Although this study was designed completely in accordance with the 3Rs principles (replacement, refinement and reduction) of the use of animals in research, replacing rats were not possible with nor lower species, neither alternative laboratory techniques. As this study had been planned to evaluate the probable effects of CART peptide on learning and memory processes in the brain, there was no suitable alternative way to gain the same quality of results, and rationalize the benefits of proposed research compared with the costs to the animals. Attributable to complex neuroanatomical organization of the prefrontal cortex and the topographic ordering of cortical and subcortical connections, our current knowledge of reward pathway, primary and secondary reinforcement and the effects of reinforcement on behavioral and neurobiological bases of reward-induced learning and memory formation as well as place preference or aversion all arise significantly from animal research and thus using in vivo methods are still the best available method for gaining meaningful and detailed data about complex cortical and subcortical networks of the reward, learning and memory pathways.
As the second R in the 3Rs principle is Reduction, we tried to use methods which minimize the number of animals used per experiment by calculating the sample size by power analysis. Based on our laboratory previous findings of morphine-induced CPP as well as pilot studies of CART-induced place preference or aversion and also by having enough knowledge and information about effect size, standard deviation, type 1 error, power of a study, statistical tests, expected attrition or death of animals we applied G Power program to calculated the sample size, the exact number of animals which not only led to missing of any significant difference, but also resulted in any prevention from unnecessary wastage of animal and resources.
The third R is Refinement methods which about minimizing the pain, suffering, distress or lasting harm that may be experienced by research animals, and improving their welfare. In our study, refinement were applied to all aspects of animal use, from their housing and husbandry to the scientific procedures performed on them; including ensuring the animals are provided with housing and environmental enrichment that allows the expression of species-specific behaviors, using appropriate anesthesia and analgesic regimes for pain relief, and training animals to voluntarily co-operate with procedures to have greater control over the procedure in order to reduce any distress.
An enormous bulk of experiments has shown the main role of CART peptide in the action of psychostimulants and alcohol. In addition to these studies, some recent experiments have reported the association between the CART peptide and opiate compounds. Some examples may be the increase of the anti-nociceptive effect of morphine by CART  or the action of CART (85–102) in attenuating the expression of chronic morphine sensitization . Upadhya and colleagues have suggested that in the framework of NAc shell, CART 55–102 serves as the final output of the endogenous opioid-mesolimbic-dopamine system . Our previous studies demonstrated that not only the content of CART 55–102 in some reward areas in the rat brain is altered by acute and chronic morphine dependence and abstinence syndrome , but also it changes in the CSF and plasma . Still, relapse is the most important problem of opiate addiction, which is related to drug-induced learning and memory . In the current study, we evaluated the probable interaction between the CART peptide and morphine in producing drug-related learning and memory. Then, we tried to find out whether the NMDA receptor content in the reward system would change in response to the administration of morphine, CART, and their combination. Place conditioning studies have verified that large amounts of neuronal circuits, neurotransmitters, neuromodulators, receptors, and discrete brain sites are involved in the brain reward mechanisms . Mesocorticolimbic system, which initiates from the ventral tegmental area (VTA) and projects to the NAc, limbic system, and many cortical areas, is the most important pathway in the reward process . Our data showed that morphine administration to rats could induce CPP dose-dependently. Numerous previous studies have reported such results. The morphine dose selection in our study was based on what Rezayof and colleagues have reported in 2002 . Since many years ago, numerous studies have been done to clarify the neurotransmitters and neuroanatomical sites, which are involved in producing CPP or CPA. Now it is clear that opioid neuronal system plays an important role in CPP learning and memory . Also, the key role of the NMDA receptor in modulating reward memory reconsolidation, including drug-induced reward and memory has been reported previously [31, 32]. Our IHC results showed that by increasing the dose of morphine, the number of NR1 subunits of the NMDA receptor in all the examined parts of the reward pathway was increased. Considering the essential role of the NAc as a key structure in motivation and memory, Ahmadi and colleagues have demonstrated that systemic administration of morphine may induce passive avoidance memory through NMDA function in the NAc . In another study, it was established that NMDA receptors in the NAc are involved in the reconsolidation of aversive and positive morphine-associated memories [34–36].
Our data also indicated that the intra-NAc shell infusion of the 2.5 µg CART produced CPP, while the higher concentration of the drug (5 µg/side) induced CPA. To the best of our knowledge, there is no previous study similar to our work. There is only one study that has evaluated the ability of intra-basolateral amygdala (BLA) infusion of CART 55–102 (with different doses of 1, 2, or 4 µg/side) to induce conditioned place preference or aversion. The results of the mentioned study are so impressive and are similar to our data. Intra-BLA infusion of aCSF or CART (1 µg/side) has produced neither CPP nor CPA, while 2 µg/side of the drug has induced CPP and 4 µg/side has made CPA . Other studies have reported that intra-VTA administration of CART 55–102 could raise locomotor activity and induce CPP . Also, injection of CART 55–102 into the NAc could decrease the locomotor activity induced by the systemic infusion of cocaine and D-amphetamine. However, it has no effect on locomotor activity when it is administered alone . Also, in CART null mice, the effect of D-amphetamine in producing CPP was attenuated. The authors proposed a modulatory role for CART peptide in locomotor activity and other affective behaviors related to the D-amphetamine administration . Our results showed that different doses of CART 55–102 (infused in the NAc shell) have rewarding or aversive effects. Furthermore, our findings also revealed that administration of CART 55–102 into the NAc shell had no significant effect on locomotor activity. Previous studies have established that the NAc shell does not play a significant role in locomotor activity . Previous studies have reported that not only CART mRNA and peptides are found in several distinct brain nuclei including the NAc, a site with a robust dopamine input, but also dopamine receptors are found on CART neurons . Considerable evidence exists showing that tyrosine hydroxylase-positive nerve terminals synapse on CART peptide-containing neurons in the NAc [16, 43]. It seems that CART, released from the axonal terminals in the framework of NAc-shell, may be the final output of the endogenous mesolimbic-dopamine circuitry that processes reward . Injection of CART 55–102 peptide into the ventricles leads to the increased turnover of Dopamine . The existing data propose that in the NAc, CART 55–102 peptide functions as a homeostatic regulator of dopamine-mediated activity . In other words, as dopamine signaling or its activity in the NAc becomes high, CART peptide is released to oppose the actions of dopamine by acting like an inhibitory neurotransmitter. On the other hand, when dopamine activity is low, CART peptide should be a less inhibitory or even an excitatory molecule. Several studies have reported that the exogenously injected CART peptide may simulate the effects of endogenously released peptide. Job has suggested that the effects of CART peptide are fully dependent on the conditions it acts in it. He has claimed that under certain settings, intra-NAc CART peptide may have no effect or enhance/facilitate some dopamine-induced behavioral effects . Our results were highly compatible with this suggestion. Moreover, it seems that a reciprocal interaction between dopamine and CART peptide exists. We hypothesize that while the level of CART peptide is increased by exogenous injection of the peptide into the NAc-shell, the dopamine level is raised and CPP is induced, but this effect completely depends on the dose of the injected CART peptide. Exogenous CART in high doses not only impairs the acquisition of CPP, but also results in driving aversion. Therefore, it seems that without a comprehensive systematic understanding of the CART peptide effects, it is challenging to guess the influence of CART peptide in low dose or high dose in any given brain structure and further studies are needed to fully realize how CART peptide actually acts.
Various studies have shown the association between the CART peptide and NMDA. One study has demonstrated that CART 55–102 could increase the levels of phosphoserine 896 and 897 on the NR1 subunit of the NMDA receptor through protein kinase A and C (leading to the elevation of NMDA function) . Also, it was revealed that the NMDA-mediated nociception in the spine is potentiated by CART administration via protein kinase A and C signaling pathways . CART may be able to stimulate the sympathetic neural axis both directly and indirectly by potentiating the glutamate action on NMDA receptor . Besides, it has been reported that CART may facilitate NMDA-mediated currents in the central amygdala . All these studies show the possibility of the existence of an interaction between CART peptide and NMDA glutamate receptor. Based on these studies and the close impact of NMDA on memory induction, we can propose the probability of CART role in the induction of CPP and CPA dose-dependently.
Our data also showed that the intra-NAc shell infusion of a dose of CART that produced neither CPP nor CPA plus an ineffective dose of morphine induced CPP. It is supposed that the CART peptide could facilitate morphine reward through the NAc shell. Endogenous CART in the framework of reward anatomical regions may be an essential element for the opioid-mesolimbic-dopamine structures and the action of drugs of abuse. CART peptide secreting neurons have been discovered in the NAc. However, the exact cellular mechanisms of CART to modify drug-induced reward are not fully understood [13, 16, 51].
To our knowledge, this is the first study evaluating the possible interaction between opioid and CART in the NAc shell framework and evaluation of the NMDA involvement in reward or aversion-induced learning and memory caused by opioid and CART interaction. In our previous studies, we tried to show the homeostatic role of endogenous CART circuitry as a mediator in the action of morphine . In the brain reward system, activation of mu opioid receptors changes the level of dopamine formation, release, and diffusion in the synaptic space. In the NAc shell, administration of a mu opioid receptor agonist elevates drug consumption, seeking behavior , and conditioned reinforcement by enhancing the drug motivational properties . According to Upadhya study, it seems that in the NAc shell, CART peptide acts downstream to dopamine and mediates the morphine-induced reward and reinforcement . According to all discussed above, it is proposed that both morphine and CART have rewarding effects and when they are administered together, enforce each other via a final common pathway (dopamine). Data analysis of IHC staining showed a higher quantity of the NR1 subunit in all examined areas. As mentioned above, place preference is a good behavioral test for evaluating drug-induced learning and memory. It is not far from the mind that the level of NMDA in some parts of the reward pathway was elevated because NMDA has a potential role in memory formation .
In order to discuss whether increased NR1 synthesis is indicative of increased NR1 functional activity or not, we have to say that most NMDA receptors in the brain are heteromeric complexes with NR1 as constructive and NR2 (A–D) as functional subunits which increase the NMDA receptor-mediated current. The NR1 subunit serves as an important subunit critical for ion selectivity and agonist binding of the NMDA receptors . In our study, while morphine treatment induced dose-dependent CPP, the amount of NR1 subunit increased in several regions of the reward pathway. Moreover, when intra-NAc shell microinjection of 2.5 µl/side CART produced CPP, the level of NR1 immunopositive neurons increased in all regions of study. It was also observed that place-aversion learning with 5 µl/side intra-NAc shell infusion of CART peptide was accompanied by a significant decrease in NR1 subunit immunoreactivity in the reward pathway. Thus, according to the crucial role of NMDA receptors in the expression of drug-induced CPP or CPA, developing several forms of learning and memory, and the important structural role and pharmacological characteristics of NR1 subunit protein in the NMDA receptor building and function, it seems that increased NR1 synthesis can be indicative of increased NR1 functional activity. Confirmation of this hypothesis should be assessed by further studies in future.
Another question that may come to mind is whether there is precedent for learning processes to be accompanied by increased NR1 synthesis. There are a number of studies demonstrating that several forms of learning and memory are accompanied by increased NR1 synthesis. Cui and colleagues showed that transient NR1 knockout at the time of learning impaired consolidation and storage of nondeclarative taste memory . It was also demonstrated by Kalev-Zylinska and colleagues that knocking down of the NR1 subunit causes learning impairment, and NR1 overexpression increases fear learning . Furthermore, using striatal NR1-knockout mice, it was shown that striatal-specific deletion of NR1 subunit of NMDA receptors disrupts the development of start/stop activity and impairs sequence learning . Using RNA interference in the honeybee brain during and shortly after appetitive learning, it was indicated that acute disruption of the NMDA receptor subunit NR1 selectively impairs learning as well as mid-term and early long-term memory formation . Moreover, a study in 2009 found that in the Morris water maze test, where identifying the location of an escape platform depends on visual cue-dependent learning, NR1DATCre mice showed a significant longer latency to locate the platform . Furthermore, Ping Li and co-workers found that high-intensity ultrasound irradiation could decrease learning and memory abilities by reducing the expression of NR1 and NR2B in the hippocampal regions and damaging the structure of synapses. In contrast, low-intensity ultrasound irradiation can enhance the learning and memory abilities of the offspring rats by increasing the expression of NR1 and NR2B in the hippocampal regions . In addition, it was found in clinic that a patient with Multiple Sclerosis who developed severe cognitive impairments like learning and memory problems had intrathecal antibodies against the NR1 subunit of the NMDA receptor .
Our data also showed that CART-induced CPA could abolish the morphine CPP. It seems that CART 55–102 with aversive dose acts on NAc shell circuitry to modify the morphine reward and reinforcement (related to drug induced-learning and memory). In agreement with our data, Rademacher and colleagues have shown that intra-basolateral amygdala administration of an aversive dose of CART 55–102 before the systemic administration of a rewarding dose of amphetamine produced neither CPP nor CPA. They proposed that CART in an aversive dose acts on basolateral amygdala circuitry to block amphetamine reward . Brain-imaging studies have identified NAc and the amygdala as putative neuroanatomical regions involved in drug conditioning . The amygdala-accumbens pathway modulates drug-induced learning and memory through several neurotransmitters in this circuitry like dopamine . Based on the results of these studies, it was predictable that we get such results because amphetamine and morphine act via dopaminergic pathway and CART acts downstream to dopamine.
In our study, although the levels of the NR1 subunit in the NAc and hippocampus were reduced meaningfully in comparison to aCSF + morphine group (5 mg/kg), no significant changes were observed in the PFC. We have previously reported that the PFC has the lowest amount of CART peptide compared to the NAc, striatum, and hippocampus. Furthermore, it was much less influenced than the other sites by acute and chronic morphine administration and also acute naloxone injection to addicted rats . This may be a hypothesis for the lack of an apparent change in NR1 expression in the PFC in comparison to two other sites. Although we did not find direct evidences in the literature in support of our hypothesis, it has been claimed that exogenously injected CART peptide is able to selectively increase NMDA receptor-mediated glutamatergic neurotransmission . Altogether, it can be proposed that the NMDA receptor may interact with the CART peptide in potentiating or inhibiting morphine-induced reward, reinforcement and place conditioning. Further studies are needed in this regard to evaluate the effect of CART peptide administration on NR1 subunit expression in NMDA receptor.