不同時距之母子分離對離乳期大鼠在焦慮反應、空間記憶學習以及海馬細胞增生的影響具有兩性差異

Abstract

早年生活經驗對個體在生理與行為的表現上具有長遠的影響，將初生幼鼠進行母子分離(maternal separation)的處理常作承受為個體早年生活壓力的動物模式。在過去的研究中發現，初生幼鼠進行母子分離會影響個體於成年時對壓力的行為與生理反應以及對空間記憶學習的能力，而分離時間的長短對個體所造成的影響也不盡相同。迄今大多數的研究係以雄鼠作為實驗對象，對於母子分離在雌、雄大鼠之間影響的差異，並未深入探討。此外，初生之幼鼠進行母子分離雖已發現影響其成年期的生理、行為表現，但並不清楚此現象是否在離乳期(post-weaning period)幼鼠即可顯現。故本研究以鼠齡22-24天之Long-Evans雌、雄大鼠為實驗對象，探討在出生後進行不同時距之母子分離，對此兩種性別在離乳期之焦慮行為與生理反應，空間記憶學習行為以及海馬神經新生的影響。 本研究將出生後幼鼠分為母子分離15分鐘組、母子分離180分鐘組以及母子未分離之對照組，在出生後第4天至第21天進行母子分離。子代大鼠於出生後第22天以升高十字迷宮觀察各組的焦慮行為，結果顯示母子分離15分鐘有助減輕雌鼠於升高十字迷宮中的焦慮行為。於次日將各組動物斷頭取血，以放射性免疫檢定法分析各組大鼠血清中皮質酮(corticosterone)的濃度，並取其腎上腺稱重，計算其腎上腺重/體重的百分比，母子分離15分鐘以及180分鐘之處理可使雌鼠的腎上腺重/體重百分比與血清中皮質酮濃度較未分離之對照組為低；雄鼠而言，母子分離15分鐘處理者之其腎上腺重/體重百分比較180分鐘處理者為低，而分離15分鐘以及180分鐘處理皆可使血清中皮質酮濃度較未分離之對照組為低。在空間記憶學習方面，則以Morris水迷宮的探索測試(probe test)作為各組大鼠空間記憶學習能力的測量方式。在三組雄鼠中，以母子分離15分鐘組在標的象限(target quadrant)的停留時間最久；而三組雌鼠中則以母子分離180分鐘組於標的象限的停留時間最長，即母子分離15分鐘有助雄鼠增進空間記憶學習，但對雌鼠而言，則為母子分離180分鐘之處理才能達到此效果。在子代大鼠出生後22天給予劑量為100 mg/kg的5-bromo-2'-deoxyuridine (BrdU)之腹腔注射處理，於2小時後犧牲灌流並以免疫組織化學染色觀察其海馬齒狀回細胞增生的情形，結果發現各組大鼠的細胞增生數量並沒有統計上的差異。 綜合上述結果，顯示母子分離15分鐘與180分鐘皆可使雌、雄大鼠於離乳期的腎上腺重/體重百分比以及血清中皮質酮濃度下降。此外，母子分離15分鐘可減輕雌鼠的焦慮行為表現，並增進雄鼠的空間記憶學習能力。而母子分離180分鐘則可改善雌鼠的空間記憶學習能力。由此顯示長短不同時距之母子分離因性別的差異對個體的影響有所不同。

Early life experience appears to have long-lasting influences on an organism’s physiological functions and behavior. Neonatal maternal separation (MS) has been used to model long-term changes in neurochemical responses and behavior associated with exposure to early-life stress. Many studies have shown that neonatal MS changes the behavior and physiological responses to stress, and the spatial memory and learning ability in adulthood. The duration of MS is a critical factor of these effects. Although stress induces different physiological and behavioral responses between males and females, male rats were used as subjects in most of these studies. In addition, whether neonatal MS leads to sex differences in abovementioned behaviors remains unclear in post-weaning rats. In the present study, we used both male and female Long-Evans rats on postnatal 22-24 days which have been through different duration of MS to reveal the effects of MS on anxiety responses, spatial memory and learning and cell proliferation. Neonatal pups were divided into three groups: control, MS 15 minutes (MS-15) and MS 180 minutes (MS-180). Infant rats were deprived of maternal contact between the 4th and the 21st postnatal days. Elevated plus maze was used to investigate the anxiety-like behavior on 22nd postnatal day. MS-15 female rats showed significantly lower anxiety-like behavior. We also collected the blood to measure the basal levels of coritocosterone and weighed the adrenal glands. Our results showed that MS-15 and MS-180 female rats reduced the basal levels of corticosterone and the percentage of adrenal gland/body weight. MS-15 male rats reduced the percentage of adrenal gland/body weight and both MS-15 and MS-180 male rats had lower basal level of corticosterone than control male rats. The duration on target quadrant of Morris water maze was used to investigate the spatial memory and learning ability. MS-180 female rats stay longer on the target quadrant than control and MS-15 female rats, while MS-15 male spend more time on the target quadrant than control and MS-180 male rats. To investigate the cell proliferation in granule cell layer (GCL), 5-bromo-2'-deoxyuridine (BrdU) was given at a dose of 100 mg/kg to rats by intraperitoneal injection and the animals were perfused two hours later. The immunohistochemistry of BrdU was conducted to examine cell proliferation in the hippocampus dentate gyrus. Our results showed that there is no significant difference in cell proliferation in GCL between all groups. In summary, both MS-15 and MS-180 groups reduced the basal levels of corticosterone. In female rats, MS 15 minutes and MS 180 minutes reduced the percentage of adrenal weight/body weight. In male rats, only MS 15 minutes had the same effect. Moreover, MS 15 minutes reduced the anxiety-like behavior in female rats, and improved the spatial memory and learning ability in male rats. On the other hand, MS 180 minutes improved the spatial memory and learning ability in female rats. These results indicated that neonatal short-term and long-term MS induced different effects on post-weaning rats, and gender-difference may involve in the effects.