Adolescent and adult responsiveness to the incentive value of cocaine reward in mice: Role of neuronal nitric oxide synthase (nNOS) gene
Introduction
Adolescence is a period characterized by increasing social behavior, risk-taking, and novelty seeking; the adaptive transition to adulthood may potentially contribute to a unique susceptibility to the abuse of drugs (Spear, 2000). Animal and human studies demonstrated specific neural changes in certain brain regions during adolescence. Increase in prefrontal cortex (PFC) dopamine (DA) fiber density (Kalsbeek et al., 1988) and overproduction of striatal and PFC DA receptors in rats (Andersen et al., 1997, Andersen et al., 2000) and striatal DA receptors in humans (Seeman et al., 1987) have been reported. Synaptic elimination of glutamatergic PFC afferents in adolescent humans (Huttenlocher, 1984) and non-human primates (Zecevic et al., 1989) and reductions in glutamate and NMDA receptor binding in rat PFC (Guilarte, 1998) have also been noted.
The corticostriatal and mesolimbic DA and glutamate systems hold major roles in the effects of psychostimulants, and exposure to such drugs during adolescence may interfere with ongoing neuroplastic events. The incentive value of drug and natural reward in rodents is commonly assessed by the conditioned place preference (CPP) paradigm, which involves Pavlovian learning. Repeated pairing of a subject with unconditioned stimulus (US, e.g., psychostimulant) in a specific environment, to become conditioned stimulus (CS), elicits conditioned response (CR). Based on the CPP paradigm some evidence suggests age-dependent changes in the incentive value of drug reward (Tirelli et al., 2003; review). For instance, in CD1 mice amphetamine CPP developed on postnatal days (PD) 14–28 but not PD30–49 (Laviola et al., 1994, Adriani and Laviola, 2003). It is thought that enhanced DA activity in the PFC compared to the nucleus accumbens (NAC) during adolescence may render the organism less sensitive to psychostimulants (Spear, 2000). However, cocaine CPP developed in mice (Laviola et al., 1992) and rats (Bolanos et al., 1996, Campbell et al., 2000) on PD21–34 as in adult subjects. Therefore, it is not entirely clear if the incentive value of psychostimulant reward changes from adolescence to adulthood. Most of the studies conducted thus far on the effect of abused substances on adolescent behavior have encompassed a wide adolescent age range (PD20 through PD35) and focused on relatively short-term consequences of drug exposure (e.g., one to two weeks following cessation of drug treatment). Hence, knowledge about the long-term consequences of adolescent exposure to psychostimulants is scarce. The first goal of the present study was to investigate: (a) the acquisition and expression of cocaine CPP during the periadolescent period (PD24–45); and (b) the vulnerability of the same subjects to cocaine challenge in adulthood (e.g., PD70). Additionally, given the relative paucity of studies on the effect of psychostimulants in female rodents, compared to studies in male rodents, the second goal was to investigate potential sex-dependent differences in response to cocaine CPP during adolescence and adulthood. The third goal was to investigate the role of neuronal nitric oxide synthase (nNOS) in the neural plasticity underlying the acquisition, maintenance and reinstatement of cocaine CPP. The reinstatement of CPP following extinction is considered as a model for drug relapse.
In brain, nitric oxide (NO) is produced primarily by the conversion of L-arginine to L-citrulline via nNOS in response to stimulation of N-methyl-d-aspartate (NMDA) type of glutamate receptors (Garthwaite and Boulton, 1995). The spliced variant nNOSα, which constitutes 95% of nNOS catalytic activity, is linked to the NR2B subunit of the NMDA receptors via a postsynaptic density protein (PSD95) (Brenman and Bredt, 1997). NO has a major role in non-synaptic communication between glutamatergic and monoaminergic neurons (Kiss and Vizi, 2001). NO exerts facilitatory influence on both tonic extracellular DA levels and phasic DA neuron spike activity (Grace, 1991, West and Grace, 2000, West et al., 2002). NO also facilitates glutamate release, and stimulation of glutamate receptors on dopaminergic nerve terminals may further stimulate DA release (Moore et al., 1999). In a series of studies we found that mice lacking the nNOS gene (nNOS knockout; KO) are less sensitive to the psychomotor, rewarding and neurotoxic effects of cocaine and methamphetamine, thus supporting the role of nNOS in the neural plasticity and neurotoxicity that underlie psychostimulants effects (Itzhak and Ali, 2006, Itzhak et al., 2002; reviews).
Ontogeny studies of nNOS have shown that the expression of nNOS in the striatum and cerebellum nearly doubled between PD20 and 60 (Keilhoff et al., 1996) and that nNOS and tyrosine hydroxylase (TH) immunoreactive neurons appear within the same striatal patches at distinctive times during development (Murata and Masuko, 2003). Therefore, nNOS may influence adolescent responsiveness to psychostimulants directly through intrinsic changes or via its effects on DA and glutamate systems which are in transition. Accordingly, the third goal of the present study was to investigate the role of nNOS in the various phases of cocaine CPP in adolescent and adult mice. Results suggest that the nNOS gene is required in adolescence for the development of neuroadaptations that enable the maintenance and reinstatement of CPP, thus the nitrergic system may be critically involved in the development of persistent drug seeking behavior from adolescence through adulthood.
Section snippets
Animals
Adult male and female homozygote nNOS knockout (KO) mice (B6;129S4-Nos1; 6–8 weeks old) were purchased from Jackson Laboratories (Bar Harbor, Maine). These mice were generated on a mixed SV129 and C57BL/6 background as described previously (Huang et al., 1993). Because the breeding of nNOS KO mice that have been backcrossed to the C57BL/6 mouse strain was unsuccessful (personal communication with Jackson Laboratories and our own experience), B6;129SF2 mice (KO and wild type; WT) were used.
Immunohistochemistry of striatal nNOS
Immunostaining of nNOS was performed in order to verify that KO mice lacked the enzyme. Fig. 1 shows representative images of striatal neurons of WT and KO adult male mice (PD92–94) that stain positively and negatively for nNOS, respectively. The intensity of nNOS immunoreactive neurons in dorsal regions of the striatum was higher than in other brain regions (VTA, amygdala and frontal cortex; data not shown) of WT mice. Additional studies in adult females and adolescent mice (PD25–PD35) yielded
Discussion
The aims of the present study were to investigate: (a) the acquisition, expression, maintenance and reinstatement of cocaine CPP from periadolescence (PD24–45) through adulthood (PD70); (b) potential sexual dimorphism during adolescence and adulthood in the response to cocaine-induced CPP; and c) the role of the nNOS gene in the neural plasticity that underlie the acquisition, expression, maintenance and reinstatement of cocaine CPP.
In adolescent WT mice no significant sex-dependent differences
Acknowledgements
The work described was supported by USPHS awards DA12867 and DA19107 from the National Institute on Drug Abuse, National Institutes of Health. The authors thank Erica Givens for excellent technical support.
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