Final Report Summary - INTRICA (Development and Neuromodulation of Intrinsic Cortical Activity) Project’s ObjectivesNeurons in the brain neocortex form recurrent networks that are intrinsically active in the form of oscillating activity, visible at increasingly macroscopic neurophysiological levels: from the local field potentials (LFPs) to the clinically relevant electroencephalography (EEG). In vivo, during non-REM sleep and quite wakefulness (rest) but also in vitro, in brain slices, the cortex spontaneously engages in a slow (<1Hz) oscillation whose cellular correlates are alternating periods of sustained depolarization and hyperpolarization (Up and Down states, respectively). The spontaneous activity of the cortex is of particular neurobiological and clinical interest since it reflects the cortex’s hardwiring as shaped by genes and experience, the intrinsic properties of its cells and the dynamics of their synapses; and it consists the background upon which incoming sensory stimuli interact determining cortical responses and behavior. In the clinic, EEG recordings of spontaneous cortical activity during rest are employed for the discovery of biomarkers of psychiatric disorders, such as autism and schizophrenia. In the current study we used LFP recordings to assess Up states in spontaneously active cortical slices of mice in order to:Objective 1: Test whether and how spontaneous Up states in vitro change during cortical postnatal development and maturation. Objective 2: To investigate the cholinergic modulation of spontaneous cortical Up states.Objective 3: To study changes in spontaneous cortical Up states during postnatal development in a mouse model for autism. Overview of ResultsResult 1: In this study, we used LFP recordings to assess the emergence and maturation of recurrent network activity in spontaneously active cortical slices from the first postnatal days to 27 months. In regard with objective 1 of our initial proposal the study was expanded in order to: (a) include multiple age groups (as opposed to the original two: 40do and 6mo) in order to achieve a continuum of ages and define developmental trajectories, and (b) evaluate the changes that occur during early development in comparison to those that occur during maturation and ageing. In addition, spontaneous cortical Up states were compared in two distinct cortical areas: the primary somatosensory and the primary motor cortex. We find that spontaneous Up states in vitro change systematically with age in a region-specific manner (Fig. 1). Up states’ developmental profile delineates periods of pronounced changes, indicative of intense re-organization of cortical circuits; and also periods of constancy, which likely reflect stability in the underlying cellular and synaptic elements. We also document that the transition to mature patterns of activity between adolescence and adulthood, as well as between adulthood and old age is at least partially mediated by changes in GABAA- and GABAB-inhibition, respectively. Finally, we introduce a novel, non-parametric, way of quantifying Up state activity by means of pattern analysis using dynamical trajectories. A manuscript describing these results is under review in Cerebral Cortex. Result 2: We investigated the cholinergic control of spontaneous cortical Up states via high- and low-affinity nicotinic receptors (nAChRs) and its contribution to changes that occur during ageing in Up states’ dynamics. Using genetically modified animals and pharmacological manipulations we find that nicotinic receptors (nAChRs) modulate up state activity through both high-affinity (containing the β2 subunit, β2-nAChRs) and low-affinity receptors (containing the α7 subunit, α7-nAChRs) but through distinct mechanisms. Moreover, we discovered a cross-talk between the high-affinity nicotinic receptors (β2-nAChRs) and GABAB-mediated inhibition. A manuscript describing these results is under review in the Journal of Neuroscience. Result 3: We decided to investigate the development of spontaneous cortical Up states in two distinct mouse models of autism spectrum disorders (ASD) in order to assess the generalisability of the findings. We first examined an environmental model of autism (prenatal injections of valproic acid, or VPA). These experiments revealed no significant deviation of VPA-treated animals from normal, refuting our hypothesis that Up states may provide biomarkers of neurodevelopmental disorders. We next examined a genetic model (Fragile-X mouse model) and these experiments are still in progress. In addition, we engaged a parallel line of experiments during the second period of the project in which we asked whether spontaneous Up states in the adult brain can reflect an experience of early life seizures, and we found that indeed Up states are affected in an area-specific manner. Conclusions(1) In this study we propose that spontaneous network activity, in the form of recurring Up/Down states, recorded in acute cortical slices can serve as a reliable marker of functional cortical maturation, defining periods of intense re-organization in the underlying neuronal circuits. Our data show that developmental modifications in this activity extend past the early developmental period into early adulthood (3rd postnatal month; fig. 1), suggesting that adolescence is a period during which cortical networks are still being stabilized. We suggest this functional index reflects the combined cellular and molecular maturational changes of the cortex in the framework of network dynamics, and hence provides an integrated view of cortical development across the entire life span of the mouse. We therefore propose it can be useful as a baseline against which to compare cortical dynamics in animal models of neurological and mental disorders of cortical origin. Hence this work introduces an in vitro model for the development and maturation of the normal cortical network and sets the stage for the discovery of endophenotypes of disorders that are manifested as disruptions of the excitation-inhibition balance, such as epilepsy, mental retardation disorders (e.g. Rett or Down syndrome), autism and schizophrenia.(2) Local recurrent cortical networks exhibit spontaneous network activity in the form of Up/Down states - a prominent feature of the cortical activity during slow wave sleep in vivo. The fact that this type of persistent activity can be sustained in the absence of sub-cortical or long range inputs, has fuelled the proposal that Up/Down state activity in brain slices can serve as a model of the basic operation of the cortex and that the mechanisms that generate or modulate this type of activity may form the substrate for cognitive functions such as short-term memory, memory consolidation, or modulation of neuronal activity during attention. Here we have revealed a direct modulatory role for the high affinity nAChRs acting through GABABRs in the regulation of this network phenomenon. Our results provide a framework that integrates previously contradictory results and suggest a new role for the nicotinic modulation of intrinsic persistent cortical activity under conditions of low ACh release, that could generate novel insights for therapeutic interventions of disorders related to abnormal patterns of cortical dynamics. Socio-economic impactIncreasing numbers of people suffering from severe brain disorders, either neurological or psychiatric, is a true emotional and financial burden for their families and society, and a significant cost for the state economy. Unfortunately, drugs currently available to treat diseases of the brain are often of limited effectiveness, with considerable side-effects. Moreover, brain disorders such as epilepsy, autism, schizophrenia have a neurodevelopmental profile, which makes their understanding with longitudinal studies in humans complicated and difficult to control for. This reality stresses the need for more research of the neurobiology underlying brain pathologies by developing proper experimental models. Interestingly, accumulating evidence suggests that abnormal rhythms within the thalamocortical system are present in a wide variety of neurological and psychiatric conditions (Llinas et al., 1999; Schulmann et al., 2011). For example, in the clinic, EEG recordings of spontaneous cortical activity during rest are employed for the discovery of biomarkers of psychiatric disorders, such as autism and schizophrenia. In order for biological research of brain disorders to proceed, it is essential to ‘decompose’ complex disorders into simpler parameters that can serve as biological markers or endophenotypes of the disease. An endophenotype is the biological manifestation of a disease at a reduced level of biological organization as opposed to the macro-level of behaviour (Gottesman and Gould 2003; Hasler et al. 2004). In this perspective, animal models are essential and have been useful in identifying molecular and cellular markers relevant to behaviour, contributing to the discovery of final common functional pathways of respective disorders. To our knowledge, using spontaneous in vitro Up states of the mouse neocortex as a marker of functional cortical development has not been previously proposed and could be valuable as a baseline against which to compare developmental deficits in animal models of neurological and mental disorders, leading to the discovery of network endophenotypes that would promote their study and would pave the path for the better understanding of their underlying synaptic, cellular and molecular mechanisms (Fig. 2). Llinas, R.R. Ribary, U., Jeanmonod, D., Kronberg, E. and Mitra, P.P. (1999):Thalamocortical dysrhythmia: A neurological and neuropsychiatric syndrome characterized by magnetoenc-ephalography. PNAS, 96(26): 15222-15227Hasler G, Drevets WC, Manji HK, Charney DS. (2004). Discovering endophenotypes for major depression. Neuropsychopharmacology 29:1765-1781Gottesman, II, Gould TD. (2003). The endophenotype concept in psychiatry: etymology and strategic intentions. Am J Psychiatry 160:636-645.Schulman JJ, Cancro R, Lowe S, Lu F, Walton KD, Llinás RR. (2011) Imaging of thalamocortical dysrhythmia in neuropsychiatry. Front Hum Neurosci. 5: 1-11.
