Organotypic hippocampus slice cultures- Hasam-Handerson

Question 1

what is the optimal age for organotypic hippocampal slices? can we use old brains?

Answer 1

ideally we use young rodent brains (p6-9). it is possible to use older brains, but, since the young brain is more plastic- young slices can survive longer.

Question 2

preparation of organotypic slice culture

Answer 2

1. decapitulation (of the young animal- no anaesthesia)
2. cut the brain, the occipital lobe and separate the 2 hemispheres (midsagital cut)
3. extraction f hippocampus
4. place hippocampus on a filter paper
5. slicing - using Mcllwan tissue chopper; slices are 400 micrometer thick
6. place slices on dish
7. incubation

Question 3

what medium is used for organotypic hippocamapal slice culture?
how is the culture maintained/fed

Answer 3

• MEM (minimum essential medium)
• Maintenance: with fetal serum supplemented media; kept in sterile conditions and pre-warmed to 36 degrees;
• feeding: every second day

Question 4

how many slices per animal?

Answer 4

12-18 slices

Question 5

what are the advantages and disadvantages of organotypic brain slice culture?

Answer 5

Advantages:

• reduction of number of animals
• developmental processes can be traced in-vitro (synapse formation, connectivity, neural development)
• different developmental time points can be assessed
• pharmacological manipulations under controlled environment
• simultaneous pharmac. treatments in a single animal subject
• electrophysiological and optical methods are easy to use

Disadvantages:

• has to be extremely sterile
• development occurs ex-vivo
• works best on neonatal tissue
• properties depend on culturing technique
• results have to be interpreted carefully
• aberrant connectivity

Question 6

composition of the medium

Answer 6

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

Question 7

What are gamma oscillations and how are they associated with schizophrenia?

Answer 7

• Fast network oscillations (30-80 Hz)
• require excitation (pyramidal cells) and rapid inhibition by interneurons
• involved in higher brain functions (e.g. sensory processing, memory)
• gamma oscillations in schizophrenic patients are lower –> cognitive deficits

Question 8

what are PV+ fast spiking basket cells?

Answer 8

Parvalbumine positive fast spiking basket cells

• type of interneurons
• important for gamma oscillations
• mediate perisomatic inhibition
• fast spiking phenotype
• postnatal maturation

Question 9

Results of the schizophrenia study

Answer 9

both oxidative stress AND NMDAR hypo function result in alteration of gamma oscillations and thus in lower function of PV+ interneurons and altered cognition