Morphine Reward in Morphine Reward in Dopamine-deficient MiceDopamine-deficient Mice

Hnasko TS, Sotak BN, Palmiter RD (2005). Nature 438:854-857.Hnasko TS, Sotak BN, Palmiter RD (2005). Nature 438:854-857.

Presented by Mattia M. MigliorePresented by Mattia M. MiglioreMarch 30, 2006March 30, 2006

Introduction:Introduction:

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First recorded reference to opium use First recorded reference to opium use occurred around 300 B.C.occurred around 300 B.C.

Morphine is an analgesic first isolated Morphine is an analgesic first isolated from opium in 1806.from opium in 1806.

Morphine’s name came from Morphine’s name came from Morpheus, the Greek god of dreams.Morpheus, the Greek god of dreams.

Opioids exert their effects by binding Opioids exert their effects by binding to opioid receptors (to opioid receptors (µ, δ, and κ) which µ, δ, and κ) which then couple to G-proteins, inhibit then couple to G-proteins, inhibit adenylate cyclase, activate Kadenylate cyclase, activate K++ currents, and decrease Cacurrents, and decrease Ca+2 +2 currents. currents.

Much like other opioid analgesics, Much like other opioid analgesics, morphine has the potential to cause morphine has the potential to cause addiction.addiction.

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Introduction (cont):Introduction (cont): Addiction can be defined as Addiction can be defined as

uncontrolled, compulsive use of a uncontrolled, compulsive use of a substance despite adverse consequences substance despite adverse consequences resulting from its use.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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Introduction (cont):Introduction (cont):

Drug addiction (also Drug addiction (also termed substance termed substance dependence) affects dependence) affects millions of people world millions of people world wide.wide.

The degree of drug The degree of drug abuse ranges from just abuse ranges from just occasional use to occasional use to compulsive use compulsive use ultimately resulting in ultimately resulting in fatal consequences.fatal consequences.

www.WHO.org

(Goldstein and Volkow et., 2002).

Neurobiology of Addiction:Neurobiology of Addiction: Drugs of abuse illicit a Drugs of abuse illicit a

feeling of euphoria or a feeling of euphoria or a “high” by activating the “high” by activating the brain’s reward circuitry.brain’s reward circuitry.

Dopamine has been believed Dopamine has been believed to be responsible for feelings to be responsible for feelings of reward for the last 30 yrs, of reward for the last 30 yrs, and has been called the “feel and has been called the “feel good neurotransmitter.”good neurotransmitter.”

DA has long been implicated DA has long been implicated in the development of in the development of addiction.addiction.

Most drugs of abuse have Most drugs of abuse have been shown (via been shown (via microdialysis studies) to microdialysis studies) to increase extracellular DA increase extracellular DA levels and/or DA cell firing in levels and/or DA cell firing in the nucleus accumbens.the nucleus accumbens. Goodman and Gilman’s 11th edition.

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Synthesis of Dopamine:

Evidence supporting DA’s role in Evidence supporting DA’s role in reward:reward:

In 1954, Olds and Milner showed that direct electrical In 1954, Olds and Milner showed that direct electrical stimulation of the brain had powerful rewarding stimulation of the brain had powerful rewarding effects. Later, Olds et al. used intracranial self-effects. Later, Olds et al. used intracranial self-administration of various substances to try to identify administration of various substances to try to identify the neurotransmitters involved in reward.the neurotransmitters involved in reward.

Studies showed that DA receptor antagonists can Studies showed that DA receptor antagonists can inhibit the rewarding effects of food, and of inhibit the rewarding effects of food, and of intracranial self-stimulation (Zhou and Palmiter, intracranial self-stimulation (Zhou and Palmiter, 1995).1995).

Dopamine agonists and drugs that inhibit the DAT Dopamine agonists and drugs that inhibit the DAT have been shown to cause animals to self administer have been shown to cause animals to self administer these agents, and to develop a conditioned place these agents, and to develop a conditioned place preference (CPP) for these drugs. preference (CPP) for these drugs.

Evidence supporting DA’s role in Evidence supporting DA’s role in reward (cont.):reward (cont.):

Bilateral 6-OHDA lesions result in a severe decrease Bilateral 6-OHDA lesions result in a severe decrease of activity, and the animals will refuse to eat or drink of activity, and the animals will refuse to eat or drink (an obliteration of the natural reward cues). (an obliteration of the natural reward cues).

Schultz et al. showed that the anticipation of a reward Schultz et al. showed that the anticipation of a reward (juice) in monkeys caused an increase in firing, and a (juice) in monkeys caused an increase in firing, and a change in the pattern of DA neuron firing.change in the pattern of DA neuron firing.

Maldonado et al. showed that D2 receptor knock out Maldonado et al. showed that D2 receptor knock out mice do not show a CPP in response to morphine.mice do not show a CPP in response to morphine.

Volkow et al. used neuroimaging studies in humans Volkow et al. used neuroimaging studies in humans to show that cocaine and methylphenidate increase to show that cocaine and methylphenidate increase brain dopamine levels, and this increase was brain dopamine levels, and this increase was associated with the feeling of a “high.”associated with the feeling of a “high.”

Molecular Mechanism of Molecular Mechanism of Drug Addiction:Drug Addiction:

(Nestler and Aghajanian, 1997)

Brain regions involved in drug Brain regions involved in drug addiction:addiction:

(Golstein and Volkow, 2002).

(Volkow et al, 2003)

(Golstein and Volkow, 2002).

What makes some people become What makes some people become substance dependent?substance dependent?

Hypothesis:Hypothesis:

Dopamine is not an essential Dopamine is not an essential component of opiate responses, component of opiate responses,

and that dopamine is not required and that dopamine is not required for opioid mediated reward.for opioid mediated reward.

Methods:Methods: Dopamine deficient miceDopamine deficient mice: a : a

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

severely hypoactive, adipsic, and aphagic. Cell 83:1197-1209.

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Synthesis of Dopamine:

Methods (cont.):Methods (cont.): Mice required daily L-Dopa administration to induce them Mice required daily L-Dopa administration to induce them

to eat. Morphine was administered 18-24 hrs after L-Dopa .to eat. Morphine was administered 18-24 hrs after L-Dopa . Virally Rescued Dopamine Deficient Mice (vrDD):Virally Rescued Dopamine Deficient Mice (vrDD): In In

order to perform the behavioral tests, they used a viral gene order to perform the behavioral tests, they used a viral gene transfer to restore DA in the striatum (because DA deficient transfer to restore DA in the striatum (because DA deficient mice are slow and hypoactive).mice are slow and hypoactive).

Behavioural tests:Behavioural tests: 1. Locomotor tests were done using photo-beam activity 1. Locomotor tests were done using photo-beam activity cages. Morphine was administered IP at 0,0.25,2.5,12.5, cages. Morphine was administered IP at 0,0.25,2.5,12.5, and 25 mg/kg.and 25 mg/kg.

2. Tail flick tests were perfomed by using warm water 2. Tail flick tests were perfomed by using warm water baths. Briefly, the animal’s tail was submerged 0.5-1 cm in baths. Briefly, the animal’s tail was submerged 0.5-1 cm in the water bath, and the latency to withdraw the tail was the water bath, and the latency to withdraw the tail was recorded (with a cut off time of 15s). The animals were recorded (with a cut off time of 15s). The animals were tested three times/treatment and average used. Morphine tested three times/treatment and average used. Morphine was administered 30 min. Prior to test IP at 0,3,6,12, and 24 was administered 30 min. Prior to test IP at 0,3,6,12, and 24 mg/kg.mg/kg.

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

(Cami and Farre, 2003).

ResultsResults

Conclusions:Conclusions:

Dopamine appears to be essential in the Dopamine appears to be essential in the development of locomotor response to development of locomotor response to morphine.morphine.

Dopamine appears to play an important role in Dopamine appears to play an important role in the level of analgesia experienced after the level of analgesia experienced after morphine administration.morphine administration.

Dopamine may be required for reward Dopamine may be required for reward seeking, but does not appear to be seeking, but does not appear to be indispensable for morphine’s rewarding indispensable for morphine’s rewarding effects.effects.