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Ethanol and 3, 4-Methylenedioxymethamphetamine (MDMA) are popular recreational drugs widely abused by adolescents that may induce neurotoxic processes associated with behavioural alterations. Adolescent CD1 mice were subjected to ethanol intake using the drinking in the dark (DID) procedure, acute MDMA or a combination. Considering that both drugs of abuse cause oxidative stress in the brain, protein oxidative damage in different brain areas was analysed 72 h after treatment using a proteomic approach. Damage to specific proteins in treated animals was significant in the hippocampus but not in the prefrontal cortex. The damaged hippocampus proteins were then identified by mass spectrometry, revealing their involvement in energy metabolism, structural function, axonal outgrowth and stability, and neurotransmitter release. Mice treated with MDMA displayed higher oxidative damage than ethanol-treated mice. To determine whether this oxidative damage was affecting hippocampus activity, declarative memory was evaluated at 72 h after treatment using the object recognition assay and the radial arm maze. Although acquisition in the radial arm maze was not impaired by ethanol intake, MDMA treatment impaired long-term memory in both tests. Therefore, oxidative damage to specific proteins observed under MDMA treatment affects important cellular function on the hippocampus that may contribute to declarative memory deficits.
Adolescence is a critical developmental period in which the brain emerges from an immature state to adulthood (Spear 2000). Thus, the impact of drug abuse on the adolescent brain might have severe negative consequences. This is also a time when novel experiences involving drugs such as ethanol or psychostimulants may be sought. Many teens view risky behaviours as exciting and rewarding, ignoring any negative health consequences (Crews et al. 2007). Several reports indicate that consumption of ethanol and the psychostimulant 3, 4-Methylendioxymethampehtamine (MDMA or ecstasy) are common among adolescence and young adults (Barrett et al. 2006). It is well known that both ethanol and MDMA cause neuroinflammation and neurotoxicity in brain areas, including the prefrontal cortex, striatum and hippocampus (Izco et al. 2006; Rodríguez-Arias et al. 2011). Thus, both types of drug abuse, alone or in combination, induce glial activation (Vallés et al. 2004; Ros-Simó et al. 2012), increase reactive oxygen species and oxidative damage (Busceti et al. 2008; Alves et al. 2009; Rump et al. 2010), and proinflammatory cytokines release (Connor et al. 2001; Qin et al. 2008). Furthermore, MDMA induces neuronal terminal loss (Touriño et al. 2010). In those who consume alcohol and MDMA, these neurotoxic processes can lead to neurodegeneration in specific brain regions that may alter a variety of cognitive and performance tasks, including memory and learning processes (Fadda and Rossetti 1998; Parrott 2001). Cognitive dysfunctions are very prominent in several neuropsychiatric disorders and often diminish patients' quality of life (Millan et al. 2012). Several investigations have studied the effects of alcohol and MDMA on memory acquisition (Able et al. 2006; Kay et al. 2011) or short-term memory (Brooks et al. 2002; García-Moreno et al. 2002). However, little is known about the effects of these drugs on memory consolidation and long-term memory. The length of exposure to the drug and the pattern of consumption that will result in neurotoxicity and cognitive alterations remains a matter of debate.
Numerous studies have established that oxidative damage occurs in brain after ethanol and MDMA treatment in rodents and non-human primates (Busceti et al. 2008; Alves et al. 2009; Rump et al. 2010; Collins and Neafsey 2012), but little has been published about the specific oxidatively damaged proteins. Damage that negatively affects protein function can be measured by detecting carbonyl formation on amino acid side-chains (Levine et al. 1994; Tamarit et al. 2012). Therefore, identification of these proteins after treatment with ethanol, MDMA or both in brain areas involved in cognitive functions (Morris et al. 1982) can provide useful data to better understand how these impaired protein functions can affect these brain areas.
To test whether brain areas involved in cognitive functions become altered after ethanol and MDMA treatments, we carried out declarative memory tasks, which rely on the potentially affected areas. One of the tasks was the object recognition test, involving the hippocampus and the adjacent perirhinal cortex; which are strongly interconnected (Wixted and Squire 2011). The other task was the radial arm maze, which involves different aspects of spatial reference and working memory, involving the hippocampus and the prefrontal cortex respectively. In both tasks, memory consolidation was evaluated.
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Under our experimental conditions, both ethanol intake and acute MDMA-induced damage to specific hippocampus proteins in CD1 mice. MDMA-induced damage was more prominent than ethanol-induced effects, particularly to proteins related to structural functions and to outgrowth and stability. Indeed, an acute neurotoxic dose of MDMA affected consolidation of declarative memory in two different paradigms. Under this pattern of ethanol consumption, animals did not exhibit significant memory deficits compared to controls. The MDMA-induced memory impairments could be related to the specific oxidatively damaged hippocampus proteins.
Previous studies have revealed a clear involvement of oxidative stress in the neurotoxicity induced by MDMA and ethanol consumption (Alves et al. 2009; Rump et al. 2010). These studies led us to hypothesize that ethanol and MDMA could induce protein oxidation in discrete brain areas, and these changes could be associated with cognitive impairment. Therefore, our aim in this study was to identify oxidatively damaged proteins in different brain areas involved in learning and memory processes, specifically the hippocampus and prefrontal cortex, of mice treated with ethanol and/or acute MDMA. Oxidatively damaged proteins are known to be one of the most important causes of brain protein damage and dysfunction (Berlett and Stadtman 1997). Protein carbonyl formation is one of the most studied markers resulting from oxidative stress and can be easily detected and quantified (Levine et al. 1994; Tamarit et al. 2012). Under our experimental conditions, proteins involved in a variety of cellular functions were found oxidatively modified in hippocampus but not in prefrontal cortex (data not shown) by intake of ethanol, MDMA or its combined treatment. These included α-enolase, glyceralehyde-3P-dehydrogenase, aconitate hydratase, α and β subunit of the ATP synthase, CRMP-2, actin, α-internexin, synapsin-1 and HSC 71. Carbonylation, and thus, inactivation of proteins related to energy metabolism may contribute to an energy deficiency associated with drug use. This result could explain the observation that MDMA decreases brain ATP production, leading to membrane ionic dysregulation, calcium entry and additional free radical formation (Darvesh and Gudelsky 2005). In addition, carbonylation of proteins related to energy metabolism has been found in brains affected by Huntington's (Sorolla et al. 2010), Parkinson's (Malkus et al. 2009) or Alzheimer's (Castegna et al. 2002) neurodegenerative diseases. Furthermore, Castegna and colleagues (Castegna et al. 2002) report that in Alzheimer's disease CRMP-2 and HSC 71 proteins were oxidatively modified in the hippocampus. Moreover, synapsin-1 is involved in neurotransmitters release (Evergren et al. 2007) and hippocampal neuronal development (Fornasiero et al. 2009). Finally, actin and α-internexin (also known as neurofilament 66) are proteins involved in neuronal stability and axonal growth (Levavasseur et al. 1999). Considering the well-established MDMA-induced dopaminergic and serotonergic axon terminal loss (Sprague et al. 1998; Touriño et al. 2010), carbonylation of these proteins could well be involved in this mechanism of neurodegeneration. Our findings support protein oxidation as an additional important contributor to the mechanisms underlying the hippocampal neurotoxicity of ethanol and MDMA. Indeed, some researchers have reported attenuation of ethanol and MDMA-induced neurotoxicity by free radical scavengers and antioxidants (Colado and Green 1995; Crews et al. 2006), providing indirect evidence for the involvement of oxidative damage in the mechanism of ethanol and MDMA neurotoxicity.
To analyse whether this oxidative damage can be connected to a cognitive dysfunction, we evaluated the possible impairment in declarative memory tasks induced by the exposition to ethanol, MDMA and its combined treatment in mice. Under our experimental conditions, acute MDMA but not ethanol-induced memory impairments. No increased memory deficits were observed with the combination of both drugs of abuse indicating that such impairment is clearly related to MDMA administration. Previous studies have reported memory deficits with the administration of either drug alone without an enhancement of the deficits when ethanol and MDMA were administered together (Vidal-Infer et al. 2012).
Memory deficits induced by MDMA treatment were observed in the object recognition test 72 h. post-treatment even after three training days, which was performed to ensure a good acquisition of the task. In addition, in the radial arm maze test, animals treated with the psychostimulant performed significantly more spatial reference memory errors (Fig. 6d) than on the last day of acquisition, before MDMA administration (Fig. 6b, day 12). In contrast, those that did not receive MDMA performed the same number of spatial reference memory errors as the last training day. Several different learning and memory tasks following MDMA have been studied in rodents. For instance, non-spatial memory has been evaluated resulting in memory impairment in MDMA-treated rats (Camarasa et al. 2008). In agreement with our results, rats in the radial arm maze (Kay et al. 2011) or rats and mice in the Morris water maze (Camarasa et al. 2008) showed signs of spatial reference memory impairment without alterations in working memory, which depends more on prefrontal cortex. However, in all these studies animals received the MDMA treatment before the acquisition period; in our procedure animals had already acquired the task before the treatment. Thus, our results indicate that MDMA affects the process of consolidating previous learning. In line with these data, in previous murine studies MDMA impaired acquired tasks in behavioural paradigms, such as operant-delayed alternation task involving working memory but not spatial reference memory (Viñals et al. 2012) or active avoidance performance (Trigo et al. 2008), involving emotional memory.
There is evidence that human alcoholics show deficits in spatial memory tasks (Bowden and McCarter 1993), and similar results have been found in animal models (Kameda et al. 2007). However, some authors do not find such damage after ethanol consumption (Popović et al. 2004). In our study, ethanol consumption did not provoke deficits in the consolidation of any of the tasks evaluated, and no effect was observed in ethanol-treated adolescent animals during the acquisition period in the radial arm maze. These ethanol-induced neural changes and the potential for recovery seem to be dependent on length of ethanol exposure, volume of ethanol, degree of withdrawal signs or number of binge bouts, genetics and age (Crews and Nixon 2009). Thus, a pattern of binge exposure during adolescence impacts the developing brain and induces neural consequences, such as cognitive and behavioural dysfunctions (Guerri and Pascual 2010). However, our animals have low preference for ethanol as previously reported (Ros-Simó et al. 2012), which probably could not be considered as a pattern of binge drinking (Fig. 2). Thus, there is the possibility that the amount of ethanol ingested is not enough to produce alterations on memory consolidation, even though it can lead to long-term emotional-like behaviour alterations (Ros-Simó et al. 2012).
In agreement with this hypothesis, it has been reported that low doses of ethanol (0.5 g/kg) administered 30 min before the first training session on each day for 4 days did not impair spatial learning in rats (Acheson et al. 2001). Although acute ethanol induces memory impairments, after repeated treatment with low doses (0.6 g/kg) a tolerance develops to the amnesic effects (Kameda et al. 2007).
Interestingly, proteins carbonylated in MDMA-treated mice are involved in memory processes. In this context, CRMP-2 plays a critical role in axonal outgrowth and pathfinding through the transmission and modulation of extracellular signals (Fukata et al. 2002). Furthermore, it has been reported to be oxidatively modified in the brain in Alzheimer's disease and has been related to memory loss associated with decreased interneuronal connections and to shortened dendritic length (Coleman and Flood 1987). Other works have reported similarities between hippocampus damage related to memory deficits induced by MDMA abuse and Alzheimer's disease (Busceti et al. 2008). Actin, only carbonylated in MDMA-treated subjects, has a crucial role in cytoskeleton network integrity (Fletcher and Mullins 2010) and is concentrated in dendritic spines where it can produce changes in their shape that might be involved in memory function (Morgado-Bernal 2011). Thus, it could be suggested that both proteins may be involved in the MDMA-induced cognitive impairments observed.
We must take into account that protein carbonyl formation was also analysed in the prefrontal cortex with no significant results. Thus, our findings suggest that the impact of the MDMA-induced oxidative damage to specific proteins is distinctive for hippocampus, the brain area mainly involved in declarative memory (Morris et al. 1982) and where synaptic plasticity associated with the process of learning occurs (Malenka and Nicoll 1999). In addition, MDMA-induced energy dysfunction may enhance the loss of interneuronal connections (Butterfield et al. 2006). Ethanol-treated subjects also exhibited oxidatively damaged proteins, indicating that brain functions might be affected. However, ethanol-treated animals did not display memory deficits suggesting that other alterations may appear as a result of ethanol intake, as previously described (Ros-Simó et al. 2012).
In summary, MDMA but not ethanol intake affects consolidation of declarative memory in adolescent mice. Under this pattern of consumption, ethanol does not seem to affect learning acquisition in mice. The observed oxidative damage to specific proteins in hippocampus, especially those related to axonal and dendritic outgrowth and stability, may contribute to the cognitive deficits observed. However, we cannot discard other alterations, besides memory deficits, as a consequence of oxidative stress. Thus, studies of specific protein damage may provide new insights in understanding the MDMA and ethanol mechanisms of neurotoxicity and its behavioural consequences.