Journal: Cell reports

Article Title: Identification of a Core Amino Acid Motif within the α Subunit of GABA A Rs that Promotes Inhibitory Synaptogenesis and Resilience to Seizures

doi: 10.1016/j.celrep.2019.06.014

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-GABA A R γ2L mouse monoclonal , UC Davis/NIH NeuroMab Facility , Cat #75-442; RRID: AB_2617122.

Techniques: Staining, Recombinant, Saline, Bicinchoninic Acid Protein Assay, Western Blot, Protease Inhibitor, Sequencing, Software, Microscopy

Journal: Nature Communications

Article Title: Neurexins regulate presynaptic GABA B -receptors at central synapses

doi: 10.1038/s41467-021-22753-5

Figure Lengend Snippet: a The diagram of the calyx of Held synapse. b Strategy for selective deletions of all neurexins at the calyx of Held by crossing PV-Cre mice with triple Nrxn123 cKO mice (Chen et al. , Luo et al. ). c Representative traces of EPSC before and after application of 20 µM SKF-97541 (SKF), a potent and selective GABA B -receptor agonist, recorded in acute slices from littermate control and neurexin123 TKO mice at P12–P14. The normalized EPSCs before and after SKF are shown in inset. d Summary graphs of EPSC1 amplitudes before and after SKF for control and Nrxn123 TKO mice. P = 2.3E−7, P = 0.072, paired two-sided t -test. P = 0.0062, unpaired two-sided t -test. e Summary graphs of EPSC1 remaining unblocked by SKF application. P = 1.79E−7, unpaired two-sided t -test. f Summary graphs of the paired-pulse ratio (PPR) before and after SKF in control and Nrxn123 TKO mice. P = 2.81E−5, P = 0.09, paired two-sided t -test. P = 0.0038, unpaired two-sided t -test. Data are means ± SEM. Number of cells (from at least three mice per group) analyzed are indicated in the bars ( d – f ); Statistical differences were assessed by Student’s t -test. (* P < 0.05; ** P < 0.01; *** P < 0.001). Source data are provided as a Source Data file.

Article Snippet: Twenty micrometer thick brainstem slices containing the MNTB region were prepared as described and labeled with Homer1 (rabbit, 1:1000, Millipore, Cat#: ABN37), GABA B1 (mouse, monoclonal, 1:1000, NeuroMab Cat#: 75-183), GABA B2 (mouse, monoclonal, 1:1000, NeuroMab Cat#: 75-125) primary antibodies and secondary antibodies conjugated to Alexa647 (1:3000, ThermoFisher) or CF568 (1:3000, Biotium).

Techniques: Control

Journal: Nature Communications

Article Title: Neurexins regulate presynaptic GABA B -receptors at central synapses

doi: 10.1038/s41467-021-22753-5

Figure Lengend Snippet: a Representative confocal microscopy images of MNTB-containing brainstem slice with specific labeling of VGluT1 (green) and GABA B -receptor subunit 2 (GABA B2 , red) from both littermate control and Nrxn123 TKO mice at P12. Scale bar, 10 µm. b Summary of VGluT1 and GABA B2 immunostaining intensity (normalized to control). P = 1, P = 1.35E−9, unpaired two-sided t -test. c , d Same as a and b except for specific labeling of VGluT1 (green) and GABA B -receptor subunit 1 (GABA B1 , red). P = 1, P = 0.031, unpaired two-sided t -test. Data are means ± SEM. Number of sections/animals for immunostaining are indicated in the bars ( b , d ). Statistical differences were assessed by Student’s t -test. (* P < 0.05; *** P < 0.001). Source data are provided as a Source Data file.

Article Snippet: Twenty micrometer thick brainstem slices containing the MNTB region were prepared as described and labeled with Homer1 (rabbit, 1:1000, Millipore, Cat#: ABN37), GABA B1 (mouse, monoclonal, 1:1000, NeuroMab Cat#: 75-183), GABA B2 (mouse, monoclonal, 1:1000, NeuroMab Cat#: 75-125) primary antibodies and secondary antibodies conjugated to Alexa647 (1:3000, ThermoFisher) or CF568 (1:3000, Biotium).

Techniques: Confocal Microscopy, Labeling, Control, Immunostaining

Journal: Nature Communications

Article Title: Neurexins regulate presynaptic GABA B -receptors at central synapses

doi: 10.1038/s41467-021-22753-5

Figure Lengend Snippet: a Representative dSTORM images of MNTB-containing brainstem slice with dual labeling of Homer1 (magenta) and GABA B -receptor subunit 2 (GABA B2 , yellow) from both littermate control and Nrxn123 TKO mice. b Summary of the number of GABA B2 clusters normalized to the number of Homer1 clusters. P = 0.0051, unpaired two-sided t -test. c Distribution of Homer1 and GABA B2 localizations. d – f Similar as a – c , except for GABA B1 antibody used. P = 0.0057, unpaired two-sided t -test. Data are means ± SEM. Number of sections/animals for immunostaining are indicated in the bars ( b , e ). Statistical differences were assessed by Student’s t -test. (** P < 0.01). Source data are provided as a Source Data file.

Article Snippet: Twenty micrometer thick brainstem slices containing the MNTB region were prepared as described and labeled with Homer1 (rabbit, 1:1000, Millipore, Cat#: ABN37), GABA B1 (mouse, monoclonal, 1:1000, NeuroMab Cat#: 75-183), GABA B2 (mouse, monoclonal, 1:1000, NeuroMab Cat#: 75-125) primary antibodies and secondary antibodies conjugated to Alexa647 (1:3000, ThermoFisher) or CF568 (1:3000, Biotium).

Techniques: Labeling, Control, Immunostaining

Journal: Nature Communications

Article Title: Neurexins regulate presynaptic GABA B -receptors at central synapses

doi: 10.1038/s41467-021-22753-5

Figure Lengend Snippet: a Schematic of experimental approach for virus injection and electro-physiology recording (Top); GFP expression in AAV-transfected CA3 region of hippocampus (bottom). b Representative traces of EPSCs evoked by fiber stimulations with increased intensity, recorded in acute hippocampus slices from Nrxn123 cKO mice injected with AAV-ΔCre or AAV-Cre. c The input–output curve of EPSC amplitudes in relation to the stimulation intensity. P = 0.0113, unpaired two-sided t -test. d Example traces of paired-pulse EPSCs before and after addition of GABA B -receptor agonist SKF, the normalized EPSCs before and after SKF are shown in inset. The intensity of fiber stimulation was tuned to evoke EPSC1 at similar amplitude for each cell. e Summary graphs of EPSC1 amplitudes before and after SKF for control (ΔCre) and Nrxn123 TKO synapses (Cre). P = 0.0001, P = 0.0001, paired two-sided t -test. P = 0.99, unpaired two-sided t -test. f Summary graphs of EPSC1 rise time (left) and decay time constants (right). P = 0.0008, P = 0.6136, unpaired two-sided t -test. g Summary graphs of EPSC1 remaining unblocked by SKF application. P = 0.0013, unpaired two-sided t -test. h Summary graphs of the PPR before and after SKF in control and Nrxn123 TKO synapses. P = 0.0001, P = 0.0984, paired two-sided t -test. P = 0.2772, unpaired two-sided t -test. i Summary graphs of the CV of EPSC1 amplitude before and after SKF in control and Nrxn123 TKO synapses. P = 0.0385, P = 0.99, paired two-sided t -test. P = 0.4664, unpaired two-sided t -test. Data are means ± SEM. Number of cells (from at least three mice per group) analyzed are indicated in the graph ( c ) or bars ( e – i ); Statistical differences were assessed by Student’s t -test. (* P < 0.05; ** P < 0.01, *** P < 0.001). Source data are provided as a Source Data file.

Article Snippet: Twenty micrometer thick brainstem slices containing the MNTB region were prepared as described and labeled with Homer1 (rabbit, 1:1000, Millipore, Cat#: ABN37), GABA B1 (mouse, monoclonal, 1:1000, NeuroMab Cat#: 75-183), GABA B2 (mouse, monoclonal, 1:1000, NeuroMab Cat#: 75-125) primary antibodies and secondary antibodies conjugated to Alexa647 (1:3000, ThermoFisher) or CF568 (1:3000, Biotium).

Techniques: Virus, Injection, Expressing, Transfection, Control

Journal: Nature Communications

Article Title: Neurexins regulate presynaptic GABA B -receptors at central synapses

doi: 10.1038/s41467-021-22753-5

Figure Lengend Snippet: a Schematic of experimental setup for virus injection and optogenetic stimulation of PV + interneurons (Top); PV-Cre dependent expression of Chief-TdTomato in AAV-transfected CA1 region of hippocampus (bottom). b Example traces of paired-pulse blue light-evoked IPSCs before and after addition of GABA B -receptor agonist SKF, recorded in acute hippocampus slices from PVCre + mice (control) or PVCre + /Nrxn123 cKO mice (TKO) injected with AAV-DIO-Chief-TdTomato. The normalized IPSCs before and after SKF are shown in inset. c Summary graphs of IPSC1 amplitudes before and after SKF for control and Nrxn123 TKO synapses. P = 0.0001, P = 0.0026, paired two-sided t -test. P = 0.6, unpaired two-sided t -test. d Summary graphs of IPSC1 amplitude remaining unblocked by SKF. P = 0.0047, unpaired two-sided t -test. e Summary graphs of the PPR ratio before and after SKF in control and Nrxn123 TKO synapses. P = 0.0022, P = 0.6165, paired two-sided t -test. P = 0.5114, unpaired two-sided t -test. f Summary graphs of the CV of IPSC1 amplitude. P = 0.0143, P = 0.99, paired two-sided t -test. P = 0.99, unpaired two-sided t -test. g Summary graphs of IPSC1 rise time (left) and decay time constant (right). P = 0.4974, P = 0.063, unpaired two-sided t -test. Data are means ± SEM. Number of cells (from at least three mice per group) analyzed are indicated in the bars ( c – g ); Statistical differences were assessed by Student’s t -test. (* P < 0.05; ** P < 0.01; *** P < 0.001). Source data are provided as a Source Data file.

Article Snippet: Twenty micrometer thick brainstem slices containing the MNTB region were prepared as described and labeled with Homer1 (rabbit, 1:1000, Millipore, Cat#: ABN37), GABA B1 (mouse, monoclonal, 1:1000, NeuroMab Cat#: 75-183), GABA B2 (mouse, monoclonal, 1:1000, NeuroMab Cat#: 75-125) primary antibodies and secondary antibodies conjugated to Alexa647 (1:3000, ThermoFisher) or CF568 (1:3000, Biotium).

Techniques: Virus, Injection, Expressing, Transfection, Control

Journal: Nature Communications

Article Title: Neurexins regulate presynaptic GABA B -receptors at central synapses

doi: 10.1038/s41467-021-22753-5

Figure Lengend Snippet: a Schematic of cerebellum circuits and experimental approach for stimulating basket cell (BC)-Purkinje cell (PC) inhibitory synapse. b Example traces of paired-pulse IPSCs before and after addition of GABA B -receptor agonist SKF, recorded in acute cerebellar slices from littermate control and Nrxn123 TKO mice P35-49. The normalized IPSCs before and after SKF are shown in inset. c Summary graphs of IPSC1 amplitudes before and after SKF for control and Nrxn123 TKO synapses. P = 0.0016, P = 0.051, paired two-sided t -test. P = 0.0389, unpaired two-sided t -test. d Summary graphs of IPSC1 amplitude remaining unblocked by SKF for control and Nrxn123 TKO mice. P = 0.0028; unpaired two-sided t -test. e Summary graphs of the PPR before and after SKF in control and Nrxn123 TKO mice. P = 0.000101, P = 0.097, paired two-sided t -test. P = 0.762, unpaired two-sided t -test. f Summary graphs of the CV of IPSC1 amplitude before and after SKF in control and Nrxn123 TKO mice. P = 0.00073, P = 0.066, paired two-sided t -test. P = 0.348, unpaired two-sided t -test. g Summary graphs of IPSC1 rise time (left) and decay time constant (right). P = 0.000953, P = 0.977; unpaired two-sided t -test. Data are means ± SEM. Number of cells (from at least three mice per group) analyzed are indicated in the bars ( c – g ); Statistical differences were assessed by Student’s t -test. (* P < 0.05; ** P < 0.01; *** P < 0.001). Source data are provided as a Source Data file.

Article Snippet: Twenty micrometer thick brainstem slices containing the MNTB region were prepared as described and labeled with Homer1 (rabbit, 1:1000, Millipore, Cat#: ABN37), GABA B1 (mouse, monoclonal, 1:1000, NeuroMab Cat#: 75-183), GABA B2 (mouse, monoclonal, 1:1000, NeuroMab Cat#: 75-125) primary antibodies and secondary antibodies conjugated to Alexa647 (1:3000, ThermoFisher) or CF568 (1:3000, Biotium).

Techniques: Control

Journal: Nature Communications

Article Title: Neurexins regulate presynaptic GABA B -receptors at central synapses

doi: 10.1038/s41467-021-22753-5

Figure Lengend Snippet: a The diagram of the calyx of Held synapse. b Strategy for selective deletions of all neurexins at the calyx of Held by crossing PV-Cre mice with triple Nrxn123 cKO mice (Chen et al. , Luo et al. ). c Representative traces of EPSC before and after application of 20 µM SKF-97541 (SKF), a potent and selective GABA B -receptor agonist, recorded in acute slices from littermate control and neurexin123 TKO mice at P12–P14. The normalized EPSCs before and after SKF are shown in inset. d Summary graphs of EPSC1 amplitudes before and after SKF for control and Nrxn123 TKO mice. P = 2.3E−7, P = 0.072, paired two-sided t -test. P = 0.0062, unpaired two-sided t -test. e Summary graphs of EPSC1 remaining unblocked by SKF application. P = 1.79E−7, unpaired two-sided t -test. f Summary graphs of the paired-pulse ratio (PPR) before and after SKF in control and Nrxn123 TKO mice. P = 2.81E−5, P = 0.09, paired two-sided t -test. P = 0.0038, unpaired two-sided t -test. Data are means ± SEM. Number of cells (from at least three mice per group) analyzed are indicated in the bars ( d – f ); Statistical differences were assessed by Student’s t -test. (* P < 0.05; ** P < 0.01; *** P < 0.001). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against VGluT1 (guinea pig, polyclonal, 1:1000, Millipore, Cat#: AB5905; RRID: AB_2301751), GABA B1 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-183), and GABA B2 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-125) were used.

Techniques: Control

Journal: Nature Communications

Article Title: Neurexins regulate presynaptic GABA B -receptors at central synapses

doi: 10.1038/s41467-021-22753-5

Figure Lengend Snippet: a Representative confocal microscopy images of MNTB-containing brainstem slice with specific labeling of VGluT1 (green) and GABA B -receptor subunit 2 (GABA B2 , red) from both littermate control and Nrxn123 TKO mice at P12. Scale bar, 10 µm. b Summary of VGluT1 and GABA B2 immunostaining intensity (normalized to control). P = 1, P = 1.35E−9, unpaired two-sided t -test. c , d Same as a and b except for specific labeling of VGluT1 (green) and GABA B -receptor subunit 1 (GABA B1 , red). P = 1, P = 0.031, unpaired two-sided t -test. Data are means ± SEM. Number of sections/animals for immunostaining are indicated in the bars ( b , d ). Statistical differences were assessed by Student’s t -test. (* P < 0.05; *** P < 0.001). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against VGluT1 (guinea pig, polyclonal, 1:1000, Millipore, Cat#: AB5905; RRID: AB_2301751), GABA B1 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-183), and GABA B2 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-125) were used.

Techniques: Confocal Microscopy, Labeling, Control, Immunostaining

Journal: Nature Communications

Article Title: Neurexins regulate presynaptic GABA B -receptors at central synapses

doi: 10.1038/s41467-021-22753-5

Figure Lengend Snippet: a Representative dSTORM images of MNTB-containing brainstem slice with dual labeling of Homer1 (magenta) and GABA B -receptor subunit 2 (GABA B2 , yellow) from both littermate control and Nrxn123 TKO mice. b Summary of the number of GABA B2 clusters normalized to the number of Homer1 clusters. P = 0.0051, unpaired two-sided t -test. c Distribution of Homer1 and GABA B2 localizations. d – f Similar as a – c , except for GABA B1 antibody used. P = 0.0057, unpaired two-sided t -test. Data are means ± SEM. Number of sections/animals for immunostaining are indicated in the bars ( b , e ). Statistical differences were assessed by Student’s t -test. (** P < 0.01). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against VGluT1 (guinea pig, polyclonal, 1:1000, Millipore, Cat#: AB5905; RRID: AB_2301751), GABA B1 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-183), and GABA B2 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-125) were used.

Techniques: Labeling, Control, Immunostaining

Journal: Nature Communications

Article Title: Neurexins regulate presynaptic GABA B -receptors at central synapses

doi: 10.1038/s41467-021-22753-5

Figure Lengend Snippet: a Schematic of experimental approach for virus injection and electro-physiology recording (Top); GFP expression in AAV-transfected CA3 region of hippocampus (bottom). b Representative traces of EPSCs evoked by fiber stimulations with increased intensity, recorded in acute hippocampus slices from Nrxn123 cKO mice injected with AAV-ΔCre or AAV-Cre. c The input–output curve of EPSC amplitudes in relation to the stimulation intensity. P = 0.0113, unpaired two-sided t -test. d Example traces of paired-pulse EPSCs before and after addition of GABA B -receptor agonist SKF, the normalized EPSCs before and after SKF are shown in inset. The intensity of fiber stimulation was tuned to evoke EPSC1 at similar amplitude for each cell. e Summary graphs of EPSC1 amplitudes before and after SKF for control (ΔCre) and Nrxn123 TKO synapses (Cre). P = 0.0001, P = 0.0001, paired two-sided t -test. P = 0.99, unpaired two-sided t -test. f Summary graphs of EPSC1 rise time (left) and decay time constants (right). P = 0.0008, P = 0.6136, unpaired two-sided t -test. g Summary graphs of EPSC1 remaining unblocked by SKF application. P = 0.0013, unpaired two-sided t -test. h Summary graphs of the PPR before and after SKF in control and Nrxn123 TKO synapses. P = 0.0001, P = 0.0984, paired two-sided t -test. P = 0.2772, unpaired two-sided t -test. i Summary graphs of the CV of EPSC1 amplitude before and after SKF in control and Nrxn123 TKO synapses. P = 0.0385, P = 0.99, paired two-sided t -test. P = 0.4664, unpaired two-sided t -test. Data are means ± SEM. Number of cells (from at least three mice per group) analyzed are indicated in the graph ( c ) or bars ( e – i ); Statistical differences were assessed by Student’s t -test. (* P < 0.05; ** P < 0.01, *** P < 0.001). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against VGluT1 (guinea pig, polyclonal, 1:1000, Millipore, Cat#: AB5905; RRID: AB_2301751), GABA B1 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-183), and GABA B2 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-125) were used.

Techniques: Virus, Injection, Expressing, Transfection, Control

Journal: Nature Communications

Article Title: Neurexins regulate presynaptic GABA B -receptors at central synapses

doi: 10.1038/s41467-021-22753-5

Figure Lengend Snippet: a Schematic of experimental setup for virus injection and optogenetic stimulation of PV + interneurons (Top); PV-Cre dependent expression of Chief-TdTomato in AAV-transfected CA1 region of hippocampus (bottom). b Example traces of paired-pulse blue light-evoked IPSCs before and after addition of GABA B -receptor agonist SKF, recorded in acute hippocampus slices from PVCre + mice (control) or PVCre + /Nrxn123 cKO mice (TKO) injected with AAV-DIO-Chief-TdTomato. The normalized IPSCs before and after SKF are shown in inset. c Summary graphs of IPSC1 amplitudes before and after SKF for control and Nrxn123 TKO synapses. P = 0.0001, P = 0.0026, paired two-sided t -test. P = 0.6, unpaired two-sided t -test. d Summary graphs of IPSC1 amplitude remaining unblocked by SKF. P = 0.0047, unpaired two-sided t -test. e Summary graphs of the PPR ratio before and after SKF in control and Nrxn123 TKO synapses. P = 0.0022, P = 0.6165, paired two-sided t -test. P = 0.5114, unpaired two-sided t -test. f Summary graphs of the CV of IPSC1 amplitude. P = 0.0143, P = 0.99, paired two-sided t -test. P = 0.99, unpaired two-sided t -test. g Summary graphs of IPSC1 rise time (left) and decay time constant (right). P = 0.4974, P = 0.063, unpaired two-sided t -test. Data are means ± SEM. Number of cells (from at least three mice per group) analyzed are indicated in the bars ( c – g ); Statistical differences were assessed by Student’s t -test. (* P < 0.05; ** P < 0.01; *** P < 0.001). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against VGluT1 (guinea pig, polyclonal, 1:1000, Millipore, Cat#: AB5905; RRID: AB_2301751), GABA B1 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-183), and GABA B2 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-125) were used.

Techniques: Virus, Injection, Expressing, Transfection, Control

Journal: Nature Communications

Article Title: Neurexins regulate presynaptic GABA B -receptors at central synapses

doi: 10.1038/s41467-021-22753-5

Figure Lengend Snippet: a Schematic of cerebellum circuits and experimental approach for stimulating basket cell (BC)-Purkinje cell (PC) inhibitory synapse. b Example traces of paired-pulse IPSCs before and after addition of GABA B -receptor agonist SKF, recorded in acute cerebellar slices from littermate control and Nrxn123 TKO mice P35-49. The normalized IPSCs before and after SKF are shown in inset. c Summary graphs of IPSC1 amplitudes before and after SKF for control and Nrxn123 TKO synapses. P = 0.0016, P = 0.051, paired two-sided t -test. P = 0.0389, unpaired two-sided t -test. d Summary graphs of IPSC1 amplitude remaining unblocked by SKF for control and Nrxn123 TKO mice. P = 0.0028; unpaired two-sided t -test. e Summary graphs of the PPR before and after SKF in control and Nrxn123 TKO mice. P = 0.000101, P = 0.097, paired two-sided t -test. P = 0.762, unpaired two-sided t -test. f Summary graphs of the CV of IPSC1 amplitude before and after SKF in control and Nrxn123 TKO mice. P = 0.00073, P = 0.066, paired two-sided t -test. P = 0.348, unpaired two-sided t -test. g Summary graphs of IPSC1 rise time (left) and decay time constant (right). P = 0.000953, P = 0.977; unpaired two-sided t -test. Data are means ± SEM. Number of cells (from at least three mice per group) analyzed are indicated in the bars ( c – g ); Statistical differences were assessed by Student’s t -test. (* P < 0.05; ** P < 0.01; *** P < 0.001). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against VGluT1 (guinea pig, polyclonal, 1:1000, Millipore, Cat#: AB5905; RRID: AB_2301751), GABA B1 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-183), and GABA B2 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-125) were used.

Techniques: Control