Olivia Zanoletti

Mitochondrial function in the brain links anxiety with social subordination

Significance Within a dominance hierarchy, low social status strongly reduces individual well-being. In socially living species, rank in a hierarchy is determined through competitive encounters. Despite the numerous health consequences, the ability of personality traits to predispose individuals to a particular social rank remains largely unclear. Our work identifies trait anxiety as a predisposing factor to a subordinate rank. We demonstrate that mitochondrial function in the nucleus accumbens, a brain region relevant for motivation and depression, is a critical mediating factor in the subordinate status displayed by high-anxious rats. These findings highlight a role for cerebral energy metabolism in social behavior and point to mitochondrial function in the nucleus accumbens as a potential marker and avenue of treatment for mood disorders. Dominance hierarchies are integral aspects of social groups, yet whether personality traits may predispose individuals to a particular rank remains unclear. Here we show that trait anxiety directly influences social dominance in male outbred rats and identify an important mediating role for mitochondrial function in the nucleus accumbens. High-anxious animals that are prone to become subordinate during a social encounter with a low-anxious rat exhibit reduced mitochondrial complex I and II proteins and respiratory capacity as well as decreased ATP and increased ROS production in the nucleus accumbens. A causal link for these findings is indicated by pharmacological approaches. In a dyadic contest between anxiety-matched animals, microinfusion of specific mitochondrial complex I or II inhibitors into the nucleus accumbens reduced social rank, mimicking the low probability to become dominant observed in high-anxious animals. Conversely, intraaccumbal infusion of nicotinamide, an amide form of vitamin B3 known to enhance brain energy metabolism, prevented the development of a subordinate status in high-anxious individuals. We conclude that mitochondrial function in the nucleus accumbens is crucial for social hierarchy establishment and is critically involved in the low social competitiveness associated with high anxiety. Our findings highlight a key role for brain energy metabolism in social behavior and point to mitochondrial function in the nucleus accumbens as a potential marker and avenue of treatment for anxiety-related social disorders.

Role for MMP-9 in stress-induced downregulation of nectin-3 in hippocampal CA1 and associated behavioural alterations

Chronic stress is a risk factor for the development of psychopathologies characterized by cognitive dysfunction and deregulated social behaviours. Emerging evidence suggests a role for cell adhesion molecules, including nectin-3, in the mechanisms that underlie the behavioural effects of stress. We tested the hypothesis that proteolytic processing of nectins by matrix metalloproteinases (MMPs), an enzyme family that degrades numerous substrates, including cell adhesion molecules, is involved in hippocampal effects induced by chronic restraint stress. A reduction in nectin-3 in the perisynaptic CA1, but not in the CA3, compartment is observed following chronic stress and is implicated in the effects of stress in social exploration, social recognition and a CA1-dependent cognitive task. Increased MMP-9-related gelatinase activity, involving N-methyl-D-aspartate receptor, is specifically found in the CA1 and involved in nectin-3 cleavage and chronic stress-induced social and cognitive alterations. Thus, MMP-9 proteolytic processing emerges as an important mediator of stress effects in brain function and behaviour.

Impaired hippocampal neuroligin-2 function by chronic stress or synthetic peptide treatment is linked to social deficits and increased aggression

Neuroligins (NLGNs) are cell adhesion molecules that are important for proper synaptic formation and functioning, and are critical regulators of the balance between neural excitation/inhibition (E/I). Mutations in NLGNs have been linked to psychiatric disorders in humans involving social dysfunction and are related to similar abnormalities in animal models. Chronic stress increases the likelihood for affective disorders and has been shown to induce changes in neural structure and function in different brain regions, with the hippocampus being highly vulnerable to stress. Previous studies have shown evidence of chronic stress-induced changes in the neural E/I balance in the hippocampus. Therefore, we hypothesized that chronic restraint stress would lead to reduced hippocampal NLGN-2 levels, in association with alterations in social behavior. We found that rats submitted to chronic restraint stress in adulthood display reduced sociability and increased aggression. This occurs along with a reduction of NLGN-2, but not NLGN-1 expression (as shown with western blot, immunohistochemistry, and electron microscopy analyses), throughout the hippocampus and detectable in different layers of the CA1, CA3, and DG subfields. Furthermore, using synthetic peptides that comprise sequences in either NLGN-1 (neurolide-1) or NLGN-2 (neurolide-2) involved in the interaction with their presynaptic partner neurexin (NRXN)-1, intra-hippocampal administration of neurolide-2 led also to reduced sociability and increased aggression. These results highlight hippocampal NLGN-2 as a key molecular substrate regulating social behaviors and underscore NLGNs as promising targets for the development of novel drugs for the treatment of dysfunctional social behaviors.

Hippocampal neuroligin-2 overexpression leads to reduced aggression and inhibited novelty reactivity in rats.

Disturbances of the excitation/inhibition (E/I) balance in the brain were recently suggested as potential factors underlying disorders like autism and schizophrenia resulting in associated behavioral alterations including changes in social and emotional behavior as well as abnormal aggression. Neuronal cell adhesion molecules (nCAMs) and mutations in these genes were found to be strongly implicated in the pathophysiology of these disorders. Neuroligin2 (nlgn2) is a postsynaptic cell adhesion molecule, which is predominantly expressed at inhibitory synapses and required for synapse specification and stabilization. Changes in the expression of nlgn2 were shown to result in alterations of social behavior as well as altered inhibitory synaptic transmission, hence modifying the E/I balance. In our study, we focused on the role of nlgn2 in the dorsal hippocampus in the regulation of emotional and social behaviors. To this purpose, we injected an AAV construct overexpressing nlgn2 in the hippocampus of rats and investigated the effects on behavior and on markers for the E/I ratio. We could show an increase in GAD65, a GABA-synthesizing protein in neuronal terminals, and furthermore, reduced exploration of novel stimuli and less offensive behavior. Our data suggest nlgn2 in the hippocampus to be strongly implicated in maintaining the E/I balance in the brain and thereby modulating social and emotional behavior.

Diazepam actions in the VTA enhance social dominance and mitochondrial function in the nucleus accumbens by activation of dopamine D1 receptors

Benzodiazepines can ameliorate social disturbances and increase social competition, particularly in high-anxious individuals. However, the neural circuits and mechanisms underlying benzodiazepines’ effects in social competition are not understood. Converging evidence points to the mesolimbic system as a potential site of action for at least some benzodiazepine-mediated effects. Furthermore, mitochondrial function in the nucleus accumbens (NAc) has been causally implicated in the link between anxiety and social competitiveness. Here, we show that diazepam facilitates social dominance, ameliorating both the competitive disadvantage and low NAc mitochondrial function displayed by high-anxious rats, and identify the ventral tegmental area (VTA) as a key site of action for direct diazepam effects. We also show that intra-VTA diazepam infusion increases accumbal dopamine and DOPAC, as well as activity of dopamine D1- but not D2-containing cells. In addition, intra-NAc infusion of a D1-, but not D2, receptor agonist facilitates social dominance and mitochondrial respiration. Conversely, intra-VTA diazepam actions on social dominance and NAc mitochondrial respiration are blocked by pharmacological NAc micro-infusion of a mitochondrial complex I inhibitor or an antagonist of D1 receptors. Our data support the view that diazepam disinhibits VTA dopaminergic neurons, leading to the release of dopamine into the NAc where activation of D1-signaling transiently facilitates mitochondrial function, that is, increased respiration and enhanced ATP levels, which ultimately enhances social competitive behavior. Therefore, our findings critically involve the mesolimbic system in the facilitating effects of diazepam on social competition and highlight mitochondrial function as a potential therapeutic target for anxiety-related social dysfunctions.

Involvement of CRFR1 in the Basolateral Amygdala in the Immediate Fear Extinction Deficit.

Abstract Several animal and clinical studies have highlighted the ineffectiveness of fear extinction sessions delivered shortly after trauma exposure. This phenomenon, termed the immediate extinction deficit, refers to situations in which extinction programs applied shortly after fear conditioning may result in the reduction of fear behaviors (in rodents, frequently measured as freezing responses to the conditioned cue) during extinction training, but failure to consolidate this reduction in the long term. The molecular mechanisms driving this immediate extinction resistance remain unclear. Here we present evidence for the involvement of the corticotropin releasing factor (CRF) system in the basolateral amygdala (BLA) in male Wistar rats. Intra-BLA microinfusion of the CRFR1 antagonist NBI30775 enhances extinction recall, whereas administration of the CRF agonist CRF6–33 before delayed extinction disrupts recall of extinction. We link the immediate fear extinction deficit with dephosphorylation of GluA1 glutamate receptors at Ser845 and enhanced activity of the protein phosphatase calcineurin in the BLA. Their reversal after treatment with the CRFR1 antagonist indicates their dependence on CRFR1 actions. These findings can have important implications for the improvement of therapeutic approaches to trauma, as well as furthering our understanding of the neurobiological mechanisms underlying fear-related disorders.

Neuroligin-2 Expression in the Prefrontal Cortex is Involved in Attention Deficits Induced by Peripubertal Stress.

Emerging evidence indicates that attention deficits, which are frequently observed as core symptoms of neuropsychiatric disorders, may be elicited by early life stress. However, the mechanisms mediating these stress effects remain unknown. The prefrontal cortex (PFC) has been implicated in the regulation of attention, including dysfunctions in GABAergic transmission, and it is highly sensitive to stress. Here, we investigated the involvement of neuroligin-2 (NLGN-2), a synaptic cell adhesion molecule involved in the stabilization and maturation of GABAergic synapses, in the PFC in the link between stress and attention deficits. First, we established that exposure of rats to stress during the peripubertal period impairs attention in the five-choice serial reaction time task and results in reductions in the GABA-synthesizing enzyme glutamic acid decarboxylase in different PFC subregions (ie, prelimbic (PL), infralimbic, and medial and ventral orbitofrontal (OFC) cortex) and in NLGN-2 in the PL cortex. In peripubertally stressed animals, NLGN-2 expression in the PL and OFC cortex correlated with attention measurements. Subsequently, we found that adeno-associated virus-induced rescue of NLGN-2 in the PFC reverses the stress-induced attention deficits regarding omitted trials. Therefore, our findings highlight peripuberty as a period that is highly vulnerable to stress, leading to the development of attention deficits and a dysfunction in the PFC GABAergic system and NLGN-2 expression. Furthermore, NLGN-2 is underscored as a promising target to treat stress-induced cognitive alterations, and in particular attentional deficits as manifested by augmented omissions in a continuous performance task.

The effects of stress during early postnatal periods on behavior and hippocampal neuroplasticity markers in adult male mice

Infancy is a critical period for brain development. Emerging evidence indicates that stress experienced during that period can have long-term programming effects on the brain and behavior. However, whether different time periods represent different vulnerabilities to the programming of different neurobehavioral domains is not yet known. Disrupted maternal care is known to interfere with neurodevelopmental processes and may lead to the manifestation of behavioral abnormalities in adulthood. Mouse dams confronted with insufficient bedding/nesting material have been shown to provide fragmented maternal care to their offspring. Here, we compared the impact of this model of early-life stress (ELS) during different developmental periods comprising either postnatal days (PNDs) 2-9 (ELS-early) or PND 10-17 (ELS-late) on behavior and hippocampal cell adhesion molecules in male mice in adulthood. ELS-early treatment caused a permanent reduction in bodyweight, whereas this reduction only occurred transiently during juvenility in ELS-late mice. Anxiety was only affected in ELS-late mice, while cognition and sociability were equally impaired in both ELS-treated groups. We analyzed hippocampal gene expression of the γ2 subunit of the GABAa receptor (Gabrg2) and of genes encoding cell adhesion molecules. Gabrg2 expression was increased in the ventral hippocampus in ELS-late-treated animals and was correlated with anxiety-like behavior in the open-field (OF) test. ELS-early-treated animals exhibited an increase in nectin-1 expression in the dorsal hippocampus, and this increase was associated with the social deficits seen in these animals. Our findings highlight the relevance of developmental age on stress-induced long-term behavioral alterations. They also suggest potential links between early stress-induced alterations in hippocampal Gabrg2 expression and the developmental programming of anxiety and between changes in hippocampal nectin-1 expression and stress-induced social impairments.

The interplay of conditional NCAM-knockout and chronic unpredictable stress leads to increased aggression in mice

Abstract The neural cell adhesion molecule (NCAM) is a key regulator of brain plasticity. Substantial evidence indicates that NCAM is down-regulated by exposure to sustained stress and chronic stress seems to lead to increased aggression. In addition, constitutional NCAM deletion in mice has been shown to lead to increased intermale aggression and altered emotionality Forebrain-specific postnatal NCAM knockout was previously shown to impair cognitive function, particularly when animals were exposed to subchronic stress, but the effects on emotional and social behavior remain unclear. In this study, we investigated the potential interplay of a forebrain-specific postnatal NCAM deletion and exposure to different lengths of repeated stress (i.e. subchronic: 14 days; chronic: 29 days) on aggressive and emotional behavior. Our results show that postnatal deletion of NCAM in the forebrain leads to increased aggression and altered emotionality depending on the duration of stress, whereas conditional NCAM knockout has no basal impact on these behaviors. These findings support the involvement of NCAM in the regulation of emotional and aggressive behaviors, suggesting that diminished NCAM expression might be a critical vulnerability factor for the development of these behavioral alterations under repeated exposure to stress.

Constitutive differences in glucocorticoid responsiveness to stress are related to variation in aggression and anxiety-related behaviors

Glucocorticoids coordinate responses that enable an individual to cope with stressful challenges and, additionally, mediate adaptation following cessation of a stressor. There are important individual differences in the magnitude of glucocorticoid responsiveness to stressors. However, whether individual differences in glucocorticoid responsiveness to stress are linked to different behavioral strategies in coping with social and non-social challenges is not easily studied, owing to the lack of appropriate animal models. To address this, we generated three lines of Wistar rats selectively bred for the magnitude of their glucocorticoid responses following exposure to a variety of stressors over three consecutive days at juvenility. Here, we present findings following observations of a high level of variation in glucocorticoid responsiveness to stress in outbred Wistar rats, and the strong response to selection for this trait over a few generations. When challenged with different stressful challenges, rats from the three lines differed in their coping behaviors. Strikingly, the line with high glucocorticoid responsiveness to stress displayed enhanced aggression and anxiety-like behaviors. In addition, these rats also showed alterations in the expression of genes within both central and peripheral nodes of the hypothalamic-pituitary-adrenal (HPA) axis and enhanced reactivity to acute stress exposure. Together, these findings strongly link differences in glucocorticoid responsiveness to stress with marked differences in coping styles. The developed rat lines are thus a promising model with which to examine the relationship between variation in reactivity of the HPA axis and stress-related pathophysiology and could be employed to assess the therapeutic potential of treatments modulating stress habituation to ameliorate psychopathology.