Methods

Question 1

How would you choose which type of method to use when you have a new drug that you wanna test to see if there are receptors?

Answer 1

x

Question 2

What are behavioral measures crucial for?

Answer 2

Understanding basis of disorder and inducing changes
Developing ANIMAL MODELS
Screening newly designed drugs in preclinical and clinical studies

Question 3

What are the advantages of using animals?

Answer 3

Control of living conditions and history

Can do things that you cant do on humans

They are very similar in many ways (animals and humans)
Rats have same pyramidal cells!
- you can try treatments out in preclinical studies and see if there are side effects/symtpoms

Question 4

What’s a test?

Answer 4

Measure specific physiological, emotional, cognitive or other processes
i.e.
Water maze is an assay for spatial memory

Elevated plus for anxiety,

Conditioned place preference for reward

We never know what an animal is thinking so we have to infer that from behavior. These tests should be similar to humans.

Often use multiple tests to see a complex behavior (different parts of fear -memory or actual reduced pain)

Human tests must be applicable to real life.

Question 5

What’s an animal model?

Answer 5

Manipulation (lesion, genetics, pharmacology) that causes a change that we see is similar in a disease.

Mimic pathology or induce a drug : MAKES THE SAME BEHAVIORAL PHENOTYPE.

1 model doesn’t do it for complex disorders, so you need multiple models for different symptoms
- the anhedonia model of depression

Example for pharmacological induction: NDMA glutamate receptor antagonist given shows a similar effect, but the ppl with disease have never had that drug, so something else caused it, so its something else. but whatever caused it has the SAME TARGETS IN THE BRAIN.

Question 6

What are stereotaxic surgical permenant brain lesions?

Answer 6

1) permenant: destroy tissue through
a. aspiration (vaccum)
b. radiofrequency (implant and pass current, very precise)

A and B kill neurons, they kill the axons passing through there too so the effects might be cause of that

c. excitotoxic -glutamate antagonist that overexcite and kill intrinsic neurons (most modern)
- Example: kanic acid and ibotenic acid.

For all these, the brain reorganizes.

Question 7

What are kanic and ibotenic acid?

Answer 7

excitotoxic permenant lesion that mimics glutamate.

Question 8

Describe stereotaxic reversible lesions?

Answer 8

Inactivation. Intracranial infusion of a drug via cannula to suppress activity (anasthetics or GABA agonists)

Brain doesn’t have time to reorganize or compensate.

Intracranial infusion: more specific to drugs in specific brain regions! Maybe do this after you’ve tested a drug systemically.

Question 9

What are neurotransmitter specific neurotoxins? Examples?

Answer 9

Just like a NT but changed a bit so its toxic. Only absorbed into specific cells
Requires stereotactic surgery and injection
ONly taken up by the targetted cells (in terminals or cell bodies)

Destroys from the inside out.
Deplete an area or whole system.

6-OHDA = DA NE
57 Dihydroxytryptamine 5 HT
192 IgG saprin= ACH

Question 10

What are

6hydroxydopamine, 57 dihydroxytrpytamine and 192 igG saporin?

Answer 10

DA NE
5HT
AcH

NEurotransmitter specific neurotoxins.

Question 11

What are implanted macroelectrodes?

Answer 11

Simulate a whole area to produce action potentials, can assess whether activation of an area produces behavioral patterns.

Should be the opposite of neurotoxins, but sometimes this can also disrupt a normal pattern of behavior.

Question 12

What is microdialysis?

Answer 12

Measures NT release in a specific region WHILE SUBJECT IS DOING SOMETHING.
- probe with artificial CSF, then it’s permeable and NT enter solution in the probe via diffusion.
Slow sample every 5 mins or so.
Tiny amounts are pumped back out and analyzed with HPLC

Question 13

What is HPLC?

Answer 13

high performance liquid chromatography.

Used with microdialysis to separate sample into components depending on MOLECULAR SIZE or IONIC CHARGE.

Then determine concentration of molecules via elecrochermistry or other.

1) monoamines (DA, NE, 5HT) are electrochemical measures , count how many electrons are given off.
2) Glut, ACH different methods

Do a baseline then see what happens with a drug or behavior.

Question 14

What kinds of microelectrodes are there?

Answer 14

Intracellular/patch clamp: animal asleep or in vitro brain slice.

• glass microelectrodes
• patch clamp uses suction
• membrane potential, electrical currents. How synaptic transmission/ionic currents are regulated and changed.
• only for individual neurons

Extracelluar: metal electrodes. Goes outside the cell and sees if it fires (firing rates)

• multiple neurons at once
• live animal
• can see how changes in behaior have different firing rates

Question 15

What are the different methods that drug receptors can be located and quantified with?

Answer 15

Soup: ground up and analyzed to count how much
Slice: intact pieces slides of tissue to localize targets

Ways to label:
Radioligands
Antibodies

Question 16

Describe antibodies in more detail.

Answer 16

Very specific but hard to generate.
Give an animal the antigen (receptor you want bound to)
ANimal will create an antibody that sticks to that protein. Exact antibody. Extract this antibody in blood samples, modify it and tag with fluorescence or radioactive thing. Then use labelled antibodies to see where/how many receptors there are.

Question 17

Describe radioligands in detail?

Answer 17

Endo/exogenous. We know that it sticks to a receptor. Imped one atom that is radioactive (Spiperone (D2 antagonist, hydrogen is swapped) to make it radioactive. measure radioactivity coming out to count receptor binding (counting or photography)

Question 18

How can you use radioligand binding to determine the number of receptors in an area of interest?

Answer 18

Solution

1. containing animal tissue, OR regular cells with a specific receptor that are engineered
2. radioligand

Bath the cells with the receptor with the radioligand, then washout the unbound radioligand and remaining amount is bound. Measure radioactivity. “grind and bind”

Question 19

How does radioligand binding get used to identify if novel chemicals have affinity for that receptor?

Answer 19

Standard ligand combined with test compound.
Give cell a receptor that you care about, then give a RADIO LIGAND we already know sticks to it, then apply the NEW ligand and if it sticks some should be displaced. The amount of radioactivity is LESS if it binds more.

ie. Eticloprite (D2 antagonist) if radioactivity is zero then its bumped EVERYTHING OFF.

1. Give standard radioactive drug 
2. Then give your drug in different concentrations 
3. See how sticky it is based off displacement.  Do this for different types of receptors: shows selectivity.

Question 20

How can we visualize receptor location and distribution in the brain?

Answer 20

Probes: radioligands or antibodies)

Receptor autoradiography: needs highly sensitive drug to be specific. Radioligand goes with slices then exposed to film sensitive radiography. EX: cocaine. but you need a very selective ligand so cocaine isn’t the best

Immunocytochemistry (ICC): similar more specific approach. slices with antibodies (very specific) but antibodies are hard to get. But it is the better method)

Question 21

What is in situ hybridization?

Answer 21

Use when you don’t have an antibody or you have a small amount. Locate cells that make a protein, detects the mRNA that make that protein, (labels single strand base pair fragments)
Label MRNA for the receptor (make fluorescent things stick to it)

Amount of mRNA gives estimate to amount of protein in a cell.

Advantages: highly selective, easier to analyze vs autorediography or ICC, EXTREMELY SENSTIVE to very small amounts.

Disadvantages: misleading, just cause it has mRNA doesn’t mean its been made or inserted where it needs to go.

Question 22

What is enzyme linked immunosorbent assay? (ELISA)

Answer 22

Used to see if after you’ve done a manipulation, does this change concentration of receptors??

Antibodies linked to an enzyme that acts on substrate to make a colour. color intensity is antigen present.

Standard comparison to homogenized tissue samples.

Pros: economical, quick
can compare across regions or animals.

Question 23

How to measure drug efficacy for metabotropic vs ionotropic receptors?

Answer 23

1. Metabo
   2nd messenger, change enzymes (cAMP)
   - Measure 2nd messenger enzyme activity in different concentrations to get a dose response curve.

    - Get cells with receptor
    - When it changes camp, it changes how much colour it emits. 
    - Use enzyme assays
        § When we give drug, does it change the 2nd messenger in the same way as the endogenous ligand. 
        § Altering it the way the normal endogenous ligand does. 
        § YOU CAN HAVE A DRUG that sticks, but doesn't change the 2nd mesesnger cAMP the way that the endogenous one ususally does. 

2. Ion
   Ion channels
   Change in ionic current.
   Use patch clamp (see membrane voltage and current)
   Does this drug hyper/deoplarize a cell the way a endogenous one does.
   The example uses current. (a group of neurons)
   Example: ACH: drug enhanced ach.
   Does it cause the same depo/hypo as the endogenous one do.

Doesn’t work well for metabotropic: metabotropic is more complex, patch clamp doesn’t work well. That’s why you need enzyme assays.

Question 24

What’s PET?

Answer 24

Low temporal resolution (good for disease, bad for behavior)

Positron emission tomography: uses radioactive isotoeps (OCF to see where blood flow/glucose/o2 is happening during a task)

Also combined with radiolabelled drugs that binds NT receptors, PET can show the locations of receptors in living brian and NT release (displacement of radioligand because they are COMPETEING)

Limitations: temporal resolution, only a few (5HT, DA, GABA), only works where there are lots of receptors and NT like the striatum

Question 25

What is raclopride? What is modafinil?

Answer 25

radioactive dopamine antagonist used in PET to show the striatum

Modafinil changes how much dopamine sticks to receptors.

Question 26

Describe PET transmitter release.

Answer 26

Pet scan measures signal from radiotracer that’s bound to a receptor (number of receptors or basal transmitter levels)

Increased release of endogenous transporters reduces binding signal.

Can do this at baseline then add the new drug and see a reduction in radioactivity or nah.

Question 27

Describe fMRI

Answer 27

functional, measuring bold response (oxygenated blood has different magnetic signals because of hemoglobin?
- MUCH BETTER spatial and temporal resolution

Changes in brain activity DURING A TASK or at RESTING STATES (can see connectivity this way if things go up and down together)

Pharmacological MRI: sees what happens with drugs.

BOLD isn’t necessarily synaptic firing, its oxygenated blood in an area which usually indicated presynaptic inputs.

• inputs can also be inhibitory!
• so just cause we see more bold response doesn’t mean we have more neural firing.

BOld is just measuring oxygenated blood (more activity needs more oxygen for sodium potassium pump)

Question 28

What are genetic manipulations in neuropharmacology?

Answer 28

To determine functions of certain proteins.

Knockout: deleted gene
Knockin/transgenes: original gene removed, subsituted.
Good for when you don’t have a drug to target.

ISSUES:
Compensations by other genes might mask the effect of the mutation OR when developing it has time to changed…

Selective deletion/expression 1. conditional knockout: in only a subset of brain regions.

2: engineer gene so that it turns on/off when you give a drug (doxycycline: stops producing that receptor)
3. Viral vectors: target a type of cell (ex: pyramidal PFC D1 receptor neurons), injected in adulthood.

Question 29

What is doxycycline

Answer 29

a drug that stops genes from producing a receptor (gene manipulations)

Question 30

Describe optogenetics

Answer 30

insert light sensitive genes ion pumps/channels
- inserted via viral vectors

Very specific: either to NT system or specific brain region

Activated by light in an implanted optic fibres.

Activated with a specific light wavelength.

Very temporally specific: can do it for a few seconds, show things WITHIN A BEHAVIOR that are good or bad.

EX: chanelrhodopsin, excitatory, responds to blue light Na+ ions
Halorhodopsin: inhibitory, yellow light. pump for cl- ions

Question 31

What method do you use if you need very good temporal resolution?

Answer 31

Optogenetics, fMRI (add more?)

Question 32

What method do you use for good spatial resolution?

Answer 32

fMRI? In situ hybridization, Immunocytochemistry, slice method

Question 33

Describe chemogenetics?

Answer 33

insert genes for specific receptors (2nd messenger receptors muscarinic, opiod) DREADDs = designer receptors exclusively activated by designer drugs

Dread receptors activated by special drugs (CNo) that ONLY ACTIVATE THE SPECIAL RECEPTORS. No endogenous ligand binding.

Activation via binding inhibits or excites neurons that have those receptors.
Administered systemically but still only effects the brain regions with the receptors.
- cellular specificity

Question 34

whats the problem with the speical drug CNO?

Answer 34

liver metabolizes it to Clauzapine that actually has an effect as an antipsychotic. must use control animals that are given just clauzapine.

now there are other receptors (compound 21) that metabolize to something inert.

Question 35

What can radioligand binding tell you?

Answer 35

Number of receptors in an area or affinity of those receptors. Receptor audoradiography is used to see location of these receptors.

Question 36

What uses antibodies?

Answer 36

Eliza, ICC