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Neurohistology Manual - Part 1

For Labs on November 11, 2014

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Guide to the Neurohistology Slides

Slide List: #1 Bovine spinal cord stained with hematoxylin and eosin. #2 Three levels of human spinal cord stained to visualize nuclei acids, showing ribosomal RNA (Nissl substance), nucleolar RNA and chromatin in neurons and glia. #2a Four levels of human spinal cord stained with hematoxylin and eosin. #3 Lumbar human spinal cord stained to visualize both axonal and dendritic neurofilaments/neurofibrils and myelin. #5 Bovine spinal cord stained to visualize nucleic acids. #6 Cross section of human cerebral cortex (cortical gryrus) stained to visualize gliofibrils in astrocytes. #7 Cross section of human medulla stained to visualize myelin (blue) and nucleic acids including Nissl bodies #9 Fragment of human temporal lobe (showing hippocampus, inferior horn of the lateral ventricle, and choroid plexus which you will learn later) , stained with a trichrome mixture to enhance the visibility of the choroidal ependymal cells. #10 Cat spinal cord process to show collagenous structures a deep green color, Nissl substance dark blue, neuronal cytoplasm light blue, and nucleoli and glial cytoplasm red. #42 Coronal section of kitten forebrain (cerebral hemispheres + diencephalon) stained with thionin for cell bodies (Nissl stain) ________________________________________________________________________ #20 Horizontal section of a well preserved monkey eye (compared to #20a) stained with hematoxylin and eosin. Paraffin section. #20a Horizontal section of human eye obtained at autopsy, stained with hematoxylin and eosin. Delamination of retinal and choroidal elements in processing may facilitate analysis. Paraffin section. #22 Decalcified axial section of guinea pig cochlea and associated structures in the temporal bone stained with hematoxylin and eosin. Paraffin section.

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#27 Sagittal section of cat lower brainstem and cerebellum stained with thionin, as in slide #5. #39 Half-coronal sections of rat brain stained for neuronal expression of NeuN (Fox 3). #40 Coronal section of squirrel monkey forebrain (cerebral hemispheres + diencephalon) and ponsstained with thionin for cell bodies (Nissl stain) #41 Coronal section of kitten forebrain (cerebral hemispheres + diencephalon) stained for myelin (Pal-Weigert)

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Spinal nerve

Dorsal rootganglion

Ventral root

Dorsal rootlet

Ventral rootlets

DuramaterSubarachnoid space

Dorsal root

Piamater

Fig. 2 Spinal Cord

Fig. 3 Astrocytes

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Lessons (1-8): 1. Spinal cord - Motor neurons, Nissl substance, axon hillock: Open slide #5, containing a section of the spinal cord of the cow, stained only for nucleic acids (thionin stain). You may want to rotate the image 180 degrees into a more standard orientation, dorsal up. The stain labels Nissl substance (ribosomal RNA), chromatin (DNA) and nucleoli (nucleolar RNA). The staining is very light, but this will allow you to examine the large motor neurons of the ventral horn with great clarity, once you have located them. The staining of the motor neurons in this slide is classic. The cells cut through the plane of their nucleoli show the prominent nucleolus in the center of a large, clear nucleus, surrounded by a perikaryon filled with blue granules of Nissl substance. Find the two motor neurons in the ventral horn of the right side that have their axon hillock in the plane of the section. Recall that the beginning of the axon of large neurons differs from any of the dendrites in that it lacks Nissl substance. To what cell type do the very small nuclei that are scattered about near the motor neurons belong? 2. Spinal cord – Segmental levels: Next, go to slide #2a to recall the varying appearance of cross sections at different levels of the human spinal cord. This slide has four sections representative of the cervical enlargement, the thoracic cord, the lumbar enlargement and the sacral cord. What criteria can be used to identify each level? 3. Spinal cord – Neuropil, glial cells, meninges, dorsal root ganglion Now open slide #10high (10.1). This cross section of cat spinal cord (see Fig. 2) is stained with a three-color procedure that stains neuronal cytoplasm (including axonal cytoplasm) light blue to aqua, glial cytoplasm red, and the collagen fibers of the pia, arachnoid and dura a brilliant green. Identify the spinal gray matter at low power, center on the ventral horn, and zoom to higher powers to examine the region between the motor neuron cell bodies (you will have to know when you are looking at the cells or between them). The region containing the interlaced neuronal and glial processes is called the neuropil. Neuropil translates as nerve hair, and refers the filaments present in neuronal and glial processes. Spatially, the neuropil fills up the ventral horn and gives it its shape. Functionally, since it is where the dendrites of the motor neurons and interneurons of the ventral horn are located, it is also where most of its axo-dendritic synapses are located. Notice the small nuclei in the neuropil. To what cell type do they belong? Assuming you have observed the large motor neurons, what color is their nucleoli? What color is the neuropil in a thionin stained section? – in an H&E stained section? Examine the white matter anywhere outside the spinal gray. Note its bubbly, moth eaten, red toned matrix, containing blue-green colored dots. The dots, which are axons in cross section, are situated in clear, circular areas where the myelin has been washed out in processing. The red toned material is glial cytoplasm – of what cell type(s)? Can you find some of their nuclei? Move outward to the pial surface of the white matter to identify the collagen fibers of the pia matter. What separates the pia from the surface of the spinal cord? What is the external glia limitans? By moving circumferentially around the outline of the spinal

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cord, you should find places where the pia mater extends into the white matter as thin sheets. These partial septa often contain blood vessels. The empty space beyond the pia is the arachnoid space, and beyond that you will find the thick and densely stained dura matter. Where is the arachnoid membrane? Return to low power to identify the dorsal and ventral roots, dorsal root ganglion and spinal nerve. Note that, while all the DRG cells are approximately spherical in shape, they vary widely in size. The size difference is a function of the diameter of their axons, which correlates with conduction velocity and modality. What modality is conveyed by the largest of the ganglion cells? – by the smallest? Note that the Nissl substance of the DRG cells is fine-grained compared to that in the large motor neurons you have examined. Try to find an axon hillock. Note the fascicles of dorsal root axons that penetrate the ganglion. Each DRG cell is encircled by a single cell thick shell of peripheral glia, called satellite cells (modified Schwann cells). Given a slate of choices concerning basement membrane (basal lamina) location(s) in the DRG, would you select: a. Each ganglion cell is surrounded by a BM lying between its cell body and the satellite capsule. b. Each ganglion cell is surrounded by a BM lying just outside its satellite capsule. c. The DGR as a whole is enclosed in a single BM. The cells of the DRG are packaged closely together. Does a DRG have a neuropil? If so, where is it? If not, why not? 4. Spinal cord – stained to visualize neurofilaments, neuropil and myelin: The next slide to examine is # 3high (3.1). This is a cross section of human spinal cord, stained for myelin (blue) and neurofilament protein (pink to purple). Notice the axons and dendrites crossing through the neuropil of the ventral horn and the cross sections of the axons in the white matter. The glial component of the neuropil is unstained, and invisible. (Erythrocytes and nucleolar protein(s) are also stained with silver in this section.) At low power, center on the ventral horn in an area between the motor neurons, and zoom in to see the lacework of processes in the neuropil. Some of them may be traced back to the darkly stained cell bodies of motor neurons. Why would it be a waste of time to look for axon hillocks in this section? Moving over to the white matter, you will find many axons in perfect cross section, and some of them may be encircled by myelin rings, if well-enough preserved. 5. Spinal cord – More on myelin: Myelin rings in the spinal cord white matter are much easier to identify in slide #23, especially in the dorsal white matter. This is a cross section of human lumbar cord stained only for myelin. Also find the myelin rings in the ventral roots. Why are these roots cut in cross section? Using the split-screen function, compare cross-sectional views of the white matter in slide #23 with slide #3high. 6. Glial cells -Astrocytes: Now open slide #6, containing a cross section of a human cerebral cortex (a folding of the cerebral cortex termed cortical gyrus), stained to visualize intermediate filaments (gliofilaments) of astrocytes. Microglia and oligos are not stained. Start by

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examining the white matter core of the center of the gyrus. You will quickly find star shaped cells, whose numerous radially extending, thin processes are stained almost black with the metal. These are the classically defined fibrous astrocytes. Note that some of the astrocytic processes extend toward and contact blood vessels. The blood vessels are made visible by the end feet of the glial processes apposed to them. The coating of astrocytic endfeet is called the perivascular glia limitans (of the blood vessel). Now move gradually outward toward the pia matter to note that the astrocytes become less well stained and their processes become more sinuous. In the cortex, these are the classically defined protoplasmic astrocytes (see Fig. 3) that contain fewer glial filaments. Examining the pial surface, you may find astrocytes that extend processes to the surface, where they would be in contact with the BM of the brain. Their endfeet form the external glia limitans. 7. Glial cells - Ependymoglia: Now open slide #1 to examine the ependymal lining of the central canal. This is a cross section of the spinal cord of a cow, stained with H&E. Once again, note the appearance of the large ventral horn motor neurons, glial cell nuclei and lacy neuropil, as colored by the H&E stain. Then move to the center of the intermediate gray and zoom in on the ependyma. Note the more or less single layer of almost cuboidal ependymoglial cells surrounding the central ventricle. Using the split-screen function, compare the ventral horn motor neurons, neuropil and ependymoglial cells of slide #1 with slide #5. 8. Glial cells – Choriod plexus The last slide, #9, shows a section of a block of human temporal lobe, containing the lateral ventricle and adjoining brain tissue (hippocampal formation ,Fig. 4). It was stained by the three color method (as for slide #10, see above). At low power, locate the lateral ventricle, brain tissue (hippocampus) and choroid plexus. There are no blood vessels in the ventricle, except for those within the choroid plexus. The choroid plexus protrudes into the ventricular cavity from a thin membrane (choroidal lamina) that attaches to the brain tissue at both ends. At higher magnifications, examine the choroidal lamina. Which of its surfaces gives attachment to collagen fibers? Examine the choroid plexus. The choroidal ependymal cells are purple-colored, plump cuboidal cells, aligned in a single layer covering a core of pia mater. In other words, the choroid plexus, formed from infolded choroidal lamina is inside-out relative to the rest of the brain. Note that the blood vessels of the choroid plexus run in its connective tissue core. Finally, examine the interface between the lateral ventricle and the brain substance to identify the ordinary (i.e., non-choroidal) ependymal cells. Most of them will appear more squamous than cuboidal.

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Fig. 4 Choroid plexus in inferior horn of lateral ventricle

Inferior hornof lateralventricle

Hippocampus

Fornix

Dentategyrus

choroidplexus

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9. Nuclear and Cortical Organization in Gray Matter. Figure 5. slide 42. Nissl stain.

Examine slide 42. Gray matter (appendix p. 15) has several organizational patterns. Cortical patterns have layers of neurons and are, for the most part, on the surface of the cerebral hemisphere. The two types of cortex are depicted on the slide. Neocortex covers most of the surface and has 6 distinct cell layers. In this Nissl stained slide you can see “columns” of neurons, and apparent layering, but differences in neuronal size and shape are hard to discern. Allocortex has fewer layers. The box contains a few regions of allocortex including the hippocampus and the dentate gyrus (you’ll hear more about them later). The region with a very dense layer is the dentate gyrus. In the hippocampus, adjacent to the ventricle, you can see the pyramidal shape of the large neurons, and observe their parallel orientations. (Pyramidal cells also populate the neocortex, but it is hard to see the pyramidal shape in this material). Gray matter is said to have a nuclear organization when the cell bodies are relatively close packed, and organized into a rounded structure. Examples are three nuclear regions within the thalamus: the lateral geniculate body (lgb), the medial geniculate body (mgb) and other thalamic nuclei. Examine these. Notice similarities and differences between the nuclear patterns.

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APPENDIX

Table of Contents

Assigned and Suggested Readings 10 Neuron Doctrine 10 Types of Neurons 11 Projection Neurons vs Interneurons 11 Histological Differences between Axons and Dendrites 12 Types of Synapses and the Generation of Impulses 12 Glial Cells 13 Schwann cells, Oligodendrocytes and Myelin Astrocytes Ependymolglia Microglia The Blood-Brain Barrier and Choroid Plexuses 14 Unmyelinated Axons and Conduction Velocity 15 Gray Matter and White Matter, vs the Structure of Nerves and Ganglia 15 The Epithelial Nature of Nerve Fibers, Ganglia and the CNS 15-16 Nerve Injury, Degeneration and Regeneration 17 Assigned Readings

In the following pages, I will review only the basic histology of nervous tissue, leaving the detailed structure of the CNS (its neuroanatomy) to subsequent chapters. Martin’s “Neuroanatomy Text and Atlas” has a chapter (Chap. 1) containing information on neurohistology. Neuron Doctrine Neurons are cells that are unique to the nervous system; they are electrically excitable and transmit signals containing information and commands, often over a considerable distance, to other neurons or non-neuronal cells (i.e., muscle cells, gland cells). Some neurons, also, deliver their products directly into the blood stream, i.e., they are neurosecretory cells. The neuron is an anatomically and functionally polarized cell consisting of a globular cell body and one or more thin cellular extensions of variable length, their neurites, which serve functionally as input and output devices. The input structures can be thought of collectors, the output structures as transmitters. The concept of the neuron as a polarized cell is the basis of the “neuron doctrine” as formulated at the end of the 19th century by Ramon y Cajal in Spain and Waldeyer in Germany. The nervous system had been thought of as a syncitial network of tubes that carried nervous signals here and there, and the

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cell bodies that were evident were thought of as some kind of nodes in the network. How else could signals move over long distances if not in a continuum of protoplasm? Using special staining methods, Ramon y Cajal and others were able to show that the developing nervous system contains only individual cells, not yet a syncitium, and that individual cells are readily found in the adult. These cells, for the most part, had polarized forms, whose processes were oriented in many systems in ways consistent with a directed functional polarization, their processes appearing to serve as either input or output structures. The input structures are usually what are now termed the dendrites of the cell; the output structures are usually their axons. Unless the axon is very short, the transmitted output is carried by a brief, moving, explosive reversal of its membrane potential, called an action potential or nerve impulse. In a myelinated axon, the potential reversal occurs only at the nodes of Ranvier (below), which may be spaced as much a 1-1.5 mm apart, while in unmyelinated axons, the event moves as a continuous wave. Thus, the canonical polarized form of a neuron is – dendrite in, axon out. But, there are many exceptions and variations on this theme, in that certain types of neurons lack dendrites and others lack axons. Types of Neurons Generally, if the neuron has an axon, it has only one (unless it is a bipolar ganglion cell of the VIIIth cranial nerve; below). Neurons that lack an axon are called amacrine cells; their dendrites carry out both input and output functions. (The best characterized amacrine cells are found in the retina and olfactory bulb.) Most neurons have several or at least one dendrite. The dendrites can have elaborate branching forms that remind one of a tree. Hence, the term “dendritic tree” came into wide usage a long time ago. Motor neurons and neurons with a similar morphology are often referred to as being multipolar cells because they exhibit both an axon and several dendrites. In the types of neurons that lack dendrites altogether; their axon(s) serves for both input and output. Such neurons are found in the dorsal root ganglia and in some of the sensory ganglia of the cranial nerves. Those having only one axon can be referred to as unipolar cells. In this case, the singular stem of the axon emerges from a spherical cell body before bifurcating into a peripherally directed and a centrally directed branch. (Some sourcebooks refer to the peripheral branch of the axon as a “functional dendrite” on the notion that it carries the input signal toward the cell body to be passed on from there to the centrally directed axon. However, we will see below that the input signal in the peripheral axon of a dorsal root ganglion neuron bypasses the cell body, and that the electrical character of the signal collected by ordinary dendrites does not move in the way an action potential does. It is basically stationary and reaches through, but does not pass through, the cell body. There is also a tradition for calling these cells pseudounipolar because they begin development as bipolar in form, i.e., a neurite on each side of the cell body). Truly bipolar neurons persist in the adult in the retina and in some other locations. These cells have one dendrite and one axon on opposite sides of the soma. The sensory ganglion cells of the eighth cranial nerve are also bipolar, but in a different way. In this case, a myelinated axon emerges from opposite sides of the cell body, without a common stem. Finally, the secretory cells of the adrenal medulla (a neural derivative) are essentially apolar neurons that lack both dendrites and axons; the cell body does it all. There are two major classes of neurons in which the axon is mainly or partially in the PNS: motor neurons and sensory neurons. All other neurons, the majority, form networks located entirely within the CNS that utilize sensory data and memory to organize behavior. The motor neurons are usually described as efferent neurons because they transmit action potentials away from the CNS, while sensory neurons and their axons are afferent in orientation because they bring signals into the CNS from other parts of the body. Motor neurons, wherever they are found, are multipolar, exhibiting both an axon and several dendrites, while spinal sensory neurons and the sensory neurons of cranial nerves that innervate skin and mucosa have no dendrites, and are typically unipolar. Motor neurons innervate skeletal, smooth and cardiac muscle, as well as glands and other motor neurons located in the autonomic ganglia. Spinal and cranial nerve sensory axons convey sensate information from the skin (e.g., pain, touch, temperature), from muscles, tendons and joints (proprioception), and from the visceral organs (fullness,

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pain, nausea) to the CNS, and non-sensate input leading to reflex actions. As noted above, the axon given off from these unipolar sensory neurons bifurcates into a peripheral branch and a central branch. The peripheral branch conducts action potentials from the periphery toward the point of bifurcation and the central branch carries the action potential further into the CNS. As noted above, the sensory ganglion cells of cranial nerve VIII are bipolar in the sense that the central and peripheral axonal processes come off from opposite poles of the cell body, rather than from a common stem. Since the perikaryon lies in the middle of the conduction path, it is covered by a thin layer of myelin. Projection Neurons vs Interneurons

From all of the above, one can get the impression that neurons, especially in the CNS, come in a wide variety of shapes and sizes, many of which types have been given picturesque names or named for their discoverer, like pyramidal cells, granules cells, Purkinje cells, Deiter’s neurons, Mauthner cells, etc. The shape of neurons and their dendritic “trees” is related to their function. The variability in neuronal morphology can be simplified to some extent by classifying neurons as Golgi Type I or Type II. Golgi Type I neurons have a long axon that leaves the immediate vicinity of the cell body to reach a significantly distant target for synapsing. Such neurons are also referred to as projection neurons. An example of this type would be the alpha motor neuron, or the cortical pyramidal cell that innervates it. In contrast Golgi Type II neurons have short axons that remain within the general area of the cell to synapse on other neurons of the parent gray matter. Such neurons are also referred to as interneurons or local circuit neurons. Care should be taken with often misleading descriptions of Golgi Type neurons found in the literature. . Histological Differences Between Axons and Dendrites The neuronal cell body and its proximal dendrites contain large amounts of rough endoplasmic reticulum that is visible at the light microscopic level with an appropriate stain as clumps and flecks in the perikaryon and the proximal parts of dendrites. These organelles are referred to as Nissl substance or Nissl bodies for the individual who first described them clearly. The presence of rough endoplasmic reticulum in dendrites suggests they contain extensions of the perikaryal cytoplasm. Indeed, there is no distinct boundary between the perikaryon and the proximal part of a dendrite. The Golgi apparatus may extend some distance into the proximal dendrite in some neurons, and the dendrites and perikarya contain the same type of randomly polarized mictrotubules. Another feature unique to the dendrites of some neurons, like the pyramidal cells of the cerebral cortex, is the presence of dendritic spines distributed along the shaft and branches of the dendritic “tree.” These are small, often drumstick-shaped appendages onto which synapses are made by incoming axons. No matter how far out along a dendrite the spines are located, each of them is associated with a small Nissl body. This is viewed as an indication of the local synthesis of protein targeted to the individual dendritic spine, and is probably related to memory formation or its maintenance. In contrast, the axon presents a uniquely distinct ultrastucture. Primarily, it lacks Nissl substance, and its microtubules are homogeneously polarized. The region of the cell body (or in some cells the base of a proximal dendrite) from which an axon arises is termed the axon hillock, so-called because it usually has a conical shape. The axon hillock is also devoid of Nissl substance and Golgi tubules. The proximal part of such an axon is called its initial segment. It is never myelinated, and shows submembranous specializations related to its role as the zone of initiation of the axonal action potential. You might think of it as the first node of Ranvier (below), but a lengthy one. Indeed, it like the nodes contains a high concentration of voltage gated sodium channels. Finally, an axon can be myelinated, but there no known examples of myelinated dendrites. These ultrastructural characteristics represent the morphological differences between axons and dendrites.

Axons and dendrites differ both morphologically and functionally. In brief, dendrites are extensions of the neuronal perikaryon; axons are specialized outgrowths lacking Nissl substance and Golgi tubules. Axons can be myelinated.

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Types of Synapses and the Generation of Impulses As we already know, neurons that possess an axon communicate with other neurons or non-neuronal cells by means of synapses located either at the terminal end of the axon, usually called “boutons terminaux”, or along a region of its shaft and branches, where they would be referred to as “synapses en passant.” Such synapses may be axo-dendritic, if formed on the surface of the dendrites of another neuron, or axo-somatic, if formed directly on the perikaryon. Axo-spinous synapses are a type of axo-dendritic synapse made on dendritic spines. In some systems, the incoming axon terminal synapses on the axon hillock or the axon initial segment of the target neuron; such synapses are axo-axonic, and in most of the locations that they occur, are inhibitory. Axo-axonic synapses can also be made onto axon terminals, where they can mediate pre-synaptic inhibition. Wherever they are placed, the side of the synapse belonging to the axon is termed the presynaptic side. The postsynaptic side belongs to the other cell. Analysis of nervous tissues with the electron microscope confirmed the existence of synapses per se, and revealed the detailed structure of the various types of synapses. Two general types of synapse, known as Gray Type I and Gray Type II have come to be associated, respectively, with excitatory vs inhibitory function in the CNS.

Glial cells Schwann cells, Oligodendrocytes and Myelin

The nervous system contains other cell types in addition to neurons. Glial (neuroglial) cells, which are also unique to the nervous system, have various supportive functions. One function of glia is to form the myelin sheath of axons. In the PNS, the glial cells that myelinate axons are called Schwann cells, while oligodendrocytes do so in the CNS. The change over from oligos to Schwann cells occurs at the point of emergence of the dorsal and ventral roots from the spinal cord and the cranial nerve roots from the brainstem (except for the optic nerve, which contains oligos throughout). Because axons are usually longer than the myelinating cells are wide, many glia arranged in linear series are needed to ensheath the entire length of an axon. This should not be taken to imply that the myelinating cells are small. Myelinating Schwann cells can form segments of myelin 0.5 – 1.5 mm in length in the adult. The adjacent segments, termed internodes, are separated from each other by small gaps of about 1 µm, termed the nodes of Ranvier, which have an important role in impulse propagation along the axon. In the PNS, each segment of myelin (internode) belongs to a single Schwann cell that has wrapped itself spirally around the axon to form multiple layers of modified cell membrane, layers from which the cytoplasm has been effectively excluded (squeezed out?), and the one Schwann cell wraps only one axon. In the CNS, a single oligodendroglia can myelinate several axons in its vicinity, but contributes only one segment of myelin to each. It does this by means of dendrite-like extensions from its cell body that reach out to and enwrap the axons in question. While mostly lipid, CNS and PNS myelin differ in their protein composition, which makes them susceptible to different autoimmune diseases. CNS myelin, alone, is compromised in multiple sclerosis, while other neuropathologies affect PNS myelin, such as Guillain-Barré and Charcot-Marie Tooth syndromes.

Astrocytes Astrocytes are another type of glial cell. Astrocytes have many protoplasmic extensions, giving them a starlike or spidery appearance. These processes can be very long and can thin out into undulating sheets. Astrocytes are, topologically, space filling cells; they separate neurons from each other (except at the synapses), and their fine processes, for the most part, occupy all the spaces among and between the axons and dendrites of the gray matter, and between the axons of the white matter. They also wall-off the neural parenchyma of the CNS from the external connective tissues (i.e., the pia mater) by forming an external glia limitans (below). However, although the neurons and glia are tightly

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packed in the CNS, they are everywhere separated by a continuous extracellular space of about 10 nanometers in width. (The synaptic cleft at Gray Type I synapses is on the order of 20 nm). The intermediate filaments of the cytoskeleton of astrocytes contain a astrocyte-specific protein (not present in neurons or oligos), termed glial fibrillary acid protein (GFAP). These filaments form a network in the perikaryon of the cell, and extend outward into the proximal parts of its processes (starlike). Astrocytes in the white matter contain dense aggregates of the filaments, and are called fibrous astrocytes. The astrocytes in the gray matter contain fewer and more loosely arranged filaments, and are called protoplasmic astrocytes. One of the main functions of astrocytes is to remove potassium ions and neurotransmitter chemicals such as glutamate from the extracellular space to rebalance the electrolyte environment and help terminate transmitter action. They also help to regulate the water content of the extracellular space. Another function is to regulate access of blood borne metabolites to the neural parenchyma (see below, blood brain barrier). Ependymoglia Ependymoglial cells line the brain ventricles in one form or another. In the choroid plexuses, they are the secretory cells that contribute to the formation of the cerebrospinal fluid, and are referred to as choroidal ependymal cells. In some of the circumventricular organs, they are sensory cells that test the CSF for various metabolites and hormones. Everywhere else, they are referred to as ordinary ependymal cells, except in certain locations where the walls of the CNS are thin. There, they take on the appearance of tanycytes (below). Choroidal ependymal cells are cuboidal and filled with secretory granules. Ordinary ependymal cells can be cuboidal or flattened into a quasi-squamous shape. Ependymal cells of cuboidal, columnar, or squamous morphology resemble simple epithelia in being closely packed and held together by tight junctions. They are unlike astrocytes and oligos, which are usually spaced apart from each other and generally have longer processes. They are also ciliated and possess microvilli on their apical surface (facing into the cerebral ventricles). Unlike epithelia, there is no basement membrane the basal side of ependymoglia, and the basal surface sends out short processes like the tentacles of an octopus that interdigitate with astrocyte processes to form an internal glia limitans in various locales in the brain. Tanycytes have a cell body on the ventricular side, where they sample the contents of the CSF, and extend a slender process toward the pial surface or into certain nuclei of the hypothalamus, where their end feet form an external glia limitans or a perivascular limitans. In this respect, in addition to containing GFAP, tanycytes resemble astrocytes. They also resemble the radial glia of the developing CNS, which are precursor cells for neurons and glia (except for the microglia). Microglia These are potentially phagocytic cells that have migrated from mesoderm into the CNS during development. In the adult, they are small and stationary when at rest, but have slender, motile processes that extend out into the neighboring extracellular spaces. Upon detecting foreign or infectious material, they can transform into ameboid cells that migrate toward the site of injury to act as phagocytes. At the injury site, they can become engorged with debris, cease phagocytosis, round up and remain as “gitter cells.” The Blood-Brain Barrier and Choroid Plexuses Organic non-polar compounds of small molecular dimensions, like alcohol and ethyl ether, can pass readily from the blood stream into the parenchyma of the CNS. Similar polar compounds, like amino acids and glucose, require specific transport mechanisms. The gating systems that differentially admit or bar entry are referred to collectively as the “blood-brain barrier.” Similarly, there are blood-nerve and CSF-brain barriers. Part of the blood-brain barrier can be attributed to the absence of fenestration and the presence of tight junctions among the endothelial lining cells of CNS capillaries. The transporter mechanisms needed to pass through this physical barrier are located in the endothelial

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cells and in the adjacent astrocytes of the perivascular glia limitans. The CSF-brain barrier is attributed to the occluding tight junctions between the apical ends of the ordinary ependymal cells lining the brain ventricles. The blood-brain and CSF-brain barriers are viewed as means for regulating the internal environment of the CNS. However, there are locations in the brain specialized for sensing the contents of the blood and CSF that lack these barriers. The choroid plexuses form in specific locations in the wall of the developing CNS. In these places, the walls do not thicken, as they do elsewhere by filling up with neurons and glia; instead, they remain thin, consisting of a single layer of ependymal cells. Blood vessels pushing inward from the pia mater at these locations carry the pia and the thin layer of ependyma with them forming the complex coils and tubules of the choroid plexuses. The ependymal cells, the choroidal ependyma, then become specialized for secretion. At these locations, the capillaries in the pia adjacent to the choroidal ependymal cells lack tight junctions, and there is no blood-choroid plexus barrier. But, free access to the CSF is blocked by the tight junctions of the choroidal ependyma. Unmyelinated axons and conduction velocity

In both CNS and PNS, small diameter axons exist that are not myelinated (unmyelinated axons). In the CNS such axons do not possess any special covering, but in the PNS, they are far from being naked. They run singly or in groups partly buried in channels or grooves in the accompanying Schwann cells. In other words, there are non-myelinating as well as myelinating Schwann cells in peripheral nerve. Unmyelinated axons conduct action potentials relatively slowly compared to myelinated axons. This is related to at least two factors. The first is that unmyelinated axons usually are smaller in diameter, and the speed of action potential propagation increases with increasing diameter. Usually, axons are myelinated if they are larger in diameter than 1 µm, while smaller axons remain unmyelinated. Myelinated axons can have diameters as great as 20 µm, while unmyelinated axons can be a thin as 0.2 µm, a difference of two orders of magnitude. A second factor is that myelin segments increase the speed of propagation by extending the effective wave-length of the action potential, technically referred to as its length-constant. Other features, such as the presence of tight junctions in the paranodal region of the myelin and the concentration of voltage-gated sodium channels at the nodes of Ranvier, contribute to the salutatory character of action potential propagation in a myelinated axon. The range of conduction velocities extends from 120 m/sec in the largest myelinated axons to as little as 0.5 m/sec in the smallest unmyelinated axons. Gray Matter and White Matter, vs the Structure of Nerves and Ganglia Aggregations of functionally related autonomic (see below) or sensory neuronal cell bodies in the PNS are called ganglia, while in the CNS such a group of cells might be called a nucleus. In the CNS, such nuclei form part of the gray matter of the brain and spinal cord. CNS neurons also form into densely populated cortical sheets (layers) on the surface of the cerebral hemispheres and into loose networks of cells in the lower brainstem, collectively termed the reticular formation. In addition to neuronal cell bodies, the gray matter contains the dendrites and at least the proximal part of the cells’ axons, terminating axons, axons just passing through, and many glial cells. The white matter of the CNS is analogous to the nerves of the PNS in the sense that both consist entirely of axons (no neuron cell bodies).

Peripheral nerves-

Glial cells are located within both the gray and white matter of the CNS, as well as in the ganglia and nerves of the PNS. The axons within a PNS nerve are collected into compact bundles of axon fascicles. Each fascicle is contained within a sleeve of connective tissue called the perineurium. The connective tissue of the perineurial sleeves is predominantly cellular, consisting of a number of layers (depending on the diameter of the fascicle) of squamous mesothelial cells and some reinforcing collagen fibers. The ensleeved fascicles are collected together in a dense, more fibrous collagenous

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connective tissue, called the epineurium, that, in effect, is the nerve delineated in your gross dissections. Within each fascicle, the nerve fibers penetrate through a matrix of a fine connective tissue, termed the endoneurium, consisting of longitudinally arranged reticular fibers (type III collagen), fibroblasts, macrophages and blood vessels.

The Epithelial Nature of Nerve Fibers, Ganglia and the CNS Although the term, nerve fiber, is often used colloquially to mean axon, it technically refers to a specific constellation of structures in a peripheral nerve bounded by a tube of basement membrane. The nerve fiber consists of the basement membrane tube, the Schwann cells to which it is attached, and the one or more axons ensheathed by the Schwann cells. The tube of basement membrane runs along the entire length of the nerve fiber without interruption. It persists even at the neuromuscular junction of motor axons, where it separates the axon terminal from the muscle cell membrane. There are two types of nerve fibers, myelinated and unmyelinated. As discussed above, a myelinated fiber, seen in cross section consists of one axon surrounded by multiple spiral wrappings of the cytoplasm and membrane of one myelin-forming Schwann cell, and the axon is called a myelinated axon. An unmyelinated fiber seen in cross section, consists of several axons that run in grooves along the surface of a single normal-looking Schwann cell, and the axons nursed along this way are called unmyelinated axons. In both cases, when seen lengthwise, the Schwann cells are lined up within the fiber in serial order. One might say, concerning the nerve fiber as viewed in cross section, that the glial cell and neuronal component in a myelinated fiber stand in a one-to-one relationship, while the same relationship in an unmyelinated fiber is one-to-many.

The tube of thin basement membrane (visible only by EM) that separates the Schwann cell from (or attaches it to) the endoneurial connective tissue reminds us that neurons and glial cells are embryologically derived from the ectoderm, an epithelial tissue. You might imagine that if you follow the tube of basal lamina of a nerve fiber back toward the CNS, it would penetrate into the gray matter and eventually surround, for example, the motor neuron whose axon lies within that nerve fiber. Not so. Instead, the tube would spread out on contact with the surface of the CNS to become continuous with the basement membrane of the CNS. The CNS basement membrane separates it from (or attaches it to) its own connective tissue encasement, the pia matter. While it may appear odd at first, the brain and spinal cord together are enveloped by (attached to) a single basement membrane that is continuous everywhere under the pia mater and continuous with the basement membrane tubes extending out around each individual nerve fiber entering or leaving the CNS.

Please note that the endoneurial matrix through which the individual nerve fibers run while in the spinal (or cranial) nerve is continuous centrally with the connective tissue matrix containing the dorsal root ganglion cells and with the internal matrices of the dorsal and ventral roots. In the ganglion, it is still referred to as endoneurium, while in the roots it is referred to as pia mater.

In the CNS, glial cells and neurons are intermingled everywhere except at the internal

(ventricular) and external surfaces. The ependymoglia lining the ventricles are arranged in a tight-fitting sheet, as in a cuboidal epithelium, which, in most areas, does not leave room for any intervening neuronal processes. On the outer surface, the end-feet of astrocytes forms a compact layer against the basement membrane, termed the external glia limitans (aka. external glial limiting membrane, although it is not really a membrane). This glia limitans normally prevents contact of neuronal processes with the basement membrane. Thus glial cells, whether astrocytes or Schwann cells (and Muller glia in the retina), intervene between the local neuronal elements and the extracellular basement membrane.

The above analysis becomes a little more complicated by the vascular supply of the CNS.

Blood vessels penetrate the brain and spinal cord, which have rich capillary beds and take up a major portion of the cardiac output. However, the topological relationships described above are not fundamentally disturbed by the blood supply. The arterial supply enters from the pial (external) surface. As the blood vessel tunnels into the CNS, it carries a coating of pia mater and CNS basement

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membrane with it. The glia limitans inpockets to line the tunnel. The vessel contains its own tube of basement membrane lying just external to the endothelium. When, after branching extensively, the vessels narrow down to the capillary level, and the basement membrane of the endothelial lining of the capillary and the basement membrane of the CNS almost come into apposition but remain separated by a narrow extracellular compartment termed the perivascular space. This crevice contains a scattering of collagen fibers and connective tissue cells. Astrocyte end feet continue to surround the blood vessel and remain attached to the CNS basement membrane. The end feet in this location are referred to as the perivascular glia limitans. On their way out of the CNS, venules and veins follow the same pattern in reverse. Thus, while the CNS is extensively tunneled by branching blood vessels, the tunnels are lined by an uninterrupted sheet of basement membrane continuous with the basement membrane on the outer surface of the brain and spinal cord.

Nerve Injury, Degeneration and Regeneration Damage to a spinal nerve as a result of cutting or severe crush will cause a loss of function of those axons involved. For example, if a branch of a spinal nerve supplying a skeletal muscle is cut, that muscle will be paralyzed and no proprioceptive information will return from the muscle or its tendons. Sensation from the skin would also be abolished if the nerve contained cutaneous afferents. Furthermore, the loss of the sympathetic postganglionics will, at least initially, cause a reddening of the skin (loss of vasoconstriction), dryness (absence of sweating), and absence of any goose bump response to chilling (loss of pilo-erection).

After being cut, the part of the axon distal to the damage will decompose. This is called Wallerian degeneration. The degeneration of the axon and its terminals is the result of an interruption of anterograde axonal transport by which proteins synthesized in the cell body are normally carried into the axon and its branches. The part of axon proximal to the damage may survive as long as the cell body survives. Often, the neuronal cell body enlarges slightly and changes in appearance. The nucleus may shift to an eccentric position and the staining of Nissl bodies may become less intense. This reaction is referred to as retrograde chromatolysis. In motor neurons, the synaptic endings of its presynaptic supply may pull away from the postsynaptic membrane. These events can lead ultimately to the death of the cell. Cell death by this means is more common perinatally than in the adult, and more serious in some systems than in others. While it is sometimes referred to as retrograde degeneration, it should not be confused with Wallerian degeneration, which follows a rapid time course. In addition to neuronal changes, the skeletal muscle formerly innervated by that nerve branch will begin to atrophy unless rescued by a regeneration of its nerve supply. Atrophy and/or degeneration of the cells postsynaptic to cut axons is referred to as transneuronal or transsynaptic degeneration. Transneuronal degeneration is also more common in the young and in some systems more than others. In the PNS, unlike the CNS, regeneration of axons and re-innervation of targets is possible. This process occurs over a period of months. The degenerated distal part of the axon is resorbed by phagocytosis. After a crushing injury, the growing axon may be able to follow its original path to its target by growing out within the original basement membrane tube. But, if the nerve is grossly severed, its regenerating axons would likely end up in the wrong places by growing down the wrong tubes and/or growing outside the tubes in the endoneurial spaces. During axon regeneration chromatolysis persists because these changes in the cell body are correlated with the active production of protein for regrowth. When the axon of a motor neuron reaches its target, the muscle atrophy may reverse. The capacity for regeneration in the CNS is limited, and presents a challenging research problem.