Anterograde axonal tracing with the subunit B of cholera toxin: a highly sensitive immunohistochemical protocol for revealing fine axonal morphology in adult and neonatal brains

Angelucci, Alessandra; Clascá, Francisco; Sur, Mriganka

doi:10.1016/0165-0270(95)00155-7

Cited by 190 publications

(162 citation statements)

References 35 publications

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“…RGC axons course through layer III of the SC to contact layer II neurons retinotopically (20). These can be visualized by intraocular injection of cholera toxin β-subunit (CTB), which labels the entire retinal projection via active uptake and transport (21,22). For a 5-mo C57 and a 5-mo DBA/2, CTB labeled the entire retinotopic projection in the SC except for the retinal optic disk, which does not contain RGCs ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Distal axonopathy with structural persistence in glaucomatous neurodegeneration

Crish

Sappington

Inman

et al. 2010

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

309

482

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An early hallmark of neuronal degeneration is distal transport loss and axon pathology. Glaucoma involves the degeneration of retinal ganglion cell (RGC) neurons and their axons in the optic nerve. Here we show that, like other neurodegenerations, distal axon injury appears early in mouse glaucoma. Where RGC axons terminate in the superior colliculus, reduction of active transport follows a retinotopic pattern resembling glaucomatous vision loss. Like glaucoma, susceptibility to transport deficits increases with age and is not necessarily associated with elevated ocular pressure. Transport deficits progress distal-to-proximal, appearing in the colliculus first followed by more proximal secondary targets and then the optic tract. Transport persists through the optic nerve head before finally failing in the retina. Although axon degeneration also progresses distal-to-proximal, myelinated RGC axons and their presynaptic terminals persist in the colliculus well after transport fails. Thus, distal transport loss is predegenerative and may represent a therapeutic target.axon transport | optic neuropathy | retinal ganglion cell | glaucoma | optic nerve

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Section: Resultsmentioning

confidence: 99%

Distal axonopathy with structural persistence in glaucomatous neurodegeneration

Crish

Sappington

Inman

et al. 2010

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

309

482

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“…We examined the LGN of these animals at P8, after anterograde labeling of retinogeniculate projections at P7 with intraocular injection of WGA-HRP in one eye and CTB subunit in the other eye. Alternate sections were processed using TMB histochemistry to visualize WGA-HRP (Mesulam, 1978) or immunohistochemistry to visualize CTB (Angelucci et al, 1996). Segregation of inputs from the two eyes was evident in every case.…”

Section: Resultsmentioning

confidence: 99%

“…One series of sections was used to reveal HRP using TMB, and the series of adjacent sections was used to reveal CTB, using the method described by Angelucci et al (1996). Briefly, sections were washed in PBS, pH 7.4, rinsed in methanol and 0.3% hydrogen peroxide to quench endogenous and injected peroxidase activity, treated with 0.1 M glycine, preincubated in 2-8% normal rabbit serum (NRS, Vector Laboratories, Burlingame, CA) and 2.5% bovine serum albumin (BSA), then incubated in goat anti-CTB primary antibody (List) at a concentration of 1:4000 for 1-2 d at room temperature in a solution containing 2% NRS, 2.5% BSA, and 2% Triton X-100.…”

Section: Methodsmentioning

confidence: 99%

A Role for Nitric Oxide in the Development of the Ferret Retinogeniculate Projection

et al. 1996

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The ferret retinogeniculate projection segregates into eyespecific layers during the first postnatal week and into ON/OFF sublaminae, which receive inputs from either on-center or offcenter retinal ganglion cells, during the third and fourth postnatal weeks. The restriction of retinogeniculate axon arbors into eye-specific layers appears to depend on action potential activity (Shatz and Stryker, 1988) but does not require activation of NMDA receptors (Smetters et al., 1994). The formation of ON/OFF sublaminae is also activity-dependent and is disrupted by in vivo blockade of NMDA receptors (Hahm et al., 1991). To investigate a possible mechanism whereby blockade of postsynaptic NMDA receptors in the lateral geniculate nucleus (LGN) results in changes in the size and position of presynaptic axon arbors, we tested the role of the diffusible messenger nitric oxide (NO) in the development of the retinogeniculate pathway. We found previously that NO synthase (NOS) is transiently expressed in LGN cells during the refinement of retinogeniculate projections . In this study, treatment with N G -nitro-L-arginine (L-NoArg), an arginine analog that inhibits NOS, during the third and fourth postnatal weeks resulted in an overall pattern of sublamination that was significantly reduced compared with normal and control animals. Single retinogeniculate axon arbors were located in the middle of eye-specific layers rather than toward the inner or outer half as in normal or control animals. The effect of NOS inhibition was not a consequence of the hypertensive effect of L-NoArg. In contrast to the effect of L-NoArg on the formation of ON/OFF sublaminae, treatment with L-NoArg during the first postnatal week did not disrupt the formation of eye-specific layers. Biochemical assays indicated significant inhibition of NOS during both treatment periods. These data suggest that NO acts together with NMDA receptors in activity-dependent refinement of connections during a specific phase of retinogeniculate development. Key words: diffusible messenger; visual system; lateral geniculate nucleus (LGN); pattern formation; eye-specific layers; ON/ OFF sublaminae; neuronal activityThe precise pattern of connections underlying adult visual processing in mammals arises from the refinement of less specific connectivity present early in development. The mechanisms by which diffuse connections are refined rely at least in part on neuronal activity. In the ferret, retinogeniculate connections are refined in two distinct phases. During the first postnatal week, retinal axons within the lateral geniculate nucleus (LGN) segregate into eye-specific layers (Linden et al., 1981). During the third and fourth postnatal weeks, afferents to the A and A1 layers, which receive input from the contralateral and ipsilateral eye, respectively, further segregate into sublaminae. The inner sublamina receives inputs from on-center retinal ganglion cells, whereas the outer sublamina receives inputs from off-center retinal ganglion cells (Stryker and Zahs, 1983;Hahm and S...

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“…CTB has occasionally been used for anterograde tracing purposes. The quality of fiber labeling with CTB is poor however, compared to other standard anterograde fiber tracers, although several procedures have been devised in attempts to solve this drawback (Angelucci et al, 1996).…”

Section: Bi-directionally Transported Tracer: Ctbmentioning

confidence: 99%

“…FG can be used at a wider range of survival time, ranging from 2 days to 1 year (Schmued and Fallon, 1986;Pieribone and Aston-Jones, 1988;Schmued and Heimer, 1990;Akintunde and Buxton, 1992;Schmued, 1994;Novikova et al, 1997). Regarding CTB, one should keep in mind that a short survival time will increase the amount of anterograde CTB labeling (Trojanowski et al, 1981;Angelucci et al, 1996), while retrograde labeling with CTB seems to be enhanced with longer survival times.…”

Section: Sur6i6al Timementioning

confidence: 99%

Complex brain circuits studied via simultaneous and permanent detection of three transported neuroanatomical tracers in the same histological section

Lanciego

Wouterlood

Erro

et al. 2000

Journal of Neuroscience Methods

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Experimental neuroanatomical tracing methods lie at the basis of the study of the nervous system. When the scientific question is relatively straightforward, it may be sufficient to derive satisfactory answers from experiments in which a single neuroanatomical tracing method is applied. In various scientific paradigms however, for instance when the degree of convergence of two different projections on a particular cortical area or subcortical nucleus is the subject of study, the application of single tracing methods can be either insufficient or uneconomical to solve the questions asked. In cases where chains of projections are the subjects of study, the simultaneous application of two tracing methods or even more may be compulsory. The present contribution focuses on combinations of several neuroanatomical tract-tracing strategies, enabling in the end the simultaneous, unambiguous and permanent detection of three transported markers according to a three-color paradigm. A number of combinations of three tracers or of two tracers plus the immunocytochemical detection of a neuroactive substance can be conceived; we describe several of these combinations implemented by us using the present multitracer protocol.

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Anterograde axonal tracing with the subunit B of cholera toxin: a highly sensitive immunohistochemical protocol for revealing fine axonal morphology in adult and neonatal brains

Cited by 190 publications

References 35 publications

Distal axonopathy with structural persistence in glaucomatous neurodegeneration

Distal axonopathy with structural persistence in glaucomatous neurodegeneration

A Role for Nitric Oxide in the Development of the Ferret Retinogeniculate Projection

Complex brain circuits studied via simultaneous and permanent detection of three transported neuroanatomical tracers in the same histological section

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