Colture organotipiche Slice di studi di post-natale neurogenesi
Summary
Qui si descrive una tecnica per lo studio dell'ippocampo la neurogenesi postnatale utilizzando la tecnica organotipica cultura fetta. Questo metodo permette la manipolazione in vitro di neurogenesi adulta e permette l'applicazione diretta di agenti farmacologici per l'ippocampo in coltura.
Abstract
Qui si descrive una tecnica per lo studio dell'ippocampo la neurogenesi postnatale nel cervello dei roditori utilizzando la tecnica organotipica cultura fetta. Questo metodo mantiene la morfologia caratteristica topografica dell'ippocampo pur consentendo l'applicazione diretta di agenti farmacologici per le sviluppo giro dentato dell'ippocampo. Inoltre, culture fetta possono essere mantenuti fino a 4 settimane e, quindi, permettono di studiare il processo di maturazione dei neuroni granulari neonati. Culture fetta consentono efficace manipolazione farmacologica di fettine ippocampali escludendo variabili complesse quali incertezze relative alla posizione anatomica profonda dell'ippocampo e la barriera ematoencefalica. Per queste ragioni, abbiamo cercato di ottimizzare culture fetta organotipiche specificamente per la ricerca neurogenesi postnatale.
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
Representative Results
Discussion
Dopo CldU (o BrdU) amministrazione, la linea temporale di applicazione di agenti farmacologici può essere scelto a bersaglio DGCS neonato durante particolari finestre di sviluppo. Ad esempio, un agente ipotetico può essere applicato durante la seconda settimana post-iniezione CldU, che si propone di coincidere con l'età di neuroni immaturi che sono in fase di sviluppo in cui viene depolarizzante GABA. Futuri studi che utilizzano questo protocollo potrebbe adattare l'agente farmacologico e la finestra di espos…
Disclosures
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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