Oxytocin neurons mediate the effect of social isolation via the VTA circuits
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Evaluation Summary:
This study examined the effects of social isolation in adolescent and adult male mice, a topic timely and relevant. The work sheds light on oxytocin as a key regulator that modulates the dopaminergic midbrain imparting long-lasting effects on social interaction. A critical open question is whether these results would apply to female subjects. The findings will merit from more thorough interpretations and controls of social behavior data and synaptic plasticity. This paper will be of interest to those interested in social neuroscience and plasticity in general.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Social interaction during adolescence strongly influences brain function and behavior, and the recent pandemic has emphasized the devastating effect of social distancing on mental health. While accumulating evidence has shown the importance of the reward system in encoding specific aspects of social interaction, the consequences of social isolation on the reward system and the development of social skills later in adulthood are still largely unknown. Here, we found that 1 week of social isolation during adolescence in male mice increased social interaction at the expense of social habituation and social novelty preference. Behavioral changes were accompanied by the acute hyperexcitability of putative dopamine (pDA) neurons in the ventral tegmental area and long-lasting expression of GluA2-lacking AMPARs at excitatory inputs onto pDA neurons that project to the prefrontal cortex. Social isolation-dependent behavioral deficits and changes in neural activity and synaptic plasticity were reversed by chemogenetic inhibition of oxytocin neurons in the paraventricular nucleus of the hypothalamus. These results demonstrate that social isolation in male mice has acute and long-lasting effects on social interaction and suggest that homeostatic adaptations mediate these effects within the reward circuit.
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Evaluation Summary:
This study examined the effects of social isolation in adolescent and adult male mice, a topic timely and relevant. The work sheds light on oxytocin as a key regulator that modulates the dopaminergic midbrain imparting long-lasting effects on social interaction. A critical open question is whether these results would apply to female subjects. The findings will merit from more thorough interpretations and controls of social behavior data and synaptic plasticity. This paper will be of interest to those interested in social neuroscience and plasticity in general.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Musardo and colleagues explore the neurobiological impact of social isolation on the adolescent versus adult male mouse brain. The authors suggest that isolation alters subsequent social behaviors by causing increased excitability of Oxytocin neurons in the PVN and, subsequently, increased insertion of GluA2-lacking AMPARs in DA neurons of the VTA that project to PFC.
Strengths:
• The authors examine the acute effects of social isolation during 2 periods of adolescence and during a period of young adulthood. They show that acute social isolation in adolescent mice from P28-P35 (but not P21-P28) increases subsequent social interaction time while decreasing habituation to social novelty. In contrast, isolation during adulthood has the opposite effect in that it reduces social interactions.
• When …
Reviewer #1 (Public Review):
Musardo and colleagues explore the neurobiological impact of social isolation on the adolescent versus adult male mouse brain. The authors suggest that isolation alters subsequent social behaviors by causing increased excitability of Oxytocin neurons in the PVN and, subsequently, increased insertion of GluA2-lacking AMPARs in DA neurons of the VTA that project to PFC.
Strengths:
• The authors examine the acute effects of social isolation during 2 periods of adolescence and during a period of young adulthood. They show that acute social isolation in adolescent mice from P28-P35 (but not P21-P28) increases subsequent social interaction time while decreasing habituation to social novelty. In contrast, isolation during adulthood has the opposite effect in that it reduces social interactions.
• When adolescently-isolated mice are regrouped and tested as adults, the isolation-induced increase in social interaction persists, but not the reduction in habituation to social novelty, suggesting some effects of adolescent social isolation are long-lasting.
• The authors show acute adolescent social isolation increases activity of PVN neurons projecting to the VTA concomitant with an increased density of PVN oxytocin neurons. They also show that regrouping mice eliminates the increased activity of PVN and VTA neurons but results in a long-lasting increased rectification index for VTA neurons projecting to the PFC.
• The authors then go on to establish a causal role for oxytocin signaling in isolation-induced changes in social behavior by showing that inhibiting the activity of all oxytocin neurons using a chemogenetic approach prevents the effects of social isolation on 1) subsequent social behavior and 2) the electrophysiology of VTA neurons (although, not specifically PVN-VTA neurons). They further show that globally inhibiting GluA2-lacking AMPARs in the VTA rescues the long-lasting effects of isolation on social interaction behavior in the regrouped mice.
Weaknesses:
• It does not appear that inhibition of oxytocin neurons was specific to the PVN, leaving room for effects to be ascribed to other nuclei (e.g., supraoptic nucleus). The inhibition of GluA2-lacking AMPARs in the VTA was not specific to DA neurons nor to neurons projecting to the PFC. Nor was the VTA inhibition of these AMPARs performed in the acute social isolation condition, which exhibited more wide ranging social behavioral effects. Social isolation may affect oxytocin neurons elsewhere in the brain, and it may be inhibition there that rescues the behavior and VTA electrophysiological phenotypes. Even if it was increased activity of oxytocin neurons specifically in the PVN that was responsible, that increased excitability may well have other downstream effects beyond increasing excitability of VTA neurons. These limitations are not acknowledged in the manuscript.
• The paper describes significant differences between rodents and humans in terms of how social isolation affects the circuity of interest, yet there is no discussion of this in terms of limited applicability of the present findings to humans. This is particularly troubling when only male mice are tested.
• The social behavioral changes triggered by social isolation are interpreted with a relatively narrow and negative light. We urge caution in interpreting the present phenotypes as absolute negatives, particularly when the main phenotype associated with adolescent social isolation is a subsequent increase in social interaction. Perhaps these are better characterized as changes in preference for social interaction as opposed to social deficits-or they could be interpreted as changes in social recognition memory in some cases. Note that recent work in other mouse models has revealed that social approach behavior can significantly shift depending on whether or not the neurobiology of the subject and the stimulus mouse match. Very different patterns of social interactions might be observed if group vs. isolated mice were tested using an isolated stimulus mice as opposed to a group-housed stimulus mouse (see a cautionary tale: Smith et al., 2021 Molecular Psychiatry, https://www.nature.com/articles/s41380-021-01237-4?proof=t). Use of the term "social craving" so definitively throughout the discussion also seems unwarranted based on the type of data presented (e.g., no tests of motivation to engage in social interactions were presented). Craving implies having experienced something that is very rewarding and wanting more of that thing; however, these mice never experienced any social interactions with non-cage mates, so how can they crave it? To this end, it might be informative to test these behaviors using cage mates as stimulus mice.
• Many of the statistical analyses employed/reported were not appropriate (e.g., use of 2-Way ANOVAs when repeated measure ANOVAs should have been used; reporting main effects when a significant interaction between main effects was necessary to support the post hoc tests used and the conclusions made).
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Reviewer #2 (Public Review):
Musardo et al have evaluated the lasting effects of acute social isolation on future social interactions in juvenile mice, revealing a compelling oxytocin-mediated mechanism. The authors laid out clear hypothesis within a well-contextualized framework, and evaluated them using appropriate behavioral paradigms, chemogenetic, and pharmacological tools. The work sheds light on Oxytocin as a key regulator that modulates DA neurons in VTA projecting to the mPFC imparting long-lasting effects on social interaction. Conceptually, this work provides important information on neural signatures of social craving.
Neurobehavioral disorders that impact sociality, such as Autism spectrum disorder (ASD) are sexually dimorphic in occurrence. While the behavioral experiments in this study are well controlled, only males are …
Reviewer #2 (Public Review):
Musardo et al have evaluated the lasting effects of acute social isolation on future social interactions in juvenile mice, revealing a compelling oxytocin-mediated mechanism. The authors laid out clear hypothesis within a well-contextualized framework, and evaluated them using appropriate behavioral paradigms, chemogenetic, and pharmacological tools. The work sheds light on Oxytocin as a key regulator that modulates DA neurons in VTA projecting to the mPFC imparting long-lasting effects on social interaction. Conceptually, this work provides important information on neural signatures of social craving.
Neurobehavioral disorders that impact sociality, such as Autism spectrum disorder (ASD) are sexually dimorphic in occurrence. While the behavioral experiments in this study are well controlled, only males are used as subjects, limiting the potential impact of this work.
The study used unknown sex-matched conspecific juveniles in direct free interaction and three-chamber tasks (one week younger than the experimental animal; Page 9 line 18,31). There is no explanation for choosing this specific age and age difference. Does the exposure to same-age juvenile or adult sex-matched conspecifics elicit a different behavioral response?
Line 29-31 page 3 states that social isolation-dependent behavioral deficits are age-dependent and isolated adult mice show less social interaction compared to isolated juveniles. The authors should further elaborate on this interesting point and consider developmental differences in oxytocin system that could relate to it.
cFOS+ and Oxt+ coexpression images are needed to relate the increase in the activity of PVN (via the IEG proxy) and the density of Oxt+ cells in PVN (figure 3 and page 4, line 11-17). Also, it is unclear what is happening here-are neurons increasing Oxt production to become more efficiently labeled or changing identity of their synthesized neuropeptides?
The expression of inhibitory (Oxt-hM4Di) DREADD in the PVN should be illustrated with immunohistochemical evidence to demonstrate expression and selectivity. Some interpretation related to whether SON Oxt neurons, labeled in the cross, could be involved in the observed effects would be useful.
Page 11, lines 30-32: The authors identified putative DA neurons based on position, morphology, and capacitance, while providing sparse details in the methods.
The authors have exclusively shown scaled-up excitatory responses by pharmacologically blocking GABAA receptors, determining synaptic scaling after social isolation without considering potential effects on inhibitory post synaptic responses. Can any predictions be made?
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Reviewer #3 (Public Review):
This study investigated how one week of adolescent social isolation (p28-p35) in mice impact VTA dopamine neurons and social behavior acutely at p35 and later in the adulthood at molecular and circuit level. Authors found that isolation led to increase in social interaction in free social interaction test, but no change in sociability during 3 chamber test. At the neural level, they observed excitability of VTA DA neurons are increased at p35 but not in adulthood. However, in the adult isolated mice, they found long-lasting expression of GluA2-lacking AMPARs at excitatory inputs onto DA neurons projecting to frontal cortex. Importantly, they found that the excitability of oxytocin expressing PVN neurons projecting to VTA is enhanced by isolation, and this change seems to have a causal impact to isolation …
Reviewer #3 (Public Review):
This study investigated how one week of adolescent social isolation (p28-p35) in mice impact VTA dopamine neurons and social behavior acutely at p35 and later in the adulthood at molecular and circuit level. Authors found that isolation led to increase in social interaction in free social interaction test, but no change in sociability during 3 chamber test. At the neural level, they observed excitability of VTA DA neurons are increased at p35 but not in adulthood. However, in the adult isolated mice, they found long-lasting expression of GluA2-lacking AMPARs at excitatory inputs onto DA neurons projecting to frontal cortex. Importantly, they found that the excitability of oxytocin expressing PVN neurons projecting to VTA is enhanced by isolation, and this change seems to have a causal impact to isolation induced DA neuron and behavioral changes as chemogenetic suppression of oxytocin neuron activity during isolation period prevented these changes.
Overall, these findings support a key role of oxytocin expressing PVN neurons in regulating the adolescent isolation induced changes in VTA dopamine neuron excitability in adolescent mice, and synaptic function onto VTA neurons projecting to NAc through CP-AMPA receptor in adulthood. These findings are significant to the field as it provides the molecular and circuit mechanism mediating the effect of adolescent social isolation on social behavior. While the questions and findings are overall interesting and significant, there are some concerns with the interpretations of social behavior data and synaptic plasticity results that will benefit from more careful considerations and addition of control experiments.
Regarding the interpretation of social behavior data, as isolated mice show increased social interaction during direct social interaction, but not during 3 chamber sociability assay, it is informative to more thoroughly explore why isolated mice show different outcomes between these two tests. To better understand the cause of increased direct social interaction (social craving vs aggression), it would be informative to conduct additional behavioral testings from isolated mice (e.g. social CPP, aggression assay). In addition, more discussion on possible factors that could influence the long-lasting effects of adolescent social isolation on adult social behavior is helpful as previous studies found juvenile social isolation rather leads to reduced sociability, and reduced active approach in free direct interaction.
Regarding other concerns, the current manuscript did not test whether the deprivation induced synaptic scaling changes reported in Fig 5 only emerges slowly in the adult, or it is already acutely happening at the end of isolation at p35. This information would help to interpret whether the synaptic changes are due to chronic adaptation uniquely happening slowly or not. It is also unclear how oxytocin neuron activity contributes to synaptic plasticity onto VTA-NAc projection. One possibility is that oxytocin expressing PVN neurons projecting to VTA themselves release glutamate and contribute to synaptic plasticity. It would be informative to optogenetically examine the synaptic connectivity between PVN->VTA projections and VTA->NAc projection neurons in isolated mice. Finally, there are some concerns of the selection of statistical testings, and data presentation.
Overall, this study revealed novel contributions of oxytocin PNV neurons in regulating adolescent isolation induced changes in VTA dopamine neurons to impact social behavior. More thorough interpretation of social behavior data and control experiments related to synaptic plasticity in VTA would help better understand the interesting findings made in this study.
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