Tract tracers are used to map neuronal pathways within the nervous system. Four main categories of tracing agent can be identified: FLUORESCENT TRACERS, ANTEROGRADE TRACERS, RETROGRADE TRACERS and TRANSNEURONAL TRACERS. A fifth—RADIO ACTIVE TRACER—is described here though it is rarely used now. Such techniques have been very powerful in describing connections within the nervous system.
Fluorescent tracers. Tracing using fluorescent material (that is by using a FLUOROCHROME—a chemical that emits light [fluoresces] when light of a specific wavelength is shone on to it) can be done in several ways. Fluorescent agents such as TEXAS RED, TRUE BLUE or DIAMIDINO YELLOW can be delivered by MICROINJECTION or MICROIONTOPHORESIS into specific brain areas, where they will be taken up by neurons and spread throughout those neurons. Fluorochromes can also be combined with immunohistochemical procedures (IMMUNOFLUORESCENCE—see IMMUNOHISTOCHEMISTRY), though this is a relatively rare procedure. Another use of fluorescent agents is in intracellular injection (a procedure referred to as NEURONAL FILLING). With this, agents such as LUCIFER YELLOW are injected directly into neurons (on tissue sections rather than in whole brain) in which they spread out colouring the entire cell and giving a vivid picture of its complete MORPHOLOGY.
Anterograde tracers. An anterograde tracer is one which is transported anterogradely along a neuron: that is from CELL BODY to AXON TERMINAL. The most commonly used is PHASEOLUS VULGARIS-LEUCOAGGLUTININ (PHA-L—a LECTIN derived from the red kidney bean; other plant lectins have also been used), which is usually delivered by microiontophoresis into brain tissue. Cell bodies and dendrites take it up and transport it along axons to terminals, a procedure that takes 7–12 days typically. An alternative anterograde tracing agent is BIOCYTIN (biotinyl-lysine), which is also taken up by cell bodies but transports in 24–48 hours. There are a number of advantages and disadvantages to weigh up when deciding whether to use PHA-L or biocytin; these are discussed in Bolam (1992).
Retrograde tracers. A retrograde tracer is one which is transported retrogradely along a neuron: that is from AXON TERMINAL to CELL BODY. The first to be widely used was HORSERADISH PEROXIDASE (HRP), though this is in fact taken up by axon terminals as well as by cell bodies and dendrites. Making a conjugate of HRP and wheatgerm agglutinin (WGA—a plant lectin) to produce WGA-HRP, or producing a conjugate of CHOLERA TOXIN SUBUNIT B (CTB) and HRP to produce CTB-HRP improves the selectivity of retrograde transport. Both WGA and CTB can also be used independently of HRP. COLLOIDAL GOLD can also be used in conjunction with these (WGA-HRP-gold or CTB-HRP-gold). The significant advantage of this is that colloidal gold preparations are taken up by intact neurons but not by the cut ends of damaged fibres, which of course yields a better representation of tracing from the neurons in the injected regions. This is a general problem when using tracing agents, and anything that can help obviate it is of value.
Transneuronal tracers. These are agents that can cross a SYNAPSE, making it possible to deliver them to one part of the brain and then determine what the trans-synaptic connections of that area are. WGA-HRP is in fact to a very limited degree a transneuronal tracer (it does not present a significant problem of interpretation), but the most commonly used agents are ALPHA HERPES VIRUSES. The prescnce of these in tissue can be detected using immunohistochemistry, antibodies for herpes viruses being commercially available.
Radioactive tracers. These are tracing agents—typically one of the AMINO ACIDS such as LEUCINE—that have been tagged with a RADIOLABEL. Amino acids injected into brain are taken up by cells and used in the formation of PROTEINS at various points within them. Since they are radiolabelled they can be detected using AUTORADIOGRAPHY. Such techniques were valuable, but because it lacks the anatomical or functional specificity of other techniques it is now rarely used.
These techniques are widely used in NEUROANATOMY. They do not have to be used singly: DOUBLE or even TRIPLE LABELLING allows one to combine different procedures. For example, one could combine the use of tract tracers with immunohistochemistry to examine the relationships between specifically identified neurons in one structure (using immunohistochemistry) and projections from another structures (using an anterograde tracer). Alternatively, one could use two different fluorescent tracers, each injected into different structures, and attempt to detect neurons in a third structure that show either single or double labelling. Some care has to be exercised when attempting double or triple labelling, to make sure that the different labels can be effectively processed (they may for example require different forms of tissue fixation) and effectively visualized, showing clear differentiation between each type of label.
