Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

Summary

Organotypic slice cultures are a powerful tool to study neurodevelopmental or degenerative/regenerative processes. Here, we describe a protocol that models the neurodevelopmental death of Purkinje cells in mouse cerebellar slice cultures. This method may benefit research in neuroprotective drug discovery.

Abstract

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early postnatal development, cerebellar Purkinje cells are known to go through a vulnerable period, during which they undergo programmed cell death. Here, we provide a detailed protocol to perform mouse organotypic cerebellar slice culture during this critical time. The slices are further labeled to assess Purkinje cell survival and the efficacy of neuroprotective treatments. This method can be extremely valuable to screen for new neuroactive molecules.

Introduction

In vitro modeling is an essential tool in biomedical research. It allows investigators to study and tightly control specific mechanisms in restricted cell types, or in isolated systems/organs. Organotypic slice culture is a widely used in vitro technique, especially in the field of neuroscience¹. The method was first established by Gähwiler, who cultured brain slices using the roller tube technique², and later modified by Yamamoto et al., who introduced the use of a microporous membrane to perform cortical slice cultures³. Compared to primary cell cultures, organotypic slice cultures present ....

Protocol

All experiments involving animals were performed in accordance with Northwestern University Animal Studies committee.

1. Preparation prior to organotypic cerebellar slice cultures

1. In a cell culture hood sprayed with 70% ethanol beforehand, prepare 200 mL of culture medium in a 250 mL bottle-top vacuum filter attached to a sterile bottle receiver. Add 100 mL of Basal Medium Eagle (BME), 50 mL of Hanks' Balanced Salt Solution (HBSS), 50 mL of heat-inactivated horse serum, 1 mL of.......

Discussion

Cerebellar slice culture is a powerful tool to study programmed Purkinje cell death during postnatal development. This technique can be used to rapidly screen candidate molecules for their neuroprotective potential. The main advantage is that the setup is simple and very cost effective, and only requires a modest investment in equipment (a vibratome can cost up to 3 times more than a tissue chopper). Moreover, 10 to 15 healthy slices can be generated from one mouse pup, allowing for different assays to be performed in pa.......

Materials

Name	Company	Catalog Number	Comments
Alexa Fluor 594 Donkey anti-Mouse IgG secondary antibody	ThermoFisher scientific	A21203
Basal Medium Eagle (BME)	ThermoFisher scientific	21010046
Biosafety cabinet Class II, Type A2	NuAire	NU-540-400
Bovine serum albumin	Millipore Sigma	A2153
Brush
anti-Calbindin D-28K antibody (CB-955)	Abcam	ab82812
CO2 Incubator	NuAire	NU-5700
Corning Costar Flat Bottom 6-well Cell Culture Plates	Fisher Scientific	07-200-83
Coverslips, 22 x 50 mm	Fisher Scientific	12-545-E
Dressing forceps, straight	Harvard Apparatus	72-8949
Double edge blades	Fisher Scientific	50949411
Ethanol 200 proof	Decon Labs, Inc	2701
Eye Scissors, straight	Harvard Apparatus	72-8428
Fine forceps	Fisher Scientific	16-100-127
L-Glutamine 100X	ThermoFisher scientific	25030149
Glucose solution	ThermoFisher scientific	A2494001
Hanks' Balanced Salt Solution	ThermoFisher scientific	14025092
Hoechst 33342, Trihydrochloride, Trihydrate	Fisher Scientific	H21492
Horse Serum, heat inactivated, New Zealand origin	ThermoFisher scientific	26050088
ImageJ
McIlwain Tissue Chopper	Fisher Scientific	NC9914528
Microprobes	Fisher Scientific	08-850
Millicell Cell Culture Inserts	Millipore Sigma	PICM0RG50
Nalgene Rapid-Flow Sterile Disposable Filter Units with PES Membrane, 250 mL	ThermoFisher scientific	168-0045
Nikon A1R confocal laser microscope system	Nikon
NIS-Elements Imaging Software	Nikon
Paraformaldehyde	Acros Organics	41678-0010
Pasteur pipets	Fisher Scientific	13-678-20D
Potassium Chloride	Fisher Scientific	BP366-500
ProLong Gold Antifade Mountant	ThermoFisher scientific	P10144
Operating Scissors, straight	Harvard Apparatus	72-8403
Orbital shaker Belly Dancer	IBI Scientific	BDRLS0003
Prism 8	GraphPad
Superfrost Plus Microscope Slides	Fisher Scientific	12-550-15
Tissue Culture Dish, 60 mm w/ grip ring	Fisher Scientific	FB012921
Tissue culture plate, 24 well	Falcon/Corning	353047
Transfer pipettes, sterile	ThermoFisher scientific	13-711-21
Triton X-100	ThermoFisher scientific	BP151-500