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Histology SSN October 22, 2003 Muscle Sarah Little sjl2017 Grace Liu gcl2008 SKELETAL MUSCLE I. Organization A. General • Muscle fibers run parallel and straight • Mucle tissue = many fasiciculi • Fasicuclus = many fibers (cell) • Muscle fiber = many fibrils “Mucle fiber” is really a syncytium of small myoblasts that form a large multinucleated cell polygons shaped in cross section nucleus at the outer edge of the fiber B. Surrounding connective tissue 1) Endomysium • Loose connective tissue that surrounds each muscle fiber • Reticular fibers • Contains the smallest capillaries and neuronal branches 2) Perimysium • Connective tissue + fat + blood vessels and nerves • Group fibers into fascicles 3) Epimysium • Dense connective tissue that surrounds muscle groups. Large vessels and nerves penetrate this to reach muscle fibers II. Myofilaments & Striations A. Types: 1) Thick: myosin 2) Thin: actin B. Sarcomere = Basic Contractile Unit C. Striations: Diagramed in sarcomere. Seen in entire muscle b/c myofibrils and myofibrils are in register. • H-band = myosin not overlapping actin • A-band = all myosin (both overlapping and non-) • I-band = actin not overlapping myosin • Z disc = disc in center of I band from which actin extends 1 Histology SSN October 22, 2003 III. Function Tidbits A. Musculotendinous junction = to dense regularly-arranged connective tissue (tendon) i.e. collagen type I. B. Triad = functions to deliver intracellular Ca++ to initiate contraction. (2 triads/sarcomere) 1. (1) Transverse (T) Tubule = in-pocketing of plasma membrane 2. (2) cisternae of the Sarcoplasmic reticulum C. Motor unit = neuron + muscle cells it innervates. CARDIAC MUSCLE I. Organization • Fiber = one, mononuclear cell • Central nucleus (can be seen in cross section too) • Columns of cells are branched II. Intercalated discs A. Gap junctions • Lateral portion • Important for ionic continuity, to coordinate contraction B. Fasciae adherens • Transverse portion • Holds cells together at their ends C. Desmosomes • Lateral and transverse portions • Keeps cells together ** Tight Junction = Zona occludans is not found in cardiac muscle III. Functional A. intrinsic rhythm - does not need ACh or norepinephrine for contraction SMOOTH MUSCLE 2 Histology SSN October 22, 2003 I. Characteristics • Surrounds the walls of ducts, blood vessels, GI tract, etc. • Function to give an imprecise squeeze (staggered sheets of cells) • Nuclei • cigar-shaped • lie in the middle of the fiber • appear wrinkled or “puckered” • Plane of section determines visibility of nuclei, cell width • No striations because of diffuse contractile elements • Gap junctions allow for united contractions • Basal Lamina (Collagen IV) and Reticular Fibers (Collagen III) surround individual fibers II. Smooth muscle vs. Dense irregularly-arranged connective tissue A. Color • Smooth muscle is more purple because of combination of actin/myosin and RNA (red + blue = purple) • Connection tissue is more red because of purity of collagen B. Nuclei • Smooth muscle nuclei are inside cell • Fibroblast nuclei are in between collagen fibers C. Homogeneity • Smooth muscle is composed only of muscle • Connective tissue contains fibroblasts, macrophages, lymphocytes, etc., and will therefore have a ragged appearance Skeletal Muscle Cardiac Muscle Smooth Muscle Striated Yes Yes No T Tubule Location A-I junction Z disk Sarcoplasmic Reticulum: 2 cisternae in Triad w/ T tubule No Anastamosing network Yes Yes Yes (limited) No Yes thin filaments thin filaments thick filaments Electrically coupled via Gap Junctions? Can regenerate? level of Calcium regulation 3 Histology SSN October 22, 2003 MUSCLE QUESTIONS 1) Which of the following statements is FALSE? a. The band marked “A” does not include actin filaments. b. The M line is found in the band marked “A”. c. During contraction, the length of the band marked “A” remains the same. d. During contraction, the length of the band marked “B” is shortened. 2) Which of these statements is FALSE about the muscle type at the pointer? a. The cells in this muscle type are electrically coupled. b. This muscle type utilizes a T-tubule system for calcium delivery. c. This muscle type is not capable of regeneration. d. Calcium regulation occurs at the level of thick filaments. 3) Which figure(s) demonstrates smooth muscle? a. Figure A&C b. Figure A&D c. Figure B&C d. Figure B&D 4) The structure at the pointer is composed of all of the following except: a. desmosomes b. tight junctions c. gap junctions d. fascia adherens 5) This tissue contains… a. actin, myosin, troponin b. myosin, troponin, gap junctions c. desmin, intercalated discs, calmodulin d. actin, myosin, calmodulin 6) The structure at the pointer is a: a. site of calcium-binding protein (calsequestrin) and a Ca2+-activated-ATPase. b. site of glycogen storage and degradation to glucose c. site from while Ca2+ is passively transported during muscle relaxation d. site into which calcium is released during muscle relaxation e. form of rough endoplasmic reticulum 4 Histology SSN October 22, 2003 MUSCLE ANSWERS 1) a. This is skeletal muscle. A marks the A band and B marks the I band. The A band consists of myosin and some actin that overlaps the myosin. Thick filaments of myosin anchor at the M line, which is found in the middle of the H zone. The figure below demonstrates how the length of the A band stays the same during contraction, while the I band gets shorter. 2) d. This is cardiac muscle. The cells are electronically coupled via gap junctions, thus allowing contractile signals to pass from cell to cell and allowing cardiac muscle to behave as a syncytium. T tubules are used for calcium delivery in both skeletal and cardiac muscle. Smooth muscle cells have no T tubule system. Cardiac muscle is not capable of regeneration (mature cardiac muscle cells do not divide). If injury to cardiac muscle tissue leads to death of cells, fibrous connective tissue forms (scarring) with consequent loss of cardiac function. Since tropomyosin and troponin wind around the actin filaments, calcium regulation does not occur at the level of the thick filament. 3) b. A is smooth muscle and B is skeletal muscle (from the esophagus). C is an EM of skeletal muscle and D is an EM of smooth muscle. Skeletal muscle has striations whereas smooth muscle does not. The EM in Figure C shows a higher magnification of the H band, A band, I band, and Z line in a striated muscle fiber. Figure C also shows a triad of T tubules and flanking terminal cisternae, which is only found in skeletal muscle. Whereas skeletal muscle has multiple, peripherally-located nuclei, smooth muscle has a single, centrally located nucleus, as seen in Figure D. 4) b. Pointer is at an intercalated disc. An intercalated disc does not have tight junctions. The desmosomes are on both the transverse and lateral components of the intercalated disc, while the fasciae adherens are on the transverse. Note 5 Histology SSN October 22, 2003 from Dr. Rothman: Gap junctions can also be a component of the transverse portion of the disc but they are not as prevalent as on the lateral. 5) d. This is smooth muscle. As in striated muscle, contraction is initiated by a rise in calcium in the cytosol, but this does not act through a troponin-tropomyosin complex on the actin filament. A rise in calcium stimulates a myosin lightchain kinase to phosphorylate one of the two light chains of myosin. When this chain is phosphorylated, the myosin head can react with actin and produce contraction. Intercalated discs are specific to cardiac muscle. 6) a. This is an EM of skeletal muscle. The pointer is at the sarcoplasmic reticulum. The sarcoplasmic reticulum (SR) provides a mechanism for the muscle cell to regulate the concentration of cytosolic calcium. It is a modified smooth endoplasmic reticulum that serves alternatively as a storage site and a source of cellular calcium. Calcium is actively transported from the cytosol to the SR through the activity of a Ca2+-dependent ATPase. Calsequestrin is a calciumbinding protein found in the SR that, as it’s name implies, functions in the sequestion of calcium. Calcium is released from the SR during muscle contraction and stored during relaxation. Glycogen is stored as particles or droplets in the cytoplasm, which contains the enzymes required for the synthesis and breakdown of glycogen. 6 Histology SSN October 22, 2003 NERVE Shira Abeles, sra2013@columbia.edu Katie Butler, kc361@columbia.edu The Basics (fill in with presentation) Nervous System Classifications (specific to this Histo lab) Nervous System CNS Spinal Cord Cerebellum PNS Cerebral Cx ANS (Ganglia) 7 Somatic Enteric (Peripheral Nerve) Histology SSN October 22, 2003 Functions & Histological Features CNS Spinal Cord Cerebellum Distinguishing *Butterfly = gray matter Feature -- dorsal horn – narrow, sensory -- ventral horn – wider, motor *Purkinje Cells *Pyramidal Cells - -- specialized motor cortex; polygonal in shape, scattered - large, round cells; prominent nucleoli; wreathed in satellite cells Higher processing Sympathetic & Parasympathetic - Functions (general – you’ll learn MUCH more about this when you study Neuroanatomy) White Matter (axons) eosinophilic pear-flask shaped; UNIQUE dendrites mlcr layer f(n): outflow of cerebellum Transmits signals to & from brain; site of reflex control Motor coordination *exterior, axons traveling to & from brain - axons cut in *most interior layer – looks diffuse, w/in granular layer cross section in spinal cord preparation Gray Matter (cell bodies) Cerebral Cx *butterfly - processes from cell bodies usually exit gray matter in plane of section PNS ANS Ganglion *Site of cell bodies Peripheral Nerve *Nerve bundles -- look for axons in crosssection (round, clear/ white bodies (myelin) with central “dot”) Somatic motor & sensory (innervates smooth muscle & glands; organ function) *interior N/A N/A *exterior N/A N/A (don’t confuse w/ outer, molecular layer of gray matter (see below)) *molecular + granular layers - - - molecular layer = outermost, pale staining, mostly axons & dendrites granular layer = cellular, basophilic Purkinje cells separate the two 8 Histology SSN October 22, 2003 Extraneuronal Cells Function Support Myelin Production Cells in CNS Astroglia Cells in PNS Satellite Cells - “star-shaped” - structural support extend processes to wrap around capillaries form BBB - Oligodendrocytes Schwann Cells - form myelin sheath that - cuboidal cells, surround cell bodies IN AUTONOMIC GANGLIA Do Not Make Myelin insulates axons Connective Tissue None – NO Collagen Fibroblasts Neuronal Supporting Structures Function CNS PNS Meninges: For Peripheral Nerve Supporting/Surrounding Bundles Only: Tissues: Innermost Pia Mater – thin, direct contact Endoneurium – Vascular, Middle Outermost with brain & SC Arachnoid –“spider-web”; sends delicate trabeculae to pia (subarachnoid space is filled w/ CSF) Dura Mater – “hard”; durable outer covering arround individual nerve fibers Perineurium – Arrangers axons into ‘fascicles’ Epineurium – binds ‘fascicles’ into ‘bundles’ Figure: Schematic of connective tissue layers in peripheral nerve bundle. Epineurium binds fascicles bundles Fascicle Perineurium binds nerves fascicles Endoneurium wraps individual neurons Peripheral Nerve Bundle 9 Histology SSN October 22, 2003 High-Yield Tips: • - Peripheral Nerve vs. Smooth Muscle: nerve tissue more compact and less eosinophilic ***nerve tissue wavy & bubbly – arises from myelin leaking out upon staining • - Dendrites vs. Axons: with Nissl staining, if see process that stains dark blue, dendrite, not axon. . . axons contain no synthetic machinery and therefore do not stain w/ Nissl • - Molecular Layer of Cerebellum vs. White Matter of Cerebellum (cerebellar medulla) Look for Purkinje cells. Purkinje cells are very distinct, and they demarcate the border b/t molecular & granular layers of the cerebellum. The white matter of the cerebellum resides underneath the basophilic granular layer. • - Mnemonic: “Satellites are on the periphery of space.” In other words, satellite cells are found in the peripheral nervous system. • Mnemonic: “Pyramidal cells are in the Cerebral Cortex.” • - Mnemonic: “Schwann sounds like one” One Schwann cell myelinates one PNS axon. 10 Histology SSN October 22, 2003 NERVE QUESTIONS Questions 1-2 pertain to the structures at the pointers in Figures A and B. 1. Which of the following statements is correct regarding the cells at the pointers in Figures A and B? a. The cells are situated in white matter. b. The cells are situated in a ganglion. c. The cells are situated in the gray matter. d. The cells are situated between the white and gray matter. 2. In Figure B the processes at the arrow are: a. myelinated fibers. b. axons. c. collagen fibers. d. dendrites Question 3: 3. Which figure(s) demonstrates smooth muscle? a. Figure A b. Figure B c. both Figures A and B d. neither Question 4: 4. The region at the pointer is required for: (choose all that apply) a. protein synthesis. b salutatory conduction. c. insulation. d. production of endoneurium. Question 5: 5. All of the following characterize the organ depicted by the slide except: a. Microglia b. Myelinated axons c. Fibroblasts d. Oligodendrocytes Question 6: 6. Which of the following is TRUE of the cell at the pointer?: a. maintains stable microenvironment b. resides in spinal cord. c. is producing myelin. d. transmits signals from molecular to granular layers. 11 Histology SSN October 22, 2003 NERVE ANSWERS 1. C. Cerebellum, Pointer A on Purkinje cell, Pointer B on Purkinje cell dendrite. This is a Purkinje cell in the cerebellum, found at the junction between the molecular and granular layers. The cell body lies in the granular layer. Its dendrites branch out in the molecular layer. 2. D. The arrow points to the dendrites of the Purkinje cell in the molecular layer. 3. B. Figure A, peripheral nerve; Figure B, smooth musc. Peripheral nerve and smooth muscle are hard to tell apart. Here are some distinctions: -nerve tissue tends to be more compact, less eosinophilic, wavy and bubbly because there are regions where myelin has leaked out. -smooth muscle is more diffuse, more intense eosinophilic color. 4. B & C. Peripheral nerve, pointer on myelin sheath. The myelin sheath is layers of lipid wrapping around the axon. It is required for saltatory conduction. It is also used for insulation. 5. C. Figure depicts cerebral cortex. There are different support cells in the CNS and PNS. This is a section from the CNS. There are fibroblasts in the PNS but not the CNS. 6. A. This is a satellite cell, found in autonomic ganglia. These PNS support cells do not make myelin, while Schwann cells do. (Slide #83) 12 Histology SSN October 22, 2003 SKIN Katie: Km2101 Jamie: Jmp2035 Functions: Resists Abrasion, Immunological Defense, Prevents Fluid Loss, Prevents Fluid Absorption From Environment, and UV Protection. Refer to Chapter 14 in Ross EPIDERMIS stratum corneum • fully keratinized (dead) epithelial cells, no nuclei, low H2O content • sealed extracellular space a) prevents evaporation b) barrier against pathogens • sqames, most superficial cells, sloughed off stratum lucidum • only in thick skin; advanced keratinization stratum • keratinocytes here contain basophilic granules of keratohyalin granulosum a) leaks into extracellular space and acts to seal cell b) this aids in waterproofing and protection of skin • lamellar bodies with glycolipids are secreted (same function as keratohyalin) stratum • cells attached to each other via desmosomes which gives spiny spinosum appearance to keratinocytes under LM • spaces in between cells allow fluid from vessels in the dermis to nourish the cells (epidermis is avascular!) • Langerhans cells (involved in the immune system) found here • in upper part synthesis of keratohyalin granules and lamellar bodies begins stratum • contains stem cells from which new keratinocytes arise by mitosis germinativum a) these cells are relatively small, cuboidal to low columnar (basalis) b) look for mitotic cells with basophilic appearance • melanocytes are dendritic cells that synthesize melanin a) melanin protects from UV radiation b) can be distributed to surrounding keratinocytes DERMIS papillary layer (loose CT) • blood vessels, nerves, lymphatics found here • dermal papillae project into the epidermis • Meissner’s corpuscles (in dermal papillae); mostly in fingertips a) encapsulated; two point discrimination • bulb of the hair follicle (see page 385 in Ross) a) matrix of hair follicle is equivalent to the basal layer of epidermis b) melanocytes contribute melanin granules c) sebaceous glands (always associated with hair follicle) 13 Histology SSN October 22, 2003 i. ii. secrete sebum between shaft and follicle holocrine secretion—cell is stuffed with product and necroses d) arrector pili (smooth muscle bundle) i. hair pulled to vertical position ii. goosebumps due to skin depression Reticular layer (dense CT) • eccrine sweat glands (coiled tubular glands—not associated with hair follicles) a) secretion contains salts and IgA b) secretory portion in subcutaneous tissue i. secretory tubules: acidophilic layer of myoepithelium contracts to expel secretion ii. myoepithelium – sympathetic innervation c) ducts extend to epidermis i. excretory ducts: TWO layers of cuboidal cells ii. remove salt – final sweat hypotonic iii. corkscrew path in epidermis • Pacinian corpuscle (found in deeper dermis and hypodermis) i. encapsulated ii. senses vibration, deep pressure HYPODERMIS subcutaneous tissue • loose CT, many glands, ducts, blood vessels, lymphatics • abundant adipose tissue 14 Histology SSN October 22, 2003 SKIN QUESTIONS 1. In the stratum basalis, ________ are found. They attach keratinocytes to _________. a. b. c. d. Hemidesmosomes, basal lamina Desmosomes, basal lamina Hemidesmosomes, other keratinocytes Desmosomes, Langerhans cells 2. What is the function of the structure at the pointer? a. b. c. d. senses light touch. pain and temperature sensation. deep pressure and vibration sensation. increases rate of neuronal transmission. 3. The structure is a(n) ________; it is located in the _________ layer, and its ducts terminate in the ________. a. b. c. d. sebaceous gland, hypodermal, stratum granulosum. eccrine sweat gland, dermal, stratum granulosum. eccrine sweat gland, dermal, stratum corneum. sebaceous gland, hypodermal, stratum corneum. 15 Histology SSN October 22, 2003 SKIN ANSWERS 1. Lab 8, slide 2 a) Choice A is correct b) Choice B is not correct because desmosomes attach keratinocytes to other keratinocytes. c) Choice C is not correct because hemidesmosomes attach keratinocytes to the basal lamina. d) Choice D is not correct because Langerhans cells are involved in the immune response and does not form desmosomes with neighboring keratinocytes. 2. Lab 8, slide 17 (pointer on Pacinian Corpuscle) a) senses light touch – NO. This better describes a Meissner’s corpuscle, which senses 2 point discrimination. b) pain and temp. sensation – NO. This describes free nerve endings in the epidermis. c) deep pressure and vibration sensation – YES. d) increases rate of neuronal transmission – partly true. At best, this answer is only partly correct, as the STRUCTURE is the corpuscle. A myelin sheath (composed of Schwann cells) surrounds the nerve axon entering the corpuscle and does ensure an increased rate of transmission relative to transmission in unmyelinated nerves – but the myelin sheath only extends for one or two nodes. The inner core of the corpuscle is composed of lamellae of attenuated Schwann cells, while the outer core of the corpuscle is composed of lamellae of extra-corpuscular endoneurial cells. Lymphlike fluid and collagen sit between the lamellae; displacement of lamellae causes an action potential – so the Schwann cells are also involved in whether the transmission will happen at all (their role is not limited to ensuring speed). 3. Lab 8, slide 14 a) Choice A is not correct because sebaceous glands are only associated with hair follicles. b) Choice B is not correct because if this were true, sweat would have to percolate to the outer surface of the epidermis. c) Choice C is correct. d) Choice D is not correct because, again, sebaceous glands are only associated with hair follicles. 16