Morphine-conditioned Mice Research Articles

Ketamine Blocks Morphine-Induced Conditioned Place Preference and Anxiety-Like Behaviors in Mice.

Patients suffering from opioid use disorder often relapse during periods of abstinence, which is posited to be caused by negative affective states that drive motivated behaviors. Here, we explored whether conditioning mice with morphine in a conditioned place preference (CPP) training paradigm evoked anxiety-like behavior during morphine abstinence. To do this, mice were conditioned with morphine (10 mg/kg, i.p.) for 5 days. Twenty-four hours following conditioning, anxiety levels were tested by measuring time in the open arms of the elevated plus-maze. The next day, mice were placed in the three-compartment chamber to measure morphine-induced CPP. Our results show that following morphine conditioning, mice spent significantly less time in the open arm of the elevated plus-maze and expressed robust morphine CPP on CPP test day. Furthermore, we found that an acute treatment with (R,S)-ketamine (10 mg/kg, i.p.), a medication demonstrating promise for preventing anxiety-related phenotypes, 30 min before testing on post-conditioning day 1, increased time spent in the open arm of the elevated plus-maze in saline- and morphine-conditioned mice. Additionally, we found that the second injection of ketamine 30 min before CPP tests on post-conditioning day 2 prevented morphine-induced CPP, which lasted for up to 28 days post-conditioning. Furthermore, we found that conditioning mice with 10% (w/v) sucrose using an oral self-administration procedure did not evoke anxiety-like behavior, but elicited robust CPP, which was attenuated by ketamine treatment 30 min before CPP tests. Overall, our results suggest that the ketamine-induced block of morphine CPP may not be attributed solely to alleviating negative affective states, but potentially through impaired memory of morphine-context associations.

Differential effects of aprepitant, a clinically used neurokinin-1 receptor antagonist on the expression of conditioned psychostimulant versus opioid reward.

Neurokinin-1 receptor (NK1R) signaling modulates behaviors associated with psychostimulants and opioids. Psychostimulants, such as amphetamine (AMPH) and cocaine, bind to monoamine transporters and alter their functions. Both dopamine and norepinephrine transporters are regulated by NK1R activation suggesting a role for NK1R mediated catecholamine transporter regulation in psychostimulant-mediated behaviors. The effect of in vivo administration of aprepitant (10mg/kg) on the expression of AMPH (0.5 and 2mg/kg) and cocaine (5 and 20mg/kg)-induced conditioned place preference (CPP) as well as locomotor activation was examined in C57BL/6J mice. The effect of aprepitant on morphine (1 and 5mg/kg)-induced CPP was also examined to identify the specific actions of aprepitant on psychostimulant versus opioid-induced behaviors. Aprepitant administration significantly attenuated the CPP expression and locomotor activation produced by AMPH and cocaine. In contrast, aprepitant significantly enhanced the expression of CPP produced by morphine while significantly suppressing the locomotor activity of the mice conditioned with morphine. Aprepitant by itself did not induce significant CPP or conditioned place aversion or locomotor activation or suppression. Attenuation of AMPH or cocaine-induced CPP and locomotor activation by aprepitant suggests a role for NK1R signaling in psychostimulant-mediated behaviors. Stimulation of morphine-induced CPP expression and suppression of locomotor activity of morphine-conditioned mice suggest differential effects of NK1R antagonism on conditioned psychostimulant versus opioid reward. Collectively, these findings indicate that clinically used NK1R antagonist, aprepitant may serve as a potential therapeutic agent in the treatment of psychostimulant abuse.

Acquisition of morphine conditioned place preference increases the dendritic complexity of nucleus accumbens core neurons

Contexts associated with opioid reward trigger craving and relapse in opioid addiction. Effects of reward-context associative learning on nucleus accumbens (NAc) dendritic morphology were studied using morphine conditioned place preference (CPP). Morphine-conditioned mice received saline and morphine 10 mg/kg subcutaneous (s.c.) on alternate days. Saline-conditioned mice received saline s.c. each day. Morphine-conditioned and saline-conditioned groups received injections immediately before each of eight daily conditioning sessions. Morphine homecage controls had no CPP training, but received saline and morphine in the homecage concomitantly with the morphine-conditioned group. Morphine conditioning produced greater place preference than saline conditioning. Mice were sacrificed 1 day after CPP expression. Dendritic changes were studied using Golgi-Cox staining and digital tracing of NAc core and shell neurons. In the NAc core, morphine homecage administration increased spine density, while morphine conditioning increased dendritic complexity, as defined by increased dendritic count, length and intersections. Place preference positively correlated with dendritic length and intersections in the NAc core. The core may mediate reward consolidation and determine how context-related signals from the shell lead to motor behavior. The combination of drug and conditioning in the morphine-conditioned group produced unique morphological effects different from the effects of drug or conditioning procedures by themselves. An additional study found no differences in neuron morphology between saline-conditioned mice, trained as described earlier, and mice that were not conditioned, but received saline in the homecage. The unique effect of morphine reward learning on NAc core dendrites reflects a brain substrate that could be targeted for therapeutic intervention in addiction.

Increase of Type 2 Ryanodine Receptors in Mouse Nucleus Accumbens in the Development and Expression of Morphine-Induced Place Preference

The present study investigated the effect of ryanodine receptors (RyRs) in the development and expression of morphine-induced conditioned place preference (CPP). Type 2 RyRs (RyRs-2) in the nucleus accumbens (NAcc) significantly increased in morphine-conditioned mice, whereas type 1, 2, and 3 RyRs in the frontal cortex and ventral tegmental area showed no changes. Intracerebroventricular pretreatment with dantrolene, a RyRs antagonist, during the conditioning phase of CPP, dose-dependently inhibited morphine-induced CPP. The expression of morphine-induced CPP was abolished by dantrolene administration before the post-conditioning test. These findings suggest that RyRs-2 in the NAcc participate in the development and expression of morphine-induced CPP.

Affective taste responses in the presence of reward- and aversion-conditioned stimuli and their relationship to psychomotor sensitization and place conditioning

Anecdotal experience and empirical evidence suggest animals approach or avoid conditioned stimuli based on the ability of those stimuli to elicit affective responses or interfere with affective assessments of ongoing stimuli. Thus, this study investigated the relationship between the ability of drug-conditioned environments to induce conditioned place preference or aversion and their ability to influence palatability responses to sucrose and quinine in those same environments. Mice were conditioned to methamphetamine (2mg/kg), morphine (10mg/kg) or naloxone (10mg/kg). Following testing for the expression of place conditioning, palatability responses to sucrose and quinine in the conditioned contexts were assessed. In general, virtually no effects of exposure to drug-conditioned contexts on overall positive or aversive palatability responses were observed. However, in naloxone-conditioned mice, the strength of conditioned place aversion to the naloxone-paired context correlated with aversive taste reactivity responses to quinine in that context. In morphine-conditioned mice, positive reactions to sucrose in the morphine-paired context negatively correlated with positive reactions to sucrose in the vehicle-paired context. Interestingly, the rate of methamphetamine-induced behavioral sensitization during conditioning and positive taste responses to sucrose in the methamphetamine-paired context positively correlated. These studies suggest that conditioned stimuli interact with or modulate the affective experience of ongoing unconditioned stimuli such as tastants, and these may reflect behavioral processes that guide behavior optimally.

Role of the calcium/calmodulin-dependent protein kinase ii (CaMKII) in the morphine-induced pharmacological effects in the mouse

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a family of multifunctional protein kinases that activates signaling pathways. The present study was designed to ascertain whether CaMKII could play a substantial role in the expression of morphine-induced antinociception, hyperlocomotion and rewarding effect in the mouse. An i.c.v. pretreatment with a CaMKII inhibitor KN-93 failed to affect the antinociception and hyperlocomotion induced by s.c. administration of a prototype μ-opioid receptor agonist morphine. In contrast, the morphine-induced place preference was significantly attenuated by i.c.v. pretreatment with KN-93. The levels of phosphorylated-CaMKII (p-CaMKII) in the limbic forebrain, but not in the frontal cortex and the lower midbrain, were significantly increased in morphine-conditioned mice, whereas the levels of CaMKII in three brain regions obtained from morphine-conditioned mice were not changed. This up-regulation of p-CaMKII in the limbic forebrain obtained from morphine-conditioned mice was significantly inhibited by i.c.v. pretreatment with KN-93. These results provide evidence that the increase in CaMKII activity in the mouse limbic forebrain may contribute to the rewarding effect, but not the antinociception and the hyperlocomotion, induced by morphine.

Involvement of protein kinase Cγ isoform in morphine-induced reinforcing effects

The present study was designed to investigate the role of protein kinase C (PKC) isoform in the morphine-induced reinforcing effect in mice. An intracerebroventricular injection of calphostin C, a specific PKC inhibitor, produced a dose-dependent reduction in the morphine-induced place preference. The protein level of PKCγ was significantly up-regulated in membrane preparations of the limbic forebrain obtained from the morphine-conditioned mice compared to that from the saline-conditioned mice. However, the protein levels of PKCα, βI, βII and ε were not affected in the same preparation. By contrast, there were no changes in the protein level of all five PKC isoforms in the lower midbrain. Furthermore, we investigated the rewarding properties of morphine in mice lacking PKCγ gene. A significant place preference was observed following treatment with morphine in wild-type mice, whereas such an effect of morphine was not found in PKCγ knockout mice. These findings suggest that activated PKCγ in the limbic forebrain following the treatment with morphine may be critical for the development and/or maintenance of reinforcing effects induced by morphine in mice.

Mechanisms of morphine-induced rewarding effect: involvement of NMDA receptor subunits

The glutamate receptor contributes to excitatory synaptic transmission in the central nervous system and plays an important role in memory acquisition, learning and neurological disorders. Molecular cloning studies have revealed that NMDA receptors consist of two families, the NR1 and NR2A-NR2D subunits, and NMDA receptors are thought to be pentameric or tetrameric complexes of the NR1 subunit with one or more of the NR2 subunits. It has been proposed that NMDA receptors are implicated in the development of opioid dependence. The non-selective NMDA receptor antagonist dizocilpine has been shown to suppress not only physical but also psychological dependence produced by morphine. An intracerebroventricular (i.c.v.) treatment with a specific antibody against the carboxyl-terminal region of the NR2B subunit abolishes the morphine-induced place preference, whereas antibodies against the NR1 and NR2A subunits do not affect the rewarding effect of morphine, indicating that the blockade of the NR2B subunit suppresses the development of the morphine-induced rewarding effect. Under these conditions, the NR2B subunit protein is up-regulated in the limbic forebrain of morphine-conditioned mice. These findings suggest that the NMDA receptor, especially NR2B subunit, is an important modulator of the development and/or expression of psychological dependence on morphine.

Molecular evidence for the involvement of NR2B subunit containing N-methyl- d-aspartate receptors in the development of morphine-induced place preference

The present study was designed to clarify the role of the NR1, NR2A and NR2B subunits of N-methyl- d-aspartate receptors in the development of morphine-induced place preference using specific antibodies to N-methyl- d-aspartate receptor subunits in the mouse. An i.c.v. injection with specific antibodies against the carboxyl-terminal region of either NR1, NR2A or NR2B subunits caused the attenuation of seizures induced by i.v. infusion of N-methyl- d-aspartate in the mouse. Using western blotting, NR1, NR2A and NR2B subunits were found to be highly expressed in the cerebral cortex and hippocampus of the mouse brain, which are key areas in producing seizures regulated by N-methyl- d-aspartate receptors. These findings indicate that all three subunits of the N-methyl- d-aspartate receptor in these areas are likely to be critical for the seizure induced by i.v. infusion of N-methyl- d-aspartate. Furthermore, present data provide evidence that these antibodies when given into the brain specifically act on the target sites, resulting in the blockade of the respective N-methyl- d-aspartate receptor subunit. Under these conditions, i.c.v. treatment with the antibody against NR2B subunits abolished the morphine-induced place preference, whereas antibodies against NR1 and NR2A subunits did not affect the rewarding effect of morphine. Furthermore, the NR2B subunit protein was specifically up-regulated in the limbic forebrain of morphine-conditioned mice, whereas the protein levels of either NR1 or NR2A subunits were not altered. From these results, we suggest that the stimulation of the carboxyl-terminal region of NR2B subunits in the limbic forebrain may contribute to the development of morphine-induced rewarding effect.