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Gene targeting: Silva and Giese.
Pharmacological approaches to
the study of learning and
memory: White and Salinas
Types of memory
• There is general agreement that there are
several different types of memory, each of
which is predominantly in a different part
of the brain.
Declarative vs. procedural memory
• Declarative memory: facts, dates, events
(telephone number, birthdate)
– Hippocampus is critical
• Procedural memory: how to perform an act
(ride a bicycle)
– Basal ganglia (dorsal striatum / caudateputamen) is critical
• Patients with Alzheimer's disease are
unable to learn or remember ordinary facts
(declarative memory) but are normal or
nearly normal at learning and
remembering how to do things (procedural
memory).
Memory experiment
• Alzheimer's patients learned and
remembered how to read complex words
in a mirror as well as normal control
subjects
• Were unable to recall the training session
or the fact that they had acquired this skill.
Classical (Pavlovian) conditioning
• Another kind of memory is distinct, both
behaviorally and anatomically, from
declarative or procedural memory.
• Pavlovian conditioning is a form of
learning based on the tendency of certain
natural events (food presentation) to elicit
involuntary responses (salivation) with little
or no training.
• Initiating event: Unconditioned stimulus
(US)
• Response pattern: Unconditioned
response (UR)
• Another "neutral" stimulus (ringing a bell),
besides the US, does not usually elicit the
UR.
• If another "neutral" stimulus (ringing a bell)
is presented simultaneously several times
with the Unconditioned stimulus (food), the
"neutral" will be able to elicit the
Unconditioned response (salivation).
• The "neutral" stimulus (ringing a bell) is
then called the Conditioned stimulus (CS).
• The response (salivation) is then called
the Conditioned response (CR)
• This process of learning an association
between a CS and a CR is called
Pavlovian or classical conditioning, or
sometimes "associative learning".
• Pavlovian conditioning occurs
automatically, with no control, voluntary
participation, or (usually) even awareness
on the part of the individual to whom it
occurs.
• Evidence in animals and humans indicate
that the amygdala is critical for classical
conditioning.
• The studies indicate that the amygdala
mediated expression of conditioned
rewarding and approach behaviors as well
as conditioned aversive and escape
responses (e.g., "freezing" in mice).
Fear conditioning
• A simple form of associative learning (Pavlovian
conditioning)
• Animals learn to "fear" a previously neutral
stimulus (conditioned stimulus, CS), because the
US has been presented at the same time as an
aversive stimulus (unconditioned stimulus, US)
such as a foot shock.
• Conditioned animals, when exposed to the CS,
tend to refrain from all movement except
breathing ("freezing").
• Freezing responses can be triggered with two
different types of CS, each working via different
parts of the brain:
• - In "cued conditioning", the CS is simply a tone
(e.g., 85 dB, 2800 Hz), and lesions in the
amygdala, but not the hippocampus, appear to
disrupt this type of conditioning.
• - In "contextual conditioning", rodents become
conditioned to the "context" in which they were
exposed, such as a particular location.
Contextual conditioning is thought to depend on
both the amygdala and the hippocampus.
Information storage / memory
• The most-widely accepted theory of how
information is stored in the nervous system is
based on a concept first described by D.O.
Hebb, now called Hebbian learning.
• Start with the idea that each perception evokes a
unique set pattern of neural activity.
• The set of activated neurons are connected to
each other, and reactivate each other for a short
period of time.
• Hebb suggested that this period of recurrent
activation repeatedly activates the synapses
connecting the neurons, causing the synapses
to undergo permanent changes. These changes
facilitate future activation of the synapses.
• The pattern of permanently facilitated synapses
increases the probability that on future
occasions activation of one part of some of the
neurons will activate the rest of the neurons,
leading to recall of the information it represents.
• Changes in synapses resulting from the
simultaneous (or near simultaneous) activation
of neurons is generally thought to be the basis of
all learning, including procedural, declarative,
and conditioned learning.
• We will see that the central role of synaptic
changes in learning and memory provides the
bases for the action of neurologic drugs.
Memory consolidation
• When first acquired, memories are stored in a
labile state (represented by Hebb's recurrent
activation phase) and are subject to disruption
by external events.
• With the passage of time their storage may
become more permanent (Hebb's synaptic
changes) and are less susceptible to disruption.
• This process by which memories become
permanent is called "consolidation". The interval
during which the hypothesized process of
synaptic change occurs is called the
consolidation period.
Gene Targeting:
Methods exist to
• add, delete, or modify genes in the mouse
genome.
• restrict expression of mouse genes to specific
regions of the brain,
• restrict expression to specific experimental
conditions:
– high/low temperature
– presence/absence of antibiotic
• These methods can be used to create mouse
models of human disease, e.g., Alzheimer'
disease.
• <Figure 2. Illustration of gene targeting
techniques>
• The hippocampus has long been known to
be involved in memory.
• <describe patient>
• Genetic work on hippocampal-based learning
and memory has focused on long-lasting
changes in synaptic efficacy (long-term
potentiation and long-term depression)
• Concept: memories can be stored in neural
circuits by changing the strength of synaptic
connections in neurons that are activated
simultaneously in a learning event.
• Computer simulations have demonstrated
that information (memories) can be stored
and recalled in a "neural network" in which
the weights between "neurons" are altered
as a result of learning.
• The hippocampus is capable of longlasting changes in synaptic strength.
• Drugs that block these synaptic changes
also block memory formation.
Biochemistry of LTP
• Induction of LTP in the CA1 region of the
hippocampus involves
– NMDA receptor activation
– consequent post-synaptic increase in
calcium
– activation of protein kinases and other
enzymes
– a partially-characterized sequence of events
leading to increased synaptic strength
Mutants in genes in this pathway
cause changes in learning ability
• <Table 1. Gene mutants that impair LTP in
hippocampus>
• The first gene-targeting study of LTP and
learning used mice with a null mutation for
the alpha CaMKII gene.
• This gene responds to changes in calcium
(Ca) ion changes related to memory
formation.
• Alpha CaMKII mutants showed impaired
LTP and LTD in the hippocampus and
neocortex.
• Although the alpha CaMKII mutant mice
were severely impaired in the
hippocampal-dependent version of the
water maze, they were able to learn the
"visible-platform" version of this task,
which is known not to depend on
hippocampal function.
• alpha CaMKII mutants
– can learn that the platform is the only escape
in the pool
– have the motivation to escape the water
– have the motor coordination and sensory
perception required to efficiently swim to the
escape platform,
– but they are unable to learn the spatial
relationships required to guide them to the
hidden platform.
• CaMKII appears to be involved in the early
stages of memory formation (during initial
learning), but not in long-term memory
formation.
Gene transcription, translation, and
memory
• DNA is transcribed to produce RNA
• RNA is translated to produce protein
• DNA -> RNA -> protein
• Transcription factors are proteins that regulate
what genes are transcribed (expressed).
• Transcription factors typically bind near the
promoter region of a gene (the on/off switch).
• Studies in the 1980's showed using drugs
that inhibit protein synthesis also inhibit
long-term memory formation.
• Several inhibitors of RNA synthesis or
protein synthesis block long-term memory,
but do not affect short-term memory.
• The Aplysia snail: a favorite model
organism for memory research
• Experiments with Aplysia showed that long term
memory required the activation of transcription
factors such as CREB (Cyclic AMP Response
Element Binding protein).
• To trigger transcription, CREB binds to a specific
regulatory DNA sequence (TGACGTCA) in the
promoter region of certain genes.
• This sequence is the Cyclic AMP Response
Element (CRE).
• When DNA or RNA fragments with the
CRE sequence are injected into Aplysia,
so that they bind to any available CREB,
they block long-term but not short-term
memory formation.
CREB in long-term memory in
Drosophila
• Dominant-negative mutation of Drosophila
CREB block long term memory, but do not affect
other memory stages.
• Studies were performed using temperature
sensitive CREB mutants, which were only
inactivated in high temperature.
• Wild type and mutant CREB flies grew up in the
permissive (low) temperature, and were then
given memory tasks at high temperature. Only
flies with the mutant CREB showed long-term
memory deficits.
• A different CREB mutant expressed CREB at
high levels at high temperature (under the
control of a heat-shock promoter).
• These flies could learn in a single training trial
(super memory), where wild type flies required
multiple spaced trials.
• These results indicate that CREB is required for
long-term memory, and is the rate-limiting factor
in the nuclear events leading to long-term
memory in flies.
Problems in gene knockout studies
of memory
•
•
•
•
Compensatory effects of other genes
Genetic background effects
Developmental effects
Impact of unknown physiological or
environmental factors
Altering memory using drugs
• Certain post-training treatments can
modulate memory storage in ways the
enhance retention.
• First observed with stimulant drugs
– strychnine (very low doses)
– amphetamine
– caffeine
Example experiment
• Rats were put into a cage where they could drink
water.
• After being put in the cage, the rats heard a
series of 10 second tones, each terminated with
a brief foot shock.
• The shock caused the animals to stop moving
(freeze).
• After several such tone-shock pairings, the rats
acquired a conditioned freezing response, which
lasted for several minutes each time the tone
was presented.
• The next day, the rats were placed in the
drinking cage.
• Tone came on when they began to drink.
• Animals froze
• Duration of freezing was used as a measure of
the rats' memory for the tone-freezing
association.
• Rats that experiences more pairings (12) froze
significantly longer than rats that had fewer
pairings (2).
• Some rats got drug injections immediately after
their experience of the tone-shock pairings.
• Rats that got two pairings followed immediately
by a saline injection froze for slightly longer than
rats that got two pairings but no injection.
• However, rats that got two pairings followed
immediately by an amphetamine injection froze
for about the same length of time as rats that got
12 pairings (but no drug).
• Another group of rats got 2 pairings
followed by amphetamine injection 2 hours
later.
• These rats froze for the same length of
time as rates that received saline or no
injection, that is, the drug had no effect.
• The results indicate that the immediate
amphetamine injections improved the rats'
memory for the tone-freezing association.
• The fact that the delayed drug injection
had no effect is consistent with the idea
that the memory was susceptible to
modulation only during a consolidation
period that lasted less than two hours.
How might drugs that affect
memory work?
• Access to the brain from the circulatory
system is controlled by the blood-brain
barrier (BBB).
• This barrier is made up of a layer of cell
surrounding the blood vessels that supply
the brain.
• These cells determine the degree to which
substances in the blood can enter the
brain.
• Fat-soluble substances (e.g., alcohol) cross the
BBB more easily than water –soluble
substances.
• Drugs and hormones with large molecular
weights do not easily pass the BBB.
• Some substances, including glucose and insulin,
are actively transported into the brain.
• The degree to which drugs cross the BBB is
critical to their effects on memory.
How drugs act on synapses
• Neurons communicate with each other at
synapses using chemical neurotransmitters.
• This provides the bases for drugs (and poisons)
to affect synaptic transmission.
• Drugs with chemical properties similar in some
way to those of neurotransmitters can act on
synapses to alter behavior and thoughts
(psychotropic or psychoactive drugs)
• Drugs that increase synaptic transmission
are "agonists".
• Drugs that block or reduce synaptic
transmission are "antagonists".
• About 25 neurotransmitters are known in
the mammalian brain.
• Most psychoactive drugs act on the
synapses of a single neurotransmitter.
• These synapses often occur in different,
functionally unrelated parts of the brain,
controlling many different behaviors
• The psychological actions of drugs can be
quite complex and difficult to predict