Complex brain circuits studied via simultaneous and permanent detection of three transported neuroanatomical tracers in the same histological section

Abstract

Experimental neuroanatomical tracing methods lie at the basis of the study of the nervous system. When the scientific question is relatively straightforward, it may be sufficient to derive satisfactory answers from experiments in which a single neuroanatomical tracing method is applied. In various scientific paradigms however, for instance when the degree of convergence of two different projections on a particular cortical area or subcortical nucleus is the subject of study, the application of single tracing methods can be either insufficient or uneconomical to solve the questions asked. In cases where chains of projections are the subjects of study, the simultaneous application of two tracing methods or even more may be compulsory. The present contribution focuses on combinations of several neuroanatomical tract-tracing strategies, enabling in the end the simultaneous, unambiguous and permanent detection of three transported markers according to a three-color paradigm. A number of combinations of three tracers or of two tracers plus the immunocytochemical detection of a neuroactive substance can be conceived; we describe several of these combinations implemented by us using the present multitracer protocol.

Vesicular glutamate (VGlut), GABA (VGAT), and acetylcholine (VACht) transporters in basal forebrain axon terminals innervating the lateral hypothalamus

“…Confirming its documented utility as an anterograde tracer (Veenman et al, 1992;Wouterlood and JorritsmaByham, 1993;Lanciego et al, 2000;Reiner et al, 2000), we found that BDA-10,000 provided discrete labeling of BF cell bodies and reliable anterograde labeling of BF fibers and varicosities. Although we noticed some labeled cells in areas surrounding the injection site in the MCPO-SI, which might have been retrogradely labeled, we did not find cells retrogradely labeled at a distance in the prefrontal cortex or LH, confirming that BDA-10,000, in contrast to BDA-3000 MW, results in neglible retrograde labeling (Wouterlood and Jorritsma-Byham, 1993;Reiner et al, 2000).…”

“…Confirming the utility of BDA as an anterograde tracer that can be readily used along with immunostaining for elucidating complex neural circuits (Lanciego et al, 2000), we were able to apply double staining for BDA and the VTPs in order to identify the neurotransmitters utilized by the BF neurons projecting to the LH. We found that with the use of Triton (0.1%), we obtained complete penetration in material double stained for VTP antibodies and streptavidin (through 25-m-thick sections).…”

Efferent Projections of the Anterior and Posterodorsal Regions of the Medial Nucleus of the Amygdala in the Mouse

“…On the other hand, BDA10k is highly recommended as an anterograde tracer, and it does yield some retrograde labeling [Reiner et al, 2000, and references therein]. Other benefits of BDA10k are that it might be combined with established retrograde tracers, such as cholera toxin B and Fluoro-Gold [Coolen and Wood, 1998;Coolen et al, 1999;Lanciego et al, 2000], and that BDA tracing at ultrastructural level is also possible, in particular in combination with transmitter immunocytochemistry [Pickel et al, 1996;Reiner et al, 2000;Miyashita et al, 2007]. We confirm that BDA10k might be the tracer of choice for anterograde tracing experiments.…”

Role of neuronal activity and kinesin on tract tracing by manganese-enhanced MRI (MEMRI)

“…Typically tracers injected into the brain enter neurons locally and are picked up by the intracellular transport machinery and then carried along neuronal processes to reach distant synapses or cell bodies (Fritzsch, 1993;Grafstein and Forman, 1980;Lanciego et al, 2000). Transport from the synapse back to the cell body is termed "retrograde," and from the cell body towards the synapse "anterograde".…”