Stress plays a crucial role in adaptation, but prolonged stress can negatively impact brain function and contribute to psychiatric disorders like anxiety, PTSD, and depression. Individual responses to stress vary widely, with some exhibiting resilience and others showing susceptibility. The diathesis-stress hypothesis suggests that biological predispositions increase vulnerability to stress. The brain's serotonergic network, a complex system regulating various behaviors, has been implicated in neuropsychiatric disorders due to dysregulation of serotonergic neurotransmission. Serotonin deficiency has been observed in some depression patients, and genetic mutations affecting serotonin transporters increase depression risk following stressful events. Many antidepressants and anxiolytics target the serotonergic system. The majority of serotonergic neurons reside in the dorsal raphe nucleus (DR), whose activity is crucial for reward-associated and emotional behaviors. Low serotonin levels increase susceptibility to stress-induced depression. The ventral tegmental area (VTA), involved in motivation and reward, undergoes aberrant dopamine neuronal adaptations after chronic stress, leading to depression-like behavior. This study investigated the role of the DR-VTA serotonergic circuit in mediating the effects of social stress and its electrophysiological adaptations in response to stress.

Existing research highlights the roles of stress and serotonergic dysregulation in psychiatric disorders, but the specific serotonergic circuit involved in stress vulnerability remained unclear. Studies indicate that the DR serotonergic system plays a critical role in reward-associated and emotional behaviors, and that low serotonin levels are linked to increased vulnerability to depression. The VTA, critical for motivation and reward, is also implicated in the development of depression-like behavior after chronic stress. While studies show that DR serotonergic neurons project to the VTA, their involvement in the stress response was largely unaddressed. This research builds upon existing knowledge by focusing on a specific subpopulation of DR serotonergic neurons projecting to the VTA.

The study used Sert-Cre mice, where Cre recombinase is selectively expressed in adult serotonergic neurons. A combination of viral vectors (AAVs) were employed to label and manipulate specific neuronal populations. To identify projection targets of DR serotonergic neurons, a double-floxed (DIO) Cre-dependent AAV vector expressing enhanced archaerhodopsin (eArch) fused with GFP was injected into the DR. Retrograde tracing using AAVretro expressing Cre-dependent fluorescent proteins (eYFP or mCherry) was performed in the mPFC, NAc, and VTA to identify VTA-projecting serotonergic neurons (5-HTDR→VTA neurons). An intersectional strategy using AAVretro expressing Cre-dependent Flp recombinase and AAV expressing eYFP in a Flp-dependent manner was used to comprehensively analyze axonal collateralization. Electrophysiological recordings (whole-cell patch-clamp) were used to investigate the electrophysiological properties of 5-HTDR→VTA neurons in control, susceptible, and resilient mice after chronic social defeat stress (CSDS). Optogenetic techniques (using ChR2 and eNpHR) were employed to bidirectionally manipulate the activity of 5-HTDR→VTA neurons, followed by behavioral assessments (social interaction, open field, elevated plus maze, tail suspension, and sucrose preference tests) to evaluate the impact on stress susceptibility. Immunohistochemistry was performed to validate viral expression and fiber placement. Statistical analyses included paired t-tests, one-way ANOVA, and two-way ANOVA.

The study identified a distinct subpopulation of DR serotonergic neurons projecting specifically to the VTA (5-HTDR→VTA neurons). These neurons showed significantly reduced spontaneous firing rates and intrinsic excitability in mice susceptible to CSDS compared to controls and resilient mice. Optogenetic inhibition of 5-HTDR→VTA neurons following subthreshold social defeat stress promoted stress vulnerability in stress-naive animals, while chronic silencing induced vulnerability in mice initially resilient to CSDS. Conversely, enhanced excitatory synaptic inputs onto 5-HTDR→VTA neurons were observed in resilient mice, and activation of this pathway conferred stress resilience. Bidirectional manipulation of the 5-HTDR→VTA pathway demonstrated that it is both necessary and sufficient for regulating vulnerability to social stress. The study also found that VTA neurons received both glutamatergic excitatory and serotonergic inhibitory inputs from the DR. In resilient mice, there was an increase in the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in 5-HTDR→VTA neurons, suggesting a potential mechanism for resilience.

The findings demonstrate that the 5-HTDR→VTA serotonergic circuit plays a crucial role in mediating susceptibility to social stress. Decreased activity in this circuit is associated with vulnerability, while increased activity promotes resilience. The results support both the serotonergic neurotransmission and diathesis-stress hypotheses of psychiatric disorders. The study clarifies that treating DR serotonergic neurons as a homogenous population may lead to conflicting results, as different subpopulations may exhibit distinct functional roles. The findings support the notion that specific serotonergic circuits, such as the 5-HTDR→VTA pathway, might be key targets for developing novel therapies for stress-related disorders. The co-release of glutamate with serotonin from 5-HTDR→VTA neurons highlights the complex neurochemical interactions involved in regulating stress responses. Further research is needed to identify the precise upstream pathways influencing this circuit and the specific downstream effects within the VTA.

This study identified a discrete serotonergic circuit, 5-HTDR→VTA neurons, as a critical regulator of vulnerability to social stress. The bidirectional manipulation of this circuit demonstrates its crucial role in determining stress resilience. These findings suggest that therapeutic strategies targeting this specific circuit could be effective in preventing or treating stress-related psychiatric disorders. Future research should focus on identifying the upstream brain regions modulating this circuit and the specific VTA cell types involved.

The study primarily used male mice, limiting the generalizability to female mice. The chronic social defeat stress model, while widely used, may not fully capture the complexity of human stress experiences. The optogenetic manipulations, while precise, might not entirely mimic the natural dynamics of serotonergic neurotransmission. Further investigation is needed to fully elucidate the complex interplay of neurotransmitters and synaptic plasticity within this circuit in various stress models and in both sexes.