Kupferschmidt David A, Cody Patrick A, Lovinger David M, Davis Margaret I
Section on Synaptic Pharmacology and In Vivo Neural Function, Laboratory for Integrative Neuroscience, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health Bethesda, MD, USA.
Front Neuroanat. 2015 Feb 4;9:6. doi: 10.3389/fnana.2015.00006. eCollection 2015.
Optogenetic constructs have revolutionized modern neuroscience, but the ability to accurately and efficiently assess their expression in the brain and associate it with prior functional measures remains a challenge. High-resolution imaging of thick, fixed brain sections would make such post-hoc assessment and association possible; however, thick sections often display autofluorescence that limits their compatibility with fluorescence microscopy. We describe and evaluate a method we call "Brain BLAQ" (Block Lipids and Aldehyde Quench) to rapidly reduce autofluorescence in thick brain sections, enabling efficient axon-level imaging of neurons and their processes in conventional tissue preparations using standard epifluorescence microscopy. Following viral-mediated transduction of optogenetic constructs and fluorescent proteins in mouse cortical pyramidal and dopaminergic neurons, we used BLAQ to assess innervation patterns in the striatum, a region in which autofluorescence often obscures the imaging of fine neural processes. After BLAQ treatment of 250-350 μm-thick brain sections, axons and puncta of labeled afferents were visible throughout the striatum using a standard epifluorescence stereomicroscope. BLAQ histochemistry confirmed that motor cortex (M1) projections preferentially innervated the matrix component of lateral striatum, whereas medial prefrontal cortex projections terminated largely in dorsal striosomes and distinct nucleus accumbens subregions. Ventral tegmental area dopaminergic projections terminated in a similarly heterogeneous pattern within nucleus accumbens and ventral striatum. Using a minimal number of easily manipulated and visualized sections, and microscopes available in most neuroscience laboratories, BLAQ enables simple, high-resolution assessment of virally transduced optogenetic construct expression, and post-hoc association of this expression with molecular markers, physiology and behavior.
光遗传学构建体彻底改变了现代神经科学，但准确、高效地评估其在大脑中的表达并将其与先前的功能测量相关联的能力仍然是一项挑战。对厚的固定脑切片进行高分辨率成像将使这种事后评估和关联成为可能；然而，厚切片通常会显示自发荧光，这限制了它们与荧光显微镜的兼容性。我们描述并评估了一种我们称为“Brain BLAQ”（阻断脂质和醛淬灭）的方法，该方法可快速降低厚脑切片中的自发荧光，从而能够使用标准落射荧光显微镜在传统组织标本中对神经元及其突起进行高效的轴突水平成像。在小鼠皮质锥体神经元和多巴胺能神经元中进行病毒介导的光遗传学构建体和荧光蛋白转导后，我们使用Brain BLAQ评估纹状体中的神经支配模式，在该区域自发荧光常常会掩盖精细神经突起的成像。用Brain BLAQ处理250 - 350μm厚的脑切片后，使用标准落射荧光体视显微镜可以在整个纹状体中看到标记传入纤维的轴突和小点。Brain BLAQ组织化学证实，运动皮层（M1）投射优先支配外侧纹状体的基质成分，而内侧前额叶皮层投射主要终止于背侧纹状体小体和伏隔核的不同亚区域。腹侧被盖区多巴胺能投射在伏隔核和腹侧纹状体内以类似的异质模式终止。使用最少数量的易于操作和观察的切片以及大多数神经科学实验室都有的显微镜，Brain BLAQ能够能够对病毒转导的光遗传学构建体表达进行简单、高分辨率的评估，并将这种表达与分子标记、生理学和行为进行事后关联。
