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Methamphetamine (METH) is a psychostimulant drug that causes irreversible brain damage leading to several neurological and psychiatric abnormalities, including cognitive deficits. Neuropeptide Y (NPY) is abundant in the mammalian central nervous system (CNS) and has several important functions, being involved in learning and memory processing. It has been demonstrated that METH induces significant alteration in mice striatal NPY, Y1 and Y2 receptor mRNA levels. However, the impact of this drug on the hippocampal NPY system and its consequences remain unknown. Thus, in this study, we investigated the effect of METH intoxication on mouse hippocampal NPY levels, NPY receptors function, and memory performance. Results show that METH increased NPY, Y2 and Y5 receptor mRNA levels, as well as total NPY binding accounted by opposite up- and down-regulation of Y2 and Y1 functional binding, respectively. Moreover, METH-induced impairment in memory performance and AKT/mammalian target of rapamycin pathway were both prevented by the Y2 receptor antagonist, BIIE0246. These findings demonstrate that METH interferes with the hippocampal NPY system, which seems to be associated with memory failure. Overall, we concluded that Y2 receptors are involved in memory deficits induced by METH intoxication.
Methamphetamine (METH) is a highly addictive psychostimulant that may lead to neurological and psychiatric abnormalities. Indeed, METH abusers show structural brain abnormalities, specifically in the hippocampus and prefrontal cortex, which are associated with cognitive deficits (Thompson et al. 2004; Salo et al. 2009). Similarly, contingent and non-contingent METH regimens lead to impairment of spatial, short- and long-term recognition and perceptual memories in rodents (Cherng et al. 2007; Lee et al. 2011; O'Dell et al. 2011; Reichel et al. 2012). In accordance with these observations, we previously showed that an acute METH administration alters the expression of several rat hippocampal ionotropic glutamate receptor subunits, which seems to be correlated with the hippocampal-dependent memory impairment observed (Simões et al. 2007). It is known that METH induces neuropathology via several mechanisms, including monoaminergic system damage, excitotoxicity, and neuroinflammation (Silva et al. 2010). In fact, METH intoxication triggers hippocampal gliosis and cytokine production (Gonçalves et al. 2008, 2010), together with significant cytoskeleton, synaptic, and axonal protein alterations (Gonçalves et al. 2010), but without evidence of cell death.
Neuropeptide Y (NPY) is a neuromodulator widely distributed in the hippocampus (Gehlert 2004) and acts via NPY Y1, Y2, and Y5 receptor subtypes (Silva et al. 2005a; Xapelli et al. 2006). This peptide has several important functions such as the regulation of appetite and circadian rhythms (Berglund et al. 2003), cognitive processing (Thorsell et al. 2000; Karl et al. 2008; Sørensen et al. 2008a,b), and neuroprotection (Silva et al. 2005b). Moreover, seizures up-regulate hippocampal NPY levels, and it has been consistently considered an endogenous antiepileptic agent (Woldbye et al. 1996; Silva et al. 2003a, 2005a; Xapelli et al. 2007). However, increased NPY levels could also affect other hippocampal functions, including learning and memory. In fact, some authors have clearly demonstrated that hippocampal NPY over-expression was accompanied by hippocampal activity-dependent plasticity reduction in excitatory synapses, which was associated with acquisition and retention deficits in spatial memory (Thorsell et al. 2000; Sørensen et al. 2008a). Furthermore, in vitro studies showed that hippocampal NPY reduces calcium influx and glutamatergic transmission mainly via presynaptic Y2 receptors (Silva et al. 2003b), and these effects result in the suppression of long-term potentiation (LTP) (Sørensen et al. 2008a,b). There are limited data available regarding the effects of METH on NPY and its receptors. It is known that multiple high doses of METH produce an increase of rat striatal pre-pro-NPY mRNA-expressing neurons (Horner et al. 2006). Similarly, Thiriet et al. (2005) demonstrated an up-regulation of striatal NPY mRNA, together with a down-regulation or biphasic changes in Y1 or Y2 receptor expression, respectively, following METH administration. The authors also reported neuroprotective effects of NPY against METH-induced striatal neurotoxicity mediated via Y1 and Y2 receptors (Thiriet et al. 2005). Nevertheless, as far as we know, the role of the hippocampal NPY system under METH consumption has never been addressed before.
Thus, the aim this study was to investigate the possible changes in mouse hippocampal NPY expression, as well as the expression and functionality of NPY receptors triggered by METH intoxication. Moreover, memory performance and the signaling pathway underlying such alterations were also evaluated. We concluded that METH increases hippocampal NPY levels and differentially affects the levels and functionality of NPY receptors. Moreover, we suggested that augmented Y2 receptor activation may be implicated in METH-induced memory impairment. Overall, our findings demonstrate that METH interferes with the hippocampal NPY system that may be associated with memory impairment.
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- Materials and methods
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The present work reveals that hippocampal NPY system alterations are centrally involved in memory impairment induced by METH intoxication. Specifically, METH-induced memory deficit was abolished by Y2 receptor blockade, and we further unraveled the involvement of the AKT/mTOR signaling pathway in these effects.
METH is an illicit psychostimulant drug that affects several brain regions, leading to neurological changes that include damage to monoaminergic neurons, gray-matter loss, white-matter hypertrophy, and excitotoxicity (reviewed by Cadet and Krasnova 2009). Recently, we verified that an acute METH treatment leads to astrogliosis, microglia activation, and tumor necrosis factor-∝ production, accompanied by neuronal dysfunction, namely disturbance at cytoskeletal, synaptic and axonal protein levels, but without evidence of hippocampal cell death (Gonçalves et al. 2010). Furthermore, the same METH regimen increases the blood–brain barrier permeability in the hippocampus, without affecting the striatum and frontal cortex (Martins et al. 2011), which led us to conclude that hippocampus is particularly susceptible to METH. Concerning the impact of METH on the NPYergic system, Thiriet et al. (2005) showed that METH (4 × 10 mg/kg, each 2 h) increases striatal NPY mRNA levels after 3 and 7 days. Moreover, the same study reported that METH induced a down-regulation of Y1 receptor mRNA and a contrasting up-regulation of Y2 receptor mRNA levels (Thiriet et al. 2005). Nonetheless, these authors did not show if changes in the striatal NPY system led to behavioral alterations. Here, we hypothesized that the observed changes in the hippocampal NPY system could underlie METH-induced mnemonic deficits. In fact, neuroplastic changes in the NPY system are prominent in several brain pathologies, such as epilepsy, Parkinson's disease, brain ischemia, or drug abuse (Goodman and Slovitter 1993; Cannizzarro et al. 2003; Duszczyk et al. 2009; Olling et al. 2009). Here, we verified that expression levels of NPY and its receptors, as well as receptor function, were differentially altered by METH, depending on hippocampal subregion and the time-point analyzed. However, further studies are needed to clarify if these transient effects are benign or will lead to long-lasting brain injury. Nevertheless, other studies have demonstrated that distinct insults differentially modulate the NPYergic system. Olling et al. (2009) described that ethanol-treated rats (5× 20% ethanol daily, during 4 days) only changed Y2 receptor mRNA levels in the DG subregion. In contrast, repeated electroconvulsive seizures led to an up-regulation of Y1, Y2, and Y5 receptor mRNA levels in the DG subfield together with a decrease in Y2 receptor mRNA levels in the CA3 of mouse hippocampus (Christensen et al. 2006). NPY receptor localization in the hippocampal subregions may be one possible explanation for these differences. In fact, Y1 receptors are highly expressed in the DG, Y2 receptors show strong expression in CA3 and CA1 pyramidal cell layers, and Y5 receptors are mainly expressed in DG and CA1 subfields (Naveilhan et al. 1998). Several authors have suggested that NPYergic system changes can be an adaptive mechanism for counteracting degeneration and/or cell death (reviewed by Vezzani and Sperk 2004; Xapelli et al. 2006), but NPY signaling is not only involved in neuroprotection. Indeed, there are studies showing that NPY receptors regulate voluntary ethanol consumption and its toxic effects (Thiele et al. 2002; Schroeder et al. 2003; Rimondini et al. 2005). Moreover, the activation of Y1 receptors can have proconvulsive effects (Olesen et al. 2012), while Y2 receptors have an important role in the generation of anxiety- and stress-related behaviors (Tschenett et al. 2003).
Multiple strands of evidence indicate an important role for the hippocampus in the formation and retrieval of episodic and contextual memories in humans and animals (Wiltgen et al. 2010; Vann and Albasser 2011), and it is well established that hippocampal dysfunction produces pronounced amnesia for newly acquired information (reviewed by Frankland and Bontempi 2005). Our group previously described that acute subcutaneous injection of METH (30 mg/kg) impairs rat spatial working memory, one of the hallmarks of hippocampal integrity (Simões et al. 2007). Besides, it has been demonstrated that acute and chronic METH exposure leads to both spatial and recognition memory deficits in rodents (Belcher et al. 2005, 2008; O'Dell et al. 2011; Reichel et al. 2012). Additionally, human studies also proved that METH use leads to memory impairment (Scott et al. 2007). In fact, a single dose of amphetamines has a long-lasting effect in the human body, which may contribute to memory deficits shown in METH abusers (Marshall and O'Dell 2012). It is also known that the storage and update of memory also require hippocampal plasticity mechanisms, such as LTP (reviewed by Bruel-Jungerman et al. 2007; Neves et al. 2008). In fact, it was shown recently that rats (Hori et al. 2010) and mice (Swant et al. 2010) chronically exposed to METH exhibited a decrease of both membrane potential and LTP magnitude in the CA1 pyramidal cell layer. So, it is plausible to hypothesize that defective hippocampal synaptic plasticity could trigger METH-induced cognitive performance deficits.
It is well established that METH consumption interferes with several neurotransmitters, including glutamate (reviewed by Krasnova and Cadet 2009; Silva et al. 2010) that plays a pivotal role in LTP (reviewed by Bliss and Collingridge 1993). In fact, METH-injected rats (7.5 mg/kg every 2 h over a period of 6 h; i.p.) exhibited augmented hippocampal extracellular glutamate levels (Raudensky and Yamamoto 2007), and we previously demonstrated that an acute METH treatment (30 mg/kg, s.c.) up-regulates both N-methyl-D-aspartate and α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors in the rat hippocampus (Simões et al. 2007). Additionally, it is well known that METH causes pronounced increases in extracellular levels of monoamines that may lead to activation of an array of brain circuits, such as the perirhinal cortex-prefrontal cortex-hippocampal circuitry, which is essential for memory tasks (Reichel et al. 2012). Specifically, Reichel et al. (2012) suggested that alterations in the function of transporters for serotonin in the object-in-place circuitry may underlie memory deficits independently of overt neurotoxic effects. Here, we describe that METH increases the levels of hippocampal NPY and Y2 receptors, as well as Y2-mediated functional activity. Additionally, it was previously reported that the activation of metabotropic glutamate receptors stimulates NPY mRNA expression in granule cells and interneurons of rat dentate gyrus (Schwarzer and Sperk 1998). Moreover, presynaptic Y2 receptor activation mediates NPY-induced inhibition of hippocampal glutamate release (Silva et al. 2001), which can explain LTP impairment at CA1-subicular cell synapses (Sørensen et al. 2008a). Thus, we hypothesized that the observed changes in the hippocampal NPY system could underlie METH-induced mnemonic deficits. To clarify this question, we examined spatial working memory using a Y-maze test (Cherng et al. 2007), and taking into consideration that the integrity of the hippocampus is essential not only to spatial working memory but also to recognition memory (Broadbent et al. 2004), we also performed the novel object recognition test. The contribution of NPY and its receptors to cognitive behaviors has been poorly explored. Nevertheless, Thorsell et al. (2000) demonstrated that transgenic rats over-expressing NPY in the hippocampus show spatial memory impairment. Supporting this evidence, it was shown that vector-mediated hippocampal NPY over-expression in rats impaired LTP in the CA1 subfield and, consequently, the animals exhibited a delay in hippocampal-dependent spatial discrimination learning (Sørensen et al. 2008b). Moreover, we observed an early up-regulation in both mRNA and protein levels of Y2 receptors, as well as in their functional activity. These alterations persisted until 3 days post-METH injection and were significant in the CA1 subregion that has a prominent role in hippocampal plasticity. Additionally, it is well established that METH triggers the release of monoamines, but the relationship between both monoamine and NPY systems is poorly understood, with no references to cognitive functions. Nevertheless, data from Meurs et al. (2007) indicate that NPY-induced increases in hippocampal dopamine may be mediated via sigma 1 receptors and NPY effects occur via increased activation of hippocampal D2 dopamine receptors. On the other hand, hippocampal D2 dopamine receptor binding showed positive linear correlations not only with memory function but also with frontal lobe functions, which supports the conclusion that D2 dopamine receptors contribute to local hippocampal functions (long-term memory) (Takahashi et al. 2008). Here, we did not explore the possible involvement of the dopamine system, but we clearly showed that the blockade of Y2 receptors completely prevented METH-induced memory impairment.
Synaptic plasticity, and consequently memory formation, requires new protein synthesis (Kandel 2001; Bruel-Jungerman et al. 2007; Costa-Mattioli et al. 2009). Furthermore, protein synthesis underlying the formation of long-lasting memories is highly regulated at the translation level, where the AKT/mTOR pathway regulates the translation rate and the integration of information from diverse synaptic inputs (reviewed by Hoeffer and Klann 2010). Indeed, the AKT/mTOR signaling cascade was identified as being crucial to the induction of protein synthesis-dependent synaptic plasticity required for hippocampus-dependent learning and memory processes (Cammalleri et al. 2003; Opazo et al. 2003). Hence, to unravel the signaling pathways mediating the anti-mnemonic effects of METH, we explored the potential involvement of the AKT/mTOR pathway. We found that an acute dose of METH disrupted the AKT/mTOR pathway in the hippocampus through Y2 receptor activation. Moreover, we have previously shown that NPY via Y2 receptor activation inhibits Ca2+ influx (Silva et al. 2003b), which, in turn, can restrain the AKT/mTOR cascade compromising new protein synthesis and, consequently, inhibiting LTP (Opazo et al. 2003). Indeed, the impairment of protein synthesis machinery and LTP results in memory deficits. Thus, it is reasonable to postulate that our observations could be explained by Ca2+ depression mediated by activation of the hippocampal NPY system.
In summary, this study provides a new explanation for the memory deficits induced by METH intoxication. Our work supports the potential importance of the hippocampal NPY system, and the functional relationship between Y2 receptors and AKT/mTOR pathway activation in memory impairment induced by METH. Thus, we may suggest that targeting this specific pathway or the modulation of Y2 receptor activity could provide interesting therapeutic approaches.