Lec 2: Research methods in hormones and behaviors Flashcards
BEHAVIORAL ENDOCRINOLOGY
Scientific study of the bidirectional interactions between hormones and behavior.
Hormone: organic chemical messenger released from endocrine cells that travels through the blood system to interact with cells via very specific receptors.
Endocrine glands: ductless glands from which hormones are released into the blood stream (produce here aswell) in response to specific physiological signals. Can be direct or indirect release.
- Hormones can affect behavior, and behavior can influence hormones. Hormones achieve this by affecting individuals’ sensory systems, integrators, and/or effectors (output systems). Hormones can impact the 3 systems important for behaviors (metabotropic vs ionotropic).
- They change gene expression or the rate of cellular function affecting behavior by increasing the probability that a given behavior will occur in the presence of a specific stimulus. ie: people take testosterone to gain muscle mass, we are seing a transcriptional effect.
The effects of hormones are notable not only in behavior but also in several human attributes and characteristics
- Lance armstrong won tour de france 7 times but was stripped winnings because he used steroids (ATP) that improve perfomrmance. Increasing the circulation of red blood cells and giving him an energic/oxygen advantage.
- Testosterone treatment (TRT) - banned the use of TRT - his physique changed a ton. Use of testosterone = gain of body mass. Also leads to higher levels of paranonia, agressive behaviors.
HISTORICAL ROOTS OF BEHAVIORAL ENDOCRINOLOGY
- The study of the interaction between hormones and behavior has been remarkably multidisciplinary since its beginning.
- Relationships among the endocrine glands, their hormone products, and behavior have been implicitly recognized for centuries.
- For example: male sex organs, or testes, produce and secrete a hormone called testosterone that influences sexual behavior, aggression, territoriality, as well as many other behaviors that differentiate males from females. Removing these male sex organs (casteration) will have an influence.
- Casastro: Would get their testies removed to keep the tone of their voice (super high pitch).
How we determine if a gland affects behavior?
Ablation and Replacement
Ablation (castration) and Replacement
1. Gland that is suspected to be the source of the hormone affecting behavior is surgically removed;
2. Effects on behaviour are observed; observe if there is a change in behavior
3. Hormone (or gland) is replaced by reimplantation, injection of an extract from the gland, or injecting a purified hormone;
4. Determination is made whether the observed consequences of removal are reversed by the hormonal replacement therapy. Determine if these changes have a consequence. Determine if this gland is the source of the hormone. Is the surgically removed gland the hormonal source?
Berthold’s three major conclusions:
1 - the testes are transplantable organs;
2 - transplanted testes can function and produce sperm;
3 - because the testes functioned normally after all nerves were severed, there are no specific nerves directing testicular function.
Seperated animals in 3 groups.
- animals in group 1 (castrated in early development) were smaller than normal and failed to engage in classical male bird behavior (ie, singing, trying to engage in sexual activity).
- birds in group 2 were also castrated but they had them reimplanted with testies to see if this would reimplant the behavior. Lead to normal male behavior.
- third group, they catrated and switched the testies (gave one of them a testies). These birds developed the similar characteristics
From this we see that the testies are functionning normally after all the nerves were severed so we can say that another form of communication is happening. Probably something in the blood is communicating with the testies making the animals produce sperm and have sexual characteristics developed.
statistical test: ANOVA (3 groups)
2 groups = t-test
Conclude that testies are transferable/transplantable organs. Ie, transplanting testies can produce sperm.
HOW CAN WE SCIENTIFICALLY APPROACH HORMONES AND BEHAVIOR?
Most research in behavioral endocrinology involves only a few types of simple behavior. This narrow focus on only a few behavioral measures is partially a response to the enormous variation inherent in observations of complex behaviors.
There are advantages and disadvantages to this approach. These are 4 types of inquiries in this field:
Immediate causation: The physiological mechanisms
underlying behavior.
Development: The role of experience in individual behavior.
Evolution: The perspective(s) adopted by biologists who assume that evolutionary processes are central to issues in ecology, systematics, and behavior.
Adaptive function: The role of any structural, physiological, or behavioral process that increases an individual’s fitness to survive and reproduce.
ie: testosterone makes birds singe because it makes them reproduce
CLASSES OF EVIDENCE FOR DETERMINING
HORMONE-BEHAVIOR INTERACTIONS
What sort of evidence would be sufficient to establish that a particular hormone affected a specific behavior or that a specific behavior changed hormone concentrations?
3 premises:
1. A hormonally dependent behavior should disappear when the source of the hormone is removed, or the actions of the hormone are blocked. We do not to necessarily remove a gland to block it, we have blockers to do that.
2. After the behavior stops, restoration of the missing hormonal source or its hormone should reinstate the absent behavior.
3. Finally, hormone concentrations and the behavior in question should be covariant; that is, the behavior should be observed only when hormone concentrations are relatively high and never or rarely when hormone concentrations are low.
Experimental method review
independent variable: group variable.
dependent variable: behavior
changes observed from experiments = outcomes
examine the differences between the groups confer from these changes
Quasi-experimental Designs - to investigate cause and effect (causation). Lies between the experimental and non-experimental designs.
Quasi-experimental Designs
* This approach falls between experimental and non experimental designs. It aims to investigate cause-and-effect relationships like experimental designs but often lack some key elements, such as random assignment.
Comparison Groups: Researchers might compare groups that naturally differ in hormone levels (such as individuals with naturally high vs. low testosterone) or groups influenced by external factors (such as individuals using hormone replacement therapy vs. those not using it).
** Natural Observations: **These designs often rely on naturally occurring variations in hormone levels rather than manipulating them artificially. to try not to influence or bias the result.
Non-Random Assignment: Participants are not randomly assigned to groups due to limitations in controlling or manipulating hormone levels in a controlled manner. Instead, researchers observe and compare existing groups. But we do try to account for counfounders in our experiments.
Use of Statistical Controls: Quasi-experimental designs employ statistical techniques to control for confounding variables (factors other than hormones that could influence behavior) to strengthen the validity of the findings.
The lack of random assignment makes it harder to establish a clear cause-and-effect relationship between hormones and behavior.
Correlational design
Correlational Designs
Examination of relationships between variables without intervening or manipulatingm them. Instead of seeking causation, these studies aim to identify associations or correlations between variables to understand how they relate to each other.
1.Measurement of Variables: Researchers measure two or more variables to determine if they are related. These variables could be anything measurable, such as height, age, test scores, hormone levels, etc.
2.Assessment of Relationships: Statistical analyses, likelly correlation coefficients, are used to assess the strength and direction of relationships between variables. Remember correlation coefficients range from -1 to 1 with a positive values indicating a positive relationship, and negative value indicates a negative relationships.
3.No Manipulation of Variables: In contrast to experimental designs, correlational studies do not manipulate variables or control experimental conditions. Instead, they observe and analyze existing data.
4.Identifying Associations: These studies can reveal if changes in one variable are associated with changes in another variable. For instance, a correlational study might examine whether there’s a relationship between testosterone levels and aggression.
Longitudinal design
Longitudinal Designs
* Study changes in variables over an extended period within the same individuals or groups. Unlike crosssectional studies that capture data at a single point in time, longitudinal studies follow participants over time
Key aspects
1.Data Collection Over Time;
2.Tracking Changes;
3.Identification of Trends and Patterns;
4.Analysis of Stability and Change;
5.Challenges and Attrition;
6.Resource-Intensive:
immunoassays.
immunoassays: analytical techniques used for the quantification of an analyte(hormone) based on the antigen-antibody reaction.
Antigen: a molecule that can bind to a specific antibody
(can be the hormone we want to study)
Antibody: a protein made by plasma cells (a type of white blood cell) in response to an antigen (a substance that causes the body to make a specific immune response).
use antibodies to quantify concentration of antigens.
The body produces very specific antigens which allows us to have specificity in terms of hormones
Bioassays
Bioassay: test the effects of the hormone and measure its biological activity on a living animal
* rabbit pregnancy test: use these animals to see a reaction in these animals. This test allowed scientist to identify the chorionic gonadotropin (hCG) which is there when women are pregnant. ‘CG’ = hormone when women are pregnant = in placenta.
- ends in ‘tropin’ = involved in development/nurishment of certain cell or tissue. ie: BDNF = develops neuroplasticity
- enlargement of the ovaries and they would show follicular maturation= pregnant
- Use animals to produce antibodies, because we want to produce antibodies that are specific to certain antigens.
Radioimmunoassays
All types of immunoassays use an antibody that binds to something.
Radioimmunoassay (RIA): is based on the principle of competitive binding of an antibody to its antigen (hormone) that results in change in radioactivity.
- first step: labelling an antigen/antigen molecule with radioactivity and then comparing different proportions of this labeled antigen with an unlabeled antigen.
- We separate several tubes with different known concentrations of the hormone (the antigen) that is not labeled and the antigen that is labeled. We see how they bind to the antibody. We quantify that and then create a curve that will be used to benchmark the hormonal concentration.
- ie: in the 1st tube, you have very low concentration of the known hormone that is not labeled and you know the concentration of the labelled hormone (which is small) meaning you will have a radiation signal that is higher. So, low concentrations of thre actual hormone is identified by higher levels of the labeled hormone.
- The labeled and unlabeled antigens will fight to bind to the antibody.
- cold and hot antigens (hot=radioactive one, cold = unlabeled one).
- Having this different concentration between labeled and unlabeled hormones can be used as parameters for our own quantification.
- The higher the concentration of the unlabeled hormone will be seen at the end of the curve (negative association). The more unlabeled hormone we have, the less radiation we have.
radioimmunoassays steps
If you have more of the hormone, it will competwe with the radioactive (hot antigen) and so you will see less radiation.
Step 1: Establishing norms to interpret concentration. Preparing the standard curve.
Step 2: Compare the experimental sample to the standards. We label the antigens using an immune reaction. We see the competition between the labeled and unlabeled antigens and see the displacement of this.
Step 3: Measure the change in the ration between the emission by the radioactive and measure this.
We have:
* The hormone of interest = the cold one
* We have a seperate quantified proportion of antigens that are labeled.
We both these both in a tube (the right amount) and then the readings of the radiation will be processed by the machine. We can use this reading to see what the proportion is based on the standard curve. Extrapolate to the actual concentration in picograms the concentration of the hormone.
Essentially, we have competitive binding between things that we know (labeled) and something that we do not know (unlabeled).
Enzyme immunoassay (EIA) or Enzyme linked immunosorbent assay (ELISA)
- Commonly used to detect and quantify hormone.
- Very similar principles as we saw in Radioimmunoessays.
- Major difference is that instead of using radioactive tags, these antigens use an enzyme. An antibody tagged with an enzyme that changes the optical density (colour) of a substrate or molecule.
- this process of seing a colour is common in COVID test, pregnancy test. These are antigen tests.
Same idea as radioimmunoassay:
1) the antibody is attached to the bottom of the well and this antibody is specifically designed to bind a specific molecule (the antigen) or in our case the hormone that we want to measure.
2) We add then antigen (the sample we want to measure). The antigen will bind to the antibody and another antibody is provided that is conjugated to an enzyme.
3) When we add the substrate for this enzyme, a chemical reaction occurs and the enzyme converts the substrate to a colorful product which can be measured.
Example: for a covid or pregnancy test, we do not quantify it, we just show the colour. Gradient of the colour has to be standardized like in radioimmunoassay (standards with different concentrations).
What do we use enzyme immunoassay (EIA or ELISA) for?
This method can be used to detect a hormone (change of color) or quantify a hormone using a standard curve (gradient of color).
If we have a change of colour = positive (hormone present)
no change of colour = negative (no hormone present)
- Small concentrations can be an issue because sometimes we cannot detect the hormone because the concentration is too small.
3 types of ELISA
Direct: Antigen is immobilized on the bottom of the plate and the enzyme linked antibody directly binds to the antigen.
Indirect: Primary antibody binds our antigen and then a secondary antibody binds the primary antibody. The secondary antibody is enzyme linked.
Sandwich: most common. Primary antibody is immobilized and captures the antigen of a solution and another primary antibody specifically binds the same antigen.
All three of these have one thing in common: the last antibody is always conjugated to the enzyme.
problem with immunoassays
- With these tests, we do not know where the hormone is acting, where is this hormone coming from.
- In many case we want to determine the exact locus of the action of one or more hormones.
- To identify mroe specifically where this hormone is acting we can use:
1) immunocytochemistry: we collect the cells, use the solutions, drop the solutions that are used and then we can identify within the cells if a hormone is present or not.
2) Immunohystochemistry: use it if you want to look at a specific tissue. We use antibodies to determine the location of hormone in specific tissue or cells.
Immunocytochemistry (ICC)/ immunohistochemistry (IHC)
- The only difference is that our antigen or antiodies will be fixed in the slide and then we can visualize if there is a reaction or not.
Immunofluorescence
- Same technique as ICC and IHC but instrad of emitting or changing the color, it will emit fluorescence. We can capture this with a specific microscope.
- We can establish different gradients of fluorescence to establish quantity.
Autoradiography
Determining the location of hormonal action
Radiolabeled hormones (labelled with a radioactive tag) are injected into a tissue or animal to determine hormonal uptake and indicate receptor location - to see if this radiolabeled hormone will end up in a certain tissue.
* If this hormone binds to the receptor, it will emit radiation that can be detected by a film.
* The parts of the tissue that binds with this radiolabeled hormone will be seen as dark points which will show in which parts of the tissue this hormone is acting.
Steps:
1. Tissues are sliced into several very thin sections; adjacent
sections are then subjected to different treatments. ie: slice the amygdala and then drop the labeled testosterone (hormone).
2. One section of the target tissue is stained in the usual way to highlight various cellular structures.
3. The next section is placed in contact with photographic film, and the emission of radiation from the radiolabeled hormone develops an image on the film. Place the photographic film to identify the presence or not of this substance.
4. The areas of high radioactivity on the film can then be compared with the stained section to determine how the areas of highest hormone concentration correlate with cellular structures. Allows us to pinpoint exactly where a given hormone is acting.
Blot test – Western blot
**Used to: Quantifying several proteins at the same time. **
* Technique used to fractionate mixtures of proteins, DNAs, or RNAs so they can hybridize with markers that travel different distances in an electrophoretic gel based on their size.
1) homogenize the cells: break the cells so everything is loose in our solution. This mixture of proteins and organeles is placed in a gel and then its subjected to electrophoresis: a small electric current is applied through a gel and since proteins have electric charges, this current will move those proteins. This is a way of separating small proteins from big proteins.
2) Transfer the membrane/gel to an incubator with a specific antibody which will be used to identify the proteins/hormones of interest
3) We then detect the antibody by the same process using chemical immunofluorescence and then quantify the substrate that emits the colorful reaction.
remember: proteins are - charged so they are attracted to the + anode.
Instead of looking to individual hormones, we use these to identify several proteins at the same time. Then we add the specific antibodies and we detect them using the different ingredients of illumination.
**do not need to know all the steps, just the main differences between these methods.
Western blot = measures proteins
Northern blots = measure DNA
Southern blots = measures RNA.
Western blot
- Main difference from other techniques is that we can quantify several hormones at the same time.
- Common techniqe for research and clinical analysis.
In Situ Hybridization steps
I. The tissue is fixed, sliced very thin, mounted on slides, and either dipped into emulsion (complementary DNA with a label - can be an enzyme or radioactive) or placed over film and developed with photographic chemicals.
II. Tissue is also counterstained to identify specific cellular structures and see where in the cell the hromone is present.
III. A radiolabeled cDNA probe is introduced into the tissue. If the mRNA of interest is present in the tissue, the cDNA will form a tight association (i.e., hybridize) with it. So, you introduce the DNA and if this complementary DNA matches with target mRNA it will form a tight association and will hybridize.
IV. The tightly bound cDNA, and hence the mRNA, will appear as dark spots.
Note: very small amounts of mRNA sometimes are not detectable so we can incubate to replicate this pattern of cDNA in mRNA several times so that it can be detectable.
Similar to PCR.
Immunohistochemistry vs In Situ Hybridization
The immunohistochemisty identifies the protein and then using In Situ Hybridization to identify the RNA, we can confirm that his protein is prodices in the same cell because we have the mRNA.
Main immunoassays
Brain imaging
Positron Emission tomography - PET
Injection of radiotracers = radioactive tracers that are specific to a hormone.
—> PET scanner can detect the radioactivity as the compound accumulates in different regions
Pharmacological techniques
We can use specific chemical agents to stimulate or inhibit endocrine function. The use of synthetic chemicals agents can alter the action or function of a hormone. The two major groups of chemical agents are referred to as:
Agonists – mimics hormones – stimulate endocrine function
Antagonists – hormones blockers – inhibit endocrine function
Cypoterone acetate is an inhibitor of testosterone, it reduces the level of testosterone because it binds to the receptor and blocks it, which makes the cell prodcuce less testosterone.
Condulation = technique where you inject the drug directly to the tissue of the animal by fine tubes and observe the behavior effects in vivo.If you want to precisely see the action we can use small very small cannulas to direcly inhect the antagonist or agonist.
Functional Magnetic Resonance Imaging - fMRI
- Involves exposing the brain to multiple magnetic fields
- Spatial and temporal resolution to detect changes in brain activity during specific tasks or conditions
- Can be task-based (asking subjects to perform a task or thinking about something) our resting-state.
- task-based fMRI tells you what parts of the brain are active while you are doing an activity.
- resting-state shows the natural patterns of correlation between regions of the brain (functional networks). These functional patterns of activity does not tell us much about performing a task but it tells us which regions are commonly co-activated. Allows us to see the synchrony between those two regions.
- uses BOLD signal = consumption of blood oxygenation in the brain.
Good spatial resolution, poor temporal resolution (EEG is better for temporal).
Genetic Manipulation
Mutant mouse to include or remove specific genetic instructions of the DNA for a hormone or hormone receptor. So we can have an overexpression of specific gene or a knockout of the specific gene.
Gene. Discrete region of DNA within a chromosome that when expressed (transcribed), leads to the production of ribonucleic acid (RNA).
Transgenic. Relating to an animal in which a gene has been inserted, altered, or deleted.
Knockout An individual, usually a mouse, in which a specific gene has been inactivated.
In nature, we have individuals with a natural knockout of the gene so we can inbreed them and then have more subjects with the trait of the gene.
If you want to specifically focus on a given region of the DNA, we can introduce altered DNA into embryonic cells (blastocyst) and take this embryo and implant it into a surrogate mother. They will give births to chimeric mice.
alter DNA –> insert into germ line –> cells will multiply in that embryo –> chimera/mosaic
CRISPR
- Introduce knockouts or activation of genes in specific tissue in specific times.
- CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a revolutionary technology in molecular biology that allows precise editing of genes within organisms.
- CRISPR technology involves cutting DNA at specific locations using the Cas9 protein guided by a designed RNA sequence.
- This precise DNA cleavage allows researchers to introduce modifications by relying on the cell’s natural repair mechanisms (small insertions or deletions ay), allowing the insertion of a new DNA sequence.
- natural proccess that functioned as a bacterial immune system
- Uses CRISPRs and CAS (chop DNA)
- CAS protein cut out a segment of the bacteria’s CRISPR region and copies it into short pieces of RNA.
- The RNA binds to CAS-9. These complexes search for a match to the virus. If the virus invades again, the CAS-9 recognizes it and destroys the viral DNA.
1) design a guide RNA to match the gene they want to edit and attach the guide to CAS-9.
2) Guide RNA directs CAS-9 to the target gene and the protein’s molecular scissors snip the DNA.
3) Once the DNA is cut, the cell will try to repair it, if scientist add a seperate sequence of template DNA, the cellular proteins can perform a different DNA repair process called homology directed repair.
4) The template DNA is used as a blueprint to duife the rebuilding process. Repairing a defective gene or inserting a complete new one.
Hunger hormones
Ozempic: drug that people are usng to treat insulin but is also interesting to lose weight.
Leptin:
* produced at the adipose tissue (fatty tissue)
* helps to maintain normal weight and also normal weifht at a long term basis.
* Your blood levels of leptin is correlated with how much body fat you have.
* leptin resistance: hormone can no longer act due to disturbances in the endocrine system. Leptin resistance causes you to feel hungry and eat more even though your body has enough fat storage. So disregulation of this hormone is implicated in obesity.
