Abnormal fear conditioning and amygdala processing in an animal model of autism

A core feature of autism spectrum disorders is the impairment in social interactions. Among other brain regions, a deficit in amygdala processing has been suggested to underlie this impairment, but whether the amygdala is processing fear abnormally in autism, is yet not clear. We used the valproic acid (VPA) rat model of autism to (a) screen for autism-like symptoms in rats, (b) test for alterations in amygdala-dependent fear processing, and (c) evaluate neuronal reactivity and synaptic plasticity in the lateral amygdala by means of in vitro single-cell electrophysiological recordings. VPA-treated animals displayed several symptoms common to autism, among them impaired social interactions and increased repetitive behaviors. Furthermore, VPA-treated rats were more anxious and exhibited abnormally high and longer lasting fear memories, which were overgeneralized and harder to extinguish. On the cellular level, the amygdala was hyperreactive to electrical stimulation and displayed boosted synaptic plasticity as well as a deficit in inhibition. We show for the first time enhanced, overgeneralized and resistant conditioned fear memories in an animal model of autism. Such hyperfear could be caused by the hyperreactivity and hyperplasticity found in the lateral amygdala, which may in turn be due to a deficit in the inhibitory system of the amygdala. We hypothesize an 'aversive world' syndrome that could, even if not a primary cause of the disorder itself, underlie some core symptoms in autism, such as impairments in social interactions and resistance to rehabilitation

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

Elevated NMDA receptor levels and enhanced postsynaptic long-term potentiation induced by prenatal exposure to valproic acid

Henry Markram, Tania Rinaldi

Valproic acid (VPA) is a powerful teratogen causing birth defects in humans, including autism spectrum disorder (ASD), if exposure occurs during the first trimester of embryogenesis. Learning and memory alterations are common symptoms of ASD, but underlying molecular and synaptic alterations remain unknown. We therefore studied plasticity-related mechanisms in the neocortex of 2-week-old rats prenatally exposed to VPA and tested for changes in glutamate-mediated transmission and plasticity in the neocortex. We found a selective overexpression of NR2A and NR2B subunits of NMDA receptors, as well as the commonly linked kinase calcium/calmodulin-dependent protein kinase II. Synaptic plasticity experiments between pairs of pyramidal neurons revealed an augmented postsynaptic form of long-term potentiation. These results indicate that VPA significantly enhances NMDA receptor-mediated transmission and causes increased plasticity in the neocortex. Enhanced plasticity introduces a surprising perspective to the potential molecular and synaptic mechanisms involved in children prenatally exposed to VPA.

2007

Hyper-emotional neurophysiology in a rat model of autism

Mônica Regina Favre

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.