Hyper-emotional neurophysiology in a rat model of autism

Autism is a pervasive neurodevelopmental disorder of unknown etiology, characterized by a spectrum of symptoms of social impairment, abnormal communication, and unusual restricted, inflexible behaviors. Patients also frequently present various levels of sensory, learning, memory and attention abnormalities, anxiety and phobias, in addition to disturbances in gastrointestinal, sleep, metabolic and immune functions, also with higher rates of physical malformations and seizures. There is currently no empirically based consensus on the causes of symptoms and their remarkable heterogeneity. It is also unknown if associated symptoms are directly linked to autism etiology or if they represent parallel comorbidities. In consequence of our incomplete understanding of the disease process, treatment options are vast and not equally efficacious across all diagnosed individuals. Thus, autism today leads to a lifelong disability that requires significant assistance from health care providers, educational systems and family members. Ultimately, improved diagnostic indicators and personalized therapies should allow the autistic individual to live with reduced costs and more independently. Prenatal exposure to compounds such as valproic acid (VPA) has been associated with higher risk for development of autistic features. The teratogenic and neurodevelopmental effects of VPA in humans are replicated in rodents exposed early in embryonic development, at the time neural tube closure. Thus, the VPA-rat has emerged as a well validated model of autism for controlled investigations of the neurobiological basis of autistic-like symptoms and individual response to environmental interventions. Our laboratory previously demonstrated in the model that the primary sensory cortical, frontal cortical, and amygdala microcircuits are hyper-functional in the pup, in parallel to excessive conditioned fear responses, over-generalization of fear to non-conditioned stimuli, and resistance to fear extinction, in the adult. Together with the full range of autistic symptoms and increasing evidence for local hyper-functional connections and cortical minicolumnpathy in patients, data from the VPA model inspired the hypothesis of autism as an Intense World Syndrome. The theory proposes that the polygenetic background of each individual sets the threshold for susceptibility to their prenatal and post natal environments. At the event of a biological insult, be it exogenous such as VPA-exposure, or genetic, such as a rare set of polymorphisms, a cascade of molecular events would lead to hyper-plastic and hyper-reactive neural microcircuits creating a hyper-functional endophenotype. Depending on the developmental trajectory of each individual, this would lead to abnormalities in four dimensions: hyper-attention, hyper-memory and hyper-perception, and hyper-emotionality. Different patterns of hyper-functional outcomes in each individual would then create a fragmented, intense and even aversive experience of the world, and explain the wide heterogeneity in symptom severity and most, if not all, characteristics of autistic people. Thus, inherent to the theory is the description of autistic individuals as highly biologically sensitive and thus that a predictable, positive, safe and calm environment is an essential feature of early postnatal life for positive outcomes in the child. For my doctoral dissertation, I addressed if embryonic exposure to VPA in the rat leads to hyper-functional emotion system, and if this leads to differential response of individuals to postnatal environment. In Chapter 1, I introduce the clinical characteristics of autism and the brain systems involved in emotion and cognition through modulation of fear, anxiety learning and memory. Then I introduce the research rationale for this project. In Chapter 2, I present our published manuscript describing physical malformations, and other teratogenic and developmental health effects in the VPA-rat. This study further validates the VPA-rat as a model of effects in humans, and summarizes important outstanding questions in the VPA-model, in view of the variability in protocols for model generation. We emphasize that such general health parameters could serve as indicators of the precise time and location of toxic event, and thus as potential predictors of differential symptom dimensions and severities. In Chapter 3, I present our study of predictability in the environment of VPA animals as an essential feature for beneficial effects of enrichment, on autism-like hyper-emotionality. This study provides an elegant and computationally accessible method for multivariate analysis of group effects and individual-based effects. In support of the Intense World Theory, our results suggest that VPA-exposed animals are particularly sensitive to their environment, and point to the hippocampus and amygdala as candidate targets of environmental enrichment predictability effects in reducing fear and anxiety in certain VPA exposed individuals. Finally, in Chapter 4, I present some concluding remarks on the implications of this project and future perspectives.

Learning, Stress and the Role of Personality Profiles in Rats

Basira Salehi, Maria del Carmen Sandi Perez

Stress has been shown to modulate many aspects of physiology and behavior. In particular, substantial work has confirmed that stress is a strong modulator of learning and memory processes. It is more than a century that Yerkes and Dodson have shown that relationship between stress intensity and memory function follows an inverted-U-shaped curve; memory increases with stress to an optimal point, above or below which memory decreases. Despite the great popularity of the Yerkes-Dodson law, the validity of the law has been criticized due to significant methodological problems in the study performed by Yerkes and Dodson (1908) and their data being judged insufficient to substantiate conclusions, among other reasons. Moreover the existing evidence has not yet illustrated this phenomenon under the same experimental conditions for relational learning tasks. On the other hand, stress is not the only factor that modulates cognitive abilities. Different types of learning and memory might be affected by other factors such as individual differences in personality. While the relationship between personality and learning has been studied in human, animal studies addressed to understanding the relationship between personality and learning have been rare. In addition, exposure to stress during pregnancy can have deleterious emotional effects on women. However, the mechanisms underlying these emotional effects are not well understood, and the development of suitable animal models for stress-induced depression in females still remains scarce. The goal of this Thesis was to investigate how stress affects cognitive and emotional processes in different animal models, and to explore the importance of individual differences in the mediation of stress effects. On a first study, we investigated the relationship between stress intensity and memory function. For this purpose cognitive abilities of rats to learn a radial arm water maze were assessed under different stress intensities, using behavioural and endocrine approaches. Our results confirm, for the first time, the existence of an inverted-U-shape memory function according to stressor intensity during the early learning and memory phases in a hippocampus-dependent task. Furthermore, we present evidence that these stress effects are not shown uniformly by all individuals; rather, performance at either the high or the low stress intensities is differentially affected in individuals with different personality-like profiles. In a second study, we investigated whether different personality traits in the Wistar outbred strain of rats were associated with performance in specific types of learning and memory tasks that have been reported to depend on specific brain areas, such as the amygdala, hippocampus and prefrontal cortex (PFC). Using a wide variety of behavioral tests, we identified three personality factors: anxiety, exploration and locomotion. According to our results, the locomotion trait displayed no correlation with learning and memory. However curiosity trait predicted extinction of fear memories and spatial reversal learning, and the anxiety traits was related to extinction of fear. These results suggest that behavioral traits, anxiety and curiosity, might be linked to the functioning of specific brain areas (i.e. amygdala and PFC). Our findings are consistent with recent human studies showing that certain personality traits can predict academic achievement and point out the importance of constructing learning environments that could optimize individual strengths. In a third study, we used a model of stress during pregnancy in an attempt to mirror more closely than other experimental approaches based mainly on movement restraint procedures the human condition where stress is often persistently imposed by a male aggressive partner. For that purpose we used aggressive male rats that cohabitated with (and therefore can be considered a stressor) female rats during their pregnancy and, then, characterized the behaviour of these females after weaning. Similar to effects reported in humans, mother rats displayed anxiety-like and depressive-like behaviours, with the females presenting a high anxiety trait being the more vulnerable to the deleterious effects resulting from exposure to the aggressive male. These results propose an interesting animal model to study the effect of stress during pregnancy. Taken together, our results reinforce the view that stress has a strong impact on cognitive function and emotion. They also stress the importance of taking into account individual differences to characterize individuals with different vulnerability to be affected in both cognitive and emotional domain by exposure to stress. Our approach is novel and original in that it takes into account several behavioural (personality-like) traits simultaneously and shows the importance of different traits for different aspects of the relationship between stress, emotion and cognition. The approaches and findings provide valuable models to address the neurobiological mechanisms underlying stress effects on behavior.

EPFL2010

The role of the amygdala in emotional memories

Kamila Markram, Maria del Carmen Sandi Perez

This thesis investigates the role of the amygdala for the establishment of fear memories with a multidisciplinary approach, including behavioural, psychopharmacological, genetic, molecular, and electrophysiological techniques in rats or mice, under healthy or pathological conditions. This research program aims to shed light on the acquisition and storage of emotional memories in the amygdala and closely interconnected brain areas. In one line of experiments, the molecular mechanisms leading to the establishment of fear memory traces in the amygdala were investigated. For this purpose, the functional role of the polysialylated neural cell adhesion molecule PSA-NCAM, expressed in the synaptic junction, was assessed in the amygdala – and also prefrontal cortex and hippocampus – with psychopharmacological and genetic approaches and tasks that strongly rely on these brain areas. Two lines of studies were followed: 1) amygdala-targeted cleavage and enhancement of PSA-NCAM in rats and 2) general cleavage of PSA-NCAM throughout the brain using genetically modified mice. Taken together, both approaches show that amygdaloid PSA-NCAM plays no role in the acquisition and storage of fear memories, but is rather involved in their extinction. Furthermore, the results confirm the importance of PSA-NCAM in hippocampus mediated learning and for the first time show that prefrontal cortex mediated learning depends on PSA-NCAM. These results suggest that PSA-NCAM is selectively involved in some, but not all, synaptic plasticity processes in the brain. In another line of experiments, the valproic acid (VPA) animal model of autism was used to investigate a possible contribution of the amygdala towards the autistic pathology. VPA was injected once at a specific time point during gestation, the time of neural tube closure. The offspring of such treated rats were first characterized in a broad set of behavioural tasks. It was found that VPA-treated offspring exhibited very specific behavioural anomalies closely resembling autistic symptomotology, such as impaired social interaction, exploration and recognition, enhanced repetitive behaviours, impaired sensorimotor gating and increased anxiety, while other behavioural parameters were left unharmed. Once the validity of the model was established, amygdala functionality was assessed. The results demonstrated that VPA-treated offspring exhibited highly enhanced conditioned fear memories, which generalized to other stimuli and were resistant to extinction. Electrophysiological in vitro recordings in the amygdala revealed hyper-reactivity towards stimulation and enhanced activity-induced synaptic plasticity. These results imply that enhanced activity and plasticity in the amygdala may underlie the exaggerated fear memories. Furthermore it is suggested in this thesis that a hyper-reactive amygdala may underlie some of the most basic symptoms observed in autism: reduced social interactions and resistance to rehabilitation.

EPFL2007

Psychobiological Vulnerability to Stress

Jorge Eduardo Castro Cifuentes, Maria del Carmen Sandi Perez

Several psychobiological factors are associated with vulnerability/resilience to stress-induced depression. Emerging evidence indicates behavioral traits, such as anxiety, can be related to depression-like symptoms after exposure to chronic stress. However, single traits cannot explain the variability observed in individual's vulnerability to stress, highlighting that a combination of behavioral traits might provide a better characterization of the individual's vulnerability to development of depression following prolonged stress. Hippocampal neurogenesis has been highlighted as a cellular mechanism involved in the reduction on hippocampal volume under conditions of stress and depression, suggesting that a lack of new cells and connections may underlie the cognitive and emotional symptoms of mood disorders, and modulating neurogenesis is regarded as a target/mechanism for antidepressants actions. Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that is expressed and potentially involved in cellular proliferation in the central nervous system. MIF was shown to induce cell cycle progression via activation of the extracellular signal-regulated kinase (ERK) cascade. Stress has been shown to alter the degree of activation of the multifunctional ERK 1/2 -as indicated by its phosphorylation (p) pERK1/2- and pharmacological manipulations of the ERK pathway affected anxiety and coping behavior. Nonetheless, other mediating mechanisms are involved in the course of both depression and antidepressant actions. Even though the amygdala has been related to the etiology and recovery of depression through imaging studies showing correlations between these mood conditions and amygdala's volume and functional activation, the number of studies devoted to this brain area is still scarce. We hypothesize here an essential role of the amygdala on depression and antidepressant actions based on the implication of the basolateral complex (BLA) in emotional arousal and stress-induced modulation of cognitive processes, and on recent data showing activation of corticotropin releasing factor receptors (CRFR1) as a substrate for stress-induced alterations. Interestingly, alterations in the functional connectivity between the amygdala, the hippocampus and frontal areas have been identified in individuals with genetic vulnerability to depression. Looking for such psychobiological interactions is particularly important today because of increasingly incidence of depression and stress related disorders'. Identifying and understanding psychobiological vulnerability to stress is crucial for best oriented and more effective prevention and intervention strategies. Therefore, the aim of this thesis was: i) to identify in rats behavioral traits capable of predicting differences in vulnerability/resilience to the behavioral, endocrine, neurogenic and brain activity effects of stress, ii) to explore the role of the amygdala and personality traits in the antidepressants' effects on depression-like behavior and hippocampal neurogenesis, and iii) to study the role of MIF expression in hippocampal neurogenesis and anxiety- and depression- like behaviors. Our first study indicates that both the direction and intensity of the impact of stress on behavior corresponds to different patterns of neurogenesis impairment and brain activity; moreover, these effects are dependent on the interaction between personality traits and the length of stress exposure. Chronic stress induced progressively depressive-like symptoms associated with an increase in cell proliferation and a decrease of cell survival in the hippocampus and with a decrease in ERK1/2 activity in the prefrontal cortex, the hippocampus and a U-shape effect in the amygdala. Personality-like profiles of high exploration seem to play a resilient role for the development of stress-induced depressive-like symptoms and the combination of high anxiety and low exploration seems to lead to higher vulnerability to develop depression-like behaviors. Whereas sub chronic stress increased cell proliferation in the combined profile of high anxiety and low exploration, profiles of low exploration showed a decrease in cell survival after exposure to chronic stress. Interestingly, ERK1/2 activity was decreased in control animals of the personality profile high anxiety and low exploration and subsequently increased after sub-chronic and chronic stress in the paraventricular nucleus, the amygdala and the hippocampus but not in the orbitofrontal cortex. The second study provides evidence for an important role for the amygdala on fluoxetine-stimulated cell proliferation and survival, as well as on the establishment of a link between cell proliferation and depression-like behavior. We also show an important role for anxiety in mediating the effects of basolateral amygdala and antidepressant activity on cell proliferation and survival. Chronic fluoxetine treatment had a positive effect on hippocampal cell survival only when the BLA was lesioned. Anxiety was related to hippocampal cell survival in opposite ways in sham- and BLA-lesioned animals (i.e., negatively in sham- and positively in BLA-lesioned animals). Both BLA lesions and low anxiety were critical factors to enable a negative relationship between cell proliferation and depression-like behavior. Therefore, our study highlights a role for the amygdala on fluoxetine-stimulated cell survival and on the establishment of a link between cell proliferation and depression-like behavior. It also reveals an important modulatory role for anxiety on cell proliferation involving both BLA-dependent and -independent mechanisms. The third study demonstrates the relevance of MIF expression for adult hippocampal neurogenesis. We identify MIF expression in neurogenic cells (in stem cells, cells undergoing proliferation and in newly proliferated cells undergoing maturation) in the subgranular zone of the rodent dentate gyrus. A causal role for MIF in cell proliferation was demonstrated using genetic and pharmacological approaches. Behaviorally, genetic deletion of MIF resulted in increased anxiety- and depression-like behaviors, as well as of impaired hippocampus-dependent memory. Together, our studies provide evidence supporting a pivotal role for MIF in both basal and antidepressant-stimulated adult hippocampal cell proliferation. Moreover, loss of MIF results in a behavioral phenotype that to a large extent corresponds with alterations predicted to arise from reduced hippocampal neurogenesis. These findings underscore MIF as a potentially relevant molecular target for the development of treatments linked to deficits in neurogenesis, as well as to problems related to anxiety, depression and cognition. Altogether the results presented support the idea of high anxiety trait individuals being a particularly prone phenotype and exposure to stressful life events as a requirement for individuals with this personality trait to develop depression.

EPFL2012