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Title: | 食慾素在海馬突觸可塑性中的角色及行為呈現 The role of orexins in hippocampal synaptic plasticity and its behavioral presentation |
Authors: | Guan-Ling Lu 盧冠伶 |
Advisor: | 邱麗珠(Lih-Chu Chiou) |
Keyword: | 食慾素,突觸可塑性,制約場地偏好,海馬迴, Orexin,synaptic plasticity,conditioned place preference,hippocampus, |
Publication Year : | 2018 |
Degree: | 博士 |
Abstract: | 食慾素系統包括兩種肽-食慾素A與B,以及兩種受體-食慾素受體1與2。食慾素系統被認為參與了學習與記憶的調節,但是對於其調控海馬迴路突觸可塑性仍有許多爭議。我們使用胞外電生理紀錄在小鼠海馬迴腦片Schaffer Collateral-CA1突觸測量場興奮突觸後電位 (fEPSPs) 來觀察突觸可塑性。我們同時也使用制約場地偏好 [conditioned place preference (CPP) task] 反應來觀察海馬迴相關的學習及藥物制約喜好行為。
首先,我們針對食慾素A在小鼠海馬迴腦片Schaffer Collateral-CA1 突觸的兩種場興奮突觸後電位之突觸可塑性,如長期增益 (LTP) 以及去增益 (depotentiation) 的影響加以研究。當外給食慾素A濃度超過30 nM會降低LTP,而此現象可以被食慾素受體1拮抗劑 (SB334867) 而非受體2拮抗劑 (EMPA) 所拮抗。相反地,外給食慾素A在低濃度1 pM的情況下,可以避免去增益 (depotentiation) 的程度,而這種回復突觸增益程度的情況我們稱之為再增益 (re-potentiation)。這種由極低濃度的食慾素A所產生的再增益現象僅發生在給予1赫茲而非2赫茲的低頻刺激中觀察到,而高頻刺激則不限TBS或是僵直性刺激 (tetanic stimulation)。當我們同時給予選擇性的食慾素受體1 (SB334867) 與受體2 (EMPA) 的拮抗劑甚至是雙重食慾素受體拮抗劑 (TCS1102) 都可以顯著地對抗由極低濃度的食慾素A所產生的再增益現象。此外若是事先給予磷脂酶C (phospholipase C, PLC) 或腺苷酸環化酶 (adenylyl cyclase, AC) 及蛋白激酶A (protein kinase A, PKA) 的拮抗劑都可以預防由極低濃度的食慾素A所產生的再增益現象。另外我們使用化學方法產生去增益現象,如NMDA受體與 A1 腺苷受體 (A1-adenosine receptor) 及代謝性谷氨酸受體 [type 1/5 metabotropic glutamate (mGlu1/5) receptors] 的激活劑可以取代低頻刺激來產生去增益現象,且其拮抗劑可對抗低頻刺激所產生的去增益現象,上述的受體激活劑產生的去增益現象並不會受到極低濃度的食慾素A的影響。這個結果暗示我們食慾素A雙向調控海馬迴CA1突觸可塑性,在適當濃度下經由食慾素受體1來抑制長期增益現象,而在極低濃度下藉由食慾素受體1和2及其訊息傳遞磷脂酶C或腺苷酸環化酶及蛋白激酶A來產生再增益現象。 接著我們想要探討極低濃度食慾素A所產生的再增益現象是否具有生理意義。活化多巴胺受體 (D1-like R)曾經被報導可以產生海馬迴突觸的再增益現象,而這種現象與可卡因 (cocaine)制約場地偏好行為有關,因此我們想要檢視是否食慾素所引起的再增益現象也參與在可卡因制約場地偏好行為中。我們將參與可卡因制約場地偏好測試的小鼠取出海馬迴腦片測量突觸可塑性的改變。在產生可卡因場地制約偏好的小鼠上確實如預期觀察到顯著的再增益現象。但是令人吃驚的是給予食鹽水的對照組別,卻同時觀察到具有再增益現象;當我們使用相同的藥物處理時,改用飼養的鼠籠 (home cage) 取代制約場地,則無法觀察到海馬迴腦片中的再增益現象。因此我們推論制約場地的訓練可能會引起海馬迴腦片的再增益現象發生,且此現象與可卡因藥物的給予並不相關。更進一步,我們發現到單純進入制約場地但缺少注射行為的訓練過程,並無法引起海馬迴腦片的再增益現象發生,顯示制約場地訓練引起海馬迴腦片的再增益現象發生是需要配合注射行為的操弄,但是單純的注射操弄並無法引起海馬迴腦片的再增益現象。我們假設此制約場地訓練引起海馬迴腦片的再增益現象與體內極低濃度食慾素的釋出有關,因此在進行制約場地訓練前給予雙重食慾素受體拮抗劑 (TCS1102)可以對抗海馬迴腦片的再增益現象發生。 更進一步的實驗中,我們想要解釋可卡因處理之制約場地偏好改變的小鼠所產生偏好行為與海馬迴腦片的再增益現象是否與體內食慾素的釋出有關。我們發現可卡因制約場地偏好小鼠在給予10或20 mg/kg的可卡因時,會有相同程度的偏好行為與海馬迴腦片的再增益現象。但是當我們在進行制約場地與可卡因 (10 mg/kg) 配對前,也就是獲取狀態 (acquisition phase) 給予雙重食慾素受體拮抗劑 (TCS1102) 可以對抗偏好行為及海馬迴腦片的再增益現象發生,而在較高濃度的可卡因 (20 mg/kg) 配對時,給予雙重食慾素受體拮抗劑 (TCS1102) 並不能夠對抗偏好行為及海馬迴腦片的再增益現象發生。此外我們也發現較高濃度的可卡因 (20 mg/kg) 配對時,需要給予多巴胺受體 (D1-like R) 的拮抗劑來對抗偏好行為及海馬迴腦片的再增益現象發生。 這個結果暗示我們外給食慾素A在小鼠海馬迴腦片Schaffer Collateral-CA1 突觸的突觸可塑性是雙向的,可以抑制長期增益且預防低頻刺激的去增益現象發生;同時我們針對極低濃度食慾素A所產生的再增益現象與1赫茲的低頻刺激有關係,並透過活化食慾素受體1與2,其訊息傳遞路徑為磷脂酶C或腺苷酸環化酶及蛋白激酶A。低頻刺激所誘發產生的去增益現象對於低濃度的食慾素是非常敏感的,當連結到制約場地不同空間情境及制約操作的注射時就可以被釋放而產生再增益現象。另一方面,食慾素調節的海馬迴再增益現象也可以在形成可卡因場地偏好上有貢獻,然而僅限於適當劑量的可卡因,若在高劑量可卡因下,則會由多巴胺受體所取代。總結,本文首次揭露了食慾素在低頻刺激誘發的去增益現象所扮演的角色,同時建議其在場地制約訓練下相關記憶之生理角色。 The orexin system consists of two peptides, orexin A and B and two receptors, OX1R and OX2R. It is implicated in learning and memory regulation while controversy remains on its role in modulating hippocampal synaptic plasticity in vivo and in vitro. In our studies, we used extracellular electrophysiological recording to measure the field excitatory postsynaptic potentials (fEPSPs), at the Schaffer collateral-CA1 synapse of mouse hippocampal slices. We also used conditioned place preference (CPP) paradigm to measure the hippocampal associated learning and drug primed preference behavior. First, we investigated effects of orexin A on two forms of synaptic plasticity, long-term potentiation (LTP) and depotentiation of fEPSPs, at the Schaffer collateral-CA1 synapse of mouse hippocampal slices. Orexin A (≧30 nM) attenuated LTP induced by theta burst stimulation (TBS) in a manner antagonized by an OX1R antagonist (SB334867), but not OX2R antagonist (EMPA). Interestingly, we found orexin A, at 1 pM, co-application of orexin A prevented the induction of depotentiation induced by low frequency stimulation (LFS), namely inducing re-potentiation. This re-potentiation effect of sub-nanomolar orexin A occurred at LFS of 1Hz, but not 2 Hz, and with LTP induced by either TBS or tetanic stimulation. It was significantly antagonized by SB334867, EMPA and TCS1102, selective OX1R, OX2R and dual OXR antagonists, respectively, and prevented by D609, SQ22536 and H89, inhibitors of phospholipase C (PLC), adenylyl cyclase (AC) and protein kinase A (PKA), respectively. LFS-induced depotentiation was antagonized by blockers of NMDA, A1-adenosine and type 1/5 metabotropic glutamate (mGlu1/5) receptors, respectively. However, orexin A (1 pM) did not affect chemical-induced depotentiation by agonists of these receptors. These results suggest that orexin A bidirectionally modulates hippocampal CA1 synaptic plasticity, inhibiting LTP via OX1Rs at moderate concentrations while inducing re-potentiation via OX1Rs and OX2Rs, possibly through PLC and AC-PKA signaling at sub-nanomolar concentrations. Next, we further elucidated the physiological role of our findings on the hippocampal re-potentiation induced by ultra low concentrations of orexin A. Since activating dopamine D1-like receptor (D1-likeR) has been reported to also induce hippocampal re-potentiation and this effect may contribute to the cocaine-CPP behavior, we then examined whether orexin-induced re-potentiation also contribute to cocaine-CPP. We isolated hippocampal slices from the mice going through the CPP test and examined the changes of their hippocampal synaptic plasticity. Indeed, the hippocampal slices in mice that had acquired cocaine-CPP displayed significant hippocampal re-potentiation, as expected. However, we found unexpectedly that needle injection-paired contextual exposures during the CPP task in mice resulted in re-potentiation at CA1 synapses of hippocampal slices, regardless of whether the CPP behavior is expressed or not. Simply exposing the mouse in the CPP apparatus, or giving the mouse consecutive intraperitoneal injections in its home cage or a novel cage did not lead to hippocampal re-potentiation. Besides, this CPP training procedure-induced hippocampal re-potentiation was prevented when mice were pretreated with TCS1102, a dual orexin receptor antagonist. These results suggest that orexin-mediated modulation on hippocampal depotentiation by the training process in the CPP paradigm. Finally, we examined the contribution of endogenous orexins in hippocampal re-potentiation in mice acquiring cocaine-CPP. We found that cocaine-CPP mice at 10 or 20 mg/kg were able to show the same magnitude of CPP and hippocampal re-potentiation. A dual orexin receptor (OXR) antagonist, TCS1102 was used to attenuate the effect of cocaine CPP and the hippocampal re-potentiation in cocaine-CPP mice at moderate dose (10 mg/kg) but not high dose (20 mg/kg). However, dopamine D1-likeR antagonist, SCH23390 was able to attenuate the cocaine-CPP and the hippocampal re-potentiation in cocaine-CPP mice at moderate or high dose. In summary, these results suggest that orexin A can bidirectionally modulate hippocampal CA1 synaptic plasticity; inhibiting LTP via OX1Rs at moderate concentrations while inducing re-potentiation via OX1Rs and OX2Rs, possibly through PLC and AC-PKA signaling at sub-nanomolar concentrations. LFS-induced hippocampal depotentiation is vulnerable to the low level of orexin that can be released upon there is an association between the differential spatial contexts in the CPP apparatus and needle injections in the CPP test, leading to hippocampal re-potentiation. In the other aspect, orexin-mediated hippocampal re-potentiation may also function during the formation of cocaine-CPP. However, this effect of orexins only works at moderate doses of cocaine and will be replaced by dopamine, via D1-likeRs when cocaine is at higher doses. Together, we revealed that orexins can inhibit LFS-induced hippocampal depotentiation to keep hippocampal synaptic transmission remaining potentiated. This modulatory effect on the hippocampal synaptic plasticity may play a role in CPP training-associated memory. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70083 |
DOI: | 10.6342/NTU201800358 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 藥理學科所 |
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