内侧前额叶皮层-基底外侧杏仁核投射神经元分层亚群的解剖学研究

doi:10.3969/j.issn.1674-8115.2022.09.008

上海交通大学学报（医学版） ›› 2022, Vol. 42 ›› Issue (9): 1216-1224.doi: 10.3969/j.issn.1674-8115.2022.09.008

• 创新团队成果专栏 • 上一篇

内侧前额叶皮层-基底外侧杏仁核投射神经元分层亚群的解剖学研究

何璐瑶¹^,²(), 黄东萍¹^,²(), 邵孟孟¹^,², 张凯¹^,², 任百慧¹^,², 孔庆丹¹^,², 徐天乐¹^,²(), 吕江腾¹^,²()

^1.上海交通大学基础医学院解剖学与生理学系，上海 200025
^2.上海交通大学医学院附属上海儿童医学中心儿童脑科学中心，上海 200127

收稿日期:2022-01-27 接受日期:2022-06-07 出版日期:2022-09-08 发布日期:2022-09-08
通讯作者: 徐天乐,吕江腾 E-mail:heluyao1996@163.com;xu-happiness@shsmu.edu.cn;jiangtenglu@shsmu.edu.cn
作者简介:何璐瑶（1996—），女，硕士生；电子信箱：heluyao1996@163.com
何璐瑶（1996—），女，硕士生；电子信箱：heluyao1996@163.com第一联系人：（何璐瑶和黄东萍为本文共同第一作者）
基金资助:
国家自然科学基金(31972903);上海市高水平地方高校创新团队(SSMU-ZDCX20181100);上海市科学技术委员会基础研究重点项目(18JC1420302)

Anatomical structure of laminar specific subtypes of medial prefrontal cortex-basolateral amygdala projection neurons

HE Luyao¹^,²(), HUANG Dongping¹^,²(), SHAO Mengmeng¹^,², ZHANG Kai¹^,², REN Baihui¹^,², KONG Qingdan¹^,², XU Tianle¹^,²(), LÜ Jiangteng¹^,²()

^1.Department of Anatomy and Physiology, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, China
^2.Center for Brain Science of Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China

Received:2022-01-27 Accepted:2022-06-07 Online:2022-09-08 Published:2022-09-08
Contact: XU Tianle,Lü Jiangteng E-mail:heluyao1996@163.com;xu-happiness@shsmu.edu.cn;jiangtenglu@shsmu.edu.cn
Supported by:
National Natural Science Foundation of China(31972903);Innovative Research Team of High-Level Local Universities in Shanghai(SSMU-ZDCX20181100);Basic Research Project of Science and Technology Commission of Shanghai Municipality(18JC1420302)

摘要/Abstract

摘要：

目的·从脑区特异性和皮层分层特异性水平上精细解析小鼠内侧前额叶皮层（medial prefrontal cortex，mPFC）-基底外侧杏仁核（basolateral amygdala，BLA）投射神经元亚群的解剖学结构。方法·给C57BL/6小鼠BLA注射逆向示踪腺相关病毒，21 d后，心脏灌流并取脑。使用冰冻切片机制作mPFC的连续冠状切片，利用荧光显微镜采集脑片图像，选取具有mPFC分区代表性的脑片，统计分析病毒标记的胞体在mPFC内不同亚区的分布情况。采用转基因小鼠（Tbr2-CreER：：LSL-Flp小鼠和Rbp4-Cre小鼠）与重组酶（Flp和Cre）依赖的腺相关病毒，特异性地荧光标记小鼠mPFC第2层与第5层投射神经元的胞体及其在BLA内的投射轴突；病毒注射28 d后，同样灌流、取脑，收集BLA的连续冠状切片，免疫荧光染色标记后在荧光显微镜下采集脑片图像；通过测定BLA内不同区域投射轴突的荧光强度，比较mPFC第2层与第5层神经元在BLA投射轴突的空间分布情况。结果·荧光显微镜观察及定量分析结果表明：BLA投射神经元的胞体在包含前扣带回皮层和前边缘皮层的背内侧前额叶皮层（dorsal medial prefrontal cortex，dmPFC）中分布较多，且主要分布在第2层和第5层；而在包含内侧眶额叶皮层和下边缘皮层的腹内侧前额叶皮层（ventral medial prefrontal cortex，vmPFC）中分布较少，且没有观察到明显的分层现象。随后，在研究dmPFC的2个分层投射神经元的轴突分布时发现：在BLA内，第2层投射神经元的轴突分布较为均匀，而第5层投射神经元的轴突则更多分布在背侧区。结论·mPFC-BLA投射神经元的胞体分布具有脑区分布特异性和层分布特异性，dmPFC中不同层神经元的轴突投射在BLA也具有相应的脑区特异性。

关键词: 投射神经元, 内侧前额叶皮层, 基底外侧杏仁核, 脑区特异性, 层特异性, 轴突

Abstract:

Objective ·To dissect the fine anatomical structure of medial prefrontal cortex (mPFC)-basolateral amygdala (BLA) projection neurons based on regional and laminar specificity in the cortex in mice. Methods ·The retrograde tracer of adeno-associated viral (AAV) vectors were injected into BLA of C57BL/6 mice. Twenty-one days after the injection, the mice were sacrificed and the brains were harvested. The coronal sections containing mPFC were prepared by using a cryostat microtome. The images were taken by fluorescence microscopy. Then the representative brain slices containing mPFC were selected and the distribution of virus-labeled cell bodies in different subregions of mPFC was analyzed. By using transgenic mouse lines (Tbr2-CreER::LSL-Flp mice and Rbp4-Cre mice)with Flp- or Cre-dependent AAV, layer 2 (L2) and layer 5 (L5) projection neurons in mPFC and their axons in BLA were marked, respectively. Twenty-eight days after the injection, the mice were sacrificed and the brains were also harvested. Coronal sections of the BLA were collected. The brain slices were imaged by fluorescence microscopy after immunofluorescence staining. By measuring the fluorescence intensity of projecting axons in different regions within BLA, the spatial distributions of the axons in BLA of mPFC L2 and L5 projection neurons were compared. Results ·Through fluorescence microscopy and quantitative analysis, it was found that the cell bodies of BLA projection neurons were mainly located at L2 and L5 in the dorsal mPFC (dmPFC) which contains anterior cingulate cortex and prelimbic cortex. However, the distribution of BLA projection neurons in the ventral mPFC (vmPFC) including medial orbital cortex and infralimbic cortex did not exhibit the similar distribution. When studying the axonal distribution of dmPFC projection neurons in two layers, it was found that the axons of L2 projection neurons dispersed in the BLA, while the axons of L5 projection neurons were mainly located in the dorsal region of BLA. Conclusion ·The cell body distribution of mPFC-BLA projection neurons shows regional specificity and laminar specificity. The axon projections initiated from different layers of dmPFC also exhibit regional specificity in BLA.

Key words: projection neuron, medial prefrontal cortex (mPFC), basolateral amygdala (BLA), regional specificity, laminar specificity, axon

中图分类号:

Q422

何璐瑶, 黄东萍, 邵孟孟, 张凯, 任百慧, 孔庆丹, 徐天乐, 吕江腾. 内侧前额叶皮层-基底外侧杏仁核投射神经元分层亚群的解剖学研究[J]. 上海交通大学学报（医学版）, 2022, 42(9): 1216-1224.

HE Luyao, HUANG Dongping, SHAO Mengmeng, ZHANG Kai, REN Baihui, KONG Qingdan, XU Tianle, LÜ Jiangteng. Anatomical structure of laminar specific subtypes of medial prefrontal cortex-basolateral amygdala projection neurons[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(9): 1216-1224.

图/表 5

图1 病毒注射位点及荧光蛋白在内侧前额叶神经元的表达情况Note： A. Left: the diagram of AAV-Syn-MCS-Retro-mCherry-3flag injection into BLA of C57BL/6 mice. Right: an example of injection site shown in the left with the expression of red fluorescent protein in BLA. B. An example of red fluorescent protein-labeled neurons in mPFC 21 d after injection. BLA—basolateral amygdala; CeA—central amygdala; mPFC—medial prefrontal cortex; ACC—anterior cingulate cortex; PL—prelimbic cortex; IL—infralimbic cortex; MO—medial orbital cortex. The numbers in the figure B are the specific layers.

Fig 1 Viral injection site and the expression of fluorescent protein in the neurons of mPFC

图2 逆向标记的神经元在mPFC亚区的分布Note： A. Representative brain slices of mPFC with the cell bodies of BLA projection neurons. Black dots indicate the location of retrogradely labeled cell bodies. The values below the brain slices indicate the distances from the Bregma point. B. Percentage of labeled cells in mPFC subregions (n=10). The three graphs respectively correspond to the brain slices of the three positions in the upper figure A.

Fig 2 Distribution of retro virus-labeled neurons in mPFC subregions

图3 逆向标记的神经元在mPFC各层的分布Note： A. Example images of retrogradely labeled cell bodies in ACC, PL, and MO/IL+MO. Two dotted lines indicate that the distance from pia mater is 200 μm and 400 μm, respectively. B. Statistics of labeled neurons on laminar distribution (n=10). The value below each row indicates the distance from the Bregma point. L—layer.

Fig 3 Laminar distribution of retrograde labeled neurons in mPFC

图4 标记mPFC不同层神经元的特异性病毒注射策略和荧光蛋白表达情况Note： A/C. Top panels: the diagrams of injection strategy in Tbr2-CreER::LSL-Flp mice (A) and Rbp4-Cre mice (C). Bottom panels: the schematic diagrams of the patterns of labeling L2 (A) or L5 (C) projection neurons, respectively. B/D. Left: the examples of fluorescence images of virus expression in the mPFC of Tbr2-CreER::LSL-Flp mice (B) and Rbp4-Cre mice (D). Right: the magnified images. GFP—green fluorescent protein; RFP—red fluorescent protein.

Fig 4 Specific virus injection strategy for labeling different layers of the neurons in mPFC and the expression of fluorescent proteins

图5 不同层mPFC-BLA投射在杏仁核的轴突分布及荧光强度Note：A. Examples of immunofluorescence staining of total axon projections initiated from mPFC (GFP) and the projections from L2 (Tbr2-CreER::LSL-Flp, RFP) and L5 (Rbp4-Cre, RFP) in the BLA. The value above each row indicates the distances from the Bregma point. B. Comparison of spatial distribution along the dorso-ventral axis (D-V axis) of total axon projections initiated from mPFC in BLA between two mouse lines (n=5). The value above each graph indicates the distances from the Bregma point. C?E. Left: comparison of spatial distribution of two subtypes of mPFC-BLA projections between the dorsal (D) and ventral (V) regions of BLA. Right: the axon fluorescence intensity ratio between the two groups of mice. ①P=0.043, ③P=0.006, ⑤P=0.010, compared with the dorsal region; ②P=0.037,④P=0.012, compared with Tbr2 group. BLP—basolateral amygdaloid nucleus, posterior part.

Fig 5 Axon distribution and fluorescence intensity of different layers of mPFC-BLA projection neurons in amygdala

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内侧前额叶皮层-基底外侧杏仁核投射神经元分层亚群的解剖学研究

Anatomical structure of laminar specific subtypes of medial prefrontal cortex-basolateral amygdala projection neurons

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