Fluoro-green and fluoro-red: two new fluorescent retrograde tracers with a number of unique properties

doi:10.1016/0006-8993(96)00654-3

Brain Research

Volume 736, Issues 1–2, 14 October 1996, Pages 61-67

https://doi.org/10.1016/0006-8993(96)00654-3 Get rights and content

Abstract

As a means of improving nerve tract-tracing in the peripheral and central nervous systems we experimented with two (retrograde) fluorescent emulsions, which we have tentatively named Fluoro-Green (FGr) and Fluoro-Red (FRe), and which we believe possess the following seven advantages: (1) they show little diffusion beyond the injection site; (2) their excitation/emission characteristics allow their use in double-tracing experiments; (3) they do not ‘leak’ from labeled cells; (4) their fluorescence is presented as large granules in the cytoplasm and its processes; (5) the fluorescence lasts for a sufficiently long time to permit repeated observation; (6) they may be used in combination with a wide variety of other neuroanatomical tracing methods; (7) they are economical, non-toxic and easy to utilize.

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For pathophysiological studies, it is advantageous to label specific neuronal populations in living animals. This study aimed to establish a method for stable and long-lasting fluorescent labeling of corticospinal neurons in the living animal. The two fluorescent dyes Fluoro-Red and Fluoro-Green were injected in the cervical spinal cord of anesthetized newborn rats. After a recovery period, treated rats were returned to the mother. After 24 h and 14 days, fixed brain sections revealed wide-spread fluorescence in elongated or pyramidal-shaped cell profiles in a discrete internal cortical layer, consistent with layer V pyramidal cells. Labeled neurons displayed spontaneous synaptic activity using the slice patch clamp method. These results suggest that these dyes are effective tools for pathophysiological and slice patch clamp studies focused on specific neuron groups.
On the use of fast blue, fluoro-gold and diamidino yellow for retrograde tracing after peripheral nerve injury: Uptake, fading, dye interactions, and toxicity
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The usefulness of three retrograde fluorescent dyes for tracing injured peripheral axons was investigated. The rat sciatic was transected bilaterally and the proximal end briefly exposed to either Fast Blue (FB), Fluoro-Gold (FG) or to Diamidino Yellow (DY) on the right side, and to saline on the left side, respectively. The nerves were then resutured and allowed to regenerate. Electrophysiological tests 3 months later showed similar latencies and amplitudes of evoked muscle and nerve action potentials between tracer groups. The nerves were then cut distal to the original injury and exposed to a second (different) dye. Five days later, retrogradely labelled neurones were counted in the dorsal root ganglia (DRGs) and spinal cord ventral horn. The number of neurones labelled by the first tracer was similar for all three dyes in the DRG and ventral horn except for FG, which labelled fewer motoneurones. When used as second tracer, DY labelled fewer neurones than FG and FB in some experimental situations. The total number of neurones labelled by the first and/or second tracer was reduced by about 30% compared with controls. The contributions of cell death as well as different optional tracer combinations for studies of nerve regeneration are discussed.
Chronic mitochondrial inhibition induces glutamate-mediated corticomotoneuron death in an organotypic culture model
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There is growing evidence that mitochondrial dysfunction is an important factor in a cascade of neurotoxic events as observed during pathogenesis of various neurodegenerative diseases. In the neurodegenerative disease amyotrophic lateral sclerosis (ALS) both spinal and cortical motoneurons degenerate, but in experimental studies most attention so far has been focussed on the spinal motoneurons. In order to study the role of mitochondrial dysfunction in the pathways leading to cortical (upper) motoneuron (CMN) death, a long-term culture system of rat cortical explants was used. CMNs were visualized by immunocytochemical labeling with antibodies directed against nonphosphorylated neurofilament, SMI-32, and for their identification we also used their location in layer V of the explant, their size, and their morphological appearance. In this model the effect of mitochondrial inhibition was studied through chronic malonate treatment. For 2 weeks, low doses of complex II inhibitor malonate were added to the cultures twice a week. The malonate-induced chronic mitochondrial inhibition resulted in a dose-dependent increase of CMN death in the slices. Neuroprotection was achieved with the NMDA antagonist MK-801 and the non-NMDA antagonist CNQX indicating the involvement of glutamate in the malonate-induced CMN death. Furthermore, our data indicate that chronic mitochondrial inhibition results in CMN death, which is mediated by glutamate excitotoxicity via both non-NMDA and NMDA receptors. In this respect the present in vitro approach may act as a model for understanding mechanisms underlying CMN death in ALS.
Selective stimulation of dendrite outgrowth from identified corticospinal neurons by homotopic astrocytes
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Corticospinal neurons were identified in primary cultures of cortical neurons established from rats that had been injected with a fluorescent tracer to retrogradely label the corticospinal tract. We measured neurite outgrowth from corticospinal neurons after they had been co-cultured with astrocytes derived from either the cerebral cortex (homotopic region) or spinal cord (target region) of postnatal rats. The axon length of corticospinal neurons was increased when they were cultured on astroglial monolayers compared to a control monolayer (fibroblasts). However, no difference in axon length was noted on cortical versus spinal cord-derived astrocytes. On the other hand, total dendritic length was increased on cortical compared to spinal cord astrocytes. This increase in total dendrite length was not the result of differences in the length of primary dendrites, but primarily of a higher number of dendrites and increased branching on the cortical astroglia. If the corticospinal neurons were co-cultured without physical contact with the astrocytes, axonal and dendritic outgrowth were not stimulated when compared to the fibroblast control.
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Research reportFluoro-green and fluoro-red: two new fluorescent retrograde tracers with a number of unique properties

Abstract

Neurosci. Lett.

Neurosci. Lett.

Brain Res.

Brain Res.

Exp. Neurol.

Brain Res.

Brain Res.

Brain Res.

Brain Res.

Retrograde neuronal labeling by means of bisbenzimide and Nuclear Yellow (Hoechst 5769121). Methods to prevent diffusion of the tracers of retrogradely labeled neurons

Neurosci. Lett.

Features of foreign proteins affecting their retrograde transport in axons of the visual system

J. Neurocytol.

A retrograde fluorescence double-labeling study of cat's optic nerve cell which has a bifurcating axon

Acta Anat. Nippon.

Research report
Fluoro-green and fluoro-red: two new fluorescent retrograde tracers with a number of unique properties