1st DNF SYMPOSIUM

The night life of astrocytes

We have recently described a macroscopic pathway in the central nervous system – the glymphatic system that facilitates the clearance of interstitial waste products from neuronal metabolism. Glymphatic clearance of macromolecules is driven by cerebrospinal fluid (CSF) that flows in along para-arterial spaces and through the brain parenchyma via support from astroglial aquaporin-4 water channels. The glymphatic circulation constitutes a complete anatomical pathway; para-arterial CSF exchanges with the interstitial fluid, solutes collect along para-venous spaces, then drain into the vessels of the lymphatic system for ultimate excretion from the kidney or degradation in the liver. The glymphatic system is only active during sleep. As such, this circulation represents a novel and unexplored pathway for understanding the biological necessity for sleep.

Optogenetic deconstruction of sleep-wake states.

Sleep is a primary and essential biological need for higher vertebrates and sleep-like states have been demonstrated in lower vertebrates. While the functions of sleep are still a matter of debate and may include memory consolidation, metabolism clearance and brain plasticity, the basic neurobiological mechanisms controlling sleep-wake state transitions and maintenance remain largely unknown. > Over several decades, experimental evidence has identified distinct arousal-and sleep-promoting neural populations in the brain; however, how these nuclei act individually and collectively to promote and maintain sleep-wake states is unknown. We have recently applied optogenetic technology to the repertoire of techniques used in experimental sleep research to probe the function of hypocretins/orexins and melanin-concentrating hormone (MCH) neurone in arousal and rapid eye-movement sleep, respectively. This work further revealed their communication mode and define possible networks for hypothalamic control of arousal and sleep.

The sleep-deprived brain, a view from the NMDA receptor

A major line of modern sleep research addresses the consequences of sleep loss on the expression of genes and proteins. The ultimate aim of this approach is to understand the mechanisms via which sleep affects neuronal function. However, sleep loss influences the expression of many genes and proteins. Hence, it is of obvious interest to identify specific proteins whose expression is altered by sleep loss and whose effect on defined, system-relevant, neuronal consequences can be understood in mechanistic detail. We have identified an obligatory role for NR2A-containing NMDA receptors in mediating the effects of sleep deprivation on hippocampal synaptic plasticity. Hippocampal synaptic plasticity in NR2A-knockout mice turns out to be insensitive to sleep loss, although the behavioral reaction to sleep deprivation is normal. As to the mechanistic basis of NR2A's role, we show that sleep deprivation augments the number of NR2A proteins on the spines of CA1 apical dendrites. Our work has now expanded beyond hippocampus to other brain circuits and to the situation of chronic sleep disruption.

Presenter:	Luc Stoppini
Affiliation:	Hepia, University of Applied Sciences, Geneva 4, rue de la Prairie CH-1202 Genève
Title:	3D engineered nervous tissues derived from human embryonic stem cells and IPs cells as in vitro models for neurotoxicity studies
Abstract:          The aim of our work is to develop 3D stable and long term in vitro neural cultures that may be used as a model for in vitro neurotoxicity testing. Human stem cells (H9, CH6, iPs), were differentiated to generate neuroprogenitor cells. Functional 3D neural networks were examined after up to 2 months in culture. To characterize the models we measured changes in gene- and protein expression of neural markers by real-time PCR and western blotting, as well as morphological analyses and extracellular recordings of electrophysiological activities. 3D cultures expressed markers of both mature neurons and astrocytes, and intricate neurite networks were observed in 2D cultures. Preliminary experiments on two-month old human 3D histotypic tissues, treated for 24-48 hours with either methylmercury or trimethyltin chloride (10 nM-20 microM for both molecules), showed decreased gene expression of the neuronal markers, beta-3 tubulin, synapsin-1, NeuN, as well as increased level of the glial marker GFAP. This is indicative for neuronal failure and astrogliosis. Further developing of these human in vitro models could enable screenings of toxicological profiles of chemicals or of new drug candidates.
Presenter:	Camilla Bellone
Affiliation:	Department of Basic Neurosciences, Medical Faculty, University of Geneva, 1 rue Michel Servet, CH-1211 Geneva, Switzerland
Title:	Down-regulation of Shank3 in the VTA impairs postnatal maturation of excitatory synapses onto DA neurons.
Abstract:          Shank3 is a scaffolding protein of the postsynaptic density that plays a critical role in orchestrating glutamatergic receptors at the synapse. Functionally, Shank3 links group I mGluRs to NMDARs and AMPARs through its interaction with Homer proteins and it is important in regulating synaptic transmission and plasticity. In the ventral tegmental area (VTA), mGluR1 function is required for driving postnatal maturation of AMPARs and NMDARs but the role of Shank3 is unknown. Here we show that in vitro manipulation of Shank-Homer interaction impairs mGluR1-driven switch of NMDAR subunit composition at excitatory synapses onto dopamine (DA) neurons of the VTA. In order to evaluate the role of Shank3 in the VTA in vivo, we knocked down all the major isoforms of the gene in neonatal mice. We performed whole cell path clamp technique of DA neurons on acute midbrain slices and characterized excitatory transmission at juvenile synapses. We observed that the absence of Shank3 during critical period of development disrupts postnatal maturation of AMPARs and NMDARs. Haploinsufficiency of Shank3 in human is one of the most prevalent causes of autism spectrum disorders (ASDs), neurodevelopmental pathologies characterized by impaired social interactions. Since knocked down of Shank3 specifically perturbs the postnatal development of DA neurons, our manipulation would possibly lead to impairment in social behaviours.
Presenter:	van der Kooij Michael
Affiliation:	EPFL School of Life Sciences - Brain Mind Institute Laboratory of Behavioral Genetics Station 19, 1015 Lausanne
Title:	Social ranking depends on anxiety-profile and is controlled by the mesolimbic midbrain.
Abstract:          In a social hierarchy individuals are ranked based on competitive performance. Limited knowledge is available on the determinants for social ranking which is surprising seeing that the outcome of competitive encounters has important consequences for future behavioral interactions and health. Using adult male rats, we investigated the role of trait anxiety in social dominance. The anxiety-profiles were identified based on behavior in the elevated plus maze. Animals were pair-wise matched for body-weight but differed in anxiety-profile (high- vs. low-anxiety). Pairs were introduced to a novel/neutral cage after which animals display offensive behavior; social dominance was determined by summarizing the durations of offensive behavior. High-anxious individuals predominantly ended up subordinate during social encounters. Peripheral pretreatment with the anxiolytic benzodiazepine diazepam reduced anxiety and enhanced competitive behavior for high-anxious rats. The peripheral effects of diazepam on anxiety and social dominance were recapitulated by intra-VTA (ventral tegmental area) micro-infusion. Moreover, the effects of diazepam on social dominance in high-anxious rats were subject to the participation of the nucleus accumbens (NAc). Intra-NAc micro-infusion of a D1-agonist enhanced social dominance (whereas a D2-agonist lacked behavioral effect). The effects of intra-VTA Diazepam on social dominance were blocked by intra-NAc-shell pretreatment with a D1-antagonist. Our results highlight the personality trait anxiety in social hierarchies and emphasize mesolimbic dopaminergic mechanisms for the mediation of these effects. We suggest that anxiolytics acting on the mesolimbic dopamine system could reduce the predisposition for a low rank in highly anxious individuals.
Presenter:	Bovard David
Affiliation:	Department of Pharmacology and Toxicology
Title:	CNGA4 ion channels in the mouse vomeronasal neurons : new roles in association with TRPC2
Abstract:          Mammalian pheromones are key chemical signals in the regulation of social behaviors such as aggressivity or sexual mating. The detection of pheromones, which takes place in sensory neurons of the vomeronasal organ (VNO), implies the activation of the transient receptor potential canonical channel 2 (TRPC2) as the final effector. While the role of this protein is now well understood, some other channel proteins expressed in the VNO remain without a function. This is the case for the cyclic nucleotide-gated channel 4 (CNGA4). CNGA4, forms a heteromeric cationic channel in the main olfactory epithelium with two others CNG subunits. It increases the sensitivity of this channel to cyclic nucleotides and is also important for the calmodulin-dependent odor adaptation. As no other CNG subunits are present in the VNO, the role of CNGA4 in this organ might be completely different. We therefore decided to study the role of the CNGA4 protein in the VNO. We observed the protein to be expressed in axons, dendrites and on the microvilli of the vomeronasal sensory neurons. We found that it indeed plays a role in the pheromone signaling pathway as mice lacking the CNGA4 protein display a modified social behavior. These modified behaviors were similar to those observed with TRPC2-/- mice. We thus hypothesized that CNGA4 and TRPC2 might interact and form a heteromeric channel. We further observed with in vitro experiments using HEK cells as an expression system that CNGA4 could directly interact with TRPC2 acting either as a chaperon or as a subunit of a heteromeric channel. These results give us some new insights on the combined roles of these vomeronasal transduction ion channels.

Circulatory factors modulate brain aging and plasticity

Growing evidence links neurodegeneration with altered immune responses not only in the brain but in the periphery as well. In addition, age is the main risk factor for sporadic forms of neurodegenerative diseases and aging of peripheral organs may affect brain function. How the systemic environment affects brain health is largely unknown and while some of these interactions may involve cells entering the nervous tissue it is likely that others are mediated by soluble factors. We use a combination of physiological methods to manipulate systemic aging and proteomic methods to try to identify factors that age or potentially rejuvenate the brain. Our findings point to systemic changes in immune responses and cellular signaling factors with aging and may be relevant for our understanding of age-related neurodegeneration.

Gene-circuit interactions during assembly of forebrain neurons

While the initial assembly of neurons into specific circuits largely relies on cell-intrinsic differentiation programs, reciprocally, circuit activity instructs cell-type specific gene expression programs later in development. We will discuss recent work from our laboratory illustrating how such developmental cross-talks between gene expression and circuit connectivity act to orchestrate the assembly of cognate neurons into functionally-specialized pathways.

Astrocytes regulate adult hippocampal neurogenesis

Adult neurogenesis results in the constant addition of new neurons, which have increased plastic properties and participate to mechanisms of learning and memory. Unlike during development, several steps of adult neurogenesis from cell proliferation to neuronal differentiation and synaptic integration depend on cell-extrinsic mechanisms involving neuronal activity, pathological conditions or animal behavior. Here, we will show how the neurogenic niche is involved in the regulation of several steps of adult neurogenesis and discuss the relevance of these mechanisms for brain plasticity.