Unformatted text preview: Behavioural Pharmacology
Motor activity: Infrared beam crossings automate measurement
Rotarod: Tests coordination via animal’s ability to stay on rotating rod
Stereotyped behaviour: Direct observation, record on video for later analysis
Tail flick test: Rat’s tail over heat, tail flick occurs when painful.
- If drug produces analgesia, tail flick occurs after longer time.
Spatial memory measurement
Radial arm maze: Central platform with 8 arms; arms are baited
- Mice must remember which arms it has already visited
Morris water maze: Pool of white water, one escape platform.
- Used to investigate long-term memory by retesting days/weeks after learning.
Delayed response task: Cue, delay, response.
- Different dishes (one food one empty) are shown as cue.
- After delay i.e. blinds close, response is tested.
Place preference test: Train rat to associate drug administration with particular side of cage
- Drug on one side, saline on other
- On test day, let rat choose side to go → if it chooses drug, suggests drug is reinforcing
Self administration: Drug self-administered by mice
- Give animal cocaine → food reward only if right lever pressed
- Give animal saline → food reward only if left lever pressed
- Test day: give drug X. If drug feels like cocaine, right lever will be pressed.
- Rats put in situation whereby they cannot escape from warning
- Test in situation where they could initiate escape response when given warning
- Some rats will not bother to try escaping, i.e. learned helplessness → model depression
Startle response: Used to test anxiolytic (anxiety) drugs
- Group A: Loud sound, measure startle response (force on floor to produce jump)
- Group B: Light before loud sound → produce higher force than group A
- Test drug: does it reduce extra force?
1. TH in rat brain is inhibited by end product CAs i.e. feedback inhibition → also in humans.
2. Drosophila indicated importance of CREB for long term memory → also in rodents
- Potential target for human memory studies Techniques in Neuropharmacology
Transmitter life cycle:
- Receptor interaction
- Inactivation i.e. removal from synaptic cleft
Protein presence can be determined via immunoassay, in situ hybridisation or labeled ligand
1. Tagging with antibodies and fluorescence
Specific enzyme associated with synthesis can serve as marker for that transmitter
e.g. TH used as market for catecholamines (dopamine, NE, epinephrine)
Tag antibody with fluorescent compound → use fluorescent microscope
- Antibodies can be created via injecting purifying protein into rabbits
Column chromatography for purifying proteins
- Column filled with charged resin, only some proteins bind to charge
- Column filled with resin which acts like sieve, separates molecules by size
Used to determine….
- If particular protein is located in the cell
- If a brain area contains greater amount of protein than other area
- If a disease is associated with a change in amount of protein
- Tagged antibodies used to show hypocretin (orexin), a peptide low in narcolepsy
- Somatostatin (growth-hormone inhibiting hormone) which regulated endocrine system
- Produced by jellyfish. Gene for GFP has been isolated, can be used in vivo to monitor
protein localisation, trafficking in living cell etc
E.g. GFP attached to CA synthesizing enzyme located in CA synaptic vesicles,
- Brain neurons cultured and use to test hypothesis that activation of PKC by a
phorbol ester (PMA) could alter movement of CA vesicles within neuron
- Neurites show increase in DBH 2. In situ hybridisation
Using complementary DNA to target specific area in DNA
- Detect radioactivity by putting film over tissue, radioactive energy exposes the film
- Autoradiography → hybridization of tissue slices with radioactive cDNA identifies cells
which transcribe that particular gene. Darker areas correlate to higher expression.
Measures presence of antigen by use of antibodies. Preset amount of antibody placed in all tubes
Fixed amount of radiolabeled antigen added to all tubes
Labelled antigen binds w/ antibody
Different, known amount of antigen added into selected tubes
Non-radioactive/radioactive antigens compete for antibody binding sites
Percentage of bound labeled antigen measured + plotted against quantity of unlabeled
7. Standard curve used to determine amount of unlabeled antigen in given sample
6. Can fail if…
A. Not enough tissue ligand to compete out the labeled ligand
B. Tissue ligand reduces the labeled ligand binding to zero
C. Tissue compounds other than the ligand of interest can compete out the labeled ligand
In vivo microdialysis:
- High-performance liquid chromatography (HPLC) coupled to detector can measure NT
- Can couple microdialysis (measurement of unbound analyte concentrations in the
extracellular fluid) collection to HPLC analysis
- CA and 5-HT storage revealed in vivo via formaldehyde treatment
In vitro release studies:
- Brain slices
- Synaptosomes, ie isolated synaptic terminals Receptor interaction: measuring receptor number and affinity No NB
Must correct for nonspecific binding (NB)
- Nonspecific has range in which binding seems to be non-saturable; this must be
subtracted from total binding.
- Can be measured by incubating with labeled ligand and a l arge amount of unlabeled
ligand - Bound/Free Ligand on y-axis vs. Bound on the x-axis
Slope = -1/Kd (Kd = dissociation constant; indicates receptor affinity for ligand)
- ↑ affinity = less Kd
X-intercept = Bmax (maximum # of binding sites; indicates receptor number)
e.g. Chronic DA receptor blockade by haloperidol (HLP) increases Bmax Total Binding = Specific + Nonspecific
Specific = Total – Nonspecific
- Results are most reliable when the Total is much larger than Nonspecific
Q: Would increase in receptor number (upregulation) prevent hlp treatment from blocking DA
A: Probably not, due to 1. Limit in receptor number growth
2. Can give sufficient HLP to handle increase. Measuring receptor binding in vivo
1. PET scans
Positron emission tomography (PET)
- Isotopes have short half-lives → safer
- Positron emitted → collides with electron, emitted gamma rays are detected
- Shows where + how much of labelled compound was present in brain
- Reuptake sites can also be measured using positron which binds to reuptake protein
PET scans using 15
O labelled water → greater flow to higher activity areas.
- Doesn’t require isotopes
- Measures differences in oxygenated:deoxygenated hemoglobin ratio
- More oxygenated sent to higher activity areas
- e.g. Human w/ increase blood flow in anterior cingulate cortex expected more pain from
- Measures uptake of 2-DG; greater uptake in higher activity areas
- 2-DG used as it persists in tissue, not broken down unlike regular glucose
EEG directly measures electrical activity.
1. Microarrays determine if mRNA production has changed
- Specific DNA segments put on chip
- mRNA isolated + labelled (e.g. with fluorescent tag)
- Binding to DNA determined
2. Proteomics uses 2D gels to see if new protein has been produced.
- After learning trial, protein separations on gels are reproducible
3. Knockout animals, such as mice
- Specific gene targeted for disruption, ie knocked out ...
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- Spring '13