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. Video LinkThe video component of this article can be found at

Synaptic GluN2B/CaMKII-α Signaling Induces Synapto-Nuclear Transport of ERK and Jacob

“…We next set out to confirm these results in acute hippocampal slices where we induced NMDAR-dependent chemical LTP (Otmakhov et al, 2004 ; Boehm et al, 2006 ), dissected the CA1 area of the hippocampus, extracted the nuclei and performed quantitative immunoblot analysis of nuclear panERK/panJacob as well as pERK levels. Equal loading of nuclear proteins was controlled with an antibody directed against NeuN (for protocol see Yuanxiang et al, 2014 ). Application of KN-93 had no effect on the induction and maintenance of LTP within the first hour following stimulation (Figure 4A ).…”

“…Total incubation time with KN-93 was 1 h. Potentiated and control slices were shock frozen and underwent dissection of CA1 regions. Five dissected CA1 regions from the same experimental preparation were pooled together, homogenized and underwent nuclear isolation as described previously (Karpova et al, 2013 ; Yuanxiang et al, 2014 ). Briefly, CA1 regions were homogenized in 50 μl of cold (4°C) hypotonic lysis buffer containing 10 mM HEPES, 1.5 mM MgCl 2 , 10 mM KCl (pH 7.9), protease (Complete, ROCHE) and phosphatase (ROCHE) inhibitors.…”

Potentiation of Schaffer-Collateral CA1 Synaptic Transmission by eEF2K and p38 MAPK Mediated Mechanisms

“…To this end, 5 μM A-484954 was applied to the eEF2K substrate for 8, 16, or 32 min, followed by snap freezing and storage at -80°C. On the day of analysis, the CA1 region was isolated and the resulting eEF2 phosphorylation level was examined (Yuanxiang et al, 2014). The western blots indicated that A-484954 significantly prevented the phosphorylation of eEF2 ( Figure 1A ).…”

Jacob, a Synapto-Nuclear Protein Messenger Linking N-methyl-D-aspartate Receptor Activation to Nuclear Gene Expression

“…Interestingly, Jacob is imported to the nucleus in rat hippocampal slices and neuronal cultures only after induction of NMDAR-dependent LTP but not Ltd. (Behnisch et al, 2011;Yuanxiang et al, 2014;Melgarejo da Rosa et al, 2016). Of note, it accumulates in the nucleus already within 30 min after LTP induction, a time window critical of activity-induced gene expression required for long-lasting LTP expression (Frey et al, 1996;Behnisch et al, 2011).…”