Journal: eLife

Article Title: Paradoxical network excitation by glutamate release from VGluT3 + GABAergic interneurons

doi: 10.7554/eLife.51996

Figure Lengend Snippet: ( A ) Representative images illustrating native VGluT3 expression within CA1/3 stratum pyramidale (SP) at postnatal day 5, 15, and 30 (p5,15,30) as indicated. Group data for terminal density counts in CA1 and CA3 throughout development are plotted at right (n = 6–19 sections from three to six animals per age were examined; individual observations (n) along with means are provided). ( B ) Representative high resolution Airyscan images illustrating colocalization of VGluT3 signal in CA1 perisomatic axon terminals with CB1R signal (upper row, ( B 1 ) as well as GAD1 and VGAT signals (second row, ( B 2 ) but not Syt2 labelled terminals (third row, ( B 3 ); SO, stratum oriens; SR, stratum radiatum). Widefield as well as digitally magnified images from boxed regions are provided showing merged and split channels for each set of markers, with DAPI labeling, as indicated. Filled arrowheads highlight terminals showing colocalization while open arrowheads highlight terminals lacking colocalization. Colocalization of VGluT3 with each marker is quantified in the box plots ( B 4 ) with data normalized to total CB1R + terminal counts (left plot) or total VGluT3 + terminal counts (middle and right plots; 1200–6580 terminals from n = 8–14 sections in each staining combination, from three mice were evaluated). ( C ) Representative Airyscan images and group data illustrating conserved VGluT3 expression at all putative release sites along continuous individual axon segments. Three individual axon segments are highlighted in the CA1 SP widefield image and a segment from one of these axons (boxed region, ( C 1 ) is digitally magnified. Group data plotted at right summarize the percentage of putative release sites along individual axons from CB1R + /VGluT3 + cells that are VGluT3 + (pie chart and box plot, normalized to number of boutons in each axon segment) as well as the correlation between total number of boutons scored per length of each continuous axon examined (IBI, interbouton interval; 510 boutons were evaluated from n = 28 axon segments across three mice). ( D ) An additional example of conserved VGluT3 expression across all putative release sites of a continuous axon segment within SO with merged and split channels as indicated. For statistical analysis *p<0.05, **p<0.01, ***p<0.001. Scalebar in A = 10 µm; in B, C, D = 5 µm; in C 1 = 1 µm. Figure 1—source data 1. Data plotted in .

Article Snippet: To address the homogeneity of the terminals along the same parent axon, we traced continuous CB1R + axon segments using the Imaris function ‘ Filaments ’ in ‘ manual mode ’.

Techniques: Expressing, Labeling, Marker, Staining

Journal: eLife

Article Title: Paradoxical network excitation by glutamate release from VGluT3 + GABAergic interneurons

doi: 10.7554/eLife.51996

Figure Lengend Snippet: ( A ) Representative high-resolution images illustrating colocalization of VGluT3 and RFP signal in CA1 perisomatic axon terminals of P10 and P30 VGluT3-Cre:Ai14 mice as indicated (scale bars = 5 µm). Colocalization of VGluT3 with RFP across development is quantified in the bar chart at right with data normalized to the total number of RFP + terminals examined at each age (237–1447 terminals from n = 3–8 sections in each staining combination, from 2 to 4 mice were evaluated at each age; ***p<0.001). ( B ) Representative high-resolution images illustrating colocalization of VGluT3 and CB1R signal in CA1 perisomatic axon terminals at P10 and P90 as indicated. Widefield as well as digitally magnified images from boxed regions are provided showing merged and split channels for each marker, with DAPI labelling, as indicated (scale bars = 5 µm). The group data bar charts at right illustrate the the colocalization of VGluT3 to CB1R + terminals at P10 and P90 (normalized to total number of CB1R + terminals examined) as well as overall CB1R + terminal density at these ages (1265–2205 terminals from n = 4–9 sections from three mice were evaluated at each age; *p<0.05). ( C ) Anatomical reconstructions of representative biocytin filled CA1 and CA3 VGluT3 + BCs recorded in P13-15 and P30-40 VGluT3-Cre:Ai14 mice as indicated (red axon, black dendrite). Group data summary plots at right illustrate similar overall axon length (upper bar chart) and distribution (lower Sholl plot) at the two developmental stages (n = 7–9 VGluT3 + BCs, CA1 and CA3 combined, were recovered from recordings in 2 VGluT3-Cre:Ai14 mice for each time point). Figure 2—figure supplement 2—source data 1. Data plotted in .

Article Snippet: To address the homogeneity of the terminals along the same parent axon, we traced continuous CB1R + axon segments using the Imaris function ‘ Filaments ’ in ‘ manual mode ’.

Techniques: Staining, Marker

Journal: eLife

Article Title: Paradoxical network excitation by glutamate release from VGluT3 + GABAergic interneurons

doi: 10.7554/eLife.51996

Figure Lengend Snippet: ( A ) Images of representative RFP + cells recorded in VGluT3-Cre:Ai14 mice from CA1 (upper row) and CA3 (lower row) hippocampus illustrating prototypical BCs and DTIs. Inset traces illustrate membrane responses of each representative cell (upper series of traces) to the current injection waveforms shown (lower series of traces). Image insets highlight colocalization of the somatic dye fill (biocytin) with RFP. ( B ) Basic properties of unitary GABAergic synaptic transmission between VGluT3 + BCs (black) or VGluT3 + DTIs (gray) with CA1 PCs. At left, the recording configuration is schematized above an image of a sample RFP + BC-CA1PC pair. At right, sample traces show individual trial synaptic events in postsynaptic CA1 PCs (lower traces) following presynaptic spiking (upper traces) in either RFP + BCs or DTIs as indicated. The basic properties of transmission are summarized in the group data bar charts below (n = 25 and 12 pairs, from 17 and 10 mice for BC and DTI pairs, respectively). Note that for these recordings postsynaptic CA1PC cells were filled with high [Cl - ] and held at −70 mV yielding GABAergic dominated inward currents. ( C ) Sample traces (left) from representative recordings of VGluT3 + BC-CA1PC and VGluT3 + DTI-CA1PC pairs as indicated illustrating asynchronous release of GABA during presynaptic trains of stimuli. Note the postsynaptic events are tightly synchronized with presynaptic APs early in the train and then fail to remain time locked to presynaptic APs as the train continues resulting in a progressive reduction in the synchronicity ratio plotted at right for all recordings (n = 6 and 5 pairs from 4 and 4 mice for BC and DTI pairs, respectively). ( D ) Sample traces (above) and group data bar chart summary (below) illustrating DSI of VGluT3 + BC/DTI-CA1PC pairs. For each set of traces an averaged uIPSC obtained under control conditions (ctl) is shown in comparison with an averaged uIPSC obtained following depolarization of postsynaptic CA1PCs to 0 mV for 5 s (DSI, 5–10 individual uIPSCs were averaged for each condition). The group data bar chart quantifies the magnitude of uIPSC suppression induced by DSI (n = 22 and 10 pairs from 17 and 9 mice for BC and DTI pairs respectively). ( E ) Representative sample image of a biocytin filled postsynaptic CA1PC soma decorated with VGluT3 + CB1R + presynaptic terminals consistent with DSI of VGluT3 + BC /-CA1 PC pairs. Throughout the figure summary plots provide individual observations as well as group means ± SEM. Figure 3—source data 1. Data plotted in .

Article Snippet: To address the homogeneity of the terminals along the same parent axon, we traced continuous CB1R + axon segments using the Imaris function ‘ Filaments ’ in ‘ manual mode ’.

Techniques: Membrane, Injection, Transmission Assay, Control, Comparison

Journal: eLife

Article Title: Paradoxical network excitation by glutamate release from VGluT3 + GABAergic interneurons

doi: 10.7554/eLife.51996

Figure Lengend Snippet: ( A ) Representative Airyscan images examining colocalization of VGluT3 with CB1R (left panels), VGAT and GAD1 (right panels) in terminals located from SP to the SR/SLM border. While SP and upper SR VGluT3 + terminals typically coexpress CB1R, VGAT, and GAD (filled arrowheads) a population of large VGluT3 + terminals at the SR/SLM border do not express these markers (open arrowheads). ( B ) Representative Airyscan images illustrating VMAT2 expression within many of the large VGluT3 + terminals located at the SR/SLM border. Also shown is the coexpression of VGluT3 and CB1R within a subset of terminals located within SR.

Article Snippet: To address the homogeneity of the terminals along the same parent axon, we traced continuous CB1R + axon segments using the Imaris function ‘ Filaments ’ in ‘ manual mode ’.

Techniques: Expressing

Journal: eLife

Article Title: Paradoxical network excitation by glutamate release from VGluT3 + GABAergic interneurons

doi: 10.7554/eLife.51996

Figure Lengend Snippet: ( A ) Representative high-resolution Airyscan images highlighting prominent somatic AMPAR signal within CA1 SP in close proximity to perisomatic targeting CB1R + and VGluT3 + CCKBC terminals. ( B ) Traces from a representative recording (above) and group data summary (below) illustrating a lack of light driven glutamatergic output onto CA1PCs in Nkx2.1-Cre:Ai32 mice (n = 6 cells from one mouse). ( C ) Group data summary comparing PSC Glu conductance recorded in CA1 PCs by light evoked activation of VGluT3 + INTs the absence (n = 45 cells from 22 mice) or presence (n = 21 cells from three mice) of a cocktail (AM251/CGP 55845/UBP 302/LY 341495/CTZ) designed to minimize presynaptic depression and AMPAR desensitization. Throughout the figure summary plots provide individual observations as well as group means ± SEM. Figure 4—figure supplement 1—source data 1. Data plotted in .

Article Snippet: To address the homogeneity of the terminals along the same parent axon, we traced continuous CB1R + axon segments using the Imaris function ‘ Filaments ’ in ‘ manual mode ’.

Techniques: Activation Assay

Journal: eLife

Article Title: Paradoxical network excitation by glutamate release from VGluT3 + GABAergic interneurons

doi: 10.7554/eLife.51996

Figure Lengend Snippet: ( A ) Traces from representative recordings (above) and group data summary (below) comparing light-evoked GABA and glutamate synaptic conductances observed in CA1 PCs of VGluT3-Cre:Ai32 or VGluT3-Cre:Chronos slices maintained in control (left, n = 22 cells from four mice) or SCZD supplemented ACSF (right, n = 23 cells from four mice; **p<0.01 vs. Control ACSF). ( B ) Traces from a representative recording (above) and group data plot (below) illustrating DSI of PSC Glu evaluated in a subset SCZD treated slices (n = 7 cells from two mice). ( C ) Traces from a representative VGluT3 + BC-CA1PC pair recording (above) and group data summary (below) illustrating enhanced glutamatergic, relative to GABAergic, transmission in slices maintained in SCZD (n = 4 pairs from two mice) compared to slices maintained in control ACSF (n = 8 pairs from six mice). Traces are averages of 3–5 sweeps per condition obtained by presynaptic train stimulation. ( D ) Traces from representative recordings (above) and group data plots (below) comparing light evoked GABA and glutamate synaptic conductances in CA1PCs of VGluT3-Cre:Ai32 mice maintained on B6 + diet (left, n = 24 cells from four mice) or B6 - diet (right n = 56 cells from nine mice **p<0.01 vs. B6 + ). ( E ) Example recording with GABAergic transmission blocked that displayed large polysynaptic network barrages following light evoked monosynaptic glutamatergic events (upper traces) that could be eliminated by the CB1R agonist WIN-55212–2 (lower traces). Individual trials/sweeps are shown overlayed in gray with the averaged events shown in black. ( F ) Example recording of a VGluT3-Cre:Ai32 CA1PC held in current clamp illustrating the transition from light-evoked GABAergic inhibition (upper traces) to suprathreshold glutamatergic excitation (middle traces) that is suppressed below threshold by WIN-55212–2 (lower traces). ( G ) Group data summary plot of the proportion of slices exhibiting light-evoked polysynaptic network barrages similar to those illustrated in E as well as the percentage of trials/sweeps in such recordings displaying network events in the absence (n = 25 slices from 11 mice) or presence of WIN-55212–2 (n = 17 slices, from six mice). ( H ) Images of a representative hippocampal section from a VGluT3-Cre mouse infected with AAV-mDlx-Flex-ChR2-mCherry (VGluT3-Cre:mDlxChR2) with a filled PC. ( I–J ) Averaged traces from representative PC recordings in slices from VGluT3-Cre:mDlxChR2 mice illustrating light evoked GABA and glutamate cotransmission under control ( I ) or SCZD ( J ) conditions. ( K ) Traces from a sample VGluT3-Cre:mDlxChR2 recording that exhibited light evoked polysynaptic network barrages. ( L ) Group data summary comparing PSC GABA and PSC Glu conductance ratios for VGluT3-Cre:mDlxChR2 slices maintained in control (n = 4 cells from one mice) or SCZD ACSF (n = 4 cells from one mice). Throughout the figure summary plots provide individual observations as well as group means ± SEM. Figure 5—source data 1. Data plotted in .

Article Snippet: To address the homogeneity of the terminals along the same parent axon, we traced continuous CB1R + axon segments using the Imaris function ‘ Filaments ’ in ‘ manual mode ’.

Techniques: Control, Transmission Assay, Inhibition, Infection