Gene targeting: Silva and Giese' Pharmacological approaches to the study of learning and memory: Whi

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Gene targeting Silva and Giese.Pharmacological
approaches to the study of learning and memory
White and Salinas
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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.

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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 /
  caudate-putamen) is critical

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• 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).

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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.

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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.

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• 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.

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• 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)

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• 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.

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• 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).

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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").

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• 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.

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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.

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• 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.

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• 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.

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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.

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Gene TargetingMethods 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.

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• ltFigure 2. Illustration of gene targeting
  techniquesgt

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• The hippocampus has long been known to be
  involved in memory.
• ltdescribe patientgt

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• 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.

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• 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 long-lasting
  changes in synaptic strength.
• Drugs that block these synaptic changes also
  block memory formation.

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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

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Mutants in genes in this pathway cause changes in
learning ability

• ltTable 1. Gene mutants that impair LTP in
  hippocampusgt

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• 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.

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• 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.

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• 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.

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• CaMKII appears to be involved in the early stages
  of memory formation (during initial learning),
  but not in long-term memory formation.

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Gene transcription, translation, and memory

• DNA is transcribed to produce RNA
• RNA is translated to produce protein
• DNA -gt RNA -gt 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).

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• 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.

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• The Aplysia snail a favorite model organism for
  memory research

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• 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).

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• 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.

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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.

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• 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.

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Problems in gene knockout studies of memory

• Compensatory effects of other genes
• Genetic background effects
• Developmental effects
• Impact of unknown physiological or environmental
  factors

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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

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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.

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• 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).

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• 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).

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• 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.

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• 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.

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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.

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• 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.

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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)

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• Drugs that increase synaptic transmission are
  "agonists".
• Drugs that block or reduce synaptic transmission
  are "antagonists".

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• 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