Molecular Biological and Genetic Techniques for Studying Learning and Memory

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Molecular Biological and Genetic Techniques for
Studying Learning and Memory Thomas Gould,
Ph.D. Department of Psychology Temple University
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Transgenic Techniques

• Inserts a novel gene into genome
• Developed in the early 1980s by John Gordon and
  by Ralph Brinster and Richard Palmiter and their
  co-workers.
• Although all of the cells in the body contain an
  identical set of genes, some genes are active in
  only one or a few tissues.
• The two main parts of a gene are the regulatory
  region and the protein-coding region.
• When the right combination of proteins binds to
  specific sites along the DNA in the regulatory
  region, the gene is switched on, and the
  protein-coding region becomes active.

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Transgenic Techniques

• Make your DNA
• Using recombinant DNA methods, build molecules of
  DNA containing the promoter and structural gene
  you desire
• Insert into plasmid DNA to copy
• Cloning
• Transform ES cells in culture
• Expose the cultured cells to the DNA to allow
  incorporation
• Select for successfully transformed cells
• neor (a gene that encodes an enzyme that
  inactivates the antibiotic neomycin and its
  relatives, like the drug G418, which is lethal to
  mammalian cells) is part of the vector
• Expose embyronic stem cells to G418
• Inject surviving cells into the inner cell mass
  (ICM) of mouse blastocysts.

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Transgenic Techniques

• Embryo transfer
• Prepare a pseudopregnant mouse (by mating a
  female mouse with a vasectomized male - stimulus
  of mating elicits the hormonal changes needed to
  make her uterus receptive)
• Transfer the embryos into her uterus.
• No more than one-third will implant successfully
• Test offspring
• Remove a small piece of tissue from the tail and
  examine its DNA for the desired gene
• No more than 10-20 will have it, and they will
  be heterozygous for the gene
• Establish a transgenic strain
• Mate two heterozygous mice and screen their
  offspring for the 14 that will be homozygous for
  the transgene
• Or create dominant transgene
• Mating these will found the transgenic strain.

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Knockout Mice

• DNA that has been mutated is injected into
  embryonic stem cells in cell culture
• Stem cells are injected into blastocysts that
  will incorporate the cells
• Cells need to be incorporated into the gametes to
  be useful (low probability)
• Mice born with this mutation are called chimeras
  and have one copy of the mutated DNA
• Chimeras are crossbred producing ¼ offspring with
  two copies of mutated gene

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Cerebellar Circuit
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Eye-Blink Circuit
Parallel Fibers
Climbing Fibers
Cerebellar Cortex
Granule Cells
Inferior Olive
Cerebellar Interpositus Nucleus
Mossy Fibers
Red Nucleus
Trigeminal Nucleus (US)
Lateral Pontine Nucleus(CS)
Abducens Nucleus
Reticular Formation
Eye-Blink
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Genetic Analysis of Cerebellar Plasticity

• Current goals
• Use genetic manipulation to examine effects of
  pre- and postsynaptic up and down regulation of
  PKA and CREB at the granule cell Purkinje cell
  synapse on classical conditioning of the
  eye-blink reflex and on cerebellar LTP and LTD
• Use the tetracycline system to temporally control
  gene expression
• Examine Developmental issues
• Study acquisition vs extinction
• Use Genetic Manipulation to examine age-related
  changes in learning and memory

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Temporal Control of Transgene Expression

• Tetracycline Responsive Transciptional Activator
  (tTA) is used to temporally control transgene
  expression
• tTA stimulates gene expression from its cognate
  promoter
• Doxycycline inhibits promoter activity

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Spatial and Temporal Control of Transgene
Expression
Double Transgenic Mice
tTA-Gene
Region Specific Promoter
tTA
_
Doxycycline

Effector Gene
tTA responsive Promoter
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Effector Genes

• R(AB) transgene is the dominant negative form of
  the regulatory subunit of PKA
• C(QR) transgene has a mutation in the catalytic
  subunit which up regulates PKA
• ICER (inducible cAMP early repressor) transgene
  down regulates CREB via transcription factor
  repression
• CREBY/F transgene promotes CREB gene expression
  via constitutive phosphorylation of a mutant
  polypeptide at Ser 133.

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Region Specificity

• ?6 promoter from the type A gamma-aminobutyric
  acid receptor alpha6-subunit gene is only
  expressed in cerebellar granule cells
• L7 promoter is from a Purkinje cell-specific
  gene.

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Spatial and Temporal Control of PKA Expression in
Cerebellar Granule Cells
Double Transgenic Mice
tTA-Gene
?6 Promoter
tTA
Doxycycline
_

R(AB) Transgene
tTA responsive Promoter
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Spatial and Temporal Control of PKA Expression in
Cerebellar Purkinje Cells
Double Transgenic Mice
tTA-Gene
L7 Promoter
tTA
Doxycycline
_

R(AB) Transgene
tTA responsive Promoter
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Microarrays

• Method of examining changes in gene expression
  associated with event, drug, or disease

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Probing Microarrays
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Delay Contextual Fear Conditioning
Training Day
Testing Day
Context - Shock US Association Hippocampal
Dependent
Test Freezing to Context
Clicker CS - Shock US Association Hippocampal
Independent
Test Freezing to CS in Altered Context
CS 30 sec white noise, US 0.5 mA 2 sec shock,
ITI 2 minutes, 2 trials
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Duration of Enhancement

• Nicotine must be administered on training and
  testing days for enhancement. Will enhancement
  be seen in the absence of nicotine at a second
  test?
• Groups
• Nicotine pre-training and prior to testing at 24
  hours retest one week later with no nicotine
• Saline pre-training and prior to testing at 24
  hours retest one week later with no nicotine

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Nicotine Alters Gene Expression

• Long-term memory for contextual fear conditioning
  remained enhanced at later retests in the absence
  of nicotine (Gould and Higgins, 2003)
• Long-term memory is thought to be stored in
  neurons as a result of changes in gene expression
  induced by the activation of intracellular
  signaling pathways (reviewed in Abel and Lattal,
  2001)
• Nicotine can activate cellular and molecular
  processes involved in the chain of events linking
  synaptic activity to gene expression (Berg and
  Conroy, 2002 Dajas-Bailador et al., 2002)
• Use microarray analysis to determine if
  hippocampus-dependent learning in the presence of
  nicotine results in a different pattern of gene
  expression than learning in the absence of
  nicotine

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Affymetrix Microarrays
6000 mouse genes per chip Arrayed as
oligonucleotides 20 per gene Mismatch
oligonucleotides used as controls Does nicotine
alter gene expression in the hippocampus during
fear conditioning?
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Microarray Experimental Design
Train and test mice nic/nic sal/sal Prepare
mRNA from hippocampus, amygdala, prefrontal
label cRNA prepared from mRNA hybridize to mouse
Affymetrix microarray MGU74Av2 Compare gene
expression levels compare to normal variance
in these brain regions compare to mice treated
with nicotine or saline and not
conditioned Confirm using real-time PCR
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Nicotine and Saline Mean Hippocampi Log Base 2
Expression Values

• Each dot represents the expression level of a
  single probe set (Gene) on both chips.
• Dots outside the line of (yx) are outliers
  potentially represent genes that affected by the
  nicotine manipulation.
• Manipulation does not affect most of the genome.
  The are about 20 genes that appear affected
  between the expression levels of
  5-11--representing about 1.5 fold changes.

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Changes In Gene Expression

• Differences between nicotine and saline arrays
  were specific because there were no global
  changes in average expression between the groups
• 20 Genes with Significantly Higher Expression in
  Nicotine Group
• 3 Genes with Significantly Higher Expression in
  Saline Group

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Potential Genes of Interest

• Potassium voltage-gated channel, shaker-related
  subfamily, beta member 1
• Accessory potassium channel protein which
  modulates the activity of the pore-forming alpha
  subunit alters the functional properties of
  kv1.1 and kv1.4
• Centrin 2
• Coding for calmodulin
• Annexin A3
• Inhibitor of phospholipase a2
• Nucleosome assembly protein 1-like 1
• May be involved in modulating chromatin formation
  and contribute to regulation of cell
  proliferation
• Mitogen activated protein kinase 8
• phosphorylating a number of transcription factors
  
• Mitogen activated protein kinase 10
• phosphorylating a number of transcription factors

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Conclusions

• Nicotine enhances hippocampus-dependent versions
  of fear conditioning
• Nicotine enhancement of fear conditioning is
  long-lasting and this long-lasting memory is
  expressed in the absence of nicotine
• Nicotine administration during training and
  initial testing is associated with an
  up-regulation of genes that may have a role in
  synaptic plasticity

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Acknowledgements

• Transdisciplinary Tobacco Use Research Center
  UPENN
• American Federation of Aging Research
• The PA Department of Health (TG)

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Thanks
Gould Lab Jennifer Davis Mike Lewis Olivia
Rossebo Dan Moore Steve Higgins Joel Lommock Alla
Kryss Collaborators and Colleagues Ted Abel,
Ph.D. UPENN Sheree Logue, Ph.D. Aventis
Pharmaceutical Jeanne Wehner, Ph.D. Univ
Colorado Diana Woodruff-Pak, Ph.D. Temple Univ