前腦特異性敲除Ccn 2基因的小鼠展現出不尋常的嗅覺行爲

Abstract

CCN2（又稱 CTGF, Connective Tissue Growth Factor）為一種分泌型細胞外基質蛋白，過去研究多聚焦於其在細胞增殖與細胞外基質重塑中的作用，但近期證據顯示其亦參與神經系統之調控。值得注意的是，CCN2 在囓齒類動物腦中呈現特定的區域性表現，主要分布於嗅球（OB）與前嗅核（AON）等嗅覺相關腦區，提示其可能在氣味訊息處理中扮演角色。由於全身性 Ccn2 剔除會導致新生小鼠無法存活，本研究以 Emx1-Cre × Ccn2fl/fl 小鼠建立前腦特異性 Ccn2 剔除模式（FbCcn2 KO），以探討 CCN2 缺失對社會性氣味處理歷程中神經迴路活化之影響。在嗅覺習慣化/去習慣化測試中，雌雄兩性 FbCcn2 KO 小鼠對非社交性氣味之嗅聞型態皆與對照組相近，顯示其基本嗅覺辨識能力未受損；然而 KO 小鼠對異性墊料（OS）之嗅聞時間顯著延長，對同性墊料（SS）則無明顯差異。為釐清此行為變化背後的神經機制，本研究以 c-Fos 免疫組織化學法，比較水（Water）、SS和OS三種氣味刺激下，嗅球及其下游多個嗅覺相關腦區之神經活化型態。結果顯示，CCN2 缺失所造成的影響具有明顯的性別與氣味特異性：雄性 KO 小鼠於SS刺激下，嗅球小球層（GL）、前嗅核側部（AOD）、嗅結節（TU）及前額葉 PrL/IL 等區域之活化程度降低；而在OS刺激下，差異則集中於終紋床核內側後部（BNSTMP）, 其未能有效銜接內側杏仁核（MeA）所傳遞之訊號至內側視前區（MPA）。在雌性 KO小鼠中， SS刺激後，變化見於副嗅球顆粒細胞層（GrA）、梨狀皮質（Pir）及 前額葉IL/OFC；OS刺激後，變化則出現於主嗅球顆粒細胞層（GrO）、副嗅球僧帽細胞層（MiA）、前嗅核內側部（AOM），以及下視丘腹內側核外側部（VMHVL）。嗅球 RNA 定序結果進一步支持上述性別差異：雄性 KO 嗅球以立即早期基因（如 Fos、Arc、Egr1、Npas4、Nr4a1 等）之下調為主；雌性 KO 嗅球則以原鈣黏蛋白家族基因（如 Pcdha11、Pcdhga2、Pcdhgb 系列）表現改變為主要特徵。綜合上述結果，本研究提示前腦 CCN2 並非單純調控特定行為輸出，而是廣泛地影響不同階層的嗅覺迴路對於社交氣味訊息之反應活性，且此一功能具有明確的性別特異性，這些結果為理解 CCN2 於嗅覺社會行為神經迴路中的角色，提供了行為與分子層次兼具的整合性框架。

CCN2 (also known as CTGF, Connective Tissue Growth Factor) is a secreted extracellular matrix protein whose functions have traditionally been studied in the context of cell proliferation and extracellular matrix remodeling. Recent evidence, however, indicates that CCN2 also participates in the regulation of the nervous system. Notably, CCN2 exhibits region-specific expression in the rodent brain, with prominent distribution in olfactory-related regions such as the olfactory bulb (OB) and the anterior olfactory nucleus (AON), suggesting a possible role in the processing of odor information. Because whole-body Ccn2 deletion results in perinatal lethality, the present study employed Emx1-Cre × Ccn2fl/fl mice to generate a forebrain-specific Ccn2 knockout model (FbCcn2 KO) in order to examine how CCN2 deficiency affects neural circuit activation during social odor processing. In the habituation/dishabituation test, both male and female FbCcn2 KO mice displayed sniffing patterns comparable to controls in response to non-social odors, indicating that basic odor discrimination was preserved. KO mice, however, spent significantly more time investigating opposite-sex (OS) bedding, while their investigation of same-sex (SS) bedding did not differ from controls. To clarify the neural basis of this behavioral change, c-Fos immunohistochemistry was used to compare activation patterns in the olfactory bulb and multiple downstream olfactory-related regions following exposure to water, SS bedding, and OS bedding. The effects of CCN2 deficiency proved to be both sex- and odor-specific. In male KO mice, SS exposure was associated with reduced activation in the glomerular layer (GL) of the olfactory bulb, the anterior olfactory nucleus, dorsal part (AOD), the olfactory tubercle (TU), and the prefrontal PrL/IL regions. Under OS exposure, genotype-dependent differences were instead concentrated in the medial posterior bed nucleus of the stria terminalis (BNSTMP), which appeared unable to effectively relay medial amygdala (MeA)-derived signals to the medial preoptic area (MPA). In female KO mice, SS exposure altered activation in the granule cell layer of the accessory olfactory bulb (GrA), the piriform cortex (Pir), and the prefrontal IL/OFC, whereas OS exposure altered activation in the granule cell layer of the main olfactory bulb (GrO), the mitral cell layer of the accessory olfactory bulb (MiA), the medial part of the anterior olfactory nucleus (AOM), and the ventrolateral subdivision of the ventromedial hypothalamus (VMHVL). RNA sequencing of the olfactory bulb further supported this sex-specific pattern: male KO olfactory bulbs were characterized primarily by downregulation of immediate early genes (including Fos, Arc, Egr1, Npas4, and Nr4a1), whereas female KO olfactory bulbs were characterized mainly by altered expression of protocadherin family genes (including Pcdha11, Pcdhga2, and members of the Pcdhgb subfamily). Taken together, these findings suggest that forebrain CCN2 does not regulate a single, discrete behavioral output but instead contributes to the transmission, allocation, and transformation of social odor information within olfactory circuits, a function that is clearly sex-specific. These results provide an integrated behavioral and molecular framework for understanding the role of CCN2 in the neural circuitry underlying olfactory social behavior.