1. Morphine Reward in Dopamine-deficient Mice
Hnasko TS, Sotak BN, Palmiter RD (2005). Nature 438:
Presented by Mattia M. Migliore
March 30, 2006

2. Introduction:
First recorded reference to opium use occurred around 300 B.C.
Morphine is an analgesic first isolated from opium in 1806.
Morphine’s name came from Morpheus, the Greek god of dreams.
Opioids exert their effects by binding to opioid receptors (µ, δ, and κ) which then couple to G-proteins, inhibit adenylate cyclase, activate K+ currents, and decrease Ca+2 currents.
Much like other opioid analgesics, morphine has the potential to cause addiction.
structure_of_morphine.jpg

3. Introduction (cont):
Addiction can be defined as uncontrolled, compulsive use of a substance despite adverse consequences resulting from its use.
Addiction can result from repeated exposure to a substance, which then results in neurochemical adaptations in the reward system of the brain.
People can become addicted to drugs, alcohol, tobacco, gambling, sex, and even food.
Addiction is extremely difficult to study because no animal model of addiction even comes close to the complexity of the human condition.
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4. Introduction (cont):
Drug addiction (also termed substance dependence) affects millions of people world wide.
The degree of drug abuse ranges from just occasional use to compulsive use ultimately resulting in fatal consequences.

5. Figure 1
                                                                                                                                                                                                                                                                                                   
(Nestler and Aghjanian, 1997)

6. (Goldstein and Volkow et., 2002).

7. Neurobiology of Addiction:
Drugs of abuse illicit a feeling of euphoria or a “high” by activating the brain’s reward circuitry.
Dopamine has been believed to be responsible for feelings of reward for the last 30 yrs, and has been called the “feel good neurotransmitter.”
DA has long been implicated in the development of addiction.
Most drugs of abuse have been shown (via microdialysis studies) to increase extracellular DA levels and/or DA cell firing in the nucleus accumbens.
Goodman and Gilman’s 11th edition.

8. Synthesis of Dopamine:

9. Evidence supporting DA’s role in reward:
In 1954, Olds and Milner showed that direct electrical stimulation of the brain had powerful rewarding effects. Later, Olds et al. used intracranial self-administration of various substances to try to identify the neurotransmitters involved in reward.
Studies showed that DA receptor antagonists can inhibit the rewarding effects of food, and of intracranial self-stimulation (Zhou and Palmiter, 1995).
Dopamine agonists and drugs that inhibit the DAT have been shown to cause animals to self administer these agents, and to develop a conditioned place preference (CPP) for these drugs.

10. Evidence supporting DA’s role in reward (cont.):
Bilateral 6-OHDA lesions result in a severe decrease of activity, and the animals will refuse to eat or drink (an obliteration of the natural reward cues).
Schultz et al. showed that the anticipation of a reward (juice) in monkeys caused an increase in firing, and a change in the pattern of DA neuron firing.
Maldonado et al. showed that D2 receptor knock out mice do not show a CPP in response to morphine.
Volkow et al. used neuroimaging studies in humans to show that cocaine and methylphenidate increase brain dopamine levels, and this increase was associated with the feeling of a “high.”

11. Molecular Mechanism of Drug Addiction:

12. Mu opiate receptor activation decreases GABAergic interneurons’s inhibition on midbrain dopamine neurons resulting in increased dopamine release in the nucleus accumbens.
(Nestler and Aghajanian, 1997)

13. Brain regions involved in drug addiction:
(Golstein and Volkow, 2002).

14. (Volkow et al, 2003)

15. (Golstein and Volkow, 2002).

16. What makes some people become substance dependent?

17. Hypothesis:
Dopamine is not an essential component of opiate responses, and that dopamine is not required for opioid mediated reward.

18. Methods:
Dopamine deficient mice: a complete deletion of the tyrosine hydroxylase (TH) encoding gene results in a deficiency in both DA and NE. In order to create only DA deficient mice, Hnasko et. Al used the TH encoding sequence to target the dopamine ß-hydroxylase (DBH) promoter in embryonic stem cells. Then DBH-TH +/- mice were crossed with TH +/- mice to yield TH +/- DBH-TH +/- which were then crossed with TH +/- mice to yield dopamine deficient mice capable to still producing NE.
Zhou Q-YP, Richard D. (1995) Dopamine-deficient mice are
severely hypoactive, adipsic, and aphagic. Cell 83:

19. Synthesis of Dopamine:

20. Methods (cont.):
Mice required daily L-Dopa administration to induce them to eat. Morphine was administered hrs after L-Dopa .
Virally Rescued Dopamine Deficient Mice (vrDD): In order to perform the behavioral tests, they used a viral gene transfer to restore DA in the striatum (because DA deficient mice are slow and hypoactive).
Behavioural tests:
1. Locomotor tests were done using photo-beam activity cages. Morphine was administered IP at 0,0.25,2.5,12.5, and 25 mg/kg.
2. Tail flick tests were perfomed by using warm water baths. Briefly, the animal’s tail was submerged cm in the water bath, and the latency to withdraw the tail was recorded (with a cut off time of 15s). The animals were tested three times/treatment and average used. Morphine was administered 30 min. Prior to test IP at 0,3,6,12, and 24 mg/kg.

21. Methods (cont.):
3. Conditioned Place Preference (CPP) was performed using clear plastic boxes with 3 chambers (1 neutral grey compartment in the middle, and 2 compartment with different colored walls, different textured flooring, and different scents). First, the mice were administered caffeine (18-24 hrs after L-Dopa treatment) and placed in the center and allowed to explore for 25 min. On days 3-5 (conditioning phase), the animals received saline SQ in the morning and restricted to one compartment for 25 min, and then received morphine SQ and restricted to the opposite compartment for 25 min in the afternoons. Preference was tested on the sixth day. In the L-Dopa rescue, L-Dopa was administered similarly to the caffeine.
(Cami and Farre, 2003).

22. Results

27. www.unifr.ch/.../ Neurotransmitters/peptides.htm

29. Supplementary Figures are included as pdf files
Supplementary Figures are included as pdf files. Supplementary figure 1 is 22 Kb. Supplementary figure 2 is 16 Kb.
Supplementary Figure 1. Morphine (25 mg/kg) locomotor responses after caffeine (15 mg/kg) pre-treatment. a, Locomotor timecourse shows 30 min caffeine pre-treatment of DD (n=8) or control (n=8) mice does not restore a robust locomotor response to morphine. b, The effects of morphine and caffeine are approximately additive in DD mice (n=8/group).
Supplementary Figure 2. CPP for morphine in vrDD and control mice. Locomotor activity as measured by a, number of chamber entries or b, total distance travelled by vrDD (n=24) or control mice (n=24) during the baseline and testing phases of the experiments. c, Dose-response curve to morphine in vrDD and control mice. Scores are presented as the percentage of time spent in the drug-paired side compared to the saline-paired side during the pre- and post-treatments; two-tailed paired t-test comparing time spent on drug-paired side before and after conditioning within genotypes and doses, *p< Sample sizes were: 0 mg/kg, n=7; 5 mg/kg, n=6; 20 mg/kg, n=11. All data presented as means +/- SEM. Methods: A new line of DD mice was established with a conditional Th allele. Injection of virus expressing cre-recombinase into the dorsal striatum activates the endogenous Th gene in dopamine neurons that project to the CPu, thus restoring dopamine synthesis (similar to Szczypka et al., Neuron 2001). These mice and the viral rescue strategy will be described elsewhere. Mice were tested for CPP as described for Figure 3 except that no drugs were administered during the baseline or testing phases.

30. Conclusions:
Dopamine appears to be essential in the development of locomotor response to morphine.
Dopamine appears to play an important role in the level of analgesia experienced after morphine administration.
Dopamine may be required for reward seeking, but does not appear to be indispensable for morphine’s rewarding effects.