Fig. 3: NG2 glia form pre- and postsynaptic structures with proximal hippocampal interneurons.

a Representative image showing the morphology of dual-patched interneurons loaded with Alexa Fluor 568 in the patch pipettes in hippocampal CA1 region. One of the pair-recorded interneurons is typically chosen as the close interneuron, where the proximity to its closest NG2 glia soma is <30 µm, and the other is chosen as the far interneuron, where the proximity to its closest NG2 glia soma is farther than 30 µm. Scale bar, 20 μm. b Representative mIPSCs traces recorded from the close interneuron and far interneuron, showing an increase of frequency of mIPSCs typically occuring in the close interneuron after NG2 glia photostimulation (15 Hz, 90 s). c Summary bar graph shows a significant increase of frequency of mIPSCs onto the close interneurons but no effect on the far interneurons. P = 0.0022 and 0.2841 from 9 pairs for dual-patched close and far interneurons at postnatal 4–6 weeks, respectively, two-tailed paired t test. d Summary bar graph shows the percentage of cells displaying an increase of mIPSCs frequency from dual-recorded interneurons using Kolmogorov–Smirnov two-sample test analysis. The percentage of responsive cells is increased to 78% in the close interneurons after NG2 glia photoactivation. e Representative images showing immunostainings for gephyrin (upper panel, in magenta), CaMKII (middle panel, in magenta), and vGluT2 (lower panel, in magenta) with GFP-labeled NG2 cells (in green). The arrowheads in magenta and white indicate the anatomic proximity between the interneuron’s or pyramidal cell’s soma and its closest NG2 glia soma. Scale bars, 20 μm. f Histogram and cumulative distribution graph show the connection probabilities with respect to the inter somatic distance between gephyrin/CaMKII or vGluT2-positive neurons and NG2 glia. n = 142 pairs for gephyrin (+) interneurons–NG2 glia, n = 195 pairs, and n = 188 pairs for CaMKII (+) and vGluT2 (+) pyramidal neurons to NG2 glia from 3 mice at postnatal 6–12 weeks, respectively. g Cartoon illustrating a synaptic connection between an NG2 glial terminal and its postsynaptic dendrite from C57BL/6 wild-type mouse hippocampus. Representative images on the right panel and the panel below show NG2 antibody-labeled immunoelectron microscopy in C57BL/6 wild-type mouse hippocampus at postnatal 6–8 weeks. The presynaptic, postsynaptic structures, and the gold particles are indicated by green, blue and yellow, respectively. Scale bars, 50 nm. The bar graph shows the density of NG2-to-neuron synapses in CA1 region of the hippocampus (28.18 ± 1.42/10³ µm²), n = 4 mice. h Double labeling for GABA-immunogold within NG2-DAB positive processes/terminals, which are shown making a presynaptic contact with an underlying postsynaptic structure. The darkened “patchy/cloudy” DAB reaction products are seen mostly within the NG2-positive glial terminal (containing pleomorphic synaptic vesicles), using an antibody against NG2, as indicated by the white arrows. GABA-immunogold particles are labeled either within or adjacent to many synaptic vesicles (yellow arrowheads). LT indicates an NG2-labeled terminal. Post indicates postsynaptic structure. M indicates mitochondrion. Scale bars, 200 nm. All experiments are replicated from 5 mice. Data are presented as mean values ± SEM and error bar represents SEM.