PSYC318: Lecture 17: Cellular Memory Consolidation – First Sossin Lecture:

Towards a molecular understanding of how memory works…

- For an experience to have an impact on future behaviour, it must cause a physical change.
o A trace in the brain
- Since brains are biological, this change must reflect a molecular or biochemical change in the
function of the neuron
o Analogy of memory in computers:
o Computers have memory by either “charging a capacitor” for dynamic memory – volatile
– it goes away once you turn your computer off.
o Or, they do this by making a very small magnetic field – hard drive. This is more long
lasting.
o So memory in a computer is still represented by a physical change.

- The changes in the brain must be mediated by biological mechanisms.

- Biological minds are not engineered – they are developed through evolution.
- The only input evolution has is through the proteins and molecules that make up the brain.

Throughout the lecture, we will refer

to the following slide:
These are all biological forms of
memory.
We will be talking about changes in the
phosphorylation state of proteins –
when proteins are modified by kinases
adding a phosphate group on it. This
affects how the brain works, and short
term memories are formed this way.
These are reversed easily by
phosphatases.
We will be talking about the insertion and removal of membrane proteins from the cell’s surface –
exocytosis and endocytosis – this is a major way that memories are stored – changes the strength of the
synapse by changing the number of receptor for the neurotransmitter on the postsynaptic cell.
Persistent activation of protein kinases – recall that we just said changes in the phosphorylation state to
make a short term memory. If you change this phosphorylation state long-term (persistently active
kinase), this will make the memory long term.
A lot of the distinctions between short term and long term memories have to do with protein synthesis –
making new proteins and new mRNA that make new proteins. This requires constant energy, but persists
based on the half-life of the protein or mRNA
Then, we will talk about morphological changes at pre-existing synapses and new synapses.

Aplysia is a model system used to examine molecular traces:

- Aplysia is a simple animal and used to examine molecular traces.
- They have simple behaviours and neuronal circuits
o Reductionist - allows us to ask questions in the simplest system you can find.
- Their neurons are large and identifiable between preparations
o You can see them with the naked eye.
o This allows for tracing of the circuit underlying behaviour.
- You can make primary cultures of neurons which can recapitulate the changes observed during
behaviour
o This allows for molecule analysis of memory traces.
We sill start off by talking about non-associative memories…
Non-associative memories:
- To this point, we have talked about associative memories in this class.
o Memories where there is an association between a CS and US stimulus.
- In a non-associative memory, there is no specific association between conditioned and
unconditioned stimulus
o The stimulus ALONE is strong enough to change behaviour and create a memory.
o Ex: if you repeatedly touch an animal, they will habituate.
o There is no pairing of stimuli.

- Habituation is the decrease in a defensive reflex due to repetitive non-noxious stimulation.

o This is presenting a conditioned stimulus ALONE.

- Sensitization is the increase in a defensive reflex due to a noxious stimulus

o This is presenting the unconditioned stimulus ALONE.
o Ex: give the animal a shock
This is an Aplysia – like a snail without a shell
- It normally lives under water, so it breathes through
its siphon and gill.
- When touched on its siphon, it withdraws its gill.
 What is the behavioural memory?
Sensitization – a noxious stimulus to the head or tail causes an increase in the time and extent of gill
withdrawal to a touch to the siphon.
Habituation – repetitive touches to the siphon causes a decrease in gill withdrawal. That decrease in the
defensive reflex is called habitation.

This behaviour is mediated by abdominal ganglia !

- They are easily identifiable neurons with fixed
circuitry allowing for easy identification of the
circuit.
- Each of the neurons have a name, and are recognized
by their size and position.
You can record changes in synaptic strength during memory
formation.
How to measure synaptic strength:
Excitatory postsynaptic potential (EPSP) and Inhibitory
postsynaptic potential (IPSP)
- Action potentials cause the presynaptic cell to release neurotransmitters that acts as a ligand to
open channels in the postsynaptic cell.
- This allows for ions (mostly sodium for EPSPs and chloride for IPSPs) to flow into the
postsynaptic cell.
- The postsynaptic potentials are measured as a change in voltage in the postsynaptic cell.
Thus, if one places an electrode in the postsynaptic cell, fires an action potential in the presynaptic
cell, you can measure the strength of the synapse by measuring how much the voltage changes in the
postsynaptic cell.
Cellular Analogue of Sensitization:
In Aplysia, they found the motor neuron that causes gill withdrawal (L7) and they found the sensory
neuron that responds to touch on siphon (SN)
- We can mimic touching the siphon by stimulating a nerve that comes from the siphon.
- We can also mimic a tail shock by activating the connective from the cerebral ganglion – where
there are inteneurons that detect noxious stimuli.
Keep in mind that we use different language to describe either the behaviour or the changes in
synaptic strength…
 - Habituation – a behavioural response
- Depression – the change in synaptic strength that underlies habituation.
- The goal is to link changes in synaptic strength to behaviour.
Habituation/Depression Results:
- They found that when they continuously activated the siphon nerve (which activates the sensory
neuron, SN), and recorded from the postsynaptic neuron (motor neuron L7), the action potential
didn’t change.
- However, the SIZE of the EPSP in the motor neuron decreased over time.
- There was therefore a decrease in synaptic strength (depression).
o In other words, the same action potential in a sensory neuron now gave a much smaller
EPSP in the motor neuron.
o And since the motor neuron causes the gill to withdraw, this can explain why the gill
didn’t withdraw as much after repeated touches to the siphon.
Sensitization/facilitation results:
- Now, they decided to give the Aplysia a big shock.
- Now, they saw an INCREASE in synaptic strength.
- This is called facilitation.
- This could help explain sensitization – they saw a bigger EPSP in the motor neuron, meaning it
will be MORE likely to generate an action potential and cause a gill withdrawal.
Summary:
- Depression and facilitation changed the synaptic strength between sensory and motor
neurons
- But, depression did not diminish the ability of the touch to cause an action potential in the sensory
neuron. It only decreased the SIZE of the EPSP seen in the motor neuron.
o This means that the CONNECTION changed.
- Facilitation caused an increase in the EPSP seen in the motor neuron after a single action
potential in the sensory neurons.

Serotonin is the facilitating transmitter:

What does the unconditioned stimulus do? (shock)
- The shock caused release of serotonin.
- And, applying serotonin (5-hydroxytryptamine or 5-HT) mimicked shocking the tail/
stimulating the nerve that causes and increase in synaptic strength between the sensory and motor
neurons.
o Serotonin was sufficient.
 - Furthermore, removing 5-HT containing neurons using a neurotoxin that specifically targets
serotonin neurons causes the Aplysia to sensitize less.
o Serotonin was necessary for the behaviour of sensitization.
- Lastly, 5-HT containing neurons fire during sensitization training and 5-HT is released during
sensitization.
We can now build a circuit….
The siphon has a sensory neuron and this is directly
connected to the motor neuron that controls the gill.
There are also facilitating inter-neurons (5HT
neurons) that receive information from a sensory
neuron in the tail and outputs to the sensory neuron
in the siphon and the motor neuron in the gill.
Therefore, when you shock the tail of an Aplysia,
this causes the sensory neuron to activate the
facilitating interneuron, which releases serotonin
onto the sensory neuron of the siphon and the motor
neuron of the gill.
The result is that the connection between the sensory neuron of the siphon and the motor neuron of the
gill will be STRENGHTENED, so that a touch on the siphon will now cause gill withdrawal much easier.
Reductionist culture system:
We can reduce this further…
- We can make a culture where the sensory and
motor neurons are removed from the animal and
you can re-create the synapse between them.
- Then you can add serotonin to the petri-dish to
induce all these manipulations to generate
sensitization and facilitation.
You can’t get more reductionist than this.

Summary so far: Links behind behaviours and cellular properties:

- Behavioural habituation – repeated touches leads to decrease of withdrawal
o Cellular depression – repeated firing of sensory neuron leads to less release and decrease
of EPSP to motor neuron.

- Behavioural sensitization – shocking tail or head leads to increase in withdrawal

o Cellular facilitation – shocking nerve or addition of serotonin leads to more release and
increase of EPSP to motor neuron.
The Point:
- A strong association between a cellular change and a behavioural memory allows one to then ask
the question of how memories are made and maintained by looking at the cellular question of
how changes in synaptic strength are made and maintained.

Short term vs. Long term memory:

- They found that one shock to the animal or one application of 5HT to the isolated sensory-motor
neuron culture leads to short-term facilitation that lasts about 30 minutes.

- In contrast, five spaced shocks to the animal or spaced applications of 5HT (15 minute intervals)
to the cultures leads to long-term facilitation that lasts at least 24 hours.
o If you do that again the next day, the change will last a week.
We want to know how that different from the change of synaptic strength that lasted only 30 minutes?

Really big question:

- At the level of the molecular trace, is the long term memory due to consolidation of the short term
memory?
o Is this really what happens?
o Does the short term memory gets made to last longer somehow?
- To answer this question requires understanding what the actual changes are that mediate the
increase in synaptic strength after the two different stimuli (one application vs. 5 application of
serotonin)

How is synaptic strength increased?

(1) We can increase synaptic strength by releasing MORE neurotransmitter/action potential.
o This causes a higher probability of release
o This is what happens during short term facilitation (more to come)

(2) We can increase synaptic strength by increasing the effect of releasing the same amount of
neurotransmitter by having a bigger post-synaptic response.
o Ex: more receptors present on the postsynaptic receptors.

(3) We can increase synaptic strength by having more synapses.

 o This can only happen over the long-term
You can take what we just said and change it into an equation…
M = NPQ
- M = strength of the synaptic connection
- N = number of synapses
- P = probability of release of a synaptic vesicle after an action potential (ranges from 0-1)
- Q = amplitude of the EPSP resulting from the release of one vesicle (depends on the post-synaptic
response, ex: the amount of post-synaptic receptors).
You can change the synaptic connections if you change any of these parameters.
Change in short term synaptic strength mediated by serotonin is caused by a change in P.
How to control the probability of release of a synaptic vesicle after an action potential (P)?
1) Modulating calcium channels, which changes the amount of calcium entering with an action
potential
o Synaptic vesicles are released through calcium vesicles.
o Furthermore, vesicle release is proportional to the fourth or fifth power of calcium
concentration so small changes in calcium can cause large changes in release.
o More calcium = greater chance of vesicle release.

2) Changing the number of vesicles that are ready to be released

o The combination of vesicles at the active zone and the “priming” of vesicles
o The more vesicles that are ready, means the greater the probability that one will be
released.

3) Coupling calcium entry to fusion of the vesicle

o (1) and (2) are coupled.

▪ So, you can have the same amount of calcium come in, and the same amount of
vesicles present, and you can have some change in the procedure by which
calcium causes release of the veislc.es

▪ This is called calcium secretion coupling.

o For instance, you can change how close the synaptic vesicles are to the calcium channel –
the closer they are, the more calcium they will receive, and the easier they will be to
release.
Short-term facilitation is caused by changes in probability of vesicle release (P):
- Spike broadening
 o Serotonin activates a G coupled receptor (GPCR) and activates a G protein called Gs (s
for stimulator)
o Gs activates an enzyme called adenylate cyclase to make the second messenger cyclic
AMP (cAMP)
o cAMP activates protein kinase A (PKA ! also known as cAMP-dependent protein
kinase)
o PKA phosphorylates potassium channels that are important for regulating the length of
the action potential

▪ When an action potential comes into the terminal, it goes up, and then it comes
down – and “coming down” is regulated by voltage gated potassium channels
(repolarization)

▪ If the action potential doesn’t come down fast, then voltage stays high in the
presynaptic cell for longer, and calcium channels are thus open for longer and let
in more calcium.
o in other words… Longer action potential (action potential broadening) leads to longer
activation of voltage-gated calcium channels
o And longer activation of calcium channels means more calcium comes in and more
vesicle release.
Action potential in presynaptic terminal ! lets in calcium !
causes an EPSP
If the action potential gets broadened, it lasts longer, you get
more calcium coming in, which leads to MORE vesicles
released, which leads to a greater EPSP.
For a long time, this was the explanation for short-term
facilitation. It was caused by an increase in P.
This was believed to underlie “memory”
There was a lot of effort to keep it this simple, but it isn’t.
A combination of molecular changes represent memory
PKA has additional targets that contribute to sensitization…
Protein kinases phosphorylate more than one protein.
- For instance, it turns out there was another distinct potassium channel that gets phosphorylated by
PKA and doesn’t cause re-polarization of the action potential, but causes the sensory neuron to
become more depolarized, thus increasing its excitability.
o Not all memories are stored in changes of synaptic strength, but in excitability as well
o Excitability – how many action potentials a neuron will fire to the same input.
 - Additional targets lead to an increase in the probability of release of the vesicles because more
were ready to be released.
So not only was there more calcium coming in after an action potential (due to action potential
broadening), but there was also an increase in excitability and more vesicles were ready to be released.
It wasn’t as simple as originally thought
What is a memory trace for short term facilitation?
- The PKA phosphorylation of the potassium channels and the proteins involved in the priming
would be the memory trace.
- The memory would last as long as those sites remained phosphorylated
- Short term sensitization/facilitation lasts 20-30 minutes.
- After that, cAMP is degraded, PKA is no longer active, and a phosphatase removes the
phosphate off the potassium channels.
o The memory lasts until the phosphate is removed.
o After it was removed, synaptic strength returned to normal and the animal was back to
normal.
o This biochemical trace is considered highly volatile.

What about long-term memory?

- So far we have only discussed one shock, or one application of 5HT
- But we know that multiple or longer shocks/5HT application or coupling 5HT to firing of the
sensory neuron (associative) leads to a longer-lasting memory
- These lead to similar and distinct memory traces
- They also cause post-synaptic changes (Q)
Memory Phases in Sensory Motor Neuron Plasticity:
- Short term facilitation (STF)
o Lasts 0-30 minutes
o Due to activation of protein kinases by second messengers (cAMP) that lead to increase
in transmitter release (already discussed)

- Intermediate facilitation (ITF):

o Lasts 30 mins – 3 hours
o Unlike long term facilitation, intermediate facilitation does not need gene expression.
o Instead, it depends on persistent kinase activity and translation of pre-existing mRNAs
into proteins.
 ▪ Important ** The big difference between intermediate and long term is that is
doesn’t require gene expression (new mRNA made in the nucleus)

- Long term facilitation (LTF)

o Lasts 3 hours to 1 day
o Blocked by inhibitors of RNA transcription (therefore, gene expression is needed).
o This block occurs during induction, but not the expression of long-term facilitation.

▪ Need the gene expression while serotonin is there and the memory is being
formed, but not the entire time that the memory is there.

- Late Long term facilitation (L-LTF)

o More than 1 day
o Requires gene expression
o May require the formation and stabilization of new synapses
Long term sensitization memory (1 week) is
correlated with increased number of synapses
- Morphological investigations of long term
sensitization reveals multiple changes
(increased size of active zones, increased
docked vesicles, increased number of synapses)
- However, as memory persists, only the
increased number of synapses persists, while
other changes recover to control.
This is a powerful suggestion that shows long term
memory is stored by having more synapsesg between
two neurons.
Molecular memory traces:
- At the cellular level, memories are retained by molecular, structural changes in the neuron.
- This molecular memory trace must exist and understanding its nature is key to understanding
memory.
There is a very important question to consider…
Short term vs. long term memory – are the different time frames of memory due to:
- One memory trace that is stabilized over time (serial memory trace)?
o Consolidated from short term to long term.
o Does the short term memory have something to do with the long term memory?
 OR
- Distinct memory traces that last for different periods of time (parallel memory traces)?
o The changes in short term phosphorylation of the potassium changes has very little to do
with formulation of new synapses between the sensory and motor neuron.
o This suggests that these processes are parallel.
o They have nothing to do with one another.

Gene Expression and Memory:

- A distinguishing feature between memories that lasts on a short time and memories that persist is
the requirement for gene expression
When Sossin was a graduate student, he had many objections to this conception of gene expression..
Objections to the role for protein synthesis (gene expression) for memory:
(1) Gene expression only occurs in the nucleus, but memories are stored at synapses
o How can you change synapses if gene expression is only occurring in the cell body?
o We will talk about synaptic tagging next lecture

(2) Gene expression may be necessary, but is not instructive

o Ex: turning off gene expression reduces levels of proteins needed for memory, but those
proteins are not necessarily specifically increased during learning – they do a lot of
things.
o An analogy ! if neurons have no energy (not ATP), they will not make a memory, but
generating energy is not a party of making a memory.
Why are these objections this wrong? We first need to discuss transcription factors…
Transcription factors regulate gene expression – controls which parts of DNA are converted into
mRNA:
- Genes (DNA) are transcribed into RNA only at appropriate times
o It is regulated by small DNA elements (6-10 nucleotides) that bind to transcription
factors.
- These transcription factors act by
o Recruiting the enzymes that make RNA from DNA (RNA polymerase)
o Recruiting enzymes that open up the DNA for transcription by modifying histones
Specifically…
They found that only a specific transcription factor (CREB) is required for long term facilitation
- You didn’t need to block transcription overall, just ONE transcription factor.
 - It wasn’t some general transcription that was required, but a specific, subset of mRNA that was
under control of CREB.
Since cAMP was increased by 5HT, a search was on for a cAMP dependent transcription factor
First, they found the small sequence that was necessary and sufficient for an mRNA to be transcribed
when cAMP levels were raised (the cAMP response element – CRE)
Then, the transcription factor that bound that element was found – the cAMP response element
binding protein (CREB)
***Blocking CREB blocked long term facilitation without blocking short term facilitation
Furthermore, CREB is activated by 5-HT.
How do you activate CREB?
CREB activity is regulated by two factors:
(1) CREB only recruits RNA polymerase when it is phosphorylated
o Activation of CREB kinases are therefore required for transcription.

(2) CREB acts as a dimer (2 CREB together), and can dimerize with an inhibitory protein called
CREB repressor
o When CREB acts as a dimer, it triggers gene expression.

▪ One CREB by itself doesn’t trigger gene expression – it has to bind to another
CREB.
o However, when CREB binds to the CREB repressor, it prevents gene expression
o So one way to regulate CREB is to regulate the ratio of CREB activator to CREB
repressors.
o If there is a lot of CREB repressors, there will be more heterodimers, and less gene
expression.
o If you reduce CREB repressors, CREB will be more likely to form a homodimer, and
there will be more gene expression.
CREB activation is sufficient for converting short to long term memory:
- CREB activation is regulated by the ratio of CREB repressor to the CREB activator.
o Training induces learning and this is known to reduce the level of CREB repressor.
- Reducing CREB repressor should shift the balance to easier activation of CREB, but it still
requires some stimulation (phosphorylation of CREB)
In Aplysia, when both of these conditions are met, it results in long-term facilitation from a stimulus
that normally only leads to short term facilitation.
Thus, the specific requirements for long term facilitation (ex: multiple training periods) can be
explained simply because these multiple training periods are required to activate gene
transcription.
 If you make gene expression easier to activate (by regulating CREB), the same stimulus now makes a
long term memory.
This implies that the rate limiting factor for generating a long term memory is therefore activating
gene expression.
Gene expression is VERY important.
CREB is the rate limiting factor of long term
facilitation
1 application of serotonin – there is a memory 5
minutes after and nothing after 24 hours.
5 applications of serotonin – increase in short
and long term facilitation
Injected neurons with a CREB repressor
antibody, which sequesters CREB repressors,
preventing them from binding to CREB
activators – meaning CREB will be more likely
to cause gene expression – only one
application of serotonin was needed for
induce long term changes in synaptic
strength. ***
So just by altering the ability of CREB to trigger gene expression allowed long term memories to be
formed.
Just the antibody to CREB repressor alone wasn’t enough – you still needed serotonin. This is likely
because CREB needs to be phosphorylated by PKA.
Keep in mind: Gene expression by itself doesn’t determine whether it is a serial or parallel process. It’s
what the ROLE of gene expression does that determines this.
CREB appears to be important for many long-term memories:
- Overexpression of CREB inhibitor blocks long-term odor avoidance memory is drosophila.
- Removal of CREB repressor in vertebrates allows long term memories to be formed more easily
(smarter mice)
- CREB knockouts in mice have impaired long term memory for fear avoidance, water maze tasks,
and social recognition.
- Removing CREB acutely blocks long-term taste aversion and water maze consolidation.
- Overexpression of CREB converts a stimulus that normally does not lead to long term memory of
fear avoidance (massed training) to one that does.

Summary for today:

- Molecular memory traces are the physical manifestation of a memory
o Something physical has to happen for there to be a memory.
 o Just like in a computer
- The length of time a memory lasts depends on the stability of the trace/change
o Can be serial change or parallel change
- Short term memories are due to transient activation of protein kinases
- Intermediate memories are due to more persistent activation of protein kinases
- Long term memories require gene transcription
o Creating mRNA to generate new proteins.