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. 2016 Apr 6;90(1):56-69.
doi: 10.1016/j.neuron.2016.02.040. Epub 2016 Mar 24.

GABAergic Neuron-Specific Loss of Ube3a Causes Angelman Syndrome-Like EEG Abnormalities and Enhances Seizure Susceptibility

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GABAergic Neuron-Specific Loss of Ube3a Causes Angelman Syndrome-Like EEG Abnormalities and Enhances Seizure Susceptibility

Matthew C Judson et al. Neuron. .
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Abstract

Loss of maternal UBE3A causes Angelman syndrome (AS), a neurodevelopmental disorder associated with severe epilepsy. We previously implicated GABAergic deficits onto layer (L) 2/3 pyramidal neurons in the pathogenesis of neocortical hyperexcitability, and perhaps epilepsy, in AS model mice. Here we investigate consequences of selective Ube3a loss from either GABAergic or glutamatergic neurons, focusing on the development of hyperexcitability within L2/3 neocortex and in broader circuit and behavioral contexts. We find that GABAergic Ube3a loss causes AS-like increases in neocortical EEG delta power, enhances seizure susceptibility, and leads to presynaptic accumulation of clathrin-coated vesicles (CCVs)-all without decreasing GABAergic inhibition onto L2/3 pyramidal neurons. Conversely, glutamatergic Ube3a loss fails to yield EEG abnormalities, seizures, or associated CCV phenotypes, despite impairing tonic inhibition onto L2/3 pyramidal neurons. These results substantiate GABAergic Ube3a loss as the principal cause of circuit hyperexcitability in AS mice, lending insight into ictogenic mechanisms in AS.

Figures

Figure 1
Figure 1. GABAergic Ube3a loss does not compromise synaptic inhibition onto L2/3 pyramidal neurons
(A) Immunostaining of parvalbumin (PV), Cux1, and UBE3A in V1 of ~P80 Control (A1) and Ube3aFLOX/p+::Gad2-Cre (A2) mice. Arrowheads indicate PV-positive interneurons lacking UBE3A (scale bar = 145 µm or 75 µm for zoom-ins). (B) Schematic for recording synaptic inhibition onto L2/3 pyramidal neurons in V1 of ~P80 Ube3aFLOX/p+::Gad2-Cre mice (green shading indicates presence of UBE3A). (C) Sample recordings (scale bar = 20 pA, 200 ms) and quantification of mIPSC amplitude and frequency (Control n = 11 cells; Ube3aFLOX/p+::Gad2-Cre n = 17 cells). (D) Sample recordings of eIPSCs (D1) at stimulation intensities of 2, 10, 30, and 100 µA (scale bar = 1 nA, 40 ms). (D2) Quantification of eIPSCs. Inset depicts response amplitudes to 100 µA stimulation (Control n = 22 cells; Ube3aFLOX/p+::Gad2-Cre n = 24 cells). (E) Sample recordings (E1) depicting each phase of an inhibitory synaptic depletion and recovery experiment (scale bars: baseline = 200 pA, 20 ms; depletion = 200 pA, 70 ms; recovery = 200 pA, 20 ms). (E2) Average depletion phase showing eIPSC amplitude normalized to baseline during 800 stimuli at 30 Hz. Each point (80 plotted per genotype) represents 10 consecutive responses that were collapsed and averaged per cell. (E3) Average recovery phase showing eIPSC amplitude normalized to baseline during 90 stimuli at 0.33 Hz. Each point (30 plotted per genotype) represents 3 consecutive responses that were collapsed and averaged per cell. Average depletion and recovery responses for each genotype were fit with a monophasic exponential (Control n = 11 cells; Ube3aFLOX/p+::Gad2-Cre n = 9 cells). Data represent mean ± SEM.
Figure 2
Figure 2. GABAergic Ube3a reinstatement in Ube3aSTOP/p+::Gad2-Cre mice models glutamatergic Ube3a loss and indicates an evoked IPSC amplitude deficit onto L2/3 pyramidal neurons
(A) Immunostaining of parvalbumin (PV), Cux1, and UBE3A in V1 of ~P80 Control (A1), Ube3aSTOP/p+ (A2), and Ube3aSTOP/p+::Gad2-Cre (A3) mice. Arrowheads indicate PV-positive interneurons that co-stain for UBE3A. Arrows point to PV-negative interneurons that co-stain for UBE3A (scale bar = 145 µm or 75 µm for zoom-ins). (B) Schematic for recording synaptic inhibition onto L2/3 pyramidal neurons in V1 of ~P80 Ube3aSTOP/p+::Gad2-Cre mice. (C) Sample recordings of eIPSCs (C1) at stimulation intensities of 2, 10, 30, and 100 µA (scale bar = 1 nA, 60 ms). (C2) Quantification of eIPSCs. Inset depicts response amplitudes to 100 µA stimulation (Control n = 38 cells; Ube3aSTOP/p+ n = 44 cells; Ube3aSTOP/p+::Gad2-Cre n = 40 cells). (D) Inhibitory synaptic depletion and recovery in Control (n = 9 cells), Ube3aSTOP/p+ (n = 11 cells), and Ube3aSTOP/p+::Gad2-Cre (n = 13 cells) mice, performed as in Figure 1E. Scale bars (D1): baseline = 200 pA, 20 ms; depletion = 200 pA, 70 ms; recovery = 200 pA, 20 ms. Data represent mean ± SEM. *p≤0.05.
Figure 3
Figure 3. Glutamatergic Ube3a loss selectively reduces evoked IPSC amplitude and tonic inhibitory tone onto L2/3 pyramidal neuron
(A) Immunostaining of parvalbumin (PV), Cux1, and UBE3A in V1 of ~P80 Control (A1), Ube3aSTOP/p+ (A2), Ube3aSTOP/p+::NEX-Cre (A3), and Ube3aFLOX/p+::NEX-Cre (A4) mice. Double arrows indicate PV-positive interneurons that lack UBE3A, arrowheads indicate PV-positive interneurons that co-stain for UBE3A but lack Cux1, and single arrows depict Cux1- and PV-negative interneurons that co-stain for UBE3A (scale bar = 75 µm for all panels). (B) Schematic for recording inhibition onto L2/3 pyramidal neurons in V1 of ~P80 Ube3aSTOP/p+::NEX-Cre mice. (C) Sample recordings of eIPSCs (C1) at stimulation intensities of 2, 10, 30, and 100 µA (scale bar = 800 pA, 20 ms). (C2) Quantification of eIPSCs. Inset depicts response amplitudes to 100 µA stimulation (Control n = 14 cells; Ube3aSTOP/p+ n= 15 cells; Ube3aSTOP/p+::NEX-Cre n = 16 cells). (D) Schematic for recording inhibition onto L2/3 pyramidal neurons in V1 of ~P80 Ube3aFLOX/p+::NEX-Cre mice. (E) Sample recordings of eIPSCs (E1) at stimulation intensities of 2, 10, 30, and 100 µA (scale bar = 1 nA, 40 ms). (E2) Quantification of eIPSCs. Inset depicts response amplitudes to 80 µA stimulation (Control n = 14 cells; Ube3aFLOX/p+::NEX-Cre n = 22 cells). (F) Sample recordings (F1, scale bar = 20 pA, 200 ms) and quantification of mIPSC amplitude and frequency (F2) (Control n = 15 cells; Ube3aFLOX/p+::NEX-Cre n = 11 cells). (G) Representative trace (G1) from experiments to measure tonic inhibitory currents onto L2/3 pyramidal neurons (scale bar = 150 pA, 120 s). (G2) Quantification of change in Iholding in response to the application of the δ-GABAAR agonist THIP (left) and the subsequent chase with the competitive GABAAR antagonist, Gabazine (right) (Control n = 30 cells; Ube3aFLOX/p+::NEX-Cre n = 21 cells). Data represent mean ± SEM. *p≤0.05, **p≤0.01.
Figure 4
Figure 4. GABAergic, but not glutamatergic Ube3a loss lowers the threshold for flurothyl-induced seizures
(A) UBE3A staining in Ube3aFLOX/p+::NEX-Cre (A1) and Ube3aFLOX/p+::Gad2-Cre (A2) (scale bar = 750 µm; 400 µm for zoom-ins). Ctx, cerebral cortex; Hip, hippocampus; MGN, medial geniculate thalamic nucleus; SNR, substantia nigra pars reticulata. (B) Schematic of flurothyl-induced seizure protocol. Flurothyl administration ceases upon the occurrence of a generalized seizure. (C) Latency to myoclonus (C1) and generalized seizure (C2) in Control (n = 12) and Ube3aFLOX/p+::NEX-Cre (n = 7) mice at ~P80. (D) Latency to myoclonus (D1) and generalized seizure (D2) in Control (n = 13) and Ube3aFLOX/p+::Gad2-Cre (n = 11) mice at ~P80. Comparative survival (D3) of Control and Ube3aFLOX/p+::Gad2-Cre mice following repeated once daily exposures to flurothyl. Data represent mean ± SEM. **** p≤0.0001.
Figure 5
Figure 5. GABAergic, but not glutamatergic, Ube3a loss enhances audiogenic seizure susceptibility in AS model mice
(A) Schematic of audiogenic seizure protocol. (B) Quantification of audiogenic seizure susceptibility in Control (n = 26), Ube3aSTOP/p+ (n = 13), and Ube3aSTOP/p+::Gad2-Cre (n = 13) mice at ~P80. (C) Quantification of audiogenic seizure susceptibility (Control n = 15; Ube3aFLOX/p+::Gad2-Cre n = 13). (D) Post-weaning (P21 – P90) lethality in Control and Ube3aFLOX/p+::Gad2-Cre mice. Data represent mean ± SEM. **p≤0.01; ***p≤0.001; **** p≤0.0001.
Figure 6
Figure 6. GABAergic Ube3a loss selectively enhances LFP spectral power in the delta band
(A) Schematized configuration for local field potential (LFP) recordings in non-anesthetized mice. (B) Sample V1 LFP recordings (B1, scale bar = 100 µV, 1 s) and quantification of average spectral power (B2) from Control (n = 11) and Ube3aFLOX/p+::Gad2-Cre (n = 11) mice at ~P100. (B3) Quantification of the region (3–4 Hz) encompassing the largest genotypic difference in power within the delta band. (C) Sample V1 LFP recordings (C1, scale bar = 100 µV, 1 s) and quantification of average spectral power (C2) from from Control (n = 12), Ube3aSTOP/p+ (n = 9), and Ube3aSTOP/p+::Gad2-Cre (n = 11) mice at ~P100. (C3) Quantification of the region (2–3 Hz) encompassing the largest genotypic difference in power within the delta band. Data represent mean ± SEM. *p≤0.05.
Figure 7
Figure 7. GABAergic Ube3a loss underlies presynaptic CCV accumulation at both GABAergic and glutamatergic synapses
(A) Electron micrographs of dendritic inhibitory synapses stained for GABA in Control (A1) and Ube3aFLOX/p+::Gad2-Cre (A2) mice at ~P80. Green denotes GABAergic axon terminal, blue denotes dendrite, inset highlights clathrin-coated vesicles (CCVs) (scale bar = 200 nm). Average CCV densities at dendritic GABAergic synapses (A3, Control n = 89 synapses from 3 mice; Ube3aFLOX/p+::Gad2-Cre n = 77 synapses from 3 mice), somatic GABAergic synapses (A4, Control n =78 synapses from 3 mice; Ube3aFLOX/p+::Gad2-Cre n = 81 synapses from 3 mice), and spinous glutamatergic synapses (A5, Control n = 82 synapses from 3 mice; Ube3aFLOX/p+::Gad2-Cre n = 80 synapses from 3 mice). (B) Electron micrographs of dendritic inhibitory synapses stained for GABA in Control (B1), Ube3aSTOP/p+ (B2), and Ube3aSTOP/p+::Gad2-Cre (B3) mice at ~P80. Green denotes GABAergic axon terminal, blue denotes dendrite, inset highlights clathrin-coated vesicles (CCVs) (scale bar = 400 nm). Average CCV densities at dendritic GABAergic synapses (B4, Control n = 110 synapses from 3 mice; Ube3aSTOP/p+ n = 119 synapses from 3 mice; Ube3aSTOP/p+::Gad2-Cre n = 115 synapses from 3 mice), somatic GABAergic synapses (B5, Control n = 103 synapses from 3 mice; Ube3aSTOP/p+ n = 114 synapses from 3 mice; Ube3aSTOP/p+::Gad2-Cre n = 110 synapses from 3 mice), and spinous glutamatergic synapses (B6, Control n = 108 synapses from 3 mice; Ube3aSTOP/p+ n = 113 synapses from 3 mice; Ube3aSTOP/p+::Gad2-Cre n = 113 synapses from 3 mice). Data represent mean ± SEM. * p<0.05; ** p<0.01; ****p<0.0001.

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