Exam 1 study guide
Exam 1 study guide Bil 268
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1 Chapter 1 BIL268 What is Neurobiology? Neurobiology is the study of molecular organization of neurons, and the ways that neurons are organized, through synapses, into functional circuits that process information and mediate behavior…. By Dr. Gordon Shepherd. Why do we study Neurobiology? To better understand ourselves and to cure neural and mental illness. Alzheimer’s disease: a progressive degenerative disease of the brain characterized by dementia and always fatal. Affect over 3 million Americans, $90 billion/yr. Autism: a disorder emerging in early childhood characterized by impairments in communication and social interactions, and restricted and repetitive behaviors. Cerebral palsy: a motor disorder caused by damage to the cerebrum before, during or soon after birth. Depression: a serious disorder of mood, characterized by insomnia, loss of appetite, and feelings of dejection. Epilepsy: a condition characterized by periodic disturbances of brain electrical activity that can lead to seizures, loss of consciousness and sensory disturbances. Multiple sclerosis: a progressive disease that affects nerve conduction, characterized by episodes of weakness, lack of coordination and speech disturbance. Parkinson’s disease: a progressive disease of the brain that leads to difficulty in initiating voluntary movement. Schizophrenia: a severe psychotic illness characterized by delusions, hallucinations and bizarre behavior. Affect several million Americans, $130 billion/yr. Spinal paralysis: a loss of feeling and movement caused by traumatic damage to the spinal cord. 2 Stroke: a loss of brain function caused by disruption of the blood supply, usually leading to permanent sensory, rd motor or cognitive deficit. 3 leading cause of death in US, $25 billion/yr. Levels of Analysis: In ascending order of complexity, these levels are molecular, cellular, systems, behavioral, and cognitive. Molecular neuroscience: the brain has been called the most complex piece of matter in the universe. Brain matter consists of a fantastic variety of molecules, many of which are unique to the nervous system. These different molecules play many different roles that are crucial for brain function: messengers that allow neurons to communicate with one another, sentries that control what materials can enter or leaves neurons, conductors that orchestrate neuron growth, archivists of past experiences. The study of the brain at this most elementary level is called like this. Cellular neuroscience: Focuses on studying how all those molecules work together to give neurons their special properties. Among the questions asked at this level are: How many different types of neurons are there, and how do they differ in function? How do neurons influence other neurons? How do neurons become “wired together” during fetal development? How do neurons perform computations? Systems Neuroscience: Constellations of neurons form complex circuits that perform a common function, such as vision or voluntary movement. Thus, we can speak of the “visual system” and the “motor system” each of which has its own distinct within the brain. At this level of analysis, neuroscientists study how different neural circuits analyze sensory information, form perceptions of the external world, make decisions, and execute movements. Behavioral neuroscience: How do neural systems work together to produce integrated behaviors? Where in the brain do “mind-altering” drugs act, and what is the normal contribution of these systems to the 3 regulation of mood and behavior? What neural systems account for gender-specific behaviors? These are some questions studied here. Cognitive neuroscience: Perhaps the greatest challenge of neuroscience understands the neural mechanisms responsible for the higher levels of human mental activity, such as self-awareness, imagination, and language. The scientific process: Observation, replication, interpretation and verification. Observation: Observations are typically made during experiments designed to test a particular hypothesis. Other types of observation derive from carefully watching the world around us or from introspection, or from human clinical cases. Replication: Any observation, whether experimental or clinical must be replicated. Replication simply means repeating the experimental on different subjects or making similar observations in different patients as many times as necessary to rule out the possibility that the observation occurred by chance. Interpretation: Once the scientist believes the observation is correct, he or she interprets it. Interpretations depend on the state of knowledge at the time and on the scientist’s preconceived notions. Interpretations therefore do not always withstand the test on time. Major breakthroughs sometimes occur when old observations are reinterpreted in a new light. Verification: The final step of the scientific process. It means that the observation is sufficiently robust that any competent scientist who precisely follows the protocols of the original observer can reproduce it. Successful verification generally means that the observation is accepted as fact. The process of verification, if affirmative establishes new scientific fact, or, if negative suggests new interpretations for the original observation. 4 Animal welfare: Improvements in how animals are treated in biomedical research. Animals are used only in worthwhile experiments that promise to advance our knowledge of the nervous system. All necessary steps are taken to minimize pain and distress experienced by the experimental animals use of anesthetics, analgesics, etc. All possible alternatives to the use of animals are considered. The Law: the Animal Welfare Act (AWA) in 1966 to protect certain animals from inhumane treatment and neglect. Treat experimental animals/vertebrates humanely. The AWA requires that minimum standards of care and treatment be provided for certain animals bred for commercial sale, used in research, transported commercially, or exhibited to the public. Research facilities, Dealers, Exhibitors, and Transporters. IACUC – Institutional Animal Care and Use Committee. AAALAC – Association for Assessment and Accreditation of Lab Animal Care. A private nonprofit organization to promote humane treatment of animals used in research. UM is AAALAC accredited! Animal protocol must be approved by IACUC before use of animal. 3 R’s (reduction, refinement, and replacements) and 3 tests for undergraduate research. United States Department of Agriculture -Animal and Plant Health Inspection Service (USDA-Aphis) Mission: “To ensure that animals intended for use in research facilities or for exhibition purposes or for use as pets are provided humane care and treatment” Definition of Animal (limited): Animals-all warm- blooded species except birds, rats and mice, and farm animals used for production. Majority of research animals (>90%) are rats and mice. USDA monitors the use of less than 10% of animals- primarily pigs, rabbits, guinea pigs, hamsters, goats, sheep, cattle, horses, dogs and cats 5 The AWA defines “animal” as any live or dead dog, cat, nonhuman primate, guinea pig, hamster, rabbit, or any warm-blooded animal used for research, teaching, testing, experimentation, or exhibition purposes, or as a pet. By definition, coldblooded species (amphibians and reptiles) are exempt from coverage under the AWA. The AWA further excludes the following: Birds, rats of the genus Rattus, and mice of the genus Mus, bred for use in research; Horses not used for research purposes; (Horses used for research are covered by the AWA!!!) Farm animals, including livestock and poultry, used or intended for use as food or fiber or in agricultural research; Fish; and Invertebrates (crustaceans, insects). Who oversees the use of all animals, especially rodents? Public Health Service Policy: requires institutions receiving federal funding submit an Assurance statement to the Office of Laboratory Animal Welfare (OLAW) It states that the institution is committed to following the Guide for the Care and Use of Laboratory Animals (the Guide) The Guide (8 thEd): Guide provides details as to the type of housing, veterinary medical care for animals. Guide also defines how animal care and a designated committee must review use protocols. Animal defined as “any vertebrate animal used in research, teaching or testing” Procedures to Monitor animal care and use: The Institutional Animal Care and Use Committee (IACUC) must inspect and evaluate its facilities and programs at least twice a year. The USDA inspects “covered species” annually and more frequently if there is an issue or complaint. You must allow a USDA inspector access to your facility at anytime. AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care International) accredited facilities are inspected by representatives every 3 years. Annual reports are sent to regulatory agencies and AAALAC regarding the use of animals. 6 IACUC: Institutional Animal Care and Use Committee (IACUC). A committee to oversee and evaluate the institution’s animal program, procedures and facilities. Review all protocols describing animal use; procedures may not begin until protocols are approved. IACUCs derive their authority from the law (USDA/OLAW). At a minimum it must include a minimum of 3 people: a veterinarian trained in laboratory animal medicine, one practicing scientist, at least one community member. Federally Funded (minimum of 5)-3 above, and non- scientist and Chair. The Three R’s: Reduction, Refinemen and Replacement. REDUCTION: The number of animals needed to meet research goals. Typically a “power analysis” is done to determine the minimum number of animals required per experimental group. REFINEMEN: Finding a better way to achieve a research result. Improving an assay so that it is more humane (requiring fewer blood collections or anesthetic events) REPLACEMENT: Replace animals with other models or techniques. Animal Rights: Most people accept the necessity for animal experimentation to advance knowledge, as long as it is performed humanely and with the proper respect for animals’ welfare. However, a vocal and increasingly violent minority seeks the total abolition of animal use for human purposes, including experimentation. These people subscribe to a philosophical position often called animal rights. According to this way of thinking animals have the same legal and moral rights as humans. Animals rights activists have vigorously pursued their agenda against animal research, sometimes with alarming success. They have vandalized laboratories, destroying years of hard won scientific data and 100s of 1000s of dollars of equipment. With threats of violence they have driven some researchers out of science altogether. 7 Animal Models: Nematodes (C. elegans), insects, squid, zebra fish, rodents and monkeys. 1 Chapter 2 Neurons and Glia Histology: the microscopic study of the structure of th tissue. Done in five steps: fixation (early in the 19 century, scientists discovered how to harden, or “fix” tissues by immersing them in formaldehyde, paraformaldehyde or glutaraldehyde) and they developed a special device called a microtome to make very thin slices. Stain: Nissl stain-to-stain cell bodies (Nissl bodies but not neurites) (Fig. 2.1). The Nissl stain is extremely useful for 2 reasons: it distinguishes between neurons and glia, and it enables histologists to study the arrangement, or cytoarchitecture, of neurons in different parts of the brain. Mounting and Examination using microscope. Nissl-stained neurons: a thin slice of brain tissue has been stained with cresyl violet, a Nissl stain. The clumps of deeply stained material around the cell nuclei are Nissl bodies. Camillo Golgi: In 1873, Golgi discovered that soaking brain tissue in a silver chromate solution, now called the Golgi stain, makes a small percentage of neurons become darkly colored in their entirely. Reticular theory: a neural net. Golgi’s contributions are Golgi stain, Morphological features about glial cells, Golgi I (projection) & Golgi II (local circuit) and Golgi tendon organ. Golgi stain: silver chromate stain, potassium dichromate, silver nitrate and Impregnation. This revealed that the neuronal cell body, the region of the neuron around the nucleus that is shown with the Nissl stain, is actually only a small fraction of the total structure of the neuron. The Golgi stain shows that neurons have at least two distinguishable parts: a central region that contains the cell nucleus and numerous thin tubes that radiate away from the central region. Golgi stained neurons: the neurons are entirely stained. The basic parts of a neuron are soma (the 2 central region of the neuron containing the nucleus; also called cell body or perikaryon.) and neurites (a thin tube extending from a neuronal cell body; the two types are axons and dendrites). The neuron Doctrine: Neuron is the elementary functional unit of the nervous system. Neurons communicate with each other by contact, NOT continuity. The neurites of different neurons are not continuous with each other and communicate by contact, not continuity. This idea that cell theory also applies to neurons came to be known as this. Cajal’s Contributions: Contiguity of individual neurons, Neurons are polarized and Degeneration & regeneration of the nervous system. Connectomics: A new field in neuroscience to study the map of neural connections (called connectome) at the microscale level using computer-assisted image processing, electron microscopy, and high-throughput methods. The BRAIN Initiative (ex NIH Human Connectome Project) and The Human Brain Project. Book “Connectome: How the brain’s wiring makes us who we are” by Dr. Sebastian Seung. Understanding of memory, intelligence and mental disorders Soma: The roughly spherical central part of the neuron. The cell body of the typical neuron is about 20 μ m in diameter. The watery fluid inside the cell called the cytosol is a salty, potassium rich solution that is separated from the outside by the neuronal membrane. Within the soma are a number of membrane-enclosed structures called organelles. The cell body of the neuron contains the same organelles found in all animal cells. The most important ones are the nucleus (gene transcription), the rough endoplasmic reticulum, the smooth endoplasmic reticulum, the Golgi apparatus (sorting and delivering proteins) and the mitochondria (energy center). Everything contained within the confines of the cell membrane, including the organelles but excluding the nucleus, is referred to collectively as the cytoplasm. 3 Nucleus: Its name derived for the Latin word for “nut” the nucleus of the cell is spherical, centrally located, and about 5-10 μ m across. It is contained within a double membrane called the nuclear envelope. Pores about 0.1 μ m across perforate the nuclear envelope. Within the nucleus are chromosomes, which contain the genetic material DNA (deoxyribonucleic acid). What distinguish a neuron from a liver cell are the specific parts of the DNA that are used to assemble the cell. These segments of DNA are called genes. Gene expression: it is the “reading” of the DNA. The final product of gene expression is the synthesis of molecules called proteins, which exist in a wide variety of shapes and sizes, perform many different functions, and bestow upon neurons virtually all of their unique characteristics. Gene transcription: RNA molecules are synthesized by RNA polymerase and then processed into mRNA to carry the genetic instructions for protein assembly from the nucleus to the cytoplasm. Transcription is initiated at the promoter region of the gene and stopped at the terminator region. The initial RNA must be spliced to remove the introns that do not code for protein. The process of assembling a piece of mRNA that contains the information of a gene called transcription, and the resulting mRNA is called the transcript. Interspersed between protein-coding genes are long stretches of DNA whose functions remain poorly understood. Some of these regions, however, are known to be important for regulating transcription. At one end of the gene is the promoter, the region where the RNA-synthesizing enzyme, RNA polymerase, binds to initiate transcription. Other proteins called transcription factors tightly regulate the binding of the polymerase to the promoter. At the other end is a sequence of DNA called the terminator, or stop sequence, that the RNA polymerase recognizes as the end point for transcription. 4 RNA splicing: In addition to the non-coding regions of DNA that flank the genes, there are often additional stretches of DNA within the gene itself that cannot be used to code for protein. These interspersed regions are called introns, and the coding sequences are called exons. Initial transcripts contain both introns and exons, but then, this process of RNA splicing removes the introns removed and the remaining exons are fused together. Breakthrough of 2007 of the Human genetic variation: Neurons differ from other cells in the body because of the specific genes they express as proteins. A new understanding of these genes is now possible because the human genome (the entire length of DNA that comprises the genetic information in our chromosomes) has been sequenced. We now know the 25000 words that comprise our genome, and we know where these genes can be found on each chromosome. Your genomes differs from mine. Rough endoplasmic reticulum: Neurons make use of the information genes by synthesizing proteins. Protein synthesis occurs at dense globular structures in the cytoplasm called ribosomes. mRNA transcripts bind to the ribosomes, and the ribosomes translate the instructions contained in the mRNA to assemble a protein molecule. In other words, ribosomes use the blueprint provided by the mRNA to manufacture proteins from raw material in the form of AA. In neurons many ribosomes are attached to stacks of membrane called RER. Rough ER abounds in neurons, far more than in glia or most other non-neuronal cells. In fact, we have already been introduced to rough ER by another name: Nissl bodies. Rough ER is a major site of protein synthesis in neurons, but not all ribosomes are attached to rough ER. Many are freely floating and are called free ribosomes. Several free ribosomes may appear to be attached by a thread; these are called polyribosomes. The thread is a single strand of mRNA, and the 5 associated ribosomes are working on it to make multiple copies of the same protein. Protein synthesis on a free ribosome and on rough ER: mRNA binds to a ribosome, initiating protein synthesis. Proteins synthesized on free ribosomes are destined for the cytosol. Proteins synthesized on the rough ER are destined to be enclosed by or inserted into the membrane. Membrane-associated proteins are inserted into the membrane as they are assembled. What is the difference between proteins synthesized on the rough ER and those synthesized on the free ribosomes? The answer appears to depend on the intended fate of the protein molecule. If it is destined to reside within the cytosol of the neuron, then the protein’s mRNA transcript shuns the ribosomes of the rough ER and gravitates toward the free ribosomes. However, if the protein is destined to be inserted into the membrane of the cell or an organelle, then it is synthesized on the rough ER. As the protein is being assembled, it is threaded back and forth through the membrane of the rough ER, where it is trapped. It is not surprising that neurons have so much rough ER because, as we shall see in later chapters, special membrane proteins are what give these cells their remarkable information-processing abilities. DNA nucleus transcriptionmRNA transcript translation Protein (by Ribosomes in rough ER or free ribosomes. Golgi apparatus: this complex organelle sorts newly synthesized proteins for delivery to appropriate locations in the neuron. The stack of membrane- enclosed disks in the soma that lies farthest from the nucleus is this, which was first described in 1898 by Camillo Golgi. This is a site of extensive “post- translational” chemical processing of proteins. One important function of the Golgi is believed to be the sorting of certain proteins that are destined for delivery 6 to different parts of the neuron such as the axon and the dendrites. Mitochondrion: Another very abundant organelle in the soma is the mitochondrion (plural: mitochondria). In neurons, these sausage-shaped structures are about 1 μ m long. Within the enclosure of their outer membrane are multiple folds of inner membrane called cristae (singular crista). Between the cristae is an inner space called matrix. Mitochondria are the site of cellular respiration. When a mitochondrion “inhales” it pulls inside pyruvic acid (derived from sugars and digested proteins and fats) and oxygen, both of which are floating in the cytosol. Within the inner compartment of the mitochondrion, pyruvic acid enters into a complex series of biochemical reactions called the Krebs cycle. The biochemical products of the Krebs cycle provide energy that, in another series of reactions within the cristae (called the electron-transport chain), results in the addition of phosphate to adenosine diphosphate (ADP), yielding adenosine triphosphate (ATP), the cell’s energy source. When the mitochondrion “exhales” 17 ATP molecules are released for every molecule of pyruvic acid that had been taken in. Neuronal membrane: serves as a barrier to enclose the cytoplasm inside the neuron and to exclude certain substances that float in the fluid that bathes the neuron. The membrane is about 5nm thick and is studded with proteins. Some of the membrane- associated proteins pump substances from the inside to the outside. Others form pores that regulate which substances can gain access to the inside of the neuron. An important characteristic of neurons is that the protein composition of the membrane varies depending on whether it is in the soma, the dendrites or the axon. Phospholipid bilayer: hydrophilic head and hydrophobic tails. THE FUNCTION OF NEURONS CANNOT BE UNDERSTOOD WITHOUT UNDERSTANDING THE STRUCTURE AND FUNCTION OF THE MEMBRANE AND ITS ASSOCIATED PROTEINS. 7 Cytoskeleton: Scaffolding and it gives the neuron its characteristic shape. The “bones” of the cytoskeleton are the microtubules, microfilaments and neurofilaments. The cytoskeleton is not static. Elements of the cytoskeleton are dynamically regulated and are in continual motion. Your neurons are probably squirming around in my head even as I read this sentence. Microtubules: Measuring 20nm in diameter, they are relatively large and run longitudinally down neurites. A microtubule appears as a straight, thick-walled hollow pipe. The wall of the pipe is composed of smaller strands that are braided like rope around the hollow core. Each of the smaller strands consists of the protein tubulin. A single tubulin molecule is small and globular; the strand consists of tubulins stuck together like pearls on a string. The process of joining small proteins to form a long strand is called polymerization; the resulting strand is called a polymer. Polymerization and depolymerization of microtubules and therefore of neuronal shape can be regulated b various signals within the neuron. One class of proteins that participate in the regulation of microtubule assembly and function are microtubule-associated proteins or MAPs. Among other functions, MAPs anchor the microtubules to one another and to other parts of the neuron. Pathological changes in an axonal MAP, called tau, have been implicated in the dementia that accompanies Alzheimer’s disease. Microfilaments: measuring only 5nm in diameter, they are about the same thickness as the cell membrane. They are braids of two thin strands that are polymers of the protein actin. Actin filaments are critically involved in the mechanism of muscle contraction. Like microtubules, actin microfilaments are constantly undergoing assembly and disassembly, and this process is regulated by signals in the neuron. Neurofilaments: With a diameter of 10nm, they are intermediate in size between microtubules and 8 microfilaments. They exist in all cells of the body as intermediate filaments; only in neurons are they called neurofilaments. The difference in name reflects differences in structure among different tissues. Neurofilaments most closely resemble the bones and ligaments of the skeleton. A neurofilament consists of multiple subunits building blocks that are wound together into a ropelike structure. Each strand of the rope consists of individual long protein molecules, making neurofilaments mechanically very strong. The axon: a structure found only in neurons and highly specialized for the transfer of information over distances in the nervous system. The axon begins with a region called the axon hillock (which tapers away from the soma to form the initial segment of the axon proper). Two noteworthy features distinguish the axon from the soma: No rough ER extends into the axon, and there are few, if any, free ribosomes in mature axons and the protein composition of the axon membrane is fundamentally different from that of the soma membrane. The diameter of an axon is variable, ranging from less than 1 μ m to about 25 μ m in humans and to as large as 1mm in squid. THE THICKER THE AXON, THE FASTER THE IMPULSE TRAVELS. Axon collaterals: Axons often branch and these branches are called like this and they can travel long distances to communicate with different parts of the nervous system. An axon collateral returns to communicate with the same cell that gave rise to the axon or with the dendrites of neighboring cells. These axon branches are called recurrent collaterals. Axon terminal: all axons have a beginning (the axon hillock), a middle (axon proper) and an end. The end is called the axon terminal or terminal bouton (French for button), reflecting the fact that it usually appears as a swollen disk. The terminal is a site where the axon comes in contact with other neurons (or other cells) and passes information on to them. Axon terminals form synapses with the dendrites or somata of other 9 neurons. When a nerve impulse arrives in the presynaptic axon terminal, neurotransmitter molecules are released from synaptic vesicles into the synaptic cleft. Neurotransmitter then binds to specific receptor proteins, causing the generation of electrical or chemical signals in the postsynaptic cell. The cytoplasm of the axon terminal differs from that of the axon several ways: Microtubules do not extend into the terminal. The terminal contains numerous small bubbles of membrane called synaptic vesicles that measure about 50 nm in diameter. The inside surface of the membrane that faces the synapse has a particularly dense covering of proteins. The axon terminal cytoplasm has numerous mitochondria, indicating a high-energy demand. Synapse: the synapse has two sides: presynaptic and postsynaptic. These names indicate the usual direction of information flow from “pre” to “post”. The presynaptic side generally consists of an axon terminal, whereas the postsynaptic side may be a dendrite or the soma of another neuron. The space between the presynaptic and postsynaptic membranes is called the synaptic cleft. The transfer of information at the synapse from one neuron to another is called synaptic transmission. Information in the form of electrical impulses traveling down the axon is converted in the terminal into a chemical signal that crosses the synaptic cleft. On the postsynaptic membrane, this chemical signal is converted again into an electrical one. The chemical signal called a neurotransmitter, is stored in and released from the synaptic vesicles within the terminal. Modification of this process is involved in memory and learning and synaptic transmission dysfunction accounts for certain mental disorders. The synapse is also the site of action for many toxins and for most psychoactive drugs. Axoplasmic transport: The process of transporting materials down an axon. 10 Fast axoplasmic transport: the material is enclosed within vesicles, which then walk down the microtubules of the axon. A protein called kinesin provides the legs and the process is fueled by ATP. Kinesin moves material only from the soma to the terminal. All movement of material in this direction is called anterograde transport. Anterograde Transport: Movement of molecules/ organelles outward, from the cell body also called soma to the synapse or cell membrane. Retrograde transport: There is a mechanism for the movement of material up the axon from the terminal to the soma. This process is believed to provide signals to the soma about changes in the metabolic needs of the axon terminal. Movement in this direction, from terminal to soma is called like this RT. A different protein called dynein provides the legs for retrograde transport. Slow axoplasmic transport: Recent studies have revealed that the movement of cytoskeletal "slow" cargoes is actually rapid but unlike fast cargoes, they pause frequently, making the overall transit rate much slower. The mechanism is known as the "Stop and Go" model of slow axonal transport, and has been extensively validated for the transport of the cytoskeletal protein neurofilament. Dendrites receiving synaptic inputs from axon terminals: neurons have been made to fluoresce green using a method that reveals the distribution of a microtubule-associated protein. Axon terminals have been made to fluoresce orange-red using a method to reveal the distribution of synaptic vesicles. The nuclei are stained to fluoresce blue. Dendrites spines: This is a computer reconstruction of a segment of dendrite, showing the variable shapes and sizes of spines. Each spine is postsynaptic to one or two axon terminals. A small sac of membrane that protrudes from the dendrites of some cells and receives synaptic input. 11 Dendrites: The term is derived from the Greek for “tree”, reflecting the fact that these neurites resemble the branches of a tree as they extend from the soma. Dendritic tree: they are the dendrites of a single neuron and each branch of the tree is called a dendritic branch. Postsynaptic polyribosomes: Polyribosomes can be observed in dendrites often right under spines. Neurons based on Number of Neurites: Neurons can be classified according to the total number of neurites (axon and dendrites) that extend from the soma. A neuron with a single neurite is said t be unipolar. If there are two neurites, the cell is bipolar, and if there are three or more, the cell is multipolar. Most neurons in the brain are multipolar. Neurons based on dendrites: In the cerebral cortex (the structure that lies just under the surface of the cerebrum) there are two broad classes: stellate cells (start shaped) and pyramid cells (pyramid shaped). Neurons can also be classified according to whether their dendrites have spines. Those that do are called spiny and those that do not are aspinous. These dendrites classification schemes can overlap. For examples, in the cerebral cortex, all pyramidal cells are spiny. Stellate cells on the other hand can be either spiny or aspinous. Neurons based on Connections: Information is delivered to the nervous system by neurons that have neurites in the sensory surfaces of the body, such as the skin and the retina of the eye. Cells with these connections are called primary sensory neurons. Other neurons have axons that form synapses with the muscles and command movements; these are called motor neurons. But most neurons in the nervous system form connections only with other neurons. In this classification scheme, these cells are called interneurons. 12 Neurons based on Axon Length: Some neurons have long axons that extend from one part of the brain to the other; these are called Golgi type I neurons, or projection neurons. Other neurons have short axons that do not extend beyond the vicinity of the cell body; these are called Golgi type II neurons, or local circuit neurons. Neurons based on neurotransmitters: The motor neurons that command voluntary movements all release the neurotransmitter acetylcholine at their synapses; these motor cells are therefore also classified as cholinergic, meaning that they express the genes that enable use of this particular neurotransmitter. Collections of cells that use the same neurotransmitter make up the brain’s neurotransmitter systems. Astrocytes: The most numerous glia in the brain are called like this. An essential role of astrocytes is regulating the chemical content of this extracellular space. Astrocytes envelop synaptic junctions in the brain, thereby restricting the spread of neurotransmitter molecules that have been released. Astrocytes also have special proteins in their membranes that actively remove many neurotransmitters from the synaptic cells. Astrocytes also tightly control the extracellular concentration of several substances that could interfere with proper neuronal function for example; astrocytes regulate the concentration of potassium ions in the extracellular fluid. Help determine the growth pattern for neurites. Astrocytes fill most of the space in the brain that is not occupied by neurons and blood vessels. Myelinating Glia: The glia provides layers of membrane that insulate axons. Oligodendroglia and Schwann cells differ in their location and some other characteristics. For example, oligodendroglias are found only in the central nervous system (brain and spinal cord), whereas Schwann cells are found only in the peripheral nervous system (parts outside the skull and vertebral column). Another difference is that one 13 oligodendroglial cell contributes myelin to several axons, whereas each Schwann cell myelinates only a single axon. Node of Ranvier: A space between two consecutive myelin sheaths where an axon comes in contact with the extracellular fluid Oligodendroglial cell: like the Schwann cells found in the nerves of the body, oligodendroglia provides myelin sheaths around axons in the brain and spinal cord. The myelin sheath of an axon is interrupted periodically at the nodes of Ranvier. Other Glia: Ependymal cells and microglia. Ependymal cells: A type of glial cell that provides the lining of the brain’s ventricular system. Microglia cells: A type of cell that functions as a phagocyte in the nervous system to remove debris left by dead or dying neurons and glia. They appear to be involved in remodeling synaptic connections by gobbling them up. Microglia can migrate into the brain from the blood, and disruption of this microglial invasion can interfere with brain functions and behavior. 1 Chapter 3 The Neuronal Membrane at Rest Cytosol and Extracellular Fluid: water is the main ingredient of both the fluid inside the neuron, the intracellular fluid or cytosol, and the outside fluid that bathes the neuron, the extracellular fluid. Electrically charged atoms (ions) that are dissolved in this water are responsible for the resting and action potentials. Water is a polar Solvent: Different representations of the atomic structure of the water molecule. The oxygen atom has a net negative electrical charge and the hydrogen atoms have a net positive electrical charge, making water a polar molecule. A crystal of sodium chloride dissolves in water because the polar water molecules have a stronger attraction for the electrically charged sodium and chloride ions than the ions do for one another. Water: For our purpose here, the most important property of the water molecule is its uneven distribution of electrical charge. The two hydrogen atoms and the oxygen atom are bonded together covalently, which means they share electrons. The oxygen atom, however, has a greater affinity for electrons than does the hydrogen atom. As a result, the shared electrons spend more time associated with the oxygen atom than with the two hydrogen atoms. Therefore, the oxygen atom acquires a net negative charge (because it has extra electrons), and the hydrogen atoms acquire a net positive charge. Thus, water is said to be a polar molecule, held together by polar covalent bonds. This electrical polarity makes water an effective solvent of other charged or polar molecules; that is other polar molecules tend to dissolve in water. Ions: atoms or molecules that have a net electrical charge are known as ions. Table salt is a crystal of sodium (Na) and chloride (Cl) ions held together by the electrical attraction of oppositely charged atoms. The electrical charge of an atom depends on the difference between its numbers of protons and electrons. When 2 this difference is 1, the ion is said to be monovalent; when the difference is 2, the ion is divalent; and so on. Ions with a net positive charge are called cations; ions with and negative charge are called anions. Remember, that ions are the major charge carriers in the conduction of electricity in biological systems, including the neuron. The ions of particular importance for cellular neurophysiology are the monovalent cations Na and K, the divalent cation Ca, and the monovalent anion Cl. Phospholipid Membrane: Ions and polar molecules are said to be “water loving” or hydrophilic. Nonpolar covalent bond molecules will not dissolve in water and are said to be “water fearing” or hydrophobic. A phospholipid has a polar phosphate group (a phosphorus atom bonded to 3 oxygen atoms) attached to one end of the molecule. Thus, phospholipids are said to have a polar “head” (containing phosphate) that is hydrophilic and a nonpolar “tail” (containing hydrocarbon) that is hydrophobic. The neuronal membrane consists of a sheet of phospholipids, two molecules thick. A cross section through the membrane reveals that the hydrophilic heads face the outer and inner watery environments and the hydrophobic tails face each other. This stable arrangement is called a phospholipid bilayer and it effectively isolates the cytosol of the neuron from the extracellular fluid. Phospholipid bilayer: it is the core of the neuronal membrane and forms a barrier to water-soluble ions. Protein Structure: In order to perform their many functions in the neuron, different proteins have widely different shapes, sizes and chemical characteristics. To understand this diversity, lets briefly review protein structure. Proteins are molecules assembled from various combinations of 20 different AA. The basic structure of an amino acid. All amino acids have a central carbon atom (the alpha carbon), which is covalently bonded to four molecular groups: a hydrogen atom, an amino group (NH3), a carboxyl group (COO-), 3 and a variable group called the R group (R for residue). The differences among amino acids result from differences in the size and nature of these R groups. The properties of the R group determine the chemical relationships in which each amino acid can participate. Amino acids, the building blocks of protein: Every amino acid has in common a central alpha carbon, an amino group (NH3+), and a carboxyl group (COO-). Amino acids differ from one another based on a variable R group. The 20 different amino acids that are used by neurons to make proteins. Noted in parentheses are the common abbreviations used for the various amino acids. Structure: The primary structure is like a chain in which the amino acids are linked together by peptide bonds. As a protein molecule is being synthesized, however, the polypeptide chain can coil into a spinal- like configuration called alpha helix. THE SEQUENCE OF AMINO ACIDS IN THE POLYPEPTIDE. The alpha helix is an example of what is called the secondary structure of a protein molecule. COILING OF A POLYPEPTIDE INTO AN ALPHA HELIX. Interactions among the R groups can cause the molecule to change its three-dimensional conformation even further. In this way, proteins can bend, fold and assume a complex three-dimensional shape. This shape is called tertiary structure. THREE- DIMENSIONAL FOLDING OF A POLYPEPTIDE. Finally, different polypeptide chains can bond together to form a larger molecule; such a protein is said to have quaternary structure. Each of the different polypeptides contributing to a protein with quaternary structure is called a subunit. DIFFERENT POLYPEPTIDES BONDED TOGETHER TO FORM A LARGER PROTEIN. The peptide bond and a polypeptide: peptide bonds attach amino acids together. The bond forms between the carboxyl group of one amino acid and the amino group of another. A polypeptide is a single chain of amino acids. 4 A membrane ion channel: Ion channels consist of membrane- spanning proteins that assemble to form a pore. The channel protein has five polypeptide subunits. Each subunit has a hydrophobic surface region (shaped) that readily associates with the phospholipid bilayer. Ion channels: are made from just these sorts of membrane spanning protein molecules. Typically, a functional channel across the membrane requires that four to six similar protein molecules assemble to form a pore between them. One important property of most ion channels, specified by the diameter of the pore and the nature of the R groups lining it, is ion selectivity. Potassium channels are selectively permeable to K. Likewise; sodium channels are permeable almost exclusively to Na, calcium channels to Ca, and so on. Another important property of many channels is gating. Channels with this property can be opened and closed- gated-by changes in the local microenvironment of the membrane. UNDERSTANDING ION CHANNELS IN THE NEURONAL MEMBRANE IS KEY TO UNDERSTANDING CELLULAR NEUROPHYSIOLOGY. Ion pumps: ATP is the energy currency of cells. Ion pumps are enzymes that use the energy released by the breakdown of ATP to transport certain ions across the membrane. We will see that these pumps play a critical role in neuronal signaling by transporting Na and Ca from the inside of the neuron to the outside. Diffusion: NaCl has been dissolved on the left side of an impermeable membrane. The sizes of the letters Na and Cl indicate the relative concentrations of these ions. Channels are inserted in the membrane that allow the passage of Na and Cl. Because there is a large concentration gradient across the membrane, there will be a net movement of Na and Cl from the region of high concentration to the region of low concentration, from the left to right. In the absence of any other factors, the net movement of Na and Cl across the membrane ceases when they are equally distributed on both sides of the permeable membrane. 5 Ions and molecules dissolved in water are in constant motion. This temperature-dependent, random movement tends to distribute the ions evenly throughout the solution. Therefore, there is a net movement of ions from regions of high concentration to regions of low concentration; this movement is called diffusion. Although ions typically do not pass through a phospholipid bilayer directly, diffusion causes ions to be pushed through channels in the membrane. The movement of ions across the membrane by diffusion happens when the membrane has channels permeable to the ions and there is a concentration gradient across the membrane. Concentration gradient: A difference in concentration from one region to another. Ionic concentration gradients across the neuronal membrane help determine the membrane potential. Electricity: In addition to diffusion down a concentration gradient, another way to induce a net movement of ions in a solution is to use an electrical field because ions are electrically charged particles. Remember that opposite charges attract and like charges repel. When wires from the two terminals of a battery are placed in a solution containing dissolved NaCl, there will be a net movement of Na toward the negative terminal the cathode and of Cl toward the positive terminal the anode. The movement of electrical charge is called electrical current, represented by the symbol I and measured in units called amperes (amps). Voltage is represented by the symbol V and is measured in units called volts. It is represented by the symbol R and measured in units called ohms ( Ω ). Symbol g conductance in siemens (S). Resistance is simply the inverse of conductance (i.e. R= 1/g). There is a simple relationship between potential (V), conductance (g), and the amount of current (I) that will flow. This relationship, known as Ohm’s law, may be written I = V/R = gV: Current is the product f the conductance and the potential difference. Notice that if 6 the conductance is zero, no current will flow even when the potential difference is very large. Likewise, when the potential difference is zero, no current will flow even when the conductance is very large. Membrane potential: it is the voltage across the neuronal membrane at any moment, represented by the symbol Vm. Sometimes Vm is “at rest”; at other times it is not (such as during an action potential). Vm can be measured by inserting a microelectrode into the cytosol. The resting potential of a typical neuron is about -65 millivolts (1 mV = 0.001 volts). Stated another way, for a neuron at rest, Vm = -65 mV. This negative resting membrane potential of the neuron is an absolute requirement for a functioning nervous system. Measuring the resting membrane potential: A voltmeter measures the difference in electrical potential between the tip of a microelectrode inside the cell and a wire placed in the extracellular fluid, conventionally called “ground” because it is electrically continuous with the earth. Typically, the inside of the neuron is about -65 mV with respect to the outside. This potential is caused by the uneven distribution of electrical charge across the membrane (enlargement). Establishing equilibrium in a selectively permeable membrane: An impermeable membrane separates two regions: one of high salt concentration inside and the other of low salt concentration outside. The relative concentrations of potassium and an impermeable anion A- are represented by the sizes of the letters. Inserting a channel that is selectively permeable to K into the membrane initially results in a net movement of K down their concentration gradient, from left to right. A net accumulation of positive charge on the outside and negative charge on the inside retards the movement of positively charged K from the inside to the outside. Equilibrium is established such that there is no net movement of ions across the 7 membrane, leaving a charged difference between the two sides. Equilibrium potential: The electrical potential difference that exactly balances an ionic concentration gradient is called an ionic equilibrium potential, or simply equilibrium potential and it is represented by the symbol Eion. No net movement, small change in ionic concentration causes large change in Vm, th membrane stores electrical charge. The figure 3.12 demonstrates that generating a steady electrical potential difference across a membrane is a relatively simple matter. All that is required is an ionic concentration gradient and selective ionic permeability. Before moving on to the situation in real neurons, however, we can use this example to make four important points. o Large changes in membrane potential are caused by minuscule changes in ionic concentrations. o The net difference in electrical charge occurs at the inside and outside surfaces of the membrane. o Ions are driven across the membrane at a rate proportional to the difference between the membrane potential and the equilibrium potential. The difference between the real membrane potential and the equilibrium potential (Vm-Eion) for a particular ion is called the ionic driving force. o If the concentration difference across the membrane is known for an ion, the equilibrium potential can be calculated for that ion. Establishing equilibrium in a selectively permeable membrane: An impermeable membrane separates two regions: one of high salt concentration outside and the other of low salt concentration inside. Inserting a channel that is selectively permeable for Na into the membrane initially results in a net movement of Na down its concentration gradient, from right to left. A net accumulation of positive charge on the inside and negative charge on the outside retards the movement of positively charged Na from the outside to the inside. Equilibrium is established such that there is no net 8 movement of ions across the membrane, leaving a charged difference between the two sides; in this case, the inside of the cell is positively charged with respect to the outside. Nernst equation: the exact value of an equilibrium potential in mV can be calculated using an equation derived from the principals of physical chemistry, which takes into consideration the charge of the ion, the temperature, and the ratio of the external and internal ion concentrations. Using the Nernst equation, we can calculate the value of the equilibrium potential for any ion. BOX 3.2 PAGE 70. E ion2.303(RT/zF)log([ion] /[ioo] ) i E ion ionic equilibrium potential in V R: gas constant = 8.314 joules/mole/ K o T: absolute temperature in degrees Kelvin (K) (NOT in Celsius or Fahrenheit!) o o K = 273 + C oC = ( F – 32) 5/9, body temperature = 37 C = o 98.6 Fo z: charge of the ion F: Faraday’s constant = 96496 coulombs/mole Log: base 10 logarithm [ion] o ionic concentration outside the neuron [ion] ionic concentration inside the neuron Approximate ion concentrations on either side of a neuronal membrane: Eion is the membrane potential that would be achieved (at body temperature) if the membrane were selectively permeable to that ion. The important point is that K is more concentrated on the inside and Na and Ca are more concentrated on the outside. Sodium-potassium pump: is an enzyme that breaks down ATP in the presence of internal Na. The chemical energy released by this reaction drives the pump, which exchanges internal Na for external K. The actions of this pump ensure that K is concentrated inside the neuron and that Na is concentrated outside. It has been 9 estimated that the sodium-potassium pump expends as much as 70% of the total amount of ATP utilized by the brain. This ion pump is a membrane-associated protein that transports ions across the membrane against their concentration gradients at the expense of metabolic energy. Calcium pump: it is also an enzyme that actively transports Ca out of the cytosol across the cell membrane. Additional mechanisms decrease intracellular Ca to a very low level 0.0002nM; these include intracellular calcium-binding proteins and organelles, such as mitochondria and types of endoplasmic reticulum, which sequester cytosolic calcium ions. Ion pumps are unsung heroes of cellular neurophysiology. They work in the background to ensure that the ionic concentration gradients are established and maintained. These proteins may lack the glamour of a gated ion channel, but without ion pumps, the resting membrane potential would not exist and the brain would not function. Relative ion permeability: in a real cell multiple ions, relative permeability. Goldman equation: V =61.54mlog ((Pk[k ]o+P [Na ] )/(o [k k +P INa Na)) in IV. The resting membrane potential can be calculated using this, a mathematical formula that takes into consideration the relative permeability of the membrane to different ions. If we concern ourselves only with K and Na, use the ionic concentrations and assume that the resting membrane permeability to K is fortyfold greater than it is to Na, then the Goldman equation predicts a resting membrane potential of -65 mV, the observed value (BOX 3.3) This strain of fly was designated Shaker. Detailed studies soon explained the odd behavior by a defect in a particular type of potassium channel. Using molecular biological techniques, it was possible to map the gene that was mutated in Shaker. Knowledge of the DNA sequence of what is now called the shaker potassium 10 channel enabled researchers to ding the genes for other potassium channels based on sequence similarity. The shaker potassium channel has four subunits arranged like staves of a barrel to form a pore. Enlargement: the tertiary structure of the protein subunit contains a pore loop, a part of the polypeptide chain that makes a hairpin turn within the plane of the membrane. The pore loop is a critical part of the filter that makes the channel selectively permeable to K. The importance of regulating the external potassium concentration: Because the neuronal membrane at rest is mostly permeable to K, the membrane potential is close to Ek. Another consequence of high K permeability is that the membrane potential is particularly sensitive to changes in the concentration of extracellular potassium. A tenfold change in the K concentration outside the cell, [K]o, from 5 to 50 mM, would change the membrane potential from -65 to -17 mV. A change in membrane potential from the normal resting value (-65 mV) to a less negative value is called a depolarization of the membrane. Therefore, INCREASING EXTRACELLULAR POTASSIUM DEPOLARIZES NEURONS. The sensitivity of the membrane potential to [K]o has led to the evolution of mechanisms that tightly regulate extracellular potassium concentrations in the brain. One of these is the blood-brain barrier, a specialization of the walls of brain capillaries that limits the movement of potassium and other blood borne substances into the extracellular fluid of the brain. It is important to recognize that not all excitable cells are protected from increases in potassium Muscle cells do not have equivalents to the blood brain barrier or glial buffering mechanism. Dependence of membrane potential on external potassium concentration: because the neuronal membrane at rest is mostly permeable to potassium a tenfold change in [K]o, from 5 to 50mM, causes a 48 mV depolarization of the membrane. 11 Potassium spatial buffering by astrocytes: When brain [K]o increases as a result of local neural activity, K enters astrocytes via membrane channels. The extensive network of astrocytic processes helps dissipate the K over a large area. Lethal injection: o IV injection o Sodium thiopental o Pencuronium bromide o KCl 1 Chapter 4 Action Potential Measurement of AP: recording device that can be intracellular vs. extracellular and the different parts of AP. Intracellular recording: requires impaling the neuron or axon with a microelectrode. The small size of most neurons makes this method challenging, which is why so many early studies of action potentials were performed on the neurons of invertebrates, which can be 50-100 times larger than mammalian neurons. Thankfully, recent technical advances have made even the smallest vertebrate neurons accessible to intracellular recording methods. The goal of intracellular recording is simple, to measure the potential difference between the tip of the intracellular electrode and another electrode placed in the solution bathing the neuron (electrically continuous with the earth, and thus called ground). The intracellular electrode is filled with a concentrated salt solution, then the electrode is connected to an amplifier that compares the potential difference between this electrode and ground and this potential difference can be displayed using an oscilloscope. Extracellular recording: the action potential is characterized by a sequence of ionic movements across the neuronal membrane. These electrical currents can be detected, without impaling the neuron, by placing an electrode near the membrane. We measure the potential difference between the tip of the recording electrode and ground. The extracellular action potential is characterized by a brief alternating voltage difference between the recording electrode and ground. These changes in voltage can be seen using an oscilloscope, but they can also be heard by connecting the output of the amplifier to a loudspeaker. Each impulse makes a distinctive “pop” sound. 2 The Parts of Action Potential: Rising phase, overshoot, falling phase, undershoot or after- hyperpolarization. Rising Phase: is characterized by rapid depolarization of the membrane. This change in membrane potential continues until Vm reaches a peak value of about 40 mV. In other words, the first part of an action potential, characterized by a rapid depolarization of the membrane. Overshoot: The part of the action potential where the inside of the neuron is positively charged with respect to the outside. In other words is the part of an action potential when the membrane potential is more positive than 0 mV. The falling phase: is a rapid repolarization until the inside of the membrane is actually more negative than the resting potential. In other words, is the part of an action potential characterized by a rapid fall of membrane potential from positive to negative. Undershoot or after hyperpolarization: the hyperpolarization the follows strong depolarization of the membrane. The last part of an action potential, also called undershoots. Generation of Multiple AP: threshold and firing rate, all or none, absolute and relative refractory periods. Firing frequency: of action potentials reflects the magnitude of the depolarization current. This is one way that stimulation intensity is encoded in the nervous system. Although firing frequency increases with the amount of depolarization current, there is a limit to the rate at which a neuron can generate action potentials. The max firing frequency is about 1000 Hz and once an action potential is initiated, it is impossible to initiate another for about 1 msec. This period of time is called the absolute refractory period. Relative refractory period: it can be relatively difficult to initiate another AP for several milliseconds after the end of the absolute refractory period. During 3 it, the amount of current required to depolarize the neuron to action potential threshold is elevated above normal. Depolarization: depolarization of the cell during the AP is caused by the influx of sodium ions across the membrane. Repolarization: is caused by the efflux of potassium ions. Voltage clamp: the key technical breakthrough came with a device. Invented by the American physiologist Kenneth C. Cole and used in decisive experiments performed by Cambridge Uni. Physiologists Alan Hodgkin and Andrew Huxley around 1950. The voltage clamp enabled Hodgkin and Huxley to “clamp” the membrane potential of an axon at any value they chose. They could then deduce the changes in membrane conductance that occur at different membrane potentials by measuring the currents that flowed across the membrane. They showed that the rising phase of the action potential was indeed caused by a transient increase in g Na and an influx of Na, and that the failing phase was associated with an increase in g K and an efflux of K. Membrane currents and conductance:
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