Resolving the Detailed Structure of Cortical and Thalamic Neurons in the Adult Rat Brain with Refined Biotinylated Dextran Amine Labeling

Abstract

Biotinylated dextran amine (BDA) has been used frequently for both anterograde and retrograde pathway tracing in the central nervous system. Typically, BDA labels axons and cell somas in sufficient detail to identify their topographical location accurately. However, BDA labeling often has proved to be inadequate to resolve the fine structural details of axon arbors or the dendrites of neurons at a distance from the site of BDA injection. To overcome this limitation, we varied several experimental parameters associated with the BDA labeling of neurons in the adult rat brain in order to improve the sensitivity of the method. Specifically, we compared the effect on labeling sensitivity of: (a) using 3,000 or 10,000 MW BDA; (b) injecting different volumes of BDA; (c) co-injecting BDA with NMDA; and (d) employing various post-injection survival times. Following the extracellular injection of BDA into the visual cortex, labeled cells and axons were observed in both cortical and thalamic areas of all animals studied. However, the detailed morphology of axon arbors and distal dendrites was evident only under optimal conditions for BDA labeling that take into account the: molecular weight of the BDA used, concentration and volume of BDA injected, post-injection survival time, and toning of the resolved BDA with gold and silver. In these instances, anterogradely labeled axons and retrogradely labeled dendrites were resolved in fine detail, approximating that which can be achieved with intracellularly injected compounds such as biocytin or fluorescent dyes.

Systematic comparison of adeno‐associated virus and biotinylated dextran amine reveals equivalent sensitivity between tracers and novel projection targets in the mouse brain

“…tracers such as horseradish peroxidase (HRP), Phaseolus vulgaris leucoagglutinin (PHA-L), and biotinylated dextran amine (BDA; Gerfen and Sawchenko, 1984;Glover et al, 1986). Among these, BDA currently is widely used as an anterograde tracer because of its experimental flexibility (Lanciego and Wouterlood, 2011;Ling et al, 2012). BDA is well validated and has been used to study the connectivity of a variety of brain regions across many different species, such as macaque (Tanigawa et al, 2005), rat (Thompson and Swanson, 2010), and mouse (Wang and Burkhalter, 2007;Wang et al, 2012).…”

Anterograde and retrograde tracing with high molecular weight biotinylated dextran amine through thalamocortical and corticothalamic pathways

“…In previous studies, it has shown that BDA labeling appeared as early as 3 days and remained unchanged up to 14 days after injection in rats (Rajakumar et al, 1993). In contrast, recent study indicated that the quality of BDA labeling could be changed in animals following the different post-injection survival time (Ling et al, 2012).…”

“…The use of the biotinylated dextran amine (BDA) is a common technique for neural pathway tracing in the central nervous system (Reiner et al, 2000;Veenman, Reiner, & Honig, 1992). Although BDA can be transported rapidly from the injection site anterogradely and retrogradely to yield excellent neural labeling, the direction of BDA transportation is closely related to its molecular weight (Ling, Hendrickson, & Kalil, 2012;Reiner et al, 2000). In the family of BDA, high molecular weight BDA (10 kDa) seems to be prefererentially transported anterogradely, while low molecular weight BDA (3 kDa) retrogradely (Fritzsch, 1993;Kaneko, Saeki, Lee, & Mizuno, 1996;Ling et al, 2012;Medina & Reiner, 1997;Reiner et al, 2000).…”

“…Although BDA can be transported rapidly from the injection site anterogradely and retrogradely to yield excellent neural labeling, the direction of BDA transportation is closely related to its molecular weight (Ling, Hendrickson, & Kalil, 2012;Reiner et al, 2000). In the family of BDA, high molecular weight BDA (10 kDa) seems to be prefererentially transported anterogradely, while low molecular weight BDA (3 kDa) retrogradely (Fritzsch, 1993;Kaneko, Saeki, Lee, & Mizuno, 1996;Ling et al, 2012;Medina & Reiner, 1997;Reiner et al, 2000). In addition, even though high molecular weight BDA was recommended for anterograde pathway tracing, it also leads to some retrograde labeling (Ling et al, 2012;Reiner et al, 2000;Veenman et al, 1992).…”

“…In the family of BDA, high molecular weight BDA (10 kDa) seems to be prefererentially transported anterogradely, while low molecular weight BDA (3 kDa) retrogradely (Fritzsch, 1993;Kaneko, Saeki, Lee, & Mizuno, 1996;Ling et al, 2012;Medina & Reiner, 1997;Reiner et al, 2000). In addition, even though high molecular weight BDA was recommended for anterograde pathway tracing, it also leads to some retrograde labeling (Ling et al, 2012;Reiner et al, 2000;Veenman et al, 1992). This phenomenon implies that high molecular weight BDA, in parallel with anterograde tracing, might also be potentially used for retrograde pathway tracing.…”