Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Summary

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

Abstract

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Introduction

Adult neurogenesis in the mammalian hippocampus represents a remarkable example of the brain’s innate capacity for adaptability and plasticity. Dentate granule cells (DGCs) are generated from a renewable pool of neural progenitor cells in the hippocampal dentate gyrus, which is one of the two presently well-characterized neurogenic regions in the mammalian brain, and is thought to be particularly important for learning and memory. The hippocampus is part of the limbic system and has a deep location within the mammalian brain, which makes it a difficult target for precise pharmacological manipulation. Additionally, aberrant neurogenesis has been implicated in con....

Protocol

NOTE: All animal procedures conformed to the animal health and welfare guidelines of the Department of Comparative Medicine at the University of Toronto.

1. Preparation of Hippocampal Slices

1. Sterilize the following instruments using the dry autoclave at 125 °C: Scalpel handle (#3) (2), Standard pattern forceps, large (1), Small dissector scissor (angled to side) (1), Micro spoon (spoon and flat spatula ends) (1), Micro-spatulas (rounded and rounded tapered ends) (2), Fine paintbrush (1), Fire polished Pasteur pipette (2), Gauze squares, 2 x 2 inches (5).
2. When sterile, put the instruments into a sterile container a....

Results

Determining if organotypic cultures would be suitable for adult neurogenesis research required that they satisfy two main criteria: 1) that slices maintain characteristic morphological features of hippocampal slices after 10-21 days in vitro (DIV), and 2) that newborn DGCs can be quantified using standard immunohistochemical techniques commonly employed in adult neurogenesis research. Regarding the first criterion, Figure 1A and 1B highlight the preserved hippocampal morphology........

Discussion

Following CldU (or BrdU) administration, the timeline of application of pharmacological agents can be chosen to target newborn DGCs during particular developmental windows. For example, a hypothetical agent can be applied during the second week post-CldU injection, which is proposed to coincide with the age of immature neurons that are at a developmental stage where GABA is depolarizing. Future studies using this protocol could adapt the pharmacological agent and the window of exposure to “tailor” the approac.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5-chloro-2'-deoxyuridine (CldU)	MP Biomedicals	105478	Hazardous, Carcinogenic
Cell culture inserts, 30 mm diameter, 0.4 µm pore size	Thermo scientific	140660	Nuclon delta coating on these inserts provides better tissue adhesion and improves slice quality.
Conical Centrifuge tubes (sterile)	Fisher Scientific	14-432-22
Dissector scissors (angled to side)	Fine Science Tools	14082-09
Minimum essential medium (MEM)	Gibco	11095; liquid	Store at 4 °C
Eclipse Ni-U fluorescent microscope	Nikon
Glue for tissue	Krazy Glue	KG585	Use minimum amount of glue to achieve adhesion as any tissue exposed to glue will be unusable for IHC.
Hank’s Balanced Salt Solution (HBSS) (500 ml)	Gibco	14025-092	Store at 4 °C
Horse Serum Heat Inactivated (500 ml)	Gibco	16050-122	Make 50 ml aliquots and store at -20 °C
Kimwipes	Kimberly-Clarke	TW 31KYPBX
Modified glass pipettes (bottom of Pasteur pipette removed and edge smoothed with Bunsen flame)
Petri Dish (100 mm x 15 mm) and (60 mm x 15 mm)	Fisher Brand	FB0875712 and FB0875713A
Scalpel blades #11	Fine Science Tools	10011-00
Scalpel handle #3	Fine Science Tools	10003-12
Serological Pipettes	Sorfa Medical Plastic Co.	P8050
Standard Pattern forceps	Fine Science Tools	11000-12
Sterile vacuum filter	Thermo-Scientific	565-0020
Surgical Scissors	Fine Science Tools	14054-13
Syringe driven filter unit	Millipore-Millex	SLGP033RS
Tissue chopper with moveable stage	Stoelting	51425
Fine tip paintbrush