李江娜 安書成 李 珍

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

1 問題提出

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。單胺類遞質(zhì)失調(diào)學(xué)說是研究抑郁癥發(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遞質(zhì)的濃度,但由于其抗抑郁起效時間延遲,使得單胺類遞質(zhì)失調(diào)假說不足以解釋抑郁發(fā)生的全部現(xiàn)象。單胺類遞質(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,安書成,2011,11月)。因此,研究單胺類遞質(zhì)及其受體對 Glu興奮性系統(tǒng)與GABA抑制性系統(tǒng)的調(diào)節(jié)作用,對探討抑郁癥的發(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)迫游泳不動時間(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受體的信號傳導(dǎo)能力增強(qiáng)(Savitz,Lucki,&Drevets,2009)。由此可見,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重攝取抑制劑(selective serotonin reuptake inhibitors,SSRIs)及具有雙重作用的 5-HT/NE重攝取抑制劑,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。臨床試驗表明,30%～40%的單相抑郁病人對基于單胺類遞質(zhì)的抗抑郁藥沒有反應(yīng)(Kornstein &Schneider,2001);超過90%的雙向抑郁患者會經(jīng)歷反復(fù)的狂躁發(fā)作(Sachs,2003)。越來越多的研究顯示,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 Glu能興奮性突觸調(diào)節(jié)異常,使興奮性突觸過度激活所致。

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)。與對照組相比,急性創(chuàng)傷應(yīng)激后,大鼠前額葉中Glu、GABA均無顯著變化,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,安書成,陳慧彬,李菲,2014;余伶,安書成,廉婷,2010)。可見,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,Glu在抑郁治療中的作用逐漸引起人們的注意,主要是因為其非競爭性 NMDA受體拮抗劑氯胺酮具有快速而相對持久的抗抑郁效果(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依賴的。因此,對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 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)。有報道認(rèn)為,GABA對慢性應(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GABA具有調(diào)節(jié)作用。抑郁患者5-HT缺乏會導(dǎo)致GABA能功能紊亂,SSRIs通過使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)是前額葉的一個重要亞區(qū),它與人的情緒、認(rèn)知等高級腦功能密切相關(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是否通過對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。

為了解決以上問題,本研究主要通過建立慢性不可預(yù)見性溫和應(yīng)激(chronic unpredictable mild stress,CUMS)動物模型,采用微量注射的方法分別向OFC注射5-HT1A受體激動劑8-OH-DPAT和拮抗劑 WAY100635,通過行為學(xué)檢測,并結(jié)合高效液相色譜法(high-performance liquid chromatography,HPLC)檢測OFC區(qū)5-HT、Glu和GABA的含量,以此來探討慢性應(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 實驗動物及分組

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。實驗前動物 4～5只一籠,自由進(jìn)食飲水,適應(yīng)環(huán)境一周。隨后將動物隨機(jī)分為四組：A組為正常對照組(Control),正常飼養(yǎng),在實驗的第 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,在上述時間內(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) (問黎敏,安書成,劉慧,2012);D組為 CUMS+8-OH-DPAT組,即 CUMS 處理的同時,在同一時間雙側(cè)OFC均微量注射5-HT1A受體激動劑 8-OH-DPAT各 1 μl (10 nmol) (問黎敏等,2012)。A～D 組均進(jìn)行行為學(xué)測試,并采用 HPLC檢測OFC 5-HT、Glu和GABA水平。

2.2 實驗試劑和儀器

8-OH-DPAT (5-HT1A 受體選擇性激動劑)、WAY100635 (5-HT1A受體選擇性拮抗劑)均為美國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Fisher公司);腦立體定位儀(KOPF型)為美國Stoelting公司產(chǎn)品;曠場實驗Video Mot 2黑白多目標(biāo)動物行為監(jiān)測分析系統(tǒng)(302050-BWM)為德國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)品及自動進(jìn)樣器、UV檢測器和色譜數(shù)據(jù)處理系統(tǒng)。

2.3 實驗方法

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。在實驗的第1、7、14和21天,對各組大鼠OFC進(jìn)行微量注射藥物。實驗結(jié)束后,進(jìn)行定位檢測,見圖1中A1,不準(zhǔn)確者剔除。具體方法見(慈蕾,安書成,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),避免動物出現(xiàn)適應(yīng)。建模21天后,進(jìn)行行為學(xué)檢測。

2.4 行為學(xué)檢測

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

2.4.1 糖水測試(吳帥等,2014)

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

2.4.2 曠場實驗

測試當(dāng)天,先將動物置于測試環(huán)境中適應(yīng)至少半個小時。實驗開始時,將大鼠放置在一個四周和底面均涂黑的無蓋方箱(60 cm × 60 cm × 40 cm)箱底中心。用Video Mot 2黑白多目標(biāo)動物行為監(jiān)測系統(tǒng)記錄其5 min的活動情況。主要指標(biāo)：動物的水平穿格次數(shù)、直立次數(shù)、理毛次數(shù),以此計算大鼠的水平運(yùn)動得分、豎直運(yùn)動得分及修飾得分。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試結(jié)果。

圖1 OFC藥物注射點(A1)及其示意圖(A2)注：A和A’箭頭所指的點分別代表左右兩側(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 懸尾實驗(問黎敏等,2012)

實驗在懸尾暗箱中進(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)的不動時間。

2.5 HPLC實驗(劉慧,問黎敏,喬卉,安書成,2013)

配制不同濃度的 5-HT、Glu和GABA標(biāo)準(zhǔn)品溶液,測其峰面積,繪制標(biāo)準(zhǔn)曲線。制備OFC組織樣品,檢測其峰面積。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),使用熒光檢測器進(jìn)行檢測,發(fā)射波長330 nm,激發(fā)波長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,使用紫外檢測器進(jìn)行檢測,檢測波長為360 nm。

2.6 數(shù)據(jù)處理

實驗數(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,組間差異檢驗用單因素方差分析(one-way

ANOVA

),組間多重比較行LSD檢驗。

p

<0.05時認(rèn)為差異具有統(tǒng)計學(xué)意義,

p

<0.01表示有極顯著性差異。

3 實驗結(jié)果

3.1 行為學(xué)結(jié)果

3.1.1 糖水偏愛率

3.1.2 曠場實驗

圖2 各組大鼠總液體消耗(A)與糖水偏愛率(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 懸尾實驗結(jié)果

3.2 高效液相色譜實驗結(jié)果

3.2.1 5-HT含量變化

測定5-HT標(biāo)準(zhǔn)品溶液峰面積,確定5-HT的出峰時間為9.275 min,繪制標(biāo)準(zhǔn)曲線。檢測其線性范圍為：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含量沒有顯著性差異(

p

=0.588 >0.05)。見圖5C。

3.2.2 Glu、GABA含量變化

圖4 各組大鼠在懸尾實驗中的不動時間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)見性溫和應(yīng)激抑郁動物模型因能模擬出人類抑郁的核心癥狀,而被廣泛采納。本研究建立的 CUMS模型,動物表現(xiàn)出糖水偏愛率下降,運(yùn)動緩慢,探究能力下降,自我關(guān)注程度下降以及行為絕望度提高,所測的各項行為學(xué)指標(biāo)均能代表抑郁的典型癥狀。

單胺類遞質(zhì)尤其是5-HT失調(diào)是抑郁癥發(fā)生的經(jīng)典假說。有研究報道,慢性不可預(yù)見性應(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。而長期注射5-HT1A受體激動劑能夠緩解抑郁以及因抑郁引起的痛覺遲鈍(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水平不能滿足需要所致,具體機(jī)制并不清楚。而本實驗結(jié)果顯示,給予慢性不可預(yù)見性溫和應(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)為糖水偏愛率顯著提高及懸尾不動時間顯著減少。反之,當(dāng)正常大鼠外源性給予 5-HT1A受體拮抗劑 WAY100635后,大鼠糖水偏愛率顯著降低,懸尾不動時間顯著增加。這表明,盡管應(yīng)激引起抑郁樣行為發(fā)生時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)果與之前的研究報道相一致(Li et al.,2009;Laugeray et al.,2010;Venzala et al.,2013)。由此推測,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 5-HT水平不能滿足需要所致。當(dāng)然,也有應(yīng)激時突觸后膜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)中被廣泛報道。與健康對照組相比,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,OFC在抑郁癥的發(fā)生中具有重要作用。OFC主要接受來自中縫背核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)見性應(yīng)激通過改變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,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)。本實驗結(jié)果也顯示,CUMS大鼠OFC區(qū)Glu含量顯著高于正常對照組,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),本實驗通過微量注射抗抑郁藥5-HT1A受體激動劑 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),非競爭性的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Glu能興奮性突觸調(diào)節(jié)異常,使興奮性突觸過度激活所致(Cai et al.,2013)。而興奮性突觸的過度激活,很有可能打破了 OFC區(qū)Glu和GABA原有的平衡,導(dǎo)致抑郁。另有研究報道,在治療抑郁癥及其相關(guān)的認(rèn)知紊亂中,5-HT可通過5-HT1A受體對Glu進(jìn)行調(diào)節(jié)(Ciranna,2006;Pehrson &Sanchez,2014)。慢性不可預(yù)見性應(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)。本實驗結(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Glu水平控制失調(diào),使得Glu水平過高所致。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,但在本研究整個實驗的各個項目中,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 Glu能神經(jīng)元上的5-HT1A受體抑制 Glu水平過度升高,而未明顯影響眶額葉GABA能神經(jīng)元,這可能與相對于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,并未降低OFC中5-HT的水平,GABA水平也沒有明顯變化,而是顯著升高Glu含量。外源性注射 5-HT1A受體激動劑產(chǎn)生抗抑郁作用的同時,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Glu能神經(jīng)元的有效調(diào)控,因而使OFC區(qū)Glu水平過高所致。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 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)果對解釋基于單胺類遞質(zhì)的抗抑郁藥物延遲現(xiàn)象也可能具有一定的啟示。

Almeida,P.V.G.,Trovo,M.C.,Tokumoto,A.M.,Pereira,A.C.,&Padovan,C.M.(2013).Role of serotonin 1A receptors in the median raphe nucleus on the behavioral consequences of forced swim stress.

Journal of Psychopharmacology,27

(12),1134–1140.Andolina,D.,Maran,D.,Valzania,A.,Conversi,D.,&Puglisi-Allegra,S.(2013).Prefrontal/amygdalar system determines stress coping behavior through 5-HT/GABA connection.

Neuropsychopharmacology,38

(10),2057–2067.Bambico,F.R.,Nguyen,N.-T.,&Gobbi,G.(2009).Decline in serotonergic firing activity and desensitization of 5-HT1A autoreceptors after chronic unpredictable stress.

European Neuropsychopharmacology,19

(3),215–228.Bremner,J.D.,Vythilingam,M.,Vermetten,E.,Nazeer,A.,Adil,J.,Khan,S.,...Charney,D.S.(2002).Reduced volume of orbitofrontal cortex in major depression.

Biological Psychiatry,51

(4),273–279.Cai,X.,Kallarackal,A.J.,Kvarta,M.D.,Goluskin,S.,Gaylor,K.,Bailey,A.M.,...Thompson,S.M.(2013).Local potentiation of excitatory synapses by serotonin and its alteration in rodent models of depression.

Nature Neuroscience,16

(4),464–472.Carr,G.V.,&Lucki,I.(2011).The role of serotonin receptor subtypes in treating depression: A review of animal studies.

Psychopharmacology,213

(2–3),265–287.Ci,L.,&An,S.C.(2007).Effect and mechanisms of dopamine in orbitofrontal cortex on the regulation of gastric motility.

Journal of Shaanxi Normal University(Natural Science Edition),35

(1),99–102.

[慈蕾,安書成.(2007).眶額葉區(qū)多巴胺對胃運(yùn)動的影響及其機(jī)制研究.陜西師范大學(xué)學(xué)報(自然科學(xué)版),35(1),99–102.]

Ciranna,L.(2006).Serotonin as a modulator of glutamateand GABA-mediated neurotransmission: implications in physiological functions and in pathology.

Current Neuropharmacology,4

(2),101–114.Cubala,W.J.,&Landowski,J.(2006).Serotoninergic system and limbic-hypothalamic-pituitary-adrenal axis (LHPA axis)in depression.

Psychiatria Polska,40

(3),415–430.de Almeida,J.,&Mengod,G.(2008).Serotonin 1A receptors in human and monkey prefrontal cortex are mainly expressed in pyramidal neurons and in a GABAergic interneuron subpopulation: Implications for schizophrenia and its treatment.

Journal of Neurochemistry,107

(2),488–496.Drevets,W.C.,Thase,M.E.,Moses-Kolko,E.L.,Price,J.,Frank,E.,Kupfer,D.J.,&Mathis,C.(2007).Serotonin-1A receptor imaging in recurrent depression: Replication and literature review.

Nuclear Medicine and Biology,34

(7),865–877.Frisardi,V.,Panza,F.,&Farooqui,A.A.(2011).Late-life depression and Alzheimer's disease: The glutamatergic system inside of this mirror relationship.

Brain Research Reviews,67

(1–2),344–355.Gao,J.,Wang,H.,Liu,Y.,Li,Y.Y.,Chen,C.,Liu,L.M.,...Yang,C.(2014).Glutamate and GABA imbalance promotes neuronal apoptosis in hippocampus after stress.

Medical Science Monitor,20

,499–512.Gao,S.F.(2012).

The pathological significance of GABA and NO in depressive patients and stress models

(Unpublished doctorial dissertation).Zhejiang University,Hangzhou.

[高尚鋒.(2012).神經(jīng)遞質(zhì)GABA和NO在抑郁癥患者和應(yīng)激模型中的病理意義(博士學(xué)位論文).浙江大學(xué),杭州.]

Gigliucci,V.,O'Dowd,G.,Casey,S.,Egan,D.,Gibney,S.,&Harkin,A.(2013).Ketamine elicits sustained antidepressantlike activity via a serotonin-dependent mechanism.

Psychopharmacology,228

(1),157–166.Hashimoto,K.(2009).Emerging role of glutamate in the pathophysiology of major depressive disorder.

Brain Research Reviews,61

(2),105–123.Hashimoto,K.,Sawa,A.,&Iyo,M.(2007).Increased levels of glutamate in brains from patients with mood disorders.

Biological Psychiatry,62

(11),1310–1316.He,T.,Qiao,H.,&An,S.C.(2011,November).

Glu,GABA and their receptors in depression induced by chronic unpredictable mild stress.

Paper presented at the meeting of Academic Conferences on Digestion,Endocrine Secretion and Reproduction in 2011,Beihai,Guangxi,China.

[何婷,喬卉,安書成.(2011,11月).慢性應(yīng)激性抑郁發(fā)生與海馬Glu和GABA水平變化及其受體機(jī)制.中國生理學(xué)會消化內(nèi)分泌生殖代謝生理專業(yè)委員會2011年消化內(nèi)分泌生殖學(xué)術(shù)會議,中國廣西北海.]

Huo,F.Q.,Chen,T.,Lv,B.C.,Wang,J.,Zhang,T.,Qu,C.L.,...Tang,J.S.(2009).Synaptic connections between GABAergic elements and serotonergic terminals or projecting neurons in the ventrolateral orbital cortex.

Cerebral Cortex,19

(6),1263–1272.Huo,F.Q.,Qu,C.L.,Li,Y.Q.,Tang,J.S.,&Jia,H.(2008).GABAergic modulation is involved in the ventrolateral orbital cortex 5-HTreceptor activation-induced antinociception in the rat.

Pain,139

(2),398–405.Jiang,Y.F.(2012).

The effects of Shuyu capsule and bupleurum extract serum on GABAR-mediated signal pathway of ERK and AC/cAMP/CREB in primary cultured rat hippocampus neurons

(Unpublished master’s thesis).Shandong University of Traditional Chinese Medicine,Jinan.

[姜英鳳.(2012).舒郁膠囊及柴胡提取物含藥血清對大鼠海馬神經(jīng)元GABAR介導(dǎo)的ERK通路和AC/cAMP/CREB通路的影響(碩士學(xué)位論文).山東中醫(yī)藥大學(xué),濟(jì)南.]

Jiang,Z.C.,Qi,W.J.,Wang,J.Y.,&Luo,F.(2014).Chronic administration of 5-HT1A receptor agonist relieves depression and depression-induced hypoalgesia.

Scientific World Journal,2014

,405736.Kang,H.T.,&Wang,C.W.(2010).Effect of electro-acupuncture on 5-HTreceptor of hippocampus of the rat model with chronic stress depression.

Henan Traditional Chinese Medicine,30

(1),38–40.

[康匯婷,王朝偉.(2010).電針對慢性應(yīng)激抑郁模型大鼠海馬5-HT受體的影響.河南中醫(yī),30(1),38–40.]

Kendell,S.F.,Krystal,J.H.,&Sanacora,G.(2005).GABA and glutamate systems as therapeutic targets in depression and mood disorders.

Expert Opinion on Therapeutic Targets,9

(1),153–168.Kornstein,S.G.,&Schneider,R.K.(2001).Clinical features of treatment-resistant depression.

The Journal of Clinical Psychiatry,62

(Suppl.16),18–25.Kü?ükibrahimo?lu,E.,Sayg?n,M.Z.,?ali?kan,M.,Kaplan,O.K.,ünsal,C.,&G?ren,M.Z.(2009).The change in plasma GABA,glutamine and glutamate levels in fluoxetine- or S-citalopram-treated female patients with major depression.

European Journal of Clinical Pharmacology,65

(6),571–577.Kumar,K.,Sharma,S.,Kumar,P.,&Deshmukh,R.(2013).Therapeutic potential of GABAreceptor ligands in drug addictio-n,anxiety,depression and other CNS disorders.

Pharmacology Biochemistry and Behavior,110

,174–184.Lacerda,A.L.T.,Keshavan,M.S.,Hardan,A.Y.,Yorbik,O.,Brambilla,P.,Sassi,R.B.,...Soares,J.C.(2004).Anatomic evaluation of the orbitofrontal cortex in major depressive disorder.

Biological Psychiatry,55

(4),353–358.Laugeray,A.,Launay,J.-M.,Callebert,J.,Surget,A.,Belzung,C.,&Barone,P.R.(2010).Peripheral and cerebral metabolic abnormalities of the tryptophan-kynurenine pathway in a murine model of major depression.

Behavioural Brain Research,210

(1),84–91.Levine,J.,Panchalingam,K.,Rapoport,A.,Gershon,S.,McClure,R.J.,&Pettegrew,J.W.(2000).Increased cerebrospinal fluid glutamine levels in depressed patients.

Biological Psychiatry,47

(7),586–593.Li,H.P.,An,F.L.,&An,S.C.(2009).Orbitofrontal cortex action of 5-hydroxytryptamine and its receptor in an acute forced swimming stress-induced depression model.

Neural Regeneration Research,4

(7),530–535.Liu,H.,Wen,L.M.,Qiao,H.,&An,S.C.(2013).Modulation of hippocampal glutamate and NMDA/AMPA receptor by homocysteine in chronic unpredictable mild stress-induced rat depression.

Acta Physiologica Sinica,65

(1),61–71

.

[劉慧,問黎敏,喬卉,安書成.(2013).高半胱氨酸對慢性應(yīng)激性抑郁大鼠海馬谷氨酸及其受體的調(diào)節(jié).生理學(xué)報,65(1),61–71.]

Luo,D.D.,An,S.C.,&Zhang,X.(2008).Involvement of hippocampal serotonin and neuropeptide Y in depression induced by chronic unpredicted mild stress.

Brain Research Bulletin,77

(1),8–12.Lussier,A.L.,Romay-Tallón,R.,Caruncho,H.J.,&Kalynchuk,L.E.(2013).Altered GABAergic and glutamatergic activity within the rat hippocampus and amygdala in rats subjected to repeated corticosterone administration but not restraint stress.

Neuroscience,231

,38–48.Mahar,I.,Bambico,F.R.,Mechawar,N.,&Nobrega,J.N.(2014).Stress,serotonin,and hippocampal neurogenesis in relation to depression and antidepressant effects.

Neuroscience and Biobehavioral Reviews,38

,173–192.Muller,C.P.,&Jacobs,B.(2009).

Handbook of the behavioral neurobiology of serotonin

(Vol.21).Amsterdam: Academic Press.Musazzi,L.,Treccani,G.,Mallei,A.,&Popoli,M.(2013).The action of antidepressants on the glutamate system:Regulation of glutamate release and glutamate receptors.

Biological Psychiatry,73

(12),1180–1188.Paxinos,G.,&Watson,C.(1998).

The rat brain in stereotaxic coordinates

(4th ed.).San Diego: Academic Press.Pehrson,A.,Li,Y.,Haddjeri,N.,Gulinello,M.,&Sanchez,C.(2013).P.1.g.014 Vortioxetine,a novel multimodal antidepressant,modulates GABA and glutamate neurotransmission via serotonergic mechanisms.

European Neuropsychopharmacology,23

(Suppl.2),S196-S197.Pehrson,A.L.,&Sanchez,C.(2014).Serotonergic modulation of glutamate neurotransmission as a strategy for treating depression and cognitive dysfunction.

CNS spectrums,19

(2),121–133.Quandt,G.,H?fner,G.,&Wanner,K.T.(2013).Synthesis and evaluation of N-substituted nipecotic acid derivatives with an unsymmetrical bis-aromatic residue attached to a vinyl ether spacer as potential GABA uptake inhibitors.

Bioorganic&Medicinal Chemistry,21

(11),3363–3378.Roberts,A.C.(2011).The importance of serotonin for orbitofrontal function.

Biological Psychiatry,69

(12),1185–1191.Sachs,G.S.(2003).Unmet clinical needs in bipolar disorder.

Journal of Clinical Psychopharmacology,23

(3 Suppl.1),S2-S8.Sanacora,G.,Gueorguieva,R.,Epperson,C.N.,Wu,Y.T.,Appel,M.,Rothman,D.L.,… Mason,G.F.(2004).Subtype-specific alterations of γ-aminobutyric acid and glutamate in patients with major depression.

Archives of General Psychiatry,61

(7),705–713.Santana,N.,Bortolozzi,A.,Serrats,J.,Mengod,G.,&Artigas,F.(2004).Expression of serotoninand serotoninreceptors in pyramidal and GABAergic neurons of the rat prefrontal cortex.

Cerebral Cortex,14

(10),1100–1109.Savitz,J.,Lucki,I.,&Drevets,W.C.(2009).5-HTreceptor function in major depressive disorder.

Progress in Neurobiology,88

(1),17–31.Simpson,M.D.C.,Lubman,D.I.,Slater,P.,&Deakin,J.F.W.(1996).Autoradiography with [H]8-OH-DPAT reveals increases in 5-HTreceptors in ventral prefrontal cortex in schizophrenia.

Biological Psychiatry,39

(11),919–928.Stein,D.J.,Miczek,K.A.,Lucion,A.B.,&de Almeida,R.M.M.(2013).Aggression-reducing effects of F15599,a novel sele-ctive 5-HTreceptor agonist,after microinjection into the ventral orbital prefrontal cortex,but not in infralimbic cortex in male mice.

Psychopharmacology,230

(3),375–387.Tang,Y.M.,Zhao,H.S.,Qin,L.X.,Zhang,R.S.,Chen,R.H.,&Liu,Y.(2013).Glutamate and GABA in brain tissue of chronic unpredicted mild stress-induced depression rats.

Guangdong Medical Journal,34

(20),3098–3101.

[唐亞梅,趙宏深,秦立新,張仁生,陳若虹,劉勇.(2013).慢性輕度不可預(yù)見性應(yīng)激抑郁模型大鼠腦組織谷氨酸和γ-氨基丁酸濃度的變化.廣東醫(yī)學(xué),34(20),3098–3101.]

Taylor,W.D.,Macfall,J.R.,Payne,M.E.,McQuoid,D.R.,Steffens,D.C.,Provenzale,J.M.,&Krishnan,K.R.(2007).Orbitofrontal cortex volume in late life depression:Influence of hyperintense lesions and genetic polymorphisms.

Psychological Medicine,37

(12),1763–1773.Venzala,E.,García-García,A.L.,Elizalde,N.,&Tordera,R.M.(2013).Social

vs

.environmental stress models of depression from a behavioural and neurochemical approach.

European Neuropsychopharmacology,23

(7),697–708.Wen,L.M.,An,S.C.,&Liu,H.(2012).Role of hippocampal 5-HTreceptor and its modulation to NMDA receptor and AMPA receptor in depression induced by chronic unpredictable mild stress.

Acta Psychologica Sinica,44

(10),1318–1328.

[問黎敏,安書成,劉慧.(2012).應(yīng)激性抑郁樣行為發(fā)生中海馬 5-羥色胺 1A受體的作用及其對 NMDA受體和AMPA受體的調(diào)節(jié).心理學(xué)報,44(10),1318–1328.]

Wu,S.,An,S.C.,Chen,H.B.,&Li,F.(2014).Orbital frontal cortex D1 dopamine receptor modulate glutamate and NMDA receptor in depression induced by chronic unpredictable mild stress.

Acta Psychologica Sinica,46

(1),1–10.

[吳帥,安書成,陳慧彬,李菲.(2014).慢性應(yīng)激性抑郁發(fā)生中大鼠眶額葉多巴胺D1受體對谷氨酸及其NMDA受體的調(diào)節(jié).心理學(xué)報,46(1),1–10.]

Yu,L.,An,S.C.,&Lian,T.(2010).Involvement of hippocampal NMDA receptor and neuropeptide Y in depression induced by chronic unpredictable mild stress.

Acta Physiologica Sinica,62

(1),14–22.

[余伶,安書成,廉婷.(2010).海馬NMDA受體與神經(jīng)肽Y在慢性應(yīng)激性抑郁發(fā)生中的作用及其關(guān)系.生理學(xué)報,62(1),14–22.]

Zhang,H.W.,Chen,Z.Q.,Jia,Z.Y.,&Gong,Q.Y.(2014).Dysfunction of neural circuitry in depressive patients with suicidal behaviors: A review of structural and functional neuroimaging studies.

Progress in Neuro-Psychopharmacology and Biological Psychiatry,53

,61–66.Zhong,P.,&Yan,Z.(2004).Chronic antidepressant treatment alters serotonergic regulation of GABA transmission in prefrontal cortical pyramidal neurons.

Neuroscience,129

(1),65–73.Zhou,J.S.,Cao,X.,Mar,A.C.,Ding,Y.Q.,Wang,X.P.,Li,Q.,&Li,L.J.(2014).Activation of postsynaptic 5-HTreceptors improve stress adaptation.

Psychopharmacology,231

(10),2067–2075.