FMRP regulation of local and long-range neocortical circuits in the mouse: Links with EEG phenotypes

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Project 1 (P1) investigators have identified cortical EEG phenotypes in humans with Fragile X Syndrome (FXS), such as enhanced resting state gamma power, that correlate with clinical outcomes. Remarkably, many human EEG phenotypes are conserved in the mouse model of FXS, the Fmr1 knockout (KO), as discovered by Project 2 (P2) investigators. The across-species conservation of EEG phenotypes strongly suggest similarly dysfunctional cortical circuits in mice and humans with loss of function of Fmr1. Thus, our proposed studies to understand and correct circuit dysfunction in the Fmr1 KO mouse will likely be highly relevant to humans with FXS. We have identified two major functional circuit defects in primary sensory cortex of the Fmr1 KO mouse that likely contribute to specific EEG phenotypes in Fmr1 KO mice and humans with FXS – 1) hyperexcitability of “local” circuits within a cortical region and 2) reduced functional excitatory long- range connections between cortical regions. Hyperexcitability of local circuits is observed ex vivo in brain slices with 2 key measures: 1) Prolonged Circuit Activation (PCA) of neocortical circuits, either spontaneously or in response to sensory stimulation and 2) enhanced gamma power. We hypothesize that hyperexcitability of local neocortical microcircuits underlies the increased resting state gamma power and alterations in sensory-evoked gamma entrainment of the EEG observed in Fmr1 KO mice and humans with FXS. We test this hypothesis in mice through mechanistic conservation. Using ex vivo neocortical slices of Fmr1 KO mice we have discovered 3 synaptic microcircuit changes likely to give rise to hyperexcitable local circuits; 1) Hyperconnectivity of excitatory synapses between pyramidal neurons 2) Reduced excitatory synaptic drive onto Parvalbumin-positive (PV) inhibitory neurons 3) enhanced endocannabinoid suppression of inhibitory synaptic drive – likely from Cholecystokinin-positive (CCK) neurons. In Aims 1 and 2, in close collaboration with P2, we will use optogenetic, chemogenetic, and pharmacological approaches to manipulate PV and CCK inhibitory circuits in order to determine how their dysfunction contributes to hyperexcitability of local cortical circuits, abnormal EEG phenotypes, and related behaviors in Fmr1 KO mice. In addition to hyperexcitable local circuits, our new findings reveal weak excitatory connectivity between cortical regions in Fmr1 KO mice - specifically between homotopic contralateral cortical areas (callosal connections). Dysfunctional long-range synaptic connectivity likely contributes to the abnormal long-range functional coupling between cortical areas as observed by EEG in humans with FXS). In Aims 3 and 4, we propose experiments to determine the underlying synaptic and molecular mechanisms as well as the functional circuit level consequences of these weak long- range excitatory connections. Results of Project 3 (P3) are expected to determine the specific dysfunctional cell- types, microcircuits, and biochemical mechanisms that likely mediate human relevant EEG phenotypes in FXS and provide a rationale for specific, targeted therapeutic strategies.