A “pathway” of Netrin-1 expressing cells “paves the way” for dopamine (DA) axons growing from the nucleus accumbens to the medial prefrontal cortex during adolescence. A, The brain regions containing the bundle of dopamine fibres passing to the medial prefrontal cortex are highlighted in a line drawing of a coronal mouse brain section derived from Paxinos and Franklin (Paxinos and Franklin, 2013). B, An image of a coronal section through the forebrain of an adult mouse at low magnification (4x). Green fluorescence indicates immunostaining for tyrosine hydroxylase (TH), a marker for dopamine. The smaller and larger white squares indicate the regions enlarged in panel C and panels F & G, respectively. Scale bar = 500 μm. C, The nucleus accumbens (left) is densely packed with dopamine axons (in green). Some of these axons can be observed extending from the nucleus accumbens medially into a bundle of dopamine fibres (white arrows). Scale bar = 10 μm. D, Modified stereological quantification revealed no significant difference in dopamine axon density in the fibre bundle between adolescence (21 days old) and adulthood (75 days old). Mixed-effects ANOVA, effect of age: F=1.53, p=0.22; region by age interaction: F=1.44, p=0.49. E, The average width of the fibre bundle increased significantly from adolescence to adulthood, revealing that there is an increase in the total number of fibres passing to the medial prefrontal cortex during this period. Mixed-effects ANOVA, effect of age: F=9.45, p=0.0021; region by age interaction: F=5.74, p=0.057. F, In order to quantify the Netrin-1 positive cells along the dopamine fibre bundle, the bundle was contoured in each region, and a contour of equal area was placed medial to the fibre bundle as a negative control. Scale bar = 200 μm. G, Using quantitative stereology, Netrin-1 positive cell density was determined along and adjacent to the fibre bundle for each region. Red fluorescence indicates immunostaining for Netrin-1.H, In adolescent mice there are more Netrin-1 positive cells along the dopamine fibre bundle (“TH+”) than medial to it (“TH-”). This is what we refer to as the “Netrin-1 pathway”. Along the pathway, there is a significant increase in Netrin-1 positive cell density in regions closer to the medial prefrontal cortex, the innervation target. Mixed-effects ANOVA, effect of TH: F=105, p<0.0001. Effect of region: F=9.74, p=0.021. A post-hoc Tukey Test revealed a difference (p = 0.029) between the densities of the lateral septum and infralimbic cortex, but only within the dopamine fibre bundle. I, In adult mice the Netrin-1 pathway is maintained, however there is no longer an increasing density of Netrin-1 positive cells towards the medial prefrontal cortex. Mixed-effects ANOVA, effect of TH: F=54.56, p<0.0001. Effect of region: F=1.22, p=0.75. J, The virus injection location within the mouse brain. A Netrin-1 knockdown virus, or a control virus, was injected into the dopamine fibre bundle at the level of the dorsal peduncular cortex. K, Our experimental timeline: at the onset of adolescence a Netrin-1 knockdown virus, or a control virus, was injected, and then in adulthood the mice were sacrificed and stereological measurements taken. L, Dopamine varicosity density was quantified in the region below the injection site, in the lateral septum, and in the region above the injection site, in the infralimbic cortex. There was a significant decrease in dopamine varicosity density only in the infralimbic cortex. Mixed-effects ANOVA, virus by region interaction: F=16.41, p<0.0001. M, The experimental set-up of the final (test) stage of the Go/No-Go experimental protocol. A mouse that has previously learned to nose-poke for a reward in response to a visual cue (illuminated nose-poke hole), must now inhibit this behaviour when the visual cue is paired with an auditory cue (acoustic tone). N, Mice injected with the Netrin-1 knockdown virus show improved action impulsivity than controls because they incur significantly fewer commission errors across the Go/No-go task. Mixed-effects ANOVA, effect of day: F=68.32, p<0.0001. Day by virus interaction: F=9.00, p=0.0027. A sigmoidal curve is fit to each group of mice to determine how the two groups differ. Points indicate group means and error bars show standard error means. O, During the first days of Go/No-Go testing, both groups incur commission errors with high frequency, but, the Netrin-1 knockdown group has fewer errors than the control group (t-test, t=5.18, p<0.0001. P, The ED50 – the inflection point in each sigmoidal curve – are not different between groups, indicating that all mice improve their ability to inhibit their behavior at around the same time (T-test, t=0.97, p=0.35). Q, Mice microinfused with the Netrin-1 knockdown virus incur substantially fewer commission errors in the last days of the Go/No-Go task compared to mice injected with the control virus (T-test, t=12.38, p<0.0001). For all barplots, bars indicate group means and error bars show standard error means.

The age of onset of UNC5C expression by dopamine axons in the nucleus accumbens of mice is sexually dimorphic. Images are representative of observed immunofluorescence patterns in the nucleus accumbens core (approx. location highlighted in the coronal mouse brain section Plate 19, modified from Paxinos & Franklin, 2013). For each row, six individuals were sampled. In males (A-B), UNC5C expression on dopamine fibres in the nucleus accumbens appears during adolescence. A, At the onset of adolescence (21 days old) dopamine fibres do not express UNC5C. Scale bar = 10 μm. B, By adulthood (90 days old), dopamine fibres express UNC5C. In females (C-E), UNC5C expression on dopamine fibres in the nucleus accumbens appears prior to adolescence. C, In juvenile (15 day old) mice, there is no UNC5C expression on dopamine fibres. D, By adolescence, dopamine fibres express UNC5C. E, In adulthood, dopamine fibres continue to express UNC5C.

Plasticity of adolescent development in male Siberian hamsters according to seasonal phenotype. All results illustrated in this figure refer to results in male hamsters. A, Dopamine innervation was quantified in three subregions of the medial prefrontal cortex, highlighted in blue. UNC5C expression was examined in the nucleus accumbens, highlighted in red. Line drawing of a coronal section of the mouse brain was derived from Plate 19 of Paxinos and Franlkin (Paxinos and Franklin, 2013). B, Hamsters were housed under either summer-mimicking long days and short nights (“summer hamsters”) or winter-mimicking short days and long nights (“winter hamsters”). C, In male hamsters housed under a summer-mimicking daylength there is an increase in dopamine varicosity density in the medial prefrontal cortex between 15 and 80 days old. Mixed-effects ANOVA, effect of age: F=9.6, p=0.000255. Tukey Test, 15-80 days old (do): p=0.026; 80-215do: p<0.0001; 15-215do: p<0.0001. D, In male hamsters housed under a winter-mimicking daylength there is no increase in dopamine varicosity density until hamsters have reached 215 days of age. Mixed-effects ANOVA, effect of age: F=4.17, p=0.0205. Tukey Test, 15-80do: p=0.54; 80-215do: p=0.0006; 15-215do: p=0.0085. E, At 15 days old, dopamine axons in the nucleus accumbens of male summer-daylength hamsters largely do not express UNC5C. Scale bar = 20um (bottom right). F-G, At 80 (F) and 215 (G) days old, dopamine axons in the nucleus accumbens express UNC5C. H-I, At 15 (H) and 80 (I) days old, dopamine axons in the nucleus accumbens of male winter hamsters largely do not express UNC5C. J, By 215 days old there is UNC5C expression in dopamine axons in the nucleus accumbens of male winter hamsters. E-J, Representative images of the nucleus accumbens shell, 6 individuals were examined per group. K, Male hamsters house under a summer-mimicking daylength show an adolescent peak in exploratory behaviour in the light/dark box apparatus. Instead, those raised under a winter-mimicking photoperiod show a steady increase in exploratory behaviour over the same age range. Arrows indicate the ages at which exploratory behaviour peaks in summer (orange) and winter (blue) hamsters. Polynomial regression, effect of season: F=3.551, p=0.00056. L, In male hamsters, at 215 days of age, there is no difference in exploratory behaviour between hamsters raised under summer and winter photoperiods. T-test, effect of season: t=0.975, p=0.341. For all barplots, bars indicate group means and error bars show standard error means.

Plasticity of adolescent development in female Siberian hamsters according to seasonal phenotype. All results illustrated in this figure refer to results in female hamsters. A, Dopamine innervation was quantified in three subregions of the medial prefrontal cortex, highlighted here in blue. UNC5C expression was examined in the nucleus accumbens, highlighted in red. Line drawing of a coronal section of the mouse brain was derived from Paxinos and Franlkin (Paxinos and Franklin, 2013). B, Hamsters were housed under either a summer-mimicking or winter-mimicking daylength. C, In female hamsters housed under a summer daylength dopamine varicosity density in the medial prefrontal cortex increases between 15 and 80 days of age. Mixed-effects ANOVA, effect of age: F=16.72, p<0.0001 D, In female hamsters housed under a winter daylength there is no increase in dopamine varicosity density post-adolescence. Instead, there is a steep decline in density between 80 and 215 days of age. Mixed-effects ANOVA, effect of age: F=12.33, p=0.000043. E, As our results in panel D were unexpected, we replicated them with a second cohort of hamsters and found qualitatively identical results. Mixed-effects ANOVA, effect of age: F=34.871, p<0.0001. F, To try and determine when dopamine varicosities innervate the medial prefrontal cortex, we examined a cohort of 10- and 15-day-old hamsters. We found that varicosity density increases in the medial prefrontal cortex during this time, indicating that dopamine innervation to the medial prefrontal cortex is accelerated in female winter hamsters. Mixed-effects ANOVA, effect of age: F=5.05, p=0.03. G-H, In 10- and 15-day-old female summer hamsters there is little UNC5C expression in nucleus accumbens dopamine axons. Sample size: 4 (panel G) or 6 (panel H). I-J, By 80 days old (panel I), and continuing at 215 days old (panel J), dopamine axons in the nucleus accumbens express UNC5C in female summer hamsters. Sample sizes: 6. Scale bar = 20um (panel G bottom right). K-N, At all ages at which winter female hamsters were examined dopamine axons in the nucleus accumbens express UNC5C in winter female hamsters. Sample sizes: 4 (panel K) or 6 (panels L-N). O, In female hamsters, those raised under summer and winter daylengths both show an increase in exploratory behaviour over time. The winter hamsters peak later compared to the summer daylength hamsters. Arrows indicate the ages at which exploratory behaviour peaks in summer (orange) and winter (blue) hamsters. Polynomial regression, effect of season: F=3.305, p=0.00126. P, In female hamsters, at 215 days of age, there is no difference in exploratory behaviour between hamsters raised under summer and winter photoperiods. T-test, effect of season: t=0.309, p=0.76. For all barplots, bars indicate group means and error bars show standard error means.