Early Adolescence is a Critical Period for the Maturation of Inhibitory Behavior.

Psychiatric conditions marked by impairments in cognitive control often emerge during adolescence, when the prefrontal cortex (PFC) and its inputs undergo structural and functional maturation and are vulnerable to disruption by external events. It is not known, however, whether there exists a specific temporal window within the broad range of adolescence when the development of PFC circuitry and its related behaviors are sensitive to disruption. Here we show, in male mice, that repeated exposure to amphetamine during early adolescence leads to impaired behavioral inhibition, aberrant PFC dopamine connectivity, and reduced PFC dopamine function in adulthood. Remarkably, these deficits are not observed following exposure to the exact same amphetamine regimen at later times. These findings demonstrate that there is a critical period for the disruption of the adolescent maturation of cognitive control and PFC dopamine function and suggest that early adolescence is particularly relevant to the emergence of psychopathology in humans.

Reduced dopamine release in Dcc haploinsufficiency male mice abolishes the rewarding effects of cocaine but not those of morphine and ethanol.

RATIONALE: The Netrin-1/DCC guidance cue pathway is critically involved in the adolescent organization of the mesocorticolimbic dopamine circuitry. Adult mice heterozygous for Dcc show reduced dopamine release in the nucleus accumbens in response to amphetamine and, in turn, blunted sensitivity to the rewarding effects of this drug. OBJECTIVE: Here, we tested whether the protective effects of Dcc haploinsufficiency are specific to stimulant drugs of abuse or instead extrapolate to opioids and ethanol. METHODS: We used the place preference paradigm to measure the rewarding effects of cocaine (20 mg/kg), morphine (5 or 10 mg/Kg), or ethanol (20%) in adult (PND 75) male Dcc haploinsufficient mice or their wild-type litter mates. In a second experiment, we compared in these two genotypes, in vivo dopamine release in the nucleus accumbens after a single i.p. injection of morphine (10 mg/kg). RESULTS: We found reduced morphine-induced dopamine release in the nucleus accumbens of Dcc haploinsufficient male mice, but, contrary to the effects of stimulant drugs, there is no effect of genotype on morphine-induced conditioned preference. CONCLUSION: These findings show that reduced drug-induced mesolimbic dopamine in Dcc haploinsufficient male mice protects specifically against the rewarding effects of stimulant drugs, but not against the rewarding properties of morphine and ethanol. These results suggest that these drugs exert their rewarding effect via different brain circuits.

Amphetamine disrupts dopamine axon growth in adolescence by a sex-specific mechanism in mice.

Initiating drug use during adolescence increases the risk of developing addiction or other psychopathologies later in life, with long-term outcomes varying according to sex and exact timing of use. The cellular and molecular underpinnings explaining this differential sensitivity to detrimental drug effects remain unexplained. The Netrin-1/DCC guidance cue system segregates cortical and limbic dopamine pathways in adolescence. Here we show that amphetamine, by dysregulating Netrin-1/DCC signaling, triggers ectopic growth of mesolimbic dopamine axons to the prefrontal cortex, only in early-adolescent male mice, underlying a male-specific vulnerability to enduring cognitive deficits. In adolescent females, compensatory changes in Netrin-1 protect against the deleterious consequences of amphetamine on dopamine connectivity and cognitive outcomes. Netrin-1/DCC signaling functions as a molecular switch which can be differentially regulated by the same drug experience as function of an individual's sex and adolescent age, and lead to divergent long-term outcomes associated with vulnerable or resilient phenotypes.

The netrin receptor DCC is required in the pubertal organization of mesocortical dopamine circuitry.

Netrins are guidance cues involved in neural connectivity. We have shown that the netrin-1 receptor DCC (deleted in colorectal cancer) is involved in the functional organization of the mesocorticolimbic dopamine (DA) system. Adult mice with a heterozygous loss-of-function mutation in dcc exhibit changes in indexes of DA function, including DA-related behaviors. These phenotypes are only observed after puberty, a critical period in the maturation of the mesocortical DA projection. Here, we examined whether dcc heterozygous mice exhibit structural changes in medial prefrontal cortex (mPFC) DA synaptic connectivity, before and after puberty. Stereological counts of tyrosine-hydroxylase (TH)-positive varicosities were increased in the cingulate 1 and prelimbic regions of the pregenual mPFC. dcc heterozygous mice also exhibited alterations in the size, complexity, and dendritic spine density of mPFC layer V pyramidal neuron basilar dendritic arbors. Remarkably, these presynaptic and postsynaptic partner phenotypes were not observed in juvenile mice, suggesting that DCC selectively influences the extensive branching and synaptic differentiation that occurs in the maturing mPFC DA circuit at puberty. Immunolabeling experiments in wild-type mice demonstrated that DCC is segregated to TH-positive fibers innervating the nucleus accumbens, with only scarce DCC labeling in mPFC TH-positive fibers. Netrin had an inverted target expression pattern. Thus, DCC-mediated netrin-1 signaling may influence the formation/maintenance of mesocorticolimbic DA topography. In support of this, we report that dcc heterozygous mice exhibit a twofold increase in the density of mPFC DCC/TH-positive varicosities. Our results implicate DCC-mediated netrin-1 signaling in the establishment of mPFC DA circuitry during puberty.

DCC Receptors Drive Prefrontal Cortex Maturation by Determining Dopamine Axon Targeting in Adolescence.

BACKGROUND: Dopaminergic input to the prefrontal cortex (PFC) increases throughout adolescence and, by establishing precisely localized synapses, calibrates cognitive function. However, why and how mesocortical dopamine axon density increases across adolescence remains unknown. METHODS: We used a developmental application of axon-initiated recombination to label and track the growth of dopamine axons across adolescence in mice. We then paired this recombination with cell-specific knockdown of the netrin-1 receptor DCC to determine its role in adolescent dopamine axon growth. We then assessed how altering adolescent PFC dopamine axon growth changes the structural and functional development of the PFC by quantifying pyramidal neuron morphology and cognitive performance. RESULTS: We show, for the first time, that dopamine axons continue to grow from the striatum to the PFC during adolescence. Importantly, we discover that DCC, a guidance cue receptor, controls the extent of this protracted growth by determining where and when dopamine axons recognize their final target. When DCC-dependent adolescent targeting events are disrupted, dopamine axons continue to grow ectopically from the nucleus accumbens to the PFC and profoundly change PFC structural and functional development. This leads to alterations in cognitive processes known to be impaired across psychiatric conditions. CONCLUSIONS: The prolonged growth of dopamine axons represents an extraordinary period for experience to influence their adolescent trajectory and predispose to or protect against psychopathology. DCC receptor signaling in dopamine neurons is a molecular link where genetic and environmental factors may interact in adolescence to influence the development and function of the prefrontal cortex.

Dcc haploinsufficiency regulates dopamine receptor expression across postnatal lifespan.

Adolescence is a period during which the medial prefrontal cortex (mPFC) undergoes significant remodeling. The netrin-1 receptor, deleted in colorectal cancer (DCC), controls the extent and organization of mPFC dopamine connectivity during adolescence and in turn directs mPFC functional and structural maturation. Dcc haploinsufficiency leads to increased mPFC dopamine input, which causes improved cognitive processing and resilience to behavioral effects of stimulant drugs of abuse. Here we examine the effects of Dcc haploinsufficiency on the dynamic expression of dopamine receptors in forebrain targets of C57BL6 mice. We conducted quantitative receptor autoradiography experiments with [3H]SCH-23390 or [3H]raclopride to characterize D1 and D2 receptor expression in mPFC and striatal regions in male Dcc haploinsufficient and wild-type mice. We generated autoradiograms at early adolescence (PND21±1), mid-adolescence (PND35±2), and adulthood (PND75±15). C57BL6 mice exhibit overexpression and pruning of D1, but not D2, receptors in striatal regions, and a lack of dopamine receptor pruning in the mPFC. We observed age- and region-specific differences in D1 and D2 receptor density between Dcc haploinsufficient and wild-type mice. Notably, neither group shows the typical pattern of mPFC dopamine receptor pruning in adolescence, but adult haploinsufficient mice show increased D2 receptor density in the mPFC. These results show that DCC receptors contribute to the dynamic refinement of D1 and D2 receptor expression in striatal regions across adolescence. The age-dependent expression of dopamine receptor in C57BL6 mice shows marked differences from previous characterizations in rats.

Resilience to amphetamine in mouse models of netrin-1 haploinsufficiency: role of mesocortical dopamine.

RATIONALE: Signaling through the netrin-1 receptor, deleted in colorectal cancer (DCC), in dopamine neurons controls the extent of their innervation to the medial prefrontal cortex (mPFC) during adolescence. In mice, dcc haploinsufficiency results in increased mPFC dopamine innervation and concentrations in adulthood. In turn, dcc haploinsufficiency leads to resilience to the effects of stimulant drugs of abuse on dopamine release in the nucleus accumbens and behavior. OBJECTIVES: First, we set out to determine whether increased mPFC dopamine innervation causes blunted behavioral responses to amphetamine in adult dcc haploinsufficient mice. Second, we investigated whether unc5c, another netrin-1 receptor expressed by dopamine neurons, is involved in these effects. Third, we assessed whether haploinsufficiency of netrin-1 itself leads to blunted behavioral responding to amphetamine, whether this phenotype emerges before or after adolescence and whether increased mPFC dopamine input is the underlying mechanism. RESULTS: Adult, but not adolescent, dcc, unc5c and netrin-1 haploinsufficient mice exhibit blunted behavioral responses to amphetamine. Furthermore, adult dcc, unc5c, and netrin-1 haploinsufficient mice have exaggerated mPFC dopamine concentrations in comparison to their wild-type littermates. Importantly, resilience to amphetamine-induced behavioral activation in all the three mouse models is abolished by selective dopamine depletion in the medial prefrontal cortex. CONCLUSIONS: dcc, unc5c, or netrin-1 haploinsufficiency leads to increased dopamine content in the mPFC and to resilience against amphetamine-induced behavioral activation. Our findings raise the hypothesis that DCC, UNC5C, and netrin-1 act in concert to organize the adolescent development of mesocortical dopamine innervation and, in turn, determine behavioral responses to drugs of abuse.

Adolescence: a time of transition for the phenotype of dcc heterozygous mice.

RATIONALE: Stark differences exist between adult (>PND 70) and juvenile (∼PND 21-34) rodents in how DCC (deleted in colorectal cancer) receptors and sensitization to amphetamine interact. In adults, repeated amphetamine upregulates DCC receptor expression selectively in the ventral tegmental area (VTA), an effect that is critical for sensitization. In contrast, amphetamine administered to juveniles downregulates VTA DCC expression. Moreover, whereas adult dcc heterozygous mice fail to sensitize when repeatedly treated with amphetamine, drug treatment during the juvenile period actually abolishes this adult "protective" phenotype. OBJECTIVES: We set out to determine whether adolescence (PND ∼35-55) is a period during which: (1) amphetamine-induced alterations in VTA DCC expression switch from downregulation to upregulation; (2) the "protective" phenotype of adult dcc heterozygotes against sensitization becomes evident; and (3) the adult "protective" phenotype of dcc heterozygotes can still be abolished by repeated amphetamine treatment. RESULTS: Repeated amphetamine did not significantly alter VTA DCC expression in adolescent rodents when assessed 1 week later. Both wild-type and dcc heterozygous mice exhibited sensitization at this time. Remarkably, wild-type mice, but not dcc heterozygotes, exhibited sensitization when tested during adulthood. CONCLUSIONS: Adolescence is a time of transition for dcc heterozygotes as related to sensitization. Our results support the hypothesis that DCC may be a key factor in determining age-dependent individual differences in vulnerability to sensitization. Given that exposure to drugs of abuse during adolescence can have profound consequences for adulthood, the resilience of adult dcc heterozygous mice against adolescent exposure to amphetamine is particularly salient.