Dataset Information

Dual-Viral Transduction Utilizing Highly Efficient Retrograde Lentivirus Improves Labeling of Long Propriospinal Neurons.

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).

SUBMITTER: Brown BL

PROVIDER: S-EPMC8019739 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Dual-Viral Transduction Utilizing Highly Efficient Retrograde Lentivirus Improves Labeling of Long Propriospinal Neurons.

Brown Brandon L BL Zalla Rachel M RM Shepard Courtney T CT Howard Russell M RM Kopechek Jonathan A JA Magnuson David S K DSK Whittemore Scott R SR

Frontiers in neuroanatomy 20210322

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 ret ...[more]

PMID: 33828464

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Project description:BackgroundPropriospinal neurons, with axonal projections intrinsic to the spinal cord, have shown a greater regenerative response than supraspinal neurons after axotomy due to spinal cord injury (SCI). Our previous work focused on the response of axotomized short thoracic propriospinal (TPS) neurons following a low thoracic SCI (T9 spinal transection or moderate spinal contusion injury) in the rat. The present investigation analyzes the intrinsic response of cervical propriospinal neurons having long descending axons which project into the lumbosacral enlargement, long descending propriospinal tract (LDPT) axons. These neurons also were axotomized by T9 spinal injury in the same animals used in our previous study.ResultsUtilizing laser microdissection (LMD), qRT-PCR, and immunohistochemistry, we studied LDPT neurons (located in the C5-C6 spinal segments) between 3-days, and 1-month following a low thoracic (T9) spinal cord injury. We examined the response of 89 genes related to growth factors, cell surface receptors, apoptosis, axonal regeneration, and neuroprotection/cell survival. We found a strong and significant down-regulation of ~25% of the genes analyzed early after injury (3-days post-injury) with a sustained down-regulation in most instances. In the few genes that were up-regulated (Actb, Atf3, Frs2, Hspb1, Nrap, Stat1) post-axotomy, the expression for all but one was down-regulated by 2-weeks post-injury. We also compared the uninjured TPS control neurons to the uninjured LDPT neurons used in this experiment for phenotypic differences between these two subpopulations of propriospinal neurons. We found significant differences in expression in 37 of the 84 genes examined between these two subpopulations of propriospinal neurons with LDPT neurons exhibiting a significantly higher base line expression for all but 3 of these genes compared to TPS neurons.ConclusionsTaken collectively these data indicate a broad overall down-regulation in the genes examined, including genes for neurotrophic/growth factor receptors as well as for several growth factors. There was a lack of a significant regenerative response, with the exception of an up-regulation of Atf3 and early up-regulation of Hspb1 (Hsp27), both involved in cell stress/neuroprotection as well as axonal regeneration. There was no indication of a cell death response over the first month post-injury. In addition, there appear to be significant phenotypic differences between uninjured TPS and LDPT neurons, which may partly account for the differences observed in their post-axotomy responses. The findings in this current study stand in stark contrast to the findings from our previous work on TPS neurons. This suggests that different approaches will be needed to enhance the capacity for each population of propriospinal neuron to survive and undergo successful axonal regeneration after SCI.

Project description:BackgroundCentral nervous system axons lack a robust regenerative response following spinal cord injury (SCI) and regeneration is usually abortive. Supraspinal pathways, which are the most commonly studied for their regenerative potential, demonstrate a limited regenerative ability. On the other hand, propriospinal (PS) neurons, with axons intrinsic to the spinal cord, have shown a greater regenerative response than their supraspinal counterparts, but remain relatively understudied in regards to spinal cord injury.ResultsUtilizing laser microdissection, gene-microarray, qRT-PCR, and immunohistochemistry, we focused on the intrinsic post-axotomy response of specifically labelled thoracic propriospinal neurons at periods from 3-days to 1-month following T9 spinal cord injury. We found a strong and early (3-days post injury, p.i) upregulation in the expression of genes involved in the immune/inflammatory response that returned towards normal by 1-week p.i. In addition, several regeneration associated and cell survival/neuroprotective genes were significantly up-regulated at the earliest p.i. period studied. Significant upregulation of several growth factor receptor genes (GFRa1, Ret, Lifr) also occurred only during the initial period examined. The expression of a number of pro-apoptotic genes up-regulated at 3-days p.i. suggest that changes in gene expression after this period may have resulted from analyzing surviving TPS neurons after the cell death of the remainder of the axotomized TPS neuronal population.ConclusionsTaken collectively these data demonstrate that thoracic propriospinal (TPS) neurons mount a very dynamic response following low thoracic axotomy that includes a strong regenerative response, but also results in the cell death of many axotomized TPS neurons in the first week after spinal cord injury. These data also suggest that the immune/inflammatory response may have an important role in mediating the early strong regenerative response, as well as the apoptotic response, since expression of all of three classes of gene are up-regulated only during the initial period examined, 3-days post-SCI. The up-regulation in the expression of genes for several growth factor receptors during the first week post-SCI also suggest that administration of these factors may protect TPS neurons from cell death and maintain a regenerative response, but only if given during the early period after injury.

Dataset Information

Dual-Viral Transduction Utilizing Highly Efficient Retrograde Lentivirus Improves Labeling of Long Propriospinal Neurons.

Publications

Dual-Viral Transduction Utilizing Highly Efficient Retrograde Lentivirus Improves Labeling of Long Propriospinal Neurons.

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