organ culture
TRANSCRIPT
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Organotypic slice cultures
Jens C. ReklingDepartment of Neuroscience and Pharmacology
Panum Institute
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• What is an organotypic slice culture?
• Why make slice cultures?
• Culture techniques
• Examples of use
• Some problems
• Conclusions
Headings
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Explant of nervous tissue
• Slice 100-400µm
• 1 mm3 pieces
• Typically postnatal 0-7 day old animals
What is an organotypic slice culture?
Slice culture fromhippocampus
Acute slice fromhippocampus
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• Ease of use:- Avoid daily dissections- Grab a culture
• Experimental advantages:- Functional circuits can be studied and manipulated
over time (e.g. long-term plasticity)- Chemical control of the incubating media- Several CNS diseases can be modelled in vitro- Cell death/survival can be quantified and
manipulated over time- Cells can be imaged in narrow focal planes
Why make slice cultures?
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Culture techniquesRoller-tube technique
Important parameters:Angle, speed (6-15 revolutions/hour)
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Roller-tube techniqueMacro dissection of brain region, e.g. hippocampus
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Mc Illwain tissue chopper
Roller-tube techniqueHome-made chopper Individual 400µm acute slices
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Embedding in chicken plasma clot
Roller-tube technique
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Roller-tube techniqueCytostatic drugs produce monolayers
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Roller-tube techniquePyramidal neuron in hippocampal slice culture
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Roller-tube techniqueInterneuron in hippocampal slice culture
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Stoppini – interface method
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Culture inserts
Stoppini – interface method
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Stoppini – interface method
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Parsley – Thin explants on collagen-coated culture dishes
Culture techniques
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Victorov – Free floating slices
Culture techniques
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Retina explant cultures
Examples of use
Cortex – substantia nigra – striatumco-cultures
Types of explant tissue
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Ventral mesencephalon
Types of explant tissue
TH positive neuron
Cerebellum
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Medium
25% Horse serum50% Synthetic medium, e.g. basal medium (eagle)25% balanced salt solution plus extrac glucose
This medium can be exchanged by serum-free media a few days afterexplantation
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- Neuronal organization- Stable interneurons- Myelination markers- Mossy fiber formation- Dendritic arborization- High synaptic density- Well-developed spines- Stable presynaptic and postsynaptic proteins- NCAM maturation- Stable electrophysiological parameters- Synaptic plasticity
Similarities between adulthippocampal slices and slice cultures
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ElectrophysiologyElectrical-, synaptic-properties of neurons
Examples of use
Cortex-striatum-substantia nigra co-culture
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Multisite recordingsCoupling of slice cultures to arrays of electrodes
Examples of use
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Quantification of cell death usingpropidium iodide?
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Lesion modelsStretch-induced injury
Examples of use
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Lesion modelsEndoplasmic reticulum stress in spinal cultures
Examples of use
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Lesion modelsAbeta25-35 amyloid-induced cell death(Alzheimer's disease model)
Examples of use
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Lesion modelsEthanol withdrawal-induced cell death(Alcoholisme-induced brain dysfunction model)
Examples of use
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Gene transferThree nonviral methods
Examples of use
Electroporation
Lipotransfection
Biolistics
Thy-1 promoter, GFP-M mouse
Transgenic mouse
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Growth modelsStudies of growth factor effects on dendritic trees
Examples of use
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Lesion modelsOxygen and glucose deprivation
Examples of use
Normal air 95% N25% CO2No Glucose
Interface intact Interface broken(hypoxia+hypoglycemia)
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Depolarization-induced cell death
CA1
DG
60 min 90 mM KCl
1 mm
No insult
-9 -8 -7 -6
0
25
50
75
100
EC50= 8 nM
Log [MK 801]
% R
escu
e
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Automated quantification of PI labelling
4 5 6
A
B
C
D
1 2 3
0.0938 0.1109 0.08360.1447 0.1337 0.133 0.1377 0.1273
0.1596 0.1349 0.2342 0.4091 0.1299 0.1236 0.1370.135 0.186 1.625 1.842 0.2184 0.1196 0.1397
0.1322 0.2286 1.298 1.195 0.1648 0.1251 0.1240.1344 0.2188 0.1957 0.1462 0.1259
0.1129 0.1269 0.1355
FLUORSKANFluorometer
Single well scanning
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Automated quantification of PI labelling
0.0938 0.1109 0.08360.1447 0.1337 0.133 0.1377 0.1273
0.1596 0.1349 0.2342 0.4091 0.1299 0.1236 0.1370.135 0.186 1.625 1.842 0.2184 0.1196 0.1397
0.1322 0.2286 1.298 1.195 0.1648 0.1251 0.1240.1344 0.2188 0.1957 0.1462 0.1259
0.1129 0.1269 0.1355
FLUORSKANFluorometer
Single well scanning
A B
RFU RFU
Baseline
InsultDen
sity
10 1030 3050 5070 7090 900.00
0.02
0.00
0.04
0.05
0.06
0.10
0.080.15
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4-AP-induced cell deathCarbachol-induced cell death
s1 s
4-AP (100 µM)Induce epileptiform activity
Neu
rona
l dam
age
(% o
f ins
ult)
0
25
50
75
100
125
***
4-AP-induced cell deathRetigabine (µM)
Veh. 100 µM
Neu
rona
l dam
age
(% o
f ins
ult)
0
25
50
75
100
125
***
Carbachol-induced cell deathRetigabine (µM)
Veh. 100 µM
4-AP (100 µM – 4 h) Induce cell death
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EPO rescue cells in 4-AP-induced celldeath in hippocampal slice cultures
s1 s
4-AP (100 µM) induce epileptiform activityin hippocampal slice cultures
• 4-AP applied for 4 h induce cell death
• EPO present 24 h before,during and 24 h after insult
• Cell death measured 24 h after insult
4-AP-induced cell deathin hippocampal slice cultures
Rel
ativ
e ce
ll de
ath
(% o
f ins
ult w
ith v
eh.)
0
25
50
75
100
125
******
EPO #1 (nM)
Veh.
***
0.3 30.03
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Spontaneous time-dependent pyramidal celldeath in hippocampal slice cultures
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High oxygen tension leads to acute celldeath in hippocampal slice cultures
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Synaptic reorganization in hippocampalslice cultures
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• If acute slices can be used – use acute slices
• Co-culturing of different regions has not fulfilled it’s initial promise
• Models of slowly progressing CNS disease states have not fulfilledtheir initial promise
• Slice cultures are “corpses that are not yet quite dead”
However, • Excellent for cell-death studies
• Excellent for growth studies
• Excellent basis for models of some CNS diseases, e.g. traumatic-,hypoxic-, chemical-CNS lesions
• Excellent for some types of electrophysiological studies, especially whenimaging in a single focal plane is needed
Conclusions