Overview of Neural Cell Types and Related Antibody Targets

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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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.
 

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