Genetic Deletion of GABAA Receptors Reveals Distinct Requirements of Neurotransmitter Receptors for GABAergic and Glutamatergic Synapse Development

In the adult brain GABAA receptors (GABAARs) mediate the majority of synaptic inhibition that provides inhibitory balance to excitatory drive and controls neuronal output. In the immature brain GABAAR signaling is critical for neuronal development. However, the cell-autonomous role of GABAARs in synapse development remains largely unknown. We have employed the CRISPR-CAS9 technology to genetically eliminate GABAARs in individual hippocampal neurons and examined GABAergic and glutamatergic synapses. We found that development of GABAergic synapses, but not glutamatergic synapses, critically depends on GABAARs. By combining different genetic approaches, we have also removed GABAARs and two ionotropic glutamate receptors, AMPA receptors (AMPARs) and NMDA receptors (NMDARs), in single neurons and discovered a striking dichotomy. Indeed, while development of glutamatergic synapses and spines does not require signaling mediated by these receptors, inhibitory synapse formation is crucially dependent on them. Our data reveal a critical cell-autonomous role of GABAARs in inhibitory synaptogenesis and demonstrate distinct molecular mechanisms for development of inhibitory and excitatory synapses.

The role of GABA A R-mediated signaling in neuronal development and function has been extensively studied (Ben-Ari et al., 1997;Owens and Kriegstein, 2002;Ben-Ari et al., 2007). Early pharmacological experiments have demonstrated that GABA A R activity can modulate neurogenesis, neuronal migration and differentiation, and synapse development in the immature brain (Owens and Kriegstein, 2002;Ben-Ari et al., 2007). However, pharmacological approaches do not separate the cell-autonomous function of GABA A Rs from indirect neuronal network effects associated with global blockade of the receptors, and also do not address the structural role of GABA A Rs in the regulation of synapse development. Depolarizing GABA A R activity has also been shown to functionally interact with NMDA receptors (NMDARs) to facilitate NMDAR activation and regulate synapse development in immature neurons (Owens and Kriegstein, 2002;Ben-Ari et al., 2007). In addition, by manipulating NKCC1 or KCC2 expression, the role of depolarizing GABA A R activity in synapse, and spine development has been inferred (Chudotvorova et al., 2005;Akerman and Cline, 2006;Liu et al., 2006;Wang and Kriegstein, 2008), although recent studies have shown that the regulation of synapse development by KCC2 does not require its ion transport function (Li et al., 2007;Fiumelli et al., 2013). Furthermore, synaptic GABA A Rs in knockdown or conventional knockout (KO) mice of GABA A R subunits are reduced or lost, leading to the impairment of GABAergic synapse formation and maturation (Fritschy et al., 2006(Fritschy et al., , 2012Patrizi et al., 2008;Frola et al., 2013), which provides genetic evidence for the role of GABA A R subunits in synapse development. However, in these genetic models, neurons may adapt to the global absence of the GABA A R subunits and to altered neural network activities throughout their development. Thus, the cell-autonomous role of GABA A Rs in the regulation of synapse development remains largely unclear.
Here we have utilized the CRISPR-Cas9 approach to perform single-cell genetic deletion (Incontro et al., 2014) of all functional GABA A Rs and examine excitatory and inhibitory synapse development. This is achieved by targeting the β1, β2, and β3 subunits of the GABA A Rs in individual hippocampal neurons. These β subunits are required for the receptor assembly and agonist binding (Connolly et al., 1996;Tretter et al., 1997;Baumann et al., 2001;Olsen and Sieghart, 2008;Nguyen and Nicoll, 2018). We found that in neurons lacking GABA A Rs, GABAergic synapses are strongly impaired without an accompanying change of excitatory transmission and the spine density. Furthermore, combined genetic deletion of GABA A Rs and ionotropic glutamate receptors (iGluRs), including both AMPARs and NMDARs, reveals that signaling mediated by these receptors is critical for inhibitory, but not excitatory, synapse development.

Mouse Maintenance
All experiments using mice were performed in accordance with animal protocols approved by the Institutional Animal Care and Use Committee at National Institute of Neurological Disorders and Stroke (NINDS) and National Institutes of Health (NIH). Adult C57BL/6 mice were purchased from Charles River, housed and bred under a 12-h circadian cycle. GRIA1-3 fl/fl GRIN1 fl/fl mice were generated as described previously (Lu et al., 2013). Animals of either sex were used for the experiments.

Dissociated Hippocampal Neuronal Culture
Hippocampal cultures were prepared from E18 time-pregnant mice as previously described (Gu et al., 2016a). Briefly, the mouse hippocampi were dissected out in ice-cold Hank's balanced salt solution, and digested with papain (Worthington, LK003176) solution at 37 • C for 45 min. After centrifugation for 5 min at 800 rpm, the pellet was resuspended in DNase I-containing Hank's solution, then was mechanically dissociated into single cells by gentle trituration using a pipette. Cells were placed on top of Hank's solution mixed with trypsin inhibitor (10 mg/ml, Sigma T9253) and BSA (10 mg/ml, Sigma A9647), and centrifuged at 800 rpm for 10 min. The pellet was resuspended in Neurobasal plating media containing 2% B27 supplements and L-glutamine (2 mM). Neurons were plated at a density of 150,000-200,000 cells/well on poly-D-lysine (Sigma P6407)-coated 12 mm glass coverslips residing in 24-well plates for electrophysiology recording, and a lower plating density (100,000-150,000 cells/well) was adopted when neurons were used for immunocytochemistry. Culture media were changed by half volume with Neurobasal maintenance media containing 2% B27 supplements and L-glutamine (2 mM) twice a week.

Neuronal Transfection
Hippocampal neurons were transfected at day 2-3 in vitro (DIV2-3) using a modified calcium phosphate transfection as described before (Li et al., 2017). Briefly, 5 µg total cDNA was used to generate 200 µL total precipitates, which was added to each well at a 40 µL volume (5 coverslips/group). After 2 h incubation in a 37 • C incubator, the transfected cells were incubated with 37 • C pre-warmed, 10% CO 2 preequilibrated Neurobasal medium, and placed in a 37 • C, 5% CO 2 incubator for 20 min to dissolve the calcium-phosphate particles. The coverslips were then transferred back to the original conditioned medium. The cells were cultured to DIV 14-24 before experiments.

Image Acquisition and Analysis
Fluorescence images were obtained with the Zeiss Zen acquisition software and a Zeiss LSM 880 laser scanning confocal microscope using a 63 × oil objective (numerical aperture 1.4) at room temperature. Optical sections, merged by maximum projection, were analyzed at a time using the Image J puncta analyzer program. Thresholds were set at 3 SDs above the mean staining intensity of six nearby regions in the same visual field. Thresholded images present a fixed intensity for all pixels above threshold after having removed all of those below. Labeled puncta were defined as areas containing at least four contiguous pixels after thresholding. For puncta analysis, Images from 3 dendrites (35 µm in length) per neuron were collected and quantified. For co-localization analysis, images from soma, or three secondary dendrites (35 µm in length per dendrite) per neuron were collected and quantified. For β subunit colocalization with synaptic markers, β subunits and gephyrin or vGAT were separately thresholded and confirmed visually to select appropriate clusters following a minimal size cut-off, which included all recognizable clusters. The gephyrin-or vGATpositive β subunit puncta indicate the number of β subunit puncta per 10 mm showing at least 50% pixel overlapping with thresholded gephyrin or vGAT puncta. Synaptic vs. Total ratios was calculated by the measurement of gephyrin-or vGATpositive β subunit puncta compared to total β subunit puncta. For gephyrin and vGAT co-localization in dendrites, gephyrin and vGAT puncta were separately thresholded and confirmed visually to select appropriate clusters following a minimal size cut-off, which included all recognizable clusters. The gephyrinpositive vGAT puncta indicate the number of vGAT puncta showing at least 50% pixel overlapping with thresholded gephyrin puncta and the co-localization percentage was calculated by the measurement of gephyrin-positive vGAT puncta compared to total number of thresholded vGAT puncta. The same procedure was used to calculate vGAT-positive gephyrin. For spine density analysis, GFP was immunolabeled with anti-GFP antibodies to boost the fluorescence. 2-3 secondary or tertiary dendrites (50-200 µm long, 20-100 µm from the soma) from each neuron were collected, the number of dendritic protrusions were counted manually. For spine type analysis, images from three dendrites (35 µm in length per dendrite) per neuron were collected and spine types in each dendrite were quantified. Different spine types (mushroom, thin, stubby, and filopodia) were counted manually for each dendrite, and the data were combined from three dendrites to calculate the fractions of each type of spines for that neuron.

Statistics
All data were presented as mean ± sem (standard error of mean). Direct comparisons between two groups were made using twotailed Student's t-test. Multiple group comparisons were made using one-way analysis of variance (ANOVA) with the Bonferroni test. The significance of cumulative probability distributions was assessed by Kolmogorov-Smirnov test. Statistical significance was defined as p < 0.05, 0.01, 0.001, or 0.0001 (indicated as * , * * , * * * , or * * * * , respectively). p values ≥ 0.05 were considered not significant (ns).
tonic currents were readily detected by bicuculline, little tonic currents were observed in neurons expressing β1-3 gRNAs ( Figure 1H). We further performed rescue experiments to characterize the role of individual β subunits in the regulation of GABAergic transmission. To this end, we developed gRNA-resistant β1, β2 or β3 in HEK cells through immunocytochemical experiments (β1 * , β2 * , or β3 * in Figures 2A,C,E, respectively). We then co-transfected β1 * (Figure 2B), β2 * (Figure 2D), or β3 * ( Figure 2F) with β1-3 gRNAs in hippocampal neuronal cultures and measured mIPSCs. We found that the loss of GABAergic transmission in neurons expressing β1-3 gRNAs could be rescued by co-expressing β1 * , β2 * , or β3 * (Figures 2B,D,F, respectively), indicating that individual β subunits can support basal inhibitory transmission and can substitute for each other for GABAergic transmission in hippocampal neurons. Together, these data demonstrate that we have successfully eliminated GABAergic transmission in single hippocampal neurons and individual β subunits can rescue GABAergic transmission in neurons lacking all endogenous β subunits.
The loss of functional GABA A Rs in individual hippocampal neurons allowed us to examine the cell-autonomous role of these receptors in the regulation of GABAergic synapse development. We found that in neurons expressing β1-3 gRNAs, the immunolabeling of vGAT and gephyrin, the pre-and postsynaptic markers of GABAergic synapses, respectively, at both somatic and dendritic regions, was significantly decreased by ∼50% (Figures 3A-C), indicating a reduction of GABAergic synapse density. Interestingly, there was no change of the puncta density of Neuroligin2 (NL2), a key synaptogenic cell adhesion molecule for GABAergic synapses (Graf et al., 2004;Varoqueaux et al., 2004;Chih et al., 2005;Poulopoulos et al., 2009;Li et al., 2017), in neurons lacking GABA A Rs (Figure 3D), suggesting that NL2 clustering is independent of GABA A Rs and may be an upstream event of GABA A R-mediated signaling for inhibitory synapse development. Taken together, these data demonstrate that genetic deletion of GABA A Rs at the single-cell level leads to a substantial reduction of GABAergic synapses.
Pharmacological blockade of GABA A Rs can induce homeostatic adaptation of excitatory synaptic transmission in neurons (Turrigiano et al., 1998;Shepherd et al., 2006;Anggono et al., 2011;Diering et al., 2014). Specifically, chronic inhibition of GABA A Rs reduces AMPAR-mediated excitatory transmission. We thus examined how excitatory synapses adapted to the single-cell silencing of GABAergic inhibitory transmission. Surprisingly, recording of miniature excitatory postsynaptic currents (mEPSCs) in the presence of TTX in hippocampal neurons expressing β1-3 gRNAs showed that there was no change of frequency and amplitude of mEPSCs ( Figure 4A). In addition, immunocytochemical analysis demonstrated that the surface expression of GluA1, a key AMPAR subunit in hippocampal neurons (Lu et al., 2009), and the puncta of vesicular glutamate transporter 1 (vGluT1) did not change in β1-3 gRNA-expressing neurons (Figures 4B,C), suggesting that AMPAR trafficking to the neuronal surface and excitatory synapse density were not altered in these neurons lacking functional GABA A Rs. Furthermore, we measured the spine density by immunolabeling GFP and found that the spine density or type was not significantly changed in neurons expressing β1-3 gRNAs (Figures 4D,E). Collectively, these data show that single-cell elimination of GABAergic transmission does not induce a homeostatic reduction of excitatory synaptic transmission and also leads to little change of cell biological properties of excitatory synapses, including surface AMPAR expressing levels, and the density of excitatory synapses.
We previously employed a Cre-LoxP system to genetically delete both AMPARs and NMDARs in single hippocampal neurons and found that these iGluRs were dispensable for spinogenesis (Lu et al., 2013). One possibility was that in neurons lacking both AMPARs and NMDARs, remaining GABA A Rs could generate depolarizing drive in developing neurons and thus might provide activity necessary for spine development. We thus combined β1-3 gRNAs with the conditional KO of both AMPARs and NMDARs to genetically remove functional GABA A Rs, AMPARs and NMDARs in individual neurons and examined excitatory and inhibitory synapses. In hippocampal neuronal cultures prepared from GRIA1-3 fl/fl GRIN1 fl/fl in which three genes encoding AMPAR subunits (GluA1, GluA2 and GluA3) and the gene encoding the NMDAR obligatory subunit, GluN1, are all conditional alleles (Lu et al., 2013), we coexpressed Cre-mCherry and β1-3 gRNAs through transfection. About 2 weeks after transfection, we performed mIPSCs and mEPSCs recordings in transfected neurons and found the loss of inhibitory synaptic transmission and both AMPAR-and NMDAR-mediated excitatory synaptic transmission in these neurons (Figures 5A,B).
β1-3 gRNA-expressing neurons (Figures 5C,D), as compared to control neurons expressing the empty gRNA vector. We also examined inhibitory and excitatory synapses by the measurement of gephyrin and PSD-95 puncta. We found that compared to neurons expressing the empty gRNA vector, co-expression of both Cre and β1-3 gRNAs in GRIA1-3 fl/fl GRIN1 fl/fl neurons led to a large reduction of the density of gephyrin puncta (∼90%) ( Figure 5E). However, there was no difference of the density of PSD-95 puncta between control neurons and neurons lacking GABA A Rs, AMPARs and NMDARs ( Figure 5F). Therefore, genetic deletion of both GABA A Rs and iGluRs impairs the development of GABAergic synapses but does not change the density of spines or glutamatergic synapses.

DISCUSSION
To study the role of ionotropic GABA A Rs in the regulation of GABAergic synapse development, we have utilized the CRISPR-Cas9 technology to genetically delete all three β subunits of GABA A Rs in hippocampal neurons. GABA A R β subunits are required for the receptor assembly and GABA binding (Connolly et al., 1996;Tretter et al., 1997;Baumann et al., 2001;Olsen and Sieghart, 2008;Nguyen and Nicoll, 2018), and, consistently, we found that single-cell genetic deletion of three β subunits leads to a loss of GABAergic transmission, in agreement with a recent report (Nguyen and Nicoll, 2018). The lack of inhibitory synaptic transmission at the level of individual neurons allowed us to investigate the cell-autonomous role of GABA A Rs in the regulation of GABAergic synapse development.
Our data demonstrate that GABA A Rs are critical for GABAergic synapse development at the level of single neurons. Indeed, in hippocampal neurons lacking functional GABA A Rs in a mosaic fashion, gephyrin, and vGAT puncta at both somatic and dendritic areas are significantly reduced. Our data are consistent with previous reports in which germline KO of α1 subunit of GABA A Rs abolished GABAergic transmission in Purkinje cells, and consequently impaired GABAergic synapse formation (Fritschy et al., 2006;Patrizi et al., 2008). Similarly, knockdown or KO of the GABA A R subunit, γ2, which is important for synaptic clustering of GABA A Rs (Essrich et al., 1998), impaired GABAergic innervation and reduced GABAergic synapse density (Schweizer et al., 2003;Li et al., 2005;Frola et al., 2013). In addition, GABA A R activity has been shown to be important for GABAergic synapse formation (Chattopadhyaya et al., 2007;Arama et al., 2015;Oh et al., 2016;Lin et al., 2018). It is worth noting that broad genetic deletion or widespread pharmacological inhibition of GABA A R subunits alter neural network activity, and thus does not separate the cell-autonomous function of GABA A Rs from the indirect effects on network activity in the regulation of synapse development. Our data thus provide the genetic evidence of a critical cell-autonomous role of GABA A Rs for GABAergic synapse development. Currently, the molecular mechanisms underlying the regulation of GABAergic synapse development by GABA A Rs remain largely unclear. It has been reported that the GABA A Rs interact with the synaptic adhesion molecule, neurexins (Zhang et al., 2010). In addition, GABA A Rs may induce Ca 2+ influx through NMDARs (Owens and Kriegstein, 2002;Ben-Ari et al., 2007) or voltage-gated calcium channels (Oh et al., 2016) to regulate GABAergic synaptogenesis, and may also play a synaptogenic role in GABAergic synapse development (Fuchs et al., 2013;Brown et al., 2016). Interestingly, the puncta density of NL2, a key synaptogenic cell adhesion molecule for GABAergic synapses (Lu et al., 2016), is not altered in neurons lacking GABA A Rs, similar to a previous report (Patrizi et al., 2008), suggesting that NL2 may act upstream of GABA A R signaling for GABAergic synaptogenesis. Recent studies have identified that NL2 is crucial for synaptic anchorage of GABA A Rs through binding to the GABA A R-interacting protein, GARLH/LHFPL4 (Davenport et al., 2017;Yamasaki et al., 2017;Wu et al., 2018) and is critical for GABAergic synapse development (Poulopoulos et al., 2009;Li et al., 2017;Panzanelli et al., 2017). Thus, it is plausible that during development NL2 may regulate GABA A R clustering, which in turn modulates GABAergic synapse formation and maturation. Interestingly, in GARLH/LHFPL4 KO neurons, both NL2 and GABA A R clustering are impaired (Davenport et al., 2017;Yamasaki et al., 2017;Wu et al., 2018), indicating that GARLH/LHFPL4 may function upstream of both NL2 and GABA A Rs in the regulation of GABAergic synapse development.
vs. calcium phosphate transfection in our study) and different experimental preparations (hippocampal organotypic cultures vs. dissociated neuronal cultures in our study). One surprising observation from our study is that excitatory synaptic transmission is normal in hippocampal neurons lacking GABA A Rs. It has been well-established that chronic pharmacological inhibition of GABA A Rs induces homeostatic adaptation of excitatory synapses and reduces AMPAR-mediated synaptic transmission (Turrigiano et al., 1998;Shepherd et al., 2006;Anggono et al., 2011;Diering et al., 2014). However, the role of GABA A R-mediated signaling at the single-cell level in the regulation of glutamatergic transmission remained unclear.
Our data now demonstrate that at the level of individual neurons, the complete loss of functional GABA A Rs does not impair AMPAR-mediated excitatory transmission. Indeed, glutamatergic transmission, the expression levels of surface GluA1, a major AMPAR subunit in hippocampus (Lu et al., 2009), and the number of vGluT1 puncta are not altered in neurons lacking GABA A Rs. In addition, our work reveals that GABA A Rs are not absolutely required for spinogenesis in hippocampal neurons in vitro, as the spine density in neurons lacking GABA A Rs is indistinguishable from that in control neurons ( Figure 4D). Our data are consistent with early work in α1 KO mice in which IPSCs are lost in cerebellar Purkinje cells, but EPSCs and excitatory synapses are largely intact (Fritschy et al., 2006;Patrizi et al., 2008). In addition, in GARLH/LHFPL4 KO neurons, GABAergic transmission is severely reduced without an accompanying change of glutamatergic transmission (Davenport et al., 2017;Yamasaki et al., 2017). Similarly, single-cell ablation of gephyrin in hippocampal neurons strongly reduces GABAergic transmission, but does not change glutamatergic transmission (Gross et al., 2016). However, it is important to point out that our data do not exclude the possibility that GABA A R activation can sufficiently induce spine formation (Oh et al., 2016) and can modulate glutamatergic synapse development (Ben-Ari et al., 2007).
Previous studies have also indicated a role of NKCC1 and KCC2 in the regulation of excitatory synapse and spine development (Akerman and Cline, 2006;Liu et al., 2006;Wang and Kriegstein, 2008). During development, NKCC1 and KCC2 play fundamental roles in determining the neuronal intracellular Cl − concentration and polarity of GABA A R action (Owens and Kriegstein, 2002;Payne et al., 2003;Ben-Ari et al., 2007), and regulate GABA A R subunit expression (Succol et al., 2012). Through manipulation of NKCC1 or KCC2 expression in neurons, the role of the depolarizing GABA in excitatory synapse development has been proposed (Akerman and Cline, 2006;Liu et al., 2006;Wang and Kriegstein, 2008). Interestingly, recent studies have indicated that the regulation of synapse or dendritic development by KCC2 is independent of its ion transport function (Li et al., 2007;Fiumelli et al., 2013), suggesting that the effect of KCC2 on neuronal development may be independent of GABA action. Future work toward a more complete understanding of depolarizing GABA in excitatory synapse development will be important to our understanding of the molecular mechanisms underlying synaptogenesis.
We have previously employed a Cre-LoxP system to genetically remove iGluRs, AMPARs and/or NMDARs, in individual hippocampal neurons, and found that excitatory synaptic input is not necessary for development of neuronal spines (Lu et al., 2009(Lu et al., , 2013. One possibility is that in developing neurons lacking iGluRs, remaining GABA A Rs that are activated may generate depolarizing drives and provide activities important for neuronal morphological development. Thus, to further investigate the role of these receptors in excitatory or inhibitory synapse development, we have combined the Cre-LoxP system with the CRISPR-Cas9 approach to genetically target both iGluRs and GABA A Rs. We found that in individual hippocampal neurons lacking both iGluRs and GABA A Rs, there was no significant change of the spine density and PSD-95 immunolabeling. These data corroborate a series of recent reports that neurotransmitter release, and thus the activation of postsynaptic neurotransmitter receptors, are not required for spine and excitatory synapse development (Verhage et al., 2000;Varoqueaux et al., 2002;Lu et al., 2013;Sando et al., 2017;Sigler et al., 2017). In contrast, we found that there was a nearly 90% reduction of gephyrin puncta in these neurons lacking both iGluRs and GABA A Rs. Previously, we have shown that genetic deletion of both AMPARs and NMDARs led to a strong reduction of inhibitory transmission in hippocampal CA1 pyramidal neurons (Lu et al., 2013). Recently we have further shown that the NMDAR, but not the AMPAR, acts as an important molecule for controlling GABAergic synaptogenesis during development (Gu et al., 2016b;Gu and Lu, 2018). Together, these data suggest that NMDARs and GABA A Rs may play a synergistic role in the regulation of GABAergic synapse development. Currently, it remains unclear how NMDARs and GABA A Rs work together to control the development of GABAergic connections. It is conceivable that in developing, immature neurons, GABA A R activity may facilitate NMDAR activation, inducing Ca 2+ influx and stimulating signaling pathways important for GABAergic synapse development (Owens and Kriegstein, 2002;Ben-Ari et al., 2007;Gu et al., 2016b). It is also possible that GABA A Rs and NMDARs may activate parallel pathways to regulate the formation of GABAergic connections. In the future, it will be imperative to determine the sequential action and functional interplay of signaling pathways mediated by NMDARs and GABA A Rs in the regulation of GABAergic synaptogenesis. It will also be important to determine how these neurotransmitter receptors functionally interact with cell surface molecules important for GABAergic synaptogenesis to regulate formation of inhibitory connections (Kuzirian and Paradis, 2011;Ko et al., 2015;Lu et al., 2016;Krueger-Burg et al., 2017).
In summary, through a single-cell genetic approach in vitro we have provided new insights into the cell-autonomous role of GABA A Rs in developing neurons and discovered a dichotomy in the regulation of synapse development by this prominent Cl − channel. While inhibitory synapse development is critically regulated by GABA A Rs, establishment of glutamatergic transmission and excitatory synapses are largely independent of GABA A R-mediated signaling at the level of individual neurons. Furthermore, we have managed to remove all AMPARs, NMDARs and GABA A Rs in single neurons in culture and demonstrated that iGluR-and GABA A R-mediated signaling are not essential for spinogenesis. Our data thus suggest that other developmental pathways including neurotropic factormediated signaling (Reichardt, 2006;Park and Poo, 2013), guidance cues, and their receptors (Klein, 2009;Shen and Cowan, 2010;Koropouli and Kolodkin, 2014), trans-synaptic cell adhesion interactions (Scheiffele, 2003;Dalva et al., 2007;Missler et al., 2012;de Wit and Ghosh, 2016), and other receptors or channels-mediated signaling (Komatsuzaki et al., 2005;Eroglu et al., 2009;Uemura et al., 2010;Lozada et al., 2012a,b;Kozorovitskiy et al., 2015;Sellers et al., 2015) may play key roles in spinogenesis and excitatory synaptogenesis. It is also worth noting that our data were collected in cultured neurons and there are limitations in using in vitro models to study synapse development. Thus, future experiments in vivo will help further establish the role of GABA A Rs in synaptogenesis. Nevertheless, our data demonstrate a remarkable specificity of these ionotropic receptors in mediating signaling important for synapse development in vitro. Given the prominent roles of malfunctions of these receptors in the pathogenesis of many neurodevelopmental disorders (Cellot and Cherubini, 2014;Yuan et al., 2015), our data also highlight the importance of understanding the molecular mechanisms for the regulation of GABAergic synapse development by these receptors.

ETHICS STATEMENT
All experiments using mice were performed in accordance with animal protocols approved by the Institutional Animal Care and Use Committee at National Institute of Neurological Disorders and Stroke (NINDS), National Institutes of Health (NIH).

AUTHOR CONTRIBUTIONS
JD, SP, and WL designed the experiments. JD, DC, JL, and XG cloned and characterized the gRNA constructs. JD, SP, and TL performed the immunocytochemical experiments. JD performed the electrophysiological assays. QT provided critical technical design and problem solving. JD and WL wrote the manuscript. All authors read and commented on the manuscript.