Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes (original) (raw)
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Retrograde Loading of Nerves Tracts and Spinal Roots with Flourescent Dyes
2013
Retrograde labeling of neurons is a standard anatomical method 1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons . Generally, the dyes are applied as solid crystals or by local pressure injection using glass pipettes. However, this can result in dilution of the dye and reduced labeling intensity, particularly when several hours are required for dye diffusion. Here we demonstrate a simple and lowcost technique for introducing fluorescent and ion-sensitive dyes into neurons using a polyethylene suction pipette filled with the dye solution. This method offers a reliable way for maintaining a high concentration of the dye in contact with axons throughout the loading procedure.
Electroporation Loading of Calcium-Sensitive Dyes Into the CNS
Journal of Neurophysiology, 2004
Calcium imaging of neural network function has been limited by the extent of tissue labeled or the time taken for labeling. We now describe the use of electroporation—an established technique for transfecting cells with genes—to load neurons with calcium-sensitive dyes in the isolated spinal cord of the neonatal mouse in vitro. The dyes were injected subdurally, intravascularly, or into the central canal. This technique results in rapid and extensive labeling of neurons and their processes at all depths of the spinal cord, over a rostrocaudal extent determined by the position and size of the electrodes. Our results suggest that vascular distribution of the dye is involved in all three types of injections. Electroporation disrupts local reflex and network function only transiently (∼1 h), after which time they recover. We describe applications of the method to image activity of neuronal populations and individual neurons during antidromic, reflex, and locomotor-like behaviors. We sho...
Journal of Neuroscience Methods, 2001
In this study we have used fluorescent microspheres to retrogradely label primary sensory neurons in dorsal root ganglia (DRGs). Following injection into peripheral nerves, the animals were allowed to survive up to 480 days. Simple profile count indicates that there is a substantial retention of the labeling still after at least 480 days, i.e. about two-thirds of a rat's life span. Moreover, the appearance of the labeling remains quite distinct. Using established markers for axon damage of DRG neurons, we could detect a slight and transient effect of the peripheral nerve injection on the gene expression pattern. It is concluded that fluorescent microspheres represents an attractive means of tagging neurons in experiments covering long time periods.
Scientific Reports, 2017
The delivery of tracers into populations of neurons is essential to visualize their anatomy and analyze their function. In some model systems genetically-targeted expression of fluorescent proteins is the method of choice; however, these genetic tools are not available for most organisms and alternative labeling methods are very limited. Here we describe a new method for neuronal labelling by electrophoretic dye delivery from a suction electrode directly through the neuronal sheath of nerves and ganglia in insects. Polar tracer molecules were delivered into the locust auditory nerve without destroying its function, simultaneously staining peripheral sensory structures and central axonal projections. Local neuron populations could be labelled directly through the surface of the brain, and in-vivo optical imaging of sound-evoked activity was achieved through the electrophoretic delivery of calcium indicators. The method provides a new tool for studying how stimuli are processed in peripheral and central sensory pathways and is a significant advance for the study of nervous systems in nonmodel organisms. For neuroanatomical studies and functional imaging the targeted delivery of dyes and indicators into neuron populations remains a fundamental challenge. In some animals the gene-targeted expression of fluorescent proteins in specific neuronal populations has become the dominant in-vivo labelling method 1,2. In most experimental animals however, these genetic tools are not available. The classical technique for labelling the central or peripheral projection of neurons is the diffusion of dyes into cut nerves 3,4. As this approach destroys the functional integrity of the nerves, simultaneous labelling in both directions is prevented and neuronal activity cannot be recorded. These shortcomings prompted us toward the development of an alternative dye delivery method that can be used in a variety of animals and maintains the integrity of the tissue. In this study we focused on the nervous system of locusts and crickets, which are widely used to study auditory processing 5-7. Inspired by methods of iontophoretic transdermal medication delivery 8 , in which an external electric field is used to deliver drugs through the skin, for anatomical and functional studies of insect auditory pathways we aimed to deliver tracers across the neural sheath, i.e. the neural lamella 9 and the perineurium that form the outermost layer of connective tissue and glial cells covering nerves and ganglia. We initially focused on the locust auditory nerve, attaching the tip of a suction electrode (50 μ m inner diameter) to the surface of the intact nerve halfway between the metathoracic ganglion and the hearing organ (Fig. 1a). The electrode was filled with the polar tracers Lucifer yellow or Texas Red-3,000 MW dextran. Whole-nerve field potentials and extracellular spike activity in response to acoustic stimuli indicated a good contact and tight seal between the electrode tip and the surface of the nerve. Pulsing current through the electrode (− 40 μ A, 250 ms pulse width at 1 Hz for 30 seconds) caused electrophoretic transfer of the dye from the pipette through the sheath into the auditory nerve (see Supplementary Fig. 1 for a diagram of the dye delivery apparatus). As the current pulses transiently and locally electroporated the sheath and the axonal membranes of the sensory neurons, these became permeable and the tracers were successfully delivered into the population of auditory afferents. Following the procedure, the specimens were kept at 4 °C and the dye allowed to spread for 24 hours. After dissection and standard histological processing, fluorescent imaging of the auditory organ and the CNS demonstrated the simultaneous anterograde
A fluorescence-based double retrograde tracer strategy for charting central neuronal connections
Nature Protocols, 2007
Microspheres (beads) tagged with different fluorescent markers can be used for double retrograde axonal tracing of CNS connections. They have several advantages over other double tracer techniques, including ease-of-use, high transport efficiency, distinctive cell labeling and the ability to produce well-defined injection sites. In this protocol we describe the basic procedure for their use, some common problems and how these can be overcome. The protocol, including animal surgery, preparation and delivery of tracer can be completed in approximately 0.5 d. Subsequent histological processing (excluding survival time) can be completed in 0.5-1 d.
Long-lived retrograde fluorescent labeling of corticospinal neurons in the living animal
Brain Research Protocols, 2004
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 widespread 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.
Dil and DiO: versatile fluorescent dyes for neuronal labelling and pathway tracing
Trends in Neurosciences, 1989
a [m JSA and J i ~ ~ J \ \ Carbocyanine dyes (structure shown in Fig. i) are a family of intensely fluorescent molecules, with a number of useful and unique applications as neuron~ Two of the 18 carbon chain dyes, dil-Cls-( or 'dil' for short, and diO-C1s-(3) or 'diO', which fluoresce red and green, respectively, have been the mos chains make these molecules lipophilic, so that cells exposed to these dyes rapidly incorporate them into their plasma membranes. Once in the membran, of native lipids. As a consequence, even when dye is applied to only a portion of a neuron's surface (such as the axon or cell body), the entire cell sl labelled. Thus these dyes are very suitable for both retrograde and anterograde tracing (Honig and Hume, 1986). Carbocyanine dye labelling techniques were initially developed to allow the identification of different types of neurons in dissociated cell cultures (Hot strategy was to retrogradely label a single population of neurons in vivo, prior to culturing. For example, motoneurons can be labelled by injection of, when the spinal cord cells are cultured, the motoneurons can be distinguished from other types of spinal cord neurons also present in the cultures (Fi! second population of neurons, for example those in peripheral gangliou, can be labelled by incubating the cells directly in dye during the dissociation pro is incorporated into the plasma membrane and during the first few days in culture, it brightly labels not only the cell body, but also the neurites that gro~ label one group of neurons selectively, and diO to label a second group of neurons selectively, one can readily distinguish between the two types of neurc in culture, as membrane is endocytosed, the dye becomes internalized but is still readily visualized for long times in culture . The dyes do not apl in vivo during the dissociation process, or in culture, although a small amount of trans-cellular labelling has been detected under some conditions (I Bonhoeffer, 1987). Furthermore, the dyes do not seem to interfere with the growth or electrophysiological properties of the neurons. Thus, with this the properties of individual identified neurons or their processes and to study interactions between different types of neurons in culture ; Hu ~ration bar = 50 ~rn O'L~
Fluorescent dextrans as sensitive anterograde neuroanatomical tracers: Applications and pitfalls
Brain Research Bulletin, 1990
.-We have examined five conjugated 10,000 mol.wt. dextrans as potential anterograde tract tracers: Lucifer Yellow, Texas Red, fluorescein, Cascade Blue and tetramethylrhodamine. Pressure injections were made into the brain, dorsal root ganglia or footpads of adult rats. The retrograde tracer Fluoro-Gold was injected alone or mixed with the dextrans before injection. Three-14 days after injection, animals were perfused and sections cut with a freezing microtome. Texas Red-, fluorescein-and tetramethylrhodamine-conjugated dextrans produced intense labeling of neuronal cell bodies, axons and dendritic processes at the injection site and were transported by neurons predominantly in an anterograde direction to yield terminal and pretenninal labeling. Relative to the fluorescein and tetrametlrylrhodamine conjugates, the quality and intensity of the anterograde labeling produced by Texas Red was variable. Results with Lucifer Yellow and Cascade Blue conjugates were negative. Optimal results were produced by slow-pressure injections via glass micropipettes. In comparison with Fluoro-Gold, retrograde transport by the dextran conjugates was present, but limited in its extent. Injections of the tetramethylrhodamine conjugate into dorsal root ganglia produced anterograde labeling of afferent fibers in visceral organs and injections into the nucleus ambiguus labeled motor fibers in the esophagus. Double/triple labeling was observed in the brain and spinal cord following multiple injections of fluorescein, tetramethylrhodamine and Fluoro-Gold. Also, Fluoro-Gold could be mixed with one of the dextrans in order to produce specific retrograde and anterograde labeling from the same injection site. The conjugates were compatible with fluorescent immunocytochemical procedures, but proved unsuitable for peripheral injections. All central injections produced intensely dye-labeled macrophage or pericytes associated with blood vessels, but these cells were easily distinguished from labeled neuronal sttuctures. The potential for labeling fibers of passage represents a major pitfall with these dextran conjugates and must be considered when using these sensitive tracers.
Cellular, subcellular and functional in vivo labeling of the spinal cord using vital dyes
Nature Protocols, 2013
Here we provide a protocol for rapidly labeling different cell types, distinct subcellular compartments and key injury mediators in the spinal cord of living mice. this method is based on the application of synthetic vital dyes to the surgically exposed spinal cord. suitable vital dyes applied in appropriate concentrations lead to reliable in vivo labeling, which can be combined with genetic tags and in many cases preserved for postfixation analysis. In combination with in vivo imaging, this approach allows the direct observation of central nervous system physiology and pathophysiology at the cellular, subcellular and functional level. surgical exposure and preparation of the spinal cord can be achieved in less than 1 h, and then dyes need to be applied for 30-60 min before the labeled spinal cord can be imaged for several hours.
Fluoro-gold: a new fluorescent retrograde axonal tracer with numerous unique properties
Brain Research, 1986
A new fluorescent dye, Fluoro-Gold, has been demonstrated to undergo retrograde axonal transport. Its properties include (1) intense fluorescence, (2) extensive filling of dendrites, (3) high resistance to fading, (4) no uptake by intact undamaged fibers of passage, (5) no diffusion from labeled cells, (6) consistent and pure commercial source, (7) wide latitude of survival times and compatibility with all other tested neuro-histochemical techniques.