Glial Cells and Myelination in the CNS

Techniques for Visualizing and Phenotyping Nervous System Cells

• This includes microscopy, cell stains, tissue clearing, immunohistochemistry, in situ hybridization, and genetic/viral approaches.
• The significance of each technique in assessing different cellular aspects needs to be understood.

Light Microscopy/Brightfield

• Resolution indicates the sharpness of an image.
• Magnification refers to the image size relative to the actual specimen size.
• It is an inexpensive method.
• Specimens must be pigmented or stained and can be either dead or alive.
• Visualization occurs as light passes through the specimen using glass lenses.
• The image formed relies on light absorption.
• It has a limited resolution of 0.2 μm.
• Has a limited magnification power of ≤1500x.

Fluorescence Microscopy

• Uses specific light wavelengths to illuminate fluorophores.
• A second wavelength emitted by the fluorophore is detected through an optic filter.
• Can be used on both dead and alive specimens.
• It falls in the moderately expensive range.
• Produces 2D images.
• Resolution is limited to 0.2 μm.
• Magnification is limited to ≤1500x.
• It can produce artifacts, such as autofluorescence, photobleaching, and phototoxicity.
• Confocal microscopy can be used for a sharper image than regular fluorescence microscopy.
• Confocal microscopy can be used for 3D imaging.

Confocal Microscopy

• Reduces scatter and uses two pinhole apertures.
• Can construct 3D images from Z stacks.
• It has a limited resolution of 0.2 μm (based on the wavelength of visible light).
• It has a limited magnification of ≤1500x.
• Artifacts and bleaching toxicity can be worse with this technique.

CLARITY

• CLARITY is a process that makes tissue transparent.
• This process involves clearing lipid-exchanged acrylamide-hybridized rigid tissue.
• It's compatible with imaging, immunostaining, and in situ hybridization in a hydrogel.

Electron Microscopy

• Employs electrons to produce a visual.
• An electron beam illuminates a stained sample, generating the image.
• Utilizes electromagnets to detect electron scatter, which is then converted into a visible image.
• Achieves a resolution of 0.2 nm (based on the shorter electron wavelength).
• It has a magnification of ≤1,000,000x.
• Samples of dead/stained cells are stained with heavy metal atoms.
• Mostly useful for revealing structural targets.
• Characterized as expensive and technically challenging.

Cell Stains: Early Pioneers

• Camillo Golgi created a method to observe the structure of brain grey matter.
• The method involved metallic impregnations to observe elements of the nervous tissue.
• Camillo Golgi supported reticular theory.
• Santiago Ramón y Cajal was a supporter of neuron doctrine.

Visualizing Whole Cells

• Golgi stain labels random cells with silver salt impregnation.
• The Golgi stain can be used for discovery of spines, labeling 5-10% of all neurons.
• Is is clumpy and incompatible with electromicroscopy.
• Golgi-Cox stain uses Mercury impregnation, clumping less.
• Golgi-Cox stains are okay to use with electron microscopy.

Visualizing Cellular Components

• Cresyl Violet labels ribosome-rich areas, such as endoplasmic reticulum and nucleolus.
• Cresyl Violet does not label dendrites/axon.
• Cresyl Violet is good for visualizing laminae and cytoarchitecture.
• Cresyl Violet stains dead/degenerating cells via the redistribution of the stain internally.
• DAPI and Hoechst stains label helical DNA, not RNA.
• They require fluorescence microscopy because they are excited by UV and emit blue light.
• Propidium lodine (PI) can be used as a nuclear stain, but cell impermeable.
• PI is a red cell death marker.

Visualizing Specific Proteins

• Immunohistochemistry uses a primary antibody produced against an antigen or protein.
• A secondary antibody may be used against the primary to amplify the signal .
• Microscopy techniques include LM, FM, and EM, or autoradiography based on the label on the secondary antibody.
• Can be used to detect neuronal activation and plasticity signals.
• This may include detecting neuronal activation (e.g., Fos) and plasticity (e.g., Zif268, Arc).

Visualizing Nucleic Acids

• In-situ hybridization uses RNA or DNA probes with a complementary NA sequence.
• This technique is used to label the target RNA.
• Microscopy: LM, FM, EM, or autoradiography can be used.
• RNAscope uses in-situ hybridization with amplification (100x sensitivity).
• The process uses RNA or DNA probes, then a preamplifier, then an amplifier, then a fluorescent or enzyme probe.
• LM and FM can both be used as microscopy types.

Visualizing Genetically Defined Cell Populations/Connections

• Requires using transgenic approaches.
• Two techniques include constitutive and inducible approaches.
• Constitutive approaches involve inserting a sequence for fluorescent proteins into the DNA and is heritable.
• Offspring can have multiple transgenes after crossing different types of transgenic animals.
• Inducible approaches involve the use of viruses (or bacterial artificial chromosomes).
• This step is to infect specific cell populations and introduce a sequence for fluorescent proteins.
• This does result in heritability.
• This approach can target specific cell populations or pathways.
• AAV5→←AAV6 can be used.

Optogenetics/Chemogenetics

• Optogenetics introduces light-sensitive ion channels or pumps
• These are activated using light.
• Cell specificity comes from the promoter of inserted viral vectors containing the opsin or DREADD.
• These can target dopamine or glutamate neurons.
• Chemogenetics introduces modified Gi/o, or Gq-coupled mutated human muscarinic cholinergic or kappa opioid receptor (DREADD)
• They are stimulated using selective agonists like CNO.

Viral Vectors

• Are used in basic research.
• Can be used as tools for gene therapy.
• AAV-GDNF's glial version promotes the survival of many types of neurons.
• Prosavin uses lenti-TH and two other synthetic enzymes for dopamine.

Neuron Diversity: Types and Coverage

• The brain contains around 100 billion total neurons.
• It isn't clear exactly how many different types exist, but there is law of coverage.
• Structural categories include: unipolar, bipolar, pseudounipolar and mulitpolar neurons.
• Functional categories of neurons exist.
• Neurons include the cerebellar Purkinje cell.

Neuron Diversity: Source

• Sources of this diversity includes somatic mosaicism.
• This is composed of errors during DNA replication and DNA rearrangement via transposons.
• Epigenetic mechanisms are sources, too.
• Environmental factors such as nutrients during development and mutagen/toxin exposure are sources.
• The structural and functional manifestations include: enzymes, structural proteins, membrane constituents, secretory products.

Cerebellar Neuron Types

• The granule cells are bifurcated axons.
• Granule cells excite Purkinje cells, which can be inhibited by Golgi cells (large interneurons).
• Golgie cell arbors span Ctx and inhibit granule cells.
• Basket and Stellate cells are small interneurons that can inhibit Pukinje cells.
• Purkinje have inhibitory projection neurons.
• Purkinje cells have 2D arborization.

Cortical Neuron Types

• Pyramidal neurons account for 80% of the neurons.
• These are all except those in layer 1.
• Pyramidal cells are multipolar with a large arbor and long branching axons.
• Interneurons accounts for 20% of the neurons.
• Basket cells innervate the dendrites of pyramidal cells.
• Chandelier cells contain cartridges with boutons and they innervate pyramidal cells at the axon hillock.

Glial Cell: Types

• Includes ogligodendrocytes, astrocytes, ependymal cells and microglia.
• Also included are schwann cells and satellite cells.

The Oligodendrocytes

• 30% of all glial cells are oligodendrocytes
• Myelinating oligodendrocytes insulate and nourish axons
• One can produce approximately 30 myelin sheaths.
• Non-myelinating oligodendrocytes nourish short axons.
• Life-long oligodendrogenesis can cause alteration of neuroplasticity because this is the synthesis of these from precursor cells with myelin remodelling.
• Multiple sclerosis is a disease that effects the structure of the oliodendrocytes.

Astrocytes

• The most abundant glial cell.
• GFAP is a tell-tale sign on microscope.
• Protoplasmic- In gray matter. BBB. structural. clear glutaMate+ion, synaptogenesis
• Fibrous- White matter endfeet BBB nourishment

Radial Glia and Ependymal cells

• Radial Glia guides cell migration in the cortext
• Cons include berggman and muller glia
• Guide cell migration
• Ependymal: lines the ventricular system
• Leaves lateral-> Interventrical-> third-> fourth ventricle CSF
• Marginal: barrier between parenym and meninges
• Perivascular: part of Blood brian baties- velate: chemical/structural barrier

Ependymal Cells

• Ependymocytes: ventricular: with cilia- circulate CSF, microvilli-reabsorb
• Eg choral piexus: form
• Tanycytes: Regulate blood-CSF -> Brain Transport of Molocules of 3rd ventricle

Glial Importance

• BBB Failure AD PD HD
• Prot accumulation->AD and A disease
• Abn prunitng->Frgaile X;
• Developement - - Rett + frag+ a disease
• CsFAbnorm -> Hydrocephaly

Microglia

• Three 15%
• Baseline- support neuron synapsi and phayo stripping
• Active -Present anitgen to lym and promed
• AD, strok -> enuro- Inflan

PNS Glia

• Schwann -Two tyeps
• Myelinating -Conduct support regen NonM- support nourich P dead

Explore the roles of glial cells, focusing on oligodendrocytes and myelin formation in the central nervous system. This includes their function in nourishing axons and diseases associated with myelin sheath damage. Also covered are astrocytes, Bergmann glia, and the functions of non-myelinating Schwann cells.