Neural stem cells and neural progenitor cells (NSCs/NPCs) can differentiate and give rise to several types of other neural cells in a process called neurogenesis. The resulting cell types include glial cells, oligodendrocytes, astrocytes, intermediate progenitors, immature neurons, mature neurons, glutamatergic neurons, GABAergic neurons, dopaminergic neurons, cholinergic neurons, and motor neurons. These neural cells express various proteins, some of which, are specifically related to certain neuronal phenotypes. Therefore, they can be used as “markers”. Cell specific neuronal markers have emerged as one of the most valuable tools available to neuroscientists. Using antibodies against various cell components, investigators can:

  • Identify cells and distinguish neurons from other cell types in the nervous system (e.g. microglia, astrocytes, and oligodendrocytes)
  • Analyze the phenotype and morphology of neurons
  • Assess protein co-localization
  • Visualize synaptic connections
  • Measure protein expression levels
  • Evaluate the cell health
     

This page provides an overview of neural cell marker antibody targets that allow for specific identification of neuronal cells that are involved in various stages of neural development and neurological disorders.

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Neurogenesis

Neurogenesis is the formation of new neurons from neural stem and progenitor cells which occurs in various regions of brain. The incredible diversity of neurons in the brain, resulting from regulated neurogenesis, is crucial during embryonic development and continues in certain brain regions after birth and throughout our lifespan. During this process, stem cells can divide indefinitely to produce more stem cells, or differentiate to give rise to more specialized cells, such as neural progenitor cells. These progenitor cells then, differentiate into specific types of neurons—at specific times and regions in the brain.


Neural stem cells

Neural stem cells (NSCs) are a group of ectodermal progenitor cells that are self-renewing and, as shown in the schematic below, can differentiate into all types of neurons in the mammalian nervous system.

Neurogenesis of mammalian nervous system

Figure 1. Neurogenesis of mammalian nervous system.

NSCs express critical markers like Nestin, NOTCH1, and SOX2. Nestin is a widely used marker because, although it is expressed predominantly in stem cells of the central nervous system (CNS), its expression is absent from nearly all mature CNS cells. The transcription factor SOX2 is known to be expressed at high levels in the neuroepithelium of the developing CNS and is thought to be centrally important for neural stem cell proliferation and differentiation. The figure below shows antibodies against Nestin and SOX2. Antibody specificity was demonstrated by detection of differential basal expression of the target across cell models owing to their inherent genetic constitution. Other markers of interest include: SOX1, PAX3, and PAX6.

Figure 2. Nestin antibody in ICC and SOX2 antibody in WB: (A) Immunofluorescence analysis using Nestin Recombinant Rabbit Monoclonal Antibody (SN06-27) (Cat. No. MA5-32272) shows increased expression of nestin in Neural Stem Cells (NSC) when compared to iPSC. (B) Western blot analysis was performed on modified whole cell lysate (1% SDS) (30 µg lysate) of NCCIT (Lane 1), NTERA-2 (Lane 2), and HeLa (Lane 3). The blot was probed with SOX2 Monoclonal Antibody (Btjce) (Cat. No. 14-9811-82, 1 µg/mL) and detected by chemiluminescence using F(ab')2-Rabbit anti-Rat IgG (H+L) Secondary Antibody, HRP (Cat. No. PA1-29927, 0.25 µg/mL, 1:4,000 dilution). A 35 kDa band corresponding to SOX2 was observed in NCCIT and NTERA-2 cells, but not in HeLa, which is reported to be negative for SOX2 expression, verifying antibody specificity.

Glial cells

Glial cells are non-neuronal cells and the most abundant cell type in the central and peripheral nervous system. They do not produce electrical impulses. However, glial cells have far more cellular diversity and functions than neurons and glial cells can respond to and manipulate neurotransmission in many ways. Neurons would be unable to function without the vital roles that are fulfilled by glial cells. They are cells of neuroepithelial origin and driven primarily by NOTCH1 signaling. They differentiate to related cell types such as radial glia and Schwann cells.

In the peripheral nervous system, glial cells include Schwann cells and satellite cells which provide nutrients and structural support for neurons in the peripheral nervous system (PNS). Immature Schwann cells exhibit upregulation of the neural cell adhesion molecule (NCAM). Upon further maturation, Schwann cells demonstrate increased expression of myelin-associated genes (e.g. myelin basic protein, MBP) to support their role as sheath-producing glial cells for PNS motor and sensory neurons.

In the central nervous system, glial cells include:

  • Oligodendrocytes—help in the formation of myelin sheaths around axons in the CNS. They also support and insulate neurons. Important markers that can be used to identify oligodendrocytes are:
    Markers Function
    NG2 A membrane chondroitin sulfate proteoglycan expressed by oligodendrocyte precursor cells
    OLIG1, OLIG2 Transcription factors necessary for oligodendrocyte development
    PDGFRA A cell surface tyrosine kinase receptor and a marker of oligodendrocyte precursor cells
    MOG A glycoprotein found on the surface of oligodendrocytes
  • Astrocytes—provide nutrients and other substances to neurons, regulate the concentrations of ions and chemicals in the extracellular fluid, and provide structural support for synapses. It also forms the blood-brain barrier—a structure that blocks entrance of toxic substances into the brain. Astrocytes express specific markers that can be used to differentiate from other glial progenitor cells.
    Markers Function
    GFAP An intermediate filament and major component of the astrocyte cytoskeleton
    ALDH1L1 An enzyme that catalyzes the conversion of 10-formyltetrahydrofolate, NADP+, and water to tetrahydrofolate, NADPH, and CO2
    EAAT2/GLT-1 An astrocyte-specific glutamate transporter
    Glutamine synthetase An enzyme involved in the metabolism of nitrogen. In the brain, it is primarily found in astrocytes.
    S100B A calcium binding protein also found in oligodendrocyte precursor cells (OPCs). Double labeling with NG2 will distinguish the OPCs from the astrocytes.
  • Microglia—plays a major role in the immune system and central nervous system to activate pro-inflammatory cytokines. It scavenges and degrades dead neurons. Microglia are the macrophages of the brain, and due to the shared lineage of microglia and macrophages, many markers are common to both cell types. Below is a specific list of markers expressed in microglia.
    Markers Function
    SALL1 Sall1 is a transcriptional regulator defining microglia identity and function. It is not expressed in other members of the mononuclear phagocyte system or by other CNS-resident cells.
    TMEM119 TMEM119 is a cell-surface protein and a specific microglial marker. Unlike other microglial markers, this marker has the advantage that it isn’t expressed by macrophages or other immune or neural cell types.
    CX3CR1 It is a fractalkine receptor and is found on the surface of both microglia and macrophages in the CNS where it responds to CX3CL1 secreted by neurons.
    IBA1 Ionized calcium-binding adaptor molecule 1 is a microglial and macrophage-specific calcium-binding protein that is involved with the membrane ruffling and phagocytosis in activated microglia.
    CD68 CD68 is a lysosomal protein expressed in high levels by macrophages and activated microglia and in low levels by resting microglia.


The figure below shows antibodies against different glial cells markers which allow researchers to effectively identify and characterize cells of interest. Antibody specificity was demonstrated by detection of differential basal expression of the target across different tissue samples owing to their inherent genetic constitution.

WB, ICC, and Flow analysis of MOG, EEAT2, and SALL1, respectively

Figure 3. MOG antibody in WB, EEAT2 (GLT-1) antibody in ICC, and SALL1 antibody in flow cytometry: (A) Relative expression of MOG was observed in mouse and rat brain and cerebellum as compared to heart and liver using a MOG Polyclonal Antibody (Cat. No. PA5-19602) in western blot. (B) Immunofluorescence analysis was performed on fixed and permeabilized U-87 MG cells for detection of endogenous EAAT2/GLT-1 using a GLT-1 Recombinant Rabbit Monoclonal Antibody (9H9L17) (Cat. No. 701988, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor 488 (Cat. No. A27034, 1:2,000), clearly demonstrating localization of EAAT2/GLT-1 in the membrane. The images were captured at 60X magnification. (C) Staining of mouse brain cells–as expected, the Sall1 Monoclonal Antibody (NRNSTNX) (Cat. No. 48-9729-82) stains mouse microglia (CD11b+ CD45 intermediate; purple) but not CD45 high CD11b+ cells (blue) in the brain. Demyelinated mouse brain single cell suspension was stained with Fixable Viability Dye eF450 and either Rat IgG2a isotype control (left histogram), or clone NRNSTNX (right histogram). Viable cells were used for analysis.


Intermediate progenitor and immature neurons

Arising from radial glial cells are intermediate progenitors, which act as the source of most mature neurons in the CNS. Intermediate progenitors are characterized by their expression of paired box 6 (PAX6), a multifunctional transcription factor regulating NSC proliferation and differentiation. During later stages of neurogenesis, these cells upregulate doublecortin and neurogenic differentiation factor 1 (NeuroD1), the latter serving as a neuronal fate determinant. The figures below show antibodies against PAX6 and NeuroD1. Antibody specificity was demonstrated by detection of differential basal expression of the target across cells owing to their inherent genetic constitution.

Mature neurons

Upon reaching maturity, mammalian neuronal cell bodies are defined by expression of neuron-specific enolase (NSE), microtubule-associated protein-2 (MAP2)—that promotes assembly and stability of the microtubule network–and post-mitotic neuron-specific, RNA binding nuclear protein (NeuN). Other specific markers can be seen in the table below.

Markers Function
Neurofilaments (NEFH, NEFM, NEFL) Major intermediate filaments found in neurons
SYP Synaptophysin: Synaptic vesicle protein that regulates vesicle endocytosis in neurons
TUBB3 Tubulin is the major constituent of microtubules. TUBB3 plays a critical role in proper axon guidance and maintenance.
GAP43 This protein is associated with nerve growth. It is a major component of the motile "growth cones" that form the tips of elongating axons.


The figure below shows antibodies that detect neurofilaments–NEFH, NEFM, and NEFL. Antibody specificity was demonstrated by detection of differential basal expression of the target across cell lines tested owing to their inherent genetic constitution and CRISPR-Cas9 mediated knockout.

Western blot and ICC analysis of NEFH, HEFM, and HEFL in various cell lines

Figure 5. NEFH antibody in WB, NEFM antibody, and NEFL antibody in ICC: (A) Relative expression of NEFH was observed in SH-SY5Y and IMR-32 in comparison to LNCaP and MCF7 with an NEFH Monoclonal Antibody (RMdO-20) (Cat. No. 13-1300) in western blot. (B) Antibody specificity was demonstrated by CRISPR-Cas9 mediated knockout of target protein. A loss of signal was observed for the target protein in a NEFM KO cell line compared to the control cell line using NEFM Recombinant Rabbit Monoclonal Antibody (JM11-20) (Cat. No. MA5-32613). (C) Relative expression of NF-L was observed in PC-12 cells that were differentiated to neuronal lineage in intermediate filaments of cytoskeleton and neurite extensions in comparison to undifferentiated PC-12 cells using NEFL Monoclonal Antibody (T.400.5) (Cat. No. MA5-14981) in immunofluorescence.
 

Mature neurons can further be subdivided based on direction, action on other neurons, targets, and neurotransmitter production. Neurotransmitter-secreting neurons can be distinguished by the enzymes and transporters required for chemical synthesis and secretion which are described below.

  • Glutamatergic neurons—produce glutamate, which is one of the most common excitatory neurotransmitters in the CNS. Some of the markers specifically expressed in these neurons are as follows:
    Markers Function
    VGLUT1, VGLUT2 A glutamate transporter that transports cytoplasmic glutamate into vesicles. May be involved in neuronal Na+ transport.
    NMDAR1, NMDAR2B An essential subunit of all NMDA receptors. This forms the glutamate binding site.
  • GABAergic neurons—these neurons generate gamma aminobutyric acid (GABA), an inhibitory neurotransmitter in the CNS. GABA is a chemical messenger that is widely distributed in the brain. GABA’s natural function is to reduce the activity of the neurons to which it binds. Some researchers believe that one of the purposes that GABA serves is to control the fear or anxiety experienced when neurons are overexcited. The following markers are expressed in GABAergic neurons.
    Markers Function
    GAT-1 A transporter on the cell membrane that moves GABA into the cell.
    GAD67 A 67 kDa isoform of glutamate decarboxylase that is CNS-specific, unlike GAD65 which is also expressed in pancreas.
    Somatostatin Functions as a neurotransmitter or neuromodulator with mainly inhibitory action. It is also an important regulator of cell proliferation and differentiation. In the cerebral cortex, somatostatin is a co-transmitter of GABAergic inhibitory neurons.
  • Dopaminergic neurons—dopaminergic neurons of the midbrain are the main source of dopamine (DA) in the mammalian CNS. Their loss is associated with one of the most prominent human neurological disorders, Parkinson's disease (PD). Below is the list of markers expressed in dopaminergic neurons.
    Markers Function
    Nurr1 A transcription factor that induces TH expression and subsequently dopaminergic neuron differentiation.
    Tyrosine hydroxylase (TH) The rate-limiting enzyme of catecholamine biosynthesis. It uses tetrahydrobiopterin and molecular oxygen to convert L-tyrosine to -3,4-dihydroxyphenylalanine (L-DOPA), which is a dopamine precursor.
    Dopamine Transporter (DAT) Is responsible for the re-accumulation of dopamine after it has been released. DAT antibodies are widely used as markers for dopaminergic and noradrenergic neurons in a variety neurological disorders including depression, schizophrenia, Parkinson's disease, and drug abuse.
    OTX2 Plays the role of a transcription factor. Also, shown to be involved in the regional patterning of the midbrain and forebrain.
  • Cholinergic neurons—use the neurotransmitter acetylcholine (ACh) to send messages. The cholinergic system of neurons has been a focus of research in aging and neural degradation, specifically as it relates to Alzheimer's disease. The dysfunction and loss of basal forebrain cholinergic neurons and their cortical projections are among the earliest pathological events in Alzheimer's disease. Following are the markers specific to cholinergic neurons.
    Markers Function
    ChAT A transferase enzyme responsible for the synthesis of the neurotransmitter acetylcholine. ChAT catalyzes the transfer of an acetyl group from the coenzyme acetyl-CoA to choline, yielding acetylcholine.
    VAChT Uses a proton gradient established by the vacuolar ATPase to transport ACh into secretory vesicles
  • Motor neurons—a motor neuron’s primary function is to synapse muscle fibers and glands to convey signals from the brain. Degeneration of these neurons leads to loss of control in the muscle activity. The most common condition associate with this degeneration is Amyotrophic Lateral Sclerosis (ALS, also known as Lou Gehrig's disease), in which patients suffer from loss of voluntary muscle control and movement. Established markers of differentiated motor neurons are listed below.
    Markers Function
    ISL1, ISL2 Are members of a family of homeodomain-containing transcription factors. They are expressed in all islet cells in the pancreas and are early markers for motor neuron differentiation.
    OLIG2 Oligodendrocyte transcription factor is a basic helix-loop-helix transcription factor expressed in the motor neuron progenitor domain of the spinal cord that generates motor neurons and oligodendrocytes. OLIG2 is essential for establishing motor neuron progenitor identity downstream of Shh signaling. Subsequently, OLIG2 directly promotes neuronal differentiation in motor neuron progenitors by suppressing the expression of Hes genes, negative regulators of neuronal differentiation.
    Neurogenin 2 A member of the neurogenin subfamily of basic helix-loop-helix transcription factor genes that play an important role in neurogenesis


The figure below shows antibodies that can be used to detect some of these markers. Antibody specificity was demonstrated by detection of differential basal expression of the target across tissues tested owing to their inherent genetic constitution and modulation in expression of target protein by cell treatment.

Western blot, immunofluorescent, and immunocytochemistry analysis of mature neuron markers in various cell lines

Figure 6. VGLUT1 antibody WB, Somatostatin antibody in IF, Tyrosine Hydroxylase antibody in IF, ChAT antibody in WB, and Neurogenin 2 antibody in ICC: (A) Relative expression of VGLUT1 was observed in mouse brain and rat brain in comparison to mouse spleen using VGLUT1 Polyclonal Antibody (Cat. No. OSV00006W) in western blot. (B) Immunofluorescence was performed on fixed and permeabilized SH-SY5Y cells for detection of somatostatin using Somatostatin Recombinant Polyclonal Antibody (9HCLC) (Cat. No. 710968, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Recombinant Secondary Antibody, Alexa Fluor 488 (Cat. No. A27034, 1:2,000) which clearly demonstrates cytoplasmic localization of somatostatin. (C) Dexamethasone, a synthetic glucocorticoid, is known to increase the expression of tyrosine hydroxylase in neuronal cells. Detection of altered expression of the target protein by cell treatment demonstrates antibody specificity. Immunofluorescence analysis of tyrosine hydroxylase using Tyrosine Hydroxylase Polyclonal Antibody (Cat. No. PA1-4679), shows increased expression of tyrosine hydroxylase in a PC-12 cell line upon dexamethasone treatment at a concentration of 5 ug/mL for 6 days. (D) Expression of ChAT was observed specifically in brain and cerebellum in comparison to other tissues tested using ChAT Recombinant Rabbit Monoclonal Antibody (13H9L16) (Cat. No. 703789) in western blot. (E) Modulation of expression of target protein by cell treatment to demonstrate antibody specificity. Immunofluorescence analysis of neurogenin 2 using Neurogenin 2 Recombinant Rabbit Polyclonal Antibody (15HCLC) (Cat. No. 711595) shows induction of neurogenin 2 in retinoic acid treated SH-SY5Y cells.


Synaptic markers

The synapse is where information is transmitted from one neuron to another. There are two types of synapses: chemical and electrical. Synapses usually form between axon terminals and dendritic spines, and sometimes axon-to-axon, dendrite-to-dendrite, and axon-to-cell body synapses. The neuron transmitting the signal is called the presynaptic neuron, and the neuron receiving the signal is called the postsynaptic neuron.

The co-localization of immediately adjacent pre- and post-synaptic proteins is frequently used as a surrogate marker for the identification of synapses by microscopy. The nature of the synapse (GABAergic, glutamatergic, etc.) can also be determined by careful selection of both pre- and post-synaptic antibodies.

Neurons Markers Function
Presynaptic neurons Synapsin I, Synapsin II Synapsins are a family of vesicle-associated proteins that have been implicated in the regulation of neurotransmitter release at synapses.
Synaptotagmin Synaptotagmins are integral membrane proteins of synaptic vesicles thought to serve as Ca2+ sensors in the process of vesicular trafficking and exocytosis. Calcium binding to synaptotagmin 1 participates in triggering neurotransmitter release at the synapse.
Postsynaptic neurons HOMER1 HOMER1 is a member of the homer family of dendritic proteins. Members of this family regulate group 1 metabotrophic glutamate receptor function. It is also a postsynaptic density (PSD) scaffold protein that is involved in synaptic plasticity and calcium signaling.
PSD-95 Synaptic protein that associates with receptors and the cytoskeleton.


Most of neurons are both presynaptic and postsynaptic, which carry specific markers. The figure below shows data for presynaptic marker synapsin 1 and postsynaptic marker PSD-95. Antibody specificity was demonstrated by detection of differential basal expression of the target across tissue tested owing to their inherent genetic constitution.

Neurons are the main structural and functional units of the nervous system. Consequently, any attempt to understand how the brain works lies in the investigation of functionally distinct neuronal cell types. To study these cell types, it is very important to have antibodies that are specific to neural cell markers. Thermo Fisher Scientific offers a comprehensive library of highly specific antibodies for neuroscience research that have been validated for multiple applications like western blot, flow cytometry, immunofluorescence, ELISA, and immunohistochemistry.
 


Recommended reading

  1. Gotz M and Huttner WB (2005) The cell biology of neurogenesis. Mol Cell Biol 6: 777-88.
  2. Ladran I, Tran N, Topol A, and Brennand KJ (2013) Neural stem and progenitor cells in health and disease. WIREs Syst Biol Med 5: 701-15.
  3. Ming GL and Song H (2005) Adult neurogenesis in the mammalian nervous system. Ann Rev Neurosci 28: 223-50.
  4. Redwine JM and Evans CF (2002) Markers of central nervous system glia and neurons in vivo during normal and pathological conditions. Curr Top Microbiol Immunol 265: 119-40.
  5. Doettsch F, Caille I, Lim DA, Garcia-Verdugo JM, and Alvarez-Buylla (1999) Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 97: 703-16.
  6. Garden GA and Campbell BM (2016) Glial biomarkers in human central nervous system disease. Glia 64(10), 1755-71.
  7. Ginhoux F and Prinz M (2015) Origin of microglia: current topics and past controversies. Cold Spring Harb Perspect Biol 7(8): a020537.
  8. Jessen KR, Mirsky R, and Lloyd AC (2015) Schwann cells: development and role in nerve repair. Cold Spring Harb Perpsect Biol 7(7): a020487.
  9. Redwine JM, Buchmeier MJ, and Evans CF (2001) In vivo expression of MHC molecules on oligodendrocytes and neurons during viral infection. Am J Pathol 159: 1219-24.