Tag Archives: Nueroscience

Methods in Systems Neuroscience: Histological Stains and Tract Tracing

Histological Stains

Cells and major fibre tracts make up the basic structure of the brain and are observed using histological procedures. The most common histological procedures involve stains such as the Nissl and Weil stains. This particular cell stain stains the Nissl substance (granular bodies) found in neuronal cytoplasms. The Nissl bodies are composed of rough endoplasmic reticulum and free polyribosomes and as such are the site of protein synthesis. For a Nissl stain, neurones are held in a parofromaldehyde or formalin fixed tissue. The selective stain uses aniline dye, which colours the somas and dendrites of neurones blue, or more specifically their ribosomal RNA. The Weil fibre stains is regressive, requiring differentiation. Weil fibre stains use a hematoxylin based stain, which dyes myelin and red blood cells dark red. Weil is unique in that is can be used on a frozen section of tissue.

NisslHippo2

Tract Tracing

Anterograde tract tracing is used to identify projections than run from cell bodies to axon terminals. Various tracer molecules are used including but not limited to the green fluorescent protein, lipophilic dyes and radioactively tagged amino acids. Genetic tracers are also used including viruses and proteins. The most common genetic tracers are the Herpes complex virus type 1 (HSV) and the Rhabdoviruses. Lipophilic dyes are commonly used in electrophysiology as anterograde tracer; however, they are not selectively unidirectional nor are they actively transported across the synaptic cleft. An example of an anterograde projection is the transmission of visual information from the superior colliculus to the substantial nigra.

Retrograde tract tracers are used to identify projections from axonal terminals to somas. The most common retrograde tracers are viral stains such as the modified rabies virus or pseudorabies virus (PRV) or Batha stain. The PRV infection spreads upstream through a pathway of linked neurones. An example of retrograde projections is the transmission of nociceptive information from the parabrachial nucleus to dopaminergic neurones in the midbrain. 

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Also, bidirectional tracers, as the name suggests, can work both in an anterograde and retrograde fashion. Common bidirectional tracers include WGA-HRP, biotinylated dextran and cholera toxin subunit b. A major pitfall of bidirectional tracers or dyes is that they can move retrograde then anterograde along branches axon collateral falsely indicating an anterograde tracing. In other words, one might falsely observe that A projects to C (see diagramme above). It is important to correctly identify the direction of projections as it helps identify the morphology of a cell. Cells with different morphologies, unsurprisingly, have different processing capacities. An example of a branched projection is the tectonigral projection.

Finally, simultaneously anterograde and retrograde tracers do exist; however, not as common as one or the other. The use of tracers can be accompanied by TH immunochemistry to help identify the locations of certain neurones.

Bibliography:

Anterograde tracing. (n.d.). Retrieved January 13, 2015, from http://en.wikipedia.org/wiki/Anterograde_tracing

Coizet, V., et al. (2010). “The parabrachial nucleus is a critical link in the transmission of short-latency nociceptive information to midbrain dopaminergic neurons.” Neuroscience 168(1): 263-272.

Comoli, E., et al. (2003). “A direct projection from superior colliculus to substantia nigra for detecting salient visual events.” Nat Neurosci 6(9): 974-980.

Fung, K. (n.d.). Stains in Neuropathology. Retrieved January 15, 2015, from http://moon.ouhsc.edu/kfung/JTY1/NeuroHelp/ZNEWBS12.htm

QBI Histology and Microscopy. (n.d.). Retrieved January 15, 2015, from http://web.qbi.uq.edu.au/microscopy/cresyl-violet-staining-nissl-staining/

Redgrave, P. (Director) (2014, November 11). Methods in Systems Neuroscience. BMS224 Brain and Behaviour 1 . Lecture conducted from University of Sheffield , Sheffield.

Retrograde tracing. (n.d.). Retrieved January 13, 2015, from http://en.wikipedia.org/wiki/Retrograde_tracing