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

2. 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.

3. 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.

4. 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 1:4 that will be homozygous for the transgene Or create dominant transgene – Mating these will found the transgenic strain.

6. 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

7. Cerebellar Circuit

8. Eye-Blink Circuit Cerebellar Cortex Cerebellar Interpositus Nucleus Red Nucleus Lateral Pontine Nucleus (CS) Granule Cells Inferior Olive Reticular Formation Abducens Nucleus Trigeminal Nucleus (US) Parallel FibersClimbing Fibers Mossy Fibers

9. 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

10. 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

11. Spatial and Temporal Control of Transgene Expression Double Transgenic Mice Region Specific Promoter tTA-Gene tTA responsive Promoter Effector Gene _ +

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

13. 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.

14. Spatial and Temporal Control of PKA Expression in Cerebellar Granule Cells Double Transgenic Mice  6 Promoter tTA-Gene tTA responsive Promoter R(AB) Transgene _ +

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

16. Microarrays Method of examining changes in gene expression associated with event, drug, or disease

17. Probing Microarrays

18. Delay Contextual Fear Conditioning Training DayTesting Day Context - Shock US Association Hippocampal Dependent Clicker CS - Shock US Association Hippocampal Independent Test Freezing to Context Test Freezing to CS in Altered Context CS = 30 sec white noise, US = 0.5 mA 2 sec shock, ITI 2 minutes, 2 trials

19. 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

21. 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

22. 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?

23. 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

24. 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 (y=x) 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.

25. 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

26. 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

28. 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

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

30. 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