The early transport-tracing methods leaned on cellular centripetal transport mechanisms. The need to expand the repertoire of anterograde techniques beyond radioautographic methods (see the section “
Tritiated amino acids and radioautography”) and (bidirectionally) transported WGA-HRP (see the section “
Non-fluorescent and fluorescent retrograde tracing”) was satisfied in 1984 with the introduction of PHA-L (Gerfen and Sawchenko
1984,
1985). Research continued afterwards to find alternative tracers. After Glover et al. (
1986) had introduced dextran amine conjugated to selected fluorescent dyes as a novel, yet retrograde, neuroanatomical tract-tracing macromolecule, dextran amine again came into the spotlight when a major anterograde transport component was reported (Nance and Burns
1990; Schmued et al.
1990; Fritzsch and Wilm
1990; Chang et al.
1990). Veenman et al. (
1992) publication, immediately followed by one authored by Brandt and Apkarian (
1992), brought biotinylated dextran amine (BDA) to center stage as an anterograde neuroanatomical tracer. Dextran molecules, dextran amines, and their conjugates are taken up via an unknown mechanism by dendrites and neuronal cell bodies, and transported to a large degree in the anterograde direction (Reiner et al.
2000; Reiner and Honig
2006) (Fig.
2, schematic). Biotinylated dextran amine conjugated to lysine, MW 10 kD (supplier Invitrogen-Molecular Probes, Eugene, OR) is the most popular among the dextran amines. BDA has been used as tracer in all classes of vertebrates, including fish species (Xue et al.
2001; Northcutt
2011).
BDA tracing protocol
Application
BDA is dissolved prior to use in 10-mM phosphate buffer, pH 7.25 (5–10%, rodents; 10%. primates). This solution is usually injected under stereotaxic guidance into the CNS via iontophoresis or through a mechanical injection (Reiner et al.
2000).
For small rodents, a glass micropipette (tip-opening diameter 20–40 μm) is filled with BDA solution. Typical iontophoretic application into rat brain is through a 5-μA positive pulsed direct current (7 s on/off) on. The same equipment as for PHA-L injections can be used.
Survival time is in proportion to the length of the projection under study. Transport is estimated to span 15–20 mm of tract in 1 week (Reiner et al.
1993). In rats, we use 1 week of survival time. BDA remains stable in the rodent brain up to 4 weeks post-injection, while in primates, it may remain detectable up to 7 weeks after application.
Fixation and detection
BDA tolerates a wide variety of fixatives. This is important if electron microscopy is on the horizon. Because BDA detection depends on penetration of tagged streptavidin into the tissue sections instead of (much larger) antibodies, a better compromise between penetration and preservation of ultrastructure can be obtained than with PHA-L (Wouterlood and Jorritsma-Byham
1993). In rodents, we prefer for light microscopy a fixative that allows additional immunohistochemistry: buffered 4% formaldehyde, 0.1% glutaraldehyde, and 0.25% of a saturated picric acid solution. After fixation, the brain can be cut with any of the available sectioning methods. We have processed sections with thickness ranging between 40 and 400 μm.
Detection of transported BDA is very simple and straightforward: incubation with one of the many commercially available tagged (strept)avidins. An irreversible reaction occurs between biotin and avidin that results in a stable product. The tag can be a fluorochrome, horseradish peroxidase, or even biotin (followed by incubation with biotinylated streptavidin to amplify signal). While fluorochrome-tagged, bound avidin can be seen directly in a fluorescence microscope, visualization of bound horseradish peroxidase needs an extra diaminobenzidine-peroxide chromogen reaction.
If application fails for some reason, the sections usually look similar to failed PHA-L application: a vague ‘cloud’ of brown reaction product or some disappointing diffuse fluorescence in the injection spot instead of discrete, labeled neurons, and neuronal processes. However, BDA in tracing experiments less frequently fails in our experience than PHA-L.
Recipe for one-dimensional BDA detection
The main incubation vehicle is TBS-TX (Tris-buffered saline, pH 8, 0, with 0.5% Triton X-100 added). During all incubations, rinses, and reactions, the vials with the sections are gently rocked on a rocking plateau. Rinse between each step 3 × 10 min with TBS-TX unless otherwise specified.
-
Incubate with streptavidin-peroxidase 1:400: overnight at room temperature, or 24 h in a refrigerator.
-
Rinse twice in TBS, pH 7.6, to remove the detergent.
-
Rinse in 50-mM Tris, pH 7.6.
-
Conduct a monitored incubation in diaminobenzidine (DAB). DAB solution is freshly prepared by dissolving 5 mg 3-3′ diaminobenzidine-HCl (Sigma) in 10-ml 50-mM Tris–HCl, pH 7.6. After filtering, 3.3-μl 30% H2O2 (Merck) is added just before use.
-
Rinse and park in 50 mM Tris, pH 7.6.
Mounting and coverslipping are identical to the procedure with sections in PHA-L tracing.
Results with BDA tracing
In a neuron that has taken up BDA, the tracer fills the matrix homogeneously. Background staining usually is negligible (this is extremely helpful in high-resolution confocal laser scanning microscopy where every emitted photon that reaches the microscope’s detector counts). All the details of the labeled neurons in the injection sites, e.g., dendritic spines, are available for inspection. Most important is that for the eye, the tracer appears equally distributed along the entire trajectories of fibers. This feature allows extremely precise mapping of fiber tracts, the analysis of the compartmentation of large fascicles and association bundles, and the study of terminal projection patterns. In the electron microscope, the label generated by BDA processing occurs in cytoplasmic compartments of the perikaryon, the main dendrites, their branches, branchlets, and spines, and in fibers, varicosities, and axon terminals. Nuclei sometimes contain BDA-reaction product. When utmost care is taken, ultrastructural detail can be preserved so well that synaptic vesicles and pre- and post-synaptic membrane densities of labeled axon terminals can be appreciated (Wouterlood and Jorritsma-Byham
1993). Retrograde transport of BDA may occur, resulting in a granular deposit of the tracer in a limited number of neuronal perikarya but sometimes complete or ‘dense’ labeling is present of cell bodies and dendrites in areas known to project to the site of injection (Reiner et al.
1993). In particular, pyramidal neurons in cerebral cortex show a tendency to accumulate retrogradely transported BDA. For instance, in experiment 2012-08 shown in Fig.
3a, b, cortical pyramidal neurons became retrogradely labeled after injection of the tracer into the caudate putamen. When such retrograde labeling occurs, the investigator should be aware of the possibility of ‘false’ anterograde labeling of collateral fibers of these, initially retrogradely labeled, neurons (see also Chen and Aston-Jones
1998).
Advantages and disadvantages
BDA tracing has a high success rate and the detection of transported tracer does not require antibodies. In addition, the tracer is very tolerant to fixative composition. These three features render BDA highly efficient and make it attractive to apply BDA in multi-dimensional tracing studies, even in spite of the disadvantage of the retrograde transport component. The latter was even exploited by Bácskai et al. (
2010) who applied two fluorescent dextran amine derivatives: tetramethylrhodamine dextran amine (RDA) and fluorescein-dextran amine (FDA), contralateral to each other to the cut ends of the hypoglossal nerve in the frog,
Rana esculenta. The experiment was conducted to study the relationships of hypoglossal motoneurons across the midline of the brain stem.
Its versatility, together with the ease of application, the straightforwardness, and speed of the staining protocol, and on top of this the reliability, even compared with PHA-L, has made BDA a widely applied and successful neuroanatomical tracer. At present, BDA undoubtedly represents the first choice anterograde tracer (Lanciego and Wouterlood
2011). BDA also compares good with modern, recombinant virus-based tracing (Wang et al.
2014).
The availability of multiwavelength confocal laser scanning microscopes with good signal separation has made it possible to conduct, for instance, three-dimensional experiments, e.g., application of BDA and PHA-L as tracers plus an additional marker pinpointing a neurotransmitter or other neuroactive substance. Injection of BDA in locus A and, in the same surgical session, of PHA-L in locus B (Herrera et al.
1994) opens ways to study in detail the anatomical convergence or divergence of neuronal connectivity. It also allows the study of connectivity in long neuronal circuits (reviewed in Lanciego and Wouterlood
2006). In non-human primates, and in contrast to its efficacy in rodents, regardless of transport distances, anterograde labeling with PHA-L seems primarily valuable to study short-terminal projections (Morecraft et al.
2009).
Multi-dimensional BDA tracing: neurochemical fingerprinting
Once a bundle of fibers is labeled and the distribution of individual fibers in an area where fibers project to is made visible, it may be worth to determine whether a particular neurotransmission-related marker is present inside the labeled fibers and terminals. This type of functional identification is further called ‘neurochemical fingerprinting’. Noteworthy, neurochemical fingerprinting
avant la lettre was conducted in 1995 by Aarnisalo and Panula on possible colocalization in PHA-L-labeled medial hypothalamic neuron fibers of neuropeptide FF. The researchers used available, classical FITC–TRITC double immunofluorescence histochemistry, while imaging was achieved with standard fluorescence microscopy. Two conditions have to be met, however, to convincingly conduct this type of research. The double immunohistochemistry should be done with robust fluorochromes, while second and most importantly, high-resolution confocal microscopy is required, because only this kind of microscopy is capable of imaging colocalization with high confidence in very small, diffraction-limited objects such as axon terminals. Both PHA-L and BDA can be used as the projection fiber marker. For instance, Gautier et al. (
2017) combined tracing with PHA-L as axonal tracer with 5HT immunohistochemistry to study serotonergic bulbospinal projections in rats. We prefer BDA as the axonal tracer, because its visualization does not depend on immunohistochemistry, leaving us with maximum immunohistochemical maneuverability to detect the second or third marker, while we operate at the limits of light microscope resolution.
The direct streptavidin-based detection of transported BDA detection represents added value, because it greatly facilitates application of BDA in multi-dimensional studies. Experiments may include multiple tracing paradigms as well as several molecular biology protocols. An example of a multiple tracing paradigm is the combination of anterograde BDA tracing with dual-retrograde tracing with cholera toxin subunit B (CTB) and Fluoro-Gold (FG), both in rodents and in non-human primates (Lanciego et al.
1998a,
b; Erro et al.
1999; Lanciego et al.
2000,
2004; Castle et al.
2005). Furthermore, BDA performs nicely when combined with PHA-L (double anterograde tract-tracing, see Lanciego and Wouterlood
1994), or with PHA-L as two anterograde tracers in combination with retrograde tracing with rabies virus (triple neuroanatomical tracing; Fig.
7a, b; see Salin et al.
2008; López et al.
2010). Finally, it is noteworthy that BDA can also be combined with newly available tools for molecular biology, such as the so-called in situ proximity ligation assay (PLA). Initially introduced by Söderberg et al. (
2008), PLA was designed to disclose interactions between two proteins when located very close to each other, enabling the accurate localization of places where these interactions are taking place. In the past few years, PLA has become increasingly popular for the visualization of heterodimeric complexes made of two different G protein-coupled receptors (GPCRs). In this regard, the combination of PLA and retrograde tracing with BDA allows the identification of GPCR heteromers within neurons innervating a given brain area where BDA was delivered (example E; Fig.
8A,B; Rico et al.
2017).
Example C: VGluT1 or -2 in BDA-labeled fibers and endings Three vesicular glutamate transporters (VGluTs) so far have been identified in brain. These are proteins located in the walls of synaptic vesicles in glutamatergic neurons. A VGluT binds cytoplasmic glutamate, carries it across the membrane and releases it into the lumen of the vesicle (review in Liguz-Lecznar and Skangiel-Kramska
2007). VGluTs thus can be said to be in charge of payloading synaptic vesicles with neurotransmitter. In the case of VGluT1 and VGluT2, the payload exclusively is glutamate, while with VGluT3, several neurotransmitters have been associated (Liguz-Lecznar and Skangiel-Kramska,
2007). The existence of a VGluT1 or VGluT2 in an axon terminal can be exploited as it signals excitatory neurotransmission. VGluT1 has been associated with neurons involved in corticofugal projections, whereas VGluT would be present in neurons involved in subcortical connectivity (Fremeau et al.
2004). VGluT1 and VGluT2 are seldomly co-expressed by neurons. Anterograde neuroanatomical tracing combined with VGluT detection provides a two-dimensional tool (‘neurochemical fingerprinting’) through which one can study excitatory, glutamatergic projections in detail (Fig.
5a, schematic). Prerequisites for successful application of neurochemical fingerprinting are high-resolution anterograde tracing, good fixation, highly specific immunostaining, and high-resolution double-fluorescence confocal microscopy. Here, we investigated whether amygdalostriatal connectivity contains VGluT1 or VGluT2, that is, can be associated with excitatory amygdaloid action upon the striatum.
In rats, BDA was injected into nuclei of the amygdaloid complex (Fig.
6a). Following the above post-surgery procedure, sections containing BDA-labeled fibers were, after rinsing and blocking (5% donkey normal serum) steps, incubated overnight at room temperature in cocktails of two antibodies: guinea pig anti-VGluT1 and rabbit anti-VGluT2 [1:1000; next, the sections were rinsed three times in TBS-TX and incubated with a cocktail made up of streptavidin-Alexa Fluor
® 546 (1:400; Molecular Probes) donkey anti-rabbit-Alexa Fluor
® 488 and donkey anti-guinea pig-Alexa Fluor
® 633 (1:400; Molecular Probes)]. After rinsing the standard BDA mounting-coverslipping procedure was followed. In essence, we had three tags: VGluT1 green, BDA-labeled fiber red, and VGluT2 infrared fluorescence.
These slides were scanned in a Leica TCS-SP2 AOBS confocal laser scanning microscope equipped with 488 nm, 546 nm, and 647 nm lasers. We found extensive distribution of amygdaloid fibers in the ventral striatum in cases with BDA injection in the basomedial nucleus (example injection site in Fig.
6a). BDA-labeled amygdalostriatal fibers contained immunofluorescence signal associated mostly with VGluT1 (Fig.
6b–f, inset Fig.
6f) and, to a lesser degree, with VGluT2. Colocalization of VGluT1 and VGluT2 was extremely rare (Wouterlood et al.
2018a).
Example D: dual-anterograde tracing with PHA-L and BDA combined with retrograde tracing with rabies virus Here, our goal was to elucidate (glutamatergic) afferents reaching striatofugal neurons projecting to the substantia nigra pars reticulata (SNr) in rats. For this purpose, a multi-tracing paradigm was designed comprising iontophoretic delivery of PHA-L into primary motor cortex, iontophoretic delivery of BDA into the parafascicular thalamic complex, and deposit of rabies virus (pressure-injected) into the SNr. After 1 week post-surgery survival, animals were perfused and stained using a goat-anti-PHA-L antibody followed by a donkey–anti-goat Alexa Fluor
® 633 conjugated IgG; an Alexa Fluor
® 546-conjugated streptavidin (for BDA detection); and a non-commercial rabbit anti-rabies antibody followed by an Alexa Fluor
® 488-conjugated donkey–anti-rabbit IgG (Fig.
7). Details can be found in López et al. (
2010).
Example E: detection of GPCR heteromers within identified projection neurons
GPCR heteromeric complexes are made of two different individual GPCRs. A GPCR complex represents a molecular entity with binding and signaling characteristics different from those of each individual (monomeric) GPCR. The recent arrival of the in situ proximity ligation assay technique (PLA; Söderberg et al.
2008) made it possible for the very first time to attempt the morphological identification of the localization of GPCR heteromers with hitherto unprecedented precision. Here, we combined PLA-based GPCR heteromer detection together with retrograde tracing with BDA. Striatal medium-sized spiny neurons in non-human primates were retrogradely labeled with BDA after tracer delivery into the internal division of the globus pallidus. Briefly, the PLA protocol was carried out first and GPCR heteromers were identified as red fluorescent spots (each one made up of combination of two different individual GPCRs). Once the PLA protocol was completed, the BDA protocol was conducted by taking full advantage of an Alexa Fluor
® 488-conjugated streptavidin (Fig.
8). Details can be found in Rico et al. (
2017)