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Method Article
We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.
The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.
1. Before Starting the Preparation of Hippocampal Slices.
2. Hippocampal Slices Preparation.
3. Hippocampal Slices Culture
4. Solutions
For 500 mL | For 1000 mL | Final Concentration | |
CaCl2 (1 M) | 0.5 mL | 1 mL | 1 mM |
D-Glucose | 0.901 g | 1.802 g | 10 mM |
KCl | 0.149 g | 0.298 g | 4 mM |
MgCl2 (1 M) | 2.5 mL | 5 mL | 5 mM |
NaHCO3 | 1.092 g | 2.184 g | 26 mM |
Sucrose | 40 g | 80 g | 234 mM |
Phenol Red Solution 0.5% in DPBS | 0.5 mL | 1 mL | 0.1% v/v |
For 500 mL | For 1000 mL | Final Concentration | |
MEM Eagle medium | 4.2 g | 8.4 g | 8.4 g/l |
Horse serum heat inactivated | 100 mL | 200 mL | 20% |
L-Glutamine (200 mM) | 2.5 mL | 5 mL | 1 mM |
CaCl2 (1 M) | 0.5 mL | 1 mL | 1 mM |
MgSO4 (1 M) | 1 mL | 2 mL | 2 mM |
Insulin (1 mg/ mL), dissolved in HCl 0.01 N | 0.5 mL | 1 mL | 1 mg/l |
Ascorbic Acid, solution (25% w/v) | 0.024 mL | 0.048 mL | 0.00125% |
D-Glucose | 1.16g | 2.32g | 13 mM |
NaHCO3 | 0.22g | 0.44g | 5.2 mM |
Hepes | 3.58g | 7.16g | 30 mM |
5. Representative Results:
Slices should look white under a dissecting scope without black spots and well defined and undamaged CA1, CA3, and Dentate gyrus regions. Bacterial contamination is easily seen as moving black specks in the medium or turbidity of the SCM. When placed under the microscope, the surface of the slice should look clean after 4 days in culture with clear and discernibly cell bodies. If no clear cell bodies are seen and much debris covers the surface after 4 days, then is not a healthy slice.
This method is based on the method first described by Stoppini et al. 11 and offers a rapid manner to culture hippocampal slices. The most important aspect of this protocol is to maintain slices sterile; therefore it is critical to use appropriate sterile techniques and to properly disinfect and sterilize all the material in contact with the tissue.
Different serums sources can influence the quality of the slices. We recommend testing several batches first. If contaminat...
No conflicts of interest declared.
This work was funded by NINDS - NIH R01NS060756
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Name | Company | Catalog Number | Comments | |
Cell culture inserts | Millipore | PICM03050 | ||
6 well plates | BD Falcon | 353046 | ||
Tissue Slicer | Stoelting | 51425/51415 | ||
Microscope | Olympus | SZX7-ILLD2-100 | ||
Hippocampus dissecting tool | F.S.T | 10099-15 | ||
Large utility scissors. Perfection | F.S.T | 37500-00/37000-00 Right/ Left handed | ||
Iris Spatula | F.S.T | 10093-13 | ||
Straight spatula | F.S.T | 10094-13 | ||
Rounded spoon micro spatula | VWR | 57949-039 | ||
Dissecting single cutting edge needle | Electron Microscopy Science | 72946 | ||
Dissecting tweezers | Dummont | #2 | ||
Small dissecting scissors | F.S.T | 14060-10 | ||
MEM Eagle medium | Cellgro | 50-019 PB | ||
Horse serum heat inactivated | Invitrogen | 26050-88 | ||
L-Glutamine (200 mM) | Invitrogen | 25030081 | ||
CaCl2 (1 M) | Sigma | C3881 | ||
MgSO4 (1 M) | Sigma | M2773 | ||
Insulin (1 mg/ml), dissolved in HCl 0.01 N | Sigma | I0516 | ||
Ascorbic Acid, solution (25%) | Sigma | A4544 | ||
D-Glucose | Sigma | G5767 | ||
NaHCO3 | Sigma | S6014 | ||
Hepes | Sigma | H7523 | ||
Sucrose | Sigma | S5016 | ||
Phenol Red Solution 0.5% in DPBS | Sigma | P0290 | ||
KCl | Sigma | P3911 | ||
MgCl2 (1 M) | Sigma | M9272 |
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