1. Revival of long-term memory after synaptic eradication in Aplysia; can
lost memory be restored?
By Paulina Kamburowski
The Ohio State University, Data Analytics
kamburowski.3@osu.edu
ABSTRACT
Ever since Cajal and Ramon(1894) introduced the idea of synaptic
plasticity, it has been widely accepted that long term memory(LTM) is
stored in the brain as changes in synaptic connections (Kandel ER).
Recent studies, however, challenge this theory and propose that
memories are stored within the nuclei of individual neurons, thus
erasure of a memory’s synaptic expressions is not synonymous with
permanent memory loss. This discovery brings to light new possible
treatments for memory-loss related diseases, such as Alzheimer’s. In
early Alzheimer’s, only synaptic connections, not neurons themselves,
are destroyed. Thus, if it is true that such degradation has not
permanently erased a memory, induced formation of new synaptic
connections, as can be done with serotonin treatment, could possibly
restore lost memories, even though these new connections will be
much different from the originals.
METHODS
Experiment1(ChenSetal):
• FourgroupsofcellculturesofAplysiacellsareprepared.Sensoryneuronsarelabeledwith
dextranfluoresceinandmotorneuronswith dextranrhodamineviapressureinjection
• Initialimaging usinglaserscanningconfocalfluorescencemicroscopyshowstheoriginal
varicositiesbetweenthecells
• Overacourseof2days,eachoffourgroupsreceivesdifferenttreatments,butevery24
hourstheyarereimaged
Group1:Control
Thecultureisleftuntreated,justimaged
Group2:5X5HT
After24hours,thecellcultureistreatedwith5pulsesofserotonininordertoinducelong-term
facilitation
Group3:5X5HT-Anisomycin
After24hours,thecellcultureistreatedwith5pulsesofserotonin,andafteranother24hours,
itistreatedwiththeproteinsynthesisinhibitorAnisomycin
Group4:5X5HT-1X5HT-Anisomycin
After24hours,thecellcultureistreatedwith5pulsesofserotonin,andafteranother24hours,
itisfirstremindedwith1pulseofserotoninandimmediatelyaftertreatedwith Anisomycin
Experiment2(CaiDetal):
• Fourgroupsof Aplysiaanimalsareprepared.
• Overacourseof4days,eachoffourgroupsreceivesdifferenttreatments,butevery24
hourstheanimalsareadministeredatailshockandthelengthofthesiphon-withdrawal
reflex(inseconds)isrecorded
Group1:Controlà3XTrained
Theanimalsareuntreateduntilrightbeforethefinal96-hourmeasurement,whentheyare
trainedwith3shockstothetail
Group2:5XTrained-Aniso
Theanimalsarepre-trainedwith5shockstothetailandreceiveanAnisomycininjectionright
afterthe48-hourmeasurement
Group3:5XTrained-1XTrained-Aniso
Theanimalsarepre-trainedwith5shockstothetailandrightafterthe48-hourmeasurement,
areadministeredaremindershockimmediatelypriortoanisomycininjection
Group4:5XTrained-1XTrained-Aniso-3XTrained
Theanimalsarepre-trainedwith5shockstothetailandrightafterthe48-hourmeasurement,
areadministeredaremindershockimmediatelypriortoanisomycininjection. Beforethefinal
96-hourmeasurementtheyaretrainedwith3shockstothetail
RESULTS
Experiment 1:
As expected, cell cultures treated with serotonin formed new monosynaptic
sensorimotor connections. Anisomycin treatment alone(Group 3) does not
destroy these connections, because there first must be a reminder pulse of
serotonin to trigger reconsolidation of the memory(Group 4). The ultimate cell
varicosities imaged in Group 4 are of most interest. Rather than the
Anisomycin treatment simply destroying the new varicosities induced by the
serotonin as expected, the synaptic eradication seemed to occur randomly,
destroying some old and some new connections, but still overall yielding the
same number of varicosities. This result challenges the popular theory of
memories being stored in stable synapses, as the arrangement of the
connections does not seem to dictate the strength of the memory.
Experiment 2:
As expected, the shock-pre-training induced sensitization of the siphon-
withdrawal reflex in the 3 experimental groups. Similarly as in Experiment 1,
Anisomycin only had an effect on the duration of SWR when animals were
injected immediately after a reminder stimulus, implying that altering of
memory can only occur when the brain is actively expressing the memory.
The most remarkable results, however, can be drawn from the changes in
SWR duration in Group 4. After anisomycin destroyed synaptic connections
which were facilitated with the shock-training, previous research would
CONCLUSIONS
TheBigPicture:
These results challenge the long-accepted idea that memories are stored as
sequential firing patters between neurons, which was proposed by Cajal and
Ramon. Rather than being stored in stable synapses, memories seem to be
stored within the nuclei of particular neurons themselves, thus erasure of
synaptic expression is not synonymous with erasure of long-term memory.
Ultimately, memories may be far more refractory to modifications or
elimination than previously supposed.
FutureImplications:
Ifconfirmedandextendedtomammals,theseresultscouldleadtoimportantimplicationsfor
treatingdisordersrelatingtoLTMsuchasAlzheimer’s.PerhapseffortstotreatAlzheimer’swill
shiftfromtryingtoprevent furthermemory-lossandstrengtheningcognitiveabilitytoactually
attemptingtorestorethememorieswhichwerelost.InearlystagesofAlzheimer’s,only
degenerationofsynapticconnectionsoccurs,thustheindividualneuronsarestillintact.If
patientsareremindedofamemoryandimmediatelyaftertheirneuronsaresomehow
primedtogrownewvaricosities,perhapswiththeuseofserotoninasinExperiment1,
theoreticallyalostmemorycouldberestored.
REFERENCES
1 Chen S, Cai D, Pearce K, Sun PY-W, Roberts AC, Glanzman DL. 2014. Reinstatement
of long-term memory following erasure of its behavioral and synaptic expression in
Aplysia. eLife(2014). [accessed 3 December 2015]. doi: 10.7554/eLife.03869.:
2 Cai D, Pearce K, Chen S, Glanzman DL. 2012. Reonsolidation of long-term memory in
Aplysia. Current Biology 22:1783-1788. [accessed 3 December 2015]. doi: 10.1016/j.cub.
2012.07.038
3 Citron M. 2002.Alzheimer’s disease: treatments in discovery and development. Nature
Neuroscience 5:1055-1057. [accessed 3 December 2015]. doi: 10.1038/nn940.
4 Kandel ER. 2009. The Biology of Memory: A Forty-Year Perspective. The Journal of
Neuroscience 29(41): 12748-12756. [accessed 3 December 2015]. doi: 10.1523/
JNEUROSCI.3958-09.2009
INTRODUCTION
Previous Knowledge of Long-Term Memory:
In 1894, Cajal and Ramon proposed the famous idea that learning results
from changes in the strength of the synapse, later referred to as synaptic
plasticity. This theory has been widely accepted in the scientific
community, thus the popular belief has been that LTM is expressed in the
brain by sequences of firing neurons. When a memory is recalled, the
release of neurotransmitters, notably serotonin, determines whether the
memory is weakened (habituated) or strengthened (sensitized). By
increasing serotonin concentration to a neuron after being reminded of a
memory, the serotonin inspires growth of varicosities, a process known as
long-term facilitation, which strengthens the expression of the memory.
Current treatments for memory-loss:
Current treatments for memory loss focus not on restoring lost memory,
but on preventing further memory loss and improving cognitive function
with acetylcholine-esterase inhibitors. Much research is spent studying
extracellular amyloid plaques and intraneuronal tangles of the tau protein,
which result from the AB42 domain of the large transmembrane protein
APP. The problem with trying to target AB42 is that it could have some
essential function which has not yet been discovered, or inhibiting it could
reduce other important APP metabolites.
Aplysia:
Aplysia californica is a marine mollusk often used to study memory
because of its simple nervous system. The sensitization of the gill-and-
siphon withdrawal reflex (SWR) of Aplysia is well understood due to the
simple monosynaptic connections between sensory and motor neurons
that mediate the reflex. This monosynaptic sensorimotor connection can
be reconstituted in dissociated cell culture by training with pulses of
serotonin, which leads to long-term facilitation.
Instructors:
Adriana Dawes &
Valerie Coffman
MolGen
5660
Experiment2MathematicalAnalysis
Data Given:
Below are charts of the mean durations (in seconds) and standard
deviations of the SWR for groups 1(control) and 4(the group of most
interest) at the three different time allotments
n
μ1
σ1
48hr
12
2.5s
0.9s
72hr
12
2.5s
0.9s
96hr
12
10.4s
3.9s
n
μ4
σ4
48hr
11
53.7s
2.9
72hr
11
4.8s
3.2
96hr
11
56.7s
2.7
Group
1
Group
4
Paired Sample T-test:
In group 4 after the synapses were destroyed with anisomycin, the 3 tail
shocks seemed to induce a longer SWR than before synaptic eradication.
Is this difference between means at 48hr and 96hr significant?
𝐻0: 𝜇d=0 𝐻a: 𝜇d≠0
df =(n-1)=(11-1)=10 degrees of freedom
Using a t-table to look up the critical value for 10 degrees of freedom with
an alpha-value of .01, we obtain a value of 3.17. And since our t-value of
3.554>3.17, we reject the null hypothesis. Therefore, there is a significant
difference between the means, implying that synaptic eradication did not
erase LTM because the animal’s response was still sensitized(even more
so) after anisomycin treatment.
554.3
11
8.2
07.537.5604896
=
−−
=
−−
=
n
t hrhr
σ
µµ
IndicatethattheLTMshouldhavebeenpermanentlyerasedaswell,andthefinalshock
treatmentshouldhaveyieldedameandurationsimilartonaïvecontrolanimals.This,however,
clearlywasnotthecase,asthefinalmeanSWRdurationforGroup4was56.7scomparedto
thecontrolgroupwithameanof10.4s,adifferencesosignificantthatstatisticaltestingseems
ratherpetty.Ratherthancomparingthe96hrcontrolmeanandGroup4mean,itseemsmore
interestingtoanalyzethedifferencebetweenmeanswithinGroup4at48hr(beforeanisomycin
treatment)andat96hr(aftersynapticerasureandadministrationoffinalshocks).After
conductingapaired-samplet-testandrejectingthenullhypothesis,onecanconcludenotonly
thatLTMwasfullyrestored,butthatitwasevenmoresensitizedthanbefore.Thisindicatesthat
eradicationofthevaricositiesdidnoteraseLTM,butratherhadnoeffectonitsexpression.
SuchanobservationchallengesCajalandRamon’stheorythatmemoriesarestoredinstable
synapses,becauseLTMseemstobeindependentofsynapticplasticityandpersistsevenafter
eliminationof varicosities.