Orexins對小鼠海馬迴神經突觸可塑性之研究

Effect of orexins on synaptic plasiticity of mouse hippocampal slices

李家旭(Chia-Hsu Lee)

指導教授 ： 邱麗珠

國立臺灣大學/醫學院/藥理學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.01301

摘要

分佈於下視丘為主的orexin neurons會經由腦部廣泛投射的軸突釋出orexins， 可分為orexnin A (hypocretin 1)和orexin B (hypocretin 2)，會與orexin receptors結合。這個受體是在1998年被去孤兒化的G蛋白偶合受體，又分做兩種亞型，orexin 1 receptor (OX1R)和orexin 2 receptor (OX2R)。OX1R 對於 orexin A 的親和力比orexin B高，但是OX2R對於兩者的親和力則相似。這兩種orexin receptors的在腦中分佈極廣，並與自下視丘orexin neurons投射出的軸突所釋放的orexins接合，被証實與許多生理病理的機轉有關。hippocampus 中也有orexin neurons投射的軸突和orexin receptor的分布，且在許多以大鼠實驗對象的in vivo實驗中被證實會影響hippocampus 的突觸可塑性以及學習和記憶的表現。而以大鼠和小鼠的hippocampus 腦切片為實驗材料的in vivo實驗中，亦發現orexins有影響神經基礎突觸傳遞和突觸可塑性的現象。但orexin的角色仍眾說紛紜。因此，我們利用小鼠hippocampus 切片，觀察Schaffer Collateral-CA1區域中，外給orexins對基礎突觸傳遞和神經可塑性，包括長期增益現象和去增益現象，有何種影響。藉由在Schaffer Collateral至CA1的傳遞路徑上給予電刺激，CA1區域中的stratum radiatum進行胞外紀錄，所得到的場興奮性突觸後電位(fEPSPs)進行分析。長期增益現象(long term potentiation, LTP)是由高頻所引發刺激，我們所用的高頻刺激是由三個相隔15秒的theta頻率構成之刺激組成，每個 theta頻率的刺激是由4段100 HZ的高頻刺激構成，每段相隔200秒。去增益現象(depotentiation)則是在高頻刺激後四十分鐘，由連續的低頻(1 Hz)刺激15分鐘所引發。
灌流oirexin A (30, 100, 300 nM) 10分鐘後，所測得fEPSPs的斜率，並沒有影響基礎傳遞突觸。但orexin A會有意義的抑制LTP且具濃度相依性。高頻刺激後4分鐘內的fEPSPs的斜率變化，即後強直增強效應(post-tetanic potetanic, PTP)，亦有輕微濃度相依的抑制情形，但不具統計上的差異。而各個濃度對去增益現象的影響，亦無統計上顯著差異。
灌流[Ala11, D-Leu15]-orexin B (3, 10, 30, 100, 300, 1000 nM) 10分鐘後，並沒有發現影響基礎傳遞突觸，在低濃度即可有意義的抑制LTP，但隨濃度的升高並無法增加抑制LTP的程度。觀察PTP，各組間不具統計上的差異。而各濃度對depotentiaion，亦無統計上的顯著影響。
將3 μM專一性OX1拮抗劑SB 334867與100 nM orexin A同時灌流，發現可部分反轉orexin A對LTP所造成的抑制。對PTP的輕微抑制亦有些許的反轉，但皆未達統計上顯著的差異。而各個濃度對depotentiation的影響，亦無統計上顯著差異。單獨投予SB 334867對基礎突觸傳遞、LTP、PTP和depotentiation並無影響。
再將3 μM專一性OX1拮抗劑SB 334867與300 nM [Ala11, D-Leu15]-orexin B同時灌流，發現可部分反轉[Ala11, D-Leu15]-orexin B對LTP所造成的抑制。對PTP並無影響。各組間對depotentiaion的影響，則無統計上顯著差異。
將30 μM專一性OX2拮抗劑 Compound 29與100 nM orexin A同時灌流，發現可部分反轉orexin A對LTP所造成的抑制，對PTP並無影響。各濃度對depotentiation的影響，則無統計上顯著差異。單獨投予Compound 29對基礎突觸傳遞、LTP、PTP和depotentiation並無影響。
再將30 μM專一性OX2拮抗劑 Compound 29 與300 nM [Ala11, D-Leu15]-orexin B同時灌流，發現可部分反轉[Ala11, D-Leu15]-orexin B對LTP所造成的抑制。對PTP並無影響。各組間對depotentiation的影響，則無統計上顯著差異。
在hippocampus CA1突觸會因極短的成對刺激(50 ms)後，鈣離子於突觸前堆積，而出現paired pulse facilitation的特性。計算paired pulse ratio (PPR)，由第二個fEPSP斜率除與第一個fEPSP斜率，來評估突觸前神經傳遞物釋放的變化。我們發現在給予高頻刺激後40分鐘會有PPR下降(1.62±0.04 vs. 1.51±0.03, n=4)，即突觸前神經傳遞物釋放增加。一般認為LTP是以突觸後NMDAR開啟來為主，而我們認為LTP應同時也有增加突觸前神經傳遞物釋放。orexin A (100 nM)並不影響基礎刺激下的PPR，但有輕微減少PPR下降程度的現象。
根據我們目前的實驗結果，在小鼠hippocampus CA1區域，發現orexin A和[Ala11, D-Leu15]-orexin B皆會部分抑制LTP，但並不影響depotentiation。對LTP的抑制效果，可能同時經由OX1R和OX2R，但OX1R可能佔有較大的比重。再者，突觸前的抑制效果可能很輕微，推測可能是在突觸後，藉由活化OX1R和OX2R活化PLC/PKC的途徑，造成抑制NMDA所媒介的電流為主。

關鍵字

海馬迴 ； Orexin ； 神經突觸可塑性 ； 長期增益現象 ； 去增益現象

並列摘要

Orexins are a novel family of two hypothalamic peptides, consisting of orexin A and orexin B, which were also named hypocretin 1 and hypocretin 2, respectively. They were identified to be the endogenous agonists of an orphan GPCR in 1998, which was, therefore, named orexin receptor and consists of OX1 and OX2 receptors. OX1 receptors display higher affinity for orexin A than orexin B, while OX2 receptors have similar affinity for both orexin A and orexin B. Orexin-containing neurons are mostly localized in the lateral hypothalamus and send projections widely through the brain, including the hippocampus. Orexin A had been reported to affect learning and memory tasks and hippocampal synaptic plasticity in vivo, mostly in the rat, as well as affected synaptic plasticity of hippocampal slices of rats and mice in vitro. However, controversial results were reported. We, therefore, investigated the effects of orexins on the synaptic plasticity, long-term potentiation (LTP) and depotentiation, at Schaffer Collateral-CA1 synapses of mouse hippocampal slices. Field excitatory postsynaptic potentials (fEPSPs) were evoked by stimulating the Schaffer collateral pathway and recorded in the stratum radiatum of the CA1 region of hippocampal slices. LTP was induced by high frequency stimulation (HFS) which consisted of 3 trains of theta burst stimulation (TBS), separated by 15s. Each TBS consisted of 4 pulses at 100 Hz, separated by 200 ms. Depotentiation was induced by low frequency stimulation (LFS, 1Hz, 15 min) 40 min after HFS.
Orexin A (30-300 nM) had no effect on the baseline fEPSPs after 10 min treatment. Orexin A treated 10 min before and during HFS, decreased HFS-induced LTP dose-dependently. The magnitude of LTP (% of the baseline fEPSPs) 40 min after HFS was reduced by pretreatment with orexin A 100 nM and 300 nM. The magnitude of PTP (% of the baseline fEPSPs) 40 min after HFS was mild dose-dependently reduced by pretreatment with orexin A 30 nM, 100 nM and 300 nM. Depotentiation induced by LFS (expressed by the % restoration of the magnitude of LTP) 40 min after LFS was not different with orexin A.
[Ala11, D-Leu15]-orexin B (3~1000nM) also had no effect on the baseline fEPSPs after 10 min treatment. [Ala11, D-Leu15]-orexin B treated 10 min before and during HFS, decreased HFS-induced LTP dose-dependently. The magnitude of LTP (% of the baseline fEPSPs) 40 min after HFS was reduced by pretreatment with [Ala11, D-Leu15]-orexin B 30 nM, 300 nM and 1000 nM. The magnitude of PTP (% of the baseline fEPSPs) 40 min after HFS had no different by pretreatment with [Ala11, D-Leu15]-orexin B. Depotentiation induced by LFS (expressed by the % restoration of the magnitude of LTP) 40 min after LFS was not different with [Ala11, D-Leu15]-orexin B.
SB 334867 (3 μM), a selective OX1 receptor antagonist, partially antagonized the effect of orexin A. The magnitude of LTP (% of the baseline fEPSPs) in slices treated with orexin A+ SB-334867 was higher in slices with 100 nM orexin A, which was significantly lower than the control slices. The magnitude of PTP (% of the baseline fEPSPs) in slices treated with 100 nM orexin A had no significantly difference in slices with orexin A+ SB-334867, which was not significantly different with the control slices. SB-334867 alone had no effect on basal transmission, PTP, LTP and depotentiation.
SB 334867 (3 μM) slightly antagonized the effect of [Ala11, D-Leu15]-orexin B. The magnitude of LTP (% of the baseline fEPSPs) in slices treated with 300 nM [Ala11, D-Leu15]-orexin B + SB 334867 was higher in slices with [Ala11, D-Leu15]-orexin B, which was significantly lower than the control slices. The magnitude of PTP (% of the baseline fEPSPs) in slices treated with 300 nM [Ala11, D-Leu15]-orexin B had no statistically significant difference in slices with [Ala11, D-Leu15]-orexin B + SB 334867, which was not significantly different with the control slices. SB 334867 alone had no effect on basal transmission, PTP, LTP and depotentiation.
Compound 29 (30 μM), a selective OX2 receptor antagonist, partially antagonized the effect of 100 nM orexin A. The magnitude of LTP (% of the baseline fEPSPs) in slices treated with 100 nM orexin A + Compound 29 was higher in slices with 100 nM orexin A, which was significantly lower than the control slices. The magnitude of PTP (% of the baseline fEPSPs) in slices treated with 100 nM orexin A was no different in slices with orexin A + Compound 29, which was no significantly difference with the control slices. Compound 29 alone had no effect on basal transmission, PTP, LTP and depotentiation.
Compound 29 (30 μM) slightly antagonized the effect of [Ala11, D-Leu15]-orexin B. The magnitude of LTP (% of the baseline fEPSPs) in slices treated with [Ala11, D-Leu15]-orexin B + Compound 29 (133±7.1%, n=5) was higher in slices with 300 nM [Ala11, D-Leu15]-orexin B (120.8±7.2%, n=5), which was significantly lower than the control slices (141.9±3.5%, n=16). The magnitude of PTP (% of the baseline fEPSPs) in slices treated with 300 nM [Ala11, D-Leu15]-orexin B (146.8±8.7%, n=5) was not significantly different in slices with [Ala11, D-Leu15]-orexin B + Compound 29 (186.5±28%, n=5), which had no significantly difference with the control slices (181.5±10.4 %, n=16). Compound 29 alone had no effect on basal transmission, PTP, LTP and depotentiation.
Paired-pulse facilitation (PPF) of fEPSPs, a presynaptic phenomenon resulting from Ca2+ accumulation in the presynaptic terminals in response to stimulation by a pair pulses with short interval is induced by paired-pulse (50 ms interval) stimulation. The PPR during LTP decreased, as compared to basal PPF ratio (PPR), suggesting that increased presynaptic transmission plays a role in the LTP evoked by TBS in CA1 region of mouse hippocampal slices although this LTP is mostly mediated by NMDA receptors. Orexin A (100 nM) did not affect basal PPR but slightly reversed the PPR decreased during LTP.
It is concluded that orexin A and [Ala11, D-Leu15]-orexin B decreased LTP but not affect depotentiation in CA1 region of mouse hippocampal slices. The impairment of LTP is mediated by both OX1 and OX2 receptors. The efficacy mediated by OX1 receptors is higher than that by OX2 receptors. It is suggested that presynaptic inhibition plays a little role in orexin-induced impairment; instead, postsynaptic NMDA receptor inhibition might contribute to this impairment through PLC/PKC pathway activated by OX1 and OX2 receptor activation.

並列關鍵字

hippocampus ； orexin ； neural plastisity ； long term potentiaion ； depotentiation

參考文獻

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