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BIO482

GNU General Public License v3.0 licensed. Source available on github.com/zifeo/EPFL.

Fall 2017: Neurosciences II Cellular Mechanisms of Brain Function

[TOC]

Single-cell electrophysiology

• classification

• patch-clamp
  • pros : relevant to a neuron, time resolution $\mu s$, good signal-to-noise, inexpensive, quantitative ($I$, $V$ amplitudes, frequencies, slopes), deliver useful agents into the cell (dyes, proteins, DNA)
  • cons : spatial resolution, hard to record from > 1 channel, in-vivo challenge, need compensated capacitance
• eletric circuit : round isopotential resting cell, RC circuit
• ions
  • calcium : $Ca^{2+}$
  • potassium : $K^+$
  • sodium : $Na^+$
  • chlore : $Cl^-$
• current-clamp : membrane potential $V_m$ in response to stimulus current (controlled), measure passive properties, excitability, native $V_m$ at $I=0$

• voltage-clamp : transmembrane current $I$ needed to maintain command voltage $V_m$ (controlled), measure ion channel properties, synaptic current

• amplifier : $V_{out}=\epsilon (V_+-V_-)$
  • high impedance : $R_{in}$ ~$G\Omega$
  • gain : $\epsilon$ between $10^5-10^6$
  • command : $V_+$
  • negative feedback mode : $V_-=V_{out}$ giving $V_-\to V_+$
• two electrodes

• single electrode

• intracellular vs whole-cell : higher seal resistance ($10G\Omega > 500M\Omega$), small tip resistance ($4M\Omega < 100M\Omega$), fast ionic exchange, small cell
• glass pipettes : hard or soft glass, outer diameter 1.5-2mm, round smooth tip of diameter 1-2$\mu m$, low dielectric materal to reduce pipette capacitance
• configuration
  • whole-cell : suck, broken
  • cell-attached : suck, not broken
  • outside-out : suck, detached and closed
  • inside-out : suck, detached and opened
• filling solution ?

Transmitter release & presynaptic function

• efflux : eject compounds (neurotransmitters, toxic substance) out of the cell

• action potential : triggered by 40 mV depolarization above resting, with 10-fold increase as depolarization grows until a threshold

• vesicle : small structure of liquid enclosed by lipid bilayer

  • slowly releaseable pool
  • fast releaseable pool : ready to use

• E/IPSP : excitatory/inhibitory postsynaptic potential caused by E/ISPC current or spontaneous

• neuromuscular junction

• chromaffin cell : equivalent to postganglionic cells of sympathetic limb of autonomous nervous system
• admittance : equivalent of conductance in frequency domain, impendance the inverse
• endocytosis : active transport in which cell transports molecules into
• $Ca^{2+}$ channels

• postsynaptic receptors at excitatory synapses

  • AMPA : fast
  • NMDA : slow

  

• vesicular gluatamate transporter

• antagonist : molecule that interacts with membrane receptor increasing/decreasing another mocule effect

• ligand : molecule that can attach to another playing a precise role

• different proetins involved in the vesicle cycle

• giant synapse of squid : big enough to insert electrodes both on pre and post synaptic

• transmitter release : influx of Na+, efflux of K+, intracellular

  • Na+ : not required, but increase depolarisation
  • K+ : not required, repolarize the membrane
  • size of presynaptic deploarization : controls magnitude of transmitter release
  • Tetrodotoxin TTX : block voltage-gated Na+ channels, successive action potentials become smaller
  • Tetraethylammonium TEA : block voltage-gated K+ channels

• triggered by calcium influx

  • Ca2+ : must enter the cell to influence neurotransmitter release, porpotional to synaptic transmission, more abundant at presynpatic terminal, low concentration, voltage-gated, do not diffuse long distances because of calcium-binding proteins
    • two-fold increase : can increase transmitter released by 16-fold
    • intracellular : $10^{-7}$ M
    • extracellular : 2mM, four order of magnitude higher
  • calcium spike
  • dual function : carrier of depolarizing and special messenger
  • active zones : neurotransmitter released opposite to postsynaptic receptor, single per synapse
  • at some site, cooperative binding of several Ca2+ required to trigger release
  • Calyx of Held : large synapse in mammalian brain in auditory pathway specialized into rapid and reliable transmission, 1000 active zones
  • synaptic delay : lag between pre and post potential, 1-2 ms
    • Ca2+ : slower channels, start only when membrane begin repolarization, release in hundred $\mu s$
  • requiring ready state : Ca2+ must be ready to release beforehand
  • caging Ca2+ : light release and fluorescent
    • 1 $\mu M$ : enough induce release
    • $10-30\mu M$ : normal release
    • non linear

• released in quantal units : depends highly non-linearly on extracellular $Ca^{2+}$ concentration

  • quanta : discrete amount, produce fixed sized quantal synaptic potential, 1/s spontenous release
  • minitature end-plate potentials : spontanous noise, 0.5mV, can be enhanced by prostigmine or abolished by agents that clock ACh receptor, single ACh receptor $0.3\mu V$
  • action potential : 150 quanta of each 0.5mV in 1 ms
  • everywhere : except synpase between photoreceptors and bipolar neurons in retina

• stored and released by synaptic vesicles

  • vesicles : storage transmitter, each one stores one quantum, all-or-none in active zones, 40nm diameter
  • release : random, failure = no release, only small fraction (10%) of readily-releasable pool RRP
    • quantal output : $Np$ with $N$ quanta (usually 100-300) in nerve muscle, $p$ between 0.1 and 0.9
    • quantal size : $q$ in mV or pA if $I_{EPSC}$
    • mean : $E=V_{EPSP}=Npq$
  • docked vesciles : bound to active zone, immediate available for release
  • active zone : $0.1\mu m^2$

• synapse : each classical neuron only few synapse with any post cell

• Purkinje neuron : cerebellum, receive 10'000 terminals from single climbing fiber

• exocytosis of synaptic vesicles relies on highly conserved protein machinery, not in the exam

  • modulation of transmitter release underlies synaptic plasticity
  • fusion : 0.1-0.5 ms to empty, 10s to recycle
  • SNARE proteins : essential for mediating membrane fusion
    • dissaemble SNARE complex : SNP (solulable NSD-attachmed proetin), NSP (N-ethylmaleimide-senstive factor)

  

• calcium channel : CA, 6 blocks, 4 repetitions

• • discover : mussel cells, voltage clamp conditions while blocking potassential channel
  • equilibrium : there is a reverse at 50vm
  • n-type :
  • l-type :
  • genes : $\alpha$-subunit used in medecin to regulate blood pressure
  • Know by what their blocked and react to
  • High voltage activated HVA : expressed at higher density
  • Low voltage activated LVA
  • what is a taxonin, gene, protein
  • Exam : please give me the name of 4 ca channel, give me their type and which toxin blocks them, where cell ?

• potassium channel : K, 6 blocks

• sodium channel : NA, fast answer after SPS

• inwards : when positive current flow into the cell or negative ions that flows out, drawn downwards

• glutamate : most important excitatory

• small synaptic vessicle : 30-40nm

  • ATP : large protein, take a proton (H+ really?) and put its into vessicle

• description : dopamine et cie

• evoked EPSC

  • noise : passive vescile fusion, even in the absence of a stimulus

• active zone : group of vesciles, 3-5 dock, 0.1-0.2^2 $\mu m$

  • exclusion zone : around dock to get same response distribution, a possible model not proven
  • by chance between docks : produces low slope between release probability and $Ca^{2+}$ influx

• DM-nitrophen : capture Ca and release under light (uncage)

  • outside cell : 2mM Ca
  • Inside cell : 50nM
  • vesicle : 30-35nm
  • $E_{Ca}=\frac{RT}{zF}\ln\frac{[Ca]_0}{[Ca]_i}$ gives $+100mV$

• kinetic model : chain of action to trigger full fusion, each step can be reversed, $\gamma=6000Hz$

Electrical properties of cell membranes

• 1.1 Introduction

• 1.2 The cell membrane

  • stable intracellular environment
  • transmembrane currents : chaning eletrical field across plasma membrane
  • phospholipid
    • phosphate : head group, polar, hydrophilic, carry strong negative charges
    • hydrocarbon tails : non-polar, liphilic, hydrophobic
    • forms : spontaneous bilayer in water
    • permeable : gases, lipids, small non-polar molecules, water (limited)
    • impermeable : ions, charged molecules
  • typical cell
    • inside : high K+, $10^{11}$
    • outside : high Na+
    • area : $10\mu m$
    • elementary charge : $e=1.6\cdot 10^{-19}C$
    • volume : $1pl$
    • avogadro : $N_A=6\cdot 10^{23}mol^{-1}$
  • membrane : < 3-5nm, allow ions interaction, acts as capacitor
    • potential : $V=Q/C$ with $Q$ charges, $C$ capacitance, $1pC$ needs to move to generate $-100mV$ (6 millions ions), $I=dQ/dt$
    • capacitance : ~$1\mu F/cm^2$ (lipide), $10pF=10\cdot 10^{-12}F$ for area

• 1.3 Ion channels

  • transmembrane proteins : string of amino acids
    • can be charged : interact with lipid ?
    • alpha helix : need to have side hydrophilic, lipophilic part in the middle, per 5-6 bundled
      • subunits ?
    • hydrophobic domains
    • aquerons pore : allowing K+, Na+, Cl- (chloride) to pass
  • patch-clamp : electrodes with $1\mu m$ tip diameter with inoic solution attache to amplified
    • suck plasma : elecritc seal has only a leak of $1-10G\Omega$
    • study : suck ions channels
  • states : open/close state, as delta, $1\mu s$ timescale
    • Influence by : potential (eletric field), concentration of the ions (diffusion)
    • Voltage-current : can be linear or nonlinear
    • Ohm law : $V=IR$
    • conductance $G=1/R$ in siemens, usally $0.1-100pS$
  • highly selective : cations/anions or ions specific, $10^7$ ions/s
    • hydrated ions : need to dissassemble to get filtered
  • whole-cell current : add all ions channels, $I=NiP$ with $N$ number of channels (hundreads), single flow $i$ and $P$ open probability (ms by ms)
  • transporter : much slower, able to transport against electrochemical/concentration gradients sometimes using ATP,

• 1.4 Membrane potential

  • 2 forces :
    • membrane potential : attraction/repulsion
      • gradient : $F=-q\frac{dV}{dx}$
    • ionic concentration : $[K+]_o$
      • Nernst equation : $E_{K+}=\frac{RT}{zF}\ln\frac{[K+]_o}{[K+]_i}$ with $R$ Avogadro gas constant, $T$ temperature in Kelvin, $F$ Faraday constant, $z$ number of charge (+1 K+, -1 Cl-, +2 Ca2+)
    • reverse potential : no net flux
    • GHK equation : $V_m=\frac{RT}{F}\log_{10}{\frac{P_{K^+}[K^+]o + P{Na^+}[Na^+]o + P{Cl^-}[Cl^-]i}{P{K^+}[K^+]i + P{Na^+}[Na^+]i + P{Cl^-}[Cl^-]o}}$ with for example $P{K^+}=1$, $P_{Na^+}=0.04$, $P_{Cl^-}=0.45$
    • dynamic : $V=E_k(1-e^{-t/\tau})$ with $\tau=RC$ (1-5 ms)
      • depolarization : make more positive
      • hyperpolarization : make more negative

• 1.5 Cable properties

  • isopotential : small round, all parts same eletrical potential, not true for neurons
  • neurons : membrane potential differs according to location
    • arborisation : outgrowths filled cytoplasm, covered by membrane, diameter $0.1-1\mu m$
    • leaky cable :
      • steady-state : $\frac{d^2 V}{dx^2}=\frac{R_{axial}}{R_m}V$ giving $V=V_0e^{-(x/\lambda)}$ with $\lambda = \sqrt{R_m/R_{axial}}$ (can be $500\mu m$)
      • time-dependent : $\frac{R_m}{R_{axial}}\frac{\partial^2 V(x,t)}{\partial x^2}-R_mC_m\frac{\partial V(x,t)}{\partial t}-V(x,t)=0$ variying over time and space
        • time constant : $\tau=R_mC_m$, open many ions channel influence, 10ms
        • length constant : $\lambda=\sqrt{R_m/R_{axial}}$, thinkness influence, $100\mu m$

Excitability

• 2.1 Voltage-gated channels

  • linear channel : resistance, ohmic conductance
  • non-linear channel : depends on membrane potential
    • Four similar sub-units : sometime same or different proteins
    • S1-S3 : outside of protein, interact with bilayer
    • S4 : voltage sensing region, can move, positive charge (arginine amino acids)
    • S5-S6 : inwards, pole lining, in between is the selectivity filter

  

  • electric field interracts with S4
    • hyperpolarize : strong electric field from outside to inside, pushes doward towards interior of cell, close gate
    • depolarize : strong eletric field from inside to outside, opens gate
  • most important : sodium, potassium
  • values
    • $g_{Na}, g_K$ : ~20pS
    • leak : $R_{in}=50M\Omega$, $G_{in}=20nS$ dominated by K+
    • 1000 sodium channels need to have an impact

• 2.2 Voltage-gating kinetics

  • classical opening / closing : $1\mu s$
  • voltage gated opening/closing : longer, 200$\mu s$ for Na, long lasting for K+ delayed by 200$\mu s$
  • recovery for Na : 0.5ms small, 5s fully recovered
  • diversity : varies across differents celles in same species, impact activation
    • Sodium : encoded nine genes NaV1.1 to NaV1.9
      • N terminal
      • amino acids with sub-units
      • C terminal
      • alpha sub-unit
      • beta sub-unit : small, 4 different
    • location : impact on functionality
    • Calcium : 4 sub-units
    • Potassium : 80 genes, non-voltage gated, voltage-gated, calcium-gated (BK, SK), G-protein-gated (GIRK), Tandem pore channels
      • encode only 1 sub-unit : can mix gene in channels

• 2.3 The action potential

  • timescale all-or-none : 1ms
  • giant squid axon : 1000 larger in diameter (normal 100nm), 1mm
  • Hodgkin and Huxley : measures and models

  

  • refactoriness : available sodium voltage gated
  • threshold : $I_{Na} &gt; I_K$ densitives, activation and inactivation
  • AP : threshold varies following trajectory, typical -45mV

• 2.4 Action potential propagation

  • axon initial segment : AP initied at one unique location, 50$\mu m$ from the soma, high density of sodium voltage-gated channels

  • lowest voltage activation threshold

  • binds to : ankyrin G that binds to cytoskeleton, allowing clustered molecular scaffolds

  • axon : maintains spike by using self-reinforcing Na+ channels further forwards, backforward are blocked by refractoriness

  • speed : 1 m/s, 1mm/ms

  • increase : higher membrane resistance, low axial resistance, low membrane capacitance

  • myelination : specialised glial cells (oligodendrocytes and Schwann cells) wraps axon with 80% lipid (increase by 5000 thousands the resistance and decrease by 50 axonal capacitance), often 1mm long, can speed up to 100m/s

  • nodes of Ranvier : in between myelinated part, AP is reinforced

• 2.5 Whole-cell recordings : slices 300$\mu m$

  • in the exam ?
  • prepare brain slices : anestheise mouse, extract brain, place in ice-cold slicing solution
  • image neurons : incubate at 35 celisuis for 30 minutes, transfert to room temperature, place slice in microscope
  • prepare recording electrodes : fill pipette with solution
  • perform whole-cell patch-clamp recordings
    • search
    • cell-attached
    • whole-cell

Synaptic transmission

• 3.1 Synaptic transmission
  • neurotransmitters : one per axon, continuously discovered
    • glutamate : excitatory
    • $\gamma$-aminobutyric acid : GABA, inhibitory
    • acetylcholine : heart function, neuromuscular junction, muscle activation
    • dopamine : reward based, deficit important in Parkison/Schizophrenia, family of catecholamines
    • Met-enkephalin : 5 aminoacids
    • Oxytocin : 9 aminoacids
  • peptide
  • bouton : pre-synaptic specialization in the axon
    • calcium channels : activated by action potential and cause release of neurotransmitter
    • vesicle : by quanta, 40nm
  • snypatic cleft : neurotransmitter diffuse and binds to receptor, 50 nm
  • ligand-gated ion channels : open probability gated by presence or absence of neurotransmitter
  • postsynaptic potential : within 1 ms of action potential
  • excitatory synapse : release glutamate, activate postsynaptic ionotropic glutamate receptors permeable to Na+ (more) and K+ with reversal potential 0mV
  • inhibitory synpase : release GABA, activate postsynaptic ionotropic GABA receptors permeable to Cl- with reversl potential -70mv
  • synapse : 200-500nm
    • dark stuff : electron & protein dense that hold pre/post specialisation
    • chemical : ions, most of the time, point-point & fast
    • electrical : rare in mature brains, 10x drop of depolarization, formed by gap junction, proteins on both side of synapse where current can flow directly
    • leak : neurotransmittor in extracellular space causing other interactions
  • volume transmition : dopamine has no postsynpatic specialization and simplfy diffuse
  • retrograde signaling : endocannabinoids, nitric oxide neuotransmitters, regulate transmission the other direction round
    • dendrodendritic release : when on dentrite release vescile triggering other dentride release
  • exocytosis : vesicle release neuotransmitter content
• 3.2 Neurotransmitter release : involve 50 proteins
  • vesicle : need to dock and be release ready
  • calcium : peak 100$\mu s$ later voltage peak (duration of 500$\mu s$)
  • release rate : vesicle/ms, $4$ power (Hill coerfficient) depending on calcium concentration
  • fast fusion : need ready machinery, pull vescile close to plasma membrane, maybe prebound of membrane
    • SNARE complex : Synaptobrevin, SNAP-25, Syntaxin
    • Synaptotagmin : calcium sensor (five binding site for calcium), change its configuration and interacts with phospholipid and SNARE complex, open fusion pore
  • refilling : ATP depend process
    • kiss and run : 50-100ms
      • vacualar proton pump in ATPase : pump protons (hydrogen atoms H+) into vesicles
      • vesicular membrane potential : created by H+
      • neurotransmitter transports : take energy from electrical potential and acidification (e.g. VGlut)
    • kiss and stay : 50-100ms, after fusion, reseal and replenishes its content
    • fuse complety : part of membrane, can be endocytosed in KLAF independent manner
  • controls calcium influx
    • change action potential waveform : increase duration by reducing K+ current (will enduce a delay)
    • calciums channels : increase open probability
  • controls vesicle filling
    • vesicle pH and electrical potential : efficacy H+ pumping or transporters
    • vesicule size : 40nm
    • vesicle docking and priming : phophorilation events, change settings (distance between calcium), sensitivity
    • number of vesicle
• 3.3 Presynaptic dynamics
  • firing rate : silent for minute then burst, continuously firing at 100Hz all their life
  • activity history : change the amount of neurotransmitter, synpatic plasticity
  • short-term dynamics : 10 to 100 ms, immedialty depends on calcium
    • facilitation : accumulation of calcium (binding protein, buffers), less sucking out so calcium is not fully evacuated from specialization and go higher
    • depression : less time to replenish
  • post-tetanic potentiation PTP : minutes, complex signaling and calcium, only occurs at certain synpapses
    • need burst : 100 Hz for 1 s
    • protein kinase C, PKC : rised by calcium, targets protein Munc-18 by phosphorylation that increases the rate
  • long-term presynaptic plascticity : hours, days, complex signaling and calcium, only occurs at certain synpapses
    • need burst
    • adenylate cyclase : activated by calcium, turns ATP to cyclic AMP, that activates protein kinase A PKA that activates synaptotagmin 12 causes more releases
  • modelling :
    • refill : exponential recovery time constant
    • facilitation : exponential calcium decay
• 3.4 Presynaptic modulation
  • extracellular space : neurotransmitter diffusion, low time scale, 100ms with longer lasting effect (seconds, hours), 5$\mu m$ away
  • metabotropic receptors : also on button, seven-transmembrane with alpha-helicice, no ions but acts on internal GTP-binding proteins (beta and gamma), diffuse and acts upon ions channels or other enzymes
    • phospholipase C, PLC : split in two, inositol trisphosphate (release calcium from stores), diacylglycerol (active kinase C)
    • adenylate cyclase : increase cyclic ANP
  • presynaptic inhibition : almost all synapse, near the release site (not at active zone), G beta/gamma subunit can block Ca2+ channel or the release vesicle machinery
  • more neurotransmitter : acetylcholine, serotonin, dopamine
  • behavioural context
  • postsynpatic neuromodulation : has also metabotropic receptors, 0.5s timescale
    • G protein inwardly-rectifying potassium channels strongly affect by G protein
    • K_ir channel : strongly affect by G protein
  • clinical relevance : location highly specific, amenable by neuropharmcology
    • depression : fluoxetine/sertraline, selective serotonin reuptake blockers
    • antipsychotic : risperidone, blocking specific DA, 5HT, adrenergic receptors
• 3.5 Electron microscopy

Glutamatergic excitation

• 4.1 Glutamate receptors

  • 80% neuron rely on glutamate
  • vescile
    • per vescile : 10mM of glutate
    • fusion : per vescile, increase 1mM for 1 ms concentration in synaptic cleft
  • ligand-gated neurotransmitter ionotropic glutamate receptors : open probrobability depends on glutamate
    • four alpha helix : M1-M4, but M2 do not cross the bilayer
    • terminals : N, C not one same side
    • binding : encoded on the large domain near N terminal
    • Why four if not crossed?
    • permeable : sodium, potassium and sometime calcium

  

  • AMPA : Na+ (more), K+, reversial potential 0mV, conductance 5pS
    • kinetics : 1 pulse 1mM 1ms, giving only a few (2) ms current response, very difficult to have longer response
    • voltage insensitive : linear voltage-current relation
    • four genes : gria1-4 give rise to subunit GluA1-4, most of the time GluA1-2 or GluA1-3
    • GluA2
      • presence : linear voltage-current
      • absence : inwards rectification, saturate current at 0 for positive voltage, don't like current going outwards, permeable to calcium
  • kainate : receptors familiy, ressemble to AMPA but less defined function, smaller current, slower, also presynaptic specialization
    • five gene : grik1-5
    • subunits : GluK1-5
  • NMDA : Na+ (more), K+, Ca2+, need glycine or d-serinie presence in extracellular space (usually they are, but it could be regulating), reversial potential 0mV, conductance 50pS
    • calcium postsynapticaly : important in synaptic plasticity
    • kinetics : 1 pulse 1mM 1ms, 20ms delay to get maximum. remains active for hundread of ms after closing
    • voltage sensitive : conducatance blocked at negative potential, increase to maxium, but 0 voltage at 0 current
    • magnesium block
      • absence : linear voltage current
      • presence : Mg2+ block ion channel with 1mM Mg2+ with negative potential
    • seven genes : grin1 (required to work), grin2A-D, grin3A,B (common grin1, grin2A,B)
    • subunits : GluN1, GluN2A-D, GluN3A,B
      • GluN2A,B : strong Mg2+ block
      • GluN2C,D : weak Mg2+ block
      • GluN2A : fast, 100ms
      • GluN2B,C : medium, 300ms
      • GluN2D : slow, 1s
  • metabotropic glutamate receptors : 7 transmembrane receptors, coupled through G-protein activate sickling pathways like phospholipase or inihibt adenylate cyclase, not fully explored,
    • eight genes : slow sickling function, grm1-8
    • subunits : mGluR1-8
  • piezo actuator : two fluid with different concentration can be used to trigger glutamate pulse of 1ms with 1mM

• 4.2 Postsynaptic potentials

  • reliability : common-shape depends on the number of synapse
  • excitatory postsynaptic potential : can be 5mV or 0.5mV change

• 4.3 Glutamatergic circuits

  • mouse whisker map : same organisationa across individuals
  • barrels : cortex units processing a specific whisker on somatotopic map
    • representation : also in the brain stem and thalamus
    • layers : 1mm thick, 200-300$\mu m$ for one barrrel (C2)
      • L1
      • L2 : small pyramidal cells
      • L3
      • L4 : some small cells, main sensory input arrives there
      • L5a
      • L5b : much larger cells
      • L6
  • red light : impacts optical properties of the cortex, dark is active
  • record some neuron in a given layer : analyze probability of connection in the same layer and other layers
    • L2/3 : process information from middle layers
    • L5/6 : "integrator" processed information and send it elsewhere in the brain
    • distribution : few very strong EPSP, lots of weak EPSP
      • remove weak : small impact on computational properties
      • coefficient of variation : standard deviation / mean, strong ESPS are reliable
      • maybe genetics factors that skew the wiring diagram
      • maybe experience alters strengh of connections

• 4.4 Synaptic plasticity

  • presynpatic : ms, second timescale
  • postsynpatic : days, live
  • LTP induction
    • experimental protocol : typically 1-4x 100Hz during 1s of many axons
    • injecting depolarising current through whole-cell current postsynpatic at 0mV
    • pairing postsynpatic action potential firing with EPSP input, short current pulse at same time
    • requires : NDMA receptor activation, postsynaptic cytosolic calcium increase, activation of protein kinases (CaMKII)
  • protein kinases (CaMKII) : high density in postsynpatic region, adds phosphate groups to differents targets
    • change conductance of AMPA receptor
    • recruitement of additional AMPA receptor : floating around in plasma membrane, bind and hold them
  • LTD induction : similar opposite but not the exact opposite
    • experimental protocal : typically 1 Hz for 5-10 minutes stimulation of many axons
    • injecting weak depolarising current through whole-cell current postsynaptic to -50mV
    • requires : NMDA receptor activation, activation of protein phosphatases (calcineurin), at least cytosolic Ca required, unclear, maybe protein-protein interactions between NMDA receptor or other cycling proteins
    • protein phosphatases (calcineurin) : take phosphate away, dephosphorylation of AMPA receptors
  • spike timing-dependent plasticity
    • induction : 100 pairs repetition
    • positive time delta : strong effect 10-20ms
    • negative time delta : strong effect 50-100ms

• 4.5 Dendritic spines

  • protrusions : 1$\mu m$, 1fl, grow from parent dendrite, 1000 spines for typical cells
    • spine neck : 100-200 nm diameter
  • pyramidal cell : when mature, no glutamatergic input on main dendrite, only on spines
  • different shapes
    • mushroom : $1\mu m$
    • thin : same as diameter
  • post-synaptic density PSD : area contains dense matrix of proteins
    • PSD95 : scaffolding molecules to hold ligand-gated channels with other molecules
    • signal molecule
  • spine apparatus ER : not in all spines, endoplasmic reticulum, local protein synthesis, calcium store (buffered, can suck Ca2+ or release for plasticity)
  • spin function : limit diffusion (for biochemical reaction), might help synaptic plasticity to remain synapse specific
    • resistance : at some synapse, 1gigaohm, lead to different in potential at that location
  • highly motile : move around because of actin
    • microtuble : MAP2, dentrite full of it
    • actin : in detritic spines, polymerie and depolymerize, formation of spines
    • rewiring : can be shown by learning experience, changes of spines

GABAergic inhibition

• 5.1 GABAergic inhibition

  • avoid excitation explosion

  • GABA : gamma-aminobutyric acid

  • very specific : target speicfic ompartments like distal dendristes, axon initial segment, soma

  • vescile

    • synthesisis : take glutamate and transform it into GABA by cutting CO2 with enzyme GAD (cytosol or directly on vescile membrane)
    • package : by protein versicular GABA transporter VGAT by using proton and electical gradient to sucks GABA inside, 100mM

  • ligand-gated ion channels : outwards current

    • GABA_A : permeable to anions, chloride conductance, ms timing
    • chloride concentration : strong gradient between outside (120mM) and inside (5mM), reversial potential of -85mV

  • leaky chloride channels : on all cell membrane

  • active process to keep chloride concentration low : at soma, 12 transmembrane protein transport (KCC2) slowly chloride outside (transported together with potassium, 1-to-1 stoichiometry), energy provided by strong potassium gradient, different in dentritic and axonal membrane

  • early development : chloride concentration different, can polarize the cell, high 30mM inside, 120mM outside, because KCC2 expression low

    

  • GABA_B metabotropic receptor : via 7-transmembrane, G-protein activate potassium conductance, slow, at some distance from actuall synapse

    • GIRK : G-protein inward rectifying potassium conductance, delay 50ms, last longer 200ms (diffuse, multistep activation), question
    • inhibit : calcium influx, both postsynaptically and presynaptically on buttons, play important that reduce neurotransmitter release

  • glycine : GABA less used in brainstem and spinal cord, glycine works the same way as GABA_A

• 5.2 Inhibitory synaptic conductances

  • GABA_A channel : 10-30pS conductance, brief pulse, 1mM concentration, 10-20ms duration of 1ms pulse (solver than AMPA), 5 subunits
  • reverse potential : -75mV
  • to mesure current-clamp : resting potential is to close -75mV, need to depolarize the cell to see, inject 100pA of current
  • shunting inhibition : even if resting close to chloride reversal potential and no flow of current, there is a conductance which that act as if it reduces membrane resistance (membrane potential changer faster and is small)
  • target structure GABA_A
    • dentrites
    • soma
    • axon initial segment : form editing function, can output an AP or not
    • synapse : only in the spinal cord

• 5.3 Benzodiazepines

  • neuropharmycology : treating brain diseases

  • experimentally use : not useful for therapy

    • AMPA : CNQX, MBQX
    • NMDA : APV, MK-801
    • GAMA-A : picrotoxin, bicuculline, gabazine (antagonists)

  • Benzodiazepines : extensively use therapy, increase affinity of GABA-A receptor for GABA, total charge transfered is larger, highly specific

  • GABA binding site : alpha-beta

  • Benzodiazepine binding site : between alpha and either gamma2 or gamma3

    

  • amino acid sequences of GABA-A receptors : no effect upon a4 or a6 (a1-2-3-5 is fine) if there is not histidine but argine at position 101

    • sedation action : relax/sleep mediated by a1
    • anxiolysis : reduction in anxiety mediated by a2

• 5.4 GABAergic projections

  • neo-cortex : most short axon, a few hundreads of microns
  • other part : long range
  • neurons in reticular nucleus of thalamus : whiskers trigger glutamate in brainstem, propagate again in thalamus and then to the primary somatosensory cortex
    • nucleus reticularis : fast feedback inibition to thalamus
    • prevent AP firing outside a very narrow time window : if enough fired, trigger an inhibitory AP, millisecond precision
  • striatal projection neurons of basal ganglia
    • striatum : full of gabagertic neurons : receive both excitation from cortex and from higher thalamic areas
    • inhibit substantia nigra (SN) : tonicaly active gabargertic neurons, inhibit brain stem motor area and prevent the animal of moving, also send dopamine to striatum from (SN compacta)
      • dopamine : diffuse in extra cellular space to D1(direct path that inhibit SN, go pathway) to D2 (indirect path that inhibit globus pallidus that propagate to SN, no go pathway) on different neurons with different plasticity (learning signal)
      • Parkinson : caused by dopaminergic neurons degeneration, bradykinesia, deficit in D1
  • purkinje cells of cerebellum (little brain) : useful integrators that coordinate sensory motor actions
    • cerebellum cortex : inhibit deep cerebellum

• 5.5 Neocortical inhibition

  • neocortex : 85% excitatory, 15% inhibitory, local microcircuits
  • paravalbumin expressing PV : fast-spiking, hundreads of Hz maximum rate, AP half width 300us, 50% chance connectivity with excitatory neurons
    • what is expressing ?
    • glutamatergic pyramidal neuro : lower maximum firing rate, an order of magnitude lower
    • Parvalbumin expressing basket cell : sends axon close to soma in basket shape, fast hyperpolarizing IPSP mediated by GADA-A, large majority
    • Parvalbumin expressing chandelier cell : target axon initial segments, edit whether cell able to output AP
  • somatostatin expressing Sst : target specific distal dentritic, mostly in L1, receive pre-synpatic facilitation from nearby pyramidal cells
  • vasoactive intestinal peptide expressing VIP : inhibit other gabaergic neurons, dis-inhibition
  • neurogliaform cells : small compact cell with extremely dense axonal arborization close to soma, no obvious postsynaptic partner, release in extra cellular space
    • ​

Measuring and controlling brain function

• 6.1 Brain function and behavior

  • causal understanding of behavior : investigate brain functions at level of individual neurons and synaptic interaction, measure correlation (does not implies causality)
  • electrophysiology : recording electrodes, extracellular recordings of action potentials perturbations but invasive quality tradeoff
  • optical imaging : fluorescence microscopy, high resolution structural and functional mesures
  • optogenetics : optical control (light-sensitive protein), high spatiotemporal resolution, use genetics to put optogentic actuators in specific cell-types, ms resolution
  • small world networks : subnetwork
  • "We can falsify hypotheses, but we cannot prove them."

• 6.2 Man and mouse

  • 99% of genes coding for proteins : similar between mouse and human, ~80 mio years ago
  • neural cortex : same layers

  

  • subcortical area : similar as well, similar locations, similar genes, same receptors
    • striatum
    • thalamus
  • mouse brain : 1g, 10^8 neurons
  • human brain : 1500g, 11^11 neurons
  • mouse library : jax.org, mutants
  • differential gene expression : differentiate cell from brain, muscle by expressing different proteins, brain-map.org
  • recombinase : combine different DNA elements and make different genetic manipulations in higly targetted way
    • (Cre-)LoxP site : 34 nucleotide slong, surrounding another gene (flox genes) and it will be removed, use promoters that express in specific cell types
    • Flp-FRT
  • Morris water maze
  • introns ? : part of genes that are not expressed exons

• 6.3 Imaging the brain in action

  • voltage-sensitive flurescent dye VSD : linear, $\mu s$, inside/outside cell, not in vivo
  • population of neurons : whiskers via craniotomy
  • brain scatters light strongly : maximum depth 1mm
    • blue light, 450nm : ~50um scattered from surface
    • near infrarred, 900nm : mean path length of ~300um
  • single-photon excitation : amount of fluorescence proportional to photon density, amount of fluorescent will be the same at the focal point and neighborhood, leading causing out of focus light, impossible to get high resolution imaging, 450nm photon energy
  • two-photon excitation : nonlinear using two low enegy photo ~900nm, 1 fs, amount of fluorescence proportional to photon density squared
  • cranial window : in vivo
  • spine plasticity : comes and go
  • calcium fluorescence sensors dyes : low fluorescence at 100nM, strong at 1uM
  • summary
    • epifluorescence : ~100um, 1ms with voltage-sentive dyes
    • two-photon : ~1um, 100ms
    • limted depth : invasive alternative, endoscopic techniques

• 6.4 In vivo electrophysiology

  • resolution : temporal kHz, spatial precise

  • extracellular space : 20% volume, not exactly zero but some pertubations caused by small conduits involving some resistance

    • local field potential : synpatic potential, from extracellular space to cell
    • action potential
    • recording : one tip around 50um around
      • electrode array : utah array, space by 400um
      • silicon probes : neuronexus, space by 50um, like a sword, triangulate

  • in vivo : mechanical stability, 1um movements

    • whole-cell : fill cell with dye, AP mean 1Hz and median 0.1Hz (resting mices), synchronized state correlation (resting mices) of membrane but no AP correlation (slow fluctuation 1-5Hz with amplitude of 10mV)

  • cell-type specific membrane potential dynamics : 85% excitatory (10x more) vs 15% inhibitory (but fires 10x more)

    • excitatory pyramidal : slow membrane fluctuation, low firing rates
    • parvalbium-expressing fast-biting GABAergic interneurons : slow membrane flucutation, high firing rates
    • non-fast GABAergic : slow fluctuaitions, intermediate firing rates
    • somatostatin expressing GABAergic : no slow fluctuation, high firing rates, maybe receive strongly facillitating glutamatergic inputs, very high input resistance 200 MOhm

    

• 6.5 Controlling brain function

  • optogenetics
  • light-activated cation channel : Channelrhodopsin-2 ChR2, cloned from green algae, retinal, light gates the channel, ms timescale, rather no selective (K+, inward Na+, inward Ca2+), excitatory with resting at 0mV
    • 10ms light pulse, low jitter, reliable reaction
  • single-cell stimulation : two-photon excitation, ms precision
  • inhibition : NpHR (chloride pump), Arch (proton pump), iC1C2 (chloride channel), halorhodopsin (chloride pump good with sst neurons as high input resistance)
  • ongoing research : 1ms pulse give longer response, need to shorten this, different light turns off/on, spectral variances, target (dendrite, soma, axon)

Mechanis of brain (dys)-function

• 7.1 Sensorimotor interactions

  • behaviour : controls of muscles contractions
  • sensory input : key
  • active senstive : select sensory information we want to obtain by goal directed movements
  • loop : neuronal computation, motor output, real world interaction, sensory input
  • whisker movements : chemical synpase, acetylcholine receptors, muscle excitable (AP)
    • protraction : intrinsic muscles, wM1 cortex involves exporatory whisking
    • retraction : extrinsic muscle nasolabialis, wS1 cortex involves negative feedback

  

  • locate motor neurons : use genetic engineered virus, inject in muscle and transported to neuro, expressing mCherry/GFP colorization,
  • motor neuron : generally clustered
  • premotor neuron : complement with missing factor HSV-G that allows to jump one synapse back, widely distributed
  • active touch : cell dependent encode
  • cortical sensorimotor integration

  

• 7.2 Sensory perception

  • we learn to see the world
  • sensory percept : must be reported through motor output
  • psychophysical threshold : stimulus strength half-maximal
  • optogenetic whisking : first trained, then responsed at light, or trained with light and worked out in the classical situation
    • activity in S1 : necessary and sufficient for whisker stimulus
    • membrane potential : correlates of perception
    • late depolarisation contributes to perception

• 7.3 Learning

  • reward-based learning : sensory input (context) -> synaptically-connected neuronal network -> motor output (action)
  • midbrain dopamine neurons : AP firing following up a reward, move to the earliest predictor, project on striatum, diffuse DA
    • VTA : ventral tegmental group, signal to frontal cortex
    • SNc : substantia nigra compacta group
  • striatum : receive lots of excitation from neocortex
    • ventral area : nucleus accumbens, targetted by VTA
    • dorsal area : targetted by SNc
  • dopamine receptors : trough G-proteins
    • type 1 D1 : Gs, Goff, stimulate adenylate cyclase that increase pdouction of second messenger cyclic AMP, promotes AMPA/NMDA insertion, LTP
    • type 2 D2 : Gi/o, inhibit adenylate cycle, decrease AMP, prevent calcium entry through NMDA, LTD
  • striatal project :
    • D1Rs receptor : expressed via D1-cre recombinase, direct pathway (dSPNs) to inhibit substantia nigra reticulata, go signal
    • D2Rs receptor : expressed via D2-cre recombinase, indirect pathway (iSPNs) to inhibit globus pallidus that then inhibit also substantia nigra reticulata, no-go signal

• 7.4 Brain dysfunction

  • Parkinson : 1.2mio people, cost 14 bio euros, age-related, slow, progressive neurodegenerative disorder
    • symptoms : slowness of movements, diffuclty in walking, saking, rigidity, dementia, depression
    • degenarting neurons : cytoplasmic, protein-rich inclusions termed Lewy bodies which contaion alpha-synuclein
    • less of dopamine neurons : 60% SNc loss, 80% striatal dopamine loss
  • environmental influences : intravenous drugs contaminated with methyl-phenyl-tetrahydropyridine MPTP induce Parksinon-like, metabolised to cation 1-methyl-4-phenylpyridinium MPP+, pesticides and insecticides in rural areas
  • genetics influence : increased chance associated
    • alpha-synuclein : mutation A53T, REP1 dinucleotide
    • LRRK2 : leucine-rich repeat kinase 2, mutations giving aberrantly increased kinase activity
  • treatments
    • L-DOPA : cross blood-brain barrier and increase dopamine, serious side-effect (unwanted movements, change character)
    • deep brain stimulation : subthalamic nucleus STN, high frequency, mysterious, side effects, 100K people implanted

• 7.5 Concluding remarks