Simultaneous identification of unmyelinated and myelinated primary somatic afferents by co-injection of isolectin B4 and Cholera toxin subunit B into the sciatic nerve of the rat

Shehab, Safa; Hughes, David I.

doi:10.1016/j.jneumeth.2011.04.002

Cited by 28 publications

(39 citation statements)

References 34 publications

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“…S2). These data confirm that labeling peripheral nerves with CTB and WGA is a suitable method for differentiating large and small DRG sensory neurons (16,17). Dissociated sensory neurons prelabeled with WGA and CTB were sorted into separate fractions by fluorescence-activated cell sorting (FACS) (Fig.…”

Section: Gfrα3 Is Present In Both Myelinated and Unmyelinated Sensorysupporting

confidence: 74%

“…Populations of small and large neurons were identified by injecting peripheral nerves with wheat germ agglutinin conjugated to Alexa Fluor 647 (WGA-647), labeling a subpopulation of small sensory neurons, and cholera toxin B subunit conjugated to Alexa Fluor 488 (CTB-488), labeling large, myelinated sensory neurons (16,17).…”

Section: Gfrα3 Is Present In Both Myelinated and Unmyelinated Sensorymentioning

confidence: 99%

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Artemin promotes functional long-distance axonal regeneration to the brainstem after dorsal root crush

Wong

Gibson

Arnold

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Recovery after a spinal cord injury often requires that axons restore synaptic connectivity with denervated targets several centimeters from the site of injury. Here we report that systemic artemin (ARTN) treatment promotes the regeneration of sensory axons to the brainstem after brachial dorsal root crush in adult rats. ARTN not only stimulates robust regeneration of large, myelinated sensory axons to the brainstem, but also promotes functional reinnervation of the appropriate target region, the cuneate nucleus. ARTN signals primarily through the RET tyrosine kinase, an interaction that requires the nonsignaling coreceptor GDNF family receptor (GFRα3). Previous studies reported limited GFRα3 expression on large sensory neurons, but our findings demonstrate that ARTN promotes robust regeneration of large, myelinated sensory afferents. Using a cell sorting technique, we demonstrate that GFRα3 expression is similar in myelinated and unmyelinated adult sensory neurons, suggesting that ARTN likely induces long-distance regeneration by binding GFRα3 and RET. Although ARTN is delivered for just 2 wk, regeneration to the brainstem requires more than 3 mo, suggesting that brief trophic support may initiate intrinsic growth programs that remain active until targets are reached. Given its ability to promote targeted functional regeneration to the brainstem, ARTN may represent a promising therapy for restoring sensory function after spinal cord injury.artemin | GFRα3 | regeneration | dorsal root crush | cuneate nucleus S pinal cord (SC) injury results in permanent paresis and paralysis, owing in large part to the failure of axons to regenerate. In the adult SC, axons do not regenerate because of myelin-and injury-associated inhibitory barriers and a limited intrinsic regenerative ability (1). Although there has been some success in removing extrinsic barriers and providing neurotrophic factors to promote functional regeneration over short distances (2-4), meaningful functional recovery requires that damaged axons regenerate and reconnect with their original targets, often centimeters away from the lesion. Studies in which damaged sensory axons were induced to regenerate to the brainstem have failed to show reestablishment of synapses (5). These findings cast doubt on whether sensory axons can regenerate functionally to the brainstem. Dorsal root (DR) crush provides a useful model for studying long-distance axon regeneration without affecting the architecture of the SC. Fine touch and proprioceptive neurons with cell bodies in the DR ganglion (DRG) provide monosynaptic input to neurons in the dorsal column nuclei. These neurons can be traced using transganglionic labeling methods and are easily studied electrophysiologically, making this an ideal injury model for investigating functional regeneration from the brachial SC to the brainstem.Previous studies demonstrated that a 2-wk systemic treatment with the neurotrophic factor artemin (ARTN) promotes topographically specific regeneration of both myelinated and unmyelinated s...

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Section: Gfrα3 Is Present In Both Myelinated and Unmyelinated Sensorysupporting

confidence: 74%

Section: Gfrα3 Is Present In Both Myelinated and Unmyelinated Sensorymentioning

confidence: 99%

Artemin promotes functional long-distance axonal regeneration to the brainstem after dorsal root crush

Wong

Gibson

Arnold

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Further investigations from our group will focus on the representation of corneal afferent fibers in a cleared brain using a mixture of tracers that discriminate between myelinated and unmyelinated fibers. It is well known that each fiber type projects preferentially to different laminae of the dorsal horns of the medulla and spinal cord; CTB mainly labels terminals in laminae I and IIIeV, but not in lamina II (Baseer et al, 2014;Shehab and Hughes, 2011). The full range of ocular surface afferents has been previously described from animals whose corneas were treated with a mixture of HRP-CTB and HRP-WGA, using classical histological methods (Panneton et al, 2010).…”

Section: Discussionmentioning

confidence: 99%

Combined 3DISCO clearing method, retrograde tracer and ultramicroscopy to map corneal neurons in a whole adult mouse trigeminal ganglion

Launay

Godefroy

Khabou

et al. 2015

Experimental Eye Research

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Tissue clearing and subsequent imaging of intact transparent tissues have provided an innovative way to analyze anatomical pathways in the nervous system. In this study, we combined a recent 3-dimensional imaging of solvent cleared organ (3DISCO) procedure, light-sheet microscopy, fluorescent retrograde tracer, and Imaris software to 3D map corneal sensory neurons within a whole adult mouse trigeminal ganglion (TG). We first established the optimized steps to easily and rapidly clear a fixed TG. We found that the 3DISCO procedure gave excellent results and took less than 3 h to clear the TG. In a second set of experiments, a retrograde tracer (cholera toxin B Alexa 594-conjugated) was applied to de-epithelialized cornea to retrograde-labeled corneal sensory neurons. Two days later, TGs were cleared by the 3DISCO method and serial imaging was performed using light-sheet ultramicroscopic technology. High-resolution images of labeled neurons can be easily and rapidly obtained from a 3D reconstructed whole mouse TG. We then provided a 3D reconstruction of corneal afferent neurons and analyzed their precise localization in the TG. Thus, we showed that neurons supplying corneal sensory innervation exhibit a highly specific limited dorsomedial localization within the TG. We report that our combined method offers the possibility to perform manual (on 20 μm sections) and automated (on 3D reconstructed TG) counting of labeled cells in a cleared mouse TG. To conclude, we illustrate that the combination of the 3DISCO clearing method with light-sheet microscopy, retrograde tracer, and automatic counting represents a rapid and reliable method to analyze a subpopulation of neurons within the peripheral and central nervous system.

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“…This observation has mainly been based on the utilization of the retrograde and transganglionic neuronal tracer CTB-HRP. As mentioned above, normally it is mainly taken up and transported retrogradely by large diameter myelinated A-fibres and labels the somatotopically related areas of the dorsal horn (laminae I and III-VI) (LaMotte et al, 1991;Oszlács et al, 2015;Robertson and Grant, 1985;Shehab and Hughes, 2011;Trojanowski et al, 1982). However, 2 weeks after a peripheral nerve lesion CTB-HRP labelling was apparent not only in the deeper layers of the dorsal horn but also in lamina II, a region which normally receives only C-fibre input (Lekan et al, 1996;Shehab et al, 2004;Shortland et al, 1997;Woolf et al, 1992;Woolf et al, 1995).…”

Section: Methods For Demonstration Of the Central Projections Of Perimentioning

confidence: 94%

“…HRP conjugated to wheat germ agglutinin (WGA-HRP) is proved to be a selective marker of unmyelinated sensory neurons, hence injection of WGA-HRP into a peripheral nerve gives rise to transganglionic labelling of C-fibres, mostly nociceptors (small-and medium-sized neurons) and the somatotopically related superficial laminae (mainly lamina II) of the spinal cord dorsal horn (LaMotte et al, 1991;Molander and Grant, 1986;Shehab and Hughes, 2011). The lectin WGA specifically binds N-acetyl glucosamine residues of the carbohydrate chains of glycoconjugates of the plasma membrane (Macsween and Fox, 1975;Sharon and Lis, 1972).…”

Section: Methods For Demonstration Of the Central Projections Of Perimentioning

confidence: 99%

Capsaicin-induced activation and chemical injury of nociceptive primary sensory neurons

Oszlács

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Simultaneous identification of unmyelinated and myelinated primary somatic afferents by co-injection of isolectin B4 and Cholera toxin subunit B into the sciatic nerve of the rat

Cited by 28 publications

References 34 publications

Artemin promotes functional long-distance axonal regeneration to the brainstem after dorsal root crush

Artemin promotes functional long-distance axonal regeneration to the brainstem after dorsal root crush

Combined 3DISCO clearing method, retrograde tracer and ultramicroscopy to map corneal neurons in a whole adult mouse trigeminal ganglion

Capsaicin-induced activation and chemical injury of nociceptive primary sensory neurons

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