We thank Angela Kralemann-K&#246;hler for excellent technical support. Experimental work described herein was performed in our laboratory and was supported by grants of the German Research Council (DFG) to CK and BK&#160;and a grant of the German Ministry of Research and Education (BMBF) to BK.

Ethics statement
This study was carried out in strict accordance with the regulations of the governmental animal and care use committee, LANUV of the state North Rhine-Westphalia, (D&#252;sseldorf, Germany). All animal experiments were approved by LANUV, D&#252;sseldorf under the license number 8.87&#8211;51.04.20.09.317 (LANUV, NRW). All efforts were made to minimize distress and the number of animals required for the study.
1. Animal Care and Housing
<ol>
	<li>All animals used in the protocols described herein should be kept under specific pathogen-free conditions, as defined by the Federation European Laboratory Animal Science Association (FELASA).</li>
	<li>Mice should be kept in standard cages in a temperature- and humidity- controlled (22 &#176;C) room under diurnal conditions (12 hr light/dark cycle) with HEPA filtered air.</li>
	<li>Standardized food and water should be provided ad libitum.</li>
	<li>If I&#954;B/tTA and I&#954;B /- control mice are used, PCR-based genotyping should be performed for each animal, as described in7,18.</li>
	<li>Male animals with an age difference of less than four days should to be used to reduce individual variability.</li>
	<li>(OPTIONAL): For NF-&#954;B reactivation experiments, doxycycline must be administered in drinking water (2 mg/ml with&#160;2.5% sucrose) for at least 14 days.</li>
</ol>
2. Spatial Pattern Separation-Barnes Maze (SPS-BM)
<ol>
	<li>All behavioral testing should be carried out according to international and local guidelines.</li>
	<li>IMPORTANT! Use only male mice with an age of six months or older. The age difference between the animals of one test series should be less than four days. The testing must be performed by the same operator for each series. The mice should receive a standard diet prior to the testing to further increase the motivation partially driven by a sweet food reward.</li>
	<li>Set up a white circular plate made from hard plastic (diameter 120 cm, see Figure 5A) in a humidity- and temperature- controlled room (22 &#176;C), illuminated with at least 4 x 80 W and 3 x 215 W neon fluorescent lamps. IMPORTANT! Ensure the correct illumination is used, as the motivation of the mice to enter the food houses is partially driven by their aversion to bright, exposed places.</li>
	<li>Set up the video-tracking system. The camera should be placed 115 cm above the center of the plate (see Figure 5A).</li>
	<li>Attach multicolored extra-maze cues (EMC) to a white-colored cloth in positions easily visible for the animals, approximately 100 cm from the border of the plate (see Figure 5A).</li>
	<li>Carefully clean the plate with a rapid disinfectant that can remove any odor from the experimental set-up.</li>
	<li>Place seven identical yellow food houses (12 cm x 7 cm x 8 cm, see Figure 5A) on the white plate. The positions should be unequivocally marked (see Figure 5A).</li>
	<li>Place sweet food pellet rewards (a quarter of a Kellogs`s Froot Loop/food house) inside all food houses on defined positions (see Figure 5A) with only one defined food house being freely accessible to the animal (location F, see Figure 5A). Close the nontarget food houses with transparent foil.</li>
	<li>Prior to the test, perform habituation (one day before starting the task).Make all food houses freely accessible and allow the mice to explore the maze freely and to retrieve a food pellet reward (10 min/animal).</li>
	<li>Switch the computer and the camera on and start the video-tracking system software.</li>
	<li>Start the recording.</li>
	<li>Place the animal at the defined start point on the circular plate (Figure 5A, S: start position) and allow the mice to search for the target food house for 10 min.</li>
	<li>Stop the video-tracking.</li>
	<li>IMPORTANT! Clean the circular plate and the food houses after each trial with rapid disinfectant.</li>
	<li>Repeat the test daily for seven consecutive days (for each animal).</li>
	<li>Analyze the results (latency, distance covered and errors) using appropriate statistics software. Define errors as approaching the wrong food house and / or contact with the proper box without entering and retrieving the food pellet reward. For grouped analysis use two-way ANOVA with Bonferroni post-hoc test.</li>
	<li>(OPTIONAL)&#160;Sacrifice the mice and analyze the hippocampi as described below.</li>
</ol>
3. BrdU Labeling
<ol>
	<li>Inject intraperitoneally 50 mg/kg i.p. BrdU once daily for 3 days (analysis of differentiation and integration) or 200 mg/kg i.p. for a single injection (analysis of proliferation).</li>
	<li>Sacrifice the animals, dissect the hippocampus and prepare 40 &#181;m sections as described below.</li>
	<li>Denaturate the sections with 2 M HCl for 10 min and incubate in 0.1 M borate buffer for 10 min.</li>
	<li>Label a one-in-twelve series of 40 &#956;m sections (240 &#956;m apart) from each animal immunohistochemically, as described below using antibodies directed against BrdU.</li>
	<li>Quantify the labeled cells by confocal microscopy analysis throughout the rostrocaudal extent of the granule cell layer and subgranular zone. Multiply the resulting numbers by 12 to obtain the estimated total number of BrdU-labeled cells per hippocampus and divide by two to obtain the total number of labeled cells per DG.</li>
</ol>
4. Removal of the Brains and Preparation of Cryosections from Nonperfused Animals
<ol>
	<li>Observe locally approved procedures for euthanizing animals. Mice may be directly euthanized by cervical dislocation.</li>
	<li>(OPTIONAL) Animals can be anesthetized before euthanization according to local and international guidelines, e.g. by intraperitoneal injection of 0.8 ml Avertin for a 33 g mouse (freshly made by mixing 150 ml stock solution made of 2.2 mg 2,2,2-tribromoethanol in 1 ml isoamyl ethanol with 1.85 ml of saline or physiological buffer).</li>
	<li>Sterilize the head with Betadine (10% povidone-iodine)-soaked gauze and swab subsequently with gauze soaked in 70% ethanol.</li>
	<li>Decapitate the animal using appropriate surgical scissors and pull the skin aside.</li>
	<li>(OPTIONAL) Fixators can be applied to avoid folding back of the retarded skin.</li>
	<li>Make a midline incision in the skull and carefully pull the skull fragments aside.</li>
	<li>Carefully remove the whole brain using an appropriate surgical instrument (e.g. Moria Spoon).</li>
	<li>Precool 25 ml of 2-methylbutane in a 50 ml beaker (e.g. Schott Duran) to -30&#160;to -40 &#176;C on dry ice. 
	Note: For free-floating staining of &#34;thick&#34; sections which are ideally suited for confocal laser scanning microscopy, remove brain, wash 3 times in buffer and store at 4 &#176;C in phosphate buffered 30 % sucrose solution in 50 ml tube until brain has sunk down to bottom (typically overnight).</li>
	<li>Carefully place brain on a piece of Nescofilm (Parafilm) and cover with ample amount of TissueTek OCT compound, freeze on Nescofilm in 2-methylbutane, store at -80 &#176;C until use. 
	Note: For long-time storage at -20 &#176;C store in 9% sucrose, 7 mM MgCl2, 50 mM phosphate buffer, 44% glycerol.</li>
	<li>Cut the brain into 10-12 &#956;m thick sections on appropriate cryomicrotome. 
	Note: For thick, free-floating sections, freeze brain on cryotome stage of appropriate cryotome (e.g. Reichert Jung, Frigomobil) and cut 40 &#956;m horizontal sections at -20&#176; to - 25 &#176;C. Collect sections from knife with fine brush and collect in buffer or keep in storage solution ( step 4.9) at -20 &#176;C for long-time storage.</li>
	<li>Carefully mount two slices on single microscope slides. The use of Superfrost UltraPlus slides is highly recommended to maximize adhesion of the sections.</li>
	<li>Dry the slides for 5 min at room temperature. Slides can be stored at -80 &#176;C until use.</li>
</ol>
5. Preparation of Sections from Perfused Animals
<ol>
	<li>Anesthetize the animals as described above (step 4.2).</li>
	<li>Carefully perfuse the animal transcardially with phosphate buffered saline (PBS) containing heparin (0.025 g/100 ml PBS) and procaine (0.5 g/100 ml PBS) for 2-4 min, followed by 4% paraformaldehyde in PBS for 10-15 min. Perfusion is optimally performed by perfusing via the left ventricle and opening of the right atrium using a hydrostatic pressure of approximately&#160;1.2-1.4 m or utilizing a peristaltic pump set to approximately&#160;15 ml/min. Optimal perfusion results in a pale white brain with no red blood vessels being visible.</li>
	<li>Dissect and post-fix the brains in 4% paraformaldehyde at 4 &#176;C for 24 hr.</li>
	<li>Cryoprotect the brains in 30% sucrose in PBS at 4 &#176;C for at least 24 hr.</li>
	<li>Freeze the brains on the cryotome stage.</li>
	<li>Prepare 40 &#181;m horizontal sections from entire forebrains using an appropriate cryotome.</li>
	<li>The slides can be stored in cryoprotectant solution (0.1 M phosphate buffer, 50% glycerol, 0.14% MgCl2, 8.6% sucrose) at -20 &#176;C until use.</li>
</ol>
6. Immunohistochemistry of Brain Sections of Nonperfused Animals
<ol>
	<li>Post-fix the cryosections, prepared as described above, using -20 &#176;C cold methanol for 10 min.</li>
	<li>Block with 5% normal serum containing 0.3% Triton-X (from the species which was used for raising the secondary antibody) overnight at 4 &#176;C.</li>
	<li>Rinse 3x with 1x PBS and incubate with primary antibodies overnight at 4 &#176;C.</li>
	<li>Rinse 3x with 1x PBS and incubate with secondary antibodies at room temperature for one hour.</li>
	<li>Rinse 3x with 1x PBS</li>
	<li>Counterstain the section for DNA using SYTOX green, or DAPI (or an alternative DNA dye).</li>
	<li>Rinse 3x with 1x PBS</li>
	<li>Embed the sections in an aqueous embedding medium.</li>
	<li>Let the embedding medium polymerize for at least 48 hr.</li>
</ol>
7. Immunohistochemistry of Brain Sections of Perfused Animals
<ol>
	<li>Block as described in step 6.2 and subsequently incubate the fixed brain sections in primary antibody solution diluted in PBS containing 0.3% Triton-X (free-floating) overnight at 4 &#176;C.</li>
	<li>Rinse three times with 1x PBS and incubate in secondary antibody solution diluted in PBS containing 0.3% Triton-X (free-floating) at room temperature for 3 hr.</li>
	<li>Rinse 3x with 1x PBS</li>
	<li>Counterstain the section for DNA using SYTOX green&#160;or DAPI (or an alternative DNA dye).</li>
	<li>Rinse three times with 1x PBS</li>
	<li>Embed the sections in an aqueous embedding medium.</li>
	<li>Let the embedding medium polymerize for at least 48 hr.</li>
</ol>
8. Investigation of Mossy Fiber Projections
<ol>
	<li>Sacrifice the animals, prepare 40 &#181;m coronal sections as described above and stain the hippocampal section using antibody for neurofilament M.</li>
	<li>Scan the sections at the appropriate wavelength using a confocal laser scanning microscope to visualize mossy fibers and nuclei. Start the scanning at low magnification.</li>
	<li>Use the low magnification images for orientation and target the hippocampus with mossy fiber projections.</li>
	<li>Scan the sections (z-sectioning) at high resolution and high magnification.</li>
	<li>Analyze the morphological appearance and connectivity of the mossy fibers visualized via staining for NF-M.</li>
	<li>(OPTIONAL) Analyze the size and volume of hippocampal blades. Use the DNA signal for the measurements.</li>
</ol>
9. Fluoro-Jade C Assay (Neuronal Cell Death)
<ol>
	<li>Sacrifice the animals, dissect the hippocampus, and prepare 12 &#181;m sections as described above.</li>
	<li>Let the sections dry for 30 min at room temperature.</li>
	<li>Fix the sections in 4% PFA for 40 min at room temperature.</li>
	<li>Briefly wash the sections 3x with ddH2O.</li>
	<li>Incubate the sections with 0.06 % potassium permanganate (KMnO4) for 10 min under continuous, gentle shaking.</li>
	<li>Wash the sections 3x with ddH2O.</li>
	<li>Incubate the sections with 0.002% Fluoro-Jade C solution for 20 min at room temperature.</li>
	<li>(OPTIONAL) For simultaneous nuclear stainings, 0.002% Fluoro-Jade C solution can be supplemented with 10 &#181;g/ml DAPI.</li>
	<li>Briefly wash the sections 3x with ddH2O.</li>
	<li>Let the sections dry for 30 min at room temperature.</li>
	<li>Incubate the sections with Xylene (1 min)</li>
	<li>Mount the sections using a Xylene-based mounting medium (D.P.X.).</li>
</ol>

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The authors declare that they have no conflict of interest.

Adult neurogenesis, and the possibility of its manipulation via inhibition of NF-&#954;B in neurons, and its later reactivation via doxycycline,&#160;offers a fascinating system for investigations into newborn neurons in the adult brain, as well as into neuronal de- and re-generation. The beauty of this system is that NF-&#954;B signaling pathway inhibition in neurons not only results in changes in neuronal cell death, progenitor proliferation and migration, and severe structural and anatomical changes, but also in obvio...

Ontologically, the hippocampus is one of the oldest anatomical brain structures known. It is responsible for diverse complex tasks, such as pivotal functions in the regulation of long-term memory, spatial orientation, and formation and consolidation of the respective memory. Anatomically, the hippocampus consists of pyramidal cell layers (stratum pyramidale) including the cornu Ammonis (CA1, CA2, CA3, and CA4) regions and the dentate gyrus (gyrus dentatus), which contains granule cells and a few neuronal progenitors within its subgranular zone. The granule cells project towards the CA3 region via the so-called mossy fibers (axons of granule cells).
Until the end of the last century, the adult mammalian brain was believed to be a static organ lacking cellular plasticity and neurogenesis. However, during the last two decades, a growing amount of evidence clearly demonstrates adult neurogenesis taking place in at least two brain regions, the subventricular zone (SVZ) and the subgranular zone of the hippocampus.
Our previous studies, and those of other groups, have shown that the transcription factor NF-&#954;B is one of the crucial molecular regulators of adult neurogenesis, and that its de-regulation results in severe structural hippocampal defects and cognitive impairments1-6. NF-&#954;B is the generic name of an inducible transcription factor composed of different dimeric combinations of five DNA-binding subunits: p50, p52, c-Rel, RelB, and p65 (RelA), the latter three of which have transactivation domains. Within the brain, the most abundant form found in the cytoplasm is a heterodimer of p50 and p65, which is kept in an inactive form by inhibitor of kappa B (I&#954;B)-proteins.
To study and directly manipulate NF-&#954;B-driven neurogenesis, we use transgenic mouse models to enable simple inhibition of all of the NF-&#954;B subunits, specifically in the forebrain7 (see Figure&#160;1). For this purpose, we cross-bred the following transgenic mouse lines, I&#954;B/- and -/tTA. The transgenic I&#954;B/- line was generated using a trans-dominant negative mutant of NF-&#954;B-inhibitor I&#954;Ba (super-repressor I&#954;Ba-AA1)8. In contrast to the wild-type I&#954;B&#945;, I&#954;B&#945;-AA1 has two serine residues mutated to alanines (V32 and V36), which hinder the phosphorylation and subsequent proteasomal degradation of the inhibitor. For forebrain neuron-specific expression of the I&#954;Ba-AA1-transgene, I&#954;B/- mice were cross-bred with mice harboring a calcium-calmodulin-dependent kinase II&#945; (CAMKII&#945;)-promoter that can be driven by tetracycline trans-activator (tTA)9.
p65 knock-out mice have an embryonic lethal phenotype, due to massive liver apoptosis10, so the approach shown here provides an elegant method for investigating the role of NF-&#954;B in postnatal and adult neurogenesis.
The classic behavioral test to study spatial learning and memory was described in the 1980s by Richard Morris, a test known as the Morris water-maze (MWM)11. In this open-field water-maze, animals learn to escape from opaque water onto a hidden platform based on orientation and extra-maze cues. A dry variant of MWM is the so-called Barnes maze (BM)12. This test utilizes a circular plate with 20 circular holes arranged at the border of a plate, with one defined hole as an escape box, and visual extra-maze cues for orientation. Both experimental paradigms rely on the flight behavior induced by a rodent`s aversion to water, or open, brightly illuminated spaces. Both tests allow an investigation of spatial orientation, and the related memory performance. Although the hippocampus plays a general and essential role in the spatial memory formation, the hippocampal regions involved differ depending on the test applied. The memory tested in BM arises from neuronal activity between the enthorinal cortex (EC) and the pyramidal neurons located in the CA1-region of the hippocampus without a contribution of the DG13-16. In particular, the classic BM mainly relies on navigation via the monosynaptic temporo-ammonic pathway from EC III to CA1 to EC V. Importantly, the DG is crucially involved in the so-called spatial pattern recognition17, which implies not only the processing of visual and spatial information, but also the transformation of similar representations or memories into dissimilar, nonoverlapping representations. This task requires a functional tri-synaptic circuit from EC II to DG to CA3 to CA1 and EC VI, which cannot be tested in the BM15.
To address these challenges, we have devised SPS-BM as a behavioral test to specifically test dentate gyrus-dependent cognitive performance in control animals, and in the I&#954;B/tTA super-repressor model following NF-&#954;B inhibition. Importantly, in contrast to the MWM or the BM, the SPS-BM can reveal subtle behavioral deficits resulting from impairment of neurogenesis. Since spatial-pattern-separation is strictly dependent on a functional circuit between EC II and DG and CA3 and CA1 and EC VI, this test is highly sensitive to potential changes in neurogenesis, modifications of the mossy fiber pathway or alterations of tissue homeostasis within the DG.
Technically, the set-up of our test is based on the study by Clelland et al., in which the spatial separation pattern was tested using a wooden 8-arm radial arm maze (RAM)19. In our modified set-up, the eight arms were replaced by seven identical yellow food houses. In summary, the methods shown here, including analysis of doublecortin-expressing (DCX+) cells within the hippocampus, the mossy fiber projections, neuronal cell death and particularly the SPS-BM presented here, can be applied to investigations of other mouse models incorporating transgenes that have an impact on adult neurogenesis. Further applications may include the study of pharmacological agents and measuring their impact on DG and spatial pattern separation.

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Moria MC17 Perforated Spoon&#160;</td>
			<td style="width:127px;">FST</td>
			<td style="width:132px;">10370-18</td>
			<td style="width:167px;">removal of the brains</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dissecting microscope</td>
			<td>Carl Zeiss</td>
			<td>Stemi SV8</td>
			<td>removal of the brains</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical scissors&#160;</td>
			<td style="width:127px;">FST</td>
			<td>14084-08</td>
			<td>removal of the brains</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical scissors&#160;</td>
			<td style="width:127px;">FST</td>
			<td>14381-43</td>
			<td>removal of the brains</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dumont #5 forceps</td>
			<td style="width:127px;">FST</td>
			<td>11254-20</td>
			<td>removal of the brains</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">SuperFrost Slides</td>
			<td>Carl Roth&#160;</td>
			<td align="right">1879</td>
			<td>slides for immunohistochemistry</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Paraformaldehyde</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td>P6148</td>
			<td>fixative</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">TissueTek OCT compound</td>
			<td>Sakura Finetek</td>
			<td style="width:132px;">1004200018</td>
			<td>embedding of the brains</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:447px;">Normal Goat Serum</td>
			<td style="width:127px;">Jackson Immunolabs</td>
			<td style="width:132px;">005-000-001</td>
			<td>blocking in IHC</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:447px;">Normal Rabbit Serum</td>
			<td style="width:127px;">Jackson Immunolabs</td>
			<td style="width:132px;">011-000-001</td>
			<td>blocking in IHC</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Normal Donkey Serum</td>
			<td style="width:127px;">Jackson Immunolabs</td>
			<td style="width:132px;">017-000-001</td>
			<td>blocking in IHC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-Neurofilament-M antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>2H3</td>
			<td>IHC, Dilution 1:200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-doublecortin&#160;antibody</td>
			<td>sc-8066</td>
			<td>Santa Cruz</td>
			<td>IHC, Dilution 1:800</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">anti-GFP antibody</td>
			<td>ab290</td>
			<td>abcam</td>
			<td>IHC, Dilution 1:2000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">anti-BrdU antibody</td>
			<td>OBT0030G</td>
			<td>Accurate Chemicals</td>
			<td>IHC, Dilution 1:2000&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Fluoro Jade-C</td>
			<td>FJ-C</td>
			<td>HistoChem</td>
			<td>Determination of neuronal cell death</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Betadine</td>
			<td style="width:127px;">&#160;MUNDIPHARMA</td>
			<td>D08AG02&#160;</td>
			<td>disinfectant</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">cryomicrotome</td>
			<td>Leica</td>
			<td>CM1900</td>
			<td>preparation of brain slices</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Heparin sodium salt</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:132px;">H3393</td>
			<td>perfusion</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">circular plate made from hard-plastic (diameter 120 cm)</td>
			<td style="width:127px;">lab made</td>
			<td style="width:132px;">none</td>
			<td>plate for SPS-BM, diameter 120cm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Buraton rapid disinfectant&#160;</td>
			<td style="width:127px;">Sch&#252;lke &#38; Mayr</td>
			<td style="width:132px;">113 911</td>
			<td>disinfectant</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">video-tracking system TSE VideoMot 2 with Software Package VideoMot2</td>
			<td>tse systems</td>
			<td style="width:132px;">302050-SW-KIT</td>
			<td>tracking and analysis of SPS-BM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100&#160;</td>
			<td>Sigma Aldrich</td>
			<td>T8787</td>
			<td>permeabilisation/IHC</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:447px;">cryotome</td>
			<td style="width:127px;">Reichert&#160; Jung/Leica</td>
			<td style="width:132px;">Frigomobil 1206</td>
			<td>preparation of 40&#181;m brain slices</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Mowiol 4-88</td>
			<td style="width:127px;">Carl Roth&#160;</td>
			<td>Art.-Nr. 0713</td>
			<td>embedding of the slides</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">SYTOX green</td>
			<td style="width:127px;">Invitrogen</td>
			<td>S7020</td>
			<td>Nuclear staining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Food pellets (Kellog`s Froot Loops)</td>
			<td>Kellog`s</td>
			<td style="width:132px;"></td>
			<td>SPS-BM</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:447px;">Prism, Version 3.0</td>
			<td style="width:127px;">Graph Pad Software, San Diego, USA&#160;</td>
			<td style="width:132px;"></td>
			<td>Statistical evaluation of SPS-BM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Zen 2008 or Zen 2011 Software</td>
			<td style="width:127px;">Carl Zeiss</td>
			<td style="width:132px;"></td>
			<td>Software (Confocal microscope)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">D.P.X</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:132px;">317616</td>
			<td>mounting medium for Fluoro Jade C staining</td>
		</tr>
	</tbody>
</table>

methods for the modulation and analysis of nf-&#954;b-dependent adult neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-&#954;B plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-&#954;B activity can be used.
In this study, methods are described for the analysis of NF-&#954;B-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.

Cross-breeding of the I&#954;B/- and tTA transgenic mouse lines leads to conditional inhibition of NF-&#954;B activity in the hippocampus. 
To investigate the expression of the I&#954;B&#945;-AA1-transgene in the double transgenic mouse (Figure 1A), brains were isolated, cryosectioned and stained using an antibody against GFP (green fluorescent protein). Confocal laser scanning microscopy revealed high expression of the transgene in the CA1 and CA3 regions, an...

Watch this Scientific Journal Video about Methods for the Modulation and Analysis of NF-ÎºB-dependent Adult Neurogenesis at JoVE.com

Methods for the Modulation and Analysis of NF-&amp;#954;B-dependent Adult Neurogenesis

Methods for the manipulation and analysis of NF-&#954;B-dependent adult hippocampal neurogenesis are described. A detailed protocol is presented for a dentate gyrus-dependent behavioral test (termed the spatial pattern separation-Barnes maze) for the investigation of cognitive outcome in mice. This technique should also help enable investigations in other experimental settings.

Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

methods-for-modulation-analysis-nf-b-dependent-adult

Research

JoVE Journal

Neuroscience

14.2K Views.  University of Bielefeld.  שיטות למניפולציה והניתוח של neurogenesis בהיפוקמפוס המבוגר NF-κB תלוי מתוארות. פרוטוקול מפורט מוצג לבדיקה משוננת gyrus תלוי התנהגותי (שמכונה במבוך ההפרדה בארנס דפוס המרחבי) לחקירת תוצאה קוגניטיבית בעכברים. טכניקה זו צריכה גם לעזור לאפשר חקירות בהג?...

Video: Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). Animal models are necessary for studying the etiopathology and potential therapies of cerebrovascular diseases. Imaging the vasculature in large animals is relatively easy. However, developing vessel imaging methods of murine brain disease models is desirable due to the cost and availability of genetically-modified mouse lines. Imaging the murine cerebral vascular tree is a challenge. In humans and larger animals, the gold standard for assessing the angioarchitecture at the macrovascular (conductance) level is x-ray catheter contrast-based angiography, a method not suited for small rodents.
In this article, we present a method of cerebrovascular casting that produces a durable skeleton of the entire vascular bed, including arteries, veins, and capillaries that may be analyzed using many different modalities. Complete casting of the microvessels of the mouse cerebrovasculature can be difficult; however, these challenges are addressed in this step-by-step protocol. Through intracardial perfusion of the vascular casting material, all vessels of the body are casted. The brain can then be removed and clarified using the organic solvent methyl salicylate. Three dimensional imaging of the brain blood vessels can be visualized simply and inexpensively with any conventional brightfield microscope or dissecting microscope. The casted cerebrovasculature can also be imaged and quantified using micro-computed tomography (micro-CT)1. In addition, after being imaged, the casted brain can be embedded in paraffin for histological analysis.
The benefit of this vascular casting method as compared to other techniques is its broad adaptation to various analytic tools, including brightfield microscopic analysis, CT scanning due to the radiopaque characteristic of the material, as well as histological and immunohistochemical analysis. This efficient use of tissue can save animal usage and reduce costs. We have recently demonstrated application of this method to visualize the irregular blood vessels in a mouse model of adult BAVM at a microscopic level2, and provide additional images of the malformed vessels imaged by micro-CT scan. Although this method has drawbacks and may not be ideal for all types of analyses, it is a simple, practical technique that can be easily learned and widely applied to vascular casting of blood vessels throughout the body.

In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for Computer tomography-guided focal irradiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.

The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis.

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral ...

Heat-Induced Antigen Retrieval: An Effective Method to Detect and Identify Progenitor Cell Types during Adult Hippocampal Neurogenesis

Traditional methods of immunohistochemistry (IHC) following tissue fixation allow visualization of various cell types. These typically proceed with the application of antibodies to bind antigens and identify cells with characteristics that are a function of the inherent biology and development. Adult hippocampal neurogenesis is a sequential process wherein a quiescent neural stem cell can become activated and proceed through stages of proliferation, differentiation, maturation and functional integration. Each phase is distinct with a characteristic morphology and upregulation of genes. Identification of these phases is important to understand the regulatory mechanisms at play and any alterations in this process that underlie the pathophysiology of debilitating disorders. Our heat-induced antigen retrieval approach improves the intensity of the signal that is detected and allows correct identification of the progenitor cell type. As discussed in this paper, it especially allows us to circumvent current problems in detection of certain progenitor cell types.

During adult hippocampal neurogenesis, a distinct set of genetic markers are expressed when a quiescent neural stem cell sequentially progresses and develops into a functionally integrated neuron in the circuit. Using heat-induced antigen retrieval, progenitor cell types that are otherwise difficult to detect are identified with improved effectiveness.

Traditional  methods of immunohistochemistry (IHC) following tissue fixation allow visualization  of various cell types. These typically proceed with the ...

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

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 ...

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During this process, a portion of neural stem cells undergo differentiation and give rise to the first populations of differentiated primary neurons within the nascent central nervous system. Several vertebrate model organisms have been used to explore the mechanisms of neural cell fate specification, patterning, and differentiation. Among these is the African clawed frog, Xenopus, which provides a powerful system for investigating the molecular and cellular mechanisms responsible for primary neurogenesis due to its rapid and accessible development and ease of embryological and molecular manipulations. Here, we present a convenient and rapid method to observe the different populations of neuronal cells within Xenopus central nervous system. Using antibody staining and immunofluorescence on sections of Xenopus embryos, we are able to observe the locations of neural stem cells and differentiated primary neurons during primary neurogenesis.

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

This article presents a convenient and rapid method for visualizing different neuronal cell populations in the central nervous system of Xenopus embryos using immunofluorescent staining on sections.

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

The Mouse Hindbrain As a Model for Studying Embryonic Neurogenesis

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded forebrain neuroepithelium is not amenable to wholemount analysis to examine organ-wide neurogenesis patterns. Moreover, defining the mechanisms of forebrain neurogenesis is not necessarily predictive of neurogenesis in other parts of the brain; for example, due to the presence of forebrain-specific progenitor subtypes. The mouse hindbrain provides an alternative model for studying embryonic neurogenesis that is amenable to wholemount analysis, as well as tissue sections to observe the spatiotemporal distribution and behavior of neural progenitors. Moreover, it is easily dissected for other downstream applications, such as cell isolation or molecular biology analysis. As the mouse hindbrain can be readily analyzed in the vast number of cell lineage reporter and mutant mouse strains that have become available, it offers a powerful model for studying the cellular and molecular mechanisms of developmental neurogenesis in a mammalian organism. Here, we present a simple and quick method to use the mouse embryo hindbrain for analyzing mammalian neural progenitor cell (NPC) behavior in wholemount preparations and tissue sections.

This article demonstrates how the mouse embryonic hindbrain can be used as a model for studying developmental neurogenesis in both whole organ and tissue section preparations.

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...