Week 8 - AMPAR trafficking then vs now

theta burst stimulation protocols

instead of using very high frequency bursts of activity, use smth more physiological, use patterns normally found in the brain particularly within HPC when an animal is moving, a theta burst is bursts of activity but within 200ms intervals between bursts,

primed burst stimulation

variation of theta burst, one large depolarization 1st then theta burst after that

measuring theta burst stimulation

the stimulating electrode is in shafffer collateral axons stimulating at different activity patterns, you do a test stimulation 1st to get baseline communication between both neurons then high frequency theta burst happens then next stimulaton is successful if you see LTP, LTP in HPC CA1 neurons depends on NMDA receptor activation allowing Ca2+ influx into post synaptic neuron, (no nmda = no ca2+ = no ltp), end result of this is bigger response from same recording neuron

TAT

when you attach to it wtv is bound will diffuse into the cell via micro-pinocytosis via argining rich residues (attach to GluN2A), enters into cell so excess allows PDZ to not interact with GluN2A so gets released, it acts as a molecular decoy derived from HIV, mainly acts as a carrier

quantum dot

fluorophore that allows us to see NMDA receptor with GluN2A attaching to outside of receptor, tracks movement of NMDA, its on antibody that recognies N terminal of N2RA

PDZ domain

locks NMDA in place so it wont move, when NR2A (same as GluN1) binds instead it competes out post synaptic density so NMDA is no longer locked in place and you get lots of movement (not locked to PDZ motif anymore), TAT basically scrambled sequence so you get no binding to Glu2NA region,

N2RA

its involved in a synapse, mature version of NMDA, when bound to TAT it allows lots of movement to happen

post synaptic density

proteins that anchor NMDA (ex: PDZ motifs)

Glu2NA

would increase TAT surface expression following tetanic stimulation

negative LTP regulators

low frequency stimulation induces LTP, KO gene causes LTP to appear with low frequency stimulation

calcineurin

promotes dephosphorylation of proteins in LTD, KO removes brake causing LTP (inhibits LTP)

PKMzeta

maintains LTP so KO blocks LTP maintenance, it gets transcribed but only at active synapses will it cause a signal to stay at the synapse long enough (acts as a long lasting signal)

GluA1

AMPAR for LTP expression, KO prevents LTP not enhancing it

GluA2

AMPA receptor affecting synaptic plasticity, not explaining low frequency stimulation

post synaptic densities and tracking

lateral movement and diffusion model allows us to see receptors are actually moving, no one has observed insertion of receptors reliably in the synapse, moving into the lateral portions first suggests receptors are binding outside of synapse then moving in but this is not actually proven

AMPA delivery

the reason why we get strengthening of the synapse

how do you visualize AMPA and NMDA synaptic plasticity in vivo?

AMPAR trafficking to and from synapses is a highly dynamic and regulated process that mediates certain kinds of LTP (move towards membrane) and LTD (move away from membrane), increases in AMPAR function/numbers at synapses are thought in LTP while removal of synaptic AMPAR leads to LTD

whiskers, barrels and movement

manipulation of chronic whisker experience through stimulation previously shown to regulate AMPAR content and subunit composition in the mouse barrel cortex in ex vivo slices but not in an intact animal model, so if you repeatedly stimulate whiskers, every single one has been mapped out to an area of the cortex, change in activity + synaptic strengthen after repeated stimulation of the cortex (easier than HPC cause at brain surface)

electroporation

used it in utero to transfect layer 2/3 pyramidal neurons in mouse barrel cortex with AMPAR GluA1 subunit tagged with a ph-sensitive form of GFP (Super ecliptic phluorin SEP), the AMPAR GluA2 subunit tagged with myc (identifies injected forms of AMPA) and the morphological marker dsRed2, electroporation basically delivers cells in the nucleus using electricity, so did the DNA injection of GluN1, then electroporation of HPC and motor cortex putting tiny electrodes on sides of cortex driving DNA across it, cells then uptake it to deliver different genes

electroporation whisker experiment findings

at acidic pH of approx 5.5, phluorin fluorescence is quenched, when vesicles fuse to the plasma membrane the lumenal tag is exposed to the extracellular pH resulting in marked increase of fluorescence, so if you have GFP in trafficking vesicles, its more acidic so not expressed in cell surface cause more neutral

whisker stimulation

increase in SEP fluorescence within dendritic spine of neurons due to increased insertion and surface expression of AMPARs at post synaptic membrane

how to visualize labeled AMPARs in vivo

leave cortex open so they visualize in real time, stimulate whiskers with 5hz which is enough to elicit strengthening of mice barrel cortex, they used in vivo two photon microscopy in the mouse somatosensory barrel cortex, authors found acute whisker stimulation led to significant increase in intensity of surface AMPAR GluA1 subunit of both spines and dendritic shafts and a small increase in spine size relative to prestimulation values, using that pHluorin

mouse primary somatosensory cortex

same as the barrel cortex, contains a somatotopic map in which each individual whisker is represented as discrete anatomical unit or barrel, allowing precise delineation of functional organization development and plasticity, deflection of the D1 or B1 whisker evokes a localized change in reflected red light resulting from coupling of blood flow to neural activity, so you inject DNA then do craniotomy removing part over cortex to visualize it observing blood flow changes, the spine is where we see AMPA insertion so no fluorescence inside just on cell surface

AMPARs stability

the ½ life of the protein receptor is 12-24hrs, it says the same, strengthening after LTP leading to preservation up to 28 days indicating long term stability of GluA1

NMDARs

the induction of LTP is dependent ion activation of NMDA, injecting CPP inhibits NMDAR blocking at the synapses so you dont get increase in cell surface expression, this blocks AMPA insertion even after stimulation so get no fluorescence,

what are the 3 possible mechanisms AMPARs reach synapses during LTP?

direct insertion into the synapse, extrasynaptic insertion followed by lateral diffusion (happens before being captured by postsynaptic cells) and vesicle delivery targeted near dendritic spines

what is halotag?

self-labeling protein tag engineered from bacterial enzymes, forms a covalent bond with synthetic fluorescent ligands, allows bright, stable labelling of proteins in living cells, compatible with high resolution and single molecule imaging (easy to make, compatible cause microscopy has advanced a lot)

halotag in endogenous receptor tagging

CRISPER inserts the HaloTag sequence into native AMPAR gene (ex: GluA1-HaloTag created), receptors expressed at natural levels so all were doing is adding tag, avoids artifacts from receptor overexpression,

labelling halotagged receptors

add halotag ligand conjugated fluorescent dye, ligand binds halotag covalently to some binding group like chloroalkane so it wont come off once bound, sparse labelling allows visualization of individual receptors, compatible with super-resolution microscopy (seeing things at individual molecule level), fluroescent ligands are dif colors and can be cell permeable or non permeable, surfaces include nonmagnetic or magnetic resin and glass slides, reactive ligands will attach functional group of choice

why use viral CRISPER in neurons?

neurons are post-mitotic and hard to transfect using traditional methods (ex; lipofectin/calcium phosphate), viral vectors efficiently deliver DNA, viruses are neurotropic, AAV is commonly used for neuronal gene delivery, allows editing in specific brain regions (use in HPC, cortex, etc)

CRISPER components needed

viral DNA enters nucleus, Cas9 expressed, Cas9 + gRNA target gene, DNA cut and donor template inserted, Cas9 enzyme cuts DNA, guide DNA targets gene locus, donor template inserts halotag

SpCas9 mouse

most common mouse type used in CRISPER, its preferred because each enzyme homes within specific sequence, have dif DNA types in virus delivering Cas9 that releases it

packaging CRISPER into viruses

the AAV cargo capacity is limited to 4.7kb (pretty small), often have to use a dual virus system where the first virus expresses Cas9 and the second is a guide RNA + donor template sequence for halotag, both viruses infect neuron, deliver into specific area of the brain, applies to optogenetics and lineage tracing

how do we deliver virus to the brain?

stereotaxic injection into brain regions, infection of cultured neurons or slices, enables targeting of specific neuronal populations to express halotag resulting in endogenously tagged AMPARs (GluA1 gene after CRISPER tagged form GluA1-Halotag protein, tagged receptors expressed physiological levels and receptors can now be labeled with fluorescent dyes)

single particle tracking

label halotag receptors with bright dyes, sparse or low numbers of molecular labelling reveals individual particles therefore individual receptors, high speed imaging captures receptor trajectories and enables measurement of receptor diffusion, it provides better info on receptor diffusion in membranes, synaptic tracking at dendritic spines, insertion + lateral diffusion and changes during LTP or LTD

AMPA receptor visualization methods

SEP imaging (detects surface insertion), single particle tracking (tracks individual receptors and reveals nanoscale receptor dynamics

electrophysiological AMPAR generation

field EPSPs and whole cell recordings, detect LTP/LTD changes in synaptic strength, indirect inference about receptor changes

SEP-GluA1 imaging

ph sensitive fluorescent tag reports receptor surface insertion, visualizes bulk AMPAR trafficking in dendritic spines

CRISPER + Halotag + Single Particle Tracking

endogenous receptor tagging using CRISPER, track individual receptor movement and synaptic tagging

systems consolidation

where synaptic strengthening that has occurred within the HPC is then transferred to other parts of the brain as a memory trace, it translates short term memories into long term ones, HPC is good for learning but memory storage happens somewhere else

how does systems consolidation work?

first a novel experience is brought into HPC then sent to other parts of the cortex (ex: fimbria-fornix), as the info or experience is used more frequently, passing info between HPC to other regions is strengthened, then at some point memories are permanently associated with these cortical neurons only, becoming HPC independent, this process is protein synthesis dependent, also requires activation of CaMKII and CaMKIV

what we know and don’t know about systems consolidation

believed that HPC will activate these particular groups of neurons in the cortex when it retrieves a memory but we dont know how, some think HPC region is only involved in consolidating memories that last less than 10yrs but others argue entorhinal cortex (damaged in AD) as being more important in LTMs lasting a life time, also evidence that entorhinal cortex is involved in more repetitive or incremental types of learning

current long term memory hypothesis

current hypothesis is that long term memory formation requires LTP, synaptic tagging and eventual protein synthesis, use of actinomycin D (transcription inhibitor blocking new mRNA production affecting LTM, tests if new gene transcription is required)

what is a memory trace/engram?

current view of memory is that it is a network of synapses that store a memory that has been encoded within a discrete group of neurons, this network of neurons and the group of synapses that is associated with a memory is the memory trace/engram, disrupting it altered specific forms of memory

bZIP

beta-PKMzeta inhibitory peptide), measures maintenance of synaptic strengthening,

STDP

occurs during LTD not strengthening, NMDAR downstream, requires CAMKII activation, involves internalization of AMPA, form of synaptic weakening cause it decreases EPSP amplitude

PIN1

overexpression would inhibit local PKMzeta mRNA translation