Quels sont les mécanismes neurobiologiques à la base de la peur et de l'anxiété ? Pourquoi un bruit dans la nuit dans une maison écartée nous donne des frissons tandis qu'entre copains dans la campagne ensoleillée ce même bruit reste presque inaperçu ? Quels sont les mécanismes qui règlent les seuils de la peur et les réactions de notre corps ? Pourquoi chez certains individus des troubles d'anxiété se manifestent souvent ensemble avec des problèmes cardiovasculaires, tandis que chez d'autres, le système gastrointestinal commence à poser des problèmes ?
C'est à ce genre de questions que notre groupe essaie de trouver des réponses. Nous savons depuis lors qu'il y a une région du cerveau particulièrement active pendant les moments d'angoisse et de peur: "l'amygdale". L'amygdale se situe dans le lobe temporal, dans la région antérieure de l'hippocampe avec lequel elle a de fortes connections. L'amygdale reçoit une grande diversité d'input sensoriels (auditif, visuel, olfactif, sensoriel) et envoie des projections vers des noyaux dans le tronc cérébral qui contrôlent les réactions de nos viscères. Pour étudier la régulation de ces fonctions par l'amygdale nous utilisons le rat comme modèle animal.
Le rat est un animal très interactif et qui a une grande vie sociale. Il nous ressemble dans beaucoup d'aspects. L'approche que nous avons choisie consiste en une combinaison d'expériences in vitro et in vivo. Pour la partie in vitro, nous avons établi à Cery un laboratoire équipé d'installations d'électrophysiologie pour enregistrer avec des approches extracellulaires, intracellulaires ou patch-clamp des signaux électriques dans des tranches de cerveau.
Pour la partie in vivo, nous utilisons des capteurs télémétriques, qui permettent d'enregistrer des réponses physiologiques déclenchées par des légers stimuli de peur. Ainsi, nous pouvons suivre les augmentations de battement du cœur, de la respiration, de la pression sanguine ou de la motilité intestinale chez le rat en permanence: Avec cette méthode, nous pouvons déjà identifier le moindre bruit suspect pour le rat.
Oxytocin (OT) appears to play an important role in social behavior. This could be mediated through OT-induced decreases in fear in combination with increases in affiliative behavior that are mediated through OT receptors in respectively the central (CeA) and medial (MeA) nuclei of the amygdala. Interestingly, electrophysiological in vitro studies have shown that neurons in the MeA are differently sensitive to OT compared to neurons in the CeA and more rapidly desensitize in the CeA.
We here hypothesize that these differences in sensitivities and desensitization to OT are the result of activation of intracellular signaling pathways that are mediated by distinct G proteins and associated recruitment of beta-arrestin and that an imbalance between both OT signaling systems may underlie certain psychiatric disorders such as autism.
To test our hypothesis, we compare increases in spiking activity measured by cell-attached patch clamp in CeA and MeA following 3-4 consecutive applications of oxytocin agonists and blocking effects by the G-protein specific OT antagonist Atosiban. Our preliminary results showed responses in 29% of CeA neurons to first TGOT application that subsequently rapidly desensitized. In the MeA 18% of neurons responded that exhibited significantly less desensitization upon consecutive applications. Responses in the MeA could selectively be blocked by Atosiban. These findings are consistent with different G proteins underlying OT signaling in the CeA and MeA and open the possibility to modulate OT signaling selectively in different brain regions by specific pharmacological intervention.
Temporal lobe epilepsy (TLE) is the most common form of epilepsy and remains until today resistant to long-term pharmacological treatment. Moreover, a large number of patients that suffer from this epilepsy often also exhibit signs of anxiety disorders. Symptoms of these manifest themselves in between crises and can be more hindering then the crises themselves. The two structures that are mostly affected by TLE are the hippocampus and the amygdala. The amygdala is also important for attributing emotional values to our sensory stimuli. Our laboratory is interested in assigning which role the amygdala plays in emotional disorders, and more precisely anxiety and fear disorders.
More specifically, we are interested to examine which changes, on the synaptic level, can be found in the amygdala following epileptic activity. We think these changes are at the basis of the development of anxiety disorders in patients that suffer from TLE. To this purpose, we have developed a horizontal slice preparation of the rat brain that contain amygdala and hippocampus and interconnections between these structures. In these slices we can induce epileptiform activity by adding the GABA(A) receptor antagonist bicuculline.
To address the above questions we make use of different electrophysiological techniques such as extracellular, intracellular and patch-clamp recordings in slices of amygdala and hippocampus, followed by immunohistochemical measurements. With these techniques it is possible to study the speed and extent of propagation of epileptiform bursting. Furthermore we are currently developing an in vivo model in which local, short term application of bicuculline in the amygdala of rodents leads to long-term enhancement of anxiety and fear in rodent. To this purpose we maintain a close collaboration with the department of neurosurgery in the Lausanne University Hospital, through which we regularly participate in meetings and seminars, but also evaluations and operations on epileptic patients.
Recently, selective bilateral BLA damage was shown in a group of subjects from the Northern Cape of South Africa, caused by a rare, i.e. faulty, mutation of the ECM1 gene (Urbach-Wiethe disease; UWD). Using neuroimaging techniques it was also shown that in these UWD subjects the CMA remains intact and functional. Data of the group of Prof. Jack van Honk at UCT correspond to animal models in showing that innate as well as social fear in these UWD subjects has shifted from instrumental behaviors (sub-served by the BLA) to impulsive mechanisms (sub-served by CMA). Furthermore, recent high-resolution fMRI studies using acute hormone administration in humans (testosterone and oxytocin) have indicated that the effects of these hormones are also mediated by amygdala sub regions. Indeed, these findings also agree with recent rodent research from our group Within this collaboraiton we will use a selection of paradigms measuring fear learning, fear responses to distant and nearby threat, and social fear. These will be used with neuroimaging and measurements of the physiological expression of fear in human subjects with selective BLA damage and healthy controls (at UCT), and compared to data from a project wherein similar paradigms are used in rodents (at UniL). ECM1 knock out, pharmacological and optogenetic methods for selective inactivation of neuron populations will be applied to influence amygdala sub regions (BLA and CMA) in rodents, to study the corresponding effects on the behavioral (e.g. freezing vs. fight/flight) and physiological expression of fear (parasympathetic vs. sympathetic activation). Furthermore, in both humans and rodents we will apply placebo-controlled acute hormone administration (oxytocin and testosterone) to investigate and compare effects on these same fear behaviors and the underlying brain functions. With these approaches we aim to construct a translational amygdala-centered neurobehavioral model of fear.
Studies on the development of autism have suggested that an imbalance between excitatory and inhibitory circuits plays an important role in the development of autism. Recently, these findings were expanded to include a faulty excitatory to inhibitory switching GABAergic signaling during development in Fragile X Syndrome (FXS) and valproate acid animal models of autism. Oxytocin, a molecule important for social behavior, was found to play an important role in this development, and its absence during birth causes similar changes as in these established animal models of autism. This suggests a mechanism through which oxytocin can play an important protective role during birth and possibly beyond against the development of the autistic phenotype. While the readouts of the effects of oxytocin at the circuit level have up to now mainly focused on its effects in the hippocampus, similar changes in the amygdala, a structure important for social and fear behavior, may play an important role in autistic phenotype. Moreover, oxytocin receptors are strongly expressed in the central nucleus of the amygdala, where their activation has been shown to lead to strong decreases in anxiety and fear responses. We here propose to study in a new fragile X rat model of autism the morphological and physiological changes at the cellular and circuit level in the amygdala and to test the precise involvement and effects of oxytocin in the development and later compensation of these changes.
The objective of this project is the characterization and optimization of a new class of drug candidates based on allosteric modulation for neurodegenerative and psychiatric diseases. Specifically, allosteric modulators targeting Group III of metabotropic glutamate receptors will be assessed in animal models replicating human anxiety and fear disorders, whereas electrophysiological studies will be carried out on allosteric modulators targeting mGluR4 and mGluR7 in slices of the amygdala, in order to investigate their role in modulating synaptic transmission and further characterize their potential therapeutic application in anxiolytic disorders. Project results will contribute in the selection of the best candidate compounds for clinical development.
This project is carried out in collaboration with Addex Pharmaceuticals, an emerging global leader in allosteric-based drug discovery and development, who has developed a track record of successfully addressing “undruggable” targets, i.e. where other approaches have failed to date. Addex has developed proprietary assays for these targets that allowed the identification of small-molecule allosteric modulators for various targets as summarized earlier in their pipeline discussion. They now have advanced multiple programs involving a whole range of targets of high pharmaceutical interest, such as GPCRs, cytokine receptors and tyrosine kinase receptors.
Oxytocin (OXT) is an evolutionarily ancient hypothalamic neuropeptide, which plays a crucial role in the neuroendocrine control of parturition and lactation in mammals. Recently, applying cell-type specific targeting of OXT neurons by recombinant adeno-associated virus (rAAV), we showed that OXT release from long-range axons in the central nucleus of amygdala (CeA) suppresses freezing behavior in fear-conditioned virgin rats. This finding raised a question on how effects of local, intraamygdaloid OXT release co-interacting with volume transmission of OXT originate hypothalamic nuclei in the context of fear. This question is especially important for the period of lactation when the dendritic OXT release from hypothalamic neurons is drastically enhanced.
In our proposal we will pursue three goals: (1) To compare – by employing recombinant viruses and conventional tracers – the anatomical connections between hypothalamic OXT neurons and the central amygdala (CeA) in lactating and virgin rats; (2) to compare – by employing virally mediated expression of light sensitive channels, channelrhodopsin2 and halorhodopsin, – the effects of axonal OXT on the CeA microcircuit activity in virgin and lactating rats; (3) to compare effects of axonal OXT release within the CeA with effects of OXT originated from the hypothalamus in fear conditioning paradigms applied for virgin and lactating rats. In addition, we will combine optogenetics with the microdialysis technique and live imaging to confirm that the illumination of axonal OXT terminals in the CeA and somas/dendrites of OXT neurons in the hypothalamus indeed provokes changes/alterations in actual OXT release. Thus our results should help to answer a fundamental question about the role of volume transmission of OXT within the brain vs. addressed OXT release within distinct brain regions, recently described by us. Furthermore, our data will be highly relevant to human health, namely psychophysiology of breast-feeding women and pathogenetic mechanisms of postpartum depression.
In patients suffering from Alzheimer's disease (AD), "insecurely attached" individuals may develop more severe behavioural and psychological symptoms of dementia (BPSDs) which include affective, psychotic, and behavioural disorders. Individual attachment styles can in turn be influenced significantly by social contact at a young age. The precise cellular biological mechanism(s) that underlie(s) the development of BPSD still remain(s) unclear. These last several years, an increasing number of studies has started to reveal an important role of oxytocin (OT) in brain structures involved in social behavior. Changes in OT signaling might thus underlie these more severe symptoms in "insecurely attached" Alzheimer's patients. The current proposal is aimed at addressing this role of OT in a rodent animal model.
We here hypothesize that BPSD in patients with Alzheimer’s disease is related to differential attachment styles that are in turn tightly linked to central oxytocin signaling. We will test our hypothesis in a rat model that combines changes in attachment style induced by post-weaning social isolation with a subsequent pharmacological challenge to induce AD-like symptoms. In this model we will monitor changes in OT signaling and development of BPSD along with neurocognitive changes indicative of developing AD. To assess a causative role of OT signaling we will use various in vitro and in vivo tools and techniques (optogenetic release of oxytocin, electrophysiology & telemetry) which we have recently developed in-house to assess the effects of changes in OT signaling in the rat brain (Huber et al., Science 2005; Viviani et al., Science 2011, Charlet et al., Neuron, submitted).
This project inscribes itself in a translational collaboration with the local psychogeriatrical clinic (Dr. von Gunten). We expect to validate oxytocin in rats as a new and promising pharmacological target to improve behavioral and psychological symptoms in patients with Alzheimer's disease.