Isolation, Purification, and Culture of Primary Murine Sensory Neurons

Katzenell, Sarah; Cabrera, Jorge Rubén; North, Brian J.; Leib, David A.

doi:10.1007/978-1-4939-7237-1_15

Cited by 36 publications

(36 citation statements)

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“…Half a million neurons are typically required to load one MFC (Tsantoulas et al, 2013 ; Jia et al, 2016 , 2018 ), and while this large number is not an issue for typical CNS neurons, it is a challenge for neurons of the PNS, especially when working with PNS neurons from adult mice. Per mouse, for instance, one can obtain at most 100,000 sensory neurons from dorsal root ganglia (DRG; Heinrich et al, 2016 ), and significantly fewer still from trigeminal ganglia (TG; Katzenell et al, 2017 ). Performing a series of experiments with such small-population neurons in MFC devices is therefore impractical, if not unfeasible.…”

Section: Introductionmentioning

confidence: 99%

Microfluidics of Small-Population Neurons Allows for a Precise Quantification of the Peripheral Axonal Growth State

Jocher

Mannschatz

Offterdinger

et al. 2018

Front. Cell. Neurosci.

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Neurons are morphologically the most complex cell types and are characterized by a significant degree of axonal autonomy as well as having efficient means of communication between axons and neuronal cell bodies. For studying the response to axonal injury, compartmentalized microfluidic chambers (MFCs) have become the method of choice because they allow for the selective treatment of axons, independently of the soma, in a highly controllable and reproducible manner. A major disadvantage of these devices is the relatively large number of neurons needed for seeding, which makes them impractical to use with small-population neurons, such as sensory neurons of the mouse. Here, we describe a simple approach of seeding and culturing neurons in MFCs that allows for a dramatic reduction of neurons required to 10,000 neurons per device. This technique facilitates efficient experiments with small-population neurons in compartmentalized MFCs. We used this experimental setup to determine the intrinsic axonal growth state of adult mouse sensory neurons derived from dorsal root ganglia (DRG) and even trigeminal ganglia (TG). In combination with a newly developed linear Sholl analysis tool, we have examined the axonal growth responses of DRG and TG neurons to various cocktails of neurotrophins, glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF) and leptin. Precise quantification of axonal outgrowth revealed specific differences in the potency of each combination to promote axonal regeneration and to switch neurons into an intrinsic axonal growth state. This novel experimental setup opens the way to practicable microfluidic analyses of neurons that have previously been largely neglected simply due to insufficient numbers, including sensory neurons, sympathetic neurons and motor neurons.

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Section: Introductionmentioning

confidence: 99%

Microfluidics of Small-Population Neurons Allows for a Precise Quantification of the Peripheral Axonal Growth State

Jocher

Mannschatz

Offterdinger

et al. 2018

Front. Cell. Neurosci.

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“…To begin to address this, we developed a protocol with which adult mouse TG neurons could recapitulate their compartmentalization within microfluidic devices. In this system, neurites sprout into a compartment distal from the cell bodies, eventually forming a dense, polarized, axonal network (26). Growth of the majority of neuronal subtypes of the TG was promoted by providing a combination of neurotrophic factors (nerve growth factor [NGF], brain-derived neurotrophic factor [BDNF], glial cell-derived neurotrophic factor [GDNF], and soluble GDNF family receptor ␣1 [GFR␣1]).…”

Section: Resultsmentioning

confidence: 99%

“…Microfluidics culture of adult mouse TG neurons. TG neurons from adult mice were isolated and cultured largely as previously reported (26). Microfluidic devices (Standard Neuron Device 150 [SND150]; Xona Microfluidics) were attached to 24-by 60-mm coverslips.…”

Section: Methodsmentioning

confidence: 99%

“…The devices were then washed three times with HBSS and coated with natural mouse laminin (Invitrogen) at a concentration of 18 g/ml in HBSS overnight. TG neurons were isolated as described previously, with a few modifications (26). Mice 5 to 10 weeks old were euthanized using approved methods and transcardially perfused with phosphate-buffered saline (PBS; HyClone).…”

Section: Methodsmentioning

confidence: 99%

“…(i) In vitro. Infections in microfluidic devices were performed based on an average of 1,000 neurons growing from the cell body compartment to the distal axon compartment (26). Infection of neurons in culture for 3 days was performed at a multiplicity of infection (MOI) of 10 at the distal axon in a volume of 200 l with KOS/62.…”

Section: Methodsmentioning

confidence: 99%

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Neuronal Subtype Determines Herpes Simplex Virus 1 Latency-Associated-Transcript Promoter Activity during Latency

Cabrera

Charron

Leib

2018

J Virol

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Herpes simplex virus (HSV) latency in neurons remains poorly understood, and the heterogeneity of the sensory nervous system complicates mechanistic studies. In this study, we used primary culture of adult trigeminal ganglion (TG) mouse neurons in microfluidic devices, and an model, to examine the subtypes of sensory neurons involved in HSV latency. HSV-infected neurofilament heavy-positive neurons (NefH) were more likely to express Latency-Associated Transcripts (LATs) relative to infected neurofilament heavy-negative neurons (NefH). This differential expression of the LAT promoter correlated with differences in HSV-1 early infection that manifested as differences in the efficiency with which HSV particles reached the cell body following infection at the distal axon. , we further identified a specific subset of NefH neurons which co-expressed Calcitonin Gene Related Peptide α (NefH CGRP) as the sensory neuron subpopulation with the highest LAT promoter activity following HSV-1 infection. Finally, an early-phase reactivation assay showed HSV-1 reactivating in NefH CGRP neurons, although other sensory neuron subpopulations were also involved. Together, these results show that sensory neurons expressing neurofilaments exhibit enhanced LAT promoter activity. We hypothesize that reduced efficiency of HSV-1 invasion at an early phase of infection may promote efficient establishment of latency in NefH neurons due to initiation of the antiviral state preceding arrival of the virus at the neuronal cell body. While the outcome of HSV-1 infection of neurons is determined by a broad variety of factors neuronal subtypes are likely to play differential roles in modulating the establishment of latent infection. Two pivotal properties of HSV-1 make it a successful pathogen. Firstly, it infects neurons which are immune-privileged. Secondly, it establishes latency in these neurons. Together, these properties allow HSV to persist for the lifetime of its host. Neurons are diverse and highly organized cells, with specific anatomical, physiological and molecular characteristics. Previous work has shown that establishment of latency by HSV-1 does not occur equally in all types of neurons. Our results show that the kinetics of HSV infection and levels of latency-related gene expression differ in certain types of neurons. The neuronal subtype infected by HSV is therefore a critical determinant of the outcome of infection and latency.

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Optimized Workflow for Proteomics and Phosphoproteomics With Limited Tissue Samples

Hu,

Wang

2024

Current Protocols

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Proteomics and phosphoproteomics play crucial roles in elucidating the dynamics of post‐transcriptional processes. While experimental methods and workflows have been established in this field, a persistent challenge arises when dealing with small samples containing a limited amount of protein. This limitation can significantly impact the recovery of peptides and phosphopeptides. In response to this challenge, we have developed a comprehensive experimental workflow tailored specifically for small‐scale samples, with a special emphasis on neuronal tissues like the trigeminal ganglion. Our proposed workflow consists of seven steps aimed at optimizing the preparation of limited tissue samples for both proteomic and phosphoproteomic analyses. One noteworthy innovation in our approach involves the utilization of a dual enrichment strategy for phosphopeptides. Initially, we employ Fe‐NTA Magnetic beads, renowned for their specificity and effectiveness in capturing phosphopeptides. Subsequently, we complement this approach with the TiO2‐based method, which offers a broader spectrum of phosphopeptide recovery. This innovative workflow not only overcomes the challenges posed by limited sample sizes but also establishes a new benchmark for precision and efficiency in proteomic investigations. Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Protein extraction and digestionBasic Protocol 2: TMT labeling and peptide cleanupBasic Protocol 3: IMAC Fe‐NTA magnetic beads phosphopeptide enrichmentBasic Protocol 4: TiO2 enrichmentBasic Protocol 5: Fe‐NTA phosphopeptide EnrichmentBasic Protocol 6: High pH peptide fractionationBasic protocol 7: LC‐MS/MS analysis and database search

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Isolation, Purification, and Culture of Primary Murine Sensory Neurons

Cited by 36 publications

References 10 publications

Microfluidics of Small-Population Neurons Allows for a Precise Quantification of the Peripheral Axonal Growth State

Microfluidics of Small-Population Neurons Allows for a Precise Quantification of the Peripheral Axonal Growth State

Neuronal Subtype Determines Herpes Simplex Virus 1 Latency-Associated-Transcript Promoter Activity during Latency

Optimized Workflow for Proteomics and Phosphoproteomics With Limited Tissue Samples

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