Organotypic slice cultures

Jens C. ReklingDepartment of Neuroscience and Pharmacology

Panum Institute

• What is an organotypic slice culture?

• Why make slice cultures?

• Culture techniques

• Examples of use

• Some problems

• Conclusions

Headings

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

• 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?

Culture techniquesRoller-tube technique

Important parameters:Angle, speed (6-15 revolutions/hour)

Roller-tube techniqueMacro dissection of brain region, e.g. hippocampus

Mc Illwain tissue chopper

Roller-tube techniqueHome-made chopper Individual 400µm acute slices

Embedding in chicken plasma clot

Roller-tube technique

Roller-tube techniqueCytostatic drugs produce monolayers

Roller-tube techniquePyramidal neuron in hippocampal slice culture

Roller-tube techniqueInterneuron in hippocampal slice culture

Stoppini – interface method

Culture inserts

Stoppini – interface method

Stoppini – interface method

Parsley – Thin explants on collagen-coated culture dishes

Culture techniques

Victorov – Free floating slices

Culture techniques

Retina explant cultures

Examples of use

Cortex – substantia nigra – striatumco-cultures

Types of explant tissue

Ventral mesencephalon

Types of explant tissue

TH positive neuron

Cerebellum

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

- 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

ElectrophysiologyElectrical-, synaptic-properties of neurons

Examples of use

Cortex-striatum-substantia nigra co-culture

Multisite recordingsCoupling of slice cultures to arrays of electrodes

Examples of use

Quantification of cell death usingpropidium iodide?

Lesion modelsStretch-induced injury

Examples of use

Lesion modelsEndoplasmic reticulum stress in spinal cultures

Examples of use

Lesion modelsAbeta25-35 amyloid-induced cell death(Alzheimer's disease model)

Examples of use

Lesion modelsEthanol withdrawal-induced cell death(Alcoholisme-induced brain dysfunction model)

Examples of use

Gene transferThree nonviral methods

Examples of use

Electroporation

Lipotransfection

Biolistics

Thy-1 promoter, GFP-M mouse

Transgenic mouse

Growth modelsStudies of growth factor effects on dendritic trees

Examples of use

Lesion modelsOxygen and glucose deprivation

Examples of use

Normal air 95% N25% CO2No Glucose

Interface intact Interface broken(hypoxia+hypoglycemia)

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

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

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

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

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

Spontaneous time-dependent pyramidal celldeath in hippocampal slice cultures

High oxygen tension leads to acute celldeath in hippocampal slice cultures

Synaptic reorganization in hippocampalslice cultures

• 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