animal models of addiction

Question 1

3 Different types of animal models- construct, face, and predictive

Answer 1

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 known to influence the pathological state should have similar effects in the model (drugs)

example of construct vadility- in parkinson’s disease there is a lack of dopamine produced by the striatum. If the animal model does not have an intact striatum it may not be wise to use

face validity- eg in parkinson’s disease a cardinal symptom is having tremors/hypokinesia, so having tremors in a mouse is necessary

predictive valitidty- drugs used to help humans should also be somewhat effective in improving symtpoms in the mouse eg. L dopa would have same efficacy in mouse as in humans

we mostly only have 2

Question 2

animal model for showing substance abuse

Answer 2

Intravenous, usually the jugular vein.

Rat can tap a lever or poke a nose-hole with infra-red beam to get the drug reward.

Also self-admin by oral, in drinking fluid, can be forced (1 bottle) or choice (2 or more bottles)

graph can be plotted to show active or inactive nose pokes in group that were given the drug intravenously in the first place to show whether the drug induces drug seeking behaviour

Number of lever taps?
Schedule achieved?
Post-reinforcement pause?

Question 3

different ways the drug can be administered

Answer 3

intravenous (not so common)

sub-cutaneous (SC, common)

intraperitoneal (IP, common)

intra-cerebral (quite common)

intramuscular (not so common)

Question 4

animal model for drug seeking behaviour

Answer 4

Conditioned Place Preference/Avoidance

Initial test for side-preference
Usually give the drug in non-preferred side
Give saline in preferred side
Test for CPP after 5-10 days

Time spent in the heroin paired compartment was measured on day 2 and day 7 (pre-, post- conditioning)

so give drug before day 2 and day 7 and saline in between, measure difference in CPP
measure the time in each compartment at different concentrations of heroin

Question 5

how can inbred mice strains be used to show drug abuse vulnerability

Answer 5

eg. two different groups of mice- C57BL/6J and DBA/2J
these different groups of mice naturally show different
affinity ie morphine analgelsia, morphine preference, alcohol, amphetamine preference, cocaine preference

Question 6

how to model increase in tolerance/sensitization

Answer 6

Tolerance is commonly found in people when taking drugs such as opiates, benzodiazapines and psychostimulants
Metabolic tolerance = change in the metabolism of the drug (e.g. enzyme that degrades the drug such as alcohol dehydrogense)
Cellular tolerance = change in a receptor or reuptake site e.g. dopamine transporter

Method: Male 7-8 week old C57BL/6J and DBA/2J mice

Chronic “intermittent” escalating dose heroin (s.c.) treatment of C57BL/6J and DBA/2J mice
Intermittent Heroin: 2 injections daily (9 am and 5 pm)
Total activity measured after injection

Question 7

model for sensitization- what it is and locomoter sensitization?

Answer 7

sensitization? 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

Locomoter experiment to show sensitization:
mouse injected with cocaine at 50mg/kg every hour
at 0 60 and 120 mins
measured locomoter activity

after day 1 increase of locomoter behaviour that increased and then worse off (up and down)
same protocol on day 2 and 3

you can see the same dose of cocaine induces a much higher effect on day 3 compared to day 1
this is experiment showing sensitisation which is what is assocated with drug craving and relapse

Question 8

Mechanism of sensitization

Answer 8

D1 is post synaptic, D2 is pre and post synaptic, D1 is post synaptic

In sensitization,
Increase in dopamine transmission
Increased D2 receptor activity
Increased D1 receptor numbers and activity

Question 9

Withdrawal symptoms and how to model them

Answer 9

Physical, characterised by abstinence syndrome (LC)
Sweating, gooseflesh (cold turkey), irritability, aggression
Psychological, craving to avoid withdrawal effects

Physical symptoms of cocaine withdrawal not really 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

we can model these opioids withdrawal symptoms
by adminstering opioids like morphine or heroine in animals for a period of 2-3 weeks to make the mouse or rat dependent
inject nalaxone which is an opioid receptor antagonist. the mouse would lose weight, shaking, diarrhoea- the same withdrawal symptoms. Mice always get jumping behaviour which doesn’t happen in humans

Question 10

emotional withdrawal effects and how to model them

Answer 10

Anxiety
Irritability
Drug craving
Cramps
Hypo-locomotion
Anhedonia
Depression

Mouse model of emotional impairment during opioid abstinence- method:
Chronic Saline or Escalating-Dose Morphine administration
Assessed emotional-like behaviour (sociability, anxiety, depression) following protracted morphine abstinence

anxiety, depression, hedonia, social withdrawal last for a long period of time and can cause relapse

we left mouse wihtout drug for 7 days and measured emotive response of anxiety, depression etc

Question 11

Evidence that long term administration abolishes social preference

Answer 11

3 chambers sociability test!!!!!!

The three-chambered box as it can be seen on the screen assesses social interaction behaviours in rodents. The whole test consists of 3 phases.

During the habituation phase the mouse had free access to explore the three empty chambers for 10 minutes.

During the second phase, called the social interaction phase, two identical small cages were placed in each of the end-chambers and an unfamiliar mouse, called the stranger 1, was placed in one of the two small cages while the other cage remained empty. The test mouse was allowed to explore the whole apparatus for a period of another 10 minutes.

During the last phase, the preference for novelty phase, another unfamiliar mouse, the stranger 2, was introduced in the previously empty cage while the now familiar “stranger 1” remained in its original cage. The test animal was permitted to explore the box for another 10 minutes.

Social interaction was determined by calculating the time that the test mouse spent in the chamber containing the unfamiliar mouse (stranger 1) vs. the chamber containing the empty cage. Saline withdrawn mice, shown with the clear bars, exhibited a significant preference for the chamber containing stranger 1 vs the empty chamber as you can see here. Morphine withdrawan animals did not show any preference between the chamber containing the stranger 1 and the empty chamber demonstrating a lack of social preference.

Similarly, saline withdrawn animals showed significant preference for the chamber containing the unfamiliar mouse (stranger 2) vs the now familiar stranger 1 demonstrating social novelty preference. However, morphine withdrawn mice did not show any preference between the 2 chambers demonstrating a luck of social novelty preference.

Therefore, morphine withdrawal induced social interaction deficits in mice which is in agreement with the situation of opioid abstinent humans. This data clearly shows clear causality btween opioid abstinence and social impairment

Question 12

Evidence for increased anxiety like behaviour in long term opioid abstinence (animal model)

Answer 12

We then assessed anxiety-related behaviour with the use of the elevated plus-maze. This plus-shaped apparatus consisted of two open arms and two closed-arms and it was elevated 40cm from the floor. This test measures the conflict between the natural tendency of mice to explore a novel environment versus 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. Consisten in humans

Question 13

Evidence for Increase in depressive-like behaviour following long-term opioid abstinence (animal model)

Answer 13

SENSITIVE TO ANTIDEPRESSANTS

Forced swim test:
5 litre beaker, put water into out at specific temperature
place mouse in water and let it swim
after a period of time it likes to get out
some keep their head above the water and doesn’t bother moving- forced- swim test
gives the despair like behaviour

Question 14

what is relapse and reinstatement

Answer 14

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 15

stress induced reinstatement (animal model)

Answer 15

Stress-induced Reinstatement:

Saline or Carbetocin injection (6.4mg/kg, i.p.)
Forced-swim stress (6 minutes)
Test for place preference

animal model of stress induced relapse:
first condition animal to be dependent on drug
give period of absitinence
re-introduce a trigger such as stress that re-introduces the strug starting behaviour

conditioning, extinction (only give saline), trigger re-instatement behaviour
eg. the forced swim test for 6 mins

can do similar experiment with morphine:
during pre-conditioning- spends about 350 seconds in the morphine compartment
following extinguishing goes back to pre-conditioning levels
but stress completely re-instates and the mouse relapses towards morphine

Question 16

Link behaviour with microdialysis

Answer 16

Measuring neurotransmitter release in vivo
Association with behaviour parameters

implant probe in specific brain region in animals
probe connected to HPLC, so whatever is released from this brain region will be uptaken by HPLC
analyse metabolites- dopamine, serotonin etc.

you can measure the release of neurotransmitters from the brain WHILE the mouse is moving
so you can correlate any metabolite release in the mouse with any changes in their behaviour real time

can use:
MRI, PET scans, Receptor autoradiography
Imuunocytochemistry
In situ hybridisation

Question 17

Contribution of mouse genetics to the field of addiction

Answer 17

We have:
Wild type animals
Comparison of different strains of animals with different vulnerability to addiction
Gene knockout/knock in mice

Mice have helped:
Identified and confirmed the role of genes in addiction

Help with the development of novel pharmacotherapy for the treatment of addiction and 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 polpulation with 3-4 repeats of binding site on ppdyn gene have increased risk of cocaine addiction

Question 18

morphine in knockout mice

Answer 18

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 demontrating 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 annimals, 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.

As you can see here 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 19

Uses of KOP knockout mice

Answer 19

Now lets look a the situation in mice lacking the KOP. Again these were done in Briggite Kieffers KOP KO. As you can see the CPP was used in order to investigate the motivational properties of KOP agoinists. As we can see here KOP agonist U50488H compound induced a place aversion in the CPP paradigm in the WT mouse

. It actually spent 400 secs more in the chamber not associated with the KOP agonist injection. This effect was completely abolished in the KOP KO mouse suggesting that activation of the KOP system produces an aversive/dysphoric effect. Further freely moving microdialysis data in the nucelus accumbens of naïve WT, het and KO KO mice.

As you can see there were significantly higher levels of dopamine in KO mice vs WT indicating that KOP activation decrease dopamine release in the brain. Together this data indicates that activation of the KOP system produce an aversive/dysphoric/antirewarding effect. Indeed this is a well known effect of KOP agonist is the prime reason that enalodine which was developped as a KOP selective analgesic failed in clinical trials together with its psychotomimetic effects.

Question 20

Limitations of knockout mice

Answer 20

Rats vs mouse

Developmental compensation

Background strain and the transgene

Of course there are limitation with the use of KO mice. First they are mice and their cognitive functions are not as developped than rats which might affect behavioural studies of addiction so many people are sceptical. Moreover there is the issue of 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. Perhaps of greater concern is the problems of the background strain as for many knockouts the donor and recipient are genetically different. However xtending back crossing could limit this problem

Question 21

Current developments in mouse genetics

Answer 21

Site specific knockout mice (Cre x flox mice, SiRNA)

Epigenetics

Mechanisms of gene x environment interaction

I want to finish by saying something about 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 beleibe this is a future direction of mouse genetics.