Glial Cell Biology ILO 4 - Describe research techniques used to identify specific glial cells in the brain

Question 1

What are the three main ‘types’ of sample and give examples

Answer 1

in vivo - mice
in situ - acute brain slices
in vitro - organotypic slice cultures, isolated glial cells

Question 2

Name 5 types of studies you may do to investigate glia.

Answer 2

Morphological characterisation
Colocalisation study
Gene expression
Electrophysiology
Measuring physiological or behavioural output

Question 3

What are some of the challenges of studying astrocytes?

Answer 3

Large morphological heterogeneity
No universal marker - genetic expression varies
Need to differentiate between ‘silent’ and active
Some markers also expressed in progenitor cells

Question 4

What are the challenges of studying microglia?

Answer 4

Need to differentiate between ‘silent’ and active

Genetic markers expressed by other cells e.g. CD11b expressed in macrophages

Question 5

What are the challenges of studying oligodendrocytes?

Answer 5

Need to differentiate between progenitor and mature

Genetic markers expressed by other cells

Question 6

What technique would you use to study proteins in a brain slice?

Answer 6

Immunohistochemistry

Question 7

What technique would you use to study DNA/mRNA in a brain slice?

Answer 7

In situ hybridisation

Question 8

What is TgH?

Answer 8

Transgene homologous recombination - insert of a gene into an exact place (knockout/knock-in)

Question 9

What is TgN?

Answer 9

Transgene non-homologous recombination - random insertion, can modify expression of multiple genes so important to map expression of gene of interest

Question 10

What is constitutive labelling?

Answer 10

Insertion of a reporter gene (GFP, mCherry) downstream of a promotor of interest e.g. GFAP promoter for astrocytes, PLP promoter in oligodendrocytes

Question 11

Describe the CreLoxP system in mice.

Answer 11

Cells with active Cre recombinase disrupt gene that is flanked by LoxP, reporter gene is transcribed instead
Cells lacking Cre recombinase transcribed gene as normal

Question 12

Briefly describe two ways to achieve brain area specific temporal resolution with the CreLoxP system in mice.

Answer 12

Cre mouse line + virus expressing fluorescent protein under cell specific promoter, in LoxP system.
LoxP mouse line + virus expressing Cre recombinase under cell specific promoter, in LoxP system