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How does neurotransmitter release affect the postsynaptic membrane potential?
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How does neurotransmitter release affect the postsynaptic membrane potential?
-causes EPSP/IPSP
-glutamate binding to AMPA/NMDA receptors causes depolarization (EPSP)
-binding to GABAA/GABABR causes hyperpolarization (IPSP)
Explain the basis for a miniature EPSP or IPSP.
-neurotransmitter release from vesicles is probabilistic
-even without stimulation, vesicles of neurotransmitter are released
How do the mechanisms for short-term facilitation and short-term depression differ?
Short term facilitation:
-rapid increase in synaptic weight
-Ca2+ is slowly cleared from the synapse
-more neurotransmitter is released with each subsequent action potential if they are back-to-back
-mediated by synaptotagmin-7
Short term depression:
-rapid decrease in synaptic weight
-neurotransmitter release declines with depleting vesicle pools
-higher release rate causes more depletion and more depression
How do the mechanisms of short-term plasticity and long-term plasticity differ?
-change in the number/function of postsynaptic receptors (long term)
-change in amount of released neurotransmitter (short term)
-morphological changes in axon sprouting or spine size (long term)
What is spike timing-dependent plasticity?
-spike timing-dependent plasticity
-sign and magnitude of LTD/LTP depends on order and timing of pre/postsynaptic spikes
Why is long-term plasticity likely to underlie learning & memory?
-long-lasting synaptic changes (months or decades)
List two examples of changes that can occur in the postsynaptic cell to produce long-term synaptic plasticity.
-Change in number/function of postsynaptic receptors
-morphological changes to dendritic spines/synapses
List two examples of changes that can occur in the presynaptic cell to produce long-term synaptic plasticity.
-Axonal sprouting (number of outputs)
-Change in the amount of released neurotransmitter (e.g., activation of CB1Rs decreases neurotransmitter release probability)
-Alterations in the order/timing of presynaptic spikes changes (Hebb)
Where do transcription and translation take place in neurons?
-Transcription occurs in the nucleus, translation occurs in the soma, dendrites, & axons
Why does translation also occur outside of the soma in neurons?
-axons transport and translate mRNAs to proteins
-mRNA translation to proteins is modulated by synapse activity signals
What are immediate early genes and what distinguish their expression in neurons?-IEG are transcribed during activation, meaning depolarization-dependent -example is Fos as a marker of cell activation -normal genes are transcribed as neededWhy is that IEG can be used as a proxy for neuronal activity?-Fos is an IEG that marks cell activation -Fos expression appears and increases in a depolarization-dependent fashionWhat is the key second messenger that couples excitation and transcription in neurons?-Calcium is a second messenger that mediates changes in gene expression.What are two major ways by which extracellular Ca2+ gets into neurons in an activity-dependent fashion?-activation of VGCC or NMDAR allow calcium ions to activate CAM, a precursor to the CREB cycle -Creb cycle leads to expression of immediate early genes (IEG)What are the major intracellular storage organelles for Ca2+ in cells?-mitochondria -endoplasmic reticulumHow is Ca2+ concentration kept so low within cells and why?-to allow sensitivity to depolarization -maintained by sodium/calcium exchangerWhat are some of the mechanisms how activity modulates gene expression that we covered in this lecture?-Neuron firing opens calcium channels -This leads to transcription regulation and changes in protein expression -Many mechanisms of Ca2+ influx are depolarization-dependent -Changes in gene expression are largely dependent on activityYou have discovered a new gene and you named it Aggie. You suspect Aggie is an immediate early gene. Design a simple experiment in cell culture that tests whether the expression of Aggie is: (A) activity-dependent, and (B) Ca2+-dependent. Briefly explain your answer.-cell cultures can be divided into two groups: experimental and control -experimental neurons can be activated with an agonist (nicotine if activated by nAChR) -differences in Aggie transcription level over time can be compared between 2 groups -if activity dependent, initial timepoint will show highest expression -In the second experiment, EGTA chelates with calcium -If Aggie expression is abolished after this treatment, gene is calcium-dependentDistinguish between dynamic and undynamic functions of the cytoskeleton-Static: structure and support with actin filaments and microtubules -Dynamic: allows contractility and motility with growth coneMake sure you know the key properties of microtubules, microfilaments and intermediate filaments-Microfilaments: actin cytoskeleton elements, solid center, thinnest (8 nm across), made of g- actin, F-actin (microfilaments), asymmetrical for motor protein recognition, growth only on the plus end -Microtubules: hollow filaments (20 nm across), made of tubulin, also have a plus and minus end to create polarity for motor protein recognition -Intermediate filaments: only present in axons or glia, symmetric (no polarity for motor protein recognition)What are the common features of microtubule- and microfilament-specific motor proteins?-head to recognize filament, tail to carry cargoWhat key cellular features make having a functional cytoskeleton so important for neurons?-drives polarization and maintains neuron structure/functionWhy is that nobody has found a motor protein specific for intermediate filaments?-no polarity, motor protein cannot recognize which direction to carry cargoCytoskeletal motor proteins have two general domains regardless on which filaments they act on. What are these domains and what do they do?-head (binds microtubule filament with specificity to determine direction of travel) -tail (binds and carries cargo)You need to differentially label by immunostaining dendrites and axons in neurons you are growing in culture. What cytoskeletal proteins are the preferred targets for labeling these subcellular compartments in neurons?-MAPs are microtubule associated proteins -Tau labels axons and MAP2 labels dendritesWhat are the five basic elements of synaptic integration?1) Amplitude of unitary PSP (Bigger=more plasticity) 2) temporal summation (frequency of signals must be enough for Mg removal and LTP facilitation) 3) spatial summation (sum of multiple inputs) 4) passive and active membrane properties (dendritic trees/ion channels) 5) functional characteristic of the synapse (whether excitatory or inhibitory or able to back-propagate)What are the two active properties of dendritic integration?-Voltage-gated channels (active electrical properties similar to axons) -Dendritic spikes (enhances distal synaptic inputs)What are microglia?-Specialized CNS resident macrophages -able to self-renew -undergo structural and functional changes in response to environment -can be in an activated or resting state depending on stage of development -first line of defense against pathogens in the CNSWhat is the origin of microglia?-derived from yolk sac hematopoietic stem cells -primitive macrophages -migrate to neuroepithelium of forming CNS during early developmentList two main physiological functions of microglia-Phagocytosis and removal -Non-immune such as trophic supportWhat are activated microglia? In research papers, particularly those related to injury and diseases, we often see the term "microglia/macrophages". Why?-Less branched processes -Shorter with larger cell body -Often uni- or bi-polar -Function mostly during late embryogenesis -Both macrophages/microglia engulf and secrete inflammatory factors -macrophages/microglia are morphologically indistinguishable -share many the same cell markers used for immunolabeling (e.g., Iba1, CD11b, CD68)What is one main difference between myelin-forming cells in the CNS versus the PNS?-One oligodendrocyte can myelinate up to 40 different axons -Schwann cells can only myelinate one axonWhat are the three major functions of myelin?-Allows saltatory conduction (electric insulation & optimized AP conduction) -Metabolic support (glycolysis) -Maintains axon function & survivalAre all CNS axons myelinated?-No, some axons in gray matter are unmyelinated -Spots of myelin along an axon can be removed (plasticity)What are the major roles of astrocytes in the central nervous system?-regulate ion concentration and signaling (prevent excitotoxicity) -control blood flow -promote synaptogenesis & maintenence -astrogliosisDescribe the first experiments that demonstrated that astrocytes participate in synaptogenesis. What is the evidence that soluble factors released by astrocytes promote synaptogenesis?-Pfrieger & Barres studied retinal ganglion cell synaptogenesis w/ and w/o astrocytes -glia-free cultures formed synapses but were dysfunctional (high failure rate) -Pfrieger later found this is due to astrocyte secreted factors -single neurons were allowed to autapse -neurites still formed but only a small number of synapses (inefficient) -medium that once contained glial cells increased autapses and excitatory currentsDescribe the immune privileged status of the brain-Antigen presentation by dendritic cells -presence of anti-inflammatory modulators -protection by the blood brain barrierDescribe immune system as mediators of neurological disease-Myasthenia gravis: antibodies to AChRs -Postpartum psychosis antibodies to NMDAR (cured w/ plasma purification)Describe stress effects on the immune system-alters immune responses and the growth of tumors -induces hippocampal cell death -Organ atrophy (thymus and spleen decrease in size) -LPS and vagus nerve -Norepinephrine and Epinephrine growth factors for bacteria enhance infectivity of EnterovirusDescribe cytokine effects on the brain-Cytokines impact the hypothalamus which leads to sickness behavior -shift bodily energy from metabolism and movement to immune response -cytokine effects can persist in the absence of sickness and cause depressionWhat is potency? What is differentiation? Compare and contrast.-ability to give rise to different cell types -higher potency correlates with greater possible cell types -stem cell can be totipotent, pluripotent, or multipotent -Differentiation is when a dividing cell transforms into a specialized cell type -Terminal differentiation is when a cell can no longer divideExplain the difference between neural stem cells & neural progenitor cells.-Neural stem cells are multipotent and able to self-renew -Neural progenitor cells are multipotent but have limited proliferative capacity -NSCs are precursors to NPCsDescribe what happens during each phase of the cell cycle.-G1:cellular contents, excluding chromosomes, are duplicated -S: each of the 46 chromosomes is duplicated -G2: cell double checks for errors and executes repairs -M: cell division and cytokinesis separates cell into two daughtersWhat is interkinetic nuclear migration? How does nuclear position correlate with the cell cycle?-cells move from apical to basal side of neural tube -occurs during each cell cycle along microtubules -position is coupled to the cell cycle -exposure to signaling molecules of cycle phase controls division -crowded pub hypothesisWhat are some techniques to track the fate of progenitor cells and their progeny?-Clonal analysis labels the differentiation fate of progenitor cells and progeny -measure of cell potency -allows cell line generation -uses integrating, non-replicating viruses (e.g., lentivirus) with GFP reporter -only delivered to the original progenitors -only infects actively dividing cells -terminally differentiated cells are not infected -Another technique: birth dating -to birth date, inject labeled nucleotide analogs to track the time point a cell develops -This method revealed cortex cell layers develop from the inside outExplain evidence that different neuronal populations are born at different times.-3H-thymidine was injected into a pregnant female -offspring tissue was processed using autoradiography -At gestation day 11, the first (innermost) layer of the cortex was developed -At gestation day 13, the middle layer was developed -At gestation day 15, the outermost layer of the cortex was developed -thymidine label can only integrate into actively dividing cells (birth dating)Describe the general timeline of neurogenesis & gliogenesis.-Neural stem cells can differentiate into neurons or glia -Glia further differentiate into astrocytes or oligodendrocytes -Growth factors, morphogens, kinases, and cell-cell interactions regulate switch -During neurogenic period, neurogenesis is promoted; gliogenesis is repressedWhat are proneural transcription factors?-Basic helix-loop-helix (bHLH) transcription factors -Belong to: Math1/Atoh1, Olig1/2/3, NeuroD1/2/4/6, Ngn1/2/3, and Mash1/Ascl1 families -promotes neural gene transcription -inhibits gliogenesis by sequestering p300/CBP cofactorsWhat is unique about the developmental origin of microglia?-Microglia are derived from the embryonic yolk sac & later migrate to nervous system -all other neuron cell types are derived from neural tubeLearn cellular components of NMJ and how they are labelled-AChRs in muscle: bungarotoxin -Axons/nerve terminals: NF(neurofilament)/SV2 -Schwann cells: S-100Understand synapse formation at NMJ as a model for synaptogenesis in general-Rapsyn binds directly to AChRs to anchor them to the postsynaptic membrane. -MuSK, receptor tyrosine kinase, transduces agrin signals. -Lrp4, a muscle receptor for agrin, forms a complex with MuSK. Agrin binding to MuSK-Lrp4 complex phosphorylates MuSK and carries a retrograde signal for motor nerve terminal differentiation. -Dok-7 binds to the IC juxtamembrane region of MuSK stabilizes its dimerization and allows AChR clusteringLearn and understand the standard experimental approach to test whether a molecule is involved in synaptogenesis at the NMJ-Can be characterized by culturing muscles from young chicks -bungarotoxin is added to cultures to selectively bind AChRs -Purify the molecule and clone its gene -form a KO animal without this gene -If KO animal fails to produce postsynaptic apparati and cluster AChRs, the molecule is required for synaptogenesis at the NMJ.Brief introduction of CNS synaptogenesis-Contact initiated with cadherins -terminal branching and interactions between pre-/postsynaptic organizing molecules (NLGs and NRXNs) -Organizers recruit cytoplasmic and cytoskeletal components to form functional synapses.What distinguishes neural agrin from non-neural agrin at the molecular level?-both made by motor neurons and muscle fibers -Non-neural induces clustering of AChRs in muscle cells -Neural allows Lrp4 binding and formation of NMJDescribe the general functions and attributes of growth cones.-detect and respond to molecular guidance cues -requires adhesive surfaces to create laminin highways -repulsive function: MT severing proteins and retraction of filopodia -attractive function: Lamellipodia, filopodia, & axon/microtubules -Filopodia are actin filaments -Bundles of microtubules steer the axon terminal -Contact-dependent repulsion via growth cone collapseCompare the role of Cadherins vs. Integrins in adhesion and guidance.-Both involved in signaling for axon adhesion and guidance -Integrins bind ECM and the cytoskeleton -integrins activate Ras-MAPK pathway -Integrins are required for glial coating of laminin -Cadherin allows cell-cell binding via cadherin proteins -Cadherin allows actin cytoskeleton organization -Cadherin is calcium-dependent.How do Collagen, Laminin and Fibronectin contribute to guidance?-collagen and laminin make up the basal lamina -both have a triple helix structure -In laminin, RGD resides at the top and bottom as a target for cell binding -Laminin also often coats glia. -Fibronectin is a dimer with different functional domains to sense ECM growth signals -Fibronectin can also form an adhesive gradient for haptotaxisDistinguish contact-dependent repulsion from chemorepulsion.-Chemotaxis is the directed motion in response to chemical signals -Haptotaxis is in response to a gradient of extracellular attraction/repulsionDescribe axon guidance by Slit/Robo, Semaphorins, Netrin, & Ephrin.-prevents axon terminals for getting stuck during circuit development -netrin-2 gradient around the soma and axon of a commissural neuron -netrin-1 gradient around the floorplate (near the axon terminal) -allows commissural axons to be guided across the midline -Netrins are chemoattractants -Semaphorins are repulsive (membrane bound (Sem1)/ diffusible (Sem2, Sem3)) -Ephrins allow contact-dependent mutual repulsion via growth cone collapse -Ephrin forms lanes to repel axons and maps the tectum.What are two functions of Neurotrophins?-allow for chemotaxis (target selection) -survival (prevent apoptotic loss)Briefly describe how retinal ganglion neurons map to the tectum.-precise mapping on the tectum -axons form reversed patterns -ephrin ligands are in the caudal (posterior) brain RG region -ephrin receptors are in the eyes -tectum grows by adding new cells caudo-medially.Describe the concepts of neural competition and pruning.-most active survives to maturity -each axon branches to form a complex synapse -Interaction with the target region prevents apoptotic loss (Hebbian plasticity) -newborn kitten experiments: one eye was shut for 3 months before reopen -Kittens with one eye shut had permanent blindness in shut eye -Kittens with both eyes shut developed sight again -Shows a role of experience on neural developmentGive an example of a secreted chemoattractant.-Netrin guides commissural axons across the midline via slit-Robo signaling.What is regeneration? Sprouting?-Regeneration: growth of injured axon -Sprouting: growth of uninjured axonDescribe the neuron-intrinsic factors impacting axon regeneration potential-cell type -age -growth state (pro-regenerative transcription factors) -Microtubule stabilization -Axonal transport (Preconditioning) -High mitochondrial activityDescribe the neuron-extrinsic factors modulating regeneration-Inflammation (Early response is neuroprotective; abnormal leads to chronic disease) -Glial scar: Reactive astrogliosis, CSPG secretion -CNS myelin: inhibitors prevent neurite growth (slit/robo) -Fibrotic scar (fibroblasts): Inhibits axon regeneration -Lack of neurotropic guidance cues like BDNFHow can age impact axon growth?-Neuronal Extrinsic Factors like increased inflammation, astrogliosis, and glial scars -Intrinsic Factors: reduced neurite length, increased branches, TF, mitochondrial activityWhat are some tools used to enhance axon regeneration?-Scaffold -Stem cells -Electrical stimulationWhat are the different stem cells that could be used to enhance regeneration?-Mesenchymal stem cells (MSCs) -Olfactory Ensheathing Cells (OECs) -Schwann cells (PNS) -Induced pluripotent stem cells (iPSCs) -Embryonic stem cellsHow does the nervous system develop and what is the brain made of?-Ensemble of interconnected neurons and glia -Electron microscopy reconstruction to map connectome in drosophila -Optogenetics can reveal behavior of activating a neuron.You are performing an experiment in which you inject current into a neuron, and then record the membrane voltage changes caused by this manipulation. 1) Are you conducting a voltage-clamp or current-clamp experiment? What is the difference? 2) What information does this technique tell you about the cell's membrane?-voltage-clamp holds membrane potential at constant voltage to see changes in current -current-clamp holds current constant and measures membrane potential changes -This experiment is current-clamp. -Current-clamp measures response to current that enters the cell -also helps determine conductance/resistance after measuring voltage response -important to understand response to electrical currents and neurotransmitter activityEither lowering the sodium concentration outside a nerve cell, or exposing the cell to tetrodotoxin (TTX), will abolish action potentials. How do each of these two treatments cause the cell to become unexcitable?-depolarization causes the quick activation and inactivation of VGSC -leads to controlled influx of extracellular sodium ions -ion flow causes further depolarization of the membrane and action potential -Lowering extracellular sodium decreases sodium ions across the membrane -This prevents the generation and propagation of action potential -Tetrodotoxin is a selective blocker of VGSC and decreases sodium fluxExplain the differences between the mechanisms of saltatory and continuous action potential conduction.-Unmyelinated axons are rich in VGSC down the entire axon -allows sodium influx everywhere (not just AIS and nodes) -opens neighboring VGSC to further propagate signal -depolarization domino effect (continuous conduction) -leads to an absolute refractory period during which VGSC are inactivated -then a relative refractory period where AP can occur but requires more current -Myelination allows faster propagation of AP (saltatory conduction) -Myelin sheaths insulate to prevent ion leakage -Myelin sheaths increase the effective diameter of axons -Myelin sheaths enhance axon conductance -Oligodendrocytes myelinate the CNS, Schwann cells myelinate the PNS -Myelinated neurons can only influx sodium at the nodes of ranvier or AIS -nodes are spaced out optimal distance to keep membrane depolarized until next node -myelinated axons have fewer ion channels to speed transductionChoose the neuroscience technique that is best suited for each experimental aim: 1) Visualizing 3D structure of fluorescently-labeled neurons throughout the entire mouse hippocampus: 2) Determining the effects of induced cortical neuron activation on wheel-running behavior in mice: 3) Editing a disease-causing gene mutation to restore protein function: 4) Visualizing cortical neuron activity in awake, behaving mice: 5) Characterizing cellular diversity in the developing Drosophila brain:1) Lightsheet microscopy is also known as single plane illumination microscopy and creates intrinsic optical sections to develop 3D images 2) Optogenetic fibers can deliver laser stimulation to neurons injected with channelrhodopsin and behavioral effects can be measured 3) CRISPR/Cas9 is a gene-editing tool that can replace a targeted sequence with a desired one in a genome (homology-directed repair) 4) Calcium imaging measures changes in intracellular calcium concentrations to indicate neuronal activation in the regions they occur 5) Single-cell RNA sequencingWhat is the main evidence that ultrafast endocytosis is not kiss-and-run endocytosis?-Kiss-and-run occurs at the site of neurotransmitter release; middle of the active zone -ultrafast occurs away from release site toward the lateral edge of the active zoneWhat is the evidence that ultrafast endocytosis is not clathrin-dependent endocytosis?-Watanabe found ultrafast was stopped after 20-100 milliseconds -flash froze cells and found vesicles still formed -clathrin coating needs more time to form -Horse radish peroxidase was used to visualize the endocytosisWhat is the KEY feature that distinguishes evoked from spontaneous neurotransmitter release?-Evoked requires action potential -Spontaneous can be triggered without action potentialAccording to the SNARE hypothesis, why is it necessary to remove the protein coat that was used to generate a vesicle from a donor compartment?-protein coats are recycled for the next round of neurotransmitter release -If the coat is not removed, the vSNARE would not be accessible to the tSNAREWhat are the five criteria that have to be satisfied to identify a protein as a bonafide neurotransmitter?-Must be synthesized in the neuron being studied -released from nerve terminals in a chemically or pharmacologically identifiable form -When administered, it should mimic post-synaptic actions of the substance released by nerve stimulation -Exogenous neurotransmitter and neuron stimulation should both be blocked by drug antagonist -A specific mechanism must be present to remove the substance from the synaptic cleftList three ways in which a one-stage nervous system differs from a three-stage nervous system:-One-stage contains only one type of neuron (sensory) -Three-stage consist of three neuron types: sensory, motor, and interneurons -One-stage organisms usually have radial symmetry and nerve net -three-stage have centralization and bilateral symmetry nerve cords -one-stage organisms have omnidirectional information flow -three-stage have cephalization found in three-stage nervous systems -cephalization allows specialized neuron groups in the head of the organismDescribe two ways in which interneurons contribute to complexity of the nervous system.-Allow convergence and divergence of signal processing -Convergence is when a single neuron receives inputs from more than one neuron -Divergence is when a neuron provides multiple outputs -Interneurons can also be excitatory or inhibitory -Interneurons can cluster to form pattern generators and pacemakers for heartList the functions of each of the four different parts of a neuron:-Cell body: Metabolic center (contains nucleus and other organelles for cell functioning) -Dendrite: Receives incoming signal from other neurons; contains receptors for neurotransmitters and spines to store memory (postsynapse) -Axon: Generates and carries electrical signals (action potential) to other neuron(s) -Axon Terminal: Transmits action potential via neurotransmitters to other neurons (presynapse)For a squid giant axon at rest, the membrane potential is approximately -65 mV. What ion channel is largely responsible for this membrane polarization of around -65 mV and why?-resting membrane potential is negative due to permeability to potassium -ions enter the cell through K+ channel -This allows membrane achieve equilibrium at about -65 mV -K+ channel is closest to the resting potential of the membrane.Both the Nernst equation and the Goldman-Hodgkin-Katz equation allow for the calculation of equilibrium potentials. Under what circumstances would each equation be appropriately useful? Provide one scenario for each equation.-Nernst equation can identify the equilibrium potential -where the electrical force is equal/opposite to chemical force of ionic gradient -Nernst should be used in controlled environments when one ion is permeable -Goldman-Hodgkin-Katz equation is derived from the Nernst equation -identifies the steady-state potential of a membrane with more than one ion permeable -Should be used to calculate membrane potential with relative permeability & [gradients]Choose one of the following articles: 1) What is the central hypothesis tested? 2) Summarize two major findings. Describe the evidence collected by the authors and explain how the data supports the conclusions drawn.Group 3: Negative feedback control of neuronal activity by microglia. Hypothesis: microglia-driven negative feedback of neuronal activation serves inhibitory function to prevent excitotoxicity. Finding 1: Microglia ablation increased striatal neuron synchrony Evidence: neuron synchrony underlies seizures. Microglia function raises the threshold for neuron activation by D1 agonist ex vivo to prevent seizures. Finding 2: ATP-AMP-ADO-A1R cascade plays a critical role in microglia-mediated suppression of D1 neurons in the striatum. Evidence: microglia-deficient mice had reduced ADO and increased response to D1 agonist after inhibition of PKRY12 (longer and more severe seizures).