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1 Rachel A. Kaplan and Elbert Heng Final Exam ReviewRachel A. Kaplan and Elbert Heng
2 Announcements Your final is tomorrow; get hype! Things you should bring:A calculatorSome pencils (or pens, if you want to be bold)Your brain!
3 What this review today will cover: As your exam is tomorrow, hopefully this isn’t the first time you’re going to be reviewing materialaccordingly, we will be:going over important topics and difficult conceptsanswering any questions you haveproviding moral support
4 Musings… You should study material that was tested on previous exams You should study material that was not tested on previous examsMost importantly:You should know the big important concepts that we’ve coveredYou should also spend time to review some detailsLook at our old slides for more comprehensive reviewIn a nutshell: know everything.We’ll help you on which of the details you need to know (things that are in charts or we’ve told you to memorize in the past).
5 Be prepared to think…You have all the knowledge you need to answer these questions (all within the scope of the course) – just think hard and reason it out! The questions are designed to be answered either straight from knowledge or built from first principles.
7 THE BIG PICTURE: FIRST THIRD Fundamentals of synaptic transmission from an electrophysiological perspectiveImportant TopicsIon Channels!Membrane Potentials - The Nernst EquationThe Action PotentialMembrane PropertiesSynaptic Transmission
8 THE BIG PICTURE: SECOND THIRD A little bit of everything, but mostly synaptic transmission from a molecular and cellular biological approach with electrophysiological implicationsImportant TopicsVesicles: exo and endocytosisIndirect synaptic transmissionMechanosensationBehavioral NeurobiologyDendritesElectrical Synapses
9 THE BIG PICTURE: THIRD THIRD Plasticity and all its friendsIMPORTANT TOPICSLTP and LTDIntrinsic PlasticityLearningDevelopmentLTP and AddictionThe third paperOn Kauer’s Lecture: review the slides
10 Ion Channels Ion channels pass ions This is studied with electrophysiological techniquesVoltage ClampCurrent Clamp(and putting these two together) I/V PlotsSingle Channel vs. Whole Cell RecordingSingle channels are constantly flickering open and shut; the population of channels will reflect the state of the cellGatingModulation of Gating
11 ElectrophysiologyThis is CURRENT CLAMP – clamp current (inject current) – measure change in voltage of membrane as response.LOOK AT THE AXES
12 ElectrophysiologyTHIS IS VOLTAGE CLAMP – note the multiple voltages at which the membrane was clamped. The lines represent inward and outward currents measured as a response.This one doesn’t have axes, but you do have a clue to what’s going on as it shows the manipulations of membrane potential.
13 Electrophysiology I=Vg IV plots are extremely important to understand. They show channel characteristics. They let you see at x voltage the current will be y. MOST IMPORTANTLY: they show size or direction of current that flows at a given voltage.Which properties of the channel are shown in the plot?Reversal potential (+58mV here) – DETERMINED BY THE CONCENTRATION GRADIENTS OF IONS (NERNST EQUATION, COMING UP NEXT!)Conductance (slope of line) – conductance is a measure of how many ions a channel CAN PASS – distinct from how good a channel is at passing current. Therefore conductance depends on the ability of the channel to pass ions and the number of ions available for the channel to pass.This chart on this slide shows either a single channel’s IV plot or a non-voltage gated channel. What would a voltage gated channel’s IV curve look like? (See the next slide!)I=Vg
14 Electrophysiology This is a voltage gated channel’s IV plot. It shows that below a certain voltage (-60mV) it will not pass current in either direction.Let’s think about how an NMDAR’s I/V plot would look – it is a channel that is ligand gated (needs Glu) and voltage gated (needs depolarization to get rid of Mg++ block)It’s reversal potential would be different – because it’s permeable to both Na and K, (+58, -60mv respectively) it’s reversal potential (x-intercept would be at 0mV – the origin).
15 Gating and Modulation Gating: how the channel opens and closes S4 is the voltage sensor for VG channelse.g. Glu gates NMDARs and AMPARsModulation: changes the open probability of the channelMODULATORS:Other subunits of the protein (beta subunits)Second messengersChanges in gene expressionPhosphorylationAllosteric regulatorsThings that gate are required for channels to openModulators don’t open channels
16 Nernst Equation vs. GHK Nernst: Single ion’s equilibrium potential. Equivalent to Vrev if a channel is singly selective for that ion.GHK: Combined equilibrium potential of all relevant (permeant) ions.Can give you the RMPAlso can give you Vrev of multi-ion channels.So what could change the RMP?Altering ion concentrations!An example….Decreasing Sodium Extracellularly – decrease inward currentDecreasing Sodium Intracellularly – increase inward current
17 Membrane PropertiesAll serve to modulate the speed of an action potentialMembrane resistance (Rm)Membrane capacitance (Cm)Axial Resistance (Ra)Derived from these:Length Constant (λ)Time Constant (𝜏)All of the equations will be given to you if you would like to see the relationships written out…Membrane resistance – determined by leakyness- channel composition of membrane, presence of myelinationMembrane capacitance – don’t worry about it too much, just think of it as a place where current goes before current goes through channelsAxial resistance – an increase in axial resistance will decrease the current that can flow through the neurite (more resistant on the inside because few ions present, if the axon is thinner– has a smaller cross sectional area)Length constant – a longer length constant means that a given depolarization will cover a longer distance on the axon.Time constant – a larger time constant means that a given depolarization will travel for a longer period of time on the axon.Both of these are functions of capacitance – so the bigger the capacitance – the larger the two of these values both are.
18 Synaptic Transmission Llinas’ experimentProved that calcium was necessary and sufficient for presynaptic transmitter releaseDepolarization is not sufficient! (if no calcium, no go)Quantal HypothesisQuantum is a vesicle of neurotransmitterQuantal content - how many vesicles resleased!Quantal size – content of a single vesicle – how much NT is in itContent = mean EPP / average quantal sizeELBERT HERE
19 Mechanosensation Mechanosensitive neurons: Lots of receptor subtypes Generally: stretch-gated channels tethered to intra and extracellular matricesFast, sensitive, adaptable (so that it can transduce a wide range of inputs), and specializedLots of receptor subtypesE.g. Pacinian CorpusclesRespond to vibration because they are fast adaptingNeuron is surrounded by epithelial cells that form many layers of gelatinous membranes called lamellaePressure on causes neurons to firePressure off also causes neurons to fireIn other words, they adapt
20 More Neurons/Proteins Involved Degenerin/ENaC ChannelsRespond to stretch/mechanical stimulation – slow adaptingMeaning that they will stay open if they are continuously pokedCEP Neuron ChannelsSenses viscosity of surrounding bacteriaRapidly adapting cation channelsTRP-4: mechanosensory channelOther TRP ChannelsSense temperature, chemical tastantsTRP – transient receptor potentialTRP channels were discovered to be channels because alterations in their pore sequence caused a change in the channel’s I/v curve – so if changing the i/v curve occurs with changing the protein, the protein is responsible for that i/v curve and therefore a channel.
21 TRP ChannelsNote the structure of the channel – single protein
23 Hearing and Proprioception Vibrations of air are transduced by mechanosensory hair cellsStereocilia are deflected, links between stereocilia are stretched, allows K+ inward current to depolarize cellDeflecting the other way will hyperpolarize the hair cellStereocilia adapt by tightening tip linksMovement of head in space is transduced by similar hair cells in other organsUtricle and sacculus – linear acceleration moves gel and crystals (otoliths), causes opening of hair cellsSemicircular canals – rotational motion causes fluid in canals to move ampulla and embedded hair cells
24 Behavioral Neurobiology Responses to releasing stimulie.g. Egg RollingStimulus (egg) triggers fixed action patterne.g. Seagull Chick FeedingStimulus (spot color) triggers peckingSupernormal stimuli: allows us to study nature of what an animal is actually responding to in a stimulusReleasing stimulus: a stimulus that triggers a stereotypic behaviorFixed action pattern: stereotypic, no end point for success, no check point during behavior, once initiated, must be executed in its entiretySupernormal stimuli: a stimulus that is bigger/better/sexier than the normal stimulusWith stimuli, we can study the dimension of the releasing stimulus that actually is triggering the behavior.With egg: just that it looks like an egg, and how eggy it is (the bigger and egger it is, the more it triggers the behavior)With seagulls: REDness of the spot determines how good of stimulus it is, not how realistic the cardboard cut out of the parent is
25 Electrical Synapses Channels are composed of two Connexons Connexons are HemichannelsThey are in turn composed of 6 connexinsIf all 6 connexins are the same protein: homomericIf different: heteromericMost common connexons in the brain:Cx43 – Glial cellsCx36 – Brain neurons (perhaps the only connexon that is expressed in brain neurons!)
26 Electrical Synapse Physiology GJ provide high conductance pathway for ionic current to pass from one cell to anotherOhmic (no voltage gating)BidirectionalAlso pass small molecules like ATP, cyclic nucleotidesSo what would the electrophysiological recording of stimulation of a neuron that connected by a GJ to another neuron look like?Let’s take a closer look at the trace below… clearly shows spiking is bidirectional but greatly attenuated (diminished).
27 Gap Junction Evolution Pannexins / Innexins and Connexins are orthologuesNo sequence similarity but in teritiary structure are very similarInvertebrates do not express connexinsInnexins and connexins can form GJs or functional hemichannelsPannexins only form hemichannels
28 Learning LTP and LTD are putative cellular mechanisms Shown with lots of experiments
29 Rabbits? The process of associative learning uses this circuit Input: sensory motor – tone- parallel fibersAlso excites pons and deep nuclei directly (there are two pathways)Input: “error” signal – shock – climbing fibersOutput: motor command- eye blink – purkinje cellsLTD occurs in parallel fibers which means less inhibition of deep nuclei from purkinje cellsEasier to express blinking behavior!
30 Know this pathway! Memorize the circuit and the nature of each connection. Stimulating the parallel fibers would be a substitute for the tone. Do this with other fibers! (e.g. inferior olive, purkinje cell).
31 Lashley Searched for the engram Equipotentiality Mass Action Equipotentiality: All parts of the cortex contribute equally to learning; one part can substitute for another part.Mass Action: The cortex works as a whole; performance improves when more of the cortex is involved.
32 Development of Circuits is: Activity IndependentSperry: chemoaffinity hypothesisExperimentsEye rotationRetinal ablationStripe assayMechanismEphrins and Eph ReceptorsActivity DependentHebb: correlation based changeExperimentsRewiring of A1/V1MechanismSynapse maturationLTPDepolarizing GABAActivity dependent gene expression
33 A little bit of both…Ocular dominance columns start to develop before eye opening but require activity to segregate more completelySpontaneous retinal waves may be responsibleOcular dominance shift: Monocularly deprived animals develop ocular dominance stripes but the open eye’s stripes are much wider
34 Putting it all together: Neural development is influenced by both activity dependent and independent factorsMuch of original structure is dictated by activity dependent factorsRefinement comes from activityThis is a result of LTP-like mechanismBut in general, it’s hard to say which causes which feature…
35 Activity Independent Experiments Eye rotation in newtRotation of the eye of an adult newt will cause the newt to see the world upside-down because in the adult brain, the retino-tectal connections don’t rewire, little plasticity. Previous projections from a part of the retina now project to the “wrong” part of the tectum.Retinal ablationAblating half of the retina will cause missing connections in half of the tectum. The persisting retinal half will not rewire to take up the whole tectum.Stripe AssayNeurons from temporal retina will only grown onto membrane stripes from the anterior , and nasal retinal neurons will project through both (as it has to to get to the posterior tectum!)
36 Activity Dependent Experiments Rewiring of ferret cortexRewiring of retinal projections to the MGN (after deafening the ferret) will cause A1 to have V1’s features like orientation pinwheels and long horizontal connection.Formation of eye specific stripesThey don’t form if APV is perfused!
37 Mechanisms Ephrins and Eph Receptors Chemical gradient that guides neuronal projections from (e.g. retina to tectum) specific regions of one neural area to another specific regionAxonal Segregation / Map RefinementSynapses that fire together wire together, so synapses become refinedAccomplished via LTP (requires NMDAR activity)Synapse MaturationNMDA only synapses become unsilenced as a result of LTP (insertion of AMPARs) – change in NMDAR/AMPAR ratioDepolarizing GABA also aids in unsilencingGene Expressione.g. cpg15 is induced by neural activity and regulates synaptic maturationGABA is depolarizing in immature synapses because of high levels of Cl- intracellularly in immature neurons (negative charges flowing out would cause depolarization).