SUMMARY Because molecular mechanisms underlying refractory focal epilepsy are poorly defined, we performed transcriptome analysis on human epileptogenic tissue. Compared with controls, expression of Circadian Locomotor Output Cycles Kaput (CLOCK) is decreased in epileptogenic tissue. To define the function of CLOCK, we generated and tested the Emx-Cre; Clockflox/flox and PV-Cre; Clockflox/flox mouse lines with targeted deletions of the Clock gene in excitatory and parvalbumin (PV)-expressing inhibitory neurons, respectively. The Emx-Cre; Clockflox/flox mouse line alone has decreased seizure thresholds, but no laminar or dendritic defects in the cortex. However, excitatory neurons from the Emx-Cre; Clockflox/flox mouse have spontaneous epileptiform discharges. Both neurons from Emx-Cre; Clockflox/flox mouse and human epileptogenic tissue exhibit decreased spontaneous inhibitory post-synaptic currents. Finally, video-EEG of Emx-Cre; Clockflox/flox mice reveals epileptiform discharges during sleep and also seizures arising from sleep. Altogether, these data show that disruption of CLOCK alters cortical circuits and may lead to generation of focal epilepsy.

Fragile X Syndrome (FXS) is a neurodevelopmental disorder instigated by the absence of a key translation regulating protein, Fragile X Mental Retardation Protein (FMRP). The loss of FMRP in the CNS leads to abnormal synaptic development, disruption of critical periods of plasticity, and an overall deficiency in proper sensory circuit coding leading to hyperexcitable sensory networks. However, little is known about how this hyperexcitable environment affects inhibitory synaptic plasticity. Here, we show that in vivo layer 2/3 of the primary somatosensory cortex of the Fmr1 KO mouse exhibits basal hyperexcitability and an increase in neuronal firing rate suppression during whisker activation. This aligns with our in vitro data that indicate an increase in GABAergic spontaneous activity, a faulty mGluR-mediated inhibitory input and impaired inhibitory plasticity processes. Specifically, we find that mGluR activation sensitivity is overall diminished in the Fmr1 KO mouse leading to both a decreased spontaneous inhibitory postsynaptic input to principal cells and a disrupted form of inhibitory long-term depression (I-LTD). These data suggest an adaptive mechanism that acts to homeostatically counterbalance the cortical hyperexcitability observed in FXS.

39In mouse models of Fragile X Syndrome (FXS), cellular and circuit hyperexcitability are a 40 consequence of altered brain development [reviewed in (Contractor et al., 2015)]. Mechanisms 41 that favor or hinder plasticity of synapses could affect neuronal excitability. This includes 42 inhibitory long term depression (I-LTD) -a heterosynaptic form of plasticity that requires the 43 activation of metabotropic glutamate receptors (mGluRs). Differential circuit maturation leads 44 to shifted time points for critical periods of synaptic plasticity across multiple brain regions 45 (Harlow et al., 2010;He et al., 2014), and disruptions of the development of excitatory and 46 inhibitory synaptic function are also observed both during development and into adulthood 47 (Vislay et al., 2013). However, little is known about how this hyperexcitable environment affects 48 inhibitory synaptic plasticity. Our results demonstrate that the somatosensory cortex of the 49 Fmr1 KO mouse model of FXS exhibits increased GABAergic spontaneous activity, a faulty 50 mGluR-mediated inhibitory input and impaired plasticity processes. We find the overall 51 diminished mGluR activation in the Fmr1 KO mice leads to both a decreased spontaneous 52 inhibitory postsynaptic input to principal cells and also to a disrupted form of inhibitory long 53 term depression (I-LTD). In cortical synapses, this I-LTD is dependent on mGluR activation and 54 the mobilization endocannabinoids (eCBs). Notably, these data suggest enhanced 55 hyperexcitable phenotypes in FXS may be homeostatically counterbalanced by the inhibitory 56 drive of the network and its altered response to mGluR modulation. 57 58 Significance Statement 59Fragile X Syndrome is a pervasive neurodevelopmental disorder characterized by intellectual 60 disability, autism, epilepsy, anxiety and altered sensory sensitivity. In both in vitro and in vivo 61 recordings in the somatosensory cortex of the Fmr1 knockout mouse model of Fragile X 62 Syndrome we show that hyperexcitable network activity contributes to ineffective synaptic 63 plasticity at inhibitory synapses. This increased excitability prevents cortical circuits from 64 adapting to sensory information via ineffective plasticity mechanisms. 65 66 102016(03)1D/00039). Both Control and Fmr1 KO mice were acquired from the Jackson 113 Laboratories (Bar Harbor, ME, USA) and bred onsite. The Fmr1 KO animals obtained from these 114 crosses were tested through genotyping protocols. Male mice utilized in this study possess the 115 same congenic FVB background and only hemizygous for the X chromosome Fmr1 gene were 116 utilized for the experiments in this research. 117 118 Slice preparation 119Postnatal 19 to 25 day old control and Fmr1 KO mice were anesthetized by CO2 inhalation and 120 decapitated. Brains were quickly removed and placed in ice-cold oxygenated sucrose slicing 121 solution composed of (in mM): 234 sucrose, 11 glucose, 26 NaHCO3, 2.5 KCl, 1.25 NaH2PO4 10, 122MgSO4, and 0.5 CaCl2 (equilibrated with 95% O2 and 5% CO2, pH 7.