Histology of Nervous Tissue

Histology of Nervous Tissue
Laboratories 9 and 10

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Required Reading: Ross and Romrell: Chapter 11, pp. 256-301
Gartner and Hiatt: Chapter 7, pp. 125-146

YOUR JOB FOR THESE TWO LABORATORY SESSIONS: Following your histology labs and readings regarding nervous tissue you should be able to (a) recognize nervous tissue in routine histological sections; (b) distinguish peripheral nerves from dense CT and smooth muscle; (c) recognize the morphological differences between myelinated and unmyelinated nerves at both the light microscopic and electron microscopic levels; (d) recognize nerve cell bodies and their component parts; (e) identify and differentiate dendrites and axons; (f) understand and identify various types of neuroglia, including Schwann cells; (g) understand and identify the structural relationship of the Schwann cell cytoplasm and plasma membrane enveloping axons; (h) understand the general features of nerve synapses. You should be able to draw nerves, cell bodies, Nodes of Ranvier, synapses etc. as they would appear under both the electron and light microscopes.

NERVE CELL BODIES (SOMA)

The neuronal cell body is the site where almost all of the metabolic and biosynthetic machinery is located. Depending upon the individual type of neuron, the cell bodies may be located very far from the synaptic terminal, where nerve transmission occurs. Although nerve cell bodies themselves vary greatly, we will use two types as examples: the cell bodies of large motor neurons in the CNS, and the cell bodies of dorsal root ganglionic cells.

Images to examine neuronal cell bodies.

F1: A low magnification cross-section of the spinal cord, showing the white and gray matter. Even at this low power the motor neuron cell bodies are visible in the ventral horns of the gray matter as large dark specks. Examine the white matter regions here as well for an idea of how masses of dendrites and axons look under the light microscope. Also, the meninges enveloping the entire nerve is clearly visible. On most of your slides you will not see this structure; why?

F2: A high magnification view of a motor neuron cell body. The large very euchromatic nucleus has a very prominent central nucleolus. Do not confuse a nerve cell nucleus for the entire cell body; many nerve cell nuclei are larger by far than most other cells. The beginnings of three dendrites and the one axon can be seen emanating from this cell body. Which is which? Find the axon hillock. What is the dark blue staining material and why is it present?

F3: A high magnification view of another motor neuron cell body, surrounded by a mass of axonal and dendritic processes. A shrinkage artifact has occurred during processing, allowing you to see three or more terminal buttons; synaptic endings still adhering to the plasma membrane of the cell body.

F4: A low magnification view of a dorsal root ganglion. Neuronal cell bodies are located in the more darkly stained regions, with their axons projecting out into the lighter areas of the tissue. The cell bodies are shown to better advantage on image F5:

F5: Identify the large clear nuclei with included nucleoli. Nissl substance is stained well, as are the nuclei of satellite cells. Examine also the regions containing axons. Find the peripheral located Schwann cell nuclei. These axons are myelinated and Nodes of Ranvier can be seen. Although we will examine these further on other images and slides, try to find some now. This image is a good introduction to the general appearance of bundled myelinated axons as well as neuronal cell soma.

F6: Another view of a dorsal root ganglion, not particularly well fixed, this image shows nicely the encapsulation (arrow) and the size of the satellite cells surrounding the neuron cell bodies. The empty space between the satellite cells and neuron cell bodies is not biological. The average neuron cell body here measures 60-75 um; this is huge, with respect to almost any other cell type. (Remember, though, to compare these with oocytes much later in the course!) The asterisk (*) indicates a tangle of myelinated nerve fibers. Compare these in a moment with the unmyelinated fibers that will be seen on image F9.

Image F6 is of a "sensory" dorsal root ganglion, while image F9 will be of an autonomic ganglion, at comparable magnification. Although they look alike a lot, compare them for criteria including (a) the size of the neuron cell bodies; (b) their encapsulation by satellite cells; (c) the presence or absence of included materials; (d) the appearance and arrangement of the nerve fibers (axons/dendrites).

(a) size of neuron cell soma - DRG has greater size variation
(b) nuclear position in cell - usually central in the DRG
(c) presence of binucleated cells - fewer in the DRG
(d) encapsulation - more complete in the DRG
(e) pigment inclusion - less pigment in the DRG
(f) nerve fiber arrangement - DRG has more obvious bundles of fibers and clusters of soma

F7: Cell bodies in an autonomic nerve ganglion from the ureter. Note the eccentric nuclei and relatively uniform soma size for the neurons. Note the relative sizes of the neuronal nuclei vs. glial cell nuclei. Compare the neuronal cell nuclei with the fibroblast nuclei seen in the surrounding dense CT. Note how the CT (here stained green) surrounds, but does NOT penetrate the nerve tissue. If this were a section through smooth muscle, instead of nerve, what would the CT look like? A smaller nerve is cross-sectioned to the left, near the blood vessels. Again, note the "insulating" nature of the nearby CT, which surrounds, but does not interdigitate. Question: any thoughts as to what the large empty spaces might be?

F8: A low magnification view of an autonomic ganglion. Note that here the cell bodies are scattered randomly among the nerve fibers, and are not layered as was seen in the sensory ganglion. The cell bodies here are also smaller. The nerve fibers also should appear finer, and more closely apposed (matted). They look more tangled. These nerve cells are multipolar and their processes are unmyelinated.

F9: A similar section, but at higher magnification. Here you can see little encapsulation of the cell bodies, and an average size of only 35-50 um. The dense reddish-brown material in the cytoplasm of some of the cells is lipofuchsin, a characteristic of autonomic ganglionic cells. Compare this image with F6 already examined. Flip back and forth between these two until you can tell the differences.

Glass slides on which to examine neuronal cell bodies.

Slide #40 - spinal cord: This is a cross-section of adult spinal cord, stained with Luxol fast blue-Cresyl violet. It illustrates the cell bodies of multipolar motor neurons very nicely.

Look at this slide first by holding it up to the light and examining it with your inverted ocular. Locate the lighter internal region, looking something like an "H" - this is the gray matter. The darker blue area on this slide, with this particular stain, is the white matter. The midline cleft is on the anterior surface of the spinal cord. Locate the cell bodies of the motor neurons in the ventral horns of the gray matter. Even with your inverted ocular they are visible as dark blue dots.

Examine the slide with the microscope. Locate the cell bodies with their important constituents: nucleus, nucleolus, Nissl substance, axon hillock, axon initial segment, dendrite. Not all nuclei will necessarily show all of the features, depending upon the plane of section. Compare the nerve cell nuclei with the nuclei of surrounding neuroglial cell nuclei. These latter nuclei will appear as small round or ovoid light purple nuclei and will be seen in both the gray and the white matter.

A canal in the grey matter crossing the midline is lined by ependymal cells which are poorly preserved.

Take some time to examine the white matter, composed mainly of axons. Most of these axons are ascending or descending and are passing parallel to the long axis of the spinal cord. How, then, are most of them sectioned? Can you tell whether these are myelinated or unmyelinated? How?

Slide #41 - adult spinal cord: Stained with silver, this shows the same multipolar neuron cell bodies as large orange-brown objects, slightly pulled away from the neighboring complexity of nerve fibers, the neuropil. Axons are stained golden brown or black here. Study the white matter and compare the relative appearance of the axonal fibers. You will see some small groups of longitudinally oriented fibers; these are fibers going into or exiting from the gray matter.

Slides 45 and 46 - adult spinal cord: Stained now with H & E, these slides show the same areas as just studied. Find the cell bodies and their components. Identify processes leading from the multipolar motor neurons as either axons or dendrites. Which is an axon hillock and which a dendrite? How are they distinguished morphologically? Compare the cell bodies seen in the dorsal horns of the gray matter with those seen in the ventral horns. Find neuroglial cell nuclei and compare them as well. Blood vessels are well preserved on these slides and may be seen to good advantage in the gray matter on slide #45.

Examine the appearance of the white matter, stained with H & E. Could you distinguish it from dense connective tissue? How?

Slide #52 - cerebrum: Use this slide to study the multipolar pyramidal cells. Orient yourself first with your ocular and at low power. The gray matter of the cerebrum is on the surface (cerebral cortex), with the white matter beneath it. On this slide the gray matter is stained more lightly. (The stain here is Luxol fast blue and cresyl violet again; the Luxol blue stains the myelin sheaths of axons deeply.)

Find the cortex and note that the outermost layer has relatively few cells. Looking progressively deeper, find some roughly pyramidal-shaped neuronal cell bodies. How are they oriented with respect to the surface? The largest dendrite begins at the cell apex and points toward the surface. Smaller dendrites arise basally and extend laterally. Identify such dendrites. The axon (one per cell, remember), begins at the base of the nucleus and goes toward the white matter; these will not usually be visible here. Are all of the pyramidal cell bodies equal size?

Study the pyramidal cell nuclei carefully. Compare them with motor cell neuron nuclei carefully. Compare them with motor cell neuron nuclei with regard to the amount of heterochromatin, size, nucleoli, etc. Compare the cytoplasmic Nissl substance. Take note of the smaller neuroglial cell nuclei.

Slide #51 - cerebellum: Use this to study the cell bodies of the multipolar Purkinje cells. DO NOT CONFUSE THESE WITH THE MUSCLE PURKINJE CELLS IN THE HEART.

Images to introduce glass slide #51:

F16 and F17:

Here the gray matter is located outside (the cerebellum), with the white matter lying in the middle of many infoldings. Could you have identified the gray matter vs. white matter without reading the preceding sentence? What would your criteria be? Locate the most superficial layer of the gray matter which has relatively few neuronal nuclei. Beneath this layer, find a layer having many small, round, darkly stained nuclei. These are granule cells, a type of small neuron whose cytoplasm cannot be seen here.

Purkinje cell nuclei are found at the juncture of the outer layer (few cells) and the inner layer (many cells). They have very large, flask-shaped cell bodies and stain deeply. They have numerous large apical dendrites that branch extensively into the outer layer. With the stain used here, the extensive branching is not obvious. Examine a lot of the Purkinje cells to appreciate their ramification. Examine the Nissl substance. The Purkinje axons extend from the base of the cell into the granule layer to enter the white matter. Axons here will probably not be visible.

Slide #47 - sympathetic ganglion (autonomic): Locate the cell bodies of the multipolar ganglionic neurons. Compare them with the neuroglial cell nuclei. Find those regions on the slide containing axonal processes. Compare the axon containing regions with the dense CT surrounding the entire ganglion. Can you recognize the differences? Talk about this with your partner or others.

Compare glass slide 47 with images F8/F9 and with:

Slide #42 - dorsal root ganglion: Here, find the pseudounipolar neurons and their cell bodies. Under low power, find the nerve fiber bundles running through and around the ganglion. Interspersed between the fibers, you should then be able to find the cell bodies, here very poorly preserved and quite shrunken. Nissl substance is present, but not well seen here. Observe the concentric placed satellite cells surrounding each neuronal nucleus. External to these, find the elongated nuclei of Schwann cells or cells of the neighboring CT.

COMPARE THE BUNDLES OF AXONS WITH THE SURROUNDING DENSE CT ON THIS SLIDE. LEARN THE HISTOLOGICAL DIFFERENCES.

Compare glass slide #42 with images F4/F5/F6.

HISTOLOGY OF PERIPHERAL NERVE FIBERS

Images for introduction.

F10: A low magnification view of a cross-sectioned nerve. The epineurium is shown by (*). Surrounding the entire nerve the epineurium is continuous with the perineurium (arrow) which divides the nerve into bundles or fascicles. One such fascicle is bracketed. The endoneurium surrounding each individual nerve fiber may also be seen in some areas of this slide. Blood vessels are indicated by (a).

This is myelinated nerve. The axons cut in cross-section appear as hollow tubes, with a central "dot" (see arrowheads). The "tubes" look hollow because the myelin sheath has been largely removed during fixation; the central "dot" is the axon itself. Would you expect to find Nodes of Ranvier visible on this slide? Why or why not?

F11: A higher power view of a rat sciatic (I think) nerve. No epineurium is seen here, but the perineurium is nicely stained. The cross-sectional appearance of myelinated axons is obvious. Note how well the perineurium surrounds ("insulates") the nerve bundle, but does not penetrate it. This is a key criterion for distinguishing peripheral nerve bundles from smooth muscle bundles under the light microscope.

F12: A longitudinal view of myelinated axons. Processed normally, this tissue has also lost most of the myelin of the sheaths, but the axons (a), Schwann membranes and plasma membrane (b) has been left visible. The arrowhead points to a well-defined Node of Ranvier. Find others. Do you understand what this structure is and could you draw it in simplified form at the ultrastructural level? To what cells do the nuclei seen on this image correspond?

F13: Use Figure 11-8 in Ross & Romrell (p. 265) for orientation.

A myelinated peripheral nerve bundle, teased apart and stained with osmium tetroxide. Osmium fixes and stains lipids; accordingly, the myelin sheath here is preserved. Although the central axon is often obscured, you may be able to detect it on some regions of these axons. Find Nodes of Ranvier. Can you distinguish these from accidental "cracks" in the axons? What would you look for in terms of biology versus artifact?

Can you find any incisures of Schmidt-Lantermann? What are these? Why are they there?

F14: An EM section of a node of Ranvier. Compare this with Fig. 11.12 in Ross & Romrell (p. 268). Identify the axon, neurofilaments, plasma membrane, Schwann cell processes etc.

F15: A longitudinal section of nerve, osmicated, showing nice incisures at the arrowheads.

Glass slides on which to study peripheral nerves.

Slide #43 - nerves: (1) Study the "wavy" appearance of the nerve. Although not a really good criterion, this sometimes helps distinguish nerve from smooth muscle and dense CT. (2) Observe the perineurium. Note the shrinkage artifact where the nerve bundles have pulled away, leaving a physiological empty space. This is a very useful diagnostic tool. Note the surrounding, but non-penetrating nature of the perineurium. (3) Compare the Schwann nuclei you see here with smooth muscle cell nuclei. Similar in appearance, note things like the average number of nuclei per unit area, whether nuclei are "cork-screwed", central versus peripheral location and so forth. Practice, practice.

Find some nodes of Ranvier. You may have to look around and look hard, but some are clearly seen here.

Slides #83/83A - artery and vein: An excellent slide to study cross-sectioned peripheral nerves, in conjunction with both smooth muscle and dense CT. Look at this slide first with your ocular for orientation. Find the two large blood vessels, then look to the side to find the cross-sectioned peripheral nerve bundles. Some longitudinal views may also be present on particular slides.

Examine the nerves carefully under the microscope. Are they myelinated? Find Schwann cell nuclei. Look carefully at the dense CT forming the perineurium. Many slides will show a shrinkage artifact; use this to your advantage. Does the perineurium penetrate the nerve fascicle?

Find smooth muscle in the walls of the blood vessels. Compare this closely with the peripheral nerves. Look at the nuclei, tissue density, staining characteristics, and relationship to surrounding CT.

Find the dense CT which holds everything else on the slide together. Compare the CT with both the nerves and the smooth muscle.

Slide #177 - seminal vesicle: Scan the edges of this tissue in the loose CT containing many fat cells. Find the numerous nerves enclosed by perineurium. Shrinkage is again obvious and helpful to you. Find blood vessels, identified by their RBC content, and compare their smooth muscle with the nerves. Then, scan the area closer to the epithelium, in the denser CT. You should be able to find small peripheral nerves here as well. Is the most darkly stained material nerve, smooth muscle or dense CT? How did you decide?

Slide #109 - esophagus: Now for a true test.

This slide will take some work, but will repay your efforts. Locate the layers of smooth muscle deep within the connective tissue. Locate small nerve bundles, some of which are associated with large ganglionic cells. Use these larger cell bodies as your identification key. There may be only 1-3 neuronal cell bodies in any immediate area, but they will be visible upon close examination. The axons here are unmyelinated.

This slide is a good one for practicing your skill at distinguishing the basic tissue types. Spend some time with it.

NEUROGLIA AND SPECIALIZED NERVOUS TISSUE

Introductory images.

F18: A variety of glial cells are shown. Oligodendrocytes (arrowhead) have round medium sized nuclei and may have a few short processes (oligo means few). Microglia (arrow) are smaller and often have an angular nucleus and fibers originating at opposite poles of the cell soma. Can you identify the structure curving into the field from the upper left? It is a blood capillary with several astrocyte processes attached.

F19: Protoplasmic astrocytes stained with gold. This stain adheres to cell plasma membranes and obscures internal cell structure. The cells shown here are protoplasmic astrocytes (arrow), found in gray matter. Astrocytes send many processes which entwine blood vessels; some are shown here by the arrowheads.

F20: A neuromuscular junction showing its characteristic "hand-like" structure under the light microscope. Refer to Fawcett's figures 10-27; 10-28; and Figures 10.7 10.8, 10.9 in Ross & Romrell for comparison.

F21: A Pacinian corpuscle. Think of a cross-sectioned onion and you will never forget this one.

F22: A Meissner's corpuscle. Seen bracketed, these are found unevenly distributed in the CT of the dermal papillae (*) of the integument. This slide is from thick skin. These structures function in mediating light touch and tactile feeling. The arrow shows the dendritic nerve fiber which spirals into a spindle-shaped structure in contact with the epidermis. The entire structure is ensheathed by a connective tissue capsule.

Glass slides for glia and specialized structures.

Slides 45 and 46: Find the neuroglial cell nuclei. Oligodendrocyte nuclei are round, stain more darkly and are smaller than the nuclei of astrocytes. They are found in both white and gray matter. In gray matter they often lie close to neuronal soma.Astrocyte nuclei are larger and more pale. They are round or oval and may exhibit folded nuclear outlines. If you see any amount of cytoplasm or if the nucleus is large, you are observing a small neuronal cell body, not an astrocyte.

Only about 5% of the neuroglia are microglia. Do not waste your time trying to find one.

Slide #54 - Meissner's corpuscle (odd numbered boxes) / Pacinian corpuscle (even numbered boxes)

Share these so that you get to see both of these important sensory structures.

FINAL PRACTICE: IMAGES TO EXAMINE

F23 and F24: Peripheral myelinated nerve at low and high magnification. Can you tell the nerve from the dense CT? How? How am I sure that the nerve is myelinated anyway? This is a very typical type of section you may expect to encounter.

F25: A section, Azan stained, showing oblique and cross-sectioned nerve. Here the endoneurium (a) and other CT is blue. The nerve fibers with associated remains of the myelin sheath are stained red. One fascicle (b) is cut mainly in cross-section. Do not depend upon this type of well-demarcated color staining for your own diagnoses, but this slide is a good example of how difficult it is sometimes to distinguish small nerves from small bundles of smooth muscle. In this course we will not expect you to make every fine distinction possible; rather we are interested in the general principles and relatively "normal" appearances.

F26: A peripheral nerve bundle surrounded by dense CT and located beneath WHAT at the top of the slide? Glandular material is shown at the bottom of the slide. Soon, you should be able to distinguish a nerve such as this from dense CT and smooth muscle routinely. Have you found the smooth muscle on THIS slide?

I would advise taking some slides at random from your boxes and looking for nerves. Not all slides of course will have them, but more of them than you think should show some peripheral nerves somewhere.