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. 2020 Dec 23;9:e59545. doi: 10.7554/eLife.59545

Figure 2. Nlgn3Δ overexpression (OE) specifically potentiates VGT3+ interneuron-mediated unitary synaptic transmission.

The effects of OE of Nlgn3Δ isoform or enhanced green fluorescent protein (EGFP, control) in hippocampal CA1 pyramidal neurons on inhibitory inputs mediated by VGT3+ inhibitory interneurons. (A) Configuration of the triple whole-cell recording for VGT3+, Pv+, and Sst+ interneurons forming synapses with pyramidal neurons. (B) Sample traces of unitary inhibitory postsynaptic currents (uIPSCs). Left top, averaged sample traces of a single presynaptic action potential (AP) evoked in a VGT3+ interneuron. Left middle and bottom, superimposed averaged sample uIPSC traces (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 VGT3+ interneurons. uIPSCs are normalized to the first amplitude. Because the first uIPSC overlaps with the second uIPSC, to accurately measure the amplitude of the second IPSC, we ‘cancelled’ the first uIPSC by subtracting the traces receiving a single pulse (gray) from those receiving a paired pulse (black), both normalized to the first response. The amplitude (C) and paired-pulse ratio (PPR) (D) of uIPSCs were plotted for each pair of transfected (Trans) and neighboring untransfected (Untrans) cells (open symbols). Bar graphs indicate mean ± SEM. (E) Synaptic connectivity between presynaptic inhibitory interneuron and postsynaptic untransfected (open bars) or transfected (black) pyramidal neurons. Numbers of cell pairs: Nlgn3Δ or EGFP at VGT3+ synapses (42 pairs/23 mice and 30/21). The number of tested slice cultures is the same as that of cell pairs. n.s., not significant. Mann–Whitney U-test.

Figure 2.

Figure 2—figure supplement 1. Validation of TdTomato expression in three different cell type-specific fluorescent lines.

Figure 2—figure supplement 1.

(A–C) Images of double-labeled immunofluorescence staining against TdTomato (red, A1, B1, and C1) and cell type-specific markers (green, VGT3: A2, Pv: B2, Sst: C2) in the hippocampal CA1 region of VGT3/RFP (A), Pv/RFP (B), and Sst/RFP (C) mice. (D and E) Images of double-labeled immunofluorescence staining against TdTomato (magenta) and cell type-specific markers (green, Pv: D, Sst: E) in the hippocampal CA1 region of VGT3/RFP mice. (F) Relative proportion of hippocampal CA1 inhibitory interneurons as a fraction of neuronal populations positive for cell type-specific markers and TdTomato (+) and TdTomato-positive but marker negative (−) neurons. The TdTomato-positive neurons included for quantification were localized in the specific CA1 subregions where we performed electrophysiological recordings; VGT3/RFP mice: CA1 pyramidale, radiatum, and oriens; Pv/RFP: radiatum; Sst/RFP: oriens. Number of TdTomato-positive neurons: VGT3/RFP: 404 neurons/2 mice; Pv/RFP: 1601/2; Sst/RFP: 1789/2. (G) Relative proportion of hippocampal CA1 inhibitory interneurons in VGT3/RFP mice as a fraction of neuronal populations positive for Pv or Sst and TdTomato (+) neurons. Number of TdTomato-positive neurons: Pv/RFP staining: 1966/2; Sst/RFP: 1464/2. Scale bars, 50 µm.