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- Materials and methods
Glycogen synthase kinase 3β (GSK3β), which is abundantly present in the brain, is known to contribute to psychomotor stimulant-induced locomotor behaviors. However, most studies have been focused in showing that GSK3β is able to attenuate psychomotor stimulants-induced hyperactivity by increasing its phosphorylation levels in the nucleus accumbens (NAcc). So, here we examined in the opposite direction about the effects of decreased phosphorylation of GSK3β in the NAcc core on both basal and cocaine-induced locomotor activity by a bilateral microinjection into this site of an artificially synthesized peptide, S9 (0.5 or 5.0 μg/μL), which contains sequences around N-terminal serine 9 residue of GSK3β. We found that decreased levels of GSK3β phosphorylation in the NAcc core enhance cocaine-induced hyper-locomotor activity, while leaving basal locomotor activity unchanged. This is the first demonstration, to our knowledge, that the selective decrease of GSK3β phosphorylation levels in the NAcc core may contribute positively to cocaine-induced locomotor activity, while this is not sufficient for the generation of locomotor behavior by itself without cocaine. Taken together, these findings importantly suggest that GSK3β may need other molecular targets which are co-activated (or deactivated) by psychomotor stimulants like cocaine to contribute to generation of locomotor behaviors.
It has been well known that psychomotor stimulants like cocaine produce the increase in locomotor activity by activation of dopaminergic neurotransmission in the nucleus accumbens (NAcc) (Kalivas and Stewart 1991; Hyman 1996), which is a neuronal substrate mediating the rewarding effects of drugs of abuse (Robbins et al. 1989; Koob and Le Moal 2001; Goto and Grace 2008). Interestingly, it has been suggested that, in addition to a classic cAMP-mediated pathway, dopamine has a distinct signaling pathway involving glycogen synthase kinase 3β (GSK3β), which is a serine/threonine kinase abundantly present in the brain, and accordingly it contributes to dopamine and psychomotor stimulant-induced characteristic behaviors (Beaulieu et al. 2007, 2009).
Recently, it has been shown that inhibition of GSK3β by systemic injection of valproate or a more specific inhibitor, SB216763, attenuates hyperactivity produced by acute injection of psychomotor stimulants like cocaine or amphetamine (AMPH) (Miller et al. 2009; Enman and Unterwald 2012). Further, direct microinjection of SB216763 into the NAcc core, but not in the shell, has been shown to block the expression of locomotor sensitization induced by repeated injection of cocaine or meth-AMPH (Xu et al. 2009, 2011). These results suggest that locomotor activity produced by psychomotor stimulants is dependent on GSK3β activity in the NAcc, especially in the core. However, these studies only used inhibitors for GSK3β yet, which result in the increase of phosphorylation levels at its serine 9 residue and supposedly a subsequent reduction in its activity (Dajani et al. 2001; Frame et al. 2001). As a result, it still remains unexplored what effects the supposed increase of GSK3β activity in the NAcc in the opposite way by reducing the phosphorylation levels at the same residue will actually bring to locomotor activity. Thus, we examined the effects of direct manipulation leading to reduction of GSK3β phosphorylation levels in the NAcc core on both basal and cocaine-induced locomotor activity.
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- Materials and methods
The present results revealed that inhibition of GSK3β inactivation in the NAcc core by temporarily reducing its phosphorylation levels at serine 9 residue enhances cocaine-induced hyper-locomotor activity, while leaving basal activity unchanged. This is the first direct demonstration, to our knowledge, that the selective decrease of GSK3β phosphorylation levels in the NAcc core may actually contribute positively to psychomotor stimulant-induced locomotor activity.
In the past few years, people have shown that GSK3β activity in some brain areas is regulated by psychomotor stimulants treatment. For example, phosphorylation levels of GSK3β are reduced not only in the dorsal striatum by acute administration of AMPH or cocaine (Beaulieu et al. 2007; Miller et al. 2009) and by chronic AMPH (Mines and Jope 2012) but also in the NAcc core by chronic cocaine and meth-AMPH (Xu et al. 2009, 2011). Consistent with these results, we also observed acute cocaine decreases the ratio of phosphorylation to total levels of GSK3β distinctively in the NAcc core, but not in the shell (Fig. 1). Further, we found that these effects are more significantly expressed at later time point (60 min) than earlier (15 min), which may reflect a delayed kinetic property with a peak around between 60 and 90 min that appears in the cAMP-independent dopamine pathway through an Akt-GSK3β signaling cascade (Beaulieu et al. 2007).
Literature has affluently shown that the core and the shell are distinguished in both anatomical structures and behavioral output functions (Jongen-Relo et al. 1994; Di Chiara 2002; Zahm 2002; Meredith et al. 2008); for example, the core mediates the motivational impact of Pavlovian conditioned stimuli, while the shell does the impact of primary reinforcers (Cardinal et al. 2002). Further, psychostimulant sensitization has shown not only to increase c-fos mRNA expression or dopamine transmission but also to significantly induce structural plasticity in the core, but not in the shell (Cadoni et al. 2000; Li et al. 2004; Nordquist et al. 2008). These results suggest that our present findings, in which GSK3β differentially responds to acute cocaine in the core, may also reflect intrinsic differences in signaling pathways set wired in the core in terms of mediating psychostimulant-induced behaviors. Interestingly, it has recently been shown that specific inhibition of GSK3β activity in the NAcc core actually blocks cocaine- or meth-AMPH-induced locomotor sensitization (Xu et al. 2009, 2011), indicating that GSK3β in the core is more specifically involved in psychostimulant-induced behaviors.
In the literature, it has been consistently shown that GSK3β inhibitors such as lithium chloride or valproic acid increase phosphorylation levels for GSK3β at serine 9 residue and accordingly attenuate psychomotor stimulants-induced increase in locomotor activity (Beaulieu et al. 2008; Xu et al. 2009, 2011; Enman and Unterwald 2012). However, these studies have not investigated in the opposite way yet about the possible effects of direct inhibition of GSK3β inactivation in the NAcc on locomotor activity. Although it has previously been shown that either transgenic mice over-expressing GSK3β or more selectively GSK3β knockin mice enhance locomotor activity in response to either a novel environment or psychomotor stimulants like AMPH or methylphenidate (Prickaerts et al. 2006; Polter et al. 2010; Mines et al. 2013), these studies not only manipulated GSK3β gene affecting the whole brain but also permanently throughout animal's life-span. Thus, we have found a new way for a local and temporal inhibition of GSK3β inactivation and used in this study an artificially synthetic S9 peptide, which competes with endogenous GSK3β for the N-terminal phosphorylation site at serine 9 residue, resulting in less phosphorylation and consequent inhibition of GSK3β inactivation (Dajani et al. 2001; Frame et al. 2001). As a consequence, a bilateral microinjection into the NAcc core of this artificial peptide interestingly enhances cocaine-induced hyper-locomotor activity in a dose-dependent manner (Fig. 2) with an accompanied decrease of GSK3β phosphorylation levels (Fig. 3), which is exactly opposite result to the ones obtained when GSK3β inhibitors were used, but simultaneously consistent with the ones obtained from transgenic or knockin mice as shown in literature. Thus, our findings favorably support the notion that GSK3β activity in the NAcc core is importantly involved in cocaine-induced locomotor activity. Further, our present results also show that the decrease of GSK3β phosphorylation alone in the NAcc core without the help of cocaine is not sufficient by itself for the generation of locomotor behavior (see Figs 2 and 3). Similarly, GSK3β knockin mice data showed that a constitutive GSK3β activation enhanced locomotor activity only when a certain type of stimulus (e.g., novel environment or psychomotor stimulants) was present (Polter et al. 2010; Mines et al. 2013). In our case, we habituated rats before locomotor measurement, so that rats with compared from the ones without S9 showed no different locomotor activity unless there was cocaine present. These results importantly suggest that activation of GSK3β may become somehow workable only when other molecular targets are co-activated (or deactivated) by psychomotor stimulants like cocaine or even a novel environment (as in knockin data). It remains in the future to find out what they might be and how, though.
Rather surprisingly, our present results indicate that the ratio of phosphorylated to total GSK3β in the NAcc core is not directly proportional to the amount of locomotor activity produced; for example, rats injected with cocaine in the presence of low dose of S9 did not show less phosphorylation levels compared with rats with cocaine alone, although locomotor activity in the former was higher than the latter (see Fig. 3 and compare with Fig. 2). These results may suggest that the prior decrease of GSK3β phosphorylation by S9 in the NAcc core may just help set the stage for following cocaine to more easily produce enhanced hyper-locomotor activity similar to sensitized locomotion normally observed in rats with chronic cocaine treatment (Robinson and Berridge 1993). To confirm this hypothesis, we measured GSK3β phosphorylation levels in the NAcc at 2 weeks of drug-free withdrawal period after 7 days of daily cocaine injections and found that basal levels of GSK3β phosphorylation in the NAcc core, but not in the shell, are significantly reduced in rats with cocaine compared with saline pre-treated (Fig. 4). These results imply that reduced basal levels of GSK3β phosphorylation in the NAcc core may be related with the production of behavioral sensitization by chronic cocaine treatment, and in our case, although it is speculative yet, temporal reduction of GSK3β phosphorylation in the NAcc core by S9 may have pulled forward neuronal state to help set the stage similar to the one obtained by chronic cocaine, so that seemingly sensitized locomotor activity is produced when cocaine comes in. Taken together, our present findings indicate that GSK3β phosphorylation levels in the NAcc core may be an important regulator of cocaine-induced locomotor activity and suggest that it is worth closer examination at this molecule in terms of locomotor sensitization in the future.