Advances in Preparative and Labeling Technology for Microscopy and Imaging of the Developing Nervous System: Transport Studies of Novel Multi-Functional Correlative Probes. E. Rosa-Molinar *, ** A. Rodriguez-Cortes, *, ** J. L. Serrano Velez, *, ** M. G. Rosario-Garcia, *, *** and J. M. Rivera-Ortiz *, *** * Biological Imaging Group, University of Puerto Rico-Rio Piedras, San Juan, PR 00931-1809, ** Julio Garcia Diaz Center for Investigations in Biology, University of Puerto Rico-Rio Piedras, San Juan, PR 00931-1809, and *** Department of Chemistry, University of Puerto Rico-Rio Piedras, San Juan, PR 00931-3346 A novel multi-functional correlative probe prepared by the conjugation of mono-sulfo-NHS- Nanogold® to 10,000 molecular weight (MW) amino-functionalized dextran conjugated to Texas Red® [“Nanogold TDA”] (see Figure 1A) has been used as a retrograde neuronal tracer to label secondary motor neurons (Smn) in the lateral lateral motor column innervating the sexually dimorphic musculature (e.g. m. erector analis major [mEAM]) of a median unpaired fin, the anal fin in female and male Western Mosquitofish, Gambusia affinis affinis [1, 2]. This novel multi-functional correlative probe has a polymeric core (amino dextran); all other polymeric materials are polydisperse. Polydisperse compounds exist as a mixture of closely related but different compounds and the molecular weight given by the manufacturer is only an average. Some repercussions of polydispersity are (a) heterogeneity of morphology and size and (b) heterogeneity of number and distribution of surface functional groups. The latter makes region specific functionalization difficult or impossible, giving multi-functional correlative probes such as Nanogold TDA undesired biophysical properties. The nerves of the ano-urogenital plexus (“AUP”) of G. a. affinis were used as an easily accessible test system. The multi-functional correlative probe, Nanogold TDA did not progress as far as 10,000 MW amino-functionalized dextran conjugated to Texas Red® (10,000 MW TDA). To elucidate the mechanism for the Nanogold TDA transport we treated G. a. affinis with either 1.0 µM colchicine or 10 µgm/ml nocodazole prior to the application of the Nanogold TDA or 10,000 MW TDA. The results from this study suggests that a diffusion model best explains the mechanism by which Nanogold TDA and 10,000 MW TDA are retrogradely transported to reveal the most fine axonal fibers, dendritic branches and dendritic arborization (DA). Transmission electron microscopy (TEM) is underway and remains to be completed. The results from this study show many of the limitations in using polymeric-based polymers for multi-functional correlative probes. We have recently overcome this by using dendrimers (see Figure 1B), “star-shaped” polymers that are globular in nature and highly monodisperse, existing usually as single compounds [3]. TEM and intravital studies using these multi-functional dendrimer (“MuD”) remain to be conducted to test an hypothesis that retinoic acid originating from protoplasmic astrocytes is essential in the specification of sexually dimorphic motor neurons that populate the L LMC [4, 5]. References [1] Rosa-Molinar, E., et al., Proc. Microscopy and Microanalysis 9 (2003):182. [2] Rosa-Molinar, E. Proc. of the First International Symposium on Livebearing Fishes, New Life Publications, Inc., Homestead, Florida (book chapter in press) 2004. Microsc Microanal 10(Suppl 2), 2004 Copyright 2004 Microscopy Society of America DOI: 10.1017/S1431927604887385 1214