Methods for the Modulation and Analysis of NF-κB-dependent Adult Neurogenesis

Summary

Methods for the manipulation and analysis of NF-κB-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.

Abstract

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-κB plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-κB activity can be used.

In this study, methods are described for the analysis of NF-κB-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.

Introduction

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-κB is one of the crucial molecular regulators of adult neurogenesis, and that its de-regulation results in severe structural hippocampal defects and cognitive impairments^1-6. NF-κB 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 (IκB)-proteins.

To study and directly manipulate NF-κB-driven neurogenesis, we use transgenic mouse models to enable simple inhibition of all of the NF-κB subunits, specifically in the forebrain⁷ (see Figure 1). For this purpose, we cross-bred the following transgenic mouse lines, IκB/- and -/tTA. The transgenic IκB/- line was generated using a trans-dominant negative mutant of NF-κB-inhibitor IκBa (super-repressor IκBa-AA1)⁸. In contrast to the wild-type IκBα, IκBα-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 IκBa-AA1-transgene, IκB/- mice were cross-bred with mice harboring a calcium-calmodulin-dependent kinase IIα (CAMKIIα)-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-κB 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 DG^13-16. 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 IκB/tTA super-repressor model following NF-κB 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.

Protocol

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, (Düsseldorf, Germany). All animal experiments were approved by LANUV, Düsseldorf under the license number 8.87–51.04.20.09.317 (LANUV, NRW). All efforts were made to minimize distress and the number of animals required for the study.

1. Animal Care and Housing

1. 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).
2. Mice should be kept in standard cages in a temperature- and humidity- controlled (22 °C) room under diurnal conditions (12 hr light/dark cycle) with HEPA filtered air.
3. Standardized food and water should be provided ad libitum.
4. If IκB/tTA and IκB /- control mice are used, PCR-based genotyping should be performed for each animal, as described in^7,18.
5. Male animals with an age difference of less than four days should to be used to reduce individual variability.
6. (OPTIONAL): For NF-κB reactivation experiments, doxycycline must be administered in drinking water (2 mg/ml with 2.5% sucrose) for at least 14 days.

2. Spatial Pattern Separation-Barnes Maze (SPS-BM)

1. All behavioral testing should be carried out according to international and local guidelines.
2. 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.
3. Set up a white circular plate made from hard plastic (diameter 120 cm, see Figure 5A) in a humidity- and temperature- controlled room (22 °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.
4. Set up the video-tracking system. The camera should be placed 115 cm above the center of the plate (see Figure 5A).
5. 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).
6. Carefully clean the plate with a rapid disinfectant that can remove any odor from the experimental set-up.
7. 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).
8. 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.
9. 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).
10. Switch the computer and the camera on and start the video-tracking system software.
11. Start the recording.
12. 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.
13. Stop the video-tracking.
14. IMPORTANT! Clean the circular plate and the food houses after each trial with rapid disinfectant.
15. Repeat the test daily for seven consecutive days (for each animal).
16. 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.
17. (OPTIONAL) Sacrifice the mice and analyze the hippocampi as described below.

3. BrdU Labeling

1. 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).
2. Sacrifice the animals, dissect the hippocampus and prepare 40 µm sections as described below.
3. Denaturate the sections with 2 M HCl for 10 min and incubate in 0.1 M borate buffer for 10 min.
4. Label a one-in-twelve series of 40 μm sections (240 μm apart) from each animal immunohistochemically, as described below using antibodies directed against BrdU.
5. 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.

4. Removal of the Brains and Preparation of Cryosections from Nonperfused Animals

1. Observe locally approved procedures for euthanizing animals. Mice may be directly euthanized by cervical dislocation.
2. (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).
3. Sterilize the head with Betadine (10% povidone-iodine)-soaked gauze and swab subsequently with gauze soaked in 70% ethanol.
4. Decapitate the animal using appropriate surgical scissors and pull the skin aside.
5. (OPTIONAL) Fixators can be applied to avoid folding back of the retarded skin.
6. Make a midline incision in the skull and carefully pull the skull fragments aside.
7. Carefully remove the whole brain using an appropriate surgical instrument (e.g. Moria Spoon).
8. Precool 25 ml of 2-methylbutane in a 50 ml beaker (e.g. Schott Duran) to -30 to -40 °C on dry ice.
   Note: For free-floating staining of "thick" sections which are ideally suited for confocal laser scanning microscopy, remove brain, wash 3 times in buffer and store at 4 °C in phosphate buffered 30 % sucrose solution in 50 ml tube until brain has sunk down to bottom (typically overnight).
9. 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 °C until use.
   Note: For long-time storage at -20 °C store in 9% sucrose, 7 mM MgCl₂, 50 mM phosphate buffer, 44% glycerol.
10. Cut the brain into 10-12 μm 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 μm horizontal sections at -20° to - 25 °C. Collect sections from knife with fine brush and collect in buffer or keep in storage solution ( step 4.9) at -20 °C for long-time storage.
11. Carefully mount two slices on single microscope slides. The use of Superfrost UltraPlus slides is highly recommended to maximize adhesion of the sections.
12. Dry the slides for 5 min at room temperature. Slides can be stored at -80 °C until use.

5. Preparation of Sections from Perfused Animals

1. Anesthetize the animals as described above (step 4.2).
2. 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 1.2-1.4 m or utilizing a peristaltic pump set to approximately 15 ml/min. Optimal perfusion results in a pale white brain with no red blood vessels being visible.
3. Dissect and post-fix the brains in 4% paraformaldehyde at 4 °C for 24 hr.
4. Cryoprotect the brains in 30% sucrose in PBS at 4 °C for at least 24 hr.
5. Freeze the brains on the cryotome stage.
6. Prepare 40 µm horizontal sections from entire forebrains using an appropriate cryotome.
7. The slides can be stored in cryoprotectant solution (0.1 M phosphate buffer, 50% glycerol, 0.14% MgCl₂, 8.6% sucrose) at -20 °C until use.

6. Immunohistochemistry of Brain Sections of Nonperfused Animals

1. Post-fix the cryosections, prepared as described above, using -20 °C cold methanol for 10 min.
2. Block with 5% normal serum containing 0.3% Triton-X (from the species which was used for raising the secondary antibody) overnight at 4 °C.
3. Rinse 3x with 1x PBS and incubate with primary antibodies overnight at 4 °C.
4. Rinse 3x with 1x PBS and incubate with secondary antibodies at room temperature for one hour.
5. Rinse 3x with 1x PBS
6. Counterstain the section for DNA using SYTOX green, or DAPI (or an alternative DNA dye).
7. Rinse 3x with 1x PBS
8. Embed the sections in an aqueous embedding medium.
9. Let the embedding medium polymerize for at least 48 hr.

7. Immunohistochemistry of Brain Sections of Perfused Animals

1. 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 °C.
2. 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.
3. Rinse 3x with 1x PBS
4. Counterstain the section for DNA using SYTOX green or DAPI (or an alternative DNA dye).
5. Rinse three times with 1x PBS
6. Embed the sections in an aqueous embedding medium.
7. Let the embedding medium polymerize for at least 48 hr.

8. Investigation of Mossy Fiber Projections

1. Sacrifice the animals, prepare 40 µm coronal sections as described above and stain the hippocampal section using antibody for neurofilament M.
2. 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.
3. Use the low magnification images for orientation and target the hippocampus with mossy fiber projections.
4. Scan the sections (z-sectioning) at high resolution and high magnification.
5. Analyze the morphological appearance and connectivity of the mossy fibers visualized via staining for NF-M.
6. (OPTIONAL) Analyze the size and volume of hippocampal blades. Use the DNA signal for the measurements.

9. Fluoro-Jade C Assay (Neuronal Cell Death)

1. Sacrifice the animals, dissect the hippocampus, and prepare 12 µm sections as described above.
2. Let the sections dry for 30 min at room temperature.
3. Fix the sections in 4% PFA for 40 min at room temperature.
4. Briefly wash the sections 3x with ddH₂O.
5. Incubate the sections with 0.06 % potassium permanganate (KMnO₄) for 10 min under continuous, gentle shaking.
6. Wash the sections 3x with ddH₂O.
7. Incubate the sections with 0.002% Fluoro-Jade C solution for 20 min at room temperature.
8. (OPTIONAL) For simultaneous nuclear stainings, 0.002% Fluoro-Jade C solution can be supplemented with 10 µg/ml DAPI.
9. Briefly wash the sections 3x with ddH₂O.
10. Let the sections dry for 30 min at room temperature.
11. Incubate the sections with Xylene (1 min)
12. Mount the sections using a Xylene-based mounting medium (D.P.X.).

Results

Cross-breeding of the IκB/- and tTA transgenic mouse lines leads to conditional inhibition of NF-κB activity in the hippocampus.

To investigate the expression of the IκBα-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, and in the DG (

Discussion

Adult neurogenesis, and the possibility of its manipulation via inhibition of NF-κB in neurons, and its later reactivation via doxycycline, 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-κB 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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Moria MC17 Perforated Spoon	FST	10370-18	removal of the brains
Dissecting microscope	Carl Zeiss	Stemi SV8	removal of the brains
Surgical scissors	FST	14084-08	removal of the brains
Surgical scissors	FST	14381-43	removal of the brains
Dumont #5 forceps	FST	11254-20	removal of the brains
SuperFrost Slides	Carl Roth	1879	slides for immunohistochemistry
Paraformaldehyde	Sigma-Aldrich	P6148	fixative
TissueTek OCT compound	Sakura Finetek	1004200018	embedding of the brains
Normal Goat Serum	Jackson Immunolabs	005-000-001	blocking in IHC
Normal Rabbit Serum	Jackson Immunolabs	011-000-001	blocking in IHC
Normal Donkey Serum	Jackson Immunolabs	017-000-001	blocking in IHC
anti-Neurofilament-M antibody	Developmental Studies Hybridoma Bank	2H3	IHC, Dilution 1:200
anti-Doublecortin antibody	sc-8066	Santa Cruz	IHC, Dilution 1:800
anti-GFP antibody	Abcam	ab290	IHC, Dilution 1:2,000
anti-BrdU antibody	OBT0030G	Accurate Chemicals	IHC, Dilution 1:2,000
Fluoro-Jade C	FJ-C	HistoChem	Determination of neuronal cell death
Betadine	Mundipharma	D08AG02	disinfectant
Cryomicrotome	Leica	CM1900	preparation of brain slices
Heparin sodium salt	Sigma-Aldrich	H3393	perfusion
Circular plate made from hard-plastic (diameter 120 cm)	lab made	none	plate for SPS-BM, diameter 120 cm
Buraton rapid disinfectant	Schülke Mayr	113 911	disinfectant
Video-tracking system TSE VideoMot 2 with Software Package VideoMot2	TSE Systems	302050-SW-KIT	tracking and analysis of SPS-BM
Triton X-100	Sigma Aldrich	T8787	permeabilization/IHC
Cryotome	Reichert Jung/Leica	Frigomobil 1206	preparation of 40 µm brain slices
Mowiol 4-88	Carl Roth	Art.-Nr. 0713	embedding of the slides
SYTOX green	Invitrogen	S7020	Nuclear staining
Food pellets (Kellog`s Froot Loops)	Kellog`s		SPS-BM
Prism, Version 3.0	Graph Pad Software, San Diego, USA		Statistical evaluation of SPS-BM
Zen 2008 or Zen 2011 Software	Carl Zeiss		Software (Confocal microscope)
D.P.X	Sigma-Aldrich	317616	mounting medium for Fluoro-Jade C staining