| 摘要: | 自閉症譜系障礙 (autism spectrum disorder, ASD) 為一種以社會互動障礙、溝通困難與重複刻板行為為核心特徵的神經發展異常，已知其病理機制涉及多種基因與環境因子，並造成神經突觸連結的異常。部分單基因遺傳疾病臨床上與ASD在異常行為表現上具相似性，並確實存在更高的 ASD 共病率，因此歸類為 syndromic autism，此類疾病模型有助於我們理解ASD背後的病理機制，神經纖維瘤第一型 (neurofibromatosis type 1, NF1) 便是其中之一，患者不僅表現出多種認知與行為缺陷，也具有高於一般人群的ASD共病比例，並且同樣以男性呈現更高患病率與較嚴重的行為障礙，使Nf1基因缺陷小鼠被視為探討自閉症神經機制的潛在研究模型。然而，目前缺乏對於Nf1缺陷以及自閉症對神經結構影響的系統性探討，鑑於本實驗室許璨庭博士近期已雄性Nf1+/-小鼠之分析，故本研究著重於雌性個體，藉此補足性別面向，並進一步比較兩性在神經結構改變上的異同。
本研究採用雌性 Nf1+/- 小鼠模型結合Thy1-YFP-H神經細胞標記，期望利用Thy1神經細胞的投射特性探討以Nf1缺陷背景下的神經連結變化。我們使用高內涵影像系統 (high-content imaging system) 搭配影像分析技術，量化 Thy1-YFP 細胞與突觸投射在全腦層面的分布差異，作為評估投射連結強度與完整性的依據。進一步結合YFP+細胞量與YFP訊號量的空間分布相關性，篩選出可能高度受軸突投射支配的腦區，反推出可能受到Nf1缺陷影響的異常神經連結。
結果顯示，雌性 Nf1+/- 小鼠在多個皮質與皮質下腦區呈現顯著的細胞數與訊號量異常。細胞數方面，初級體感皮質 (SSp) 、初級視覺皮質 (VISp) 與海馬結構 (HPF) 等重要功能區塊皆出現明顯上升，尤以左側海馬結構最為集中；中隔內側核 (MS) 則未呈現細胞數改變，但在訊號量分析中顯示顯著上升，為全腦訊號增加幅度最高之目標腦區。相對地，丘腦的核區VPM和VPL在細胞數無顯著差異的情況下，分別於雙側與單側出現明顯的 YFP 訊號下降，反映出VP核區相關的上游神經投射減弱。上述異變區域分布具高度空間選擇性，並呈現左右半腦的側化趨勢，顯示 Nf1 缺陷可能分別擾動神經細胞本體與突觸連結的特定路徑，進一步影響感覺處理、記憶與動機等神經功能模組。此外，搭配雄性Nf1+/- 小鼠的分析資料後，亦發現左右半球各有一處公母皆異變的共通目標腦區，分別為左腦的背內側丘腦核 (MD) 與右腦的背側梨狀內核 (EPd) ，可能涉及嗅覺導向行為與動機選擇的共同神經基礎。
本研究結合 Nf1+/- 基因突變背景與 Thy1-YFP-H 小鼠品系，利用該系統特異性標記皮質與海馬結構等區之投射神經元的特性，作為觀察神經結構連結變化的窗口。透過YFP+神經細胞本體與YFP訊號兩項定量資訊，得以大範圍評估潛在的結構性連結改變。研究結果顯示，Nf1+/- 雌鼠於多個皮質區與海馬結構出現YFP+神經細胞數量增加；而易受軸突支配的腦區多位於皮質下區域，其中YFP訊號量下降者（如 VPM）推測為受外來軸突投射接收量改變牽動，後續可搭配Allen Brain Atlas Mouse Connectivity追查上游腦區，評估跨腦區連結強度的改變。此外，搭配許璨庭博士的研究發現，可提供自閉症和NF1於性別差異或共通的神經迴路機制的重要線索，為未來整合神經結構與行為的性別比較研究奠定基礎。;Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by social deficits, communication difficulties, and repetitive behaviors. It is widely recognized that both genetic and environmental factors contribute to its pathophysiology, frequently leading to abnormal synaptic connectivity. A subset of monogenic disorders that display behavioral phenotypes overlapping with ASD and demonstrate increased comorbidity rates are categorized as syndromic autism. These models offer unique insights into the biological underpinnings of ASD. Neurofibromatosis type 1 (NF1) is one such condition; patients often exhibit cognitive and behavioral impairments, show a higher prevalence of ASD, and display sex-specific differences—namely, a higher male-to-female ratio and more severe symptoms in males. Accordingly, Nf1-deficient mice have been regarded as a promising model for investigating ASD-related mechanisms. However, a systematic understanding of how Nf1 deficiency and ASD impact brain structure remains lacking. As Dr.Hsu from our lab has recently completed a brain-wide analysis of male Nf1+/- mice, this study aims to focus on female subjects to address sex-specific differences in structural connectivity.
We utilized female Nf1+/- mice crossed with the Thy1-YFP-H line, which selectively labels projection neurons predominantly located in the cortex and hippocampus. This genetic combination enabled us to visualize and quantify structural connectivity alterations within the Nf1-deficient background. High-content imaging coupled with automated image analysis was employed to quantify the distribution of YFP-labeled neurons and axonal projections throughout the brain. By examining the spatial correspondence between YFP+ somata and axonal signal densities, we identified candidate regions that may receive dense long-range input and are thus susceptible to projection-level disruptions under Nf1 deficiency.
Our results revealed that Nf1+/- female mice exhibit significant alterations in both cell density and axonal signal intensity across multiple brain regions. At the cellular level, increased numbers of YFP+ neurons were observed in primary somatosensory area (SSp), primary visual area (VISp), and hippocampal formation (HPF), with the most prominent changes localized to the left hippocampus. Notably, the medial septal nucleus (MS) did not show changes in cell count but exhibited the highest increase in axonal signal, suggesting enhanced incoming projections. Conversely, the ventral posteromedial (VPM) and ventral posterolateral (VPL) nuclei of the thalamus showed a significant decrease in YFP signal despite no changes in local YFP+ cell numbers, implying a reduction in upstream cortical inputs. These abnormalities displayed both spatial selectivity and hemispheric lateralization, suggesting that Nf1 deficiency may affect neuronal populations and axonal connectivity along distinct anatomical routes, potentially disrupting sensory processing, memory, and motivational circuits. Moreover, when integrated with data from male Nf1+/- mice, we identified two brain regions where both sexes exhibited consistent alterations: the left mediodorsal thalamic nucleus (MD) and the right dorsal endopiriform nucleus (EPd). These areas may constitute common circuit-level substrates related to olfactory-guided behavior and motivational selection.
Taken together, this study combines the Nf1+/- genetic mutation model with the Thy1-YFP-H mouse line, utilizing its ability to label projection neurons in cortical and hippocampal structures as a window for observing structural connectivity changes. Through quantification of both YFP+ neuron number and YFP signal intensity, we were able to evaluate potential connectivity alterations on a large scale. The results showed increased YFP+ neuron numbers in several cortical and hippocampal regions, while regions likely receiving dense axonal input—mostly subcortical—showed decreased YFP signal (e.g., VPM), suggesting changes in axonal input strength. These upstream connections can be further traced using the Allen Brain Atlas Mouse Connectivity database to assess alterations in cross-regional connectivity. Combined with the findings from Dr. Hsu’s study on males, this work provides structural clues to sex-specific and shared neural circuit mechanisms in ASD and NF1, forming a basis for future sex-comparative studies integrating structure and behavior. |
