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

Abstract

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 x 10(-7) cm2 sec-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. (J Histochem Cytochem 46:901-910, 1998)

A Novel Method for Simultaneous Anterograde and Retrograde Labeling of Spinal Cord Motor Tracts in the Same Animal

“…Although DiI can be used to label fixed tissue, the distance DiI travels in the fixed spinal cord plateaus at around 2.9 cm after 12-15 weeks in humans and guinea pigs (Lukas et al 1998). We have confirmed that DiI is an excellent postmortem tracer in rat spinal cord.…”

Development of banded afferent compartments in the inferior colliculus before onset of hearing in ferrets

“…Extending incubation times beyond 6 weeks appeared to have no consistent effect on the degree of labeling although this parameter can be important for visualizing projections (Lukas et al, 1998;Sparks et al, 2000). Perhaps myelination is more advanced for projections to the contralateral IC than for ipsilaterally projecting axons (e.g., sequential myelination of corpus callosum, Looney and Elberger, 1986; differential myelination of spinal tracts, Hamano et al, 1998) and, thus, interferes with DiI confusion or detection.…”

Thalamic afferents influence cortical progenitors via ephrin A5-EphA4 interactions

“…For this, we performed anterograde labeling of thalamic axons with the lipophilic dialkylaminostyryl dye (DiA) as an alternative approach. Labeling in living tissue is 6 mm/day faster than in fixed tissue (0.2-0.6 mm/day) due to active dye transport (Lukas et al, 1998). Thus, we injected DiA into the thalamus of freshly prepared brains at E13.5, which were then kept in culture for 10 h. After fixation, the tissue was stored for 7 days at 37°C to ensure complete axonal labeling.…”