The scheduling of adolescence with Netrin-1 and UNC5C

Reviewer #1 (Public Review):

In this study, Hoops et al. showed that Netrin-1 and UNC5c can guide dopaminergic innervation from nucleus accumbens to cortex during adolescence in rodent models. They found that these dopamine axons project to the prefrontal cortex in a Netrin-1 dependent manner and knocking down Netrin-1 disrupted motor and learning behaviors in mice. Furthermroe, the authors used hamsters, a seasonal model that is affected by the length of daylight, to demonstrate that the guidance of dopamine axons is mediated by the environmental factor such as daytime length and in sex dependent manner. While this study highlighted the important roles of two neurodevelopmental markers, netrin-1 and UNC5C, in the projection of dopaminergic azons in the adolescence/adult brain, the major weakness is that the data are quite superficial and do not establish any definitive evidence to support the causal relationship between the expression of netrin-1 and UNC5C in the projection of dopaminergic axons remain unclear.
Below are several major concerns regarding the data presented in this manuscript:

1. Despite the well-established role of Netrin-1 and UNC5C axon guidance during embryonic commissural axons, it remains unclear which cell type(s) express Netrin-1 or UNC5C in the dopaminergic axons and their targets. For instance, the data in Figure 1F-G and Figure 2 are quite confusing. Does Netrin-1 or UNC5C express in all cell types or only dopamine-positive neurons in these two mouse models? It will also be important to provide quantitative assessments of UNC5C expression in dopaminergic axons at different ages.

2. Figure 1 used shRNA to knockdown Netrin-1 in the Septum and these mice were subjected to behavioral testing. These results, again, are not supported by any valid data that the knockdown approach actually worked in dopaminergic axons. It is also unclear whether knocking down Netrin-1 in the septum will re-route dopaminergic axons or lead to cell death in the dopaminergic neurons in the substantia nigra pars compacta?

3. Another issue with Figure1J. It is unclear whether the viruses were injected into a WT mouse model or into a Cre-mouse model driven by a promoter specifically expresses in dorsal peduncular cortex? The authors should provide evidence that Netrin-1 mRNA and proteins are indeed significantly reduced. The authors should address the anatomic results of the area of virus diffusion to confirm the virus specifically infected the cells in dorsal peduncular cortex.

4. The authors need to provide information regarding the efficiency and duration of knocking down. For instance, in Figure 1K, the mice were tested after 53 days post injection, can the virus activity in the brain last for such a long time?

5. In Figure 1N-Q, silencing Netrin-1 results in less DA axons targeting to infralimbic cortex, but why the Netrin-1 knocking down mice revealed the improved behavior?

6. What is the effect of knocking down UNC5C on dopamine axons guidance to the cortex?

7. In Figures 2-4, the authors only showed the amount of DA axons and UNC5C in NAcc. However, it remains unclear whether these experiments also impact the projections of dopaminergic axons to other brain regions, critical for the behavioral phenotypes. What about other brain regions such as prefrontal cortex? Do the projection of DA axons and UNC5c level in cortex have similar pattern to those in NAcc?

8. Can overexpression of UNC5c or Netrin-1 in male winter hamsters mimic the observations in summer hamsters? Or overexpression of UNC5c in female summer hamsters to mimic the winter hamster? This would be helpful to confirm the causal role of UNC5C in guiding DA axons during adolescence.

9. The entire study relied on using tyrosine hydroxylase (TH) as a marker for dopaminergic axons. However, the expression of TH (either by IHC or IF) can be influenced by other environmental factors, that could alter the expression of TH at the cellular level.

10. Are Netrin-1/UNC5C the only signal guiding dopamine axon during adolescence? Are there other neuronal circuits involved in this process?

11. Finally, despite the authors' claim that the dopaminergic axon project is sensitive to the duration of daylight in the hamster, they never provided definitive evidence to support this hypothesis.

Reviewer #2 (Public Review):

In this manuscript, Hoops et al., using two different model systems, identified key developmental changes in Netrin-1 and UNC5C signaling that correspond to behavioral changes and are sensitive to environmental factors that affect the timing of development. They found that Netrin-1 expression is highest in regions of the striatum and cortex where TH+ axons are travelling, and that knocking down Netrin-1 reduces TH+ varicosities in mPFC and reduces impulsive behaviors in a Go-No-Go test. Further, they show that the onset of Unc5 expression is sexually dimorphic in mice, and that in Siberian hamsters, environmental effects on development are also sexually dimorophic. This study addresses an important question using approaches that link molecular, circuit and behavioral changes. Understanding developmental trajectories of adolescence, and how they can be impacted by environmental factors, is an understudied area of neuroscience that is highly relevant to understanding the onset of mental health disorders. I appreciated the inclusion of replication cohorts within the study.

Reviewer #3 (Public Review):

This study from the Flores group aims at understanding neuronal circuit changes during adolescence which is an ill-defined, transitional period involving dramatic changes in behavior and anatomy. They focus on DA innervation of the prefrontal cortex, and their interaction with the guidance cue Netrin-1. They propose DA axons in the PFC increase in the postnatal period, and their density is reduced in a Netrin 1 knockdown, suggesting that Netrin abets the development of this mesocortical pathway. In such mice impulsivity gauged by a go-no go task is reduced. They then provide some evidence that Unc5c is developmentally regulated in DA axons. Finally they use an interesting hamster model, to study the effect of light hours on mesocortical innervation, and make some interesting observations about the timing of innervation and Unc5c expression, and the fact that females housed in winter day length conditions display an accelerated innervation of the prefrontal cortex. While this work is novel and on an interesting, understudied topic, several aspects need to be further consolidated, to make it more persuasive.

Main comments
1. Fig 1 A and B don't appear to be the same section level.
2. Fig 1C. It is not clear that these axons are crossing from the shell of the NAC.
3. Fig 1. Measuring width of the bundle is an odd way to measure DA axon numbers. First the width could be changing during adult for various reasons including change in brain size. Second, I wouldn't consider these axons in a traditional bundle. Third, could DA axon counts be provided, rather than these proxy measures.
4. TH in the cortex could also be of noradrenergic origin. This needs to be ruled out to score DA axons
5. Netrin staining should be provided with NeuN + DAPI; its not clear these are all cell bodies. An in situ of Netrin would help as well.
6. The Netrin knockdown needs validation. How strong was the knockdown etc?
7. If the conclusion that knocking down Netrin in cortex decreases DA innervation of the IL, how can that be reconciled with Netrin-Unc repulsion.
8. The behavioral phenotype in Fig 1 is interesting, but its not clear if its related to DA axons/signaling. IN general, no evidence in this paper is provided for the role of DA in the adolescent behaviors described.
9. Fig2 - boxes should be drawn on the NAc diagram to indicate sampled regions. Some quantification of Unc5c would be useful. Also, some validation of the Unc5c antibody would be nice.
10. "In adolescence, dopamine neurons begin to express the repulsive Netrin-1 receptor UNC5C, and reduction in UNC5C expression appears to cause growth of mesolimbic dopamine axons to the prefrontal cortex".....This is confusing. Figure 2 shows a developmental increase in UNc5c not a decrease. So when is the "reduction in Unc5c expression" occurring?
11. In Fig 3, a statistical comparison should be made between summer male and winter male, to justify the conclusions that the winter males have delayed DA innervation.
12. Should axon length also be measured here (Fig 3)? It is not clear why the authors have switched to varicosity density. Also, a box should be drawn in the NAC cartoon to indicate the region that was sampled.
13. In Fig 3, Unc5c should be quantified to bolster the interesting finding that Unc5c expression dynamics are different between summer and winter hamsters. Unc5c mRNA experiments would also be important to see if similar changes are observed at the transcript level.
14. Fig 4. The peak in exploratory behavior in winter females is counterintuitive and needs to be better discussed. IN general, the light dark behavior seems quite variable.