animal models of addiction

Question 1

what are things to consider when choosing an animal model?

Answer 1

• Do you just want to model a condition?
• Are you interested in only certain symptoms/pathologies?
• Are you interested in treatments?
• Do you want to examine the disease process?

Question 2

describe the validity of animal models

Answer 2

• Construct validity: The model has a sound theoretical rationale (neurobiological or psychological mechanisms, aetiology)
• Face validity: Phenomenological similarity between the model and the disorder being modelled (symptoms)
• Predictive validity: Manipulations know to influence the pathological state should have similar effects in the model (drugs)

Paul Willner - Behavioural Pharmacology 2002; 13:169–188

Question 3

what can we model

Answer 3

different models are used to investigate the different stages of the addiction phases.

(1) substance abuse – drug taking
(2) Drug seeking behaviour
(3) Drug addiction/drug dependence
(4) increase in tolerance/sensitisation
(5) acute withdrawal symptoms
(6) chronic (long-term) withdrawal
(7) Compulsion to use
(8) relapse
(9) context dependency

Question 4

How is drug abuse/taking tested?

Answer 4

Taken by the animal (self-administration)
• Intravenous, usually the jugular vein.
• Rat can tap a lever or poke a nose-hole with infra-red beam to get the drug reward.
• Self-admin by oral, in drinking fluid, can be forced (1 bottle) or choice (2 or more bottles).
Note: the pharmacotherapy you test should decrease drug taking but not other behaviours e.g. food.

Given by the scientist
•	intravenous (not so common)
•	 sub-cutaneous (SC, common)
•	 intraperitoneal (IP, common)
•	 intra-cerebral (quite common)
•	 intramuscular (not so common)
•	
Given by another animal (yoked)
•	Rat 1 – can self-administer at will -usually likes the drug
•	Rat 2 – gets drug at same time as rat 1, but has no control -usually finds drug aversive (USV)

Question 5

How was baclofen investigated to see if it reduces the self-administration behaviour of cocaine?

Answer 5

• Scientists wanted to investigate if baclofen (an anti-spasticity drug also used to treat alcohol) can be used to reduce/block cocaine administration
• Baclofen= Gaba B receptor agonist
• When mouse was treated with baclofen= no longer pressed the lever anymore i.e. the reinforcing effect of the lever disappears
• Scientists wanted the mouse to block the reinforcing effect of cocaine but don’t want to block the reinforcement effect of natural rewards like food.
= Have to be careful and look!
- Baclofen didn’t affect the natural rewards!

Question 6

what is the advantage and disadvantage of the drug being administered by the experimenter to the animal?

Answer 6

Advantage: better accuracy on how much/dose the mouse is getting.

Disadvantage: doesn’t demonstrate the drug seeking behaviour

Question 7

how can drug seeking behaviour be investigated?

Answer 7

Conditioned place preference/avoidance:

1. Let mouse explore both chambers of the CPP paradigm and measure how much time it spent on the white box and black box
   Conditioning phase:
2. Take mouse and inject it with saline in the morning= let it explore just the white box for 30 mins= saline injection is associated with the white box
3. Same afternoon: take mouse and inject it with the drug and put it in the black side= leave it there for 30 mins= mouse associates the drug with the black box
4. Do this for 4-5 days
5. Post-conditioning phase: remove the door–> let mouse explore both chambers for a period of 30 mins–> prefer to stay in the black chamber= associates with reward

Mouse associates the effect of the drug with one compartment and the saline (control) in the other box

Question 8

describe how drug addiction/dependence is investigated

Answer 8

To model “dependence”:

1) Compulsive drug taking/ administer drug over a period of time
2) Develop tolerance

Mouse lives up to 2 years= chronic administration of the drug will take 15 days (3-5 years in a human situation)

Method:
• Male 7-8-week-old C57BL/6J (black) and DBA/2J (white) mice
• Chronic “intermittent” escalating dose heroin (s.c.) treatment of C57BL/6J and DBA/2J mice
- To measure and mimic tolerance

• Intermittent heroin: 2 injections daily (9am and 5pm)
Effect of heroin lasts 4-5 hours= don’t have to administer every hour- mimicking human situation.
Different to cocaine; half-life of 30 mins.

• Both on heroin over a period of 7 days whilst dose increased
• White mouse; nothing happens i.e. not sensitive compared to the black mouse

Question 9

define tolerance

Answer 9

• When a person’s reaction to a drug decreases such that larger doses are needed to achieve the same effect
• Need higher dose of drug due to pharmacokinetics (increase in enzymes that metabolise the drug quicker= shorter half-life) + pharmodynamics (desensitisation of the receptor)
• Tolerance is commonly found in people when taking drugs such as opiates, benzodiazapines and psychostimulants

Question 10

define metabolic tolerance

Answer 10

change in the metabolism of the drug (e.g. enzyme that degrades the drug such as alcohol dehydrogense)

Question 11

define cellular tolerance

Answer 11

change in a receptor or reuptake site e.g. dopamine transporter

Question 12

what is opioid tolerance?

Answer 12

• Tolerance from long-term use
• Decreased sensitivity of neurons to opiates
• Number of opiate receptors does not change
• Intracellular change – cAMP-CREB

Question 13

describe what sensitisation to a drug is

Answer 13

• When a person’s reaction to a drug increases such that smaller doses are needed to achieve the same effect

• Behavioural sensitization is the augmented motor-stimulant response that occurs with repeated, intermittent exposure to most drugs of abuse, including cocaine
• Sensitization, which is a long-lasting phenomenon, is thought to underlie drug craving and relapse to drug use
Anderson and Pierce, 2005

Question 14

what is the molecular mechanism of sensitisation?

Answer 14

• Increase in dopamine transmission
• Increased D2 receptor activity
• Increased D1 receptor numbers and activity

Question 15

name some emotional withdrawal effects of cocaine

Answer 15

• Anxiety
• Irritability
• Drug craving
• Cramps
• Hypo-locomotion
• Anhedonia
• Depression

Question 16

how can withdrawal/dependence be investigated?

Answer 16

• Physical symptoms of cocaine withdrawal not very obvious in mice and rats.
• Naloxone precipitated opioid withdrawal - naloxone injection in chronic morphine treated mice will precipitate acute physical withdrawal.
• Mecamylamine precipitate nicotine withdrawal.

Symptoms of withdrawal in mice – jumping, weight loss, increased defecation, diarrhoea, ‘wet dog’ shakes, face washing, paw tremor, writhing and increased ptosis score.

This can be used to evaluate the role of certain receptors in withdrawal – knockout mice.

Question 17

what were the 3 mouse models used to analyse emotional impairment during opioid abstinence?

Answer 17

3-chamber sociability test
radial arm maze test
forced swim test

Question 18

what did the 3 chamber sociability test show regarding mouse abstaining from morphine on a long-term basis

Answer 18

Long-term morphine abstinence abolishes social preference

3 chamber sociability test:

• Box separated into 3 chambers with doors to them
• Took control saline and opioid withdrawal mice
• Let them investigate the 3 chambers for a period of 30 mins
• Measured the time spent in social chamber compared to the other chambers
• The control mouse spent a lot of time in social chamber compared to the opioid withdrawal mouse–> no care for social interaction

Question 19

what did the radial arm maze test show regarding mouse on long-term opioid abstinence

Answer 19

Increase of anxiety- like behaviour following long-term opioid abstinence:

• Anxiety-related behaviour assessed using the elevated plus-maze which contains 2 open arms and 2 closed arms
• Measures the conflict between the natural tendency of mice to explore a novel environment Vs the tendency to avoid exposed areas
• The animals were positioned in the centre of the apparatus and left to explore for 5 mins. Anxiety-like behaviour was determined by calculating the amount of time spent and the number of entries each mouse made in the open and closed arms.
• Morphine withdrawn animals spent significantly less time and entries in the open arms compared to saline withdrawn animals. These data clearly show that prolonged withdrawal from morphine induces anxiety-like behaviour.

Question 20

what did the forced swim test show?

Answer 20

Increase in depressive-like behaviour following long-term opioid abstinence:
• Let mouse swim for six minutes (As they like they swim) in beaker so can’t escape
• In normal saline mouse: spent 150 seconds out of 6 minutes immobilised (not swimming)
• Morphine withdrawal mouse spent way more time  depressant like phenotype
• Heavily criticised as it doesn’t mimic a person who is depressed–? Usually have social defeats etc

Question 21

how can compulsive drug taking after prolonged cocaine use be tested?

Answer 21

Rat self-administers cocaine for many days.

It is now so “Addicted” that it will suffer electric shocks just to get some cocaine-compulsive drug taking.

Question 22

how can relapse be investigated?

Answer 22

In drug abuse/dependency this means that a person starts taking the drug again after a period of abstinence

• Cue could be a light that comes on when they get i.v cocaine.
• Drug could be a small dose of cocaine or similar drug.
• Stress could be tail pinch, foot shock, food or water restriction

Question 23

describe the different types of brain imaging

Answer 23

• MRI scan (brain activity)
• Positron emission tomography (Receptors)
• Receptor autoradiography
  (Can see receptor localisation and distribution)
• Immunocytochemistry
• In situ hybridisation
  (Target is an antibody, measuring the mRNA level of gene of interest)

Question 24

what are the disadvantages of imaging?

Answer 24

• Expensive
• Animal has to be immobilised= any changes occurring whilst in scanner is different (time-lapse problem)
• Resolution of images not that great
• Have to kill the animal to do autoradiography, immunocytochemistry, in situ hybridisation= not real time of neurochemical changes with behavioural changes

Question 25

what are the animal models used in addiction research?

Answer 25

• Wild type animals
• Comparison of different strains of animals with different vulnerability to addiction (i.e. looking at genetics)
• Gene knockout/knock in mice:
Lack a specific receptor (e.g. opioid receptor) expressed from a gene–> to understand the specific role of the gene e.g. in withdrawal behaviour–> observe opioid withdrawal effect on animal

Question 26

what happened when scientists knocked out MOP in mice?

Answer 26

These KO mice were immediately used to identify the role of these receptors and peptides in opioid reinforcement. These are the first studies done in Kieffers Mop KO mice demonstrating that MOP are the primary molecular target for the rewarding effects of morphine. In order to investigate the rewarding effects of morphine in WT and MOP KO animals, they used the CPP paradigm which can see here, is a paradigm based on pair associating of the drug with one compartment and saline with the other. We can see here that the WT mice seen in yellow has very high preference to the morphine paired compartment, but in the knockout mice that preference was completely abolished suggesting that the rewarding, the positive reinforcement effect of opioid is mediated via the MOP. Further freely moving microdialysis studies were carried out in the nucelus accumbens which is this region here of morphine treated miceWT and MO KO mice. 20 mg/kg of morphine increased dopamine in the nucleus accumbens of WT mice but not in Ko mice. This really shows that activation of MOP induces DA release. Together these data suggest that theMOP is central in mediating the positive reinforcement.

Question 27

how have mouse genetics contributed in the field of drug addiction?

Answer 27

• Identified and confirmed the role of MOP and KOP in cocaine addiction
• Help with the development of novel pharmacotherapy for the treatment of cocaine addiction and prevention of relapse
• Identified genes involved in the vulnerability to develop addiction and relapse

  -MOP SNP: 118A ->G associated with increased risk of opioid and cocaine addiction
  -Hispanic population with 3-4 repeats of binding site on ppdyn gene have increased risk of cocaine addiction strong association has been repeatedly found between people carrying A1-18G SNP variant of the MOP which corresponds to decreased sensitivity to opioids, with increased of opioid and cocaine addiction. Moreover, an association between ppdyn gene SNPS has been associated with increased risk of cocaine addiction in a Hispanic population.

Question 28

what are the limitations of using animal models?

Answer 28

•Rats vs mouse:
Use more mouse studies but their cognitive and decision making functions are not as developed as in rats= need more rat KO studies

• Developmental compensation: as the gene has been deleted throughout embryonic and postnatal development, and thus it is possible that what is being observed reflects indirect compensatory changes rather than the direct effect of the gene deletion.
• Background strain and the transgene: for many knockouts the donor and recipient are genetically different= species difference= phenotypic differences. However, xtending backcrossing (to 2-4 generations) could limit this problem

Question 29

what are the current directions in mouse genetics?

Answer 29

two emerging technologies that build on the usefulness of the traditional gene knockout approach. First the development of conditional knockout mice which enables temporal and spatial control over the gene deletion. To an extent this circumvents the concern over developmental compensation, but most importantly allows region specific gene deletion. This will surely allow more fine tuning in our understanding of receptors and regions involved in addictive biology. We are currently characterising the first conditional opioid receptor knockout mouse. Epigenetics modification is a new direction that animal genetics is moving towrds with great work from Eric Nestler. As both environment and genes are the 2 most important factors which contribute towards drug addiction vulnerability, there is a lot of evidence that those two factors interact. However, very little is known on the mechanism of gene x environment interaction and I believe this is a future direction of mouse genetics