A common method for identifying collateral projections is to inject different retrograde tracers into two targets and examine labeled cells for the presence of both tracers. Double-labeled cells are considered to have collateral projections to the two injection sites. This method is widely considered to underestimate the extent of collaterals. To test the efficiency of double-labeling, we mixed equal volumes of two tracers, injected them into one site in a guinea-pig brain, and counted the resulting labeled cells. Ideally, the tracers would have precisely overlapping injection sites and all labeled cells would contain both tracers. We tested several combinations of tracers: 1) Fast Blue and fluorescein dextran; 2) fluorescein dextran and FluoroGold; 3) fluorescein dextran and FluoroRuby; 4) FluoroGold and green beads; 5) FluoroGold and red beads; 6) FluoroRuby and green beads; and, 7) green beads and red beads. For each combination, a mixture was injected into the left inferior colliculus. After 1 week to allow for transport, labeled cells were counted in the right inferior colliculus and the left temporal cortex. For each mixture, the results were similar for the two areas. The percentage of cells that were double-labeled varied from 0% to 100%, depending on tracer combination. The highest efficiencies (>96%) were observed with red beads and green beads or with FluoroRuby and fluorescein dextran. The limited efficiency of other mixtures could be accounted for only in part by incomplete overlap of the two tracers at the injection site. The results indicate that the specific combination of tracers used to search for collateral projections can greatly affect the findings.

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Polymerization induced self-assembly (PISA) using methacrylate-based macromonomers as RAFT agents is an unexplored, attractive route to make self-assembled colloidal objects. The use of this class of RAFT-agents in heterogeneous polymerizations is however not trivial, because of their inherent low reactivity. In this work we demonstrate that two obstacles need to be overcome, one being control of chain-growth (propagation), the other monomer partitioning. Batch dispersion polymerizations of hydroxypropyl methacrylate in presence of poly(glycerol methacrylate) macromonomers in water showed limited control of chain-growth. Semi-continuous experiments whereby monomer was fed improved results only to some extent. Control of propagation is essential for PISA to allow for dynamic rearrangement of colloidal structures. We tackled the problem of monomer partitioning (caused by uncontrolled particle nucleation) by starting the polymerization with an amphiphilic thermoresponsive diblock copolymer, already "phase-separated" from solution. TEM analysis showed that PISA was successful and that different particle morphologies were obtained throughout the polymerization. gy, with the big advantage that conventional organosulfur based RAFT agents can be omitted. ASSOCIATED CONTENTSupporting Information. Materials and methods. Reaction schemes for the macromonomer synthesis and its chain extension via RDRP polymerization. 1 H-NMR traces of the PGMA and PGlyMA macromonomers. Additional GPC traces and DLS data for HPMA chain-extensions. This material is available free of charge via the Internet at