Adolescence as a Critical Window for Drug Abuse - Research Study of Neural Correlates of Adolescent Sensitivity and Novel Targets for Pharmacotherapy
Project location: ITALY, Milan
Project start date: January 2011 - Project end date: December 2012
Project number: 2010-68
Beneficiary: Università degli Studi di Milano Dipartimento di Scienze Farmacologiche
Cocaine is a psychostimulant drug that has long-lasting behavioral and neurobiological consequences. Very often, drug abuse develops during adolescence since adolescents tend to be more impulsive towards exciting events in life. This may depend, at least in part, on the fact that a part of the cerebral cortex, i.e. the prefrontal cortex, which contributes to the decision making, is still maturing during adolescence and therefore adolescents are more vulnerable to exciting actions, such as the experience of abusing of illicit drugs. Among the proteins that may play a role in the action of cocaine as well as during brain development, neurotrophic factors such as basic Fibroblast Growth Factor (FGF-2) represent important candidates.
Animal treatment and stress procedure
Male Sprague Dawley rats (100-125 gr) (Charles River, Calco, Lecco) were maintained under a 12-h light/dark cycle with food and water available ad libitum. Animals were allowed to adapt to laboratory conditions for one week and handled 5 minutes a day during this period. Animals were treated intraperitoneally with cocaine (20 mg/kg) or saline during adolescence, i.e. from postatal day (PND) 28 to PND 42. Then animals were divided in two groups and left undisturbed in their home cages. Animals belonging to group 1 were exposed to an acute swim stress (consisting in 5 min of swim with sacrifice 15 min after the end of stress) on PND45, i.e. three days after the end of cocaine treatment whereas animal belonging to group 2 were exposed to stress on PND90, i.e. forty eight days after the end of cocaine treatment. Immediately after sacrifice, brain regions of interest (prefrontal cortex, striatum, nucleus accumbens and hippocampus) were dissected out, frozen on dry ice and stored at -80°C.
Data are presented as means and standard errors, with each individual group comprising 8-10 samples. Changes produced by treatments were analyzed in the different brain regions, using a two-way analysis of variance (ANOVA), with stress and cocaine treatment as independent variables. When appropriate, further differences between groups were analyzed by Single Contrast Post-Hoc Test (SCPHT). However, where there was no interaction between stress and cocaine, only the main effect was reported, rather than proceeding inappropriately to subtesting of each individual treatment combination. Statistical significance was assumed at p<0.05.
Figure 1 shows no effect of cocaine on baseline FGF-2 mRNA levels in prefrontal cortex when the animals were sacrificed 3 days after the end of adolescence, i.e. on postnatal day 45 (PND45), but the trophic response to an acute stress is reduced in PND45 rats treated with cocaine during adolecence as opposed to PND45 rats treated with saline, suggesting that adolescential exposure to cocaine influences the trophic response to stress.
Figure 2 shows increased baseline FGF-2 mRNA levels in the prefrontal cortex of adult rats, i.e. PND90 rats, but, as previously observed in PND45 rats, FGF-2 mRNA levels are reduced following acute stress in cocaine-treated PND90 rats at variance from saline-treated PND90 rats, again suggesting that exposure to cocaine during adolescence influences the trophic response to stress in adult rats.
Figure 3 shows increased baseline FGF-2 mRNA levels in the hippocampus of PND45 rats; interestingly this effect is potentiated when PND45 rats were exposed to a challenging event whereas, in PND45 rats treated with saline during adolescence, stress promoted only a slight increase of FGF-2 mRNA levels.
Figure 4 shows a similar trend, although lower in magnitude, in PND90 rats. Baseline FGF-2 mRNA levels are slightly increased in PND90 rats and exposure to acute stress further increases such effect, whereas the response to stress in saline-treated PND90 rats is attenuated.
Figure 5 and Figure 6 show the effects of adolescential exposure to cocaine and the response to a challenging event in striatum. Unexpectedly, no effect of any kind was observed in this brain region
Figure 7 and Figure 8 show the effects of adolescential exposure to cocaine and the response to a challenging event in nucleus accumbens. A different effect was observed when measuring the effects of cocaine and stress on FGF-2 mRNA levels in adoelscent or adult rats. In fact, in adolescent animals, exposure to cocaine during development influenced the response to stress, since FGF-2 mRNA levels were reduced after stress in saline-treated rats while slightly increased in cocaine-treated rats. Conversely, at adulthood, no effect of both cocaine or stress was observed in this brain region.
Three major conclusions can be drawn from our data: 1) prefrontal cortex appears to be the brain region mostly affected by the combination of adolescential exposure to cocaine and stress when measuring FGF-2 mRNA levels whereas other brain regions, such as striatum and nucleus accumbens, traditionally considered to be the major targets of the action of the psychostimulant, seem to be less affected; 2) following repeated exposure to cocaine in adolescence, baseline levels of FGF-2 mRNA in adult rats were markedly increased in prefrontal cortex, suggesting a long-lasting imprinting exerted by cocaine since adult animals were sacrificed several weeks after the last exposure to cocaine and 3) the effect of the adolescential exposure to cocaine is dynamic, since it alters the subsequent trophic response to a further stress.
Taken together, these results suggest that adolescential cocaine treatment can permanently alter basal levels as well as stress-induced changes of the trophic molecule FGF-2. This is in line with a general statement which implies adolescence as a crucial period for brain development. The take home message of the experiments of the first year is that exposure to cocaine during adolescence alters the basal neurotrophic response, thus leading to a non physiological expression of this protein. In addition, exposure to cocaine alters also the response to an acute challenge such as swim stress; this is important since addictive behaviors are driven by stressful situations in humans, perhaps through changes in different proteins, one of which might be FGF-2.
Indeed, there appear to be fundamental, regional differences in the trophic factor response to repeated exposure to cocaine during adolescence. In the prefrontal cortex, FGF-2 mRNA levels are increased at adulthood suggesting a permanent effect of the adolescential exposure to the psychostimulant; interestingly, such effect is not observed at PND45, when FGF-2 mRNA levels are similar between cocaine- and saline-treated animals. This evidence suggests that the protracted withdrawal from the psychostimulant may have ‘incubated' the trophic response and point to FGF-2 changes as adaptive mechanisms set in motion by the long abstinence. Interestingly, it has been previously demonstrated that FGF-2 mRNA levels were increased 3, but not 14, days following repeated cocaine treatment at adulthood (Fumagalli et al., 2006), suggesting that permanently elevated levels of FGF-2 mRNA may represent a signature of cocaine-withdrawal from adolescential exposure.
Notably, although the baseline FGF-2 mRNA levels are different in cocaine-treated PND45 or PND90 rats, exposure to cocaine during adolescence reversed the trophic response pattern, causing a reduction after exposure to acute stress at both ages, whereas, in animals treated with saline during adolescence acute stress leads to enhanced FGF-2 expression. These results led us to hypothesize that the global alterations seen at both baseline levels and after stress may be the result of cocaine-induced dysregulation of cortical networks which begin to occur at adolescence.
Thus, adolescential exposure to cocaine distorts or impairs the mechanisms responsible for cortical FGF-2 regulation under acute challenging situations. The functional relevance of such dysregulation may be manifold; in fact, if increased FGF-2 mRNA levels represents a homeostatic, neuroprotective response to acute stress, then cocaine exposure during adolescence could enhance the susceptibility to cocaine by preventing FGF-2 mRNA elevation. Alternatively, the loss of FGF-2 up-regulation after cocaine treatment might rather be indicative of a reduced neuronal responsiveness to the drug of abuse as opposed, for instance, to the effects of cocaine exposure during gestation which results in increased stress responsiveness of meso-prefrontal dopamine neurons (Elsworth et al., 2001). If this interpretation holds true, it appears that cocaine differently affects the response to a challenge, depending on the developmental window of the exposure to the challenge. This information is interesting in view of the evidence that alterations of the FGF system appears to contribute also to disorders such as depression and schizophrenia, two psychiatric disorders known to be developmental in origin (Riva et al., 2005).
Interestingly, a different effect was observed in the hippocampus where, although dopaminergic terminals are scarce, cocaine seems to modulate FGF-2 mRNA levels. Interestingly, in this brain region, FGF-2 gene expression appears to be regulated by cocaine on PND 45, i.e. soon after the end of adolescence, whereas such effect wanes at adulthood. Indeed, on PND45, cocaine enhances FGF-2 mRNA levels, an effect that seems to be potentiated by stress. At adulthood, the trend appears to be the same but, since the magnitude of the effect is reduced, the effect is not statistically significant. Since in the hippocampus glucocorticoid receptors are highly espressed, the potentiation observed could be, at least in part, explained by high responsivity to stress which, instead, vanishes at adulthood.
Interestingly, neither cocaine nor stress affect FGF-2 mRNA levels in striatum, a quite surprising result since evidence exists that adult treatment with cocaine does affect the neurotrophic factor expression (Fumagalli et al., 2006) suggesting that the timing of cocaine treatment is important for FGF-2 expression. At variance from striatum, FGF-2 mRNA levels were affected in the nucleus accumbens of PND45, but not on PND90, rats. Notably, on PND45, although baseline levels were unchanged in the nucleus accumbens of cocaine-treated rats, however cocaine seems to influence the trophic response to stress since FGF-2 mRNA levels are not slightly reduced as in saline-treated rats.
In conclusion, these results point out how repeated exposure to cocaine at adolescence influences the basal expression of the trophic factor FGF-2, as well as its response to external stimuli delivered later in life. Finally, the dysregulation of FGF-2 responses to adult stress in animals that had been exposed to cocaine during adolescence points to specific mechanisms underlying lifelong susceptibility to adverse environmental conditions set in motion by cocaine treatment during adolescence.
Fumagalli F, Di Pasquale L, Racagni G and Riva MA. Dynamic regulation of fibroblast growth factor (FGF-2) gene expression in the rat brain following single and repeated cocaine administration. J. Neurochemistry 96 (4):996-1004, 2006.
MA. Riva, R.Molteni, F.Bedogni, G. Racagni and F. Fumagalli Emerging role of the FGF system in psychiatric disorders Trends in Pharmacological Sciences, 26:228-231, 2005