Elsevier

Brain Research

Volume 1540, 2 December 2013, Pages 1-8
Brain Research

Research Report
Morphine could increase apoptotic factors in the nucleus accumbens and prefrontal cortex of rat brain's reward circuitry

https://doi.org/10.1016/j.brainres.2013.09.045Get rights and content

Highlights

  • Apoptotic factors were increased after development of morphine rewarding effects.

  • Morphine increased the Bax/Bcl2 ratio, caspase3 and PARP levels in the NAc.

  • Morphine increased the Bax/Bcl2 ratio, caspase3 and PARP levels in the PFC.

  • The maximum change in apoptotic factors appeared with low dose morphine in the NAc.

  • The maximum change in apoptotic factors appeared with effective morphine in the PFC.

Abstract

The nucleus accumbens (NAc) and prefrontal cortex (PFC) are two parts of neuronal reward circuit involved in motivated and goal-directed behaviors. Some data suggest that morphine is toxic to neurons and induces apoptosis, while other evidence shows that morphine could have beneficial effects against cell death. This study was designed to evaluate the effect of morphine on apoptosis by measuring the expression of apoptotic proteins in two important regions, the NAc and PFC, in the rat brain's reward circuitry. Morphine subchronic administration in different doses (0.5, 5 and 10 mg/kg) in conditioned place preference (CPP) paradigm (3 times in 3 days, for each dose in each group of rats) was used to induce its rewarding effect. Then, the expression of four apoptotic factors; Bax, Bcl2, caspase3 and PARP, in the NAc and PFC were assessed using the Western blot technique. All of morphine-treated groups showed increase of apoptotic factors in these regions. In the NAc, morphine significantly increased the Bax/Bcl-2 ratio, caspase3 and PARP in the lowest dose (0.5 mg/kg) but in the PFC considerable increase was seen in dose of 5 mg/kg. Elevation of apoptotic factors in the NAc and PFC implies that morphine can affect the molecular mechanisms which interfere with apoptosis through different receptors. Our findings suggest that the NAc and PFC may have a different distribution of receptors which become active in different doses of morphine.

Introduction

Morphine, well known extract of opium poppy, has become one of the most potent pain relievers. Morphine also activates the brain's reward circuitry. The ability of morphine to strongly activate brain reward mechanisms and its ability to chemically alter the normal functioning of these systems can produce morphine dependency (Zhang and QCaL, 2008). Nucleus accumbens (NAc) is a critical element of the mesocorticolimbic system, a brain circuit implicated in reward and motivation. This basal forebrain structure receives glutamatergic inputs from regions including prefrontal cortex (PFC), amygdala (AMG), and hippocampus (HIP) (Carlezon and Thomas, 2009), and receives the dopaminergic inputs from the ventral tegmental area (VTA) of the midbrain. Lots of evidences support the involvement of VTA dopaminergic system in drug induced reward as well as natural rewards (Nestler and Carlezon, 2006). Morphine can modulate cellular functions through modulation of molecular mechanisms. Until recently, little was known about the changes in molecular mechanisms underlying morphine effects.

Morphine may act as the modulator of cell proliferation and cell death. It has been shown that opioids can protect astrocytes from apoptosis triggered by apoptosis promoting agents (Kim et al., 2001), delay neuronal death in the avian ciliary ganglion (Meriney and Gray, 2001), and promote the growth of tumor cells (Gupta et al., 2002, Ishikawa et al., 1993, Moon, 1988). On the other hand, opioids have also been demonstrated to induce apoptosis of immunocytes (Singhal et al., 1997, Singhal et al., 1998), cancer cells (Hatzoglou et al., 1996, Tegeder et al., 2003), neuroblastoma cells such as SK-N-SH, NG108-15 and PC12 cells (Kugawa et al., 1998, Oliveira et al., 2003, Yin et al., 1997) and neuronal cells (Boronat et al., 2001, Mao et al., 2002), as well as human microglia (Hu et al., 2002). Accumulating evidence has demonstrated that opiates can cause apoptosis and cell injury of neuronal cells and other cells such as immunocytes (Singhal et al., 2001, Wang et al., 2001) and cancer cells (Yoshida et al., 2000).

In the molecular mechanisms of apoptosis various key proteins are involved in the regulation of programmed cell death (Kinloch et al., 1999, Sastry and Rao, 2000). Some members of the Bcl-2 family of proteins, such as Bcl-2 and Bcl-xL, suppress apoptosis, while the expression of others, such as the homologs Bax and Bak, are pro-apoptotic (Adams and Cory, 2007). Hochenbery et al. (1990) showed that the Bcl-2 oncoprotein, localized mainly to the mitochondrial membranes and prevents the release of cytochrome c (induced by Bax) play an important role in protecting neurons from apoptosis. Signals from either intrinsic or extrinsic agents can trigger apoptotic cascade; activation of caspases which degrade their cellular targets until cell dies (Garcia-Fuster et al., 2007, Hatzoglou et al., 1996, Singhal et al., 1999, Yin et al., 1997). The activation of effecter caspases such as caspase-3 leads to downstream cleavage of various cytoplasmic or nuclear substrates including PARP. These downstream cleavage events mark many of the morphological features of apoptotic cell death (Huppertz et al., 1999). Other studies show that different addictive drugs, such as heroin, can induce apoptosis in several cultured cell lines. Heroin induces mitochondrial malfunction, caspase activation, poly-ADP ribose polymerase (PARP) cleavage, and DNA fragmentation in PC-12 cells (Meriney and Gray (2001)).

According to the above mentioned studies, we set out to determine whether morphine would affect apoptosis in the nucleus accumbens and the prefrontal cortex by assessing the apoptotic proteins involved in the regulation of programmed cell death Bcl-2 (anti-apoptotic), Bax (pro-apoptotic), Caspase-3 and PARP in rats.

Section snippets

Behavioral test

In conditional place preference paradigm as a behavioral test, our results showed that administration of morphine in conditioning phase could induce place preference (Fig. 1). One-way ANOVA followed by Dunnett’s test [F(3,23)=15.33, P<0.0001] revealed that there were significant differences in conditioning scores among the vehicle (saline control group) and experimental groups that received morphine in 5 and 10 mg/kg. The most effective dose of morphine was 5 mg/kg (P<0.01). On the other hand,

Discussion

The present study demonstrated that administration of morphine dose-dependently induces reward and changes the level of apoptotic factors in the brain structures involved in drug reward. The major findings of this study were (i) significant increase in the amount of apoptotic factors in the NAc and PFC after application of morphine during conditioned place preference model and (ii) the most effective doses of morphine on alterations of Bax/Bcl-2 ratio, caspase-3 and PARP were 0.5 and 5 mg/kg in

Animals

Thirty-two adult male Wistar rats, (Pasteur Institute, Tehran, Iran) weighing 220–320 g, were used in these experiments. Animals were housed in groups of three per cage in a 12/12 h light/dark cycle (lights on between 7:00 AM and 7:00 PM) with free access to chow and tap water. The animals were randomly allocated to different experimental groups. Rats were habituated to their new environment and handled for 1 week before the experimental procedure was started. All experiments were executed in

Acknowledgments

This study was supported by the grant (No. 90-821-A) from Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

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