Julius-Robert Lukas

Carbocyanine Postmortem Neuronal Tracing: Influence of Different Parameters on Tracing Distance and Combination with Immunocytochemistry

SUMMARY Carbocyanines (DiI, DiA, DiO) are able to travel along membranes by diffusion and therefore have been used as postmortem neuronal tracers in aldehyde-fixed tissues. Surprisingly, detailed data on the influence of different parameters on tracing distances are still missing. This study was carried out to optimize tracing procedures and to reveal the validity of the combination of postmortem tracing with immunocytochemistry. Carbocyanine crystals were applied to the cervical spinal cord, sciatic nerves, and brachial plexuses of humans and guinea pigs. Incubation in the dark at 37C for 12-15 weeks proved optimal to achieve longest tracing distances (28.9 ± 2.2 mm) in human and animal tissues. Longer incubation times and incubation temperatures higher than 37C did not result in longer tracing distances. No differences were evident between adult and newborn animals and between central and peripheral nervous system. The diffusion coefficient for DiI was calculated to be 2.5 × 10-7 cm2sec-1. After application of DiI to nerves of guinea pig extraocular muscles, DiI-positive afferent perikarya were observed in the anteromedial part of the trigeminal ganglion. These perikarya were identified by calcitonin gene-related peptide immunoreactivity (CGRP-IR). The percentage of CGRP-IR neurons after tracing was concordant with the percentage of CGRP-IR in trigeminal ganglia exclusively processed for CGRP-IR without previous postmortem tracing. These results demonstrate carbocyanines to be specific tracers for exact neuronal mapping studies.

Muscle spindles and Golgi tendon organs in bovine calf extraocular muscle studied by means of double-fluorescent labeling, electron microscopy, and three-dimensional reconstruction

In the present study muscle spindles (MSps) and Golgi tendon organs (GTOs) in bovine extraocular muscles (EOMs) were analyzed in detail. The innervation pattern of these proprioceptors was investigated with transmission electron microscope and confocal laser scanning microscope after double-fluorescent labeling. Three-dimensional (3D) reconstructions were performed of GTOs. Muscle spindles. MSps are numerous, each containing two nuclear bag fibers and up to eight nuclear chain fibers. In the equatorial region and paraequatorial region thin axons enwrapping the intrafusal muscle fibers form numerous nerve contacts on the muscle fiber surface. Double staining of such nerve terminals with synaptophysin and alpha-bungarotoxin and their fine structural features confirm their sensory nature. In the encapsulated part of the polar region neuromuscular contacts have structural features of motor nerve terminals and stain positively with alpha-bungarotoxin. Golgi tendon organs. GTOs are numerous in bovine EOMs. Each GTO contains collagen bundles but frequently also intracapsular muscle fibers. Intracapsular muscle fibers either terminate inside the GTO in collagen bundles or pass through the proprioceptor. GTOs are richly supplied with sensory nerve terminals which intermingle with the collagen bundles. Nerve terminals on intracapsular muscle fibers exhibit fine structural characteristics of motor nerve terminals and are alpha-bungarotoxin positive. The 3D images of GTOs show the detailed spatial arrangement of the GTO tissue components. These new insights in the complex and specific morphology of MSps and GTOs in bovine EOMs indicate that we deal with highly developed proprioceptors. These are supposed to provide important information for EOM innervation.