Summary

Organotypiske skivekulturer for Studier af Fødselsdepression Neurogenese

Published: March 04, 2015
doi:

Summary

Her beskriver vi en teknik til at studere hippocampus postnatal neurogenese anvendelse af organotypisk skive dyrkningsteknik. Denne metode giver mulighed for in vitro-manipulation af voksne neurogenese og giver mulighed for en direkte anvendelse af farmakologiske midler til den dyrkede hippocampus.

Abstract

Her beskriver vi en teknik til at studere hippocampus postnatal neurogenese i gnaverhjerne hjælp af organotypisk skive dyrkningsteknik. Denne metode bevarer den karakteristiske topografiske morfologi af hippocampus, samtidig med at direkte anvendelse af farmakologiske midler til udviklingslandene hippocampus gyrus dentatus. Derudover kan skive kulturer opretholdes i op til 4 uger og dermed tillader en at studere modningsprocessen af ​​nyfødte granula neuroner. Skivekulturer mulighed for effektiv farmakologisk manipulation af hippocampusskiver mens undtagelse af sammensatte variabler som usikkerheder omkring den dybe anatomiske placering af hippocampus samt blodhjernebarrieren. Af disse grunde har vi forsøgt at optimere organotypiske skive kulturer specielt til postnatale neurogenese forskning.

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 conditions, such as epilepsy, schizophrenia, and Alzheimer’s disease, which has prompted interest in understanding the influence of various pharmacological agents during the maturation and survival of newborn neurons. The distinction between postnatal and adult neurogenesis is blurred and previous studies have shown that many features of in vivo neuronal development in the early postnatal period and adulthood are similar25. Here we emphasize postnatal neurogenesis and suggest possible applications to adult neurogenesis.

Organotypic slice cultures provide an efficient in vitro method for studying various physiological properties of the mammalian hippocampus. The value of slice cultures prepared from rodent brains can be summarized in three main qualities: 1) the protocol is straightforward and requires readily available materials; 2) slice cultures allow for pharmacological studies that eliminate complex variables such as the deep anatomic location of the hippocampus and circumvents the blood brain barrier1; and 3) the well characterized structure of the hippocampus and tri-synaptic circuit is preserved2. Previous investigators have used the organotypic hippocampal culture to study synaptic development and physiology3,4, gliogenesis5-7, ischemic brain damage8,9, neuroprotection and neurorepair10-12 as well as epilepsy13-15.The slice cultures could also provide a useful model system allowing for the monitoring of cell development in conjunction with labeling of cells with green fluorescent protein (GFP) or other vital markers.

Slice cultures have also been previously employed to study postnatal hippocampal neurogenesis16-19, but one important factor in the majority of these studies is the well-characterized degeneration that results from explanting tissue from adult animals after approximately 2 weeks in vitro20,21. For this reason, slice cultures are typically prepared from early post-natal (P5-P10) mice or rat pups, which utilizes the improved viability of early postnatal brain tissue for culturing22. While previous studies have shown that the early postnatal and adult hippocampus differ with regards to synaptic physiology and the expression of specific neuronal subtypes23,24, there is substantial conservation of the choreographed developmental program that newborn dentate granule cells proceed through during maturation25. Additionally, recent studies have suggested that the physiological characteristics of newborn DGCs in culture are very similar to immature neurons in the acute hippocampal slice preparation16.

Protocol

BEMÆRK: Alle dyreforsøg var i overensstemmelse med de for dyresundhed og -velfærd retningslinjer for Afdeling for Medicin ved universitetet i Toronto. 1. Fremstilling af hippocampusskiver Steriliseres følgende instrumenter ved hjælp af tørre autoklave ved 125 ° C: Skalpel håndtag (# 3) (2), standardmønster pincet, store (1), Lille dissektoren saks (vinklet til side) (1), Micro ske (skeen og flad spatel ender) (1), mikro-spatler (afrundet og afrundede tilspidsede ender) (2…

Representative Results

1) at skiver opretholde karakteristiske morfologiske træk hippocampusskiver efter 10-21 dage in vitro (DIV), og 2) at nyfødte DGCs kan kvantificeres: at de opfylder to hovedkriterier Bestemmelse om organotypiske kulturer ville være egnet til voksne neurogenese forskning kræves anvendelse af standard immunhistokemiske teknikker almindeligvis anvendes i voksen neurogenese forskning. Hvad angår det første kriterium, figur 1A og 1B fremhæve bevarede hippocampus morfologi. Ka…

Discussion

Efter CldU (eller BrdU) administration, kan tidslinjen for anvendelse af farmakologiske midler vælges til at målrette nyfødte DGCs i bestemte udviklingsmæssige vinduer. For eksempel kan en hypotetisk middel påføres i den anden uge efter CldU injektion, der foreslås at falde sammen med en alder af umodne neuroner, der er på et udviklingstrin, hvor GABA depolariserende. Fremtidige undersøgelser med anvendelse af denne protokol kan tilpasse det farmakologiske middel og vinduet for eksponering for "skræddersy…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

This work was supported by a research grant MOP 119271 to JMW by the Canadian Institute of Health Research. The authors would like to thank Ms. Yao Fang Tan for her outstanding technical assistance.

Materials

Name of Reagent/ Equipment Company Catalog Number Comments/Description
5-chloro-2'-deoxyuridine (CldU) MP Biomedicals 105478 Hazardous, Carcinogenic
Cell culture inserts, 30mm diameter, 0.4µm 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 (100mm x 15mm) and (60mm x 15mm) 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

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Citar este artículo
Mosa, A. J., Wang, S., Tan, Y. F., Wojtowicz, J. M. Organotypic Slice Cultures for Studies of Postnatal Neurogenesis. J. Vis. Exp. (97), e52353, doi:10.3791/52353 (2015).

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