Neurobiology of Learning and Memory Prof. Stephan Anagnostaras Lecture 9: Molecular-Genetic Approaches to Learning & Memory

CamKIIa and CREB are required for LTP

Synaptic Kinase activation Nuclear CREB activation Mechanisms of Learning and Memory NMDA Synaptic Kinase activation D1 AMPA A Ca Cyclase cAMP CaMKII PDE CREB Nuclear CREB activation

Gene replacement and transgenic animals Some genes are identified through mutant analysis Forward Genetics (mutant phenotype---> genotype) To determine the function of these genes, it is possible to replace an organism’s wild type gene with an inactive gene to create a “gene knockout” Reverse Genetics (mutant genotype--->phenotype) It is also possible to introduce additional genes (transgenes) to create a transgenic organism Epigenetics Associate gene expression or polymorphisms with phenotype

In mice, two main kinds of animals Transgenic A novel gene, the transgene, either man-made or borrowed from another animal is added to the genome. Works in many species - easy Knockout A gene is deleted; in practice the gene is replaced in the same location (homologous recombination) with a null mutation of the gene. Only possible in mice, difficult Related mutants: Dominant negative: a massively overexpressed transgene that interferes with the endogenous gene (easy) Knockin: Homologous recomination of a transgene (very difficult and rare)

In vitro mutagenesis of a cloned gene Gene knockout and transgenic techniques usually involve mutagenesis of cloned genes prior to transfer into the organism

Production of transgenic mice Confusing example is a dominant negative transgene (like Mayford & Kandel), not a knockout but a lot like a knockout

Creation of mice embryonic stem (ES) cells carrying a knockout mutation

Gene knockout in mice

•Disrupts autophosphorylation of Threonine at 286 Disruption of CamKIIa by T286A point mutation disrupts LTP and spatial memory •Disrupts autophosphorylation of Threonine at 286 T=Threonine (ACT, ACC, ACA) A=Alanine (GCT, GCC, GCA,GCG) Unable to switch to Ca-Calmodulin independent state Giese et al., Science,1998

Cell-type-specific gene knockouts in mice: Cre-Lox technique Spatial and/or temporal control of deletion

Tsien et al., Cell, 1996: CA1 specific NMDAR1 knockout

CA1 DG Tsien et al., Cell, 1996

McHugh et al., Cell, 1996: Place cells in CA1 NMDAR KO Abnormally large place fields Low coherent firing

An Temporally controlled inducible CREB Repressor: The LBD Fusion Approach Kida, Josselyn et al., 2002 LBD CREBr Heat Shock Protein Tamoxifen CREBr LBD CREBr LBD Cytoplasm Nucleus

The LBD-CREB-MT Vector (single Transgene)  CaMKII promoter HA LBD CREB-MT intron polyA signal HA; influenza virus hemagglutinin (HA) tag LBD ; mutant estrogen receptor ligand binding domain (G521R) CREB-MT; dominant negative CREB (CREB S133A)

Josselyn et al., 2002 - temporal control of the CREB repressor Fear conditioning

Tetracycline-regulated expression of CamKIIa-Asp286 (dominant negative, always phosphorylated T286D D= Aspartate, “constitutively active”) Double-transgenic • Cell-specific tTA • tetO + transgene to regulate Mayford & Kandel, TIG, 1999 D= GAT, GAC

Tet-Off System

Summary - Reverse Genetics •Molecular Biology Approaches Mutant Mice • Knockouts - global deletion • Transgenics - global addition Later generation: • Point mutants (PointLox method) • Region-specific deletion by Cre-Lox • Inducible deletions • Temporal and or region control of transgene - LBD fusion approach - Tet system

Other Approaches - Quantitative Genetics and Functional Genomics Forward Genetic Approaches QTL (Quantitative Trait Loci) ENU (ethylnitrosurea - random mutagenesis) Dominant & recessive screens Mapping & Positional Cloning Epigenetic approaches DNA Microarrays Current mouse array = 11k genes/ 35k Correlate expression or polymorphisms with phenotype