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Molecules and Cells 2011 Lab 5: Nervous System I. Peripheral Nerves and the Myelin Sheath A. Introduction In this laboratory we will examine nerves, neuronal cell bodies, and ganglia. Let us start with a few important definitions: Nerve fiber = multicellular, containing both an axon and surrounding myelin sheath. The axon comes from a single neuron, but the myelin sheath is made by a train of many myelinating Schwann cells. In the case of unmyelinated axons, the unmyelinated fiber shares each Schwann cell with several other unmyelinated axons. Nerve = a bundle or bundles of nerve fibers. Ganglion = clusters of neuron cell bodies in the peripheral and autonomic nervous systems, as well as associated glial cells and axons. Therefore, ganglia can be distinguished from peripheral nerves by the presence of neuron cell bodies. Axons are neuronal processes specialized for electrical impulse conduction. EMs show a cytoskeleton rich in microtubules (neurotubules) and intermediate filaments (neurofilaments). The organelles associated with protein synthesis are rare in axons, but abundant in neuronal cell bodies, where the membrane channels, ion pumps and synaptic machinery needed in axons and dendrites are synthesized. Axons may be long (spinal cord to foot) or short (many interneurons of CNS). Myelin is formed by Schwann cells in the PNS and produces segmental insulation interrupted between successive Schwann cells at the nodes of Ranvier, where ion channels and ion pumps are localized and support saltatory impulse conduction. 1 Molecules and Cells 2011 B. Slide Descriptions Webslides 0020_O and 0021_O: Sciatic nerve, monkey, cross-section and longitudinal section, TB-AF The sciatic nerve is a mixed nerve, containing sensory axons from neuron cell bodies in dorsal root ganglia and motor axons from neurons in spinal cord gray matter. Like all larger peripheral nerves bundles, it is also mixed in the sense of containing both somatic and autonomic nerve fibers. Scan the slide at low power to see the fascicular organization, identifying epineurium (dense connective tissue surrounding the nerve and filling in between nerve bundles or fascicles), perineurium (thin layers of flattened cells immediately surrounding and defining each fascicle), and endoneurium (fine connective tissue within each fascicle between nerve fibers--see your text for orientation). Around smaller fascicles, the epineurium becomes very thin, leaving the bundle surface visible as the circumferential wrapping of perineurial cells. Note that the perineurium gives nerve bundles sharply defined boundaries that makes them easy to distinguish from vessels, ducts, CT, and muscle fibers. The nuclei visible within each nerve bundle include crescent-oval Schwann cell nuclei snugly tangent to nerve fibers, and smaller, denser nuclei belonging to endoneurial fibroblasts or the endothelium of capillaries running through the endoneurium, usually parallel to nerve fibers. At higher magnification you can see the unstained rings representing individual myelin sheaths surrounding axons. Unmyelinated nerve fibers (which typically occupy at least 5% of a nerve's cross-section) can be visualized as axons without surrounding myelin sheaths. 2 Molecules and Cells 2011 Webslide 0014_O: Peripheral nerve, mammal, cross and longitudinal sections Here you see a nerve lying parallel to 1-2 medium-small blood vessels, cut from a block that presents both l.s. and c.s. views. Here myelin remains only as a pink network of residual protein. Although the endoneurium is very hard to spot, the epineurial CT and the perineurial wrapping are easily seen. Note in c.s., the axons in the center of many nerve fibers. Unmyelinated fibers are impossible to distinguish here, but you should be able to observe the nuclei of Schwann cells, endothelial cells, and endoneurial fibroblasts. Webslide 0012_O: Monkey ear Locate the peripheral nerve in association with other tissues in the bottom right quadrant and the upper right quadrant of the slide. What criteria are useful in identifying a peripheral nerve? Look for both myelinated and non-myelinated fibers. The nervous system, like the vascular system, is very pervasive. You will see it a lot in future slides. 3 Molecules and Cells 2011 II. Basic Cell Types of the Central Nervous System and Autonomic Nervous system A. Introduction In these slides, cell types can be best distinguished in specially stained slides, where a small number of cells and their processes are densely stained. However, the "empty spaces" in this material are deceptive--they contain unstained cell processes whose cell-cell relationships are poorly illustrated. Weblide #19 of this lab, on the other hand, contains practically no "empty space." It enables you to see the region of synaptic contacts (neuropil), but the appreciation of basic cellular shape and orientation is considerably more difficult. It is necessary to combine information from both kinds of preparations to get a feeling for the cellular configurations. B. Slide Descriptions Webslide 0302_O (cat): Spinal cord This section contains a cross section of the spinal cord. The tiny central canal recalls the origin of the nervous system as an infolding which closes off to form the neural tube. Locate the central canal in the middle of your section and examine the lining layer of ependymal cells. Compare the ependymal nuclei with the neuronal nuclei you observed in previous slides. Ependymal cells also line the ventricles of the brain that are continuous with the central canal of the spinal cord. During development, newly formed neurons proliferate adjacent to the canal to form a mantle layer, which becomes the gray matter in the central region of the mature spinal cord. This gray matter occupies a butterfly-shaped cross-section in this mature spinal cord. In the gray matter, examine the large motor neuron cell bodies carefully. Depending on how the cells are cut, you may see the nucleus, nucleolus, and axon hillock, as well as the Nissl bodies (rough ER) filling the cytoplasm. Note also the smaller nuclei of neuroglial cells. Neuropil refers to the regions in gray matter that lie between cell bodies, devoid of nuclei but complexly crowded with neuronal cell processes and synapses. The neurons of the mantle layer produce processes that grow outward to form the fiber tracts of white matter, which carry parallel myelinated axons for long-range interneuronal contacts in CNS. The white matter fills most of this cross-section external to the central butterfly-shaped region of gray matter. This marginal zone is white primarily because of the abundance of myelinated axons, which you can observe in your sections. 4 Molecules and Cells 2011 Webslide 0019A_O: Spinal cord, mammalian, silver stain Quickly repeat the observation of the spinal cord as described above, noting the differences that can be readily observed due to the silver staining of the neuropil. Also note the dorsal root ganglia on either side of the spinal cord that contain large neuronal cell bodies similar to those that you will observe in the next slide. Webslide 0018_O: Gasserian Ganglion, mammal, TB-AF The Gasserian ganglion (the sensory ganglion of the fifth cranial nerve) is a collection of pseudo-unipolar cells (pseudo because the single cell process arising from the soma splits into two functionally polarized processes). In response to tactile stimuli on the face a nerve impulse travels along the axon, passes through the ganglion containing the cell bodies, and finally reaches a synapse in the sensory nucleus of cranial nerve V in the brain stem. This is the first neuron (primary neuron) in a pathway whose activity will eventually result in your awareness of being touched. In this slide, you will see the large round cell bodies of these primary neurons, also called ganglion cells. The nuclei are large and stain faintly. The single nucleolus, if it happens to be in the plane of section, stains darkly, giving the nucleus a bull's eye appearance typical of many neurons. Around each neuronal soma are smaller flattened cells, whose darkly stained nuclei form a ring around the ganglion cells. These are specialized astroglial cells called satellite cells (not to be confused with satellite cells of skeletal muscle). In the same plane of section you will also see collections of light blue disks surrounded by unstained structures that look like white donuts. These are the axons and myelin sheaths of the ganglion cells passing through the plane of section. Look among the myelinated fibers for the wedge-shaped, densely staining nuclei of the Schwann cells, whose membranes form the myelin sheaths. Among the cells and fibers in the Gasserian ganglion you can observe capillaries, many still containing easily identified red blood cells. Note the connective tissue coverings of the ganglion. 5 Molecules and Cells 2011 Webslide 0032_O: Ileum, monkey, H & E In this section of the ileum, you will see autonomic ganglia where axons (whose cell bodies are in the spinal cord) synapse with intrinsic neurons (whose cell bodies are located here in the GI tract). These ganglia (called the Auerbach's or myenteric plexus) are found all along the GI tract between the circular and longitudinal layers of the outer smooth muscle. Like other parts of the peripheral nervous system, these ganglia are covered by a thin connective tissue layer, essentially a perineurium. Like CNS, but unlike other (non-enteric) autonomic ganglia, these enteric ganglia exclude connective tissue, and contain only neurons and glial (supporting) cells. Look for the typical neuronal bull's eye nuclei. Neurons are smaller and glial supporting cells fewer in autonomic ganglia than in sensory ganglia, so they rarely appear as obvious rings of satellite cells like those seen in Webslide #18. A good example of a neuron in an Auerbach’s plexus can be found using overlay D. (as a review, other identifications: A- simple columnar epithelium with microvilli, B- mast cell, and C- simple squamous epithelium) 6