Neuronal lineage marker

A neuronal lineage marker is an endogenous tag that is expressed in different cells along neurogenesis and differentiated cells such as neurons. It allows detection and identification of cells by using different techniques. A neuronal lineage marker can be either DNA, mRNA or RNA expressed in a cell of interest. It can also be a protein tag, as a partial protein, a protein or an epitope that discriminates between different cell types or different states of a common cell. An ideal marker is specific to a given cell type in normal conditions and/or during injury. Cell markers are very valuable tools for examining the function of cells in normal conditions as well as during disease. The discovery of various proteins specific to certain cells led to the production of cell-type-specific antibodies that have been used to identify cells.
The techniques used for its detection can be immunohistochemistry, immunocytochemistry, methods that utilize transcriptional modulators and site-specific recombinases to label specific neuronal population, in situ hybridization or fluorescence in situ hybridization. A neuronal lineage marker can be a neuronal antigen that is recognized by an autoantibody for example Hu, which is highly restricted to neuronal nuclei. By immunohistochemistry, anti-Hu stains the nuclei of neurons. To localize mRNA in brain tissue, one can use a fragment of DNA or RNA as a neuronal lineage marker, a hybridization probe that detects the presence of nucleotide sequences that are complementary to the sequence in the probe. This technique is known as in situ hybridization. Its application have been carried out in all different tissues, but particularly useful in neuroscience. Using this technique, it is possible to locate gene expression to specific cell types in specific regions and observe how changes in this distribution occur throughout the development and correlate with the behavioral manipulations.
Although immunohistochemistry is the staple methodology for identifying neuronal cell types, since it is relatively low in cost and a wide range of immunohistochemical markers are available to help distinguish the phenotype of cells in the brain, sometimes it is time-consuming to produce a good antibody. Therefore, one of the most convenient methods for the rapid assessment of the expression of a cloned ion channel could be in situ hybridization histochemistry.
After cells are isolated from tissue or differentiated from pluripotent precursors, the resulting population needs to be characterized to confirm whether the target population has been obtained. Depending on the goal of a particular study, one can use neural stem cells markers, neural progenitor cell markers, neuron markers or PNS neuronal markers.

History

The study of the nervous system dates back to ancient Egypt but only in the ninetieth century it became more detailed. With the invention of the microscope and a technique of staining developed by Camillo Golgi, it was possible to study individual neurons. This scientist started to impregnate nervous tissue with metal, as silver. The reaction consists in fixing particles of silver chromate to the neurilemma, and resulted in a stark black deposit in the soma, axon and dendrites of the neuron. Thus, it was possible to identify different types of neurons, as Golgi Cell, Golgi I and Golgi II.
In 1885 there was a German medical researcher called Franz Nissl who developed another staining technique now known by Nissl staining. This technique is slightly different from Golgi staining since it stains the cell body and the endoplasmic reticulum.
In 1887, a Spanish scientist called Santiago Ramon y Cajal learned the staining technique with Golgi and started his famous work of neuroanatomy. With this technique he made an extensive study of several areas of the brain and in different species. He also described very precisely the purkinje cells, the chick cerebellum and the neuronal circuit of the rodent hippocampus.
In 1941 Dr. Albert Coons used for the first time a revolutionary technique that uses the principle of antibodies binding specifically to antigens in the tissues. He created an immunoflorescent technique for labelling the antibodies. This technique continues to be widely used in neuroscience studies for identifying different structures. The most important neural markers used nowadays are the GFAP, Nestin, NeuroD antibodies and others. For the past years there are still creating new neural markers for immunocytochemistry or/and immunohistochemistry.
In 1953 Heinrich Klüver invented a new staining technique called, Luxol Fast Blue stain or LFB, and with this technique it's possible to detect demyelination in the central nervous system. Myelin sheath will be stained blue, but other structures will be stained as well.
The next revolutionary technique was invented in 1969 by an American scientist called Joseph G. Gall. This technique is called in situ Hybridization and it is used in a large variety of studies but mainly used in developmental biology. With this technique it is possible to mark some genes expressed in determined areas of the animal. In neurobiology, it's very useful for understanding the formation of the nervous system.

Techniques

In situ hybridization

This is one of the most powerful techniques to mark cells. This method consists of hybridizing a labeled complementary DNA or RNA strand to a specific DNA or RNA in the tissue. By doing this hybridization we will be able to reveal the location of a specific mRNA, giving us information about the physiological process of organization, regulation and function of the genes.
Using this technique we can now know what are the genes and proteins that are behind a certain process, like the formation of the neural crest, or a specific behavior; and what is the location of that same genes. We can also see how changes in the distribution of these genes can affect the development of a tissue, and correlate it with behavioral manipulations. Some examples are the use of, digoxigenin- or fluorophore-conjugated oligo- nucleotide probes, for the detection of localized mRNAs in dendrites, spines, axons, and growth cones of cultured neurons; or digoxigenin-labeled RNA probes and fluorescence tyramide amplification for the detection of less abundant mRNAs localized to dendrites in vivo. These examples use FISH. With this technique we can understand the physiological processes and neurological diseases.

Immunohistochemistry

Immunohistochemistry is a technique that uses antibodies with fluorescent staining tags that target a specific antigen present in a certain protein. This high specificity allows us to localize the peptidergic and classical transmitter compounds, their synthetic enzymes and other cell specific antigen in neuronal tissue.
An example of the application of this technique in neuroscience is the immunolabeling of antigens like NGF-Inducible Large External glycoprotein, choline acetyltransferase, parvalbumin, and neurofilament protein. All of these antigens are present in specific neuronal cell types. With these we can define anatomical circuits with a high degree of resolution, and understand the role of some proteins and cells in the nervous system, as well as the location of that same proteins and cells.
Although this is a very potent technique there are some drawbacks. The procedure has a nonquantifiable nature and has the occurrence of both false positives and false negatives.

Immunocytochemistry

Immunocytochemistry uses the same method that immunohistochemistry, but with the difference that this technique is used in isolated cells in culture, and the other is in tissues. The results are the same but with more resolution, once we are looking to one cell only.

Lineage markers

Neural stem cells markers

are an example of somatic stem cell found in various tissues, both during development and in the adult. They have two fundamental characteristics: they are self-renewing and upon terminal division and differentiation, they can give rise to the full range of cells classes within the relevant tissue. Hence, a neural stem cell can give rise to another neural stem cell, or to any of the differentiated cell types found in the central and peripheral nervous systems.
The standard method of isolating neural stem cells in vitro is with the neurosphere culture system, the method originally used to identify NSCs. After some proliferation, the cells are either induced to differentiate by withdrawing the mitogens or by exposing the cells to another factor that induces some of the cells to develop into different lineages. Cellular fates are analysed by staining with antibodies directed against antigens specific for astrocytes, oligodendrocytes, and neurons. In some cases, cells are plated at low density and monitored to determine if a single cell can give rise to the three phenotypes. Immunomagnetic cell separation strategies using antibodies directed against cell surface markers present on stem cells, progenitors and mature CNS cells have been applied to the study of NSCs. Other non-immunological methods have been used to identify populations of cells from normal and tumorigenic CNS tissues, which demonstrate some of the in vitro properties of stem cells, including high aldehyde dehydrogenase enzyme activity. ALDH cells from embryonic rat and mouse CNS have been isolated and shown to have the ability to generate neurospheres, neurons, astrocytes and oligodendrocytes in vitro, as well as neurons in vivo when transplanted into the adult mouse cerebral cortex. Once a stem cell divides asymmetrically, the more mature progenitor is born and migrates to regions of differentiation. As the progenitor migrates, it matures further until it reaches a site where it stops and either becomes quiescent or fully differentiates into a functioning cell. The major obstacle to identifying and discovering markers that define a stem cell is that the most primitive cells are probably in a quiescent state and do not express many unique antigens. Thus, as with other fields like haematopoiesis, a combination of positive and negative markers will be required to better define the central nervous system stem cell.
Nonetheless, changes in the expression levels of specific molecules can be used to indicate the presence of neural stem cells in studies focused on further differentiation toward specific neural lineages. Usual markers used for neural stem cells include Nestin and SOX2. Although Nestin it is expressed predominantly in stem cells of the central nervous system, its expression is absent from nearly all mature CNS cells, thus it is an efficient marker for neural stem cells. During neurogenesis, Sox2 is expressed throughout developing cells in the neural tube as well as in proliferating CNS progenitors, hence is thought to be centrally important for neural stem cell proliferation and differentiation. In addition to intracellular molecules, products are available to study proteins which are expressed at the cell surface, including ABCG2, FGF R4, and Frizzled-9.