Figure 4. Glutamate and GABA corelease from CCK⁺VGluT3⁺INTs.

(A) Schematics depicting two strategies to genetically introduce ChR2 or the Chronos variant within CCK⁺VGluT3⁺INTs by either breeding VGluT3-Cre:Ai32 mice (above) or introducing AAV-CAG-Flex-Chronos-GFP selectively into the hippocampus of VGluT3-Cre mice (below) respectively. In both cases, the optogenetic actuator is driven by illumination with blue light while recording CA1PCs as illustrated in the bottom right subpanel. Here and throughout data from both strategies have been pooled and individual recordings from the VGluT3-Cre:Ai32 and VGluT3:Chronos strategies are depicted in black and green respectively. (B) Traces from a representative recording (above) and group data summary (below) for a subset of recordings illustrating the conversion of an outward GABAergic dominated postsynaptic current (PSC_GABA) to an inward glutamatergic mediated synaptic event (PSC_Glu) following introduction of GABA_A/B receptor antagonists as indicated (n = 16 recordings from nine mice). Traces are average events (3–5 individual sweeps) obtained at the times indicated in the group plot below. Note that Cl^- reversal potential is set to ~60 mV and postsynaptic cells are initially held positive to this so that GABAergic events are outward (positive inititial slope) while glutamatergic events are inward (negative initial slope). Bar chart inset summarizes the latencies of GABAergic and pharmacologically isolated glutamatergic events for each recording making up the time course plot. (C) Averaged traces from representative recordings (above) and group data summary (below) comparing GABAergic (PSC_GABA) and glutamatergic (PSC_Glu) synaptic conductances (G, here and throughout corresponding conductances measured for each individual recording are connected in the group data plot, n = 49 recordings from 22 mice). (D) Traces from representative recordings (above) and group data plots illustrating the current-voltage relations of GABAergic and glutamatergic synaptic events as indicated (PSC_GABA, n = 12 cells from four mice; PSC_Glu, n = 10 cells from four mice). (E) Traces from representative recordings (above) and group data (plotted below) highlighting the distinct kinetics of PSC_GABA and PSC_Glu (n = 35 cells from 15 mice with corresponding GABAergic and glutamatergic kinetics, connected points). Also shown are PSC_GABA kinetics observed at −70 mV for inward GABAergic events (n = 12 cells from three mice) and PSC_Glu kinetics obtained in CTZ (n = 13 cells from seven mice). (F) Traces from representative recordings (above) and group data summaries (below) illustrating antagonism of PSC_Glu by DNQX (n = 12 cells from four mice), GYKI 53655 (n = 12 cells from seven mice) or γ-DGG (n = 9 cells from six mice, note reversible block following wash). Group data are expressed normalized to control responses obtained prior to antagonist application. (G–H) Traces from representative recordings (above) and group data summary plots (below) illustrating DSI sensitivity of PSC_GABA (G, n = 21 cells from seven mice), and PSC_Glu (H, n = 12 cells from seven mice). Absolute amplitudes of averaged PSCs before (Ctl) during (DSI) and after DSI (Recover) are plotted. (I) As for H but with BAPTA included in the recording pipette to block endocannabinoid production (n = 8 cells from three mice). (J) Averaged traces from a representative CCK⁺VGluT3⁺BC-PC pair recording (schematic inset) illustrating GABA and glutamate cotransmission. Antagonism of the outward unitary GABAergic event (uPSC_GABA) unmasks a smaller inward unitary glutamatergic event (uPSC_Glu) that is enhanced by CTZ and increased driving force and also sensitive to DNQX. (K) Group data summary of uPSC_GABA and uPSC_Glu conductances for all pairs exhibiting glutamatergic transmission (n = 12 and 17 pairs in 10 mice for uPSC_GABA and uPSC_Glu respectively; 12 recordings with corresponding uPSC_GABA and uPSC_Glu are connected by lines, in the remainder only uPSC_Glu was evaluated as pair was obtained in partial or full GABA_AR antagonism). Also plotted is antagonism of uPSC_Glu by DNQX or GYKI or DGG in a subset of recordings (n = 10 pairs in five mice). Inset image shows sample pair anatomical recovery. (L) Group data summary of the kinetic properties of uPSC_GABA (n = 12 pairs) and uPSC_Glu (n = 11) as well as uPSC_Glu in CTZ (n = 10; in five cases with corresponding control and CTZ conditions as illustrated by the connected points). Throughout the figure summary plots provide individual observations as well as group means ± SEM.

Figure 4—figure supplement 1. Additional data in support of glutamatergic output from VGluT3⁺ INTs.

(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.