Objectives:

Before doing the lab, read Appendix II. on unknown slide identification. As you go through the slides, be cognizant of those features that are identifiable at 4X, 10X and 40X. Be able to name those features and connect them with particular organs.

There are 5 nervous system "organs" that you should be able to identify if presented in a practical exam: peripheral nerve, ganglia (three types: dorsal root, sympathetic, parasympathetic), spinal cord, cerebellum, cerebrum. The level of details required in the unknown identification is summarized in Appendix 3. You should also be able to identify the two encapsulated receptors, Pacinian and Meissner corpuscles and Auerbach's (myenteric) plexus in the GI tract.

Before examining the slides, read the Differential Diagnosis that is provided before each tissue and organ and as you examine the slide, try to connect the visual impression with the name of the structure. This will help you in the practical.
 
 

 A. Peripheral nerves.

Examine first the slides of nerve bundles or fascicles. This will make it easier to look at the ganglia slides, which also have nerve fibers in them as well as neurons. The structures that you are looking for include: myelinated axons, perineurium, epineurium, vascularization and Schwann cells. Axons sectioned transversely have a "donut" appearance.

1. Encapsulated structure filled with numerous circular fibers, "donut shaped" characteristic of axons in cross section, wavy fibers in longitudinal section.

2. No large cell bodies characteristic of neurons.

3. May show several layers of encapsulation - the perineurium and epineurium.

4. Nuclei associated with the fibers - Schwann cells

In this slide collagen fibers are stained a brilliant turquoise blue, nuclei stain brown and smooth muscle fibers can be found also with a brownish color, not as intense as the nuclei. In some slides, there is a large vein in the upper left. If you can identify it will demonstrate the stained appearance of smooth muscle in the section. Adipose tissue is also in and around the epineurium. These nerves are surrounded with a perineurium and epineurium. There is a distinct staining difference between the perineurium and epineurium so that you should be able to see both. Some shrinkage has pulled the nerve tissue away form the perineurium in places. Myelinated axons have a donut shape, easily identified in this slide.
 

40a Myelinatedted nerve c.s & l.s.

This slide has both a longitudinal and a cross section through a nerve bundle of myelinated axons. In cross section, the donut profile can be seen in many of the axons. In longitudinal section, there is not too much to be seen. The Schmidt-Lanterman clefts are difficult to find. Each nerve is surrounded by a connective tissue sheath which is easy to identify. The sheath is thin enough to be just a perineurium, but you may on careful examination detect two parts to it, which would indicate both a perineurium and an epineurium. What characteristic feature is present in the epineurium that would not likely be seen in the perineurium. Each nerve fascicle has at least one very visible blood vessel which you should be able to find. Cell types you should be able to find: Schwann-cells, endothelium, smooth muscle cells, fibroblasts, adipocyte.
 

B. Ganglia of the PNS

After examining the slides of peripheral nerve, examine the slides of the two types of ganglia: sensory (spinal)  and vegetative (sympathetic or parasympathetic). You will have an additional chance of examining a dorsal root ganglia later in the lab when you look at the spinal cord. The essential features that differentiate sensory ganglia from autonomic ganglia are the size and shape of the neurons and completeness of the layer of satellite cells as well as the clustering (nesting) of the neurons and the position of the cell nucleus. The distribution of neurons with respect to fibers is perhaps the most difficult aspect to appreciate. The histological differences in summary are:

1. Encapsulated structure containing neurons and nerve fibers
2. If sensory ganglia, i.e. dorsal root and trigeminal (Gasserian) ggll.

a. Neurons (pseudounipolar) arranged in nests separated by bundles of nerve fibers
b. Neurons surrounded by numerous satellite cells.
c. Spherical/symmetrical neuron shape and centrally located nucleus.

3. If parasympathetic or sympathetic ganglia

a. Neurons (multipolar) intermingled with nerve fibers more or less randomly
b. Some neurons may display more than one cell process
c. Relatively fewer satellite cells surround neurons
d. Asymmetric neuron shape and eccentricity located nucleus

38b Spinal ganglion sec

Some of you may remember this slide from the first lab. The perineurium may be preserved on only one side of the ganglion. Neurons, satellite cells, fibroblasts and Schwann cells are easily identified. Vasculature, which must be there is not so easy to find.

35 Sympathetic ganglion

This slide shows an excellent perineurium with many ganglion cells and satellite cells. Some of these slides are better than others. There are about 6 different cell types that can be identified in this slide without too much difficulty: ganglia, satellite cells, Schwann cells, endothelial cells, smooth muscle cells, RBCs and fibrocytes. Can you find all of these?
 

14 Parasympathetic ganglia LFB PAS, human anatomy

This slide has two ganglia separated by connective tissue which also contains several peripheral nerve bundles and blood vessels. Peripheral nerves often run parallel to vasculature. Pay particular attention to the distribution of neurons, the completeness of the satellite cells, the position of the neuron cell nucleus.
 
 
 

42b Auerbach’s plexus (myenteric), human 

42a Auerbach’s plexus, intestine sec.

One of these slides is different from the other 3. The "odd " slide, of colon, is actually better and is shown above. The other slides are cross sections of jejunum and unfortunately Auerbach's plexus will be found at the periphery of the section where the stain has faded. For a review, check out the luminal epithelium, which is nicely preserved and well stained. What kind of epithelium is it? What is the name of the CT underneath it?
 

C. Receptors of the PNS
 

41 Pacinian corpuscle sec

This is a section of pancreas in which some Pacinian corpuscles can be located. They have an "sliced onion" appearance that is quite distinctive. Pacinians are deep pressure sensors. Why would they be located in the pancreas? There are also some peripheral nerve bundles in some sections. Can you find them? This is a good slide to review epithelia because there are some small and large ducts with cuboidal and columnar epithelia. Can you locate dense irregular connective tissue?
 

70 Meissner’s corpuscles, sec.
64b Plantar skin, human, H&E

This is an H&E stained section of palmar (or plantar) skin, i.e. thick skin from the palm of your hand (or sole of the foot). Up between the dermal papillae you will find structures that look like "tornadoes". These are the Meissner’s corpuscles. What are Meissner’s corpuscles supposed to detect? Not all slides of 64b show the corpuscles.
 
 

There are 3 organs of the central nervous system that you are expected to recognize: spinal cord, cerebellum and cerebrum. All of these have very distinctive arrangements of neurons and fibers so that it is unlikely that anyone will mistake them. However, the description of the histology is rather more complicated. As you look at the slides, try and associate the visual features with the names of the specific structures keeping in mind at the differential diagnosis.

A. Spinal Cord

1. Gray /white matter division, gray matter in the center, white matter on the surface.
2. Gray matter contains neurons, white matter generally free of neurons, but contains abundant nerve fibers.
3. Gray matter, if a cross section, arranged in the shape of a butterfly
4. Central canal lined with columnar cells - the ependymal cells
5. Pronounced cleft marks ventral surface of the spinal cord: ventral fissure. It is covered by the pia mater, and superficial to the pia there are blood vessels associated with it (anterior spinal artery in the subarachnoid space) ,
6. Bundles of nerve fibers may be present and exterior to spinal cord - dorsal/posterior and ventral/anterior roots.
7. Ganglia may be present - the dorsal root ganglia
8. Dense connective tissue capsule may be present - the dura mater

38a Spinal cord and dorsal root ganglion

This is a transverse section through the spinal cord with a pair of spinal ganglia connected by their dorsal roots. Gray matter occupies the butterfly shaped, darker staining region in the center. The central canal lined with "ependymal" cells is located in the middle. The gray matter can be distinguished easily from white matter by of the presence of numerous neurons. These neurons often have a very asymmetric shape. In the attached dorsal root ganglion, the neurons are spherical and interrupted by nerve fibers (identifiable by the red stain surrounded by blue endoneurium). The ganglion is cut obliquely thereby giving the nerve fibers a speckled red appearance. The perineurium has been rather badly treated in most areas. The arrangement of satellite cells around the ganglia cells is quite dense so you should have no problem identifying them. In the white matter, the shrunken appearance of the myelinated axons is due to poor fixation and extraction during preparation. The dark dot in the center is what is left of the axon cytoplasm. Each of the ganglia are attached by their dorsal roots. Look on the opposite side for the ventral fissure and some ventral roots. Pia meter surrounds the ventral fissure and the ventral roots. The presence of blood vessels will help you to identify the pia mater.
 

37a Spinal cord

This is a visually stunning slide. The stain is silver impregnation according to Cajal, which reveals the extremely complex connections between neurons. A very distinct epineurium surrounds the entire tissue, the dura mater. There is no material outside of this epineurium. At the ventral fold, the pia mater is distinct. Blood vessels can be identified by the erythrocytes that are often present. Small pieces of dorsal roots are visible at the top.
 
 

37b Spinal cord, human, c.s.
This is a slightly less exciting version of 37a  but displays the same information. The slide is apparently silver impregnated. There is no epineurium/dura mater surrounding the spinal cord, but there are numerous ventral roots. Regions of fairly dense connective tissue can be identified by their reddish-brown appearance.

39 Nissl substrance, neurons
We looked at this slide in the first lab. Now go back and look at it, with the idea of understanding what Nissl substance is and how it is arranged in the neuron. Nissl substance should appear as dark blue granules in the cytoplasm but not in the axon hillock.
 

B. Cerebellum

1. Extensively folded (foliae and sulci) , layered structure
2. Outer layer - the molecular layer - contains relatively fewer cells than next layer deep to it
3. Second is formed by large neurons - Purkinje cells
4. Third layer - the granular layer - has numerous cell bodies
5. Deepest layer contains axons of Purkinje cells and afferent (mossy and climbing ) fibers and is relatively featureless - the white matter.
6. Molecular layers of adjacent foliae are separated by a delicate connective tissue layer with numerous blood vessels - the pia mater

43a Cerebellum, rat, Toluidine blue

This slide is a (paramedian) sagittal section of the rat the brain stem and cerebellum.
The most prominent feature of this slide is an esthetically pleasing, arborized structure, the cerebellum consisting of foliae and sulci. Early anatomists called this pattern of arborization in the cerebellum "arbor vitae" (Latin, tree of life). Examine this part of the slide while looking at page 299 of your RR&K. There is an excellent description of the different layers of the cerebellum.

Ventral to the cerebellum you will find the 4^th ventricle of the brain, lined by ependymal cells and containing the choroid plexus of the 4^th ventricle. The choroid plexuses , located in all brain ventricles, produce the cerebrospinal fluid (CSF) filling the brain ventricles and the subarachnoid space. The choroid plexus is a heavily folded structure consisting of 3 main components :

lamina epithelialis [cuboidal epithel of ectodermal origin, similar to ependyme]

pia mater [CT]

vessels from the subarachnoid space

C. Cerebrum (neocortex)
 

1. Gray and white matter division - cortex =gray matter - contains neurons, medulla - white matter - has no neurons, contains mainly axons.
2. Cortical neurons arranged in 6 layers
 

44a V. cortex, LFB-PAS, human

Your atlas describes the cortex as consisting of 6 layers between the surface and the underlying white matter. In this slide, you can see all 6 layers of the gray matter as well as the white matter. This slide is just like it is described in the atlas. The layers are best identified at 4X. You should be able to identify some of the different cell types, i.e. pyramidal cells & neuroglial cells in the cortex. The stain is luxol fast blue which stains myelin green/blue and periodic acid Schiff which stains glycogen, starch and cellulose red.
 

44c Cerebral cortex, Human
I believe that you should be able to find at least some of the 6 layers even though they are not as distinct as in the previous slide. Find a region where you can see both white matter and the surface. Layer I at the surface is easy and its boundary with layer 2 is pretty distinct. Layer 3 is also pretty clear because it seems to have fewer large cells. The boundary between layer 3 and layer 4 is distinct but layers 5 and 6 cannot be distinguished from layer 4. The area of white matter is easy to see because of the lack of neurons.
 

44b Cerebrum, silver impregn, human
This is just another representation of cerebrum, but the silver gives a different impression. You should be able to determine the boundary between gray matter and white matter and at least some of the same layers that you looked for in the previous slide.
 

46 Medulla oblongata, silver impregn, c.s.
This slide is completely optional. You will not be held responsible for it. Its organization is entirely different form any of the other CNS slides that you have been looking at.
 
 

Note that the items listed do not form an all inclusive list, just examples of the types of features to look for at the different magnifications.