利用小麥凝集素進行跨突觸追蹤探討藍斑核中間神經元之分布

Abstract

跨突觸追蹤(Transneuronal tracing)可以提供主要神經元和中間神經元連結的直接證據，但現今仍沒有適合的追蹤劑能夠在微迴路尺度下標定特定的細胞群。為了解決這個問題，我們設計了cre重組酶活化小麥凝集素血清二型類腺病毒(AAV2-CMV-DIO-WGA)，此病毒可以使帶有cre重組酶的細胞表現小麥凝集素(wheat germ agglutinin)(WGA)，並在細胞內累積進行跨突觸運輸，達到追蹤的效果。搭配免疫組織化學染色法，我們發現由這隻病毒所製造的小麥凝集素在海馬迴(CA3)、網狀視丘(Reticular thalamus,RT)以及下視丘室旁核(paraventricular nucleus, PVN)中具有逆向追蹤(retrograde tracing)的特性。在轉基因小鼠的實驗裡，我們將cre重組酶活化小麥凝集素血清二型類腺病毒打入TH-cre小鼠的藍斑核(locus coeruleus)，的確僅具有cre重組酶的主要神經元會表現小麥凝集素。除此之外，我們也發現一些位於peri-LC區域的非主要神經元也有小麥凝集素免疫反應，我們推論這些細胞就是和主要神經元有連結的中間神經元，因為在橋腦區域除了藍斑核以外沒有其他的兒茶酚安類家族的神經元，且TH-cre這隻基轉小鼠在藍斑核中並沒有cre重組酶表現外漏的問題。利用TH-cre和GAD-GFP轉基因小鼠的雜交後代，我們發現這些藍斑核的中間神經元有16%是丙胺基丁酸神經元(gamma-Aminobutyric acid -ergic neuron)(GABAergic)、27%具有FoP2這個轉錄因子。在形態學上，我們的實驗結果提供了藍斑核中間神經元存在的直接證據，這些中間神經元可能在功能上，彙整來自不同腦區的訊息到藍斑核主要神經元，進而調節其電生理性質，但至於這些中間神經元的生理功能仍需近一步的實驗分析。

In morphology, trans-synaptic tracing can provide direct evidence of the connectivity between nucleus. In modern neuroscience, there are lots of trans-synaptic tracers to use, such as fluoro-gold, chlorea toxin B, pseudo-rabies virus. But none of them can perform cell-type specific tracing under the scale of microcircuit due to non-cell type specificity and leak out expression. Yoshihara Yoshihara et al. have validated the usefulness of wheat germ agglutinin, a commonly used transneuronal tracer, in several genetic approaches (Yoshihara et al., 1999). We chose the AAV-mediated WGA method described by Yoshihiro Yoshihara, and designed AAV2-CMV-DIO-WGA, a serotype 2 adeno-associative virus carrying double floxed inverted sequence of wheat germ agglutinin(WGA). Using this virus, we found the WGA expressed by this virus had retrograde trans-synaptic activity (CA3, RT, PVN) and had no leak-out-expression. We are currently interested in the microcircuit of locus coeruleus. We then used TH-cre mice to perform the cell-type specific microcircuit tracing. This resulted in the observation of interneuron pool in peri-LC region, which have not been reported in mice. Among these interneurons, there were about 16% GABAergic interneurons and 27% FoxP2 posotive interneurons. The result here provided a direct evidence for the existence of LC interneurons in peri-LC region, and these interneurons may play a role in integrating information from other brain regions, such as amygdala and pre-frontal cortex. We need more experiments to analyze the physiological function of these interneurons.