Prelab Exercise 3 – NERVE & CIRCULATION

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Prelab Exercise 3

NERVE TISSUE Nerve tissue, particularly that comprising the central nervous system (CNS, i.e., brain and spinal cord) is amazingly complex in organization as well as function. Therefore, only a few selected regions will be examined in this lab (they will be investigated in more detail later, in the neuroscience course). In this laboratory session you will also have time to study examples of nerve tissue from the peripheral nervous system (PNS). Next to the blood vascular system, nerve tissue is the most widespread in its distribution throughout the body and is the most highly specialized for electrical excitability, conductivity and synaptic transmission. The cell of major interest in this tissue is the neuron (or nerve cell), though it is not the most numerous component of the tissue. Supporting cells, neuroglia, outnumber it at least ten to one in the CNS and many times that along peripheral nerve fibers. The neuron consists of a cell body (soma or perikaryon) and all of its processes (one axon and usually many dendrites). It is the most important structural and functional unit of the nervous system.

STAINS Various types of stains emphasize different neuronal structures. The most commonly used stains for neural tissue are Nissl stains: hematoxylin, thionin, cresyl violet, methylene blue, gallocyanin. These basic dyes react preferentially with acidic components in tissue sections such as DNA, RNA, chromatin, ribonucleoprotein and phospholipids. In neural tissue this results in staining of chromatin in the cell nuclei, the nucleolus and the Nissl bodies (ribosomes). Processes of both nerve cells (axons and dendrites) and glial cells are almost totally unstained by these Nissl stains. In the CNS, there is a light-staining meshwork of these neural processes along with the processes of glial cells. Together this is called the neuropil. The staining of neuronal processes may be accomplished by the staining of neurofibrillar components through silver or other metallic stains, or by immunohistochemical means. Axonal processes (particularly myelinated ones) may be visualized by staining of myelin by, for instance, sudan or osmic acid. The Bodian method stains neuronal processes, however it may not be optimal for observing the configuration of cell processes because it stains most of the nerve cell processes in a section, making it difficult to see the tree for the forest (or to follow the processes of one neuron). There are, however, certain modified metallic stains impregnate only 1-2% of the cells in a section along with their processes, thus allowing visualization of the configuration of nerve cell processes of a single neuorn. These are the Golgi stains which impregnate tissue by reduced metallic salts such as silver, mercury, chromium, or tungsten. These methods are notoriously capricious and time-consuming and their mode of action is still not understood. They also result in some random deposition of salts, which can complicate visualization of the stained neurons and their processes.

CENTRAL NERVOUS SYSTEM

Spinal Cord

The spinal cord is roughly cylindrical in shape in cross section and is composed of an inner butterfly or H-shaped core of gray matter surrounded by an outer cortex of white matter. In general, gray matter consists mainly of nerve cell bodies and the proximal portions of their processes as well as neuroglia. The white matter consists mainly of myelinated nerve fibers also with supporting neuroglia.

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Transverse section of spinal cord at mid-lumbar level (Weigert stain for myelin)

Gray matter. Note the anterior (ventral) and posterior (dorsal) horns of gray matter, united by a thin gray commissure containing the small ependymal-lined central canal. Observe the large motor neurons (nerve cells) in the anterior horn. Many of these cells contain darkly stained neurofibrils. The neurons are also partially outlined by darkly stained nerve cell processes. Aside from the neurons and their processes the remainder of the gray matter substance consists of supporting neuroglia cells and numerous small blood vessels (both poorly stained). Keep in mind that the amount of gray matter at any level of the spinal cord is related to the amount of the body that is innervated at that level. Thus, the amount is greatest within the cervical and lumbosacral enlargements of the cord, which innervate the upper and lower limbs respectively. White matter. Note the separation of spinal cord white matter into posterior (dorsal), lateral and anterior (ventral) columns. As in other regions of the CNS, the white matter consists here of a mixture of nerve fibers (myelinated and unmyelinated), neuroglial cells, and blood vessels. Nerve cell bodies are usually absent from white matter. Many of the nerve fibers (axons) are myelinated which accounts for the white color of white matter (in fresh preparations) and the intense dark staining in stains that stain myelin or the axons themselves.

Cerebral cortex As opposed to the gray matter of the spinal cord that is on the inside, The gray matter of the cerebral and cerebellar cortex is on the surface. The purpose of examining the following slides is to introduce you to the highly complicated organization and microscopic structure of the higher brain centers. You are not required to learn these features in detail at this time, though you should be able to distinguish cerebrum from cerebellum and be able to distinguish the 3 main layers of the cerebellar cortex. Additional detail will come later in your neuroscience course.

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The pia mater and the next meningeal layer, the arachnoid have the same embryonic derivation and provide the boundaries of the subarachnoid space (which is normally filled with cerebrospinal fluid). These are often considered together as the pia-arachnoid (leptomeningies). [See diagram above]. These two meningeal layers are joined by cobweb like trabecular processes. (Hence, name arachnoid, spider). CSF circulates in the spaces between the trabeculae (subarachnoid space). The inner surface of the pia forms a firm interface with a special layer of brain glial cells to form a pial-glial (external limiting) membrane. The large blood vessels of the brain (arteries and veins) are located between the pia and arachnoid, lying within the subarachnoid spaces. The pia and arachnoid themselves, despite being intimately related with the blood vessels, are avasular. As blood vessels penetrate the brain they carry with them pia-glia tissues, which form a cuff around the vessel. At the capillary level, only processes of glial cells (astrocytes and oligodendroglia) surround the basement membrane of the capillary endothelium. The capillaries of the CNS are distinctive structurally and functionally. Review the concept of the blood-brain barrier.

Now study the gray matter, first with the 4x, then with 10x and 40x objectives. You will see many basophilic nuclei and occasional capillaries in a field of granular eosinophilic material. Although it cannot be appreciated in this preparation the eosinophilic material consists of an amazing network of the many processes of nerve and glial cells. It is sometimes called the neuropil. Review the ultrastructure of neural tissue in the “EM of Neural Tissue” module on the virtual histology site. The largest nuclei, usually with a prominent nucleolus belong to nerve cells (neurons). Except for the largest neurons, giant pyramidal cells, not much of the surrounding cytoplasm (perikaryon, cell bodies, soma) will be seen. The more numerous smaller nuclei mostly belong to glial and capillary endothelial cells. (Recall that glial cells outnumber nerve cells by about 10 to 1 in the CNS). The neurons of the mammalian cerebral cortex are not randomly distributed. Although it may not be readily apparent to you from looking at this section, it has been shown that they are arranged in six layers or laminae parallel to the brain surface (see the figure below). It is not necessary at this time that you know the names and features of these layers. Do appreciate, however, that the stratification you see in the sections does indeed correspond to different population of cells and their processes.

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ORGANIZATION OF THE HUMAN CEREBRAL CORTEX

(NEOCORTEX)

The centrally located white matter contains no neuronal cell bodies. It consists mostly of myelinated nerve fibers (going to or from the gray matter neurons), supportive glial cells (astrocytes and oligodendrocytes), and blood vessels. The gray matter can be divided into 6 layers, which vary depending on the part of the cortex in question (you are not responsible for knowing the function of the layers at this point). The excitatory cells are mostly of pyramidal shape, while there are many small, inhibitory neurons of various shapes mixed in. Of course, as in all o the CNS there are at least 10 times as many glial cells as there are neurons.

Cerebellar Cortex The cerebellum (“little brain”) lies on the brain stem, largely hidden by the massive cerebral hemispheres. It is mainly concerned with the integration and coordination of complex motor activities and procedural learning (such as learning to ride a bicycle). Like the cerebral cortex, the cerebellar cortex has a complex organization related to its functional activities. The gray matter can divided into three identifiable layers (see diagrams below). The superficial, outermost layer is the molecular layer. It contains mainly dendrites and axons from cells in the deeper layers but only a few nerve cell bodies. The middle layer is dominated by the cell bodies of peculiar giant nerve cells, Purkinje cells, arranged in a single layer of cells and is called, logically enough, the Purkinje cell layer. The deepest layer, next to the white matter, is called the granular layer because it is packed with the cell bodies of tiny neurons (granule cells), the most numerous cells in the nervous system. The Purkinje cells are the most interesting cells. Not only are they huge (estimate the diameter of the cell body) but they have fantastic candelabra like dendritic trees that ramify in single plane in the molecular layer. These processes can not be seen by this staining method, indicating why the study of neural tissues require sophisticated methods that focus on the items that you need to visualize.

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Ultrastructure of the CNS

Examine the ultrastructure of the CNS in figs. 1-8 of the “EM of neural tissue” module virtual histology. PERIPHERAL NERVOUS SYSTEM

The peripheral nervous system is composed of all the neural tissue outside the brain and spinal cord. It serves as the mediator of neural impulses traveling in both directions between the CNS and body tissues. The entire system consists of ganglia, peripheral nerves and their terminations. A peripheral nerve is defined as a group of nerve cell processes, their supporting elements (neuroglia), and their terminations, all of which are bound together by connective tissue and are located outside the CNS. A ganglion consists of a collection of nerve cell bodies and their associated neuroglial tissue that is located outside the CNS. There are two types of motor (efferent) axons in peripheral nerve. Somatic motor axons (to skeletal muscle) end as motor end plates. Visceral (autonomic) motor axons end as terminals on myoepithelial cells, gland cells, or smooth muscle cells. These visceral motor axons often form plexi within or near organs. Afferent (sensory) nerve fibers have sense organs at their distal ends that transduce stimuli. These sense organs may consist of specialized receptor cells or may be specializations of the nerve fiber itself. This transduction converts various forms of energy into action potentials. The forms of energy converted by receptors include: mechanical (touch and pressure); thermal (degrees of warmth); electromagnetic (light); and chemical energy (odor, taste and CO2 and O2 content of blood). The receptors in each of the sense organs are adapted to respond to one particular form of energy at a much lower threshold than other receptors respond to this form of energy.

NERVE CELLS (NEURONS)

Multipolar neurons – Most neurons are multipolar. These cells typically have a coarsely granular cytoplasm (due to Nissl substance), and a centrally-placed, pale staining vesicular nucleus with a prominent nucleolus. Under the electron microscope, Nissl substance corresponds with areas of rough endoplasmic reticulum and polyribosomes. The Nissl substance of these cells extends into the base of the dendritic processes. Nissl substance is lacking in the region of the cells that gives origin to the single axon process, a region known as the axon hillock. Generally, the larger the neuron, the larger the processes (and longer the axon).

Ganglion Cells - Ganglia in the peripheral nerve tissue contain neurons of two types. Sensory ganglia have pseudounipolar neurons, while autonomic ganglia have multipolar neurons.

Dorsal (sensory) root ganglion cells are pseudounipolar cells. They are globular in shape and vary tremendously in size. The small cells have unmyelinated processes and the large cells myelinated processes. A thin cellular capsule, comprised of satellite or capsule cells, surrounds the cell bodies. These cells are continuous with the neurolemmal sheaths of the cell processes. The stem process (primary neurite) bifurcates in a T or Y fashion into two secondary processes: a peripheral process (“dendrite”) which extends to some sensory receptor and a central process (“axon”) which enters the spinal cord to synapse with neurons in the gray matter.

Representation of a somatic nerve and a reflex arc. (note connective tissue investments)

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Autonomic Ganglia

The autonomic nervous system is a motor system that is involved in innervating smooth muscle, heart muscle and glands. With the exceptions of the adrenal medulla, autonomic pathways consist of a two-neuron chain (preganglionic and postganglionic neurons) extending from the CNS to the peripheral structure. It is classified according to the cells of origin in the CNS as the sympathetic (thoracolumbar) or parasympathetic (craniosacral) division. Because it is a two-neuron pathway, there must be a synapse, which occurs in a ganglion, which is also the location of the postganglionic cell body. Sympathetic ganglia are located adjacent to the vertebrae (paravertebral ganglia; sympathetic chain of ganglia) or along the aorta (prevertebral ganglia). These can be seen in gross anatomy. Cells in these ganglia, which are surrounded by satellite cells, send long postganglionic processes to the organ supplied. The small parasympathetic ganglia are either very near or actually inside the organ supplied. These include the ciliary, otic, sphenopalatine, or submaxillary ganglia in the head or ganglia in the wall of organs such as the myenteric plexus (Auerbach) or submucosal plexus (Meissner) plexi of the GI tract. Hence, parasympathetic neurons have short postganglionic processes.

PERIPHERAL NERVE TRUNKS

In looking at sections of mixed peripheral nerves remember that you are looking at the peripheral processes of nerve cells (axons and dendrites), neuroglial supporting elements (Schwann cells), connective tissue and associated small blood vessels. Most of the fibers that you can see in tissues are myelinated. However, there are many more unmyelinated fibers that can’t be seen without special staining. The different sizes of axons reflect different speeds of conduction and different functions. Peripheral nervous system myelin consists of the wrapping of a sequential chain of individual Schwann cells around the axon, with a tiny gap (the node of Ranvier) in between. This is where the axon is exposed to the interstitial environment and is a site of action potential generation. Examine the structure of myelin on the “EM of neural tissue” section of the virtual histology site.

CHECK LIST

Identify the constituents of neurons, including: (both light and electron microscope level) -cell body (perikaryon) -nucleus and nucleolus -axon hillock -Nissl bodies -dendrites and axons -synapse -myelin sheath

Be able to identify the four neuroglial cells of the CNS and the two neuroglial cells of the PNS: -microglia -ependymal -astrocytes -oligodendrocytes -Schwann -satellite (capsule)

Identify these components of the CNS: -white matter -gray matter -motor neurons -spinal cord -blood-brain barrier -cerebral cortex - pyramidal cells -meninges

-dura mater -arachnoid -pia mater

-cerebellar cortex -molecular layer

-Purkinje cells -granular layer

Identify these components of the PNS: -ganglion, both sympathetic and parasympathetic -connective tissue sheaths of peripheral nerves

-epineurium -perineurium -endoneurium -myelin sheath -nodes of Ranvier and internodal segments Review: -neuromuscular junctions -proprioceptive muscle spindles -Nissl stains

-Silver stains -Golgi stains

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CIRCULATORY SYSTEM Arteries and veins are classified according to their size and the histology of their walls. In each case, one should analyze thoroughly the tissue composition of each of the three tunicas: intima, media, and adventitia. The histology of the media and adventitia is particularly important in determining the type of vessel. Although vessels are subdivided into categories on the basis of size and composition of the wall, there are no abrupt boundaries between categories. Rather, the histology changes gradually from the larger to the smaller vessels (see summary table, below).

ARTERIES – there are several types of arteries: elastic arteries; muscular ateries and arterioles.

Large conducting (“elastic”) arteries have a

relatively thin intima (endothelium and thin layer of CT). Also, smooth muscle cells interdigitate

with prominent lamellae of elastic fibers in the thicker media. The adventitia also contains elastic fibers along with dense collagen fibers and is thinner (relatively speaking) than in muscular arteries. There are also vasa vasorum and nervi vascularis in the adventitia.

Medium-size, distributing (“muscular”) arteries. In general, the muscular arteries correspond to the smaller named arteries, which you dissect in gross anatomy lab. The intima and internal elastic lamina are well-developed. The media has a predominance of smooth muscle, with sparse elastic fibers. The adventitia is relatively thicker than in the elastic arteries (see the figure below).

Arterioles. Arterioles contain only a few layers of smooth

muscle in the media and are quite small (40-200 µm diameter). These vessels provide much of the peripheral resistance to blood flow and therefore are involved in the regulation of blood flow and pressure.

CAPILLARIES Capillaries consist of an endothelium with a basement membrane. They are specialized to permit (and in some cases restrict) the passage of things between the blood stream and the interstitial spaces. Review the fine structure of the three types of capillaries (continuous, fenestrated, and discontinuous). Especially note how capillary structure is adapted to the physiologic requirements of each organ. For example, compare the capillaries of the brain to those of the liver.

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Types of microcirculation formed by small blood vessels. (1) The usual sequence of arteriole –> metarteriole –> capillary –> venule and vein. (2) An arteriovenous anastomosis. (3) An arterial portal system, as is present in the kidney glomerulus. (4) A venous portal system, with a second capillary bed, is present in the liver.

VEINS

Veins have larger lumens, much less smooth muscle, a thinner intima (without internal elastic lamina) and a thick connective tissue adventitia. Valves appear at intervals through the venous system.

Diagram comparing the structure of a muscular artery (left) and accompanying vein (right). Note that the tunica intima and the tunica media are highly developed in the artery but not in the vein.

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CHARACTERISTICS OF BLOOD VESSELS

ARTERIES VESSEL DIAMETER INNER LAYER MIDDLE LAYER OUTER LAYER (TUNICA INTIMA) (TUNICA MEDIA) (TUNICA ADVENTITIA) Elastic artery > 1 cm Endothelium Smooth muscle Connective tissue Connective tissue Elastic lamellae Elastic fibers Thinner than media Muscular artery 2 - 10 mm Endothelium Smooth muscle Connective tissue Large Connective tissue Collagen fibers Some elastic fibers Prominent internal Relatively little Thinner than media elastic lamina elastic tissue Ext. elastic lamina Small 0.1 - 2 mm Endothelium Smooth muscle Connective tissue Connective tissue (8-10 cell layers) Some elastic fibers Internal elastic Collagen fibers Thinner than media lamina Arteriole 40-200 µm Endothelium Smooth muscle Thin, ill-defined sheath Connective tissue (1-2 cell layers) of connective tissue Capillary 4 - 10 µm Endothelium None None

VEINS VESSEL DIAMETER INNER LAYER MIDDLE LAYER OUTER LAYER (TUNICA INTIMA) (TUNICA MEDIA) (TUNICA ADVENTITIA) Venule 50 - 100 µm Endothelium Smooth muscle Connective tissue Pericytes (1 or 2 cell layers) Some elastic fibers Thicker than tunica media Small vein 0.1 - 1 mm Endothelium Smooth muscle Connective tissue (2 or 3 layers Some elastic fibers continuous with Thicker than tunica media tunica intima) Medium vein 1 - 10 mm Endothelium Smooth muscle Connective tissue Connective tissue Collagen fibers Some elastic fibers Internal elastic membrane Thicker than tunica media in some cases Large vein > 1 cm Endothelium Smooth muscle Connective tissue; Connective tissue (2-15 layers) Some elastic fibers; Cardiac muscle Much thicker than media. near heart Sm. mus. fascicles

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LYMPHATIC VESSELS

The smallest lymphatics are often difficult to distinguish from capillaries and small venules except for their greater variability in form and cross-sectional area. Larger lymphatics resemble small venous channels, but have somewhat thinner walls, less well-defined tunicas, and an abundance of valves. Lymphatic channels should contain lymph fluid (and some lymphocytes) in contrast to venous and arterial channels, which will contain all blood elements.

CHECK LIST

VESSELS: Understand the architecture of both arteries and veins. Be aware of the approximate diameter, composition, organization and relative thickness of:

-tunica intima -tunica media -tunica adventitia

Define:

-endothelium -vasa vasorum -nervi vascularis -internal elastic lamina -external elastic lamina -metarteriole -arteriovenous anastomosis -continuous and fenestrated capillaries -pericytes -lymphatic vessels with valves.

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THE HEART Cardiac muscle is found in only one site in the body, namely the wall of the heart. The structure of this type of muscle tissue was studied specifically during the Muscle lab. This lab will consider the muscle in context of the entire organ. The walls of all chambers possess similar layers: endocardium, myocardium, and epicardium, but the relative thickness of these layers and the detailed organization of the tissues composing them vary radically in different chambers. (Note the respective continuity of these layers with tunica intima, tunica media, tunica adventitia of blood vessels.) Also, there are highly specialized structures such as the valves and their reinforcing mechanisms, which require careful analysis. Because the heart is fairly complex, it becomes important in the study of any section to determine precisely where it was cut with reference to the total cardiac anatomy. The “cardiac skeleton” is dense connective tissue between the atrium and ventricles that provide an anchoring point for cardiac muscle and that limit the direct spread of electrical activity in atrial muscle to the ventricles. The thin leaflets of the atrioventricular valve intervening between the two heart chambers attach to the cardiac skeleton. The epicardium (visceral pericardium) and subepicardial fat are outside of the myocardium. This is the location of the coronary blood vessels.

The cardiac conduction system is comprised of specially modified cardiac muscle cells (Purkinji fibers). These are designed to transmit the electrical signal from the A-V node to the ventricles through various branches. Their location, and the direction of transmission of the cardiac impulse is shown in the figure below.

CHECK LIST

Know the histology of the heart, including the morphology of atrium and ventricle. Recognize the epicardium, myocardium and endocardium.

Understand the structure and importance of the cardiac skeleton.

Explain the role of Purkinje fibers in the heart and know their structures.