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).

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 Laborator....

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