Neuronal lineage marker

RNA in situ hybridization - KRT5 and housekeeping gene in human melanoma FFPE tissue section - visualized under brightfield and fluorescence microscope

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.[13]

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.[13] 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.[14] These examples use FISH (Fluorescent in situ hybridization). With this technique we can understand the physiological processes and neurological diseases.

Mouse brain slice stained by 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 tissiue.

An example of the application of this technique in neuroscience is the immunolabeling of antigens like NGF-Inducible Large External glycoprotein (NILE-GF), choline acetyltransferase, parvalbumin, and neurofilament protein.[15] 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.[16]

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.

Neural stem cells 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 (inhibitory and excitatory neurons, astrocytes and oligodendrocytes).[17]

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.[18] 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.[19] 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 (ALDH) 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.[18] 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.[19] 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 (CNS), its expression is absent from nearly all mature CNS cells, thus it is an efficient marker for neural stem cells.[20] 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.[20] 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.

The differentiation of neural stem cells is controlled, in a context-dependent manner, by intrinsic factors and extracellular signalling molecules that act as positive or negative regulators that can be used as markers.[21]

A neural progenitor cell is distinct from a neural stem cell since it is incapable of continuous self-renewal and usually has the capacity to give rise to only one class of differentiated progeny. They are tripotent cells which can give rise to neurons, astrocytes and oligodendrocytes. An oligodendroglial progenitor cell, for example, gives rise to oligodendrocytes until its mitotic capacity is exhausted.[17] Some neural progenitor markers are capable of tracking cells as they undergo expansion and differentiation from rosettes to neurons. The neural rosette is the developmental signature of neuroprogenitors in cultures of differentiating embryonic stem cells; rosettes are radial arrangements of columnar cells that express many of the proteins expressed in neuroepithelial cells in the neural tube. It has been shown that cells within rosettes express multiple cell markers, including among others Nestin, NCAM and Musashi-1, a RNA-binding protein that is expressed in proliferating neural stems cells.[22] Neuroepithelial progenitors (NEP) are responsible for neurogenesis in the neural tube and also give rise to two other types of neural progenitor cell, radial glia and basal progenitors. Radial glia are the dominant progenitor cell type in the developing brain whereas basal progenitors are specifically located at the subventricular zone (SVZ) in the developing telencephalon. Although functional studies of radial glia are increasing, it is difficult to distinguish them from neuroprogenitors and astrocytes. Like neuroprogenitors, radial glia express intermediate filament proteins nestin as well as the transcription factor PAX6 that is expressed in some neuroprogenitors in the ventral half of the neural tube. Radial glia also express proteins characteristic of astrocytes, including the widely used glial fibrillar acidic protein (GFAP), among others. Cytological markers that might be unique to radial glia include modified forms of nestin identified by the RC1 and RC2 antibodies that recognize the murine antigens.[23]

Markers can detect neurons in different stages of development from nuclear, cytoplasmic, membrane or perisynaptic products present in neurons. It is also possible to label specifically cholinergic, dopaminergic, serotonergic, GABAergic or glutamatergic neurons.[24] Pan neuron markers have multiple targets (somatic, nuclear, dendritic, spine and axonal proteins) and consequently label across all parts of the neuron. It is used to study neuronal morphology, although there are specific markers that label particular regions of the neuron.[25]

Doublecortin (DCX) is a microtubule-associated protein that is widely expressed in the soma and leading processes of migrating neurons and in the axons of differentiation neurons. Its expression is downregulated with maturation [26]

Neuron-specific Class III β-tubulin (TuJ1) is present in newly generated immature postmitotic neurons and differentiated neurons and in some mitotically active neuronal precursors.[11]

Microtubule-associated protein 2 (MAP-2) is a cytoskeletal protein. Its expression is weak in neuronal precursors but it increases during neuron development process. In general, its expression is confined to neurons and reactive astrocytes.[15]

Neuron specific enolase (NSE), also called as gamma-enolase or enolase 2, is a cytosolic protein that is expressed in mature neurons. NSE levels increase along the neuron development reaching higher level in later stages. It can be expressed in glial cells during oligodendrocyte differentiation with the same levels that have been found in neuron culture, but is repressed when cells become mature. In pathological conditions was also reported that glial neoplasms and reactive glial cells expressed this marker.[15]

Calretinin is widely distributed in different neuronal populations of vertebrate retina, being a valuable marker for immature postmitotic neurons.[11]

Neuronal Nuclei antigen (NeuN) or Fox-3 is a nuclear protein present in postmitotic cell, at the point of differentiation into mature cells.[27] It can be used to detect almost all neuronal cell types except Purkinje cells, olfactory bulb mitral cells, retinal photoreceptor and dopaminergic neurons in the substantia nigra.[15]

Calbindin is expressed by cerebellar Purkinje cells and granule cells of the hippocampus.[11] The reorganization and migration of calbindin-stained Purkinje neurons in rat cerebellum after peripheral nerve injury suggests that calbindin may be a marker for immature post-mitotic neurons, similar to calretinin.[28]

Tyrosine hydroxylase (TH) is an enzyme involved in the synthesis of dopamine and norepinephrine. Generally, it is used as a marker for dopaminergic neurons, but it can also be found in some forebrain neurons which make norepinephrine (which is the product of dopamine and the enzyme dopamine β-hydroxylase).[29]

Choline Acetyltransferase (ChAT) is expressed in cholinergic neurons of both the CNS and PNS. In the CNS, ChAT is expressed in motor neurons and pre-ganglionic autonomic neurons of the spinal cord, a subset of neurons in the neostriatum and in the basal forebrain. On the other hand, in PNS it is present in a small group of sympathetic neurons and in all parasympathetic neurons.[30]

GABA is a mature neuronal marker expressed in GABAergic interneurons (inhibitor neurons which are generally interneurons in the brain). GAD65/67 are two enzymes involved in GABA synthesis by GABAergic interneurons.[29]