Panoramic and local histological observations of biotinylated dextran amine neural tracer labeling in the motor cortex of rat brain

doi:10.3969/j.issn.1006-7795.2025.01.014

Abstract

Abstract: Objective To reveal the detailed histological characteristics of pyramidal neuron cell bodies and their axonal projections along the corticospinal tract in the primary motor cortex (M1) of the brain, by using the biotinylated dextran amine (BDA) neural tracing technique combined with panoramic and local microscopic imaging technologies. Methods A total of 100 nL of 10% BDA (10,000 molecular weight) was injected into M1 region using stereotaxic system. The distribution of BDA labeling along the corticospinal tract was continuously tracked with panoramic tissue scanning analysis system. Detailed observations of the histological characteristics of BDA labeling were carried out with laser confocal microscope. Results It is more convenient to observe the overall distribution of BDA neural labeling by using the panoramic tissue scanning analysis system. Around the injection site in M1, the BDA labeling was shown in the somas of pyramidal neurons in layer V. In the M1 region corresponding to the contralateral site of the injection site and ipsilateral primary sensory cortex, BDA showed predominantly the anterograde labeled nerve fibers accompanied by a few retrograde labeled neurons. Besides, BDA labeled nerve fibers-including bundles and terminals-projecting to regions such as the ipsilateral striatum, thalamus, internal capsule, cerebral peduncle, and pons, and further reaching the contralateral spinal cord via the brainstem pyramidal decussation. Confocal microscopy and its 3D reconstruction system facilitated detailed analysis of the local microscopic features of BDA labeling, revealing retrograde labeled neuron cell bodies, dendrites and their spines, as well as anterograde labeled nerve fibers and their terminals. Conclusions These findings demonstrated that the integration of traditional BDA neural tracing with panoramic tissue scanning analysis and confocal microscopy provided an effective approach to the observation and analysis of long-projection neural circuits from panoramic to local perspectives, with broad application prospects.

Key words: motor cortex, neural circuits, corticospinal tract, panoramic scanning, microscopic imaging, three-dimensional reconstruction

CLC Number:

Q189

Lu Jiaying, Xu Dongsheng, Cui Jingjing, Wang Yuqing, Su Yuxin, Liu Yihan, Wang Jia, Bai Wanzhu. Panoramic and local histological observations of biotinylated dextran amine neural tracer labeling in the motor cortex of rat brain[J]. Journal of Capital Medical University, 2025, 46(1): 83-90.

References

［1］ Vercelli A, Repici M, Garbossa D, et al. Recent techniques for tracing pathways in the central nervous system of developing and adult mammals［J］. Brain Res Bull, 2000, 51(1): 11－28.
［2］ Lanciego J L, Wouterlood F G. A half century of experimental neuroanatomical tracing［J］. J Chem Neuroanat, 2011, 42: 157－183.
［3］ Qiu L Y, Zhang B, Gao Z H. Lighting up neural circuits by viral tracing［J］. Neurosci Bull, 2022, 38(11): 1383－1396.
［4］ Cui J J, Zhu X L, Shi H, et al. The expression of calcitonin gene-related peptide on the neurons associated Zusanli (ST 36) in rats［J］. Chin J Integr Med, 2015, 21(8): 630－634.
［5］ Zhang Z Y, Xu D S, Wang J, et al. Correlated sensory and sympathetic innervation between the acupoint BL23 and kidney in the rat［J］. Front Integr Neurosci, 2020, 14: 616778.
［6］ Winnubst J, Bas E, Ferreira T A, et al. Reconstruction of 1 000 projection neurons reveals new cell types and organization of long-range connectivity in the mouse brain［J］. Cell, 2019, 179(1): 268－281.e13.
［7］ BRAIN Initiative Cell Census Network (BICCN). A multimodal cell census and atlas of the mammalian primary motor cortex［J］. Nature, 2021, 598(7879): 86－102.
［8］ Muñoz-Castañeda R, Zingg B, Matho K S, et al. Cellular anatomy of the mouse primary motor cortex［J］. Nature, 2021, 598(7879): 159－166.
［9］ Yao L L, Ye Q P, Liu Y, et al. Electroacupuncture improves swallowing function in a post-stroke dysphagia mouse model by activating the motor cortex inputs to the nucleus tractus solitarii through the parabrachial nuclei［J］. Nat Commun, 2023, 14(1): 810.
［10］ Cao P, Zhang M J, Ni Z Y, et al. Green light induces antinociception via visual-somatosensory circuits［J］. Cell Rep, 2023, 42(4): 112290.
［11］ Yang Y M, Huang H, Zhu M Y, et al. A neural circuit for lavender-essential-oil-induced antinociception［J］. Cell Rep, 2024, 43(10): 114800.
［12］ Xu Y L, Zhu X X, Chen Y R, et al. Electroacupuncture alleviates mechanical allodynia and anxiety-like behaviors induced by chronic neuropathic pain via regulating rostral anterior cingulate cortex-dorsal raphe nucleus neural circuit［J］. CNS Neurosci Ther, 2023, 29(12): 4043－4058.
［13］ Cui J J, Wang J, Xu D S, et al. Alexa fluor 488-conjugated cholera toxin subunit B optimally labels neurons 3-7 days after injection into the rat gastrocnemius muscle［J］. Neural Regen Res, 2022, 17(10): 2316－2320.
［14］ Xu D S, Cui J J, Wang J, et al. Improving the application of high molecular weight biotinylated dextran amine for thalamocortical projection tracing in the rat［J］. J Vis Exp, 2018(134): 55938.
［15］ Zhang W J, Xu D S, Cui J J, et al. Anterograde and retrograde tracing with high molecular weight biotinylated dextran amine through thalamocortical and corticothalamic pathways［J］. Microsc Res Tech, 2017, 80(2): 260－266.
［16］ Xu D S, Zou L, Zhang W X, et al. Comparison of sensory and motor innervation between the acupoints LR3 and LR8 in the rat with regional anatomy and neural tract tracing［J］. Front Integr Neurosci, 2021, 15: 728747.
［17］ Wang J, Xu D S, Cui J J, et al. Visualizing the calcitonin gene-related peptide immunoreactive innervation of the rat cranial dura mater with immunofluorescence and neural tracing［J］. J Vis Exp, 2021(167): 61742.
［18］ Reiner A, Veenman C L, Medina L, et al. Pathway tracing using biotinylated dextran amines［J］. J Neurosci Methods, 2000, 103(1): 23－37.
［19］ Veenman C L, Reiner A, Honig M G. Biotinylated dextran amine as an anterograde tracer for single-and double-labeling studies［J］. J Neurosci Methods, 1992, 41(3): 239－254.
［20］ Han X, Lv G, Wu H, et al. Biotinylated dextran amine anterograde tracing of the canine corticospinal tract［J］. Neural Regen Res, 2012, 7(11): 805－809.
［21］ Liu S B, Wang Z F, Su Y S, et al. A neuroanatomical basis for electroacupuncture to drive the vagal-adrenal axis［J］. Nature, 2021, 598(7882): 641－645.
［22］ Arber S, Costa R M. Connecting neuronal circuits for movement［J］. Science, 2018, 360(6396): 1403－1404.
［23］ Klaus A, Martins G J, Paixao V B, et al. The spatiotemporal organization of the striatum encodes action space［J］. Neuron, 2017, 95(5): 1171－1180.e7.
［24］ Carmona L M, Thomas E T, Smith K, et al. Topographical and cell type-specific connectivity of rostral and caudal forelimb corticospinal neuron populations［J］. Cell Rep, 2024, 43(4): 113993.
［25］ Hausmann F S, Barrett J M, Martin M E, et al. Axonal barcode analysis of pyramidal tract projections from mouse forelimb M1 and M2［J］. J Neurosci, 2022, 42(41): 7733-7743.
［26］ Canty A J, Murphy M. Molecular mechanisms of axon guidance in the developing corticospinal tract［J］. Prog Neurobiol, 2008, 85(2): 214－235.
［27］ Steward O, Zheng B H, Ho C, et al. The dorsolateral corticospinal tract in mice: an alternative route for corticospinal input to caudal segments following dorsal column lesions［J］. J Comp Neurol, 2004, 472(4): 463－477.
［28］ Zhang Y L, Xiong Y, Mahmood A, et al. Sprouting of corticospinal tract axons from the contralateral hemisphere into the denervated side of the spinal cord is associated with functional recovery in adult rat after traumatic brain injury and erythropoietin treatment［J］. Brain Res, 2010, 1353: 249－257.

[1]	Wu Jian, Gu Yakun, Liu Jia. Research progresses in neuroprotective effects of ischemic/hypoxia preconditioning [J]. Journal of Capital Medical University, 2020, 41(4): 664-670.
[2]	Yang Tianli, Yang Yongfei, Yuan Zengqiang. Sensitivity study of HT22 in ferroptosis [J]. Journal of Capital Medical University, 2020, 41(1): 87-91.
[3]	Lu Yongquan, Chen Min, Gao Ge, Duan Chunli, Lu Lingling, Yang Hui. TRPC3 participates in α-synuclein-induced mitochondrial damage [J]. Journal of Capital Medical University, 2017, 38(6): 857-862.
[4]	Chen Min, Yu Wenjiao, Li Xin, Yang Weiwei, Li Xuran, Yu Shun. α-Synuclein promotes NMDA receptor internalization by upregulation of endocytosis protein Rab5B [J]. Journal of Capital Medical University, 2017, 38(6): 863-867.
[5]	Yu Wenjiao, Yang Weiwei, Li Xin, Li Xuran, Chen Min, Yu Shun. α-Synuclein reduces surface expressions of NMDA receptors and NMDA-invoked Ca²⁺ influx and inward current by upregulation of Rab5B in cultured primary hippocampal neurons [J]. Journal of Capital Medical University, 2017, 38(6): 868-873.
[6]	Chen Yudong, Yang Weiwei, Li Xin, Wang Peng, Li Xuran, Yu Shun. Alpha-synuclein promotes internalization of surface N-methyl-D-aspartate receptors in primary neurons and transgenic mice [J]. Journal of Capital Medical University, 2014, 35(5): 596-600.
[7]	LI Xin, CHENG Fu-rong, LI Yao-hua, WAND Yu-lan, ZHANG Yan-li, YU Shun. Effect of Alpha-synuclein on N-methyl-D-aspartic Acid-induced Cytotoxicity [J]. Journal of Capital Medical University, 2010, 31(6): 726-731.
[8]	CHENG Fu-rong, LI Xin, YIN Juan-juan, LI Yao-hua, WAND Yu-lan, YU Shun. H₂O₂ and Serum of Patient with Parkinson's Disease Promote Mitochondrial and Nuclear Translocation of Alpha-synuclein [J]. Journal of Capital Medical University, 2010, 31(6): 732-736.
[9]	CHENG Fu-rong, LI Xin, LI Yao-hua, WAND Yu-lan, ZHANG Yan-li, YU Shun. Alpha-synuclein Promotes Internalization of Surface N-methyl-D-aspartate Receptors in Dopaminergic Neuronal Cells [J]. Journal of Capital Medical University, 2010, 31(6): 737-741.
[10]	LIU Guang-wei;WANG Peng;LI Yao-hua;LI Xin;HE Xin;YU Shun. α-Synuclein Promotes Neurite Outgrowth of Primarily Cultured Rat Brain Neurons [J]. Journal of Capital Medical University, 2009, 30(5): 607-610.
[11]	Zhao Chunli;Zou Xifeng;Cai Qing;Gao Erjing;Liu Yujun;Zhao Huanying;Zhang Jinlu;Xu Qunyuan. Detection of the Signal from the Mouse Y Chromosome in Situ Hybridization with Direct Tyramine Signal Amplification [J]. Journal of Capital Medical University, 2008, 29(5): 597-600.
[12]	Huang Haixia;Wang Wei;Wei Hua;Fu Xiaosuo;Liu Ping;Niu Weizhen. Arrhythmias Induced by Stretching the Left Ventricle of Rats [J]. Journal of Capital Medical University, 2007, 28(5): 600-603.
[13]	Huang Haixia;Liu Limin;Cui Qian;Hu Yingan. The Expression of Fas-L and Caspase-3 in C57black/6 Mice after Global Cerebral Ischemia [J]. Journal of Capital Medical University, 2006, 27(5): 634-638.