinChannelrhodopsin Expression Was Expression Was Selective to Cholinergic Neurons Selective to Cholinergic Neurons in the in the PPT and LDT and Functional in VitroPPT and LDT and Functional

14、in VitroResults圖1 Fig. 2A demonstrates that LDT neurons had reliable rapid-onset action potentials following 5-ms light pulses. Fig. 2B shows the ability of a cell to follow the light pulses at 5 Hz over 2 s.圖2Activation of Cholinergic Neurons in the PPT and LDT Increases the Probability of REM Slee

15、p.EEG and EMG traces demonstrate that optogenetic activation of cholinergic neurons in the PPT during non-REM (NREM) sleep induced REM sleep (Fig. 3).The probabilityof REM sleep over time increased between ChR2+ mice(n = 11) and ChR2 mice (n = 11) for all stimulations for both the PPT and LDT (Fig.

16、4).REM sleep probability was significantlyhigher nonoverlapping confidence intervals (CIs) for 3060 s beyond the stimulation for ChR2+ PPT and LDT 60-s and 80-s stimulations compared with ChR2 60-s and 80-s stimulations.1 optogenetic activation of the PPT or LDT increased REM sleep (Fig. 5 A and D).

17、2 NREM sleep decreased between ChR2+ mice and ChR2 micefor all PPT stimulations and the 60-s LDT stimulation (Fig. 5 B and E3 Wakefulness did not change for either PPT or LDT stimulation, except for a small increase in wakefulness for the 60-s PPT stimulation Fig. 5 C and FIncrease in REM Sleep Is D

18、ue to More REM Sleep EpisodesThe increase in REM sleep occurred by increasing the number of REM sleep episodes (percentage of stimulations that induced REM sleep; Fig. 6 A and D) but not the duration of REM sleep episodes(Fig. 6 B and E). The induced REM sleep was electrophysiologicallysimilar to na

19、tural REM sleep. Power spectralanalysis of the EEG during induced REM sleep was not significantly different from the power spectra during natural REM sleepin the same ChR2+ mice (Fig. 6 C and F),Fiber Optics Were Localized to the PPT and LDTFig. 7 summarizesthe results of the histological analyses demonstrating that the tipsof the fiber o