long-term memory Flashcards
steps from DNA to protein (3)
- transcription: DNA transcribed to mRNA in nucleus
- translation: mRNA transported from nucleus to cytoplasm (or distal sites); translated in cytoplasm by ribosomes
- PTMs: changes (phosphorylation) to protein
difference bw STM and LTM in terms of transcription/translation
LTM require transcription/translation, STM don’t
inducing STF and LTF with shocks/5HT
STF: one shock/one application of 5HT to isolated sensory-motor neurons -> lasts about 30 min
LTF: 5 spaced shocks/applications of 5HT -> lasts at least 24h; multiple training days leads to LTF lasting 1 week
molecular mechanisms of memories that last different times (2)
STM -> phosphorylation
LTM -> phosphorylation, translation & transcription, new synapses
arguments against ‘transcription required for memory’ (2)
- transcription only occurs in nucleus; memories stored in synapses
- transcription may be necessary, but not instructive (no transcription -> less proteins needed for memory; but proteins maybe not necessary during learning)
how do TFs regulate gene expression (2)
- recruiting enzymes that make RNA from DNA (RNA polymerase)
- recruiting enzymes that open up DNA for transcription by modifying histones
TF required for LTF
CREB
effect of blocking CREB
blocked LTF without blocking STF
what activates CREB
5HT
how did they find CREB (3)
- cAMP was increased by 5HT so looked for cAMP-dep TF
- found small sequence necessary and sufficient for mRNA to be transcribed when cAMP levels high (CRE)
- found TF that bound to CRE -> CREB
which factors regulate CREB activity (2)
- CREB activator
- CREB repressor
what proteins does CREB recruit and when does it recruit them
proteins that recruit RNA polymerase; when it is phosphorylated
activation of … and removal of … required for transcription
CREB kinases; CREB repressor
complexes formed by CREB (2)
- dimerizes with itself
- dimer with repressor
how does training that induces learning affect CREB complex
reduces level of CREB repressor
effect of reducing/removing CREB repressor
easier to activate CREB, but still requires some stimulation; LTM formed more easily
how do multiple training periods lead to LTF
multiple training periods required to activate transcription, leading to LTF
how convert stimuli that normally gives STF, to one that gives LTF
activating CREB
effects of (a) 5HT x 1 (b) 5HT x 5 (c) 5HT x 1 + anti-CREB repressor
(a) STM
(b) STM + LTM
(c) STM + LTM
effect of overexpressing CREB inhibitor
blocks long term odor avoidance memory in drosophila
effect of removing CREB
prevents LTM
effect of overexpressing CREB on stimulus
converts stimulus that usually doesn’t induce LTM to one that does
variation of neurons that encode memory in different species
aplysia -> in every animal, same neuron (invariant)
more complex nervous system -> neurons not invariant
how test if neurons that activate transcription during learning also reactivated during recall
- put marker turned on by gene expression at time of learning -> can recognize neurons later
- when animals remember (recall), do same neurons specifically turn on gene expression again
why not add marker after promoter downstream of CREB (like FOS) to study which neurons activate gene expression during recall (2)
- every time want different marker, need to make different mouse
- transcription occurs all the time, want to limit marker expression to when animal is learning
how make specific marker to study gene expression only when animal learning
replace marker with specific new TF that can be turned on/off by drug fed to animal
tet-TAG system (6)
- transgenic mouse expressing tTA
- tTA normally binds to TetO when active (cFOS) to turn on gene (transcription)
- fear conditioning task
- add DOX during task to block transcription -> tTA doesn’t binds to TetO
- memory retrieval
- brain dissection to look at coexpression of proteins
coexpressed proteins observed after tet-TAG mouse experiment
LAC and ZIF
even if LAC and ZIF coexpressed, must remember that (2)
- most neurons that turn on transcription aren’t activated by memory
- many neurons that activate transcription during recall, didn’t activate during initial memory formation
what did the tet-TAG experiment show
correlation -> not that neurons that activate transcription are required for memory
technique to test which neurons required (necessary) for memory (vague)
technique where neurons that turn on transcription can be removed and see if memory retained
effect of overexpressing CREB in 20% of LA neurons (2)
- increases memory
- increases chances that memory induces gene expression in those neurons
strategy to kill memory neurons (3)
- express CREB under control of cre -> only CREB cells contain Cre
- Cre-dep virus (loxP-STOP-loxP-DTR) -> only CREB cells with Cre will also express DTR
- inject toxin -> cells expressing diphteria toxin receptor killed
freezing in response to tone (auditory fear conditioning) (a) control-cre vs DT (b) CREB-cre vs DT (c) CREB vs DT (d) CREB-cre vs PBS
(a) normal memory (random 20% neurons killed)
(b) CREB overexpressed -> increased memory; with DT -> decreased memory than control, but higher than baseline
(c) DT has no effect
(d) PBS has no effect
result of experiment overexpressing CREB in LA neurons (vs DT) (2)
- memory is bigger, but number of neurons that turn on transcription is the same (no increased number of neurons)
- removing CREB neurons doesn’t completely eliminate memory
which technique tests if neurons are sufficient for animals to remember
optogenetics
what are halorhodopsins
channels activated by yellow light that hyperpolarize cell by letting chloride ions through
how make false memory (vague)
make neurons that activated transcription in a context express channelrhodospin/halorhodpsin -> pair firing of those neurons with a shock
how observe neuron activity in response to false memory (3)
- when activate CREB-mediated transcription, production of tTA
- when tTA present, ChR2-mCherry activates (color)
- shine blue light over dentate gyrus, observe red spiking
steps of implanting false memory
- animal in context A + no shock + off Dox
- animal in context B + shock (association) + light to activate neurons that activated transcription in context A
- test: animal back in context A + on Dox (no transcription) -> does animal freeze (because context A neurons activated during shock so also association)? -> yes, now also freezes in context A
link bw cell excitability and transcription
neurons that start out more depolarized are most likely to activate transcription
link bw CREB and cell excitability
CREB increases excitability of neurons
how test if CREB allocates neuron by increasing excitability
- need specifically block CREB-induced increases in excitability (see if CREB necessary)
- need to increase excitability independently of CREB (see if CREB sufficient)
difference bw necessary and sufficient
necessary -> required for…
sufficient -> if alone, enough for…
how neuron gets more excitable (2)
- increase membrane potential (closer to threshold), more chance of reaching threshold and firing AP
- affecting channels that prevent multiple APs from occurring (that control refractory period)
what does excitability mean
neuron fires more AP to same stimulus
proteins that regulates excitability and how
KCNQ2 -> regulates refractory period
Kir2.1 -> regulates membrane potential
molecular/cellular effect of overexpressing dnKCNQ2
inactivate endogenous channels and increase excitability by decreasing refractory period
what is KCNQ2
voltage-activated potassium channel involved in refractory period regulation
what aspect does overexpressing dnKCNQ2 channels test and why
sufficiency of excitability for allocation; increases excitability and observe increased memory
effect of overexpressing Kir2.1
blocks increase in excitability by increasing efflux of potassium from cell, lowering membrane potential and preventing cells from firing APs
what aspect does overexpressing Kir2.1 channels test and why
necessity of increasing excitability for allocation; decreases excitability and observe that there is no increase in memory
effect of overexpressing Kir2.1 with CREB
blocks CREB from increasing excitability anad prevents memory (produces normal levels of neuronal firing)
increased excitability: necessary and/or sufficient for increased learning? and why
increasing excitability is both necessary (overexpress Kir2.1 = less excitability = no increased learning) and sufficient (overexpress KCNQ2 = increased excitability = increased memory) for increased learning
what is arc
marker of transcriptional activation
when does arc turn on (2)
during memory encoding or memory retrieval
arc and auditory fear conditioning
15-20% of LA neurons activate arc after auditory fear conditioning
relationship bw arc and excitability
neurons that have increased excitability (CREB or KCNQ2) more likely to express arc (and activate transcription during recall); neurons that have less excitability (Kir2.1) -> less likely to express arc
possible mechanism that allows linking of events in time
neurons that encode memory (that activate CREB) more likely to participate in another memory within certain time frame, since CREB increases excitability (gene expression lasts longer)
what does encoding memory for the long-term involve (2)
- coincident synaptic activation
- cell-wide decision to activate gene expression
what is needed to get increased synaptic strength (2)
- tag from synaptic activation by plasticity-related proteins (which are activated by transcription)
- tag from synapses activated by experience
results of applying 5HT to specific synapse bw motor neuron and sensory neuron in aplysia (synapse strength) + conclusion
- increased synaptic strength in synapses where applied 5HT
- no increase in synaptic strength in synapse of same sensory neuron and other motor neuron (where no 5HT applied)
- conclusion: 1 x 5HT sufficient for LTM (72h) (capture)
transcription in synapse-specific facilitation (3)
- s-s facilitation still requires transcription because blocked by CREB inhibitors
- effects of transcription limited to synapses that sees 5HT
- mRNAs sent to both synapses, but only translated at active synapse (5HT)
what does adding 5HT to synapse activate (2)
- transport to nucleus to activate gene expression
- tag at synapse
what experiment can be done to determine what is required for a tag
capture experiment
what is ‘capturing’
process that captures PRPs at synapse
explain capture experiment + result/conclusions
1 capture of synapse-specific
a. 5 x 5HT vs 1 x 5HT (at different synapses of same sensory neuron)
b. produced increase in synaptic strength in both (different amplitudes but still there) -> LTF
#2 capture of cell-wide
a. 5 x 5HT at body of sensory neuron vs 1 x 5HT at synapse
b. synapse wo 5HT -> no LTF; synapse with 5HT -> LTF
conclusion: 1 x 5HT sufficient to tag and capture PRP, (but not for inducing gene expression?)
1 application of 5HT to synapse leads to (2)
- 72h increase in synaptic strength -> capture
- increase in synaptic varicosities
1 application of 5HT + activation of gene expression leads to (2)
- 72h increase in synaptic strength (LTF)
- increase in synaptic varicosities
effect of replacing multiple applications of 5HT with injection of P-CREB
only requires 1 application of 5HT at synapse (synapse specific) -> sufficient to increase strength and for morphological changes)
why can’t use CREB with S-E mutation when injecting P-CREB instead of 5HT
P creates site for binding to a protein and S-E doesn’t mimic binding site
result of injecting P-CREB at body of sensory neuron and 1 x 5HT at synapse + conclusion
a. synapse wo 5HT -> nothing happens; not sufficient to increase synaptic strength
b. synapse with 5HT -> STF and LTF
conclusion -> 1 x 5HT + pCREB sufficient for LTF
how long does tag last and what conclusion does this lead to
couple hours; tag and transcription are separated by hours
tag & transcription separated by hours -> what does this lead to believe?
can tag synapse independently of activating transcription and still get an association
which side of synapse does tagging happen
presynaptic and postsynaptic
what does LTM formation require (2)
- activation of transcription
- synaptic tagging
transcription in STM
transcription not required for STM or ITM
argument for the fact the LTM cannot support/depend on STM
LTM depends on new synapses, so how can it come from STM? must be parallel process since new synapses take hours/days to form and be functional (STM is dissipated)
requirements for behavioral tagging (3)
- task where gene expression will be activated in same brain area where other memory will be formed
- activation of gene expression by task #1 allows weak stimulation (CS) to make LTM in task #2
- requires same neuron to be in cell assembly used in both tasks
explain behavioral tagging experiment and results
- weak CS: high freezing after 1h, low freezing after 24h -> formed STM, but not LTM
- novel OF + CS -> high freezing, activates a a lot of gene expression; familiar OF + CS -> low freezing, no LTM of previous CS
- novel OF + vehicle -> high freezing (+ gene expression); novel OF + inhibit protein formation -> low freezing; no effect of novelty of OF
conclusion: OF generates gene expression and makes PRP + weak training is enough to make tag to capture PRP, but LTM isn’t formed
plausible mechanism of how LTM should be formed via behavioral tagging
- weak training in behavioral task = STM + experience tag
- novelty of OF = PRP (transcription/gene expression)
- experience tag + PRP = LTM
what is behavioral tagging
effect of PRPs on formation of LTM induced by weak training (weak training tags synapses & novelty forms PRPs)
how does tag capture PRP
experience-activated synapses make tag, which capture PRP (proteins made by transcription) at the synapse and tag it too
