Different Roles of D1/D2 Medium Spiny Neurons in the Nucleus Accumbens in Pair Bond Formation of Male Mandarin Voles

Reviewer #1 (Public review):

These experiments are some of the first to assess the role of dopamine release and the activity of D1 and D2 MSNs in pair bond formation in Mandarin voles. This is a novel and comprehensive study that presents exciting data about how the dopamine system is involved in pair bonding. The authors provide very detailed methods and clearly presented results. Here they show dopamine release in the NAc shell is enhanced when male voles encounter their pair bonded partner 7 days after co-habitation. In addition, D2 MSN activity decreases whereas D1 MSN activity increases when sniffing the pair-bonded partner.

The authors do not provide justification for why they only use males in the current study, without discussing sex as a biological variable these data can only inform readers about one sex (which in pair-bonded animals by definition have 2 sexes). In addition, the authors do not use an isosbestic control wavelength in photometry experiments, although they do use EGFP control mice which show no effects of these interventions, a within-subject control such as an isosbestic excitation wavelength could give more confidence in these data and rule out motion artefacts within subjects.

There is an existing literature (cited in this manuscript) from Aragona et al., (particularly Aragona et al., 2006) which has highlighted key differences in the roles of rostral versus caudal NAc shell dopamine in pair bond formation and maintenance. Specifically, they report that dopamine transmission promoting pair bonding only occurs in the rostral shell and not the caudal shell or core regions. Given that the authors have targeted more caudally a discussion of how these results fit with previous work and why there may be differences in these areas is warranted.

The authors could discuss the differences between pair bond formation and pair bond maintenance more deeply.

The authors have successfully characterised the involvement of dopamine release, changes in D1 and D2 MSNs, and projections to the VP in pair bonding voles. Their conclusions are supported by their data and they make a number of very reasonable discussion points acknowledging various limitations.

Reviewer #2 (Public review):

Summary:

Using in vivo fiber-photometry the authors first establish that DA release when contacting their partner mouse increases with days of cohabitation while this increase is not observed when contacting a stranger mouse. Similar effects are found in D1-MSNs and D2-MSNs with the D1-MSN responses increasing and D2-MSN responses decreasing with days of cohabitation. They then use slice physiology to identify underlying plasticity/adaptation mechanisms that could contribute to the changes in D1/D2-MSN responses. Last, to address causality the authors use chemogenetic tools to selectively inhibit or activate NAc shell D1 or D2 neurons that project to the ventral pallidum. They found that D2 inhibition facilitates bond formation while D2 excitation inhibits bond formation. In contrast, both D1-MSN activation and inhibition inhibit bond formation.

Strengths:

The strength of the manuscript lies in combining in vivo physiology to demonstrate circuit engagement and chemogenetic manipulation studies to address circuit involvement in pair bond formation in a monogamous vole.

Weaknesses:

Weaknesses include that a large set of experiments within the manuscript are dependent on using short promoters for D1 and D2 receptors in viral vectors. As the authors acknowledge this approach can lead to ectopic expression and the presented immunohistochemistry supports this notion. It seems to me that the presented quantification underestimates the degree of ectopic expression that is observed by eye when looking at the presented immunohistochemistry. However, given that Cre transgenic animals are not available for Microtus mandarinus and given the distinct physiological and behavioral outcomes when imaging and manipulating both viral-targeted populations this concern is minor.

The slice physiology experiments provide some interesting outcomes but it is unclear how they can be linked to the in vivo physiological outcomes and some of the outcomes don't match intuitively (e.g. cohabitation enhances excitatory/inhibitory balance in D2-MSNs but the degree of contact-induced inhibition is enhanced in D2-MSN).

One interesting finding is that the relationship between D2-MSN and pair bond formation is quite clear (inhibition facilitates while excitation inhibits pair bond formation). In contrast, the role of D1-MSNs is more complicated since both excitation and inhibition disrupt pair bond formation. This is not convincingly discussed.

It seemed a missed opportunity that physiological readout is limited to males. I understand though that adding females may be beyond the scope of this manuscript.

Reviewer #3 (Public review):

Summary:

The manuscript is evaluating changes in dopamine signaling in the nucleus accumbens following pair bonding and exposure to various stimuli in mandarin voles. In addition, the authors present chemogenetic data that demonstrate excitation and inhibition of D1 and D2 MSN affect pair bond formation.

Strengths:

The experimental designs are strong. The approaches are innovative and use cutting-edge methods. The manuscript is well written.

Weaknesses:

The statistical results are not presented, and not all statistical analyses are appropriate. Additionally, some details of methods are absent.