Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Summary

We provide a detailed protocol for induction of long-term potentiation in the CA1 region of the hippocampus and the subsequent isolation of nuclear enriched fractions from the tetanized area of the slice. This approach can be used to determine activity dependent nuclear protein import in cellular models of learning and memory.

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.

Introduction

Synaptic N-methyl-D-aspartate-receptors (NMDARs) play a crucial role in synaptic plasticity and cell survival signaling whereas activation of extrasynaptic NMDARs can trigger neurodegeneration and cell death. These changes depend on tightly controlled/regulated activity dependent gene expression and thus require constant communication between activated synapses or dendrites and the nucleus⁷. The MAP kinases ERK1/2 are downstream effectors of synaptic NMDARs signaling and are involved in NMDAR-activation-induced gene expression, whereas signaling via extrasynaptic NMDAR has no or an inhibitory effect on ERK1/2 activity^8,11.

Protocol

1. Preparation of Acute Hippocampal Slices from Adult Rat Brain

1. Anesthetize rats with isoflurane. CAUTION: Perform the procedure using a closed exicator, do not inhale isoflurane. Make sure that the animal is completely anesthetized.
2. Decapitate the rat, quickly isolate the brain, and immerse it in precarbonated (95% O₂ / 5% CO₂ gas mixture) ice-cold Gey’s solution (composition in mM: 130 NaCl, 4.9 KCl, 1.5 CaCl₂·2H₂O, 0.3 MgSO₄·2H₂O, 11 MgCl₂·6H₂O, 0.23 KH₂PO₄, 0.8 Na₂HPO₄·7H₂O, 5....

Results

We have previously shown that the synapto-nuclear protein messenger Jacob accumulates in the nucleus following the induction of LTP but not LTD¹. Moreover, translocation of Jacob after synaptic stimulation requires activation of MAPK ERK1/2 and phosphorylation of Jacob at Ser180 (Figure 1). Phosphorylated Jacob translocates to the nucleus in an importin-dependent manner and the phosphorylated state can be preserved over extended periods of time by association with the intermediate filament αin.......

Discussion

The steps described in the protocol above provide guidance how to prepare hippocampal acute sliced from young or adult rats, induce and record LTP, rapidly dissect stimulated area of slice, and prepare nuclear enriched fraction for studying activity dependent protein dynamics. This approach derives from combination of several different methods used independently from each other. We optimized a workflow and provide sufficient detail for the beginner to set up their own experiments to study the subcellular redistribution o.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Equipment
CED 1401 AD/DA converter	Cambridge Electronics Design, UK
Stereomicroscope	Leica S4E, Germany
Vibrotome	Leica VT1000S, Germany
Isolated pulse stimulator	A-M Systems, USA		Model 2100
Centrifuge	Thermo Scientific
SDS-PAGE system	Biorad
Cooler	Julabo
Bright field Microscope	Nikon Eclipse TS100
Cell culture incubator	Thermo Electron Corporation
Micromanipulator	Luigs & Neumann, SM-5, Germany
Blotting chamber and electric power supplier	Hoefer Scientific Instruments, San Francisco, CA
Submerged type recording chamber	custom made
U-shape and submerged type incubator	custom made
Small surgical scissors
Scalpel
Thin spatula
Plastic Pasteur pipette
Plastic culture dish
Bunsen beaker
Syringe
Reagents
NaCl	Roth	Art.-Nr.3957.1	≥99.5%, p.a., ACS, ISO
KCl	Roth	Art.-Nr.6781.1	≥99.5%, p.a., ACS, ISO
CaCl2·2H2O	Merck	1.02382.0500	pro analysis
MgSO4·2H2O	Merck	5886.05	pro analysis
MgCl2·6H2O	AppliChem	CAS-NO: 7791-18-6; EC-NO:2320946	for molecular biology
KH2PO4	Merck	12034.025	for molecular biology
Na2HPO4·2H2O	Merck	1.06574.1000	extra pure
Glucose·H2O	Roth	Art.-Nr.6887.1	for molecular biology
HEPES	Roth	Art.-Nr.9105.4	PUFFERAN, ≥99.5%, p.a.
NaHCO3	Merck	1.06329.1000	pro analysis
Protease inhibitor cocktail	Roche
Phosphostop	Roche
Bicuculline	Tocris Bioscience
Isoflurane	Baxter
Antibodies
Primary antibodies	Company	Catalog Number	Species
pJac-s180	Biogenes		rabbit (diluted 1:100)
NeuN	Milipore	MAB377	mouse (diluted 1:1,000)
NSE	Cell Signaling	D20H2	rabbit (diluted 1:1,000)
Beta-actin	Sigma	A-5441	mouse (diluted 1:5,000)
Secondary antibodies	Company	Catalog Number	Species
IgG HRP conjugated	DAKO		goat anti-mouse (1:5,000)
IgG HRP conjugated	NEB		goat anti-rabbit (1:5,000)