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Neuron. Author manuscript; available in PMC Jun 7, 2013.
Published in final edited form as:
PMCID: PMC3372864
NIHMSID: NIHMS374177

Maternal loss of Ube3a produces an excitatory/inhibitory imbalance through neuron type-specific synaptic defects

SUMMARY

Angelman syndrome (AS) is a neurodevelopmental disorder caused by loss of the maternally inherited allele of UBE3A. AS model mice, which carry a maternal Ube3a null mutation (Ube3am−/p+), recapitulate major features of AS in humans, including enhanced seizure susceptibility. Excitatory neurotransmission onto neocortical pyramidal neurons is diminished in Ube3am−/p+ mice, seemingly at odds with enhanced seizure susceptibility. We show here that inhibitory drive onto neocortical pyramidal neurons is more severely decreased in Ube3am−/p+ mice. This inhibitory deficit follows the loss of excitatory inputs and appears to arise from defective presynaptic vesicle cycling in multiple interneuron populations. In contrast, excitatory and inhibitory synaptic inputs onto inhibitory interneurons are largely normal. Our results indicate that there are neuron type-specific synaptic deficits in Ube3am−/p+ mice despite the presence of Ube3a in all neurons. These deficits result in excitatory/inhibitory imbalance at cellular and circuit levels and may contribute to seizure susceptibility in AS.

Keywords: Ube3a, Angelman syndrome, excitatory/inhibitory imbalance, autism spectrum disorders, 15q11-q13

INTRODUCTION

Angelman syndrome (AS) is characterized by severe intellectual disabilities, EEG abnormalities, gait disturbances, disrupted sleep patterns, profound language impairment, and autism (Williams et al., 2006). Seizures are present in 90% of AS patients, significantly impacting their quality of life and that of their caregivers (Thibert et al., 2009). AS is caused by deletions or loss-of-function mutations in the maternally inherited allele of UBE3A (Rougeulle et al., 1997). UBE3A encodes an E3 ubiquitin ligase that, in the brain, is expressed primarily from the maternal allele as a result of neuron-specific imprinting (Albrecht et al., 1997). Similar to humans with AS, mice lacking maternal Ube3a (Ube3am−/p+) have abnormal EEG activity and are susceptible to cortical seizures, suggesting that loss of Ube3a might disrupt the excitatory/inhibitory balance in the neocortex (Jiang et al., 1998).

Loss of maternally inherited Ube3a results in decreased excitatory synaptic drive onto pyramidal neurons in layer 2/3 (L2/3) of neocortex, as evidenced by a loss of dendritic spines (Yashiro et al., 2009). Decreased Ube3a-mediated proteasomal degradation of Arc and Ephexin5 proteins may lead to excitatory synaptic defects (Greer et al., 2010; Margolis et al., 2010). These observations suggest a mechanism for how the loss of Ube3a may cause fewer and/or weaker excitatory synapses. While these deficits may be relevant to cognitive phenotypes in Ube3am−/p+ mice, they would not on their own predict hyperexcitability and increased seizure susceptibility. We hypothesized that Ube3a loss results in more severe inhibitory deficits, with the net outcome favoring cortical hyperexcitability.

Here, we use the visual cortex as a model to study the role of Ube3a in the establishment and function of inhibitory circuits. We show that Ube3am−/p+ mice have an abnormal accumulation of clathrin-coated vesicles at inhibitory axon terminals, indicating a defect in vesicle cycling. Consistent with this observation, inhibitory synaptic transmission onto L2/3 pyramidal neurons recovers slower following vesicle depletion in Ube3am−/p+ mice, compared to wildtypes. Recovery following high-frequency stimulation of excitatory synapses onto L2/3 pyramidal neurons, however, is normal. This discrepancy among synapse types may further contribute to excitatory/inhibitory imbalance during high levels of activity. Finally, we show that synaptic inputs onto inhibitory neurons in Ube3am−/p+ mice are largely normal. We conclude that neuron type-specific synaptic deficits likely underlie neocortical excitatory/inhibitory imbalance in AS.

RESULTS

Inhibitory Deficits in Mature Ube3am−/p+ Mice

An excitatory/inhibitory imbalance in AS could arise from reduced numbers of inhibitory interneurons, abnormal inhibitory connectivity, and/or decreased inhibitory neurotransmission. To test the first possibility, we performed immunohistochemistry for three markers – parvalbumin, calretinin, and somatostatin – which together label 96% of the total GABAergic interneurons in L2/3 of mouse primary visual cortex (V1) (Gonchar et al., 2007). We compared Ube3am−/p+ mice and their wildtype (WT) 129Sv/Ev strain littermate controls at postnatal day 80 (P80), an age where AS model mice exhibit abnormal EEG patterns and are susceptible to seizures (Jiang et al., 1998). We found no differences in the density of inhibitory interneurons expressing these markers (Figure 1A-B), implying that the relative number of inhibitory interneurons is normal in L2/3 of V1.

Figure 1
Inhibitory synaptic deficits arising through development in Ube3am−/p+ mice are not due to decreased density of inhibitory interneurons

Next we investigated the strength and number of inhibitory synapses onto L2/3 pyramidal neurons. Using whole-cell voltage-clamp, we recorded miniature inhibitory postsynaptic currents (mIPSCs) in the presence of tetrodotoxin to gauge spontaneous inhibitory synaptic activity onto excitatory L2/3 pyramidal neurons. We recorded mIPSCs at two ages: P25, during the critical period for ocular dominance plasticity and when excitatory deficits have been observed previously, and P80, when the visual cortex is fully mature. We observed no difference in mIPSC amplitude between WT and Ube3am−/p+ mice at either P25 or P80, suggesting that the loss of Ube3a did not change the strength of inhibitory synapses (Figure 1D; Table S1). While we saw no genotypic differences in mIPSC frequency at P25, L2/3 pyramidal neurons in Ube3am−/p+ mice had a reduction in mIPSC frequency by P80 (Figure 1E). These observations indicate that the loss of Ube3a leads to fewer functional inhibitory synapses, or a reduction of their release probability onto L2/3 pyramidal neurons.

To further investigate the development of inhibitory inputs onto L2/3 pyramidal neurons, we recorded evoked inhibitory postsynaptic currents (eIPSCs) using L4 stimulation at different intensities in P25 and P80 Ube3am−/p+ and WT mice (Figure 1F). This type of stimulation activates diverse inhibitory inputs and, with strong stimulation, can activate most of the inhibitory inputs onto L2/3 pyramidal neurons (Morales et al., 2002). We saw no significant difference in eIPSC amplitude at P25 (Figure 1G), but a large decrease in eIPSC amplitude at P80 in Ube3am−/p+ mice compared to WT (Figure 1H). Together, these results confirm that there is a severe deficit in the amount of inhibition arriving onto L2/3 pyramidal cells in the mature visual cortex of Ube3am−/p+ mice.

In principle, a decrease in eIPSC amplitude could arise from reductions in the number of postsynaptic GABA receptors, a decrease in the release probability of inhibitory axon terminals, fewer functional inhibitory synapses, or a depolarized inhibitory interneuron action potential threshold. It is unlikely that the decrease in eIPSC amplitude at P80 is due to a decrease in the number of GABA receptors at active synapses, since the amplitude of mIPSCs was similar in Ube3am−/p+ and WT mice. To assess whether the decrease in eIPSC amplitude is due to the loss of functional synapses or to a decrease in release probability, we examined the paired-pulse ratio of inhibitory inputs. Specifically, we stimulated L4 at varying inter-pulse intervals to evoke IPSCs in L2/3 pyramidal cells, and compared the paired-pulse ratio in WT and Ube3am−/p+ mice. We observed no difference in the paired-pulse ratio between genotypes at either P25 or P80, implying that release at functional inhibitory inputs onto L2/3 pyramidal cells is normal in response to brief stimuli given at several interpulse intervals (Figure 1I). Finally, we found no difference in the action potential threshold or intrinsic excitability of FS neurons (a major class of inhibitory interneurons) in Ube3am−/p+ mice compared to WT, indicating that reduced eIPSC amplitude is unlikely to be due to reductions in evoked action potentials in inhibitory interneurons (Figure S1A-B). By exclusion, our data suggest a reduction in the total number of functional inhibitory synapses.

Neuron type-specific Reduction in Functional Synapses in Ube3am−/p+ Mice

Our previous observations of reduced inhibition were made in Ube3am−/p+ mice on the 129Sv/Ev strain, which are susceptible to spontaneous seizures (Jiang et al., 1998), making it difficult to determine whether the synaptic abnormalities were a cause or result of seizures (Sloviter, 1987). To address this concern, we tested for possible synaptic deficits in Ube3am−/p+ mice maintained on the C57BL/6J strain, which have a low incidence of evoked seizures and no reported spontaneous seizures (Jiang et al., 1998). We performed mIPSC recordings at P80 in WT and Ube3am−/p+ C57BL/6J mice to test if synaptic defects arose in the absence of spontaneous seizures. As before, we observed no genotypic differences in the amplitude of mIPSCs, but a large decrease in mIPSC frequency in Ube3am−/p+ mice (Figure 2A). We also performed mEPSC recordings in L2/3 pyramidal neurons in mice on the C57BL/6J strain to confirm previous results from the 129Sv/Ev strain (Yashiro et al., 2009). Consistent with previous results, there was a significant decrease in mEPSC frequency, but not amplitude, between Ube3am−/p+ and WT mice (Figure 2B). These observations reveal that Ube3am−/p+ L2/3 pyramidal neurons have a 50% reduction in spontaneous inhibitory synaptic activity, but only a 28% decrease in excitatory synaptic activity. Additionally, intrinsic excitability was of L2/3 pyramidal neurons was increased in Ube3am−/p+ mice compared to WT (Figure S1C). Together these data suggest that a disproportionate loss of inhibition may lead to an excitatory/inhibitory imbalance in Ube3am−/p+ L2/3 pyramidal neurons.

Figure 2
Ube3a loss leads to neuron type-specific defects in inhibitory neurotransmission

Ube3a is expressed by both excitatory and inhibitory interneurons in the cerebral cortex (Sato and Stryker, 2010). Therefore, Ube3a loss might be expected to affect both neuron classes. To assess this possibility, we recorded spontaneous synaptic activity in fast-spiking (FS) inhibitory interneurons, which we identified by membrane properties, aspinous dendrites, and characteristic high firing rates (Okaty et al., 2009). FS inhibitory interneurons in L2/3 were targeted for whole-cell recording at P80, an age at which excitatory and inhibitory neurotransmission onto L2/3 pyramidal neurons is altered. In contrast to L2/3 pyramidal neurons, the loss of Ube3a did not affect either the amplitude or the frequency of mIPSCs onto FS inhibitory interneurons (Figure 2C). Moreover, excitatory connections onto FS inhibitory interneurons appeared normal, as Ube3a loss did not alter mEPSC amplitude or frequency (Figure 2D). Similar to L2/3, Ube3a loss did not change the frequency or amplitude of mIPSCs or mEPSCs onto L5/6 FS inhibitory interneurons (Figure S2A-B). These results imply that Ube3a loss has neuron type-specific synaptic effects.

Ube3a Loss Results in Inhibitory Deficits from FS Interneurons

We examined the effects of Ube3a loss on FS inhibitory interneurons, which provide the majority of perisomatic inhibitory input to L2/3 pyramidal neurons (Jiang et al., 2010), impart feed-forward and feedback inhibition, and have been implicated in seizure susceptibility (Di Cristo et al., 2004). Despite the challenge of performing paired recordings in adult neocortical slices, we were able to investigate synaptic connectivity between 83 pairs of L2/3 FS inhibitory interneurons and L2/3 pyramidal neurons in WT and Ube3am−/p+ mice at P80 (Table S3).

We first analyzed synaptic connectivity from FS inhibitory interneurons to pyramidal neurons. Using current-clamp recordings, we evoked action potentials in FS interneurons with depolarizing current injections at 30 Hz, and simultaneously recorded the response in pyramidal neurons (Figure 3A). To measure short-term plasticity we normalized the amplitude of the evoked IPSPs to the amplitude of the first IPSP in the train. We observed no change in the short-term plasticity between genotypes (Figure 3B). However, the amplitude of the first IPSP between these pairs was significantly decreased in Ube3am−/p+ mice, indicating decreased connection strength from FS inhibitory interneurons to L2/3 pyramidal neurons (Figure 3C). We also found a 31% decrease in connection probability in Ube3am−/p+ mice compared to WT mice (Figure 3D), supporting the conclusion that the decreased IPSP amplitude is likely due to a reduction in the number of functional synapses made from FS interneurons to pyramidal neurons. Finally, we estimated the average inhibitory drive from FS inhibitory interneurons onto L2/3 pyramidal neurons, by calculating the product of connection strength and connection probability, finding that inhibitory drive was reduced by 71% in Ube3am−/p+ mice compared to WT mice (Figure 3E).

Figure 3
Synaptic deficits arise from both FS and non-FS inhibitory interneurons in Ube3am−/p+ mice

To further investigate possible effects of Ube3a loss on synaptic connectivity, we examined the connections from L2/3 pyramidal neurons to FS inhibitory interneurons. We measured short-term plasticity and found that Ube3am−/p+ mice had increased facilitation at synapses from pyramidal neuron to FS interneurons (Figure S3B). To assess connection strength in this pathway, we measured the amplitude of the first EPSP evoked in the postsynaptic FS interneuron, detecting no difference between genotypes (Figure S3C). Finally, we found no genotypic difference in the connection probability of L2/3 pyramidal to FS inhibitory interneuron pairs (Figure S3D).

These data suggest that, while excitatory connection frequency and strength onto L2/3 FS interneurons are unchanged in Ube3am−/p+ mice, excitatory inputs onto FS inhibitory interneurons have altered short-term plasticity, potentially leading to defective engagement of FS inhibitory interneurons during trains of activity. The unchanged strength of the pyramidal to FS inhibitory interneuron connections in Ube3am−/p+ mice was unexpected, since short-term plasticity measurements indicated a change in release probability at this synapse. We conclude that other factors, such as differences in calcium buffering, coupling of calcium channels to release machinery, or vesicular trafficking, must underlie the observed changes (Atwood and Karunanithi, 2002). Together, these experiments identify a specific inhibitory interneuron subtype, FS inhibitory interneurons, that is at least partially responsible for the decrease in inhibition found in L2/3 pyramidal neurons in the AS model.

Ube3a Loss Results in Inhibitory Deficits from Non-FS Interneurons

L2/3 pyramidal neurons receive inhibition from a variety of inhibitory interneuron subtypes (Markram et al., 2004). To test whether inhibitory deficits in Ube3am−/p+ mice could also be ascribed to other types of interneurons, we used agatoxin, a potent irreversible antagonist of P/Q-type voltage-gated calcium channels (VGCCs), to block release of GABA selectively from FS inhibitory interneurons (Jiang et al., 2010). Agatoxin suppressed about 90% of the total eIPSCs in both WT and Ube3am−/p+ mice 20 minutes after perfusion of the toxin (Figure 3G). The agatoxin-insensitive portion of the eIPSC had an increased latency from stimulation onset and an increased rise time, suggesting that the agatoxin-insensitive inputs targeted the distal dendrites of L2/3 pyramidal neurons (Figure S3G-H). Agatoxin-insensitive inputs also had decreased paired-pulse depression compared to the total eIPSC, a signature of non-FS inhibitory interneurons (Figure S3F) (Gupta et al., 2000). After agatoxin perfusion, we recorded eIPSCs at different stimulation intensities and again found a decrease in the strength of inhibitory inputs in the Ube3am−/p+ mice, compared to WT, demonstrating that Ube3a loss also affects inputs from non-FS classes of inhibitory interneurons (Figure 3H).

Ube3am−/p+ mice have Defects in Synaptic Vesicle Cycling

Our electrophysiological data suggest that inhibitory deficits in Ube3am−/p+ mice result from a loss of functional inhibitory synapses onto L2/3 pyramidal neurons. However, a reduction in functional synapses could arise anatomically from fewer synaptic contacts, postsynaptically by a loss of functional receptors, or presynaptically by a severe depletion of releasable synaptic vesicles rendering a subset of inhibitory axon terminals non-functional. To test for an anatomical correlate to our functional data, we used immunohistochemistry to stain WT and Ube3am−/p+ mice for the vesicular GABA transporter (VGAT), a marker for the axon terminals of inhibitory interneurons (Chaudhry et al., 1998). We were surprised to see similar densities of VGAT-positive puncta in WT and Ube3am−/p+ mice, suggesting no change in the number of inhibitory interneuron axon terminals (Figure S4A-C). However, there remained the possibility that some of these axon terminals were nonfunctional. To explore this possibility, we used electron microscopy to examine synaptic structure in WT and Ube3am−/p+ mice. Post-embedding immunogold localization of GABA was used to identify inhibitory synapses onto somata in L2/3 of V1. The area of GABA-positive axon terminals and proportion of mitochondria per terminal were not different between WT and Ube3am−/p+ mice (Figure 4A2-A3). However, there was a decrease in the number of synaptic vesicles, and a large increase in the number of clathrin-coated vesicles (CCVs), in the Ube3am−/p+ mice compared to WT (Figure 4A4-A5, S4F-G). We also tested whether the defects we observed in inhibitory synapses were generalized to excitatory synapses. Similar to inhibitory synapses, we observed a decrease in the number of synaptic vesicles, but no change in the area of excitatory axon terminals or the proportion of mitochondria per terminal (Figure 4B1-B4, S4D-E). Finally, we saw little or no decrease in the number of CCVs at excitatory synapses between genotypes (Figure 4B5, S4D-E). These data suggest a defect in synaptic vesicle cycling in inhibitory synapses of Ube3am−/p+ mice.

Figure 4
Inhibitory synapses of Ube3am−/p+ mice have presynaptic defects

Incomplete Recovery of IPSCs after High-Frequency Stimulation in Ube3am−/p+ mice

Previous studies examining synaptic vesicle cycling have identified genes whose mutation leads to increased numbers of CCVs in axon terminals (Slepnev and De Camilli, 2000). Many of these mutant synapses maintain the ability to release neurotransmitter and have normal short-term plasticity; however, during periods of high activity these synapses fail to adequately replenish their synaptic vesicle pool, resulting in a delayed recovery to baseline levels of transmitter release (Luthi et al., 2001). These studies led us to test if inhibitory synapses in the Ube3am−/p+ mice had functional deficits similar to other synaptic vesicle cycling mutants. We applied a train of 800 stimuli at 10 Hz while recording eIPSCs in L2/3 pyramidal neurons in WT and Ube3am−/p+ mice (Figure 4C). We then decreased the stimulation frequency to 0.33 Hz and recorded the recovery phase of the eIPSC (Figure 4C1). Ube3a loss had no effect on the depletion phase of the eIPSC (Figure 2C2) in agreement with our previous experiments examining short-term plasticity (Figure 1I and and3B).3B). However, we found a large decrease in the rate and level of recovery of the eIPSC in Ube3am−/p+ mice compared to WT (Figure 4C3). These data are consistent with defects in inhibitory synaptic vesicle cycling in Ube3am−/p+ mice. Specifically, the decrease in recovery of the eIPSC, combined with the increase in CCVs, suggests an inability of newly endocytosed CCVs to reenter and replenish the synaptic vesicle pool. These defects may render a subset of inhibitory synapses nonfunctional in Ube3am−/p+ mice.

Finally, we challenged excitatory synapses with the same high frequency stimulation protocol that we used to test inhibitory synapses (Figure 4D1). Unlike inhibitory synapses, Ube3a loss did not have an effect on the recovery of excitatory synapses from high-frequency stimulation (Figure 4D3). Thus, the pronounced accumulation of CCVs observed at inhibitory synapses correlates well with the selective deficits in the synaptic recovery from high-frequency stimulation. Moreover, these functional data demonstrate that following a high-frequency train of activity a period of heightened excitatory/inhibitory imbalance may occur in this circuit.

DISCUSSION

This work represents the first demonstration that maternal loss of Ube3a, as seen in individuals with AS, leads to neuron type-specific synaptic deficits. Our findings suggest that loss of Ube3a can result in an excitatory/inhibitory imbalance in the neocortex.

Earlier studies showing decreased excitatory neurotransmission in Ube3am−/p+ mice were difficult to reconcile with reports of high seizure susceptibility (Jiang et al., 1998; Yashiro et al., 2009). Our data provides clarification, showing that the loss of Ube3a causes a particularly severe decrease in inhibitory input to L2/3 pyramidal neurons. We also report that AS model mice have a synaptic vesicle cycling defect, which suggests a basis for this deficit. The vesicle cycling defects we observe are similar to those observed after deletion of the presynaptic proteins synaptojanin (Cremona et al., 1999) or endophilin (Milosevic et al., 2011), both which lead to increased CCVs at synaptic terminals, and decreased synaptic recovery from high levels of activity. Notably, inhibitory synapses may be particularly sensitive to disruptions in vesicular trafficking, due to their enhanced activity and smaller vesicle pools (Hayashi et al., 2008). These results, combined with our functional studies describing defective inhibitory synaptic transmission in Ube3am−/p+ mice, suggest a means by which a hyperexcitable cortical circuit could arise despite fewer excitatory synapses.

Ube3a is present in both excitatory and inhibitory interneurons in the brain (Sato and Stryker, 2010). Our results showing different synaptic defects onto excitatory and inhibitory neurons indicate Ube3a deficiency causes neuron type-specific deficits. Since, Ube3a targets its substrate proteins for proteasomal degradation, the consequences of Ube3a loss may depend on which substrate proteins are normally present in a cell. This hypothesis is supported by recent work showing that Arc, a protein expressed postsynaptically in excitatory but not inhibitory interneurons, is a Ube3a substrate (Greer et al., 2010; McCurry et al., 2010). Thus, the loss of Ube3a is expected to cause an inappropriate overexpression of Arc in excitatory neurons without affecting inhibitory interneurons. Given the ability of Arc to influence AMPA receptor endocytosis (Chowdhury et al., 2006), the neuron type-specific expression of Arc could partly explain the excitatory synaptic defects observed onto L2/3 pyramidal neurons and the lack of effect in FS interneurons. Conversely, our findings suggest a novel synaptic defect in Ube3am−/p+ mice at inhibitory synapses, primarily affecting presynaptic function at inhibitory synapses and resulting in fewer functional synapses. Intriguingly, the observed excitatory and inhibitory defects both involve endocytic processes, albeit at different sides of the synaptic cleft, suggesting common processes may be involved.

Altered function of GABA receptors and/or inhibitory interneurons has been hypothesized to underlie many of the phenotypes seen in AS (Dan and Boyd, 2003). While attention has focused on how defects in GABAergic neurotransmission may relate to epileptic phenotypes in AS, abnormalities in inhibition can have wide-ranging consequences, including disrupting synaptic plasticity, cortical network oscillations, and cortical circuit architecture (Cardin et al., 2009; Hensch, 2005). For example, FS inhibitory interneurons have a critical role in ocular dominance plasticity (Hensch et al., 1998), which is severely reduced in Ube3am−/p+ mice (Sato and Stryker, 2010; Yashiro et al., 2009). Our finding that inhibitory interneuron to L2/3 pyramidal neuron connections are altered in Ube3am−/p+ mice may prove important for understanding the mechanisms underlying plasticity and learning defects in AS. Understanding the specific synaptic impairments caused by the global loss of Ube3a may provide insights into the intractable nature of seizures found in many individuals with AS.

Excitatory/inhibitory imbalance has been observed in several genetic disorders that meet diagnostic criteria for autism spectrum disorders, including neuroligin-3 mutation, Fragile X, and Rett syndrome (Dani et al., 2005; Gibson et al., 2008; Tabuchi et al., 2007). Moreover, excitatory/inhibitory imbalance may be a general neurophysiological feature of autism spectrum disorders, contributing to inappropriate detection or integration of salient sensory information due to a decreased signal-to-noise ratio (Rubenstein and Merzenich, 2003). Our finding that an excitatory/inhibitory imbalance may develop in AS due to the loss of functional inhibitory synapses highlights the importance of identifying Ube3a substrates in inhibitory interneurons.

EXPERIMENTAL PROCEDURES

See Supplemental Experimental Procedures for details relating to electrophysiology and immunohistochemistry.

Animals

Ube3a-deficient mice on the 129Sv/Ev background were originally developed by Jiang and colleagues (Jiang et al., 1998) and obtained through the Jackson Laboratory (Bar Harbor, ME). Ube3a-deficient mice backcrossed onto the C57BL/6J background were obtained from Yong-hui Jiang (Duke University) and crossed with mice expressing GFP in a subset of FS inhibitory neurons (Chattopadhyaya et al., 2004) obtained through Jackson Laboratory.

Statistics

Most experiments and analyses were performed blind to genotype. Unpaired students t-tests were used on all data excluding the following; input-output, frequency-current, short-term plasticity, connection probability and for depletion and recovery experiments. Graphs represent the mean and error bars represent the SEM. For all figures p-values are as follows *p<0.05, **p<0.01, ***p<0.001. All statistics were performed in Graphpad Prism.

Supplementary Material

01

Acknowledgments

We thank Rylan Larsen and Matt Judson for critical readings of the manuscript, Paul Manis for experimental advice, Yong-hui Jiang for his generous donation of C57BL/6J Ube3a-deficient mutant mice and Kristen Phend for histological support. Imaging was supported by the Confocal and Multiphoton Imaging Core of NINDS Center Grant P30 NS045892 and NICHD Center Grant P30 HD03110. M.L.W was supported by a Neurobiology Research Training Grant from NINDS (5T32NS007431) and a National Research Service Award from NINDS (1F31NS077847). R.J.W. was supported by NINDS (5R01NS035527). B.D.P was supported by the Angelman Syndrome Foundation, the Simons Foundation, the National Eye Institute (R01EY018323), and the National Institute of Mental Health (1R01MH093372).

Footnotes

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