13. Mental Health 3: Drug Addiction

Question 1

What does alcohol cause?

Answer 1

• Alcohol is an enormous social, economic and healthcare burden
• In 2013, 8416 deaths occurred as a direct result of alcohol consumption

Question 2

How do different drugs elicit different primary effects?

Answer 2

• Different drugs bind to different receptors to cause different primary effects

Question 3

What receptors do drugs act through?

Answer 3

• Opiates - u opioid receptors
• Cannabis - cannabinoid receptors
• Alcohol - several; major being GABA and NMDA
• Most, if not all, drugs also act secondarily through dopamine receptors in the mesolimbic reward system

Question 4

What are the 3 contributing factors to drug addiction?

Answer 4

Genetics

• Some people are more/less sensitive to drugs
• Some people develop more/less tolerance to drugs

Social Environment

• During childhood
• Present friendship groups

Personality
- Trauma can increase probability of developing addiction

Question 5

Why are Drosophila a good model to study drug addition?

Answer 5

• Have a very short life cycle (10 days @ 25C) so can acquire many generations in short time
• 50% of genes in Drosophila have human homologs
• 75% of human genes implicated in disease are present in Drosophila
• Some genes involved in drug addiction are conserved (e.g. GABA, NMDA and dopamine receptors)
• Drosophila exhibit complex behaviours that can be used to identify genes involved in drug addiction

Question 6

Why aren’t Drosophila a good model for studying drug addiction?

Answer 6

• Some gene involved in drug addiction are not conserved (e.g. opioid and cannabinoid receptors)
• Environmental and personality factors are hard to model in Drosophila

Question 7

Describe the booz-o-mat experimental approach.

Answer 7

• Flies are kept in chamber which receives a supply of air that can be clean or contain ethanol fumes
• Digital cameras then records flies responses to different air types
• Movie analysis then allows fly behaviours to be analysed

Question 8

What is the WT response to ethanol fumes in the booz-o-mat approach?

Answer 8

• Flies show sudden startle response
• Then show rapid increases in locomotion for a short time
• Then locomotion decreases as flies become sedated
• Conc of ethanol that produces these responses in flies is similar to the conc that elicits the same response in humans

Question 9

Describe the inebriometer approach.

Answer 9

• Relies on the sedation of flies following prolonged exposure to ethanol
• Flies are kept in tube that receives air supply containing ethanol fumes
• When flies become sedated they fall down the ladder inside tube
• Can measure the time taken for flies to become sedated and fall down ladder following ethanol administration (elution time)
• Can then count the number of flies that elute at different time points (WT flies peak at 20mins, Sensitive flies quicker, less sensitive flies slower)

Question 10

What are the 2 independent factors that contribute to drug addiction?

Answer 10

Sensitivity - the inability to tolerate the adverse effects of a drug - start point before 1st exposure

Development of tolerance - the reduction in sensitivity when a drug is used repeatedly over time - higher dose is required to elicit same response as previous

Question 11

What is an experimental approach to measure sensitivity?

Answer 11

• Irradiate flies to induce random mutations
• Use inebriometer approach to screen for flies that elute faster than WT (sensitive) or slower than WT (less sensitive)
• Can then look at genetic differences between more/less sensitive mutants to identify genes that alter sensitivity

Question 12

What was found in the cheapdate mutant?

Answer 12

• Cheapdate mutants have increased sensitivity to ethanol compared to WT
• MET is significantly faster in cheapdate mutant (15min) compared to WT (20min)
• This decreased sensitivity was independent of ethanol conc ie present at all conc)

Question 13

What is the gene mutated in cheapdate mutant?

Answer 13

cheapdate is an allele of amnesiac gene - a neuropeptide that activates the cAMP pathway

Question 14

Describe the activation of the cAMP pathway.

Answer 14

• amnesiac binds to its receptor and activates heterotrimeric g proteins
• Activated Ga subunit activates Adenylyl cyclase which converts ATP to cAMP
• cAMP activates PKA which enters the nucleus and phosphorylates (activates) CREB
• CREB binds to CREB-binding protein and then to CREB-binding elements in DNA to activate target gene expression

Question 15

How is the cAMP pathway affected in cheapdate mutant?

Answer 15

cAMP pathway is not activated therefore target genes are not expressed

Question 16

What did researchers do following identification of cheapdate?
What were the results?

Answer 16

Wanted to see if increased sensitivity was exclusive to cheapdate mutant or if mutating other components of cAMP pathway also increased sensitivity

Measured MET in 3 mutant flies:

• Andenylyl cyclase mutant
• PKA mutant
• cAMP phosphodiesterase mutant (dunce)

Adenylyl cyclase and PKA mutants also had increased sensitivity as shown by faster MET’s

cAMP phosphodiesterase mutant did not show an effect on sensitivity

Suggests activation of cAMP pathway reduces sensitivity to ethanol

Question 17

What is an experimental approach to measure development of tolerance?

Answer 17

• Repeat inebriometer using the SAME flies

- Screen for mutants that show less/more tolerance to identify genes that alter development of tolerance

Question 18

Do Drosophila develop tolerance?

Answer 18

Yes, WT flies develop tolerance following 1st exposure

- WT flies show decreased sensitivity to ethanol on 2nd exposure (ie MET increases)

Question 19

What was found in the hangover mutant?

Answer 19

• hangover mutants have reduced tolerance to ethanol compared to WT
• Tolerance measured by % increase in MET from 1st to 2nd exposure
• Tolerance is significantly smaller in hangover mutants

Question 20

What does hangover encode?

Where is ti expressed?

Answer 20

• A Zn finger protein (involved in nucleotide binding)
• In situ hybridisation showed in WT hangover is expressed in the brain but in hangover mutant this expression is lost
• Western blot analysis confirmed that hangover protein is absent in mutant

Question 21

What is required to develop tolerance?

What evidence also supports this?

Answer 21

• Turning cAMP pathway off

- cAMP phosphodiesterase mutant (dunce) also shows reduced tolerance to ethanol

Question 22

How did researchers identify how hangover functions?

Answer 22

• Immunoprecipitated the total RNA content of cells and using AB against hangover found that Hangover co-immunoprecipitated with RNA ie it binds RNA

Question 23

How do hangover and dunce act to regulate tolerance?

Answer 23

• hangover and dunce act in a negative feedback loop

1. hangover encodes an RNA-binding protein which binds and stabilises dunce mRNA, increasing the amount of transcript
2. Increased dunce expression leads to increased cAMP degradation and pathway inactivation
3. Increased dunce expression also feedsback to inhibit hangover expression
4. Balance between hangover and dunce expression determines degree of cAMP pathway inactivate and therefore tolerance

Question 24

Why do hangover and dunce mutants have reduced tolerance?

Answer 24

They fail to turn off cAMP pathway