title: Isolation of CA1 nuclear enriched fractions from hippocampal slices to study activity-dependent nuclear import of synapto-nuclear messenger proteins.
abstract: Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) induced in acute hippocampal slices are widely accepted as cellular models of learning and memory. There are numerous studies that use live cell imaging or immunohistochemistry approaches to visualize activity dependent protein dynamics. However these methods rely on the suitability of antibodies for immunocytochemistry or overexpression of fluorescence-tagged proteins in single neurons. Immunoblotting of proteins is an alternative method providing independent confirmation of the findings. The first limiting factor in preparation of subcellular fractions from individual tetanized hippocampal slices is the low amount of material. Second, the handling procedure is crucial because even very short and minor manipulations of living slices might induce activation of certain signaling cascades. Here we describe an optimized workflow in order to obtain sufficient quantity of nuclear enriched fraction of sufficient purity from the CA1 region of acute hippocampal slices from rat brain. As a representative example we show that the ERK1/2 phosphorylated form of the synapto-nuclear protein messenger Jacob actively translocates to the nucleus upon induction of LTP and can be detected in a nuclear enriched fraction from CA1 neurons.
authors.affiliation: RG Neuroplasticity, Leibniz Institute for Neurobiology.
authors.forename: Pingan
authors.initials: P
authors.lastname: Yuanxiang
authors.affiliation: RG Neuroplasticity, Leibniz Institute for Neurobiology.
authors.forename: Sujoy
authors.initials: S
authors.lastname: Bera
authors.affiliation: RG Neuroplasticity, Leibniz Institute for Neurobiology.
authors.forename: Anna
authors.initials: A
authors.lastname: Karpova
authors.affiliation: RG Neuroplasticity, Leibniz Institute for Neurobiology.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Kreutz
authors.affiliation: RG Neuroplasticity, Leibniz Institute for Neurobiology; Department of Cell Biology, Utrecht University; mmikhayl@lin-magdeburg.de.
authors.forename: Marina
authors.initials: M
authors.lastname: Mikhaylova
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D000818:Animals; D056547:CA1 Region, Hippocampal / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D002467:Cell Nucleus / Q000378:metabolism*; D007858:Learning / Q000502:physiology; D017774:Long-Term Potentiation; D020935:MAP Kinase Signaling System; D008568:Memory / Q000502:physiology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D051381:Rats; D013569:Synapses / Q000378:metabolism*
chemical_list: D009419:Nerve Tissue Proteins; C504443:Nsmf protein, rat; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3