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Discovering Hopkins: Neuroscience Lab The Johns Hopkins University Lab Manual Objective: This class is designed to give you the “hands-on” experience of discovering how the structure of the nervous system relates to its function. Comparisons will be made between the sheep brain and the human brain (models and plastinated). General Notes Familiarize yourself with your lab station and the lab. Waste Disposal a. Biohazard waste boxes are to be used only for biohazard materials. b. Broken glass will be put in the broken glass boxes. c. Used scapel blades are to be put in the blade boxes. d. Needles are to be put in the sharps container. e. Normal trash (papers, etc.) can be disposed of in the trash in the hall. Do NOT do any dissections until you have read the procedure section AND seen the demonstration. Sheep brains are stored in Carosafe®, which is a propylene glycol based holding fluid. Gloves are to be worn at all times when handling the specimens. Materials Throughout this class, you will be using sheep brains, human brain models, skull models, plastinated human tissue, and numerous pieces of equipment. You are responsible for treating all the materials that are used in the lab with respect. You are also responsible for using the lab materials properly, as instructed. Linda Gorman -1- Last Updated 7/25/2012 Part 1: Orientation Cranial (Anterior, Rostral) Orientation of the Nervous System Orientation: Neural Tube Sagittal Section Horizontal Section Coronal (Transverse, Frontal) Section ←Medial (Proximal) →Lateral (Distal) Dorsal (Superior) Neuraxis Ventral (Inferior) Caudal (Posterior) The nervous system evolves from a neural tube and all directions are given in relationship to the neuraxis. The neuraxis is an imaginary line drawn through the spinal cord through the front of the brain. In quadrupeds (4 legged animals), directional terms are based on four basic reference points: head - up, tail - behind, back - top, and belly - down. In bipeds (e.g., humans), the neuraxis bends through a 90o angle in the skull and human directional references shift with this bend. 1. The (top part) head or front part of the animal is the anterior (front), rostral (rostrum, beak or mouth), or cranial (cranium, head). 2. The (bottom part) tail or hind part of the animal is denoted as posterior (behind), or caudal (cauda, tail). 3. The back of the animal is the dorsal (dorsum, back) or superior portion. 4. The front end is ventral (venter, belly), or inferior. 5. Medial is towards the midline (neuraxis), and lateral refers to structures away from the midline (neuraxis). 6. Proximal is closer to the midline and distal is farther away from the midline. To view structures internally the brain is sectioned into three planes. The orientation is given in relationship to the primitive neural tube. 1. Sagittal sections (parallel to the neuraxis) bisects into right and left 2. Horizontal (perpendicular to the neuraxis if neural tube stays flat) bisects into upper and lower 3. Coronal (cross section, that bends - also called transverse or frontal) bisects into front and back Linda Gorman -2- Last Updated 7/25/2012 Part 2: Meninges Ventricular System Meninges 25 mls Foramen of Luschka Foramen of Magendie 100 mls Cerebrospinal Fluid (CSF) Choroid Plexus 500 mls daily Makes (2/3) of CSF Subdural Space Subarachnoid Space Bear M.F. et al., Neuroscience: Exploring the Brain (2007) Bear M.F. et al., Neuroscience: Exploring the Brain (2007) Rosenzweig M.R. et al., Biological Psychology (2001) 1. Be sure that your sheep brain is placed in your dissection tray with the dorsal surface facing upward. 2. Make midline cut in the superior aspect of the dura in the rostral to caudal direction. (As you reach the posterior aspect of the brain, the dura is going to be harder to cut through.) 3. Make lateral cuts at the most rostral margin of the dura to facilitate the removal of the dura. 4. Peel the dura carefully from the dorsal surface of the sheep brain towards the ventral surface. Be very careful not to damage the cranial nerves, vasculature and pituitary gland . 5. Cut the dura around the pituitary and remove it from the brain. Part 3: Ventral surface of the brain Sheep Brain: Cranial Nerves Vascular System: Blood Supply Pyramidal tract 5 Rosenzweig M.R. et al., Biological Psychology (2001) 1. Carefully lift the ventral part of dura to visualize connections and then cut the connections as far away from the brain as possible. 2. Looking at the ventral surface of the brain without the dura and the human brain model, identify the major blood vessels and cranial nerves. Functionally, you can divide the 12 cranial nerves into 4 groups. Linda Gorman -3- Last Updated 7/25/2012 1. Three cranial nerve nuclei mediate pure sensory information vision, smell, and audition. Olfactory (1), Optic (2), Vestibulocochlear (8), 2. Five cranial nerves mediate motor behaviors. Oculomotor (3), Trochlear (4), Abducens (6) – these all control eye movements Spinal Accessory (11) – neck muscles Hypoglossal (12) – tongue muscles 3. Four cranial nerves are mixed sensory and motor. Trigemminal (5) – face, sinuses and teeth Facial (7) – face, salivary and tear glands Glossopharyngeal (9) – tongue, tonsils, pharynx Vagus (10) – heart, lung, gastrointestinal tract, bronchi, trachea, larynx 4. Some cranial nerves, such as the Vagus, mediate control of the autonomic functions such as respiration, heart rate, and gastrointestinal functions. Part 3: Skull 1. Using your human skull model, look to see where the vasculature and cranial nerves enter and exit. Part 4: Cerebral Hemispheres Cerebral Hemispheres Sheep Brain: Dorsal Surface Gyrus Sulcus Fissure (superior frontal sulcus) 2 Rosenzweig M.R. et al., Biological Psychology (2005) 1. Compare the cerebral hemispheres of the sheep and human brain. What you are looking at is the neocortex, which is made of 6 layers of cells. 2. The two cerebral hemispheres are separated by the medial longitudinal fissure (also known as the interhemispheric fissure). 3. Each hemisphere is divided into four major lobes, named for the overlying bones of the skull. 4. In the sheep brain, frontal lobes are limited caudally by the crucial (cruciate) sulcus. The cruciate sulcus is somewhat variable in the sheep, and sometimes difficult to locate. However, the superior frontal sulcus (coronal sulcus) is easily located. Parallelling the medial longitudinal fissure, the superior frontal sulcus divides the frontal poles into approximately equal left- and righthalves, and, if traced caudally, it is seen to "T-end" into the cruciate sulcus. In the human brain, frontal lobes are limited caudally by the central sulcus. 5. The area caudal to the cruciate sulcus is the parietal lobe whose line of separation from the more posteriorly placed occipital lobe is ill-defined. The temporal lobe in the sheep is poorly Linda Gorman -4- Last Updated 7/25/2012 developed when compared to the human brain. In the human, the Sylvian or lateral fissure separates the temporal lobe from the frontal and parietal lobes. Generally speaking, the frontal lobe is involved in cognitive & motor function, the parietal lobe is for sensory (touch) and integration of information, the temporal lobe is for auditory information, and the occiptal lobe is for visual information. Part 5: Cerebellum and Brainstem Sheep Brain: Inferior Surface Sheep Brain: “Open” Dorsal Pons Trapezoid body superior colliculus Medulla Pyramidal tract inferior colliculus cerebellar hemisphere vermis Spinal Cord 13 9 6. Now, turn your attention to the cerebellum on both the sheep specimen and human brain model. Laterally, the two large masses are the cerebellar hemispheres, joined to each other by the narrow median vermis. The laminations of the cerebellar cortex are very distinctive. Parallel fissures and sulci are interposed between the thin, transversely running folia of the cerebellar cortex. The median portion or vermis forms a low ridge from which the lobules of the hemispheres slope gently downwards and laterally. The cerebellum is concerned with modulating the rate, force and range of muscular contractions for their smooth interplay in movement and posture. 7. Gently pull back on the cerebellum to reveal the dorsal surface of the brain stem. Here you should be able to easily see the corpora quadrigemina, that is, the superior (rostral) and inferior (caudal) colliculi. The superior colliculi are considerably larger than the inferior, in the sheep. Collectively, this area is also called the tectum and forms the roof of the midbrain. The superior colliculus is an important visual center. It receives inputs directly from the retina via optic nerves. It is involved in the location of visual stimuli, orienting responses and ocular reflexes. The inferior colliculus is an important auditory center. 8. The space between the cerebellum and the brainstem is 4th ventricle. 9. Look at the inferior surface of the brain and locate the brainstem, which is made up of the midbrain, pons, and medulla. The brainstem contains structures that function to keep you alive. It takes care of all your “housekeeping” chores for your body. Linda Gorman -5- Last Updated 7/25/2012 You need your brainstem to breathe and to control your heart, pumping blood to every part of your body. Your brainstem keeps your body at a constant temperature. It also “tells” you to eat when you are hungry, drink when you are thirsty and sleep when you are tired. Part 6: 3D Dissection Connecting Fibers Association Fibers – Corona Radiata Projection Fibers – Internal Capsule Commissural Fibers 1. Gently pull the caudal end of the medulla downward to open the 4th ventricle. Cut through the cerebellar peduncles, taking care not to damage the medulla and pons. Remove the cerebellum. 2. You will now be able to see the dorsal surface of the brainstem. Again, identify the superior colliculus and inferior colliculus. You can now also see the pineal gland. 3. Open up the interhemispheric fissure to locate the corpus callosum. Remove the caudal right neocortex by cutting away the grey matter in the coronal plane. Continue until you reach the white matter of the corpus callosum. 4. Carefully cut through the corpus callosum. Make a horizontal cut through the hemisphere so that you do not rip through the callosum. 21 19 Linda Gorman -6- Last Updated 7/25/2012 5. Below the white matter lies the lateral ventricle which can be pulled open to remove the remaining overlying cortex. This will expose the hippocampal formation. The hippocampus is part of the limbic system which plays an integral role in attention, learning and memory. 6. Identify the corona radiata. 7. Bisect your brain specimen, making a sagittal cut right down the midline. 8. Locate the caudate nucleus, part of the basal ganglia, located within the lateral ventricle. Also identify the thalamus and hypothalamus. The basal ganglia functions in motor planning and is made up of the caudate, putamen, globus pallidus and substantia nigra. The thalamus processes most of the information reaching the cerebral cortex from the rest of the CNS. It has a football shape. The hypothalamus, regulates autonomic, endocrine, and visceral integration. Part 7: Brain Sections 1. Using an intact sheep brain, prepare brain sections by cutting 0.5 cm (about ¼ inch) sections using the long blade and the mitre box. Coronal Sections – Odd numbered stations Horizontal Sections – Even numbered stations 2. Try to identify some of the structures that we have looked at in these sections. Compare them to the plastinated human material. Linda Gorman -7- Last Updated 7/25/2012 caudate nucleus (7) thalamus (16)pineal gland (11), superior colliculi (12). hippocampus (13) corpus callosum (5) References http://www.vh.org/adult/provider/anatomy/BrainAnatomy/BrainAnatomy.html (human brain dissection) http://brainmuseum.org/index.html (a virtual brain museum – great for comparing species) http://sumsdb.wustl.edu/sums/index.jsp (atlases of human, macaque, rat, mouse brains) http://www.msu.edu/~brains/index.html (whole and sectioned sheep brains) http://academic.uofs.edu/department/psych/sheep/ieframerow.html (dissections with lab manual) http://vertex.biostr.washington.edu/da.html http://www.psychology.uoguelph.ca/faculty/peters/labmanual/PrintSheepBrain.html (sheep brain dissections and manual) Anderson, James, E. 1983. Grant’s Atlas of Anatomy. Baltimore: Williams & Wilkins. Bear, M.F. et al. 2007. Neuroscience: Exploring the Brain Lippincott Williams & Wilkins. Cooley, Richard K. and Vanderwolf, C.H. 2004 The Sheep Brain: A Basic Guide. London: A.J. Kirby Co. Haines, Duane, E. 2004. Neuroanatomy: An Atlas of Structures, Sections, and Systems. Lippincott Williams & Wilkins. Hollinshead, W. Henry. and Rosse, Cornelius. 1985. Textbook of Anatomy. Pennsylvania: Harper & Row. Kandel, Eric R., Schwartz, James H., and Jessel, Thomas M. 2000. Priniciples of Neural Science. New York: McGrawHill. Martin, John H. 1989. Neuroanatomy: Text and Atlas. Connecticut: Appleton & Lange. Linda Gorman -8- Last Updated 7/25/2012