Organotypic hippocampal slice cultures

Question 1

Optimal age for organotypic hippocampal slices?

Answer 1

Ideally very young (PN6-9) rodent brains

Is possible to use older brains but since younger brains are more plastic they can survive longer

Question 2

Preparation of organotypic slice culture

Answer 2

1. Decapitation of young animal
2. Cut brain along midsaggital plane & remove occipital lobe
3. Extract hippocampus
4. Place on filter paper
5. Slice using tissue chopper
6. Place 400 um slices on dish (2 per well)
7. Incubate

Question 3

How is culture maintained/fed?

Answer 3

• MEM (minimum essential medium)
• maintained w/ fetal serum supplemented media
• Keep in sterile conditions
• Warmed to 36º
• Feed every 2 days

Question 4

How many slices can you get? What does it depend on?

Answer 4

12-18 slices per animal

Depends on animal age, angle of cutting, skill, and what kind of day you’re having, even

Question 5

Chopper vs vibratome

Answer 5

Chopper is faster, has fixed angle, non-changable temperature, and is only used with young animals (compresses brain)
Vibratome is slower, has an adjustable cutting angle, is much stronger (cannot be used in young tissue), and tissue is submerged w/ changeable temperature

Question 6

Advantages of organotypic brain slice culturing?

Answer 6

• Reduces number of animals needed
• Can assess developmental processes (ex: synapse formation, connectivity, and neural developments)
• Can test same animal at different developmental timepoints
• Can do pharmacological manipulation in a controlled setting (even on the same animal)
• Can do simultaneous pharmacological treatments in a single animal subject using different slices
• E-phys and optical methods are easy to do on slice culture

Question 7

Disadvantages of organotypic brain slice culturing

Answer 7

• Conditions need to be sterile
• Development may occur ex vivo
• Works best with neonatal and juvenile tissue
• Properties of the culture depend on culturing techniques
• Results have to be interpreted carefully
• Aberrant connectivity can occur

Question 8

Composition of medium

Answer 8

-50% MEM
-25% fetal horse serum
-25% HBSS
-1 mM L-glutamine
pH set to 7.3

Question 9

What are gamma oscillations? Role in schizophrenia?

Answer 9

= Fast network oscillations
-Require excitation via pyramidal cells and rapid inhibitions by PV+ fast-spiking basket cells (interneurons)
Are involved in higher brain functions
Gamma oscillations are altered in schizophrenic patients, leading to cognitive defects

Question 10

What kind of inhibition do PV+ fast-spiking basket cells mediate? What makes them special?

Answer 10

• Mediate perisomatic inhibition, particularly in gamma oscillations
• Develop/mature post-natally (ex: much greater spiking rate at DIV 10 vs DIV 3)

Question 11

Results of schizophrenia study

Answer 11

Both oxidative stress and NMDAR hypo-function → alteration in gamma oscillations/lower functions of PV+ interneurons (down regulation?) and altered cognition