This work was funded by the DFG (SFB 779 TPB8, Kr1879/3-1 MRK), DIP grant (MRK), EU FP7 MC-ITN NPlast (MRK), Center for Behavioral Brain Sciences (CBBS, Sahsen-Anhalt), (AK and SB), MM is a recipient of European Molecular Biology Organization (EMBO) Long-Term Fellowship (EMBO ALTF 884-2011) and Marie-Curie IEF.

1. Preparation of Acute Hippocampal Slices from Adult Rat Brain
<ol>
	<li>Anesthetize rats with isoflurane. CAUTION: Perform the procedure using a closed exicator, do not inhale isoflurane. Make sure that the animal is completely anesthetized.</li>
	<li>Decapitate the rat, quickly isolate the brain, and immerse it in precarbonated (95% O2 / 5% CO2 gas mixture) ice-cold Gey&#8217;s solution (composition in mM: 130 NaCl, 4.9 KCl, 1.5 CaCl2&#183;2H2O, 0.3 MgSO4&#183;2H2O, 11 MgCl2&#183;6H2O, 0.23 KH2PO4, 0.8 Na2HPO4&#183;7H2O, 5 Glucose&#183;H2O, 25 HEPES, 22 NaHCO3, pH 7.32) for 30 min1,2,10,16.</li>
	<li>Remove the cerebellum and part of the entorhinal cortex. Separate the cortical hemispheres with a mid-sagittal cut, then place each hemisphere down on its medial surface. Thereafter make a 50-70&#176; cut (50-70&#176; transverse) along the dorsal edge of each hemisphere13,14.</li>
	<li>Glue each hemisphere with the freshly cut surface on the slicing platform of the sectioning system. The platform should be covered by precarbogenated ice-cold Gey&#8217;s solution.</li>
	<li>Cut 350 &#956;m 50-70&#176; transverse slices from anterior to posterior side using a vibratome adjusted to minimize z-axis oscillation. The hippocampal formation, subicular and entorhinal cortices, as well as the cortices that are located dorsolateral to the hippocampus will be part of the slices used for experiments13,14.</li>
	<li>Transfer hippocampal slices to a U-shape and submerged type incubator and incubate for at least 2 hr at 32 &#176;C with carbogenated artificial cerebrospinal fluid (ACSF containing in mM: 110 NaCl, 2.5 KCl, 2.5 CaCl2&#183;2H2O, 1.5 MgSO4&#183;2H2O, 1.24 KH2PO4, 10 Glucose&#183;H2O, 27.4 NaHCO3, pH 7.3)1,2,10,16.</li>
</ol>
2. Positioning of Electrodes, Baseline Recording, and Induction of LTP
<ol>
	<li>Transfer the hippocampal slice to a submerged type recording chamber mounted under a microscope. Perfuse (6-7 ml/min) with carbogenated ACSF for at least 30 min at 32 &#176;C.</li>
	<li>Prepare glass capillary microelectrodes filled with ACSF (tip resistance is 3-5 M&#937;).</li>
	<li>Place a glass microelectrodes filled with ACSF in the CA1 Schaffer-collateral fibers for stimulation and in the CA1 stratum radiatum for fEPSP recording1,2,10&#160;(Figure&#160;2). The distance between the electrodes should be about 300 &#956;m.</li>
	<li>Evoke field Excitatory Postsynaptic Potentials (fEPSPs) by stimulation of Schaffer-collateral fibers with biphasic rectangular current pulses (200 msec/polarity) in a range of 3-4 V.</li>
	<li>Perform the maximum stimulation test by measuring the input-output relationship and define the stimulation strength as 40% of maximum fEPSP-slope values obtained and keep it constant throughout the experiment.</li>
	<li>Begin the baseline recording for at least 15 min after the maximum stimulation test. Measure the responses to test stimuli every minute throughout the experiment. Perform baseline recordings with low-frequency stimulation.</li>
	<li>Record the baseline for at least 30 min.</li>
	<li>For Late-LTP induction apply high-frequency 100 Hz tetanization consisting of three 1 sec stimulus trains at 100 Hz with a 5 min intertrain interval. To increase the field of stimulation within CA1 region 5 &#956;M bicuculline can be washed in 2 min before tetanization and should be washed&#160;out immediately after last tetanization.</li>
</ol>
3. Collection of Slices after Induction of LTP and Snap Freezing
<ol>
	<li>Stop LTP recording 2 min or 30 min after three trains of tetanic stimulation inducing Late-LTP.</li>
	<li>Remove the electrodes and quickly transfer the slice onto a precold metal platform placed on dry ice. Repeat the procedure for a control slice kept in U-shaped incubator. CAUTION: Use gloves while working with dry ice.</li>
	<li>Collect each slice in a 1.5 ml tube and store at -80 &#176;C.</li>
</ol>
4. Isolation of Nuclear Enriched Fractions from the CA1 Region of the Hippocampus
<ol>
	<li>Take 1.5 ml Eppendorf tubes with the frozen slices from -80 &#176;C and keep them on ice.</li>
	<li>Add 0.5 ml of fresh cold TBS buffer containing protease (PI) and phosphatase (PS) inhibitors, into the tube. Incubate for 2-3 min and transfer to a stereomicroscope. CAUTION: Use protective gloves and a lab coat while working with PI and PS.</li>
	<li>Dissect the CA1 stratum pyramidale region of the hippocampus using two needles. Use one needle for holding the slice under the stereomicroscope and the second needle for cutting the CA1 area.</li>
	<li>Collect the dissected CA1 regions from 5 slices (for each group) into a new 1.5 ml tube containing 50 &#956;l lysis buffer (1x hypotonic lysis buffer containing in mM: 10 HEPES, 1.5 MgCl2, 10 KCl, pH 7.9, with PI and PS). Note that this amount of material will be sufficient to run 2-3 immunoblots.</li>
	<li>Homogenize collected tissue by careful pipetting up and down. Use a 200 &#956;l&#160;pipette. Incubate the lysate on ice for 5-7 min to allow the cells to swell.</li>
	<li>Take 2 &#956;l of the lysate, drop on a microscopic slide, and visualize the swelling of the cells under a bright field microscope. With proper swelling of the cells the nuclei appear as round intact structures.</li>
	<li>Collect 20 &#956;l of sample into a fresh 1.5 ml tube as &#8216;homogenate fraction&#8217;. Add 8 &#956;l of denaturing 4x SDS sample buffer.</li>
	<li>Centrifuge the remaining lysates at 11,000 rpm&#160;for 1 min.</li>
	<li>Carefully collect the supernatant from the top (&#8216;cytosolic fraction&#8217;), transfer into a new tube and add 20 &#956;l of 4x sample buffer.</li>
	<li>Resuspend the pellet in 60 &#956;l of hypotonic buffer and add 20 &#956;l of 4x sample buffer. This fraction is referred to as a &#8216;nuclear enriched fraction&#8217;.</li>
	<li>Homogenate, cytosolic, and nuclear enriched fractions can be stored at -20 &#176;C or -80 &#176;C for later immunoblotting.</li>
</ol>
5. Semiquantitative Immunoblotting for Synapto-nuclear Proteins
<ol>
	<li>Defreeze the samples and boil them for 5 min at 95 &#176;C.</li>
	<li>Take 5 &#181;l from each fraction and determine the protein concentration by amido black test or BCA test.</li>
	<li>Load equal amount of protein samples from homogenate, cytoplasmic and nuclear enriched fractions on SDS-PAGE. Samples from control and LTP slices should be placed on the same gel for direct comparison.</li>
	<li>Perform a standard western-blotting procedure (wet transfer is recommended).</li>
	<li>Probe the membranes with the antibody of choice. Subsequently the same blots (or same samples run in parallel) can be probed with a cytoplasmic marker - neuron specific enolase2 (NSE2) and nuclear marker - NeuN and a -actin antibody as a loading control.</li>
</ol>
6. Data Analysis
<ol>
	<li>Efficiency of LTP induction can be analyzed by Clampfit software. The average slope of baseline recordings was compared with the slopes after tetanization using two-way ANOVA, p &#60;0.05 was considered significantly different. The fEPSP slope values were depicted in diagrams as the mean &#177; S.E.M.</li>
	<li>For quantification of immunoblots, scan either the autoradiographic film and analyze the integrated density of protein bands by ImageJ or fluorescence bands with a Licor system. Values of immunoreactive bands should be normalized for the loading and blotting control. For comparison of the control and LTP groups a nonparametrical Mann-Whitney U-Test might be used.</li>
</ol>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Name of the buffer</td>
			<td>Reagent</td>
			<td>Concentration&#160; (mM)&#160;</td>
			<td>Amount</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Gey&#8217;s solution&#160;(pH: 7.3~7.4)</td>
			<td>NaCl&#160;</td>
			<td>130</td>
			<td>7.6 g</td>
			<td>sterile filtration</td>
		</tr>
		<tr>
			<td>1,000 ml</td>
			<td>KCl</td>
			<td>4.9</td>
			<td>0.37 g</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>CaCl2&#183;2H2O</td>
			<td>1.5</td>
			<td>0.22 g</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>MgSO4&#183;2H2O</td>
			<td>0.3</td>
			<td>0.0739 g</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>MgCl2&#183;6H2O</td>
			<td>11</td>
			<td>2.24 g</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>KH2PO4</td>
			<td>0.23</td>
			<td>0.0313 g</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Na2HPO4&#183;7H2O</td>
			<td>0.8</td>
			<td>0.2145 g</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Glucose&#183;H2O</td>
			<td>5</td>
			<td>0.9909 g</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>HEPES</td>
			<td>25</td>
			<td>5.96 g</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>NaHCO3</td>
			<td>22</td>
			<td>1.85 g</td>
			<td></td>
		</tr>
		<tr>
			<td>ACSF (pH: 7.3~7.4)</td>
			<td>NaCl&#160;</td>
			<td>110</td>
			<td>6.428 g</td>
			<td>sterile filtration</td>
		</tr>
		<tr>
			<td>1,000 ml</td>
			<td>KCl</td>
			<td>2.5</td>
			<td>0.1865 g</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>CaCl2&#183;2H2O</td>
			<td>2.5</td>
			<td>0.368 g</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>MgSO4&#183;2H2O</td>
			<td>1.5</td>
			<td>0.370 g</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>KH2PO4</td>
			<td>1.24</td>
			<td>0.169 g</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Glucose&#183;H2O</td>
			<td>10</td>
			<td>1.9817 g</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>NaHCO3</td>
			<td>27.4</td>
			<td>2.3 g</td>
			<td></td>
		</tr>
		<tr>
			<td>1x&#160;Hypotonic buffer (pH: 7.9)</td>
			<td>HEPES</td>
			<td>10</td>
			<td>0.23 g</td>
			<td></td>
		</tr>
		<tr>
			<td>100 ml</td>
			<td>MgCl2&#183;6H2O</td>
			<td>1.5</td>
			<td>0.0304 g</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>KCl</td>
			<td>10</td>
			<td>0.07455 g</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1. Buffers.

<ol>
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The authors have nothing to disclose.

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...

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 activity8,11.
There are number of proteins that have been shown to shuttle between distal dendrites and the nucleus. Many of these proteins contain a nuclear localization signal and are actively transported along microtubuli in a dynein and importin-dependent manner to the nucleus6,9. Interestingly, some of these messengers only transit to the nucleus in response to specific synaptic stimuli. For example, retrograde transport of cyclic AMP response element binding protein 2 (CREB2) is induced by chemical LTD but not LTP12. Localized NMDAR-dependent synaptic stimulation drives CREB-regulated transcriptional coactivator (CRTC1) into the nucleus, a translocation process, which is involved in long-term hippocampal plasticity4. It was recently shown that the protein messenger Jacob translocates to the nucleus after both, synaptic and extrasynaptic NMDAR activation and regulates CREB dependent gene transcription5. The synaptic or extrasynaptic origin of the signal is encoded in a posttranslational modification of Jacob. Synaptic activity induces ERK1/2 dependent phosphorylation of Jacob at a crucial serine at position 180 (pJacobS&#160;180) which is a requisite for the subsequent translocation to the nucleus in primary hippocampal culture. Moreover, in CA1 neurons of acute hippocampal slices pJacobS 180 translocates to the nucleus after Schaffer collateral LTP but not LTD1,10. pS180&#160;Jacob leads to an increased expression of plasticity related genes and this gene expression feeds back to synaptic function. In sharp contrast, Jacob that translocates to the nucleus after extrasynaptic NMDARs activation is not phosphorylated at Ser180 and might be associated with different protein complex in the nucleus causing &#8216;CREB shut off&#8217; and a retraction of synaptic contacts10.
Most published studies on the nuclear import of synapto-nuclear protein messenger have been done in dissociated neuronal primary cultures. Therefore it would be interesting to see if such findings can be reproduced in physiologically more relevant conditions using hippocampal slices where neuronal connectivity and function are much better preserved. Here we present an optimized protocol for assessing LTP-dependent nuclear translocation of protein messengers by immunoblotting. This method is also suitable for analyzing activity dependent phosphorylation of proteins in a crude nuclear fraction. Specifically, the current protocol involves preparation of acute CA1 hippocampal slices, induction, and recording of LTP. Next, CA1 region is microscopically dissected to isolate the stimulated region. We combined and modified the protocol for nuclear isolation provided by CellLytic NuCLEAR Extraction Kit with changes introduced by Zhao and colleagues17. The optimized procedure includes the lysis of dissected CA1 regions in hypotonic buffer allowing cell swelling and release of nuclei. Cell lysis and nuclei morphology can be determined by microscopic examination. Nuclear enrichment is achieved by a short centrifugation step. Immunoblotting analysis with antibodies against NeuN and NSE2, specific markers of nuclear or cytosolic fractions, indicates that this approach can be used as a fast and reproducible protocol to isolate these subcellular fractions and to study very labile posttranslational modifications like protein phosphorylation. Additionally, this method is advantageous for small tissue samples deriving from dissected CA1 regions of hippocampal slices and can be used in combination to immunohistochemistry of hippocampal slices.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 1. Equipment</td>
		</tr>
		<tr>
			<td>CED 1401 AD/DA converter</td>
			<td>Cambridge Electronics Design, UK</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Axopatch 200B amplifier</td>
			<td>Axon&#65292;USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td><a href="http://www.iciba.com/data_acquisition_system">Axon Digidata acquisition system</a></td>
			<td>Axon&#65292;USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clampex 10.0 Data acquisition and analysis software</td>
			<td>Axon&#65292;USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica microscope</td>
			<td>Leica TCS SP2&#65292;Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>P-97 Standard microelectrode puller</td>
			<td>Sutter&#65292;USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td><a href="http://www.iciba.com/stereomicroscope">Stereomicroscope</a></td>
			<td>Leica</td>
			<td>Leica S4E, Germany</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibrotome</td>
			<td>Leica VT1000S, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isolated pulse stimulator</td>
			<td>Model2100, A-M systmes, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific, HERAEUS, FRSCO17</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE system</td>
			<td>BIORAD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cooler</td>
			<td>JULABO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bright field Microscope</td>
			<td>NIKON ECLIPSE TS100</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>Thermo Electron Corporation, HERAEUS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Luigs &#38; Neumann, SM-5, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blotting chamber and electric power supplier</td>
			<td>Hoefer Scientific Instruments, San Francisco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cooler</td>
			<td>Julabo, F12</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific, Heraeus, FRESCO 17</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Submerged type Recording Chamber</td>
			<td>custom made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>U-shape and submerged type incubator&#160;</td>
			<td>custom made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small surgical scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thin spatula</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Pasteur pipette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic culture dish&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bunsen beaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. Reagents</td>
		</tr>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>ROTH</td>
			<td>Art.-Nr.3957.1</td>
			<td>&#8805;99.5%, p.a.,ACS,ISO</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>ROTH</td>
			<td>Art.-Nr.6781.1</td>
			<td>&#8805;99.5%, p.a.,ACS,ISO</td>
		</tr>
		<tr>
			<td>CaCl2&#183;2H2O</td>
			<td>MERCK</td>
			<td>1.02382.0500</td>
			<td>pro analysi</td>
		</tr>
		<tr>
			<td>MgSO4&#183;2H2O</td>
			<td>MERCK</td>
			<td>5886.05</td>
			<td>pro analysi</td>
		</tr>
		<tr>
			<td>MgCl2&#183;6H2O</td>
			<td>AppliChem</td>
			<td>CAS-NO: 7791-18-6; EC-NO:2320946</td>
			<td>for Molecularbiology</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>MERCK</td>
			<td>12034.025</td>
			<td>for Molecularbiology</td>
		</tr>
		<tr>
			<td>Na2HPO4&#183;2H2O</td>
			<td>MERCK</td>
			<td>1.06574.1000</td>
			<td>extra pure</td>
		</tr>
		<tr>
			<td>Glucose&#183;H2O</td>
			<td>ROTH</td>
			<td>Art.-Nr.6887.1</td>
			<td>for Molecularbiology</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>ROTH</td>
			<td>Art.-Nr.9105.4</td>
			<td>PUFFERAN, &#8805;99.5%, p.a.</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>MERCK</td>
			<td>1.06329.1000</td>
			<td>pro analysi</td>
		</tr>
		<tr>
			<td>Protease inhibitor Coctail</td>
			<td>ROCHE</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphostop</td>
			<td>ROCHE</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bicuculline</td>
			<td>Tocris bioscience</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Isofluran</td>
			<td>Baxter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 4. Antibodies</td>
		</tr>
		<tr>
			<td>Primary antibodies</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Species</td>
		</tr>
		<tr>
			<td>pJac-s180</td>
			<td>Biogenes/ purified</td>
			<td></td>
			<td>rabbit (dil. 1:100)</td>
		</tr>
		<tr>
			<td>NeuN</td>
			<td>Milipore</td>
			<td>MAB377</td>
			<td>mouse (dil. 1:1,000)</td>
		</tr>
		<tr>
			<td>NSE</td>
			<td>Cell Signaling</td>
			<td>D20H2</td>
			<td>rabbit (dil. 1:1,000)</td>
		</tr>
		<tr>
			<td>beta-actin</td>
			<td>Sigma</td>
			<td>A-5441</td>
			<td>mouse (dil. 1:5,000)</td>
		</tr>
		<tr>
			<td>Secondary antibodies</td>
			<td></td>
			<td></td>
			<td>Species</td>
		</tr>
		<tr>
			<td>IgG HRP Conjugated</td>
			<td>DAKO</td>
			<td></td>
			<td>goat anti - mouse (1:5,000)</td>
		</tr>
		<tr>
			<td>IgG HRP Conjugated</td>
			<td>NEB</td>
			<td></td>
			<td>goat anti - rabbit (1:5,000)</td>
		</tr>
	</tbody>
</table>

isolation of ca1 nuclear enriched fractions from hippocampal slices to study activity-dependent nuclear import of synapto-nuclear messenger proteins

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.&#160;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.

We have previously shown that the synapto-nuclear protein messenger Jacob accumulates in the nucleus following the&#160;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 filamen...

Watch this Scientific Journal Video about Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins at JoVE.c

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

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.

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular ...

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Research

JoVE Journal

Neuroscience

12.0K Aufrufe.  Leibniz Institute for Neurobiology.  Wir stellen ein detailliertes Protokoll für die Induktion von Langzeit-Potenzierung in der CA1-Region des Hippocampus und der anschließenden Trennung der Kern angereicherten Fraktionen aus dem tetanized Bereich der Scheibe. Dieser Ansatz kann verwendet werden, um abhängig Kernproteinimport in Zellmodellen von Lernen und Gedächtnis zu bestimmen Aktivität. 

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

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer&#x2019;s disease.

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic cues. Their membranes are often juxtaposed at defined contact sites, which become hubs for the exchange of signaling molecules and membrane components1,2,3,4. The inter-organellar membrane microdomains that are formed between the endoplasmic reticulum (ER) and the mitochondria at the opening of the IP3-sensitive Ca2+ channel are known as the mitochondria associated-ER membranes or MAMs4,5,6. The protein/lipid composition and biochemical properties of these membrane contact sites have been extensively studied particularly in relation to their role in regulating intracellular Ca2+ 4,5,6. The ER serves as the primary store of intracellular Ca2+, and in this capacity regulates a myriad of cellular processes downstream of Ca2+ signaling, including post-translational protein folding and protein maturation7. Mitochondria, on the other hand, maintain Ca2+ homeostasis, by buffering cytosolic Ca2+ concentration thereby preventing the initiation of apoptotic pathways downstream of Ca2+ unbalance4,8. The dynamic nature of the MAMs makes them ideal sites to dissect basic cellular mechanisms, including Ca2+ signaling and regulation of mitochondrial Ca2+ concentration, lipid biosynthesis and transport, energy metabolism and cell survival 4,9,10,11,12. Several protocols have been described for the purification of these microdomains from liver tissue and cultured cells13,14. 
	Taking previously published methods into account, we have adapted a protocol for the isolation of mitochondria and MAMs from the adult mouse brain. To this procedure we have added an extra purification step, namely a Triton X100 extraction, which enables the isolation of the glycosphingolipid enriched microdomain (GEM) fraction of the MAMs. These GEM preparations share several protein components with caveolae and lipid rafts, derived from the plasma membrane or other intracellular membranes, and are proposed to function as gathering points for the clustering of receptor proteins and for protein&#x2013;protein interactions4,15.

Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain

This procedure illustrates how to isolate from the adult mouse brain the mitochondria-associated ER membranes or MAMs and the glycosphingolipid-enriched microdomain fractions from MAMs and mitochondrial preparations.

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly committed intermediate progenitor subtypes. Each of these subtypes expresses a set of specific molecular markers that, together with specific morphological criteria, can be used for their identification. Typically, immunofluorescent techniques are applied involving subtype-specific antibodies in combination with exo- or endogenous proliferation markers. We herein describe immunolabeling methods for the detection and quantification of all stages of adult hippocampal neurogenesis. These comprise the application of thymidine analogs, transcardial perfusion, tissue processing, heat-induced epitope retrieval, ABC immunohistochemistry, multiple indirect immunofluorescence, confocal microscopy and cell quantification. Furthermore we present&#160;a sequential multiple immunofluorescence protocol which circumvents problems usually arising from the need of using primary antibodies raised in the same host species. It allows an accurate identification of all hippocampal progenitor subtypes together with a proliferation marker within a single section. These techniques are a powerful tool to study the regulation of different progenitor subtypes in parallel, their involvement in brain pathologies and their role in specific brain functions.

This video article illustrates a comprehensive protocol to detect and quantify all stages of adult hippocampal neurogenesis within the same tissue section. We elaborated a method to overcome the limitations of indirect multiple immunofluorescence that arise when suitable antibodies from different host species are unavailable.

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly ...

Investigation of Synaptic Tagging/Capture and Cross-capture using Acute Hippocampal Slices from Rodents

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity is achieved in neurons within a specific time frame. These long-term plasticity-related processes are the leading candidate models to study the basis of memory formation and persistence at the cellular level. Both STC and cross-tagging involve two serial processes: (1) setting of the synaptic tag as triggered by a specific pattern of stimulation, and (2) synaptic capture, whereby the synaptic tag interacts with newly synthesized plasticity-related proteins (PRPs). Much of the understanding about the concepts of STC and cross-tagging arises from the studies done in CA1 region of the hippocampus and because of the technical complexity many of the laboratories are still unable to study these processes. Experimental conditions for the preparation of hippocampal slices and the recording of stable late-LTP/LTD are extremely important to study synaptic tagging/cross-tagging. This video article describes the experimental procedures to study long-term plasticity processes such as STC and cross-tagging in the CA1 pyramidal neurons using stable, long-term field-potential recordings from acute hippocampal slices of rats.

This video article describes experimental procedures to study long-term plasticity and its associative processes such as synaptic tagging, capture and cross-tagging in the CA1 pyramidal neurons using acute hippocampal slices from rodents.

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Implantation of Chronic Silicon Probes and Recording of Hippocampal Place Cells in an Enriched Treadmill Apparatus

An important requisite for understanding brain function is the identification of behavior and cell activity correlates. Silicon probes are advanced electrodes for large-scale electrophysiological recording of neuronal activity, but the procedures for their chronic implantation are still underdeveloped. The activity of hippocampal place cells is known to correlate with an animal&#39;s position in the environment, but the underlying mechanisms are still unclear. To investigate place cells, here we describe a set of techniques which range from the fabrication of devices for chronic silicon probe implants to the monitoring of place field activity in a cue-enriched treadmill apparatus. A micro-drive and a hat are built by fitting and fastening together 3D-printed plastic parts. A silicon probe is mounted on the micro-drive, cleaned, and coated with dye. A first surgery is performed to fix the hat on the skull of a mouse. Small landmarks are fabricated and attached to the belt of a treadmill. The mouse is trained to run head-fixed on the treadmill. A second surgery is performed to implant the silicon probe in the hippocampus, following which broadband electrophysiological signals are recorded. Finally, the silicon probe is recovered and cleaned for reuse. The analysis of place cell activity in the treadmill reveals a diversity of place field mechanisms, outlining the benefit of the approach.

We describe the diverse steps to implant chronic silicon probes and to record place cells in mice that are running head-fixed on a cue-enriched treadmill apparatus.

An important requisite for understanding brain function is the identification of behavior and cell activity correlates. Silicon probes are advanced ...

An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central nervous system and thus, can be used to analyze the diseased state of the brain or spinal cord without directly accessing these tissues. However, in mice it is difficult to obtain from the cisterna magna due to its closeness to blood vessels, which often contaminate samples. The area for CSF collection in mice is also difficult to dissect to and often only small samples are obtained (maximum of 5-7 &#181;L or less). This protocol describes in detail a technique that improves on current methods of collection to minimize contamination from blood and allow for the abundant collection of CSF (on average 10-15 &#181;L can be collected). This technique can be used with other dissection methods for tissue collection from mice, as it does not impact any tissues during CSF extraction. Thus, the brain and spinal cord are not affected with this technique and remain intact. With greater CSF sample collection and purity, more analyses can be used with this fluid to further aid neuroscience research and better understand diseases affecting the brain and spinal cord.

This protocol describes an improved technique for the abundant collection of cerebrospinal fluid (CSF) with no contamination from blood. With greater sample collection and purity, more analyses can be performed using CSF to further our understanding of diseases that affect the brain and spinal cord.

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central ...