Truszkowski TL , James EJ , Hasan M , Wishard TJ , Liu Z , Pratt KG , Cline HT , Aizenman CD .

R01 EY011261 NEI NIH HHS , T34 GM087193 NIGMS NIH HHS , T32 NS062443 NINDS NIH HHS

                fragile X syndrome

	Fig. 1. Background swimming speeds, schooling behavior and seizure severity are affected by FMRP KD. a Background swimming is decreased in FMRP KD tadpoles (Pt < 0.05). b–c Visual avoidance behavior is unaffected. Collision-escape velocity (b, the velocity of the tadpole after collision with virtual object) and escape distance (c, the distance between the tadpole and the virtual object when the tadpole initiates avoidance response) are unaffected (Pt > 0.05 for controls and FMRP KD tadpoles in b, c). d–e FMRP KD tadpoles show reduced schooling. d FMRP KD tadpoles have fewer long and short distances and more medium distances between neighboring tadpoles, indicating more dispersed swimming and decreased aggregation (P KS < 0.05). e Control tadpoles have a higher frequency of less than 90° angles for co-orientation whereas FMRP KD tadpoles have no preference for alignment (P KS < 10−20), inset. Diagram explaining schooling behavior, with small clusters of tadpoles with more short (inter-cluster) and long (intra-cluster) distances in controls, and more medium distances in FMRP KD tadpoles. Tadpoles are also co oriented with their nearest neighbor. f–h FMRP KD tadpoles seize significantly less frequently and for longer than controls, indicating decreased seizure susceptibility. f FMRP KD tadpoles seize with significantly reduced frequency compared to control MO tadpoles (P t < 0.01), g FMRP KD tadpoles have significantly longer seizures (P t < 0.005), h Seizure length plotted against seizure frequency indicates a negative correlation between the two, and separation between experimental groups
	Fig. 2. Intrinsic properties of tectal neurons are similar between control and FMRP KD tadpoles. a Example traces showing voltage gated inward and outward currents evoked by a series of depolarizing pulses. Voltage steps, 0–90 mV in 10 mV increments, were presented for 150 ms while current was measured, from a holding potential of −60 mV. b Current–voltage relationship of inward sodium (Na) and outward potassium (K) voltage-gated currents. Dotted lines are the K current, solid lines are Na current. c Example spiking traces. Current steps, 10-200pA in 10pA increments were presented for 150 ms while voltage was measured. Voltage injections of 40pA and 180pA shown. d Current vs. spiking relationship. n = 29 per group, At 10pA injection, p = 0.002 and at 20pA injection, p = 0.002 (multiple t-tests by current step, Sidak-Bonferroni multiple comparisons correction). e Maximum number of spikes at a given current injection. n = 29 per group, PMW =0.8376
	Fig. 3. Spontaneous synaptic activity in tectal neurons is similar between control and FMRP KD tadpoles. a Example spontaneous EPSCs recorded at -60 mV in the presence of GABAA receptor blocker, picrotoxin. b Example spontaneous IPSCs recorded at +5 mV, the reversal potential for excitatory currents. c Frequency of sEPSCs. n = 28 for each group, PMW = 0.6784. d Amplitude of sEPSCs. n = 28 for each group, PMW =0.1069. e Frequency of sIPSCs. n = 18 control, n = 27 FMRP KD, PMW =0.1789. f Amplitude of sIPSCs. n = 18 control, n = 27 FMRP KD, PMW =0.5740. g Excitatory recurrent activity, defined as the presence of a barrage of activity at least 200 ms in duration with a change in holding current of at least 10pA. n = 28 for each group, PMW =0.3136. h Inhibitory recurrent activity, defined as the presence of a barrage of activity at least 200 ms in duration with a change in holding current of at least 10pA. n = 18 control, n = 27 FMRP KD, PMW =0.5101
	Fig. 4. Excitatory evoked activity is not different in FMRP KD tadpoles. a Example traces of excitatory evoked activity, used to calculate the AMPA:NMDA ratio (top, control, average of 24 traces (−65 mV) and 15 traces (+55 mV)); bottom, FMRP KD, average of 19 traces (−65 mV) and 23 traces (+55 mV)). b The AMPA:NMDA ratio quantifies the size of the response at -65 mV and at +55 mV and is not different between groups. n = 14 (control), n = 9 (FMRP KD), Pt = 0.77. c Example traces of paired pulse facilitation (single paired stimuli) collected at a holding potential of −60 mV. d The ratio of the second pulse to the first pulse in a paired pulse protocol shows the level of facilitation at the synapse. n = 9 (control), n = 12 (FMRP KD), Pt =0.82. e Example traces of recurrent activity (single stimulation). f Recurrent activity quantified over two timeframes. 0-50 ms is primarily driven by the monosynaptic visual afferents while 0-300 ms primarily measures the polysynaptic activity evoked by local tectal networks. n = 18 (control), n = 16 (FMRP KD), PMW =0.84 (0-50 ms), PMW =0.91 (0-300 ms). g Monosynapticity, the ratio of the monosynpatic response (0-50 ms) to the polysynaptic response (100-200 ms). n = 18 (control), n = 15 (FMRP KD), PMW =0.44
	Fig. 5. FMRP KD tadpole cells show increased evoked inhibitory activity. a Example traces of inhibitory evoked activity (recorded at +5 mv), and excitatory (recorded at −45 mv) used to calculate the excitation-inhibition balance (top, control; bottom, FMRP KD). b Evoked inhibitory recurrent activity at +5 mV, PMW = 0.048, n = 11 (control), n = 17 (FMRP KD). c Evoked excitatory recurrent activity at -45 mV, n = 11 (control), n = 17 (FMRP KD). d Excitation to inhibition ratio, Pt = 0.009, n = 11 (control), n = 17 (FMRP KD). e Average charge of inhibitory responses calculated over bins of 100 msec following stimulus show altered time course of inhibitory responses in FMRP KD group

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