Variations in the expression of the Netrin-1 guidance cue receptor DCC (deleted in colorectal cancer) appear to confer resilience or susceptibility to psychopathologies involving prefrontal cortex (PFC) dysfunction.

Operant chambers are widely used in animal research to study cognition, motivation, and learning processes. Paired with the rapidly developing technologies for brain imaging and manipulations of brain activity, operant conditioning chambers are a powerful tool for neuroscience research. The behavioral testing and imaging setups that are commercially available are often quite costly. Here, we present a custom-built operant chamber that can be constructed in a few days by an unexperienced user with relatively inexpensive, widely available materials. The advantages of our operant setup compared with other open-source and closed-source solutions are its relatively low cost, its support of complex behavioral tasks, its user-friendly setup, and its validated functionality with video imaging of behavior and calcium imaging using the UCLA Miniscope. Using this setup, we replicate our previously published findings showing that mice exposed to social defeat stress in adolescence have inhibitory control impairments in the Go/No-Go task when they reach adulthood. We also present calcium imaging data of medial prefrontal cortex (mPFC) neuronal activity acquired during Go/No-Go testing in freely moving mice and show that neuronal population activity increases from day 1 to day 14 of the task. We propose that our operant chamber is a cheaper alternative to its commercially available counterparts and offers a better balance between versatility and user-friendly setup than other open-source alternatives.

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.

Low miR-218 expression in the medial prefrontal cortex (mPFC) is a consistent trait of depression. Here we assessed whether miR-218 in the mPFC confers resilience or susceptibility to depression-like behaviors in adult mice, using the chronic social defeat stress (CSDS) model of depression. We also investigated whether stress-induced variations of miR-218 expression in the mPFC can be detected in blood. We find that downregulation of miR-218 in the mPFC increases susceptibility to a single session of social defeat, whereas overexpression of miR-218 selectively in mPFC pyramidal neurons promotes resilience to CSDS and prevents stress-induced morphological alterations to those neurons. After CSDS, susceptible mice have low levels of miR-218 in blood, as compared with control or resilient groups. We show further that upregulation and downregulation of miR-218 levels specifically in the mPFC correlate with miR-218 expression in blood. Our results suggest that miR-218 in the adult mPFC might function as a molecular switch that determines susceptibility vs. resilience to chronic stress, and that stress-induced variations in mPFC levels of miR-218 could be detected in blood. We propose that blood expression of miR-218 might serve as potential readout of vulnerability to stress and as a proxy of mPFC function.

The development of the dopamine input to the medial prefrontal cortex occurs during adolescence and is a process that is vulnerable to disruption by stimulant drugs such as amphetamine. We have previously linked the amphetamine-induced disruption of dopamine connectivity and prefrontal cortex maturation during adolescence to the downregulation of the Netrin-1 receptor, DCC, in dopamine neurons. However, how DCC expression in dopamine neurons is itself regulated is completely unknown. MicroRNA (miRNA) regulation of mRNA translation and stability is a prominent mechanism linking environmental events to changes in protein expression. Here, using male mice, we show that miR-218 is expressed in dopamine neurons and is a repressor of DCC. Whereas Dcc mRNA levels increase from early adolescence to adulthood, miR-218 exhibits the exact opposite switch, most likely maintaining postnatal Dcc expression. This dynamic regulation appears to be selective to Dcc since the expression of Robo 1, the other guidance cue receptor target of miR-218, does not vary with age. Amphetamine in adolescence, but not in adulthood, increases miR-218 in the VTA and this event is required for drug-induced downregulation of Dcc mRNA and protein expression. This effect seems to be specific to Dcc because amphetamine does not alter Robo1. Furthermore, the upregulation of miR-218 by amphetamine requires dopamine D2 receptor activation. These findings identify miR-218 as regulator of DCC in the VTA both in normal development and after drug exposure in adolescence.

Netrin-1, an axon guidance protein, is difficult to detect using immunohistochemistry. We performed a multi-step, blinded, and controlled protocol optimization procedure to establish an efficient and effective fluorescent immunohistochemistry protocol for characterizing Netrin-1 expression. Coronal mouse brain sections were used to test numerous antigen retrieval methods and combinations thereof in order to optimize the stain quality of a commercially available Netrin-1 antibody. Stain quality was evaluated by experienced neuroanatomists for two criteria: signal intensity and signal-to-noise ratio. After five rounds of testing protocol variants, we established a modified immunohistochemistry protocol that produced a Netrin-1 signal with good signal intensity and a high signal-to-noise ratio. The key protocol modifications are as follows: •Use phosphate buffer (PB) as the blocking solution solvent.•Use 1% sodium dodecyl sulfate (SDS) treatment for antigen retrieval. The original protocol was optimized for use with the Netrin-1 antibody produced by Novus Biologicals. However, we subsequently further modified the protocol to work with the antibody produced by Abcam. The Abcam protocol uses PBS as the blocking solution solvent and adds a citrate buffer antigen retrieval step.

The fine arrangement of neuronal connectivity during development involves the coordinated action of guidance cues and their receptors. In adolescence, the dopamine circuitry is still developing, with mesolimbic dopamine axons undergoing target-recognition events in the nucleus accumbens (NAcc), while mesocortical projections continue to grow toward the prefrontal cortex (PFC) until adulthood. This segregation of mesolimbic versus mesocortical dopamine pathways is mediated by the guidance cue receptor DCC, which signals dopamine axons intended to innervate the NAcc to recognize this region as their final target. Whether DCC-dependent mesolimbic dopamine axon targeting in adolescence requires the action of its ligand, Netrin-1, is unknown. Here we combined shRNA strategies, quantitative analysis of pre- and post-synaptic markers of neuronal connectivity, and pharmacological manipulations to address this question. Similar to DCC levels in the ventral tegmental area, Netrin-1 expression in the NAcc is dynamic across postnatal life, transitioning from high to low expression across adolescence. Silencing Netrin-1 in the NAcc in adolescence results in an increase in the expanse of the dopamine input to the PFC in adulthood, with a corresponding increase in the number of presynaptic dopamine sites. This manipulation also results in altered dendritic spine density and morphology of medium spiny neurons in the NAcc in adulthood and in reduced sensitivity to the behavioral activating effects of the stimulant drug of abuse, amphetamine. These cellular and behavioral effects mirror those induced by Dcc haploinsufficiency within dopamine neurons in adolescence. Dopamine targeting in adolescence requires the complementary interaction between DCC receptors in mesolimbic dopamine axons and Netrin-1 in the NAcc. Factors regulating either DCC or Netrin-1 in adolescence can disrupt mesocorticolimbic dopamine development, rendering vulnerability or protection to phenotypes associated with psychiatric disorders.

For some individuals, social stress is a risk factor for psychiatric disorders characterised by adolescent onset, prefrontal cortex (PFC) dysfunction and cognitive impairments. Social stress may be particularly harmful during adolescence when dopamine (DA) axons are still growing to the PFC, rendering them sensitive to environmental influences. The guidance cue Netrin-1 and its receptor, DCC, coordinate to control mesocorticolimbic DA axon targeting and growth during this age. Here we adapted the accelerated social defeat (AcSD) paradigm to expose male mice to social stress in either adolescence or adulthood and categorised them as "resilient" or "susceptible" based on social avoidance behaviour. We examined whether stress would alter the expression of DCC and Netrin-1 in mesolimbic dopamine regions and would have enduring consequences on PFC dopamine connectivity and cognition. While in adolescence the majority of mice are resilient but exhibit risk-taking behaviour, AcSD in adulthood leads to a majority of susceptible mice without altering anxiety-like traits. In adolescent, but not adult mice, AcSD dysregulates DCC and Netrin-1 expression in mesolimbic DA regions. These molecular changes in adolescent mice are accompanied by changes in PFC DA connectivity. Following AcSD in adulthood, cognitive function remains unaffected, but all mice exposed to AcSD in adolescence show deficits in inhibitory control when they reach adulthood. These findings indicate that exposure to AcSD in adolescence vs. adulthood has substantially different effects on brain and behaviour and that stress-induced social avoidance in adolescence does not predict vulnerability to deficits in cognitive performance.Significance statement During adolescence, dopamine circuitries undergo maturational changes which may render them particularly vulnerable to social stress. While social stress can be detrimental to adolescents and adults, it may engage different mechanisms and impact different domains, depending on age. The accelerated social defeat (AcSD) model implemented here allows exposing adolescent and adult male mice to comparable social stress levels. AcSD in adulthood leads to a majority of socially avoidant mice. However, the predominance of AcSD-exposed adolescent mice does not develop social avoidance, and these resilient mice show risk-taking behaviour. Nonetheless, in adolescence only, AcSD dysregulates Netrin-1/DCC expression in mesolimbic dopamine regions, possibly disrupting mesocortical dopamine and cognition. The unique adolescent responsiveness to stress may explain increased psychopathology risk at this age.