BSCI 353 Exam 1 Lecture Notes, Lecture notes of Neuroscience

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Introduction to Course Introduction to Course Dr. Lin’s Background & Research  Originally from Taiwan, came to U.S. to earn his doctorate  Research o Arthropod brain and behavior  How their brain is organized o Humans have 3 cones (red, green, blue); arthropods have up to 10 cones  Arthropods can see polarized light  Hyperiid amphipods Cellular Level: Neural Signaling  Neurons and glial cells  Components of a circuit and charge flow  Electrical properties of neurons & action potentials  Synaptic transmission  Neurotransmitter diversity  Synaptic integration and computation Circuitry Level: Sensory Receptors & Transduction Pathways  Postsynaptic molecular signaling (G-proteins, second messengers, cAMP)  Sensory receptors and transduction pathway o Vision: photoreceptors and visual circuitry o Audition: hair cells and auditory circuitry The Changing Brain: Neural Plasticity & Development  Synaptic plasticity  Long-term potentiation and long-term depression  Repair and regeneration Neurons & Glia (Chapter 1) Week 1: Neurons & Glia The Structure of a Neuron  1) Golgi’s stain o Camillo Golgi (1843-1926) o Single-neuron reconstructions with Golgi stain  Santiago Ramón y Cajal o Confirm Golgi results with fluorescent dye injections o Major features of neurons visualized with electron microscopy  o Diverse nerve cell morphologies in the human nervous system   Cortical pyramidal cell  Retinal bipolar cell  Retinal ganglion cell  Retinal amacrine cell  Neurons in mesencephalic trigeminal nucleus  Cerebellar Purkinje cell  2) Nissl Stain o Franz Nissl (1860-1919) Ionic Basis of Membrane Potentials (Chapter 2) Week 2: Ionic Basis of Membrane Potentials From Form to Electrical Signals   Electrophysiology recordings  The remarkable squid giant axon o Allowed for single neuron recording  How do neurons generate membrane potentials? o Resting potential o Electrical potential Resting Membrane Potential  –65 mV  Extracellular and intracellular ions o K+, Na+, Cl-, Ca2+ o  Active transporters (ion pumps) o Ion binds to transporter and then is transported across the membrane o Actively move selected ions against concentration gradient o Create ion concentration gradients  Ion channels o Ions diffuse through channel o Allow ions to diffuse down concentration gradient o Are selectively permeable to certain ions  What makes the membrane negatively charged when a neuron is at rest? o Na+/K+ pumps (active transporters) produce and maintain a much greater K+ ion concentration inside the neuron than outside o The neuronal membrane at rest is highly permeable to K+ ions (K+ channel)  The outflow of K+ ions, down their concentration gradient, leaves the inside of the neuron negatively charged Ionic Basis of Membrane Potentials (Chapter 2)  What makes the action potentials? o Ion concentrations o Nernst equation:  Action Potentials   Alan Hodgkin and Andrew Huxley (1936) discovered the ionic mechanisms of action potentials Summary  How do nerve cells generate electrical signals?  Resting potentials o The outflow of K+ ions, down their concentration gradient, leaves the inside of the neuron negatively charged  Action potentials o Changes in the membrane potential above the threshold trigger an inflow of sodium ions and subsequent outflow of potassium ions across the membrane, leading to a wave of positive charges down the axon Action Potentials (Chapter 3) Week 3: Action Potentials Ionic Mechanisms of Action Potentials  How do neurons generate action potentials?  How do neurons transmit action potentials? The Voltage Clamp Method  Measures ion flow at any level of membrane potential  Used to measure ion flow in giant squid axon  Current flow across a squid axon membrane during a voltage clamp experiment o  1) Voltage clamping at different potentials experiment o Currents responded differently at several different potentials o With increasingly depolarized membrane potentials:  The early inward current increases in size, then decreases  The later outward current gets larger   2) Na+ removal experiment o Dependence of the early current on sodium Action Potentials (Chapter 3) o  1963 Nobel laureates in physiology or medicine o Alan Hodgkin (1914) and Andrew Huxley (1917) o Discovered the ionic mechanisms of action potentials Transmitting Action Potentials  Action potential conduction requires both active and passive current flow o 1) Na+ channels locally open in response to stimulus, generating an action potential o 2) Some depolarizing current passively flows down axon o 3) Local depolarization causes neighboring Na+ channels to open and generates an action potential further down o 4) Upstream Na+ channels inactivate, while K+ channels open  Membrane potential repolarizes and axon is refractory at this stage o 5) The process is repeated, propagating the action potential along the axon  Can neurons increase their action potential conduction speed? o Bigger axons o Better insulating axons o Electrical synapses o Escape circuits contain these features because they must respond quickly  Saltatory action potential conduction along a myelinated axon o Nodes of Ranvier are the spaces in between the myelin sheath along the axon o Myelin increases action potential conduction speed by acting as an electrical insulator Summary  When a neuron is at rest, the outflow of K+ ions down their concentration gradient leaves the inside of the neuron negatively charged  When a neuron is stimulating, changes in the membrane potential above threshold trigger an early transient inflow of Na+ ions and subsequent prolonged outflow of K+ ions across the membrane, leading to a wave of positive potential change along the inner membrane down the axon Ion Channels & Pumps (Chapter 4) Week 4: Ion Channels & Pumps Ion Channels  The rapid changes in permeability across the insulating membrane is controlled by channels that regulate the flow of Na+ and K+  Ion channel characteristics: o Allow ions to pass at high rates o Selectively permeable: Na+ and K+ flow across the membrane independently o Voltage-dependent: Those channels are able to sense the voltage across the membrane and open only when the voltage reaches appropriate levels The Patch Clamp Method  Methods o Cell-attached recording  The recording pipette is placed with tight contact between the pipette and membrane  Intracellular mechanisms function normally o Whole cell recording  The recording pipette is placed with strong pulse of suction, rupturing the membrane and the cytoplasm is continuous with pipette interior  Exposes intracellular space where ligands or drugs can be added o Inside-out recording  Pipette attaches to a piece of cell membrane, exposing intracellular space  Used when changes are being made at the intracellular surface of the ion channels o Outside-out recording  Complementary to inside-out recording  Begins with whole cell technique, after membrane is ruptured, the pipette is retracted and the membrane protrudes as it detaches from the cell and reforms itself into a smaller compartment  Pipette is retracted, ends of membrane anneal and extracellular domain is accessible  The original outside of the membrane is now on the side  Enables studies of the inner membrane surface, including moving it to another solution bath  Experiment 1: Looking for Na+ channels in a patch of squid axon o Methods  Outside-out recording  K+ current blocker (TEA) o Results of outside-out recording using a K+ blocker  With K+ currents blocked, depolarizing a patch of membrane from squid giant axon causes tiny and transient inward currents  Events have unitary amplitude at pA  10^-12 of an ampere  A current of 2 pA reflects the flow of 2000 ions per millisecond  Close correlation between the averaged microscopic inward and macroscopic Na+ currents  Both blocked by TTX Ion Channels & Pumps (Chapter 4) o Conclusions  The microscopic inward currents are resulting from the opening of single Na+ channels  Evidence  1) The currents have a time course that matches the kinetics of macroscopic Na+ currents (i.e. mostly occur at the beginning of depolarization)  2) The currents are blocked by TTX  Experiment 2: Looking for K+ channels in a patch of squid axon o Methods  Inside-out recording  Na+ current blocker (TTX) o Results  With Na+ currents blocked, depolarizing a patch of membrane from squid giant axon causes tiny but much prolonged outward currents  Events have unitary amplitude at 2 pA  Close correlation between the averaged microscopic outward currents and macroscopic K+ currents  Blocked by TEA o Conclusions  The microscopic outward currents are resulting from the opening of single K+ channels  Evidence  1) The currents have a time course that matches the kinetics of macroscopic K+ currents (i.e. delayed and have a prolonged effect  2) The currents are blocked by TEA  Conclusions from both experiments o Individual Na+ and K+ channels are responsible for the generation of action potentials o Both channels are voltage sensitive (voltage-gated Na+ and K+ channels) o Na+ channels open immediately and close right away, while K+ channels open late and have a prolonged effect  Functional states of voltage-gated Na+ and K+ channels o  1991 Nobel laureates in physiology or medicine Exam 1 Review Week 5: Exam 1 Review Neurons  Dendrites: Input region o Place to receive information from other neurons  Cell body  Axon o Action potential travels down the axon o Axon hillock  Action potential initiation o Axon terminal  Output region  Contains NTs that are released into the synaptic cleft  Neuron is polarized; information only flows in one direction  Some neurons only contain dendrites and others contain only axons Visualizing the Neuron  Golgi staining o Nervous tissue is placed in several chemical solutions, then stained with silver nitrate o After sectioning the tissue into thin slices, the entirety of about 2% of neurons can be visualized by light microscopy o  Nissl stain o Basic dyes are used to target negatively charged DNA and RNA inside the cell body o The technique stains all cell bodies o  Fluorescent dye injections o Fluorescent dye is injected into cell body of neuron to visualize results under fluorescent microscope o Good for visualizing morphology of the entire neurons o  In situ hybridization Exam 1 Review o Chemically or radioactively-labelled nucleic acid probes are used to detect mRNAs that encode specific genes o  Antibody labeling (immunolabeling) o Design antibodies to target protein product of certain genes; also shows gene expression o  Green fluorescent protein (GFP) transgenic neural labeling o Gene-encoding GFP can be inserted into neuronal DNA, allowing us to see morphology of certain neurons o Allows for studying neurons in living animals  Brainbow o Similar to GFP, but inserted tag allows for more colors to better distinguish between neighboring neurons of the same type Reticular vs. Neural Doctrine  Neural doctrine o Santiago Ramon y Cajal o Argued that each neuron is its own independent entity and synapses exist between neurons  Reticular theory o Camillo Golgi o Argued that the nervous system is organized as a web (no synapses)  Invention of electron microscopy allowed for visualization of the synaptic cleft between neurons, making Ramon y Cajal’s view more accurate o Golgi’s view is not entirely false due to the existence of gap junctions in electrical synapses o Both are right depending on the type of neurons being viewed Studying Functions of Neurons  Electrophysiology o Records electrical signal inside the neuron with a thin electrode pin o Limited to a few neurons at a time o Through intracellular electrophysiological recordings, they determined that gigantic neurons (ganglia) in squids that control jet propulsion  Calcium imaging Exam 1 Review o Engineered dye is able to fluoresce when there is a change in calcium concentration, allowing neuron activity visualization Action Potential  Rising phase o Stimulus depolarizes neuron to threshold potential, which opens voltage-gated Na+ channels o Na+ ions rush into the cell and cause membrane depolarization  Overshoot phase o Membrane potential continues to depolarize until the peak of the action potential at about +40 mV  Repolarization/Falling phase o Voltage-gated Na+ channels become inactivated and voltage-gated K+ channels are open o K+ ions rush out of the cell and cause membrane repolarization  Hyperpolarization/Undershoot phase o The membrane potential hyperpolarizes as the membrane is still permeable to K+ and more K+ exits  Na+/K+ ATPase pump reestablishes the gradient back to resting potential  TTX and TEA  TTX: Blocks voltage-gated Na+ permeability but does not affect K+ permeability  TEA: Bocks voltage-gated K+ permeability but does not affect Na+ permeability  Exam 1 Review o Suction is applied and the deal between pipette and membrane is so tight so that no ions can flow between pipette and membrane  Isolates one channel only o All current that flows when a single ion channel opens must flow into the membrane  Record change in voltage across that one channel  Used to compare different areas of the membrane to see which regions are permeable to certain ions  3 types: o Whole cell recordings o Inside-out recordings  Shows how channels are affected by intracellular recordings o Outside-out recordings  Shows how channels are affected by extracellular signals o  Experiment 1: Looking for single Na+ channels in patch of squid axon o Outside-out recording o Found tiny and transient inward currents of Na+ o Matches transient inward Na+ current seen in voltage clamp experiments  Experiment 2: Looking for single K+ channels in patch of squid axon o Inside-out recording o Found tiny outward currents of K+  Microscopic results matched macroscopic results o Individual Na+ and K+ channels are responsible for generation of action potentials o Na+ channels open immediately and close right away, while K+ channels open late and have a prolonged effect Practice Questions 1) There are two main types of cells in the nervous system: neurons and glia. What specialized features distinguish neurons from other cell types?  Neurons have axons and dendrites, structures for transmitting electrical signals and allowing neurons to establish connections and share information Exam 1 Review  Glial cells do not have typical neuronal structures (axons) and do not participate in electrical signaling as neurons do 2) Glia in many brain areas outnumber neurons and play diverse roles in neural function. Name three roles of glia.  Release glutamate (a neurotransmitter)  Integrate neuronal inputs (e.g. modulate ion and pH levels in the environment)  Modulate synaptic transmission by controlling uptake of neurotransmitter  Some glia make myelin o Oligodendrocytes in CNS o Schwann cells in PNS  Regulate nutrient uptake from blood vessels  Remove waste o Microglia  Provide structural support for neuronal development  Formation of the blood-brain barrier o Astrocytes 3) An action potential occurs if current injected into a neuron _______ the neuron to reach _______ potential. a. depolarizes; synaptic b. hyperpolarizes; synaptic c. depolarizes; threshold d. hyperpolarizes; threshold e. hyperpolarizes; resting  C) Depolarizes; threshold 4) The amplitude of the action potential of a given neuron is a. larger in response to depolarizing currents of greater magnitude. b. dependent on the magnitude of the sensory stimulus. c. related to the number of synapses on the neuron. d. smaller if the resting potential of the neuron is lower. e. always the same.  E) Always the same  The stronger stimulus, the higher frequency at which action potentials are generated 5) Which consequence of Na+ channels staying open, instead of closing after membrane depolarization, is most plausible? a. Action potentials would spread both ways, forward and backward. b. The refractory period would be longer. c. The refractory period would be shorter. d. The membrane would not repolarize (return to a negative value of membrane potential) following depolarization. e. None of the above  D) The membrane would not repolarize (return to a negative value of membrane potential) following depolarization 6) In a cell with a Ca2+-permeable membrane, if the RMP is -58 mV at room temperature, and the solution outside the neuron has 10 mM Ca2+, how much Ca2+ is in the neuron?  -2 = log(10/[Ca]in)  10^-2 = 10/[Ca]in Exam 1 Review 7) When the brain is deprived of oxygen, the mitochondria within neurons cease producing ATP. What effect would this have on the membrane potential? Why?  Ionic concentration gradients would not be maintained across the neuronal membrane, and therefore no action potentials or neural signals can happen  This is because the primary driver for the ionic concentration gradients across the neuronal membrane is Na+/K+ pump, which requires the consumption of ATP to pump ions up their concentration gradients 8) Which statement correctly differentiates between the passive and active current in a myelinated axon? a. The passive current flows only in the nodes of Ranvier, unlike the active current. b. The active current flows only in the nodes of Ranvier, unlike the passive current. c. The passive current flows in one direction along the axon, unlike the active current. d. The action potential propagation depends on the passive current only. e. The action potential propagation depends on the active current only.  B) The active current flows only in the nodes of Ranvier, unlike the passive current 9) Based on the observations made by Hodgkin and Huxley, one can expect ion channels to a. allow ions to move across the membrane quickly. b. work with the electrochemical gradients to pass ions. c. exist in distinct variants, allowing different types of ions to pass. d. respond to changes in the membrane potential. e. All of the above f. None of the above  E) All of the above 10) The traces below show the results of a patch clamp experiment in which a patch of a neuron was depolarized from -100 mV to +50 mV. In the space below the figure, name three characteristics of the current trace that convince you that a voltage-gated potassium channel was in the patch but NOT a voltage-gated sodium channel.  1) Delayed opening  2) Outward current  3) No inactivation 11) Name the function of glial cells  Recovery and development of neuronal networks  Maintaining the ionic flow of neurons 12) What is the function of microglia?  To scavenge for debris and are active if there is an inflammation (macrophages of the brain)