Long-Distance Three-Color Neuronal Tracing in Fixed Tissue Using NeuroVue Dyes

Dissecting development of neuronal connections is critical for understanding neuronal function in both normal and diseased states. Charting the development of the multitude of connections is a monumental task, since a given neuron typically receives hundreds of convergent inputs from other neurons and provides divergent outputs for hundreds of other neurons. Although progress is being made utilizing various mutants and/or genetic constructs expressing fluorescent proteins like GFP, substantial work remains before a database documenting the development and final location of the neuronal pathways in an adult animal is completed. The vast majority of developing neurons cannot be specifically labeled with antibodies and making specific GFP-expressing constructs to tag each of them is an overwhelming task. Fortunately, fluorescent lipophilic dyes have emerged as very useful tools to systematically compare changes in neuronal networks between wild-type and mutant mice. These dyes diffuse laterally along nerve cell membranes in fixed preparations, allowing tracing of the position of a given neuron within the neuronal network in murine mutants fixed at various stages of development. Until recently, however, most evaluations have been limited to one, or at most, two color analyses. We have previously reported three color neuronal profiling using the novel lipophilic dyes NeuroVue (NV) Green, Red and Maroon (Fritzsch et al., Brain. Res. Bull. 66:249–258, 2005). Unfortunately such three color experiments have been limited by the fact that NV Green and its brighter successor, NV Emerald, both exhibit substantially decreased signal intensities when times greater than 48 hours at 37°C are required to achieve neuronal profile filling (unpublished observations). Here we describe a standardized test system developed to allow comparison of candidate dyes and its use to evaluate a series of 488 nm-excited green-emitting lipophilic dyes. The best of these, NV Jade, has spectral properties well matched to NV Red and NV Maroon, better solubility in DMF than DiO or DiA, improved thermostability compared with NV Emerald, and the ability to fill neuronal profiles at rates of 1 mm per day for periods of at least 5 days. Use of NV Jade in combination with NV Red and NV Maroon substantially improves the efficiency of connectional analysis in complex mutants and transgenic models where limited numbers of specimens are available

Three-color imaging with NV Jade, Red, and Maroon allows excellent resolution of thin fibers and discrete nerve tracts in near-adult murine brain

Nerve tracts projecting from one region of mature brain to another were labeled with NV Jade (green pseudocolor), NV Red (red pseudocolor) or NV Maroon (blue pseudocolor) and imaged by confocal microscopy after 5 days of diffusion at 37 C. Bar indicates 100 μm in all images. Despite increased green autofluorescence due to myelinization of near-adult brain, NV Jade can be used in 18-day-old cerebella to label not only parallel fibers (green lines crossing from top left to bottom right) but also individual cells (stellar neuron in the center of A) at distances of up to 5 mm away from the injection site. (Representative image selected from 12 replicate specimens.) After labeling in the pontine nuclei, cerebellar cortex and restiform body of the cerebellum with NV Jade, NV Red or NV Maroon, respectively, discrete fibers are consistently labeled and readily visualized near the deep cerebellar nuclei of P18 animals without bleed through and at distances of up to 1 cm away from the injection site over 50 animals. The same type of labeling was used on two replicate specimens, which were cut slightly differently to show fiber bundles running in parallel (B) or crossing one another (C).<p><b>Copyright information:</b></p><p>Taken from "Long-Distance Three-Color Neuronal Tracing in Fixed Tissue Using NeuroVue Dyes"</p><p></p><p>Immunological Investigations 2007;36(5–6):763-789.</p><p>Published online 27 Dec 2007</p><p>PMCID:PMC2430174.</p><p></p

Three-color neurotracing with NV Jade, Red, and Maroon allows easy visualization of nerve fibers projecting from ear to brain or brain to ear by either epifluorescence or confocal microscopy

Nerve tracts originating at discrete locations in the periphery and projecting into closely adjacent regions in the brain of E18.5 mice (A, C, D) or nerve tracts arising in the brain and innervating the inner ear (B, E) were imaged using NV Jade (green pseudocolor) in combination with NV Red (red pseudocolor) and/or NV Maroon (blue pseudocolor) after 5 days of diffusion at 37°C Scale bar indicates 100 μm in all images. Abbreviations: AC, anterior crista; HC, horizontal crista; PC, posterior crista; S, saccule; U, utricle. schematically illustrates the location of dye insertions in the inner ear (colored arrows) and the nerve fibers projecting from those locations back to the brain (colored lines), and in particular into the cochlear nuclei (red, green) and vestibular nuclei and cerebellum (blue). Epifluorescence of whole mounted brain and confocal images taken from tissue section shown by the white dotted line are shown in panel C (lateral view) and panel D (section along the dotted line shown in C), respectively. shows insertion points in the brain (red and green arrows) used to fill nerve fibers projecting to the utricle (U), a vestibular endorgan in the inner ear. The corresponding confocal image is shown in panel E. Nerve fibers originating from the base or apex of the cochlea or the central vestibule of the inner ear were labeled by insertion of filter segments coated with NV Jade, NV Red or NV Maroon, respectively. Epifluorescence imaging of a whole brain mount using standard FITC, Texas Red and Cy5 filters, respectively, showed excellent color segregation of the corresponding closely adjacent projection areas in the brain (cochlear nuclei and vestibular projection area). Image shown was corrected for bleed through in the Cy5 channel from regions having high intensity NV Red signal using Image Pro (see Materials and Methods). Dashed line indicates the level of the coronal section cut from the same preparation and imaged by confocal microscopy (panel D). Neuronal profile filling was detectable at distances of up to 5 mm away from the filter insertion sites. A coronal 100 μm thick vibratome section cut along the dashed white line shown in panel A and imaged by confocal microscopy clearly shows the expected discrete projections from the inner ear into the cochlear and vestibular nuclei in the brain without any bleed-through. Using such projections, it is possible to determine the area of the cochlear nuclei (turquoise line), distances between projection bands (yellow line) or absence of overlap of specific projections in normal mice and assess how each is affected by specific mutations. Insertion of NV Jade and NV Red filter segments into the cerebellum and the brainstem, respectively, of fixed neonatal mouse brain followed by confocal imaging of whole organ mount allows detailed visualization of segregation (green, red) or overlap (yellow) of innervating fibers within specific endorgans such as the utricle (U) of the inner ear, using a much simplified protocol compared to that require for neurotracing of the same tissue with DiA and DiI (see text).<p><b>Copyright information:</b></p><p>Taken from "Long-Distance Three-Color Neuronal Tracing in Fixed Tissue Using NeuroVue Dyes"</p><p></p><p>Immunological Investigations 2007;36(5–6):763-789.</p><p>Published online 27 Dec 2007</p><p>PMCID:PMC2430174.</p><p></p

Thermal stability of selected green dyes at 37°C in 4% neutral buffered formaldehyde

Amount of intact dye remaining on dye-coated filter segments was determined for PTIR/NeuroVue dyes with excitation maxima near 488 nm as a function of incubation time and temperature (see Materials and Methods for details). The rank order of thermal stability was PTIR 327 best, followed by NV Jade, PTIR 326, and NV Emerald (see ).<p><b>Copyright information:</b></p><p>Taken from "Long-Distance Three-Color Neuronal Tracing in Fixed Tissue Using NeuroVue Dyes"</p><p></p><p>Immunological Investigations 2007;36(5–6):763-789.</p><p>Published online 27 Dec 2007</p><p>PMCID:PMC2430174.</p><p></p