Lecture 5 Flashcards
Replication of testicle implantation study with modern methods
(measuring POA)
Pregnant mice treated with T or oil
Pups treated the same from birth for 10 days
Look at brains in adulthood
Stain cells
Blind experimenters assessed size of MPO of hypothalamus
Androgens in this pattern caused increased POA
Females treated showed no difference from males
Race for hormones
Tried to find hormone substances
Guillemin and Schaly had more and worked faster = Nobel prize
Harris was too slow
Ablation and replacement
Ablation - removal or extirpation (local extinction) of a hormone source
Observe effects
Re-implantation of hormone source
Observe reversed effects
e.g. Harris and pituitary transplants
Ablation and replacement example (roosters)
Castrated male baby roosters
How do they develop?
Caponization
Replace with reimplanted own testes - roosters
Replace with another chickens testes - rooster
Therefore any balls work
Bioassays
Test of the effects of hormones on the morphology of a being or part of a living being
Effect of hormones on glands
Effects on non endocrine tissue
Bioassay example - endocrine
What makes ovaries grow?
Glands grow in response to hormone
Estrogen
Give estrogen, ovaries grow
Do not, small ovaries
Bioassay example - non endocrine tissue
Vaginal smear
Early pregnancy test
Breast to hip ratio
Test in male chickens
No test vs test
Size of roosters comb/size of deer’s antlers
VAGINAL SMEAR
use q tip to get sample of lining cells of vagina
Look at cells in slide
We know there are 4 stages of vaginal cells that happen in response to the menstrual cycle
Can infer estrogen and progesterone levels from this
Is a bioassay for them
Early pregnancy test
Urine of pregnant women injected causes sperm release in frogs or ovulation in rabbits due to HCG
Large breasts an narrow waste correlate to 17-B estrodiol
Behavioral bioassays
Operationalizing issues
Test effect of hormone on a living thing’s behavior
Physiological changes are related to behaviors
Sexual behavior when fertile
Startle response is larger when stressed
Hard because is often confounded with other things. How do you know we are measuring something that corresponds to the hormone’s change and not something else?\You have to operationalize this - define how you are going to measure the surrogate for hormones.
These could be:
Qualitative - verbal descriptions of behaviors
Quantity - mathematical number/frequency/amplitude of a behavior
BUT what behavior to record?
If there is a known relationship between the hormone and the behavior, sue that
If you are the first, tricky. you have to guess/use anecdotal experience
Also only as good as the sample and purity of the hormone
Small concentrations of hormone in the blood therefore you can be confounded by other chemicals
How do we localize and identify hormones?
How to generate hormone responsive antibodies
Take a hormone
Inject it into an animal
Immune response
Extract antibodies
Label these antibodies
Take a tissue sample, incubate it with these antibodies
They bind to hormone of interest (or receptor)
Wash out
Antibodies left are used as a surrogate for hormone level
Immunoassay
Uses competitive binding and is based on the principle that a hormone will bind to a specific protein (antibody)
5 steps
(1) find something (an antibody) that your hormone will bind to
(2) Find a ligand that you can attach to your hormone (radioactive label)
(3) Attach ligand to your hormone (labelled)
(4) Labeled and “cold” hormone compete for binding at ligand
Remove all hormone not bound t an antibody and calculate the amount of labelled hormone left in the sample when a certain amount of unlabeled hormone us in there too
(5) Curve: labelled hormone binding with different levels of cold hormone
Can be used on many fluids (CSF, blood, urine etc)
The hormone will COMPETE for binding
If there is higher levels of unlabeled hormone in the sample, thee will be less radioactively labelled hormone bound to receptor.
Make a standard curve and use this to determine the amount of unlabeled hormone in a given sample
This works when you use a radioactive label and measure radioactivity or when you use a chromatic compound and you use a spectrometer to measure color.
Radioimmunoassays use radiation
Enzymoimmunoasseys - change colour
Immunoassay examples/problems
Blood/saliva/CSF
ISSUES
Specificity - if the antibody is not specific enough, it will bind to and cause detection of other substances
Cross reactivity - some hormones have similar structures. Could bind to these
eg cortisol and progesterone
Determining receptor distribution
(1) Autoradiography
Prepare tissue (slices) Prepare hormone (radiolabel it) Exposed unstained tissue to hormone Wash Exposed stained tissue to photographic film Takes ages - days to months
Determining receptor distribution
(1) Immunohistochemistry
Labelled antibodies used to locate proteins
Can apply these to actual brain tissue and see where it binds
Stains areas with receptors
You can zoom in and out
Can see a structure then zoom in and see the details
Pharmacological techniques
Agonists and antagonists
Activate/inhibit binding sites like affecting the effect of the hormone
Can allow us to see the effects of a hormone in a living creature
Sometimes you can target subtypes of receptor
Cannulation allows these tp be placed into a specific brain area
Imaging (for humans really)
MRI
Fat and water alter hydrogen radiofrequency signal in a magnetic field
Shows size/structure
Can show injuries
Provides a map to put a functional image on
fMRI
Activity in brain in a task
Blood Oxygen Level Dependent (BOLD) contrast
Oxygenated hemoglobin is non magnetic, deoxyhemoglobin is magnetic
Activity = drop in deoxy blood as regions requires oxygen
Decreases the disruption of the magnetic field
Less disruption = more blood flow to the region = more activity
