腦室內(nèi)注射5,7-雙羥色胺對(duì)內(nèi)側(cè)前額葉皮層錐體神經(jīng)元5-HT1A受體敏感性的影響

劉彥彤,高 捷,王 爽

(1.西安醫(yī)學(xué)院生物化學(xué)與分子生物學(xué)教研室,陜西西安710021;2.西安醫(yī)學(xué)院管理學(xué)教研室,陜西西安 710021;3.西安醫(yī)學(xué)院病理生理學(xué)教研室,陜西西安 710021)

腦室內(nèi)注射5,7-雙羥色胺對(duì)內(nèi)側(cè)前額葉皮層錐體神經(jīng)元5-HT1A受體敏感性的影響

劉彥彤1,高 捷2,王 爽3

(1.西安醫(yī)學(xué)院生物化學(xué)與分子生物學(xué)教研室,陜西西安710021;2.西安醫(yī)學(xué)院管理學(xué)教研室,陜西西安 710021;3.西安醫(yī)學(xué)院病理生理學(xué)教研室,陜西西安 710021)

目的:探討腦室內(nèi)注射5,7-雙羥色胺(5,7-DHT)對(duì)內(nèi)側(cè)前額葉皮層(mPFC)錐體神經(jīng)元5-羥色胺-1A(5-HT1A)受體敏感性的影響,闡明5-HT1A受體對(duì)錐體神經(jīng)元電活動(dòng)的作用。方法:36只雄性SD大鼠隨機(jī)分為假手術(shù)組(n＝21)和5,7-DHT損毀組(n＝15)。損毀組大鼠腦室內(nèi)注射5,7-DHT,假手術(shù)組大鼠腦室內(nèi)注射同等劑量生理鹽水,2組大鼠靜脈注射不同劑量(0.5～128.0μg·kg－1)5-HT1A受體激動(dòng)劑8-OH-DPAT,采用體細(xì)胞外電生理學(xué)方法觀(guān)察mPFC中錐體神經(jīng)元放電頻率的變化,并靜脈注射5-HT1A受體拮抗劑WAY100635,觀(guān)察損毀組大鼠對(duì)5,7-DHT激動(dòng)劑和拮抗劑的敏感性,并與假手術(shù)組進(jìn)行比較。結(jié)果:在假手術(shù)組中,不同劑量(0.5～128.0μg·kg－1)8-OH-DPAT對(duì)大鼠錐體神經(jīng)元的放電頻率均產(chǎn)生興奮-抑制式的影響,這些神經(jīng)元在低劑量(0.5～32.0μg·kg－1)時(shí)被興奮,放電頻率增加(P＜0.05);而在高劑量(128.0μg· kg－1)時(shí)則被抑制,放電頻率減少。在損毀組中,不同劑量(0.5～128.0μg·kg－1)8-OH-DPAT劑量依賴(lài)性地抑制大鼠錐體神經(jīng)元的電活動(dòng)(df＝5,F＝3.44,P＝0.003),即放電頻率減少,未見(jiàn)興奮-抑制效應(yīng); WAY10035可以反轉(zhuǎn)8-OH-DPAT的抑制作用。結(jié)論:腦室內(nèi)注射5,7-DHT可使大鼠mPFC錐體神經(jīng)元5-HT1A受體敏感性降低。

5,7-雙羥色胺;5-HT1A受體;內(nèi)側(cè)前額葉皮層;錐體神經(jīng)元;電生理學(xué)

研究[1]證明:內(nèi)側(cè)前額葉皮層(medial prefrontal cortex,mPFC)中50%～60%的錐體神經(jīng)元和20%～30%的中間神經(jīng)元上有5-羥色胺-1A (5-hydroxytryptamine-1A,5-HT1A)受體的表達(dá)。5-HT1A受體通過(guò)G蛋白與K＋通道耦聯(lián)。激活5-HT1A受體,可使細(xì)胞膜產(chǎn)生超極化,從而降低神經(jīng)元的活動(dòng)[2]。實(shí)驗(yàn)[3]表明:刺激中縫背核(dorsal raphénuclei,DRN)與中縫中核(median raphénuclei,MRN)可以通過(guò)激活5-HT1A受體而抑制m PFC中錐體神經(jīng)元的電活動(dòng)。在m PFC中,微電泳5-羥色胺(5-hydroxytryptamine,5-HT)和5-HT受體激動(dòng)劑也可以通過(guò)5-HT1A受體產(chǎn)生抑制作用。但5-HT遞質(zhì)系統(tǒng)對(duì)于mPFC錐體神經(jīng)元以及5-HT1A受體的作用并不十分清楚。本實(shí)驗(yàn)以腦室內(nèi)注射5,7-雙羥色胺(5,7-dihydroxytryptamine,5,7-DHT)大鼠為研究對(duì)象,采用神經(jīng)電生理學(xué)方法,靜脈注射5-HT1A受體激動(dòng)劑8-OH-DPAT,記錄mPFC錐體神經(jīng)元放電頻率的改變,觀(guān)察5-HT1A受體敏感性的變化。

1 材料與方法

1.1 實(shí)驗(yàn)動(dòng)物和主要藥品36只雄性SD大鼠,體質(zhì)量230～320 g,由西安交通大學(xué)醫(yī)學(xué)院實(shí)驗(yàn)動(dòng)物中心提供,許可證號(hào):SCXK(陜)2007-001。大鼠在標(biāo)準(zhǔn)環(huán)境飼養(yǎng),室溫20℃～25℃,24 h晝夜循環(huán)光照,自由攝食飲水。大鼠隨機(jī)分為假手術(shù)組(n＝21)和損毀組(n＝15)。5,7-DHT和地西帕明購(gòu)自美國(guó)Sigma公司。

1.2 腦室內(nèi)注射5,7-DHT損毀組大鼠經(jīng)4%水合氯醛(400 mg·kg－1)腹腔注射麻醉,頭部固定于立體定位儀上,根據(jù)《Paxinos-Watson大鼠腦定位圖譜》確定左側(cè)mPFC的坐標(biāo)位置:AP 1.30 mm,L 2 mm,D 3.8 mm[4]。注射5,7-DHT 20 g·L－1:在注射5,7-DHT前,先給大鼠注射地西帕明(25 mg·kg－1)以保護(hù)去甲腎上腺素能神經(jīng)元,30 min后再注射5,7-DHT;假手術(shù)組大鼠以相同的方法給予10μL生理鹽水。

1.3 電生理記錄在腦室注射5,7-DHT后第3周進(jìn)行電生理記錄。大鼠在4%水合氯醛(400 mg·kg－1)腹腔注射麻醉下行氣管及靜脈插管術(shù)后,頭部固定于立體定位儀上,根據(jù)《Paxinos-Watson大鼠腦定位圖譜》,確定右側(cè)m PFC的坐標(biāo)位置:AP 2.7～3.2 mm,L 0.1～0.6 mm,D 1.5～2.5 mm[4]。在冠狀面與中線(xiàn)成2°角處傾斜進(jìn)針,以避免損傷矢狀竇。采用玻璃微電極細(xì)胞外記錄法記錄錐體神經(jīng)元的放電頻率。將信噪比大于3∶1的、穩(wěn)定的單細(xì)胞放電經(jīng)生物電信號(hào)采集與分析系統(tǒng)(CED1401 Spike2)輸入計(jì)算機(jī)后,進(jìn)行實(shí)時(shí)觀(guān)察、儲(chǔ)存和頻率分析。采樣時(shí)間10～20 min。整個(gè)實(shí)驗(yàn)過(guò)程中監(jiān)測(cè)大鼠的心電變化和直腸溫度(維持在37.0°C±0.5°C)。記錄到錐體神經(jīng)元自發(fā)放電后,穩(wěn)定10 min,然后通過(guò)頸外靜脈注射連續(xù)給予不同劑量(0.5、2.0、8.0、32.0 和128.0μg·kg－1)的8-OH-DPAT溶液,間隔約3 min;最后1個(gè)劑量的8-OH-DPAT注射后,再靜脈注射給予5-HT1A受體拮抗劑WAY100635 (50μg·kg－1),繼續(xù)觀(guān)察放電10 min。每次注射的溶液量為0.1 m L。在藥物注射前,記錄被確認(rèn)神經(jīng)元的基礎(chǔ)電活動(dòng)5～10 min。體循環(huán)給予8-OH-DPAT和WAY100635后,觀(guān)察和分析第2～3 min的放電頻率變化。

1.4 錐體神經(jīng)元的確認(rèn)mPFC中錐體神經(jīng)元通常表現(xiàn)為自發(fā)放電;AP波寬＞1 ms;呈現(xiàn)規(guī)則、不規(guī)則或爆發(fā)式的放電形式[4]。

1.5 統(tǒng)計(jì)學(xué)分析采用SPSS 13.0統(tǒng)計(jì)學(xué)軟件進(jìn)行統(tǒng)計(jì)學(xué)分析。2組大鼠放電頻率均以±s表示,不同劑量8-OH-DPAT作用后放電頻率的變化應(yīng)用單因素方差分析,組間比較以基礎(chǔ)放電頻率為標(biāo)準(zhǔn),應(yīng)用Dunnett’s多重比較。

2 結(jié)果

在假手術(shù)組中,0.5～128.0μg·kg－18-OHDPAT對(duì)大鼠錐體神經(jīng)元的放電頻率均產(chǎn)生興奮-抑制式的影響;低劑量(0.5～32.0μg·kg－1)時(shí),錐體神經(jīng)元被興奮,放電頻率增加;而在高劑量(128μg·kg－1)時(shí)錐體神經(jīng)元被抑制,放電頻率減少。低劑量的興奮作用在8.0μg·kg－1時(shí)最強(qiáng),放電頻率增加到(3.25±0.70)Hz,與基礎(chǔ)放電頻率比較差異有統(tǒng)計(jì)學(xué)意義(P＜0.01);當(dāng)8-OHDPAT的累積劑量為128.0μg·kg－1時(shí),錐體神經(jīng)元?jiǎng)t被抑制,放電頻率下降至(0.07±0.01)Hz;這種抑制作用也可以被WAY100635 (50μg·kg－1)完全反轉(zhuǎn),使大鼠錐體神經(jīng)元的放電率恢復(fù)為(0.46±0.06)Hz(圖1A和表1)。與假手術(shù)組比較,在損毀組中相同劑量(0.5～128.0μg·kg－1)8-OH-DPAT劑量依賴(lài)性地抑制大鼠錐體神經(jīng)元的電活動(dòng),而無(wú)興奮-抑制效應(yīng)。8-OH-DPAT只有達(dá)到累積劑量128μg·kg－時(shí)才能將錐體神經(jīng)元放電頻率抑制到(0.34±0.07)Hz (df＝5,F＝3.44,P＜0.05)(圖1 B和表1)。

圖1 2組大鼠mPFC中錐體神經(jīng)元的放電頻率Fig.1 Firing rates of pyramidal neurons in mPFC of rats in two groupsA:Sham operation group;B:5,7-DHT-lesion group.

表1 5-HT 1A受體激動(dòng)劑8-OH-DPAT作用下2組大鼠mPFC錐體神經(jīng)元放電頻率Tab.1 Firing rates of pyramidal neurons in mPFC of rats in two groups after treated with 5-HT1Areceptor agonist 8-OH-DPAT(±s,Hz)

表1 5-HT 1A受體激動(dòng)劑8-OH-DPAT作用下2組大鼠mPFC錐體神經(jīng)元放電頻率Tab.1 Firing rates of pyramidal neurons in mPFC of rats in two groups after treated with 5-HT1Areceptor agonist 8-OH-DPAT(±s,Hz)

?P＜0.05,??P＜0.01 compared with basal firing rate.

Firing rate Group n Basal 8-OH-DPAT(μg·kg－1) 0.5 2.0 8.0 32.0 128.0 Sham operation 21 0.35±0.05 1.41±0.30 2.32±0.40 3.25±0.70??1.38±0.17 0.07±0.01 5,7-DHT-lesion 15 1.50±0.29 1.38±0.30 1.23±0.24 1.05±0.21 0.80±0.17 0.34±0.07?

3 討論

本實(shí)驗(yàn)中所討論的神經(jīng)元均位于mPFC內(nèi),其電活動(dòng)的特征符合錐體神經(jīng)元的鑒定標(biāo)準(zhǔn)。本文作者觀(guān)察到腦室內(nèi)注射5,7-DHT后,m PFC的錐體神經(jīng)元5-HT1A受體敏感性降低,8-OHDPAT對(duì)錐體神經(jīng)元的興奮-抑制效應(yīng)消失,可能與以下原因有關(guān):①5-HT1A受體在大鼠腦內(nèi)的廣泛分布,除了存在于錐體細(xì)胞的胞體或(和)軸丘上外,5-HT1A受體也在mPFC的γ-氨基丁酸(GABA)能中間神經(jīng)元上有表達(dá)。低劑量8-OHDPAT對(duì)錐體神經(jīng)元產(chǎn)生的興奮作用,其可能性之一是:mPFC中GABA能中間神經(jīng)元優(yōu)先被抑制,釋放GABA的量減少,結(jié)果導(dǎo)致錐體神經(jīng)元的去抑制[5]。Diaz-Mataix等[6]也報(bào)道了體循環(huán)應(yīng)用低劑量5-HT1A受體激動(dòng)劑BAY×3702能夠?qū)е洛F體神經(jīng)元的電活動(dòng)增強(qiáng),并被GABA能中間神經(jīng)元拮抗劑反轉(zhuǎn),說(shuō)明低劑量BAY×3702優(yōu)先作用于5-HT1A受體;另外,突觸前5-HT1A受體可能也參與錐體神經(jīng)元活動(dòng)的調(diào)節(jié)。皮層中也有5-HT7受體表達(dá),并且與Gs蛋白耦聯(lián)促使c AMP的產(chǎn)生[7],因此刺激5-HT7受體也可能產(chǎn)生神經(jīng)元的興奮。8-OH-DPAT對(duì)5-HT7受體有很高的親和力[8],所以小劑量應(yīng)用8-OH-DPAT對(duì)m PFC的錐體神經(jīng)元產(chǎn)生的興奮作用也可能是由于興奮了5-HT7受體所產(chǎn)生的結(jié)果。但是,現(xiàn)在并無(wú)證據(jù)表明在錐體和GABA能中間神經(jīng)元上有5-HT7受體的表達(dá),這仍需要進(jìn)一步的研究。低劑量8-OHDPAT對(duì)錐體神經(jīng)元產(chǎn)生的興奮作用可能性之二, 即8-OH-DPAT產(chǎn)生的興奮作用是因?yàn)樽饔糜谄渌X區(qū)的5-HT1A受體[9]。許多研究[10]證明:DRN 和MRN對(duì)mPFC神經(jīng)元的活動(dòng)起重要的調(diào)節(jié)作用,體循環(huán)應(yīng)用5-HT1A受體激動(dòng)劑8-OH-DPAT抑制DRN和MRN中5-HT能神經(jīng)元的電活動(dòng),繼而對(duì)mPFC錐體神經(jīng)元的抑制作用減弱,導(dǎo)致錐體神經(jīng)元的電活動(dòng)增強(qiáng)。②高劑量8-OH-DPAT對(duì)錐體神經(jīng)元產(chǎn)生的抑制作用則很有可能是由于直接激活了所記錄的錐體神經(jīng)元上的5-HT1A受體而產(chǎn)生的反應(yīng)[11]。雖然不同細(xì)胞上5-HT1A受體敏感性的差異還有待進(jìn)一步研究,但以往的研究支持這種可能性,因?yàn)椴煌窠?jīng)元上受體表達(dá)的水平有差異。③在損毀組,體循環(huán)應(yīng)用8-OH-DPAT (0.5～128.0μg·kg－1)抑制大鼠錐體神經(jīng)元的電活動(dòng),而不是產(chǎn)生興奮-抑制效應(yīng)。這種抑制作用在128.0μg·kg－1時(shí)具有顯著意義,而且可以被WAY100635完全反轉(zhuǎn),表明這種作用是通過(guò)5-HT1A受體實(shí)現(xiàn)的。對(duì)這種結(jié)果的解釋可以從以下的研究中得到答案。首先,由于腦室內(nèi)注射了5,7-DHT,導(dǎo)致了海馬結(jié)構(gòu)、中腦縫核和PFC中5-HT1A受體的密度明顯降低,因此降低了皮層內(nèi)5-HT1A受體影響錐體神經(jīng)元活動(dòng)的能力。其次,低劑量8-OH-DPAT對(duì)錐體神經(jīng)元產(chǎn)生的興奮作用在損毀組大鼠中的消失可能是由于來(lái)源于縫核和mPFC中GABA能中間神經(jīng)元的傳入增加[12],這些傳入能抑制m PFC錐體神經(jīng)元的電活動(dòng),因?yàn)?-HT1A受體在GABA能中間神經(jīng)元和5-HT能神經(jīng)元上密度降低導(dǎo)致8-OH-DPAT對(duì)GABA能和5-HT能神經(jīng)元活動(dòng)的抑制作用減弱。另外,低劑量8-OH-DPAT對(duì)錐體神經(jīng)元產(chǎn)生的興奮作用的消失也可能是由于海馬結(jié)構(gòu)向mPFC的GABA能神經(jīng)元的投射減少,這種投射可以抑制錐體神經(jīng)元的電活動(dòng)[13]。

綜上所述,本文作者推斷:低劑量應(yīng)用8-OHDPAT使錐體神經(jīng)元興奮性作用的消失可能反映了損毀組大鼠5-HT1A受體功能失調(diào)或該受體在mPFC、縫核和海馬結(jié)構(gòu)中的表達(dá)降低。

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Influence of intraventricular injection of 5,7-drhydroxytryptamine in 5-HT1Areceptor sensitivity of pyramidal neurons in medial prefrontal cortex

LIU Yan-tong1,GAO Jie2,WANG Shuang3
(1.Department of Biochemistry and Molecular Biology,Xi’an Medical University,Xi’an 710021,China; 2.Department of Management,Xi’an Medical University,Xi’an 710021,China;3.Department of Pathophysiology,Xi’an Medical University,Xi’an 710021,China)

ObjectiveTo explore the influence of intraventricular injection of 5,7-drhydroxytryptamine(5,7-DHT)in 5-HT1Areceptor sensitivity of medial prefrontal cortex pyramidal neurons in the rats,and to clarity the effect of 5-HT1Areceptor on the eletronic response of pyramidal neurons.Methods36 male SD rats were randomly divided into sham operation group(n＝21)and 5,7-DHT lesion group(n＝15).5,7-DHT was injected intraventricularly in the rats in 5,7-DHT lesion group,and the same dose saline was injected in the rats in sham operation group.The rats in two groups were intravenously injected with different doses(0.5－128.0μg·kg－1)of 8-CH-DPAT.The firing rate of mPFC pyramidal neurons was recorded with extracellular electrophysioologicalexamination.The rats in two groups were intravenously injected with WAY100635,the sensitivites of the rats to 8-OH-DPAT and WAY100635 in 5,7-DHT lesion group were observed,and compared with sham operation group.ResultsThe different doses(0.5－128.0μg·L－1)of 8-OH-DDAT had an excitatory-inhibitory effect on the firing rate of mPFC pyamidal neurons in sham operation group;the neurons were excited when the doses of 8-OH-DPAT were 0.5－38.0μg·kg－1,and the firing rates were increased(P＜0.05);the neurons were inhibited when the dose of 8-OH-DPAT was 128.0μg·kg－1,and the firing rate was decreased.The different doses(0.5－218.0μg·L－1)of 8-OH-DPAT inhibited the elecctronic response of pyramidal neurons of the rats in 5,7-DHT lesion group in a dose-dependent manner(df＝5,F＝3.44,P＝0.003),and the firing rates were reduced.WAY-100635(50μg·kg－1)reversed completely the inhibition of 8-OH-DPAT.ConclusionThe sensitivity of 5-HT1Areceptor of rat mPFC pyramidal neurons can be decreased by intraventricular injection of 5,7-DHT.

5,7-drhydroxytryptamine;5-HT1Areceptor;medial prefrontal cortex;pyramidal neurons; electrophysiology

R338.2

A

2014-04-09

陜西省科技廳科研基金資助課題(2012KJXX-43);陜西省教育廳科研基金資助課題(2013JK0761)

劉彥彤(1980－),女,陜西省咸陽(yáng)市人,講師,理學(xué)碩士,主要從事神經(jīng)退行性疾病中樞機(jī)制的研究。

王 爽(Tel:029-86177414,E-mail:wangshuang78213＠163.com)

1671-587Ⅹ(2014)05-0958-04

10.13481/j.1671-587x.20140510