The Interglomerular Circuit Potently Inhibits Olfactory Bulb Output Neurons by Both Direct and Indirect Pathways

Abstract

Sensory processing shapes our perception. In mammals, odor information is encoded by combinatorial activity patterns of olfactory bulb (OB) glomeruli. Glomeruli are richly interconnected by short axon cells (SACs), which form the interglomerular circuit (IGC). It is unclear how the IGC impacts OB output to downstream neural circuits. We combined in vitro and in vivo electrophysiology with optogenetics in mice and found the following: (1) the IGC potently and monosynaptically inhibits the OB output neurons mitral/tufted cells (MTCs) by GABA release from SACs: (2) gap junction-mediated electrical coupling is strong for the SAC→MTC synapse, but negligible for the SAC→ETC synapse; (3) brief IGC-mediated inhibition is temporally prolonged by the intrinsic properties of MTCs; and (4) sniff frequency IGC activation in vivo generates persistent MTC inhibition. These findings suggest that the temporal sequence of glomerular activation by sensory input determines which stimulus features are transmitted to downstream olfactory networks and those filtered by lateral inhibition.

Significance statement: Odor identity is encoded by combinatorial patterns of activated glomeruli, the initial signal transformation site of the olfactory system. Lateral circuit processing among activated glomeruli modulates olfactory signal transformation before transmission to higher brain centers. Using a combination of in vitro and in vivo optogenetics, this work demonstrates that interglomerular circuitry produces potent inhibition of olfactory bulb output neurons via direct chemical and electrical synapses as well as by indirect pathways. The direct inhibitory synaptic input engages mitral cell intrinsic membrane properties to generate inhibition that outlasts the initial synaptic action.

Keywords: GABA; electrical coupling; interglomerular circuit; intrinsic properties; monosynaptic inhibition; olfactory bulb.

Figure 1.

Direct and indirect transmission from SACs to MTCs. A–D, Confocal image of a horizontal OB section showing the expression of ChR2-EYFP selectively in the GL (A) and colocalization of TH protein and ChR2-EYFP (B–D) in SACs. E, F, Traces from an MTC voltage-clamped at −60 mV showing outward currents in response to brief optical stimulation (blue line and arrowhead) of short axon cells in ACSF (E) or in the presence of NBQX and APV (F). Black and red traces represent the average in each condition. B, Inset, Experimental setup. Enlargement of traces in C at the bottom shows the short, consistent latencies of outward currents. G, Quantified data from 8 cells showing the under curve area charge in conditions shown in A (yellow) and B (green) as shown by the inset. H, Scatter plot showing latencies of SAC-evoked outward currents in 8 MTCs in the presence of NBQX and APV. Blue horizontal bars represent the average value of each cell.

Figure 2.

SAC activation produces long-lasting inhibition of MTCs. A, B, Cell-attached recordings showing that brief optical stimulation of SACs produces a long inhibition of MTC firing in the absence (A) or presence of NBQX and APV (B). Raster data showing spike firing activities of 10 recording traces in each conditions. C, Quantified data from 5 MTCs in cell-attached and 11 MTCs in current-clamp recording conditions showing the potent inhibition of MTCs by optical stimulation of SACs with or without fast glutamatergic synaptic blockers.

Figure 3.

MTC intrinsic properties amplify inhibitory input impact A, B, Top, Ten superimposed current-clamp traces showing responses of a MTC to the SAC-MTC IPSC averaged from 80 traces in 8 cells (A), or a hyperpolarizing current pulse 100 pA × 50 ms (B) in ACSF (left) or in the presence of synaptic blockers (right). Bottom, Averaged PSTH from 8 cells in corresponding conditions. C, Quantified data from 8 MTCs showing the inhibition duration produced by each treatment. NAG = NBQX, APV, and gabazine.

Figure 4.

Block of ionotropic synaptic receptors switches SAC-MTC transmission from inhibition to excitation. A1–D1, Ten superimposed current-clamp traces showing MTC responses to brief optical stimulation (blue vertical line) of SACs in ACSF (A1), in the presence of NBQX and APV (B1), addition of GBZ (C1), or DA receptor antagonists SKF83566 and spiperone (D1). E1, Traces averaged from 10 individual responses in the same cell with hyperpolarizing current injection to prevent spiking reveals a biphasic response: depolarization followed by hyperpolarization in the presence of NBQX, APV, and GBZ (NAG, red) or addition of DA receptor antagonists (dark blue). A2–E2, Enlargement of traces on the left side to highlight the following: (1) blocking glutamatergic transmission with NBQX and APV (B2) shortens the SAC inhibition of MTCs compared with A2; (2) addition of GBZ revealed a brief excitation immediately following optical stimulation in forms of firing (C2) or subthreshold depolarization (E2, red trace) followed by firing inhibition or hyperpolarization; and (3) further addition of DA receptor antagonists showed no effect on the excitation-inhibition responses (D2 compared with C2 and dark blue trace compared with red trace in E2).

Figure 5.

SAC-MTC electrical transmission is ∼10-fold stronger than that of SAC-ETC. A–D, Typical averaged traces recorded from an MTC (A, C) voltage-clamped at −60 mV and an ETC (B, D) voltage-clamped at −60 mV (top) or −90 mV (bottom) in response to brief (A, B) or long (1 s; B, D) optical stimulation of SACs in the presence of NBQX, APV, and GBZ (NAG, black), addition of DA receptor antagonists SKF83566 and spiperone (green), or further addition of CBX (red). E, F, Quantified data showing that brief optical stimulation of SACs evokes much larger inward currents in MTCs (n = 7; E) than ETCs (n = 5; F) when they are held at −60 mV in the presence of NBQX, APV, and GBZ (NAG, black symbols). Response of ETCs held at −90 mV (magenta and black symbols) is stronger than that held at −60 mV (black symbols). F, Inset, Same graph with an expanded abscissa scale. This response in MTCs is insensitive to DA receptor antagonists (E, green symbols) but abolished by gap junction blocker CBX (E, red symbols) where the response in ETCs is abolished by DA receptor antagonists (F, green symbols). G, H, Quantified data showing that 1 s optical stimulation of SACs evokes a larger inward currents in MTCs (n = 7; G, black symbol) but a smaller response mixed with inward or outward currents in ETCs (n = 5; H, black symbols) when they are held at −60 mV in the presence of NBQX, APV, and GBZ (NAG). When ETCs are held at −90 mV, this stimulation evokes larger inward currents (H, black symbols). Responses of ETCs, but not MTCs, are sensitive to DA receptor antagonists (green symbols). Addition of CBX abolishes residual currents in both MTCs (G, red symbols) and ETCs (H, red symbols).

Figure 6.

ON-evoked MTC responses are truncated by IGC input. A–F, Averaged PSTHs of current-clamp data from 12 MTCs showing MTC response to electrical stimulation (E-stim, red arrow) of ON (A), brief optical stimulation (O-stim, blue arrow) of the IGC (B), and E-stim followed by O-stim at different time intervals ranging from 10 to 300 ms (C–F). A, Inset, Schematic of experimental design. ONL, Olfactory nerve layer; EPL, external plexiform layer; Rec: recording electrode. G, Quantified data showing the average ON-evoked spiking activity within the 300 ms time window following E-stim (black square) or within the 300 ms time window following O-stim (green square) in 12 MTCs showing the impacted of subsequent activation of the IGC by O-stim at different time intervals (0–300 ms). Spiking is normalized to the baseline spontaneous level within a 700 ms time window before E-stim or to the ON-evoked spiking within a 300 ms time window following E-stim.

Figure 7.

SAC activation potently inhibits MTC responses to subsequent ON input. A–E, Averaged PSTHs of current-clamp data from 12 MTCs showing MTC responses to electrical stimulation (E-stim, red arrows) of A or brief optical stimulation of SACs (O-stim, blue arrow) followed by E-stim at different time intervals (B–E). A, Same as Figure 6A. F, Quantified data showing the average MTC (n = 12) spiking within a 300 ms analysis time window following E-stim and the impact of prior optical activation of the IGC at different time intervals ranging from 0 to 600 ms. MTC spiking is normalized to the spontaneous baseline level within a 700 ms time window before O-stim (left axis) or to the ON-evoked spiking within a 300 ms time window following ON stimulation (right axis).

Figure 8.

Single or repetitive activation of IGC potently inhibits MTCs in vivo. A, Experimental setup of in vivo single-unit recordings of MTCs from TH-Cre mice injected with Cre-inducible ChR2 virus in the OB. B₁, Brief optical stimulation of SACs produces prolonged inhibition of MTC spiking. Top, Single trace showing inhibition. Bottom, Raster plots of 10 sweeps at 0.1 Hz. B₂, Expanded trace from B₁ showing MTC spontaneous spiking. C, Averaged PSTH of 24 recording units from 8 mice showing that single brief optical stimulation consistently produces prolonged inhibition of MTCs. D, Averaged PSTHs of 14 single units from 5 mice showing that a train of 5 brief (blue arrows) optical stimuli at the sniffing frequencies (2–8 Hz) or 50 Hz (ETC intraburst spiking frequency) produce potent and additive inhibition of MTC firing activity. Bin size: 10 ms.

Figure 9.

Schematic of mechanisms underlying the IGC impact on OB output. A, Simplified illustration of the interglomerular circuit formed by SACs. B, Circuit connection diagram shows the direct and indirect synaptic pathways mediating interglomerular inhibition of MTCs, the principal OB output neurons. gap junct, Gap junction; glut, glutamate.