Histology of the CNS Jean-Pierre Louboutin 7/2/20 + Histology of the CNS - Definitions - Type of neurons - Axonal transport - Different types of synapses - Neurohistological techniques - Neurons in: . Cerebral cortex (i.e., Pyramidal cells) . Cerebellum (i.e., Purkinje cells) . Spinal cord - Glial cells + Objectives + Histology of the CNS - Definitions - Type of neurons - Axonal transport - Different types of synapses - Neurohistological techniques - Neurons in: . Cerebral cortex (i.e., Pyramidal cells) . Cerebellum (i.e., Purkinje cells) . Spinal cord - Glial cells + Objectives Definitions Four basic types of animal tissues: . Nervous tissue . Epithelia . Connective tissue . Muscle tissue - Central Nervous System (CNS) consists of brain and spinal cord - Peripheral Nervous System (PNS) consists of cranial nerves and spinal or peripheral nerves which are located outside the CNS - Cells of the nervous tissue are neurons - Neurons are the core components of the nervous system - Each neuron consists of: + a cell body, or soma, containing: . a nucleus . a nucleolus (function: transcribe ribosomal RNA) . the surrounding cytoplasm (called perikaryon in neurons) . numerous different organelles + numerous dendrites that form a dendritic tree + a single axon - Surrounding the CNS neurons are supportive cells called neuroglia (or glia). These cells form the nonneural component of the CNS N: nucleus NS: Nissl substance A: axon AH: axon hillock D: dendrites G: glial cells - A number of specialized types of neurons exist: + Sensory neurons respond to touch, sound, light and numerous other stimuli affecting cells of the sensory organs. Sensory neurons then send signals to the spinal cord and brain + Motor neurons receive signals from the brain and spinal cord. Cause muscle contractions and affect glands + Interneurons connect neurons to other neurons within the same region of the brain or spinal cord - Axons arise from region called axon hillock - A neuron is an electrically excitable cell that processes and transmits information by electrical and chemical signaling - Chemical signaling occurs via synapses, specialized connections with other cells. Neurons connect to each other to form neural networks - Dendrites are covered with dendritic spines making synapses with other neurons - Dendrites receive and integrate information from dendrites, neurons and axons + Histology of the CNS - Definitions - Type of neurons - Axonal transport - Different types of synapses - Neurohistological techniques - Neurons in: . Cerebral cortex (i.e., Pyramidal cells) . Cerebellum (i.e., Purkinje cells) . Spinal cord - Glial cells + Objectives Types of neurons 1. Unipolar neurons: Only one process leaving the cell body. Found in numerous sensory ganglia and peripheral nerves. Were initially bipolar during embryonic development 2. Bipolar neurons: Rare. Purely sensory neurons. Single dendritic tree and single axon. Found in retina, organs of hearing and equilibrium of the ear, olfactory epithelium of nose 3. Multipolar neurons: Most common type in the CNS. Include all motor neurons and interneurons of the brain, cerebellum and spinal cord. Numerous branched dendrites are projecting from the cell body. On the opposite side is a single axon 4. Pseudounipolar neurons: Sensory neuron in the PNS. One axon with 2 branches: one central (from cell body to spinal cord) and one peripheral (from cell body to periphery: skin, joint, muscle). No dendrites. Soma located in dorsal root ganglia + Golgi type I neurons: - Long axons (1 m or more in length) - Axons form long fiber tracts of brain and spinal cord - Examples: Pyramidal cells of cerebral cortex, Purkinje cells of cerebellum, motor cells of spinal cord + Golgi type II neurons: - Short axon terminating in neighborhood or cell body or absent - More numerous than type I neurons - Short dendrites give starshaped appearance - Often inhibitory in function + Histology of the CNS - Definitions - Type of neurons - Axonal transport - Different types of synapses - Neurohistological techniques - Neurons in: . Cerebral cortex (i.e., Pyramidal cells) . Cerebellum (i.e., Purkinje cells) . Spinal cord - Glial cells + Objectives Different types of chemical synapses Different types of chemical synapses + Histology of the CNS - Definitions - Type of neurons - Axonal transport - Different types of synapses - Neurohistological techniques - Neurons in: . Cerebral cortex (i.e., Pyramidal cells) . Cerebellum (i.e., Purkinje cells) . Spinal cord - Glial cells + Objectives Neurohistological techniques H&E Pyramidal cell of monkey Cresyl-Violet: Nissl stain - Binds to DNA and RNA Demonstrate nuclei of all cells and cytoplasmic Nissl substance (RNA of rough endoplasmic reticulum) Reduced silver methods - Produce dark deposits of colloidal silver in various structures, notably filaments inside axons Example: Cajal’s silver nitrate staining Golgi method - Study of neuronal morphology, especially dendrites - Insoluble salts of silver or mercury precipitated within cells in block - Axons typically unstained - Random staining of only a small proportion of cells, enabling resolution of structural details of dendritic trees of individual neurons Arrows: spines Histochemical and immunohistochemical methods - Have currently replaced the traditional silver methods for staining axons and glial cells - Neurotrace: similar as Nissl staining (stains DNA and RNA) but visible with fluorescence microscope Staining of the rat hippocampus using Neurotrace in control animals and animals presenting neuronal loss after experimental seizure (due to kainic acid, KA) Loss of neurons in the hippocampus 7 days after KA-induced experimental seizure Louboutin et al., FASEB J., 2011 Immunocytochemistry - Goal: to localize substances (e.g., enzymes, neurotransmitters) contained in specific populations of neurons - Substances in tissues detected by binding of specific antibodies NeuN Dopamine Glutamate decarboxylase (produces GABA) Gene transfer of green fluorescent protein (GFP) into cortex of monkey by injection of adeno-associated virus-GFP into the cisterna magna Neuronal marker Marker of transgene Merged images Myelin staining Weigert staining Luxol Blue Combined staining Cresyl-Violet and Fast Luxol Blue Electron microscopy - Reveals detailed structure of neurons and specializations existing at synaptic junctions - May be combined with immunohistochemical or Golgi methods + Histology of the CNS - Definitions - Type of neurons - Axonal transport - Different types of synapses - Neurohistological techniques - Neurons in: . Cerebral cortex (i.e., Pyramidal cells) . Cerebellum (i.e., Purkinje cells) . Spinal cord - Glial cells + Objectives Structure of cerebral cortex - Composed of gray matter - 16 billions neurons - Surface increased by gyri, separated by fissures or sulci - Cortex composed of mixture of neurons, supportive cells or neuroglia (glia) and blood vessels - Three types of cortex recognized based on phylogeny: + paleocortex: olfactory system + archicortex: hippocampal formation + remainder of cerebral cortex: neocortex Neurons of cerebral cortex - Different types of cells seen in the cerebral cortex: + Pyramidal cells - Shape of bodies is pyramidal; 10 to 50 micrometers long - Some giant pyramidal cells: Betz cells in motor precentral gyrus of frontal lobe - Apex oriented towards pial surface of cortex - From apex, thick apical dendrite giving collateral branches - Each dendrite possesses numerous dendritic spines for synaptic junctions with axons of other neurons - Axon arises from base of cell body and terminates in deeper cortical layers or more often enters white matter as projection, association or commissural fiber Pyramidal cells stained by different types of silver impregnation Apical dendrite Apical dendrite Perikaryon Perikaryon Axon Pyramidal cells of Layer V Pyramidal cell of Layer V Pyramidal cell of Layer V Pyramidal cells of Layer V 3D- reconstruction Nucleolus inside the nucleus H&E staining Silver staining P: pyramidal cells P: pyramidal cells A: astrocytes Giant pyramidal cells of Betz + Stellate cells: Granule cells - Small size - Polygonal in shape; cell bodies measure 8 micrometers - Multiple branching dendrites and short axons terminating on nearby neuron - - + Fusiform cells Long axis vertical to surface ; concentrated mainly in deepest cortical layers Dendrites arise from each pole of cell body Inferior dendrite branches within same cellular layer Superficial dendrite ascends towards surface of cortex and branches in superficial layers Axon arises from inferior part of cell body and enters white matter as projection, association or commissural fiber + Horizontal cells of Cajal Small, fusiform, horizontally oriented Found in most superficial layers of cortex Dendrite emerges from each end of cell Axon runs parallel to surface of cortex making contact with dendrites of pyramidal cells + Basket cells - Axons branch laterally and embrace cell bodies of pyramidal cells + Cells of Martinotti - Small, multipolar cells, present throughout the levels of cortex. Short dendrites - Axon directed toward pial surface of cortex where it ends in most superficial layer Main types of neurons found in cerebral cortex Horizontal cell (arrows) P.C. B.C. F.C. G.C. P.C.: pyramidal cell; B.C.: basket cell; F.C.: fusiform cell; G.C.: granule cell Nerve fibers of cerebral cortex - Nerve fibers arranged both radially and tangentially + Radial fibers run at right angles to cortical surface - Include: . afferent fibers including association and commissural fibers terminating in the cortex . axons of pyramidal, stellate and fusiform cells, leaving the cortex to become projection, association and commissural fibers of white matter + Tangential fibers run parallel to cortical surface - For the most part: collateral and terminal branches of afferent fibers - Include also axons of horizontal and stellate cells and collateral branches of pyramidal and fusiform cells - Most concentrated in layers 4 and 5, where they are named: outer and inner - terminal parts of thalamocortical fibers In visual cortex, outer band of Baillarger called stria of Gennari bands of Baillarger - Bands of Baillarger well developed in sensory areas due to high concentration of Layers of cerebral cortex (neurons on left, fibers on right) Cortical layers - Numbers of cortical layers in paleo and archicortex varies according to region - As many as five layers in paleocortex - Largest number in archicortex is three layers - In neocortex : 6 layers always recognizable at some stage of embryonic or fetal development. In adult, typical six layers cannot always all be discerned - Thickness of neocortex varies from 4.5 mm in primary motor area of frontal lobe to 1.5 mm in visual area of occipital lobe + Layer I: Molecular layer. Most superficial layer. Covered by pia mater. Consists mainly of tangentially oriented nerve fibers deriving from apical dendrites of pyramidal cells and fusiform cells, axons of stellate cells and cells of Martinotti. Afferent fibers originating in the thalamus also present. Large numbers of synapses between different neurons. Peripheral portion of layer I consists of glial cells and horizontal cells of Cajal + Layer II: External granular layer. Glial cells and small pyramidal cells and stellate cells. Apical dendrites directed towards molecular layer. Axons enter deeper layers where they terminate or enter white matter + Layer III: External pyramidal layer. Medium-sized pyramidal cells. Apical dendrites in molecular layer. Axons extend from the cell bases as projection, commissural or association fibers + Layer IV: Internal granular layer. Contains mainly small granules/stellate cells, glia and some pyramidal cells. External (outer) band of Baillarger + Layer V: Internal pyramidal layer. Glial cells and medium-sized to largest pyramidal cells (especially in the motor area: Betz cells). Stellate cells and cells of Martinotti. Inner band of Baillarger + Layer VI: Multiform layer. Deepest layer. Adjacent to the white matter. Contains intermixed cells of varying sizes and shapes (granule cells, fusiform cells, modified pyramidal cells…). Bundles of axons leave or enter the white matter. Layers of cerebral cortex (neurons on left, fibers on right) Different layers in the neocortex 3 different types of silver staining A B C Cerebral cortex: gray matter. Silver impregnation Layers of cerebral neocortex (neurons on left, fibers on right) Cerebral cortex: gray matter. Cresyl-Violet + Luxol Fast Blue Variations in cytoarchitecture - Not all areas of cerebral cortex have six layers - Areas of cortex in which six layers cannot be recognized: heterotypical - Majority of areas that possess six layers: homotypical - Two examples of heterotypical areas: + Granular type: granular layers well developed and contain numerous stellate cells - Layers 2 and 4 well developed and layers 3 and 5 poorly developed, so layers 2 through 5 merge into single layer of predominantly granular cells - Found in postcentral gyrus, superior temporal gyrus + Agranular type: granular layers poorly developed and layers 2 and 4 almost absent - Pyramidal cells in layers 3 and 5 are densely packed - Found in precentral gyrus and other areas of frontal lobe Layer V of cerebral cortex. Silver impregnation - Variations in arrangement of cells according to regions of cortex, but distinct layers always present - Example: less pyramidal and more granular cells in sensory cortex (parietal lobe) Neuronal connections of cerebral cortex with afferent and efferent fibers Intrinsic organization and circuitry of the hippocampal formation: archicortex - Three areas or sectors: CA1, CA2 and CA3 (CA: cornu ammonis) - CA1 adjacent to subiculum, CA3 close to dentate gyrus - Three layers in hippocampus: archicortex: 1. Molecular layer - Interacting axons and dendrites. Located in center of hippocampal formation, surrounding hippocampal sulcus - Continuous with molecular layers of dentate gyrus and neocortex 2. Pyramidal cell layer (stratum pyramidale) - Prominent layer composed of large neurons, many pyramidal in shape (principal cells of hippocampus - Layer continuous with layer 5 of neocortex - Dendrites extend into molecular layer and axons traverse alveus and fimbria on their way to fornix - Branches called Schaffer collaterals pass through polymorphic and pyramidal layers to synapse in molecular layer with dendrites of other pyramidal cells 3. Polymorphic layer (stratum oriens) - Similar to layer 6 of neocortex CA1 Dentate Gyrus CA2 CA3 Pyramidal cells in hippocampus Dentate gyrus - Three layers - Pyramidal cell layer replaced by granule cell layer of small neurons - Efferent fibers: mossy fibers - Many branches that synapse with pyramidal cells of CA2 and CA3 - One of the three structures with olfactory bulb and subventricular zone (SVZ) to exhibit neurogenesis in adult Brain areas where neurons are able to regenerate/proliferate in adults 1. DG: dentate gyrus 2. SVZ: subventricular zone RMS: rostral migratory stream 3. OB: olfactory bulb Structure of cerebellum Cerebellum - Numerous convoluted folds called cerebellar folia separated by sulci. Covered by pia mater - Consists of an outer gray matter or cortex and an inner white matter - 3 different layers: + Outer molecular layer with fewer and smaller neurons but many fibers + Central Purkinje cell layer. Pyriform (pear-shaped), or pyramidal cells with ramified dendrites (tree) that extend into the molecular layer + Inner granular layer with numerous small neurons - White matter: core of each cerebellar folium. Consists of myelinated fibers or axons - Nerve axons are the afferent and efferent fibers of the cerebellar cortex - Purkinje cells 3 different layers: + Outer molecular layer with fewer and smaller neurons but many fibers + Central Purkinje cell layer. Pyriform (pear-shaped), or pyramidal cells with ramified dendrites (tree) that extend into the molecular layer + Inner granular layer with numerous small neurons Hematoxylin Hematoxylin Silver staining Immunocytochemistry P: Purkinje cells CB: cell body D: dendrite DS: dendritic spine Cerebellum (Toluidine blue) Cerebellum (Toluidine blue) Structure of spinal cord Spinal cord Midthoracic region of spinal cord (transverse section) Spinal cord: anterior gray horn, motor neuron and adjacent white matter Spinal cord: anterior gray horn, motor neuron Motor neuron (H&E staining) N: nucleus Np: neuropil G: glial cells Cervical spinal cord Motor neuron (staining for neurofibrils) N: nucleus Np: neuropil G: glial cells + Histology of the CNS - Definitions - Type of neurons - Axonal transport - Different types of synapses - Neurohistological techniques - Neurons in: . Cerebral cortex (i.e., Pyramidal cells) . Cerebellum (i.e., Purkinje cells) . Spinal cord - Glial cells + Objectives Supporting cells in the CNS: Glia or neuroglia - Highly branched supportive nonneuronal cells in the CNS - Surround neurons, axons and dendrites - They do not become stimulated or conduct impulses - Different morphologically and functionally from the neurons - Can be distinguished by much smaller size - CNS contains tenfold more glial cells than neurons - Four types of glial cells are: + astrocytes + microglial cells + oligodendrocytes (role in myelin formation) + ependymal cells Astrocytes - Small cell body, large oval nucleus, dark stained nucleolus - Long, thin and smooth radiating processes extending from the cell body (star-shaped like) - Two types: fibrous (few and straight processes) and protoplasmic (numerous branching processes) Silver staining Immunocytochemistry Marker: glial fibrillary acidic protein (GFAP) Gold staining Anti-GFAP immunoperoxidase P: cytoplasmic process A: fibrous astrocyte PF: perivascular feet S: soma or cell body Silver staining A: astrocyte C: capillary Astrocytes in the cerebral cortex immunostained for Glial Fibrillary Acid Protein (GFAP) - Astrocytes provide contact between neurons and capillaries. Support metabolic exchanges between neurons and capillaries - Perivascular feet of astrocytes cover the capillary basement membrane: form the blood-brain barrier which restricts the movement of molecules from the blood into the CNS - Astrocytes contain reserves of glycogen from which they release glucose contributing to energy metabolism of CNS - Astrocytes control chemical environment by reuptake of excessive potassium ions and some neurotransmitters like glutamate (then conversion of glutamate into glutamine). Role in detoxification - Role in brain inflammation and brain scar tissue (gliosis) EM MCL Purkinje cell GCL PCL GFAP in the cerebellum: Bergmann glia (radial glial cells) Microglial cells - - Part of the mononuclear phagocyte system of the CNS originating from precursors in the bone marrow Vary in shape, irregular contours; small deeply stained nucleus almost fills the entire cell Cell processes are few, short and slender Smallest glial cells; found in gray and white matter Macrophages/phagocytes of the CNS: when tissue is damaged, microglia migrate to the area, proliferate, become phagocytic and remove dead or foreign tissue Silver staining - Marker: Iba-1 Microglial cells in the cerebral cortex immunostained for Iba-1 Microglial cells in the cerebral cortex immunostained for Iba-1 (red) and DAPI (blue) for nuclei Oligodendrocytes - Small cells with few, thin, short processes without excessive branching Found in both gray and white matter In white matter, they form the myelin sheaths around several axons at one time by contrast to the Schwann cells in the PNS- Role in myelination Silver staining Silver staining EM Axon Myelin sheath Ependymal cells - Line the ventricles in the brain and central canal of the spinal cord - Ciliated cells move the CSF through the central canal of the spinal cord Lateral ventricle Central canal Functions of ependymal cells 1. Lines ventricular cavities and central canal . Moves CSF with ciliae . Produces CSF and absorption of CSF through villi 2. Provides nutrients to stem cells of subventricular zone (SVZ) . Cells of SVZ composed of ependymocytes, astrocytes.. 3. Role in immune defense . Production of cytokines/chemokines, toll-like receptors . Protection against viruses, bacteria Detection of a transgene (GFP) carried by AAV1 virus injected in cisterna magna stained in brown in ependymal cells of monkeys n =1824; 38.1 % n =1825; 53.1 % n = 4113; 53.9 % n = 181; 45.8 % Choroid Plexus - Simple cuboidal epithelium - Production of CSF Meninges D: dura mater A: arachnoid SA: subarachnoid space T: tissue trabeculae BV: blood vessels P: pia mater WM: white matter + Histology of the CNS - Definitions - Type of neurons - Axonal transport - Different types of synapses - Neurohistological techniques - Neurons in: . Cerebral cortex (i.e., Pyramidal cells) . Cerebellum (i.e., Purkinje cells) . Spinal cord - Glial cells + Objectives Objectives - To know the definitions - To know the different types of neurons (unipolar…) - To know and recognize the neurons in cerebral cortex, cerebellum and spinal cord - To know and recognize the different glial cells in the CNS
