Techniques in Neuropharmacology Flashcards
What is the ‘In vitro’ approach?
Performed with tissue cells, biological molecules etc. outside of their normal biological context. In which brain area(s) do the drugs bind? Outside of the normal biological context (e.g. tissue slice).
What is the job of the transporter?
The job of the transporter is to remove the excess of neurotransmitter that is there. Removes excess dopamine back into the presynaptic terminal.
What are histological methods?
Slicing brains. Use a rat or a mouse. Animal had to be anaesthetised, a hole is cut into the heart, makes brain tissue very hard (preserves integrity of brain tissue). 4% Paraformaldehyde (fixative) ‘fixes’ brain tissue, preserves mechanical integrity of brain. Sectioning - Cryostat or Microtome (horizontal or vertical). Typical thickness is 20-50 micro meters.
How do you identify where the drug binds?
Autoradiography - have radioactive substance, and place it over brain sections. Put film on it so radioactivity is detected on the film. Area where drug is binding is where dopamine transporters are. Can also make a computerised heat map - red means more radioactive signal, blue means none/a lot less. Area active is the striatum.
What is the ‘In vivo’ approach?
The effects of various biological entities are tested on whole, living organisms, usually animals, including humans. Test on function, in-tact brains.
What is brain imaging in vivo?
Use radioactive substance, but very weak. Inject substance into blood stream, passes blood-brain barrier. Light beams find where the radio ligand is situated. Positron Emission Tomography (PET) can be used to visualise e.g. receptors in live human brains.
Where are dopamine receptors?
Look at human brain at horizontal level. Red/yellow (using PET with radioactive dopamine receptor ligands) indicates high concentration of dopamine receptor in the striatum. Striatum is the key site of dopamine action - dopamine in normal life is really important for striatal function, e.g. moving. Can also look at where dopamine receptors are in situ (on site) - look at horizontal section of human brain. One side is stained for dopamine 1 receptor, the other for dopamine 2 receptor. Find the same - dopamine receptors are especially important for striatal function.
What is hybridisation of labelled nuclei acids?
Studied in situ - targeting the nucleus. DNA contains instruction to build proteins, this has to be transcribed to make a protein that is functional, and a transporter that is functional etc. It is a probe that detects messenger RNA. Used to quantify how much gene expression there are. mRNA - vast majority found in the nucleus. But also label certain cell types at the soma.
Are these molecular targets required for the drug effect?
Study this using genetic manipulation in vivo. Make mutant mouse, where genes are knocked out. Inactive gene so working copy isn’t there anymore. Can also have knock in mice - take piece of foreign genetic material and insert it at a location of a specific gene. Can see compensation effects = if knock out particular receptor, other receptors may compensate and then you can wrongly conclude that that receptor is not needed for that particular function. With one technique alone, therefore, don’t want to make strong conclusions.
What are intracranial drug injections used for?
Used to manipulate receptor signalling locally in the brain. Directly inject drug into the brain using guide cannula. Liquid is pumped in using caterer. Internal cannula locally infuses drug and affects any brain areas you are targeting. While this happens the rat is moving freely, and then see whether cue related to the drug is being reacted to more or less. How you can manipulate discrete brain areas and test its effects on behaviour. More specific method on whether the drug is important in a specific behaviour. Fix head, inject drug. Use stereotaxic surgery – allows accurate targeting of specific brain structures. Locally manipulating a brain area is usually done through stereotaxic surgery.
How to detect mRNA?
In situ hybridisation in brain slices. Bind to soma area of neuron, and see if neuron is expressing certain molecule of interest. Label certain neurons expressing certain types of receptors, enzymes, activity-regulated genes (immediate early genes). Increased/decreased mRNA may indicate changes in levels of certain molecules.
What are pyramidal cells and inhibitory interneurons?
Use in situ to find phenotype of receptor. e.g. using ISH to examine neuronal activity in excitatory pyramidal cells and inhibitory interneurons in the cortex following drug exposure. Pyramidal cells express the vesicular glutamate transporter (Vglut). Inhibitory interneurons express the vesicular GABA transporter (VGAT). Activated neurons express the immediate early gene (IEG) - c-fos or Fos.
What colours indicate activated pyramidal cells and activated interneurons.
Fos + VGLT1 (pyramidal) - green and red cell means activated pyramidal cell. Fos + VGAT (interneuron) - green and purple cells means activated interneuron.
What is immuno(cyto)histochemistry?
Protein detection, another way to look at activated neurons. Detecting protein, not mRNA. First need an antibody, immunise animal with antigen – protein of interest, antibodies are released. Purify the antibodies and these detect antigen (e.g. dopamine receptor), and primary antibody detects primary protein, secondary detects secondary protein. Enzyme converts colourless substrate into colour, to show area of interest. When stained, it is stained permeantly. Can also do this with fluorescence. Whether cells are glowing, this is where protein of interest is.
How to visalise cellular expression of target molecules?
Immunocytochemistry. Binding of specific antibodies for enzymes, kinases, transcription factors, etc. Stained dark, this is where dopamine neurons are located.
