Abstract
The nervous system coordinates pathways and circuits to process sensory information and govern motor behaviors. Mapping these pathways is important to further understand the connectivity throughout the nervous system and is vital for developing treatments for neuronal diseases and disorders. We targeted long ascending propriospinal neurons (LAPNs) in the rat spinal cord utilizing Fluoro-Ruby (FR) [10kD rhodamine dextran amine (RDA)], and two dual-viral systems. Dual-viral tracing utilizing a retrograde adeno-associated virus (retroAAV), which confers robust labeling in the brain, resulted in a small number of LAPNs being labeled, but dual-viral tracing using a highly efficient retrograde (HiRet) lentivirus provided robust labeling similar to FR. Additionally, dual-viral tracing with HiRet lentivirus and tracing with FR may preferentially label different subpopulations of LAPNs. These data demonstrate that dual-viral tracing in the spinal cord employing a HiRet lentivirus provides robust and specific labeling of LAPNs and emphasizes the need to empirically optimize viral systems to target specific neuronal population(s).
Highlights
Mesoscale connectomics–characterizing a single population of neurons and/or connectivity of those neurons–has made similar progress (Lanciego and Wouterlood, 2020; Ugolini, 2020), but traditional tracers such as horseradish peroxidase, cholera toxin subunit B (CTB), hydroxystilbamidine, and conjugated dextran amines [which include biotinylated dextran amine (BDA), and rhodamine conjugated dextran amine (RDA), known as Fluoro-Ruby (FR) or Mini Ruby depending on molecular weight] remain the most widely used technique
The total number of labeled neurons was significantly higher in the FR and highly efficient retrograde (HiRet) groups compared to the retroAAV group (Figure 2A)
These results indicate that tracing long ascending propriospinal neuron (LAPN) with FR or target-defined projection labeling utilizing HiRet lentivirus provide robust labeling of LAPNs, while target-defined projection labeling using retroAAV significantly reduced labeling of LAPNs
Summary
Understanding the complexity and specificity of neural pathways and circuits in the mammalian nervous system is a major goal for neuroanatomists and is vital to understand and treat nervous system injuries and disorders (Nassi et al, 2015; Zeng, 2018; Horn and Fox, 2020; Lanciego and Wouterlood, 2020; Ugolini, 2020; Wong et al, 2020). Mesoscale connectomics–characterizing a single population of neurons and/or connectivity of those neurons–has made similar progress (Lanciego and Wouterlood, 2020; Ugolini, 2020), but traditional tracers such as horseradish peroxidase, cholera toxin subunit B (CTB), hydroxystilbamidine (known commercially as Fluoro-Gold), and conjugated dextran amines [which include biotinylated dextran amine (BDA), and rhodamine conjugated dextran amine (RDA), known as Fluoro-Ruby (FR) or Mini Ruby depending on molecular weight] remain the most widely used technique These traditional tracers allow for anterograde and retrograde tract-tracing, are valuable for revealing the locations of neurons projecting to or from and area of interest, and have been widely used throughout the nervous system for more than three decades (Lanciego and Wouterlood, 2011; Wouterlood et al, 2014; Nassi et al, 2015). Dextran amines can be taken up by axons damaged during injection procedures (Glover et al, 1986), longterm exposure to Fluoro-Gold can be neurotoxic (Naumann et al, 2000), Fluoro-Gold and CTB can inadvertently be taken up by fibers of passage (Dado et al, 1990; Chen and Aston-Jones, 1995), and biotin conjugates of CTB can be transneuronal (Lai et al, 2015), all of which may hinder mesoscale connectomic analyses
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