Lecture 3 - Research Methods

observational studies

approach is similar to correlational studies but you cant make any causal relationships, it serves as a starting point for a more expansive theory, no experimental manipulations are done

how do we observe NT systems?

using localization and quantification of system elements like receptors and enzymes, we can measure transmitter levels with metabolites, meaure gene-trait associations or cell activity

quantification and localization

you could use amino or radioligand assays to figure out where in the brain the receptors are located

quantification

this refers to how much of something is present, considers baseline gene and protein expression, this can change for instance social isolation can increase mRNA and protein levels, the control and baseline condition are being compared to some active condition and you wanna look at GABA receptor subunit expression, want to have a baseline measure and one in response to a specific element (how responsive is the system?)

localization

where the target is located, this study shows GABA receptor subunit signals, the dark part of the image shows a strong signal and lots of receptors present throughout the brain including beta 3, receptors with alpha 4 are restricted in expression, this is done for receptor subunits and can be done for any of the others

positron emission tomography

allows the measurement of receptor occupancy/availability in the live human brain under different conditions

fragile X syndrome (PET scan)

there is lower GABAa receptor occupancy, this is a correlate of the disorder and the scan allows exploration of its involvement in further studies

addiction (PET scan)

there is reduced DA2 receptor availability, this is the main piece of evidence that is used in the dopamine hypothesis, it is not true of all substance use problems but true for most that are dopaminergic, this is a correlation that is of interest, NOT a cause 

PET for exploring drug mechanisms

it can show the relationship between drugs, receptor binding and psychological function, for example the subjective intensity of psilocybin experience is correlated with 5-HT2 receptor occupancy, some of the psychedelic effects might be linked to serotonin receptor binding so the idea is the more that are bound, the stronger the effect is, see how much of the endogenous agonist do we have available

microdialysis

this is only done in animals because it is a highly invasive method, the measurement of neurotransmitter levels is very rare because it is not only very invasive but also very labor intensive

fiber photometry

done in animals, more common method, you can do it in many circumstances and it involves looking at sucrose consumption or footshock context and measuring the levels in specific cases

less invasive human research methods

magnetic resonance spectroscopy, metabolites focused measurement and CSF measurement

magnetic resonance spectroscopy

this only gives insight in certain neurotransmitters and you get poor resolution because its not very precise

metabolites focused measurement

easier to measure cause you can measure levels in the blood and the urine, hope that the levels of serotonin and its metabolites correlate, metabolite is more readily accessible, however downside is that they are correlates and not the brain so you are looking at a peripheral compartment and assuming that the brain levels are the same

gene variant-trait correlations

assess links between gene variants and traits in humans, for example the link between the DA system and substance use so if you have a certain form of a gene there is higher risk for a certain disorder, most links are weak and non reproducible, almost every behavior you are interested in is explained by 1000s of genes

transgenic knockouts

this is a very common and useful approach to studying genes, the KO mouse is a lab mouse where researchers inactivate a gene by replacing it or disrupting it with an artificial piece of DNA and observe what happens, assess gene function

where do we record cell activity?

this depends, if we want to understand the DA system, we could record from cells in DA pathways, these same areas are good targets for manipulations, there are 4 dopaminergic pathways so we can measure cell activity in different circumstances by targeting dif pathways, its informative but highly invasive so this is only done in animals

electrophysiology

this is defined at the study of electrical and physiological properties of neurons, we can study individual cells or large groups of cells, this is done in vivo or in vitro

in vivo

studying a sample in the live brain

in vitro

this is an easier method, where you study a sample in a petri dish/in cell culture, like a brain slice

DA neuron recording

DA neuron signaling changes with most rewards, not just drugs of abuse, you can observe the neuron before and after feeding, if activity increases after feeding the cell contributes to the post feeding state. certain damage related processes are more likely when the cells are heater/warm

when can we study human brains?

when an operation is already being performed, already doing surgery to open up the skull so a human study might be possible 

calcium imaging

this requires a number of steps in advance, modified system that has a fluorescent signal, it is a crude measurement of activity so better resolution in terms of temporal resolution

micro level

individual cells are pooled groups of cells

macro level

 the intact brain and many areas of it, collecting info at the same time from many cells (ex; fMRI)

tryptophan

its associated with depression, related to the monoamine theory so if we change the diet to increase tryptophan levels and availability it can potentially increase serotonin levels and help prevent and manage depression

drug treatment

giving various doses of a drug so an agonist or antagonist and observing behavior, lets say for instance you’re studying receptor Q, you’d give varying doses of an antagonist for Q and measure behavior, systemic methods are less ideal, the focused and systemic methods are more ideal

selective drug delivery 

if we wanted to understand the DA system, we could look at the effect of DA drugs in certain areas via a cannula

cannula

you implant it over the region of interest, it provides a tube to deliver the drug to the region below to determine how that region reacts to it, so for instance if the cannula is implanted into the amygdala you would shut that area off so the tube goes to it and affect it first, delivering a GABA agonist like mucimol and that would impair fear conditioning

targeted electrodes

you could put an electrode on one end and turn the fibers on to activate it in one specific area

electrodes in the VTA

this would simulate what you would see when VTA is activated, lesions of DA projections prevent this behavior so you could fix it by stimulation via the electrodes, 

intracranial self stimulation

this is an operant paradigm pairing lever presses with electrical stimulation of discrete brain pathways, rats will press the lever for stimulation of brain regions in the mesolimbic dopamine system like the VTA

lesion

this is when an area in the brain is damaged and we observe what happens, for example, the drug 6-hydroxydopamine (OHDA) destroys noradrenaline and dopamine fibers while also impairing memory, this is often used to model parkinson’s

optogenetics

working on an animal that's been engineered to express light sensitive cells in its brain, they exhibit normal activity until they are exposed to light which is done by the experimenter, they create a cell population that only you can activate selectively, the experimenter turns them off or on and sees what happens, this approach has much better temporal resolution and fewer compounds

chemogenetics

approach for selectively activating cells but not as good as optogenetics, engineer the animal with cells that are drug sensitive to a drug only you have 

activating dorso raphae neurons with chemo genetics

activating dorso raphe neurons with a drug that only you have and see what happens, chronic activation of their neurons is antidepressant (SSRIs)

activatiing LC neurons with optogenetics

stimulating locus coeruleus cells, affecting arousal and movement, stains them dark blue

transgenic modification

remains a major strategy to understand neurotransmitter systems, genes encode for the type of protein and amounts

knockout mouse

remove a gene and see what happens

KI mouse

add a gene and see have happens

global modification

remove the gene in all cells

selective modification

remove the gene in certain cells and see what happens

inducible modification

the gene can be turned on at a certain time

delta GABA receptor KO mice

when this receptor was removed they saw depression like behavior and abnormal maternal behavior post partum, suggesting there is something about the maternal window that this receptor is important, hormone metabolites act on it, deletion of the receptor is associated with depression like behavior in post partum

test subjects

humans are less frequently used, use more animal models due to invasive methods and its more cost effective, mice are cheaper to use so more common, easy to produce and maintain, genome is well understood, have human disease and similar conditions in mice + they bread very quickly 

animal model limitations

the cost is high, modelling human behaviors is hard because many sophisticated behaviors are not investigates at all and not all findings are generalizable

reliability

similar results are obtained every time you repeat the test

predictive/construct validity 

animal tests predict the appropriate human behavior, so for example anxiety tests in rates identify anxiolytics in humans

selectivity

test performance mostly reflects the appropriate behavior, for example a test for pain is not affected by cognitive ability

drug tests

the range of behavioral responses can be influenced by drugs in a dose dependent manner so like low doses of anxiolytic drugs are less effective for anxiety and vice versa

anxiety 

a feeling of worry, nervousness or unease about something, its a trait and adaptive to a point in order to avoid danger, survive and reproduce, it can be useful when it is properly regulated

elevated plus maze

the close arms of the maze are safe/unexposed and the open arms are unsafe/exposed, the animals prefer the close arms because it is dark and more akin to an area where they would usually build a home, it is the industry standard test for anxiety

elevated plus maze implications for anxiety

avoidance of the open arms is an indicator of anxiety, anxiety should not be so high that it prevents the animal from exploring to find food and shelter, the researchers found that closed arm time is high in both group but much higher in the transgenic mice demonstrating more anxiety like behavior

depression

a loss of interest or pleasure even when performing activities normally enjoyed, it is characterized by decreased energy, feelings of guilt or low self worth, disturbed sleep, appetite and activity, inability to concentrate, suicidal ideation, hard to model all of these factors

behavioral despair test

includes forced swim test and tail suspension test

anhedonia test

sucrose preference test

forced swim test

this is when you put an animal in a beaker filled with water and see how long it swims for until it gives up, looking at latency and how long they stay immobile, depression is associated with stopping earlier and for a longer period of time, its more of a stress coping measure, with antidepressant treatment the animals swim longer

memory

a process where info is stored, consolidated and retrieved, several types of memory are proposed like sensory, working/short term and long term memory , these stores work over different time periods, the

fear conditioning test

a type of animal model for memory where long term memory is modeled, used as a learning/memory test cause conditioning is learning, its an associative learning task where a shock is paired with an environment + tone, the box and the tone is eventually associated with the shock so when the animal is re-exposed to the box of tone days later, the animal displays freezing because they expect another shock, as the test is conducted days later it assess LTM and the amount of freezing reflects memory strength

novel object recognition

a type of animal memory model for long term and short term memory, this is a type of non aversive memory test that occurs in 3 stages, a training stage where the animal is exposed to a set of identical object, a break stage where the animal is removed from the chamber for a variable amount of time, then a testing statge where the animal is returned to the change and one object is replaced with a new one, animals recalling the familiar objects will show increased exploration of the novel object (indicates memory)

why do we use fear to test memory?

fear is a feeling induced by perceived danger that is learned and associated with physiological and behavioral changes, if exposed to aversive stimulus, animals will remember that experience so memory is key, if they are re-exposed to it the animals will show fear so expression of learned fear indicates memory so assessment of it is the assessment of a type of memory 

fear conditioning sample data

the data shows that LTM is resistant to decay cause the association was still present for months later, maybe its just that they are more anxious, show stronger pain response and frustratingly looking at your own response and seeing what happens

object recognition test sample data

deletion of delta GABAa receptor subunit is associated with poor performance in this task 

5 choice serial response time task

tests attention and impulse control, has to wait and nose poke at a particular location

puzzle box test

meant to test cognitive flexibility

3 chambered social interaction test (SIT)

first the animal is exposed to the chambers for about 20 mins (habituation), then they take the animal back and put them in the same chamber and measure the interaction between the test mouse and the novel one, sociability measure should be pretty high, when the new mouse gets added to the chamber the mouse should prefer that one indicating social novelty, they measure the interaction with mice to make sure they are social then measure novelty preference, they also counterbalance for position to make sure preference isnt just for a the left or right side but for the new mice

road to a new drug

takes a long time and a lot of money, need to assume the drug works and is safe in humans, even after its available it can be pulled from the market at any time, animal testing here is preclinical then they move to humans if they can justify it, failure is inherent to the process

can we tell if a given drug has high potential for abuse?

drugs that are cleared quickly tend to have higher indicators for abuse

addiction

defined as a compulsive engagement in a behavior (drug use) despite harmful consequences), this term is being phased out and the term substance use disorder is now preferred, some drugs might have high risk for abuse like recreational and therapeutic drugs, to gauge the addictive potential of a drug we can test its value as a reinforcer

fixed ratio schedule

drugs given with a certain number of bar presses (fixed ratio schedule, the number of presses is key to testing a drug’s reinforcing properties, if you require too many they will stop performing for the drug (break point), a higher breakpoint suggests reinforcing properties are present in a given drug

free new drugs

developed based on rational drug design but most of the drugs we use have been discovered by luck, lack of progress is due to a lack of valid measures for disorders, focus on traits rather than disorders, research practices and species differences