恐懼條件學習改變得麻效鎮靜狀態大鼠之血氧濃度信號與功能性連結

Abstract

在古典恐懼條件學習過程中，條件刺激(conditioned stimulus, CS)與非條件刺激(unconditioned stimulus, US)配對出現使得一個原先微弱、無法引發恐懼反應的CS逐漸獲得預測US即將來臨之能力，並控制恐懼行為之展現。杏仁核被認為是恐懼條件記憶生成之樞紐，因其接受來自CS與US訊息之投射，此兩訊息在杏仁核內部交會使得突觸前後產生耦合興奮性，透過海伯法則(Hebbian rule)留下連結學習之記憶痕跡。許多研究證據指出CS與US在感覺徑路上有許多匯聚之處，甚至在初級感覺訊息徑路上所產生的神經可塑性(neural plasticity)會進一步影響下游腦區之記憶痕跡生成。本論文企圖透過大鼠功能性核磁共振顯影來定位CS與US可能匯聚之處，透過一連串實驗分別建立行為典範、選擇刺激參數與尋找感覺通路，最後讓大鼠接受MRI掃描同時進行CS-US連結學習。行為層次上，大鼠在得麻效(dexmedetomidine, 0.1 mg/kg/hr)引發之鎮靜狀態下進行古典條件學習，在訓練後隔日接受恐懼增強驚跳反應作業(fear potentiated startle task)來測試記憶，行為結果顯示學習前給予周邊腎上腺素注射(epinephrine, 0.1 mg/kg)可增強一個微弱電擊 (0.63 mA)引發之學習，然直接使用強電擊作為US (1.25 mA)可使得大鼠在記憶測試中展現強烈的恐懼反應。在感覺刺激層面上，中等亮度的閃爍燈光(5 Hz, 2.13 cd/m2)可在視覺徑路上引發強烈的血氧濃度信號(blood oxygenation level-dependent signal)，但不會影響學習或處理嫌惡刺激相關之腦區，故被選為CS。最後大鼠在得麻效鎮靜狀態下進行古典條件學習，並同時執行功能性影像掃描，透過血氧濃度信號的改變來探測學習當下CS與US匯聚之腦區。影像結果顯示視覺徑路從皮質下結構到高等聯結區域都有學習引發之信號改變，且學習事件結束後視覺徑路與觸覺/痛覺徑路之間的功能性連結上升，甚至次級視覺皮質(secondary visual cortex, V2)與次級觸覺皮質(secondary somatosensory cortex, S2)之功能性連結可預測隔日記憶表現之好壞。本論文之研究結果顯示CS與US在腦中之匯聚場所不只一處，這些腦區形成一個表徵恐懼記憶的細胞集團，學習事件後CS與US徑路間神經活動震盪上升有助於後續的記憶穩固。

In Pavlovain fear conditioning, a neutral event noted as the conditioned stimulus (CS) acquires the ability to predict a forthcoming aversive event noted as the unconditioned stimulus (US), if the presence of US is frequently preceded by CS. The amygdala is the key substrate for fear memory as it receives the convergent inputs from the CS and US pathways to induce learning-related plasticity via a cellular mechanism based on the Hebbian rule. Several thalamic nuclei and cortical areas have also been proposed to be substrates of fear memory rather than serving as purely sensory analyzers. Even the early stage of sensory process may receive the CS-US convergent input and exerts its influence on the downstream circuit to enable the formation of engram. To delineate the cell assembly subserving learning and unveil the locus of CS-US convergence and/or interaction throughout the brain, rats treated with dexmedetomidine (0.1 mg/kg/hr) were trained to acquire CS-US association and functional images sensitive to blood oxygenation level-dependent (BOLD) contrast were obtained simultaneously. A series of experiments were designed to build a behavioral paradigm to train rats to associate the CS with US under dexmedetomidine treatment, set stimulus parameters to probe the brain pathways for the CS and US, and obtain functional images during CS-US association. First, behavioral results showed that an intense foot shock (1.25 mA) yielded a significant fear potentiated startle response, while a weak shock (0.63 mA) yielded minimal learning that can be enhanced by pre-training injection of 0.1 mg/kg epinephrine. Second, the 5 Hz flash with a median level of luminance (2.13 cd/m2) can serve as a good CS as it evoked robust visual activity without causing much impact on regions involved in aversion or learning. Finally, the imaging results reveal fear conditioning altered the BOLD activity in the visual pathway and the foci of CS-US convergence were found in both the subcortical and cortical levels. Functional connectivity change revealed that the interaction between the CS and US pathways was enhanced after the termination of the training trials, and this learning-induced functional coupling was detected at each level of the sensory process. The functional connectivity change of the V2 and the secondary somatosensory cortex (S2) bear positive correlation with the memory performance. In conclusion, the CS and US interact in multiple sites along the sensory pathways and the increment of the functional connectivity after learning may promote subsequent memory consolidation process.