應(yīng)激性抑郁樣行為發(fā)生中眶額葉5-HT1A受體對(duì)谷氨酸和γ-氨基丁酸的調(diào)節(jié)*

李江娜 安書(shū)成 李 珍

(陜西師范大學(xué)生命科學(xué)學(xué)院,西安 710119)

1 問(wèn)題提出

抑郁癥是一種比較普遍,且危害極大的心理與精神障礙性疾病。單胺類(lèi)遞質(zhì)失調(diào)學(xué)說(shuō)是研究抑郁癥發(fā)生機(jī)制及治療的主要理論之一?；诖硕粡V泛使用的一些抗抑郁藥雖能快速增加突觸間隙單胺類(lèi)遞質(zhì)的濃度,但由于其抗抑郁起效時(shí)間延遲,使得單胺類(lèi)遞質(zhì)失調(diào)假說(shuō)不足以解釋抑郁發(fā)生的全部現(xiàn)象。單胺類(lèi)遞質(zhì)及其受體,尤其是5-羥色胺(5-hydroxytryptamine,5-HT)及其受體失調(diào)可能是抑郁發(fā)生的基本原因(Carr &Lucki,2011)。而大腦谷氨酸(glutamic acid,Glu)能興奮性系統(tǒng)與 γ-氨基丁酸(gamma-amino-butyric acid,GABA)能抑制性系統(tǒng)的失調(diào)可能是抑郁癥發(fā)生的重要原因(Sanacora et al.,2004;何婷,喬卉,安書(shū)成,2011,11月)。因此,研究單胺類(lèi)遞質(zhì)及其受體對(duì) Glu興奮性系統(tǒng)與GABA抑制性系統(tǒng)的調(diào)節(jié)作用,對(duì)探討抑郁癥的發(fā)生機(jī)制可能更有意義。

5-羥色胺能系統(tǒng)失調(diào)與抑郁癥發(fā)生關(guān)系密切。研究發(fā)現(xiàn),抑郁的發(fā)生與 5-HT能突觸傳遞失調(diào)有關(guān)(Cubala &Landowski,2006),海馬注射5-HT具有抗應(yīng)激性抑郁的作用(Luo,An,&Zhang,2008)。激活中縫核的5-HT1A受體,能夠降低大鼠強(qiáng)迫游泳不動(dòng)時(shí)間(Almeida,Trovo,Tokumoto,Pereira,&Padovan,2013),眶額葉5-HT經(jīng)5-HT1A受體發(fā)揮抗抑郁作用(Li,An,&An,2009)。也有研究發(fā)現(xiàn),抑郁大鼠海馬5-HT1A受體的結(jié)合位點(diǎn)數(shù)量明顯下降,經(jīng)電針治療后可顯著提高其數(shù)量(康匯婷,王朝偉,2010)。激活突觸后膜上的5-HT1A受體能夠改善慢性應(yīng)激引起的抑郁樣行為(Zhou et al.,2014)?？挂钟羲幹委熀?突觸后膜5-HT1A受體的信號(hào)傳導(dǎo)能力增強(qiáng)(Savitz,Lucki,&Drevets,2009)。由此可見(jiàn),5-HT及其1A受體在抑郁發(fā)生和抗抑郁治療中具有重要作用。新一代的單胺能抗抑郁藥,如選擇性5-HT重?cái)z取抑制劑(selective serotonin reuptake inhibitors,SSRIs)及具有雙重作用的 5-HT/NE重?cái)z取抑制劑,雖能改善抑郁病人的治療效果和生活質(zhì)量,卻具有一定的臨床局限性,表現(xiàn)在抗抑郁作用起效慢,引起認(rèn)知障礙等副作用。臨床試驗(yàn)表明,30%～40%的單相抑郁病人對(duì)基于單胺類(lèi)遞質(zhì)的抗抑郁藥沒(méi)有反應(yīng)(Kornstein &Schneider,2001);超過(guò)90%的雙向抑郁患者會(huì)經(jīng)歷反復(fù)的狂躁發(fā)作(Sachs,2003)。越來(lái)越多的研究顯示,氨基酸能系統(tǒng)在抑郁癥病理生理和治療中起著重要作用(Kendell,Krystal,&Sanacora,2005)。Cai等(2013)研究提示,抑郁發(fā)生可能由于5-HT對(duì) Glu能興奮性突觸調(diào)節(jié)異常,使興奮性突觸過(guò)度激活所致。

Glu和GABA分別是中樞神經(jīng)系統(tǒng)中主要的興奮性遞質(zhì)和抑制性遞質(zhì),二者的協(xié)調(diào)對(duì)于維持正常情感狀態(tài)及行為表現(xiàn)至關(guān)重要,其中任何一種變化而使二者失調(diào),可引起中樞神經(jīng)系統(tǒng)多種疾病的發(fā)生(Kumar,Sharma,Kumar,&Deshmukh,2013)。研究顯示,Glu、GABA在抑郁癥中的變化不一致。重度抑郁患者血漿中GABA水平顯著下降,Glu水平明顯升高;經(jīng)抗抑郁藥物治療后,能夠反轉(zhuǎn)此效果(Kücükibrahimo?lu et al.,2009)。與對(duì)照組相比,急性創(chuàng)傷應(yīng)激后,大鼠前額葉中Glu、GABA均無(wú)顯著變化,海馬中 Glu、GABA雖均升高,但 Glu/GABA比值升高(Gao et al.,2014)。慢性應(yīng)激動(dòng)物模型研究表明,建模組大鼠內(nèi)側(cè)前額葉和海馬Glu水平顯著升高,GABA含量明顯下降(唐亞梅等,2013)。應(yīng)激引起眶額葉、海馬Glu水平升高,而谷氨酸NMDA受體拮抗劑 MK-801具有抗應(yīng)激性抑郁的作用(吳帥,安書(shū)成,陳慧彬,李菲,2014;余伶,安書(shū)成,廉婷,2010)?？梢?jiàn),Glu、GABA含量異常導(dǎo)致興奮性系統(tǒng)和抑制系統(tǒng)失衡,是抑郁發(fā)生的原因之一。近10年來(lái),Glu在抑郁治療中的作用逐漸引起人們的注意,主要是因?yàn)槠浞歉?jìng)爭(zhēng)性 NMDA受體拮抗劑氯胺酮具有快速而相對(duì)持久的抗抑郁效果(Kendell et al.,2005)。氯胺酮的抗抑郁作用能夠被5-HT耗竭而終止(Gigliucci et al.,2013)。表明其抗抑郁效果可能是 5-HT依賴(lài)的。因此,對(duì)于抑郁癥的治療策略可能需要將單胺能系統(tǒng)與Glu能系統(tǒng)共同思考。對(duì) GABA的研究顯示,中樞 GABA水平異常及GABA受體功能障礙與抑郁等多種神經(jīng)和精神疾病發(fā)生有關(guān)(Quandt,H?fner,&Wanner,2013)。越來(lái)越多的研究表明,中樞 GABA功能缺陷參與抑郁癥的發(fā)生(Frisardi,Panza,&Farooqui,2011)。研究發(fā)現(xiàn),抑郁癥患者前額葉皮層GAD65和GAD67水平均下調(diào),GABA濃度顯著降低,電休克療法和抗抑郁藥物均可使GABA水平恢復(fù)正常。此外,抑郁癥患者眶額葉皮層GABA能神經(jīng)元密度降低(Lussier,Romay-Tallón,Caruncho,&Kalynchuk,2013)。有報(bào)道認(rèn)為,GABA對(duì)慢性應(yīng)激性抑郁大鼠的認(rèn)知功能有改善作用,與抗抑郁劑聯(lián)用,這種改善效應(yīng)更顯著(姜英鳳,2012)。選擇性GABA再攝取抑制劑噻加賓也具有改善抑郁和焦慮的作用(高尚鋒,2012)。這些結(jié)果提示,GABA神經(jīng)遞質(zhì)參與抑郁癥的發(fā)生。

5-HT對(duì)GABA具有調(diào)節(jié)作用。抑郁患者5-HT缺乏會(huì)導(dǎo)致GABA能功能紊亂,SSRIs通過(guò)使5-HT受體脫敏上調(diào)5-HT水平,從而改變5-HT能系統(tǒng)對(duì)GABA能系統(tǒng)的調(diào)節(jié)作用,可能是其抗抑郁的途徑之一(Zhong &Yan,2004)。新型抗抑郁藥Vortioxetine屬于SSRIs的一種,能夠通過(guò)5-HT系統(tǒng)調(diào)節(jié)GABA能神經(jīng)傳遞(Pehrson,Li,Haddjeri,Gulinello,&Sanchez,2013)。應(yīng)激與行為研究發(fā)現(xiàn),前額葉5-HT參與調(diào)控杏仁核 GABA能神經(jīng)元,束縛應(yīng)激引起基底外側(cè)杏仁核 GABA釋放增加,而選擇性耗竭內(nèi)側(cè)前額葉5-HT,能夠減弱應(yīng)激引起的GABA變化(Andolina,Maran,Valzania,Conversi,&Puglisi-Allegra,2013)。這些研究提示,在應(yīng)激引起的行為變化中5-HT能系統(tǒng)對(duì)GABA傳遞具有調(diào)控作用。5-HT1A受體參與腹外側(cè)眶額葉引起的鎮(zhèn)痛作用,而GABA型受體拮抗劑能夠減緩5-HT1A受體介導(dǎo)的此效果(Huo,Qu,Li,Tang,&Jia,2008)。提示,OFC區(qū)5-HT1A受體與GABA能神經(jīng)元有一定關(guān)系。

眶額葉(orbitofrontal cortex,OFC)是前額葉的一個(gè)重要亞區(qū),它與人的情緒、認(rèn)知等高級(jí)腦功能密切相關(guān)。Taylor等(2007)研究發(fā)現(xiàn),抑郁癥患者OFC體積減小,結(jié)構(gòu)和功能發(fā)生改變(Zhang,Chen,Jia,&Gong,2014)。谷氨酸能神經(jīng)元在OFC大量分布,?-氨基丁酸能神經(jīng)元為OFC的中間神經(jīng)元。中縫核內(nèi)的5-HT能神經(jīng)元投射到OFC,且在谷氨酸能神經(jīng)元及中間神經(jīng)元上分布有5-HT1A受體(Huo et al.,2009;Simpson,Lubman,Slater,&Deakin,1996)。研究表明,應(yīng)激后大鼠OFC區(qū) Glu含量升高(吳帥等,2014),眶額葉5-HT經(jīng)5-HT1A受體發(fā)揮抗抑郁作用(Li et al.,2009)。

綜上所述,Glu水平過(guò)高或GABA水平的變化是應(yīng)激誘發(fā)抑郁的重要原因之一。應(yīng)激會(huì)引起OFC區(qū)Glu水平升高,眶額葉5-HT經(jīng)1A受體能發(fā)揮抗抑郁作用,然而,應(yīng)激性抑郁樣行為發(fā)生中眶額葉5-HT水平有何變化,是5-HT減少,還是其水平不能很好地調(diào)節(jié)Glu或GABA,5-HT是否通過(guò)對(duì)Glu和 GABA能神經(jīng)元調(diào)節(jié)而發(fā)揮抗抑郁作用,以及調(diào)節(jié)的受體途徑等并不清楚。

為了解決以上問(wèn)題,本研究主要通過(guò)建立慢性不可預(yù)見(jiàn)性溫和應(yīng)激(chronic unpredictable mild stress,CUMS)動(dòng)物模型,采用微量注射的方法分別向OFC注射5-HT1A受體激動(dòng)劑8-OH-DPAT和拮抗劑 WAY100635,通過(guò)行為學(xué)檢測(cè),并結(jié)合高效液相色譜法(high-performance liquid chromatography,HPLC)檢測(cè)OFC區(qū)5-HT、Glu和GABA的含量,以此來(lái)探討慢性應(yīng)激性抑郁發(fā)生中OFC區(qū)5-HT 1A受體與Glu、GABA之間的關(guān)系。

2 材料和方法

2.1 實(shí)驗(yàn)動(dòng)物及分組

健康成年雄性Sprague-Dawley (SD)大鼠32只(250～300 g,約90日齡),由西安交通大學(xué)醫(yī)學(xué)院提供。實(shí)驗(yàn)前動(dòng)物 4～5只一籠,自由進(jìn)食飲水,適應(yīng)環(huán)境一周。隨后將動(dòng)物隨機(jī)分為四組：A組為正常對(duì)照組(Control),正常飼養(yǎng),在實(shí)驗(yàn)的第 1、7、14和21天,雙側(cè)OFC均微量注射生理鹽水各1 μl;B組為CUMS模型組,即CUMS處理的同時(shí),在上述時(shí)間內(nèi)雙側(cè)OFC均微量注射生理鹽水各1 μl;C組為 WAY100635組,即正常飼養(yǎng),在相同的時(shí)間里雙側(cè)OFC均微量注射5-HT1A受體拮抗劑WAY100635各 1 μl (30 nmol) (問(wèn)黎敏,安書(shū)成,劉慧,2012);D組為 CUMS+8-OH-DPAT組,即 CUMS 處理的同時(shí),在同一時(shí)間雙側(cè)OFC均微量注射5-HT1A受體激動(dòng)劑 8-OH-DPAT各 1 μl (10 nmol) (問(wèn)黎敏等,2012)。A～D 組均進(jìn)行行為學(xué)測(cè)試,并采用 HPLC檢測(cè)OFC 5-HT、Glu和GABA水平。

2.2 實(shí)驗(yàn)試劑和儀器

8-OH-DPAT (5-HT1A 受體選擇性激動(dòng)劑)、WAY100635 (5-HT1A受體選擇性拮抗劑)均為美國(guó)sigma-Aldrich公司產(chǎn)品;5-HT標(biāo)準(zhǔn)品(sigma-Aldrich公司);Glu、GABA標(biāo)準(zhǔn)品(sigma公司);2,4二硝基氟苯(DNFB) (sigma公司);甲醇、乙腈(美國(guó)Fisher公司);腦立體定位儀(KOPF型)為美國(guó)Stoelting公司產(chǎn)品;曠場(chǎng)實(shí)驗(yàn)Video Mot 2黑白多目標(biāo)動(dòng)物行為監(jiān)測(cè)分析系統(tǒng)(302050-BWM)為德國(guó)TSE公司;微量注射器(1 μl)為上海安亭微量進(jìn)樣器廠產(chǎn)品。高效液相色譜儀為日本SHIMADZU公司產(chǎn)品及自動(dòng)進(jìn)樣器、UV檢測(cè)器和色譜數(shù)據(jù)處理系統(tǒng)。

2.3 實(shí)驗(yàn)方法

2.3.1 腦立體定位及OFC微量注射

用2%的戊巴比妥鈉40 mg/Kg腹腔注射麻醉大鼠,參照大鼠腦圖譜(Paxinos &Watson,1998)進(jìn)行腦立體定位(見(jiàn)圖1中A2),在OFC區(qū)(AP 3.7 mm;RL 1.4 mm;H 5.0 mm)之上1.5 mm處植入兩根直徑為0.9 mm,長(zhǎng)度為1.4 cm的不銹鋼套管。腦內(nèi)注射采用微量注射器(1 μl)勻速給藥1 min,停針1 min防止藥物溢出。注射時(shí)注射器伸出套管外1.5 mm,到達(dá)OFC。所用藥物均用生理鹽水溶解稀釋至所需濃度。在實(shí)驗(yàn)的第1、7、14和21天,對(duì)各組大鼠OFC進(jìn)行微量注射藥物。實(shí)驗(yàn)結(jié)束后,進(jìn)行定位檢測(cè),見(jiàn)圖1中A1,不準(zhǔn)確者剔除。具體方法見(jiàn)(慈蕾,安書(shū)成,2007)。

2.3.2 CUMS模型建立

共9種刺激,3種強(qiáng)刺激(夾尾1 min,4℃冰泳5 min,45℃熱泳5 min),6種弱刺激(禁水24 h、禁食24 h、潮濕24 h、水平搖晃5 min、晝夜顛倒12 h、傾斜24 h)隨機(jī)安排在21天內(nèi),每天一種刺激,同種刺激不能連續(xù)出現(xiàn),避免動(dòng)物出現(xiàn)適應(yīng)。建模21天后,進(jìn)行行為學(xué)檢測(cè)。

2.4 行為學(xué)檢測(cè)

各組動(dòng)物的行為學(xué)基線測(cè)試結(jié)果顯示,行為基線穩(wěn)定而正常,組間無(wú)顯著性差異。

2.4.1 糖水測(cè)試(吳帥等,2014)

實(shí)驗(yàn)前在安靜的房間內(nèi)訓(xùn)練動(dòng)物含糖飲水,每只籠內(nèi)放置同樣體積的水瓶。第一個(gè)24 h,兩瓶均裝有 1%的蔗糖溶液(稱(chēng)為兩糖適應(yīng));第二個(gè) 24 h,兩瓶液體量相等,一瓶為清水,一瓶仍為 1%的蔗糖溶液(稱(chēng)為一糖一水適應(yīng));第三個(gè) 24 h,禁水;第四個(gè)24 h,進(jìn)行糖水消耗測(cè)試,量取等量的一瓶1%的蔗糖溶液,一瓶清水(一糖一水)進(jìn)行測(cè)試。24 h后測(cè)量糖水消耗量,即同時(shí)輕輕取下兩瓶,用量筒分別測(cè)量各自剩余量,計(jì)算出清水和糖水各自消耗量,并計(jì)算動(dòng)物糖水偏愛(ài)[糖水偏愛(ài)率(%)=糖水消耗量/總液體消耗量×100%]。

2.4.2 曠場(chǎng)實(shí)驗(yàn)

測(cè)試當(dāng)天,先將動(dòng)物置于測(cè)試環(huán)境中適應(yīng)至少半個(gè)小時(shí)。實(shí)驗(yàn)開(kāi)始時(shí),將大鼠放置在一個(gè)四周和底面均涂黑的無(wú)蓋方箱(60 cm × 60 cm × 40 cm)箱底中心。用Video Mot 2黑白多目標(biāo)動(dòng)物行為監(jiān)測(cè)系統(tǒng)記錄其5 min的活動(dòng)情況。主要指標(biāo)：動(dòng)物的水平穿格次數(shù)、直立次數(shù)、理毛次數(shù),以此計(jì)算大鼠的水平運(yùn)動(dòng)得分、豎直運(yùn)動(dòng)得分及修飾得分。每只大鼠觀察結(jié)束后,都要清理箱內(nèi)殘留物,酒精去除異味,防止影響其它鼠的測(cè)試結(jié)果。

圖1 OFC藥物注射點(diǎn)(A1)及其示意圖(A2)注：A和A’箭頭所指的點(diǎn)分別代表左右兩側(cè)打藥的位置Fig.1 The injection point sites within OFC (A1) and its schematic diagram (A2).The spot of arrowA and A’ point to the left and right locations of injecting drug,respectively.

2.4.3 懸尾實(shí)驗(yàn)(問(wèn)黎敏等,2012)

實(shí)驗(yàn)在懸尾暗箱中進(jìn)行,距大鼠尾根部 1/3處用醫(yī)用膠布固定于懸尾箱內(nèi),記錄大鼠懸尾后,5 min內(nèi)的不動(dòng)時(shí)間。

2.5 HPLC實(shí)驗(yàn)(劉慧,問(wèn)黎敏,喬卉,安書(shū)成,2013)

配制不同濃度的 5-HT、Glu和GABA標(biāo)準(zhǔn)品溶液,測(cè)其峰面積,繪制標(biāo)準(zhǔn)曲線。制備OFC組織樣品,檢測(cè)其峰面積。根據(jù)標(biāo)準(zhǔn)曲線將樣品的峰面積換算為其濃度。5-HT進(jìn)樣條件為,A水相：檸檬酸-乙酸鈉緩沖體系(PH=3.8);B有機(jī)相(甲醇),使用熒光檢測(cè)器進(jìn)行檢測(cè),發(fā)射波長(zhǎng)330 nm,激發(fā)波長(zhǎng)280 nm。Glu、GABA進(jìn)樣條件,A水相：醋酸鈉緩沖液(PH=6);B有機(jī)相(乙腈水V/V=1:1),進(jìn)行梯度洗脫,使用紫外檢測(cè)器進(jìn)行檢測(cè),檢測(cè)波長(zhǎng)為360 nm。

2.6 數(shù)據(jù)處理

實(shí)驗(yàn)數(shù)據(jù)均以平均值±標(biāo)準(zhǔn)誤(Mean±

SEM

)表示,采用 SPSS 20.0軟件進(jìn)行數(shù)據(jù)分析,組間差異檢驗(yàn)用單因素方差分析(one-way

ANOVA

),組間多重比較行LSD檢驗(yàn)。

p

<0.05時(shí)認(rèn)為差異具有統(tǒng)計(jì)學(xué)意義,

p

<0.01表示有極顯著性差異。

3 實(shí)驗(yàn)結(jié)果

3.1 行為學(xué)結(jié)果

3.1.1 糖水偏愛(ài)率

3.1.2 曠場(chǎng)實(shí)驗(yàn)

圖2 各組大鼠總液體消耗(A)與糖水偏愛(ài)率(B)Fig.2 Effects of different treatment on the total fluid intake (A) and the sucrose preference (B).Results are expressed as the means± SEM (n =7～8).## p <0.01 vs Control group (n =8);** p <0.01 vs CUMS group (n =8).

圖3 各組大鼠曠場(chǎng)行為表現(xiàn)Fig.3 Effects of different treatments on locomotion (A),rearing (B) and grooming (C) in the open field test.Results are expressed as the means±SEM (n =8).## p <0.01 vs Control group (n =8);** p <0.01 vs CUMS group (n =8).

3.1.3 懸尾實(shí)驗(yàn)結(jié)果

3.2 高效液相色譜實(shí)驗(yàn)結(jié)果

3.2.1 5-HT含量變化

測(cè)定5-HT標(biāo)準(zhǔn)品溶液峰面積,確定5-HT的出峰時(shí)間為9.275 min,繪制標(biāo)準(zhǔn)曲線。檢測(cè)其線性范圍為：2 ng/mL～300 ng/mL;其線性回歸方程為：Y=25867X + 72385 (

R

=0.9996)。四組大鼠5-HT含量沒(méi)有顯著性差異(

p

=0.588 >0.05)。見(jiàn)圖5C。

3.2.2 Glu、GABA含量變化

圖4 各組大鼠在懸尾實(shí)驗(yàn)中的不動(dòng)時(shí)間Fig.4 Effects of different treatments on immobility time in the tail suspension test.Results are expressed as the means±SEM (n=8).## p<0.01 vs Control group (n=8);** p <0.01 vs CUMS group (n=8).

圖5 各組大鼠OFC區(qū)Glu (A)、GABA (B)和5-HT(C)濃度Fig.5 Effects of different treatments on orbital frontal cortex Glu (A),GABA (B) and 5-HT (C) concentrations.Results are expressed as the means± SEM (n =8).## p <0.01 vs Control group (n =8);* p <0.05 vs CUMS group (n =8).

4 討論

慢性不可預(yù)見(jiàn)性溫和應(yīng)激抑郁動(dòng)物模型因能模擬出人類(lèi)抑郁的核心癥狀,而被廣泛采納。本研究建立的 CUMS模型,動(dòng)物表現(xiàn)出糖水偏愛(ài)率下降,運(yùn)動(dòng)緩慢,探究能力下降,自我關(guān)注程度下降以及行為絕望度提高,所測(cè)的各項(xiàng)行為學(xué)指標(biāo)均能代表抑郁的典型癥狀。

單胺類(lèi)遞質(zhì)尤其是5-HT失調(diào)是抑郁癥發(fā)生的經(jīng)典假說(shuō)。有研究報(bào)道,慢性不可預(yù)見(jiàn)性應(yīng)激能夠顯著降低中縫核5-HT能神經(jīng)元的自發(fā)放電活動(dòng)及自發(fā)放電神經(jīng)元的數(shù)目,從而產(chǎn)生抑郁樣行為(Bambico,Nguyen,&Gobbi,2009)。Drevets等(2007)研究發(fā)現(xiàn),與健康人相比,抑郁癥病人顳葉內(nèi)側(cè)區(qū)及中縫核5-HT1A受體的結(jié)合力及該受體的功能均下降。而長(zhǎng)期注射5-HT1A受體激動(dòng)劑能夠緩解抑郁以及因抑郁引起的痛覺(jué)遲鈍(Jiang,Qi,Wang,&Luo,2014)。 然而,另有研究發(fā)現(xiàn),抑郁癥中5-HT的含量并未發(fā)生顯著性變化(Laugeray et al.,2010;Venzala,García-García,Elizalde,&Tordera,2013),但外源性給予5-HT卻具有抗抑郁作用(Luo et al.,2008),其原因到底是 5-HT水平降低引起抑郁,還是抑郁產(chǎn)生是因 5-HT水平不能滿(mǎn)足需要所致,具體機(jī)制并不清楚。而本實(shí)驗(yàn)結(jié)果顯示,給予慢性不可預(yù)見(jiàn)性溫和應(yīng)激之后,大鼠表現(xiàn)出抑郁樣行為,但其OFC中5-HT的含量并未發(fā)生顯著性變化。然而,當(dāng)在OFC外源性微量注射5-HT1A受體激動(dòng)劑8-OH-DPAT后,大鼠的抑郁樣行為得到顯著改善,主要表現(xiàn)為糖水偏愛(ài)率顯著提高及懸尾不動(dòng)時(shí)間顯著減少。反之,當(dāng)正常大鼠外源性給予 5-HT1A受體拮抗劑 WAY100635后,大鼠糖水偏愛(ài)率顯著降低,懸尾不動(dòng)時(shí)間顯著增加。這表明,盡管應(yīng)激引起抑郁樣行為發(fā)生時(shí)OFC區(qū)5-HT水平并未降低,但5-HT1A受體的激活可以起到抗抑郁作用,而阻斷該受體能夠使大鼠產(chǎn)生抑郁樣行為。以上結(jié)果與之前的研究報(bào)道相一致(Li et al.,2009;Laugeray et al.,2010;Venzala et al.,2013)。由此推測(cè),應(yīng)激性抑郁發(fā)生的主要原因可能不是OFC區(qū)5-HT含量的變化,而很有可能是應(yīng)激時(shí) 5-HT水平不能滿(mǎn)足需要所致。當(dāng)然,也有應(yīng)激時(shí)突觸后膜5-HT1A受體脫敏所致(Mahar,Bambico,Mechawar,&Nobrega,2014)的可能。

抑郁癥病人 OFC形態(tài)異常在神經(jīng)影像學(xué)中被廣泛報(bào)道。與健康對(duì)照組相比,重度抑郁癥患者OFC灰質(zhì)體積明顯減小(Lacerda et al.,2004)。尸檢結(jié)果發(fā)現(xiàn),抑郁癥病人大腦 OFC中神經(jīng)元及神經(jīng)膠質(zhì)細(xì)胞的密度減少,內(nèi)側(cè)眶額葉體積顯著減小(Bremner et al.,2002)。由此可見(jiàn),OFC在抑郁癥的發(fā)生中具有重要作用。OFC主要接受來(lái)自中縫背核5-HT能神經(jīng)元投射(Roberts,2011),5-HT1A受體在OFC及其它邊緣系統(tǒng)和中腦中縫核分布密度最高(Muller &Jacobs,2009),其在OFC主要作為突觸后膜異源性受體發(fā)揮作用(Stein,Miczek,Lucion,&de Almeida,2013)。免疫組化和原位雜交結(jié)果表明,大鼠(Santana,Bortolozzi,Serrats,Mengod,&Artigas,2004)、人和猴(de Almeida &Mengod,2008)的前額葉錐體神經(jīng)元和腹外側(cè)眶額葉 GABA能神經(jīng)元(Huo et al.,2009)上均存在5-HT1A受體。也有研究發(fā)現(xiàn),慢性不可預(yù)見(jiàn)性應(yīng)激通過(guò)改變5-HT1A受體功能而可能影響內(nèi)側(cè)前額葉Glu能突觸傳遞(Mahar et al.,2014)。由此可見(jiàn),OFC中 5-HT調(diào)節(jié)Glu及GABA能神經(jīng)元具有解剖學(xué)基礎(chǔ)。

眾多研究表明,Glu能突觸傳遞異常在抑郁癥的病理機(jī)制及治療中起著重要的作用(Hashimoto,2009;Musazzi,Treccani,Mallei,&Popoli,2013)。與健康對(duì)照組相比,抑郁癥病人腦脊液(Levine et al.,2000)、額葉(Hashimoto,Sawa,&Iyo,2007)及眶額葉(吳帥等,2014)中Glu水平均顯著增加。而抗抑郁藥能夠減少Glu釋放及其突觸傳遞(Musazzi et al.,2013)。本實(shí)驗(yàn)結(jié)果也顯示,CUMS大鼠OFC區(qū)Glu含量顯著高于正常對(duì)照組,大鼠表現(xiàn)出抑郁樣行為。為了證明應(yīng)激引起眶額葉Glu水平升高是否與5-HT1A受體有關(guān),本實(shí)驗(yàn)通過(guò)微量注射抗抑郁藥5-HT1A受體激動(dòng)劑 8-OH-DPAT后,CUMS大鼠OFC區(qū)Glu水平顯著降低,且能顯著改善大鼠的抑郁樣行為。此外,臨床前研究發(fā)現(xiàn),非競(jìng)爭(zhēng)性的NMDA受體拮抗劑氯胺酮能夠產(chǎn)生快速、持久的抗抑郁效果,但此抗抑郁效果會(huì)因 5-HT的耗竭而被終止(Gigliucci et al.,2013)。Cai Xiang等研究提出,抑郁發(fā)生可能由于5-HT對(duì)Glu能興奮性突觸調(diào)節(jié)異常,使興奮性突觸過(guò)度激活所致(Cai et al.,2013)。而興奮性突觸的過(guò)度激活,很有可能打破了 OFC區(qū)Glu和GABA原有的平衡,導(dǎo)致抑郁。另有研究報(bào)道,在治療抑郁癥及其相關(guān)的認(rèn)知紊亂中,5-HT可通過(guò)5-HT1A受體對(duì)Glu進(jìn)行調(diào)節(jié)(Ciranna,2006;Pehrson &Sanchez,2014)。慢性不可預(yù)見(jiàn)性應(yīng)激改變5-HT1A受體功能,可能影響內(nèi)側(cè)前額葉Glu能突觸傳遞(Mahar et al.,2014)。本實(shí)驗(yàn)結(jié)果還顯示,微量注射5-HT1A受體拮抗劑WAY100635后,正常大鼠OFC區(qū) Glu含量顯著升高,動(dòng)物也表現(xiàn)出抑郁樣行為。以上研究結(jié)果均表明,5-HT1A受體參與了對(duì)Glu水平的調(diào)節(jié),而且慢性應(yīng)激誘發(fā)抑郁樣行為,可能存在著5-HT1A受體對(duì)Glu水平控制失調(diào),使得Glu水平過(guò)高所致。然而,盡管有研究提出腹外側(cè)眶額葉GABA能神經(jīng)元(Huo et al.,2009)上存在5-HT1A受體,但在本研究整個(gè)實(shí)驗(yàn)的各個(gè)項(xiàng)目中,并未發(fā)現(xiàn)眶額葉 GABA含量的變化。該結(jié)果提示,OFC區(qū) 5-HT可能主要是通過(guò) Glu能神經(jīng)元上的5-HT1A受體抑制 Glu水平過(guò)度升高,而未明顯影響眶額葉GABA能神經(jīng)元,這可能與相對(duì)于Glu能神經(jīng)元上5-HT1A受體,GABA能神經(jīng)元上5-HT1A受體敏感度較低有關(guān)(Pehrson &Sanchez,2014)。

綜上所述,CUMS在引發(fā)大鼠抑郁樣行為的同時(shí),并未降低OFC中5-HT的水平,GABA水平也沒(méi)有明顯變化,而是顯著升高Glu含量。外源性注射 5-HT1A受體激動(dòng)劑產(chǎn)生抗抑郁作用的同時(shí),OFC中Glu水平顯著降低。以上結(jié)果提示,應(yīng)激性抑郁樣行為發(fā)生,可能是 5-HT水平不能起到對(duì)Glu能神經(jīng)元的有效調(diào)控,因而使OFC區(qū)Glu水平過(guò)高所致。但也有應(yīng)激使OFC區(qū)Glu能神經(jīng)元上5-HT1A受體功能降低,導(dǎo)致其對(duì) Glu釋放的抑制作用減弱,從而使Glu水平異常升高的可能。研究結(jié)果對(duì)解釋基于單胺類(lèi)遞質(zhì)的抗抑郁藥物延遲現(xiàn)象也可能具有一定的啟示。

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