Skip to main content
. 2020 Dec 23;9:e59545. doi: 10.7554/eLife.59545

Figure 5. Lack of presynaptic Nrxns reduces inhibitory synaptic transmission and abolishes the potentiation effect of Nlgn3Δ in VGT3+ inhibitory synaptic transmission.

Effects of Nlgn3Δ isoform overexpression (OE) in hippocampal CA1 pyramidal neurons in the absence of presynaptic Nrxn input in VGT3+ interneurons. (A) Validation of the NrxnTKO/VGT3/RFP mouse line. Expression of Nrxn genes were compared in TdTomato-positive VGT3+ neurons in organotypic hippocampal slice cultures prepared from wild-type (WT) (VGT3/RFP) and knockout (KO) (NrxnTKO/VGT3/RFP) mice. qPCRs against Nrxn 1, 2, 3 and Gapdh (internal control) were performed for single-cell cDNA libraries prepared from TdTomato-positive neurons. Number of neurons: WT (N = 9, three mice) and KO (7, 2). ***p<0.001, ****p<0.0001 (Student’s t-test). (B–E) Effect of Nrxn triple KO on VGT3+ interneuron-mediated inhibitory synaptic transmission. (B) Left top, averaged sample traces of a single presynaptic action potential (AP) evoked in a NrxnTKO VGT3+ interneuron. Left bottom, superimposed averaged sample traces of unitary inhibitory postsynaptic current (uIPSC) (Untrans: black; trans: dark gray) induced by an AP. Right, superimposed averaged sample traces of uIPSCs evoked by single (dark gray) and double (black) APs in NrxnTKO VGT3+ interneurons. Summary of uIPSC amplitude (C), paired-pulse ratio (PPR) (D), and connectivity (E). Number of cell pairs: WT (N = 23, six mice) and KO (35, 7). *p<0.05 (two-way ANOVA with Sidak’s post hoc test). (F–I) Effect of Nlgn3Δ OE on NrxnTKO VGT3+ interneuron-mediated inhibitory synaptic transmission. (F) Left top, averaged sample traces of a single presynaptic AP evoked in a NrxnTKO VGT3+ interneuron. Left bottom, superimposed averaged sample traces of uIPSC (Untrans: black; trans: dark gray) induced by an AP. Right, superimposed averaged sample traces of uIPSCs evoked by single (dark gray) and double (black) APs in NrxnTKO VGT3+ interneurons. Summary of uIPSC amplitude (G), PPR (H), and connectivity (I). Open circles connected with bars represent individual pairs of cells (C, D, G, and H). Bar graphs indicate mean ± SEM. N = 18 cell pairs (three mice). The number of tested slice cultures is the same as that of cell pairs. Mann–Whitney U-test.

Figure 5.

Figure 5—figure supplement 1. Membrane excitability is not altered in VGT3+ interneurons in NrxnTKO/VGT3/RFP mice.

Figure 5—figure supplement 1.

(Top) Sample traces of VGT3+ interneurons in organotypic hippocampal slice cultures prepared from VGT/RFP (wild-type [WT], left) and NrxnTKO/VGT3/RFP (knockout [KO], right) mice. The superimposed traces were elicited by current injections of 200, 500, and 900 pA for 200 ms. (Bottom) Summary graph of the frequency of action potentials in WT and KO VGT3+ interneurons. The input–output relationship (number of spikes elicited vs. amount of current injection over a 200 ms duration) was plotted for WT and KO neurons. Neurons were held at resting membrane potentials. Two-way ANOVA with Sidak’s post hoc test: number of cells tested: untrans, 14 cells from five mice (14/5); trans, (14/5). n.s., not significant. Resting membrane potentials were not significant between WT and KO VGT3+ interneurons: WT, −56.3 ± 0.46; KO, −56.0 ± 0.28 mV (Student’s t-test).