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(A) Excitatory synapse density in 14 DIV hippocampal neurons for a given condition normalized to the synapse density of the negative control condition. Synapse density is defined as the overlap of <t>PSD95/Synapsin</t> I puncta on GFP-transfected neurons. Neurons were co-transfected with a GFP expressing vector and either an empty vector control, a shRNA targeting Sema4A, a myc-tagged Sema4A cDNA, or co-transfection of myc-Sema4A RNAi resistant cDNA with a shRNA targeting Sema4A. In this case, the negative control is neurons co-transfected with an empty vector and a GFP-expressing vector. The dotted line at 100% indicates the control condition normalized to itself. (A, right) Representative stretches of GFP-transfected dendrite immunostained for PSD95 (red) and Synapsin I (magenta). (B) Excitatory synapse density in 14 DIV hippocampal neurons in response to Sema4A-Fc and Sema4D-Fc treatment normalized to the synapse density of the negative control condition. Synapse density is defined by the overlap of PSD95/Synapsin I puncta on MAP2-positive dendrites. In this case, the negative control is neurons treated with Fc control. The dotted line at 100% indicates the control condition normalized to itself. (B, right) Representative stretches of MAP2-positive dendrite immunostained for PSD95 (red), Synapsin I (magenta), and MAP2 (green). (C) Excitatory synapse density in 14 DIV hippocampal neurons for a given condition normalized to the synapse density of the negative control condition. Synapse density is defined as the overlap of PSD95/Synapsin I puncta on GFP-transfected neurons. Neurons were co-transfected with a GFP expressing vector and either an empty vector control, a shRNA targeting Rem2, a shRNA targeting Plexin-B1, or a shRNA targeting Plexin-B2. In this case, the negative control is neurons co-transfected with an empty vector and a GFP-expressing vector. The dotted line at 100% indicates the control condition normalized to itself. (A, right) Representative stretches of GFP-transfected dendrite immunostained for PSD95 (red) and Synapsin I (magenta). (D) Excitatory synapse density in 14 DIV hippocampal neurons in response to Sema4A-Fc normalized to the synapse density of the negative control condition. Synapse density is defined by the overlap of PSD95/Synapsin I puncta on GFP-transfected neurons. Neurons were co-transfected with a GFP expressing vector and either an empty vector control, a shRNA targeting Plexin-B1, or a shRNA targeting Plexin-B2 and were treated with either Fc control or Sema4A-Fc. In this case, the negative control is neurons treated with Fc control and co-transfected with an empty vector and a GFP-expressing vector (gray bar, far left). The dotted line at 100% indicates the control condition normalized to itself. The error for the negative control condition is contained within the error bar for each experimental condition (see Materials and Methods). (D, right) Representative stretches of GFP transfected dendrite immunostained for PSD95 (red) and Synapsin I (magenta). For treatments: 2nM Fc control, Sema4A-Fc or Sema4D-Fc were added to 13 DIV hippocampal neurons overnight (12–16hrs). n ≥ 55 neurons per condition from three experiments; n = 44 neurons for Rem2 RNAi. *p < 0.05, Two-Way ANOVA with Tukey’s post hoc Test. Error bars are ± standard error of the average ratio. Scale bars, 5µm.
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(A) Excitatory synapse density in 14 DIV hippocampal neurons for a given condition normalized to the synapse density of the negative control condition. Synapse density is defined as the overlap of PSD95/Synapsin I puncta on GFP-transfected neurons. Neurons were co-transfected with a GFP expressing vector and either an empty vector control, a shRNA targeting Sema4A, a myc-tagged Sema4A cDNA, or co-transfection of myc-Sema4A RNAi resistant cDNA with a shRNA targeting Sema4A. In this case, the negative control is neurons co-transfected with an empty vector and a GFP-expressing vector. The dotted line at 100% indicates the control condition normalized to itself. (A, right) Representative stretches of GFP-transfected dendrite immunostained for PSD95 (red) and Synapsin I (magenta). (B) Excitatory synapse density in 14 DIV hippocampal neurons in response to Sema4A-Fc and Sema4D-Fc treatment normalized to the synapse density of the negative control condition. Synapse density is defined by the overlap of PSD95/Synapsin I puncta on MAP2-positive dendrites. In this case, the negative control is neurons treated with Fc control. The dotted line at 100% indicates the control condition normalized to itself. (B, right) Representative stretches of MAP2-positive dendrite immunostained for PSD95 (red), Synapsin I (magenta), and MAP2 (green). (C) Excitatory synapse density in 14 DIV hippocampal neurons for a given condition normalized to the synapse density of the negative control condition. Synapse density is defined as the overlap of PSD95/Synapsin I puncta on GFP-transfected neurons. Neurons were co-transfected with a GFP expressing vector and either an empty vector control, a shRNA targeting Rem2, a shRNA targeting Plexin-B1, or a shRNA targeting Plexin-B2. In this case, the negative control is neurons co-transfected with an empty vector and a GFP-expressing vector. The dotted line at 100% indicates the control condition normalized to itself. (A, right) Representative stretches of GFP-transfected dendrite immunostained for PSD95 (red) and Synapsin I (magenta). (D) Excitatory synapse density in 14 DIV hippocampal neurons in response to Sema4A-Fc normalized to the synapse density of the negative control condition. Synapse density is defined by the overlap of PSD95/Synapsin I puncta on GFP-transfected neurons. Neurons were co-transfected with a GFP expressing vector and either an empty vector control, a shRNA targeting Plexin-B1, or a shRNA targeting Plexin-B2 and were treated with either Fc control or Sema4A-Fc. In this case, the negative control is neurons treated with Fc control and co-transfected with an empty vector and a GFP-expressing vector (gray bar, far left). The dotted line at 100% indicates the control condition normalized to itself. The error for the negative control condition is contained within the error bar for each experimental condition (see Materials and Methods). (D, right) Representative stretches of GFP transfected dendrite immunostained for PSD95 (red) and Synapsin I (magenta). For treatments: 2nM Fc control, Sema4A-Fc or Sema4D-Fc were added to 13 DIV hippocampal neurons overnight (12–16hrs). n ≥ 55 neurons per condition from three experiments; n = 44 neurons for Rem2 RNAi. *p < 0.05, Two-Way ANOVA with Tukey’s post hoc Test. Error bars are ± standard error of the average ratio. Scale bars, 5µm.

Journal: Molecular and cellular neurosciences

Article Title: Class 4 Semaphorins and Plexin-B receptors regulate GABAergic and glutamatergic synapse development in the mammalian hippocampus

doi: 10.1016/j.mcn.2018.06.008

Figure Lengend Snippet: (A) Excitatory synapse density in 14 DIV hippocampal neurons for a given condition normalized to the synapse density of the negative control condition. Synapse density is defined as the overlap of PSD95/Synapsin I puncta on GFP-transfected neurons. Neurons were co-transfected with a GFP expressing vector and either an empty vector control, a shRNA targeting Sema4A, a myc-tagged Sema4A cDNA, or co-transfection of myc-Sema4A RNAi resistant cDNA with a shRNA targeting Sema4A. In this case, the negative control is neurons co-transfected with an empty vector and a GFP-expressing vector. The dotted line at 100% indicates the control condition normalized to itself. (A, right) Representative stretches of GFP-transfected dendrite immunostained for PSD95 (red) and Synapsin I (magenta). (B) Excitatory synapse density in 14 DIV hippocampal neurons in response to Sema4A-Fc and Sema4D-Fc treatment normalized to the synapse density of the negative control condition. Synapse density is defined by the overlap of PSD95/Synapsin I puncta on MAP2-positive dendrites. In this case, the negative control is neurons treated with Fc control. The dotted line at 100% indicates the control condition normalized to itself. (B, right) Representative stretches of MAP2-positive dendrite immunostained for PSD95 (red), Synapsin I (magenta), and MAP2 (green). (C) Excitatory synapse density in 14 DIV hippocampal neurons for a given condition normalized to the synapse density of the negative control condition. Synapse density is defined as the overlap of PSD95/Synapsin I puncta on GFP-transfected neurons. Neurons were co-transfected with a GFP expressing vector and either an empty vector control, a shRNA targeting Rem2, a shRNA targeting Plexin-B1, or a shRNA targeting Plexin-B2. In this case, the negative control is neurons co-transfected with an empty vector and a GFP-expressing vector. The dotted line at 100% indicates the control condition normalized to itself. (A, right) Representative stretches of GFP-transfected dendrite immunostained for PSD95 (red) and Synapsin I (magenta). (D) Excitatory synapse density in 14 DIV hippocampal neurons in response to Sema4A-Fc normalized to the synapse density of the negative control condition. Synapse density is defined by the overlap of PSD95/Synapsin I puncta on GFP-transfected neurons. Neurons were co-transfected with a GFP expressing vector and either an empty vector control, a shRNA targeting Plexin-B1, or a shRNA targeting Plexin-B2 and were treated with either Fc control or Sema4A-Fc. In this case, the negative control is neurons treated with Fc control and co-transfected with an empty vector and a GFP-expressing vector (gray bar, far left). The dotted line at 100% indicates the control condition normalized to itself. The error for the negative control condition is contained within the error bar for each experimental condition (see Materials and Methods). (D, right) Representative stretches of GFP transfected dendrite immunostained for PSD95 (red) and Synapsin I (magenta). For treatments: 2nM Fc control, Sema4A-Fc or Sema4D-Fc were added to 13 DIV hippocampal neurons overnight (12–16hrs). n ≥ 55 neurons per condition from three experiments; n = 44 neurons for Rem2 RNAi. *p < 0.05, Two-Way ANOVA with Tukey’s post hoc Test. Error bars are ± standard error of the average ratio. Scale bars, 5µm.

Article Snippet: The following antibodies were used: ms α GAD65 (1:1000; Millipore Cat# MAB351, RRID:AB_2263126), rb α GABA A R γ2 , (1:150; Millipore Cat# AB5559, RRID:AB_177523), rb α Synapsin I (1:1000; Millipore Cat# AB1543, RRID:AB_2200400), ms α PSD95 (1:500; UC Davis/NIH NeuroMab Facility Cat# 75-028, RRID:AB_2307331), ms α MAP2, (1:1000; Sigma-Aldrich Cat# M4403, RRID:AB_477193), ms α 488 (1:1000; Jackson ImmunoResearch Labs Cat# 115-545-003, RRID:AB_2338840), ms α Cy3 (1:500; Jackson ImmunoResearch Labs Cat# 715-165-150, RRID:AB_2340813), and rb α Cy5 (1:500; Thermo Fisher Scientific Cat# A10523, RRID:AB_2534032).

Techniques: Negative Control, Transfection, Expressing, Plasmid Preparation, Control, shRNA, Cotransfection