Zoltán Kristóf Varga

Despite the central role attributed to serotonin in anxiety, its involvement and therapeutic efficacy are inconsistent, raising the possibility that serotonergic recruitment depends on the stress history from which anxiety emerges. Using larval zebrafish, we combined graded glucocorticoid exposure with chemogenetic DRN manipulation, whole-brain activity mapping, and pharmacology to test whether stress intensity determines serotonergic involvement in anxiety. Increasing stress intensity did not simply increase anxiety severity but generated distinct anxiety phenotypes. Lower glucocorticoid exposure produced context-general anxiety that required the serotonergic DRN, whereas higher exposure produced context-dependent, DRN-independent anxiety. Whole-brain mapping identified a posterior tubercular/hypothalamic dopaminergic region associated with DRN-dependent anxiolysis, while D1, but not D2 receptor antagonism abolished the anxiolytic effect of DRN ablation. Finally, environmentally relevant nanomolar concentrations of methylphenidate reduced anxiety-like behavior, further supporting dopaminergic modulation of anxiety. Together, our findings identify stress intensity as a determinant of serotonergic recruitment during anxiety, reveal a downstream contribution of D1-dependent dopaminergic signaling, and provide a framework for understanding the mechanistic heterogeneity of anxiety and its variable response to serotonergic treatments. GRAPHICAL

Stress elicits variable systemic and neural changes, with outcomes ranging from adaptive to pathological. Several studies have implicated the dorsal raphe nucleus (DRN), a brainstem nucleus containing a heterogeneous population of serotonergic (5-HT) neurons, in the adaptive stress response and the pathological changes resulting from chronic stress. However, it is not known whether early life chronic stress affects the developing DRN activity, or whether the stress-induced changes affect 5-HT DRN neurons in a subregion- or phenotype-specific manner. To answer these questions, we use in vivo 2-photon calcium imaging of 5-HT DRN neurons in larval zebrafish exposed to chronic unpredictable stress during early life. Here, we show that early life chronic stress prevents the habituation of the serotonergic system to repeated stress-exposure by altering the balance of excitatory/inhibitory responses within the DRN. These changes are most pronounced in a subset of serotonergic cells co-expressing GABAergic markers. Using chemogenetic ablation of 5-HT DRN neurons, we show that stress-induced plasticity of the DRN contributes to changes in startle response habituation and in locomotive activity, but not in anxiety-like behaviors. Collectively, our results emphasize the role of stress-induced plasticity of DRN neurons in the selective regulation of maladaptive behavioral outcomes.

The dorsal raphe nucleus (DRN) is the main source of serotonin (5-HT) in the central nervous system. 5-HT DRN neurons modulate a wide range of physiological functions and brain states via their widespread and heterogeneous projections. Yet the organizational logic, developmental dynamics, and experience-dependent plasticity of these projections remain poorly understood. By reconstructing the whole brain projections of 81 individual 5-HT DRN neurons across developmental stages in the larval zebrafish, we found that these neurons exhibit diverse projection patterns, ranging from broadly distributed to spatially confined, and identified two projection-defined subpopulations, targeting either the telencephalon or the spinal cord. Longitudinal analysis revealed that the gross architecture of 5-HT neurons remained stable during early development, although region-specific remodeling occurred in all neurons. Furthermore, exposure to early life stress during the first week of development, but not thereafter, altered the density of 5-HT DRN projections in a region-specific manner, suggesting a critical window of increased plasticity. Altogether, our study sheds new light on how the serotonergic circuitry develops, matures and adapts to stress, which is critical for better understanding the pathophysiology of mental disorders.

Posttraumatic stress disorder (PTSD) is a chronic psychiatric condition that develops in susceptible individuals exposed to traumatic stress. Identifying predisposing risk factors and mechanisms presents a significant challenge for prevention and therapy development. Here, we aimed to identify behavioral predictors of excessive fear generalization - a core symptom of PTSD - and its neural correlates in rats using a longitudinal design. Prior to trauma, rats underwent extensive behavioral test batteries to assess their emotional and cognitive traits. They were then exposed to a single traumatic experience via inescapable footshocks. Twenty-eight days later, fear generalization was measured in a neutral/safe context, differentiating vulnerable (high freezing) and resilient (low freezing) subpopulations. Reduced pre-trauma operant learning performance emerged as the strongest predictor of excessive fear generalization. Neuronal activity mapping revealed altered medial prefrontal cortex (mPFC) dynamics in vulnerable animals, characterized by activity changes and less coordinated activity-coupling between subregions. Gene expression analysis and cell-specific activity labeling pointed to VIP/CRH+ interneurons as potential mediators of excessive fear. As a molecular intervention, silencing prefrontal Crh expression after fear memory consolidation markedly enhanced mPFC activation and reduced fear expression, supporting a regulatory role of this interneuron population in fear processing. As a behavioral intervention, post-trauma operant training facilitated the reduction of generalized fear and led to changes in plasticity-related gene expression in the mPFC, indicating overlapping neural circuits governing operant learning and fear regulation. These findings together highlight operant learning and mPFC network functions as vulnerability markers and mediators of excessive fear generalization, with implications for prevention and targeted therapy in PTSD.

The reliability and validity of preclinical anxiety testing is essential for translating animal research into clinical use. However, the commonly used anxiety tests lack inter-test correlations and face challenges with repeatability. While translational animal research should be able to capture stable individual anxiety traits - the core feature of anxiety disorders - the conventional approach employs a single type of test at a single time, which primarily reflects transient states of animals that are heavily influenced by experimental conditions. Here, we propose a validated, optimized test battery capable of reliably capturing trait anxiety in rats and mice of both sexes. Instead of developing novel tests, we combined widely used tests (elevated plus-maze, open field and light-dark test) to provide instantly applicable adjustments for better predictive validity. We repeated these tests three times to capture behavior across multiple challenges, which we combined to generate summary measures (SuMs). Our approach resolved inter-test correlation issues and provided better predictions for subsequent outcomes under more anxiogenic conditions or fear conditioning. SuMs were also shown to be more sensitive markers of stress-induced anxiety following social isolation. Finally, we tested our method’s efficacy in discovering anxiety-related molecular pathways through RNA sequencing of the medial prefrontal cortex. SuMs revealed four-times more molecular correlates of trait anxiety than transient states, highlighting novel gene clusters. Furthermore, 16% of these correlates were also found in the amygdala. In summary, we provide a novel approach to capture trait anxiety in rodents, offering improved predictions for potential therapeutic targets for personalized medicine. We also provide recommendations to enhance feasibility without compromising validity or animal ethics, tailored to various scientific goals.

Stress elicits variable systemic and neural changes in vertebrates, with outcomes ranging from adaptive to pathological. Several studies have implicated the dorsal raphe nucleus (DRN), a brainstem nucleus containing a heterogeneous population of serotonergic (5-HT) neurons, in the adaptive stress response and the pathological changes resulting from chronic stress. However, it is not known whether early life chronic stress affects the developing DRN activity, or whether the stress-induced changes affect 5-HT DRN neurons in a subregion- or phenotype-specific manner. To answer these questions, we used in vivo 2-photon calcium imaging of 5-HT DRN neurons in larval zebrafish exposed to chronic unpredictable stress during early life. We found that early life chronic stress prevented the normal habituation of the serotonergic system to a repeated acute stressor by altering the balance of excitatory/inhibitory responses within the DRN. Interestingly, these changes were most pronounced in a subset of stress-vulnerable serotonergic cells co-expressing GABAergic markers. Further, using chemogenetic ablation of 5-HT DRN neurons, we showed that stress-induced plasticity of the DRN contributed to changes in startle response habituation and in locomotive activity, but not in anxiety-like behaviors. Collectively, our results emphasize the role of stress-induced plasticity of DRN neurons in the selective regulation of maladaptive behavioral outcomes.

Zebrafish have revolutionised physiological screening in vertebrates, however, their strong sociality present challenges for interpreting behavioural assays conducted on individual subjects. To retain the advantages of the zebrafish model while addressing its limitations, we propose the use of a solitary species—the paradise fish—as a complementary model system. We compared paradise fish and zebrafish of late larval stage in social and non-social exploratory tasks, anxiety tests and in a working memory assay to assess their performance in these individual-based challenges. We found that in contrast to zebrafish, paradise fish did not show social approach in sociability tests, their exploratory behaviour was unaffected by the presence of a conspecific, and social isolation did not impair their performance during anxiety tests. Intra- and intertest variability measures of different anxiety tests revealed that, compared to zebrafish, paradise fish express more consistent, repeatable patterns of exploratory and risk-avoidance behaviour across time and contexts. We also showed that paradise fish exploration of the Y-maze is dominated by arm alternations, suggesting advanced working memory. Considering the results of this systematic comparison and the natural history of the two species we recommend prioritizing zebrafish in social tasks, while favouring paradise fish in individual-based behavioural assays.

Freezing behavior, characterised by attentive immobility as a reaction to a perceived threat, is widely studied in the context of fear, anxiety and stress. To uncover the genetic factors underlying this behavior, we conducted a genome-wide association study (GWAS) in kennel-housed beagle dogs. Our analysis identified a single-nucleotide polymorphism (SNP) in intron 5 of the KCNQ3 gene on chromosome 13 associated with freezing behavior in response to unfamiliar environments and people. To validate this finding, we used a zebrafish model, where CRISPR/Cas9-induced kcnq3 deficiencies led to heightened fear and arousal in two behavioral tests. KCNQ3 is implicated in several neurodevelopmental and psychiatric disorders, and our results highlight its evolutionarily conserved role in modulating fear responses. In dogs, an enriched environment can mitigate the adverse effects of KCNQ3 deficiency by reducing threat perception, highlighting the role of gene-environment interactions in shaping behavioral responses. Teaser: KCNQ3 influences fear responses across species, with gene-environment interactions shaping freezing behavior in dogs.

The reliability and validity of preclinical anxiety testing is essential for translating animal research into clinical use. However, the commonly used anxiety tests lack inter-test correlations and face challenges with repeatability. While translational animal research should be able to capture stable individual anxiety traits, the current approach employs a single type of test at a single time that only measures transient states of animals, heavily influenced by experimental conditions. Here, we propose a validated, optimized test battery capable of reliably capturing trait anxiety in rats and mice of both sexes. Instead of developing novel tests, we combined widely used tests (elevated plus-maze, open field and light-dark test) to provide instantly applicable adjustments for better predictive validity. We repeated these tests three times to capture behaviour across multiple challenges, which we combined to generate summary measures (SuMs). Our approach resolved between-test correlation issues and provided better predictions for subsequent outcomes under anxiogenic conditions or fear conditioning. SuMs were also able to reveal differences in anxiety in an etiological stress model. Finally, we tested our method’s efficacy in discovering anxiety-related molecular pathways through RNA sequencing of the medial prefrontal cortex. Using SuMs, we identified four-times more molecular correlates of trait anxiety as compared to transient anxiety states, pointing out novel functional gene clusters. Furthermore, 16% of these molecular correlates of anxiety were replicated in amygdalar samples, as well. In summary, we provide a novel approach to capture trait anxiety in rodents, offering improved predictions for potential therapeutic targets for personalized medicine.

Forming effective responses to threatening stimuli requires the adequate and coordinated emergence of stress-related internal states. Such ability depends on early-life experiences and, in connection, the adequate formation of neuromodulatory systems, particularly serotonergic signaling. Here, we assess the serotonergic background of experience-dependent behavioral responsiveness employing a zebrafish ( Danio rerio ) model. For the first time, we have characterized a period during the behavioral metamorphosis in which zebrafish are highly reactive to their environment. Absence of social stimuli during this phase established by isolated rearing fundamentally altered the behavioral phenotype of post-metamorphic zebrafish in a challenge-specific manner, partially due to a decline in responsiveness and an inability to develop stress-associated arousal state. In line with this, isolation differently affected whole-brain 5-HT signaling in resting and stress-induced conditions, an effect that was present at the level of the dorsal pallium and was negatively associated with responsiveness. Administration of the 5HT1AR partial agonist buspirone prevented the isolation-induced serotonin response to novelty in the forebrain and rescued stress-induced arousal along with challenge-induced behaviors, which altogether indicates a functional connection between these changes. In summary, there is a consistent negative association between behavioral responsiveness and serotonergic signaling in zebrafish, which is well recognizable through the modifying effects of developmental perturbation and pharmacological manipulations as well. Our results imply a conserved serotonergic mechanism that context-dependently modulates environmental reactivity and is highly sensitive to experiences acquired during a specific early-life time-window, a phenomenon that was previously only suggested in mammals. Significance statement: The ability to respond to challenges is a fundamental factor in survival. We show that zebrafish that lack appropriate social stimuli in a sensitive developmental period show exacerbated alertness in non-stressful conditions while failing to react adequately to stressors. This shift is reflected inversely by central serotonergic signaling, a system that is implicated in numerous mental disorders in humans. Serotonergic changes in brain regions modulating responsivity and behavioral impairment were both prevented by the pharmacological blockade of serotonergic function. These results imply a serotonergic mechanism in zebrafish that transmits early-life experiences to the later phenotype by shaping stress-dependent behavioral reactivity, a phenomenon that was previously only suggested in mammals. Zebrafish provide new insights into early-life-dependent neuromodulation of behavioral stress-responses.