Long-Range GABAergic Inhibition Modulates Spatiotemporal Dynamics of the Output Neurons in the Olfactory Bulb

Abstract

Local interneurons of the olfactory bulb (OB) are densely innervated by long-range GABAergic neurons from the basal forebrain (BF), suggesting that this top-down inhibition regulates early processing in the olfactory system. However, how GABAergic inputs modulate the OB output neurons, the mitral/tufted cells, is unknown. Here, in male and female mice acute brain slices, we show that optogenetic activation of BF GABAergic inputs produced distinct local circuit effects that can influence the activity of mitral/tufted cells in the spatiotemporal domains. Activation of the GABAergic axons produced a fast disinhibition of mitral/tufted cells consistent with a rapid and synchronous release of GABA onto local interneurons in the glomerular and inframitral circuits of the OB, which also reduced the spike precision of mitral/tufted cells in response to simulated stimuli. In addition, BF GABAergic inhibition modulated local oscillations in a layer-specific manner. The intensity of locally evoked θ oscillations was decreased on activation of top-down inhibition in the glomerular circuit, while evoked γ oscillations were reduced by inhibition of granule cells. Furthermore, BF GABAergic input reduced dendrodendritic inhibition in mitral/tufted cells. Together, these results suggest that long-range GABAergic neurons from the BF are well suited to influence temporal and spatial aspects of processing by OB circuits.SIGNIFICANCE STATEMENT Disruption of GABAergic inhibition from the basal forebrain (BF) to the olfactory bulb (OB) impairs the discrimination of similar odors, yet how this centrifugal inhibition influences neuronal circuits in the OB remains unclear. Here, we show that the BF GABAergic neurons exclusively target local inhibitory neurons in the OB, having a functional disinhibitory effect on the output neurons, the mitral cells. Phasic inhibition by BF GABAergic neurons reduces spike precision of mitral cells and lowers the intensity of oscillatory activity in the OB, while directly modulating the extent of dendrodendritic inhibition. These circuit-level effects of this centrifugal inhibition can influence the temporal and spatial dynamics of odor coding in the OB.

Keywords: GABA release; dendrodendritic inhibition; magnocellular preoptic area; olfactory processing; oscillations; spike precision.

Figure 1.

GABAergic projection neurons to the OB are clustered in the MCPO region of the BF and are different from cholinergic neurons. A, Left, Diagram of the anterograde approach to label MCPO GABAergic neurons. An AAV5-Flex-tdTomato virus was injected in the MCPO of Gad2-Cre mice. Right, Confocal image of a section of the main OB (MOB; n = 6), showing the distribution pattern of Gad2-tdTomato axons (shown in white, to enhance the contrast of the staining) across the different cell layers, revealed by the nuclear dye DAPI (blue). Right, The mean normalized pixel intensity across layers. The densest distribution of Gad2 axons is found in the GCL and the GL of the MOB. MCL, MC layer. B, MCPO GABAergic axons innervating the GL (top) and GCL (bottom) exhibit numerous boutons (yellow arrowheads). C, Left, Diagram of the approach to retrogradely label the MCPO GABAergic neurons. An AAVrg-DIO-eGFP virus was injected unilaterally in the OB of Gad2-Cre mice. Right, Confocal micrograph showing that transduced Gad2-eGFP-positive neurons (green) are clustered in the MCPO. CPu, Caudate putamen; PC, piriform cortex; Tu, olfactory tubercle; SI, substantia innominata. D, High-magnification confocal micrographs of the MCPO containing GFP-transduced Gad2 neurons (green), immunostained with antibody against the cholinergic marker ChAT (magenta). Several neurons are positive for ChAT in the MCPO region; however, this representative image illustrates the lack of colocalization of the cholinergic marker and the GABAergic neurons retrolabeled from the OB (white arrowheads). Similar results were found in the accessory OB and are shown in Extended Data Figure 1-1.

Figure 2.

Inhibitory neurons are postsynaptic partners of MCPO LRGNs in the OB. A, Top drawings, Example of distinct reconstructed neurons after recording: 1, GC; 2, MC; 3, TC; 4, PGC; 5, EPL-I. The morphology of the neurons was reconstructed from confocal images of fixed cells that were filled with AlexaFluor-594 during the recordings. Bottom, Example of eIPSCs recorded at −70 mV in symmetrical chloride conditions, on stimulation of GABAergic axons expressing ChR2 with blue light (5 ms). LED stimulation elicited large inward currents in GC, PGC, and EPL-I, but not in output neurons, the MC or TC. B, Bar graph represents the total charge transferred during the GABAergic eIPSCs in distinct cell types in the OB (GC, n = 16; MC, n = 11; PGC, n = 5; TC, n = 10; EPL-I, n = 3). Responses are observed in the main inhibitory types, but not in the output neurons.

Figure 3.

Synaptic properties of the BF long-range GABAergic inputs onto GCs. A, Left, Diagram of the experimental configuration. GCs were recorded in voltage clamp, while GABAergic axons expressing ChR2 were stimulated with a brief pulse of light (0.5-1 ms). Right, Overlay of selected min-eIPSCs (gray traces) in GCs (n = 5 cells, 3 mice). Only min-eIPSCs with rise times of <4 ms are included (n = 120 events, 5 cells). Left, Short gray lines indicate the amplitude of single events. The min-eIPSC had an average amplitude of 66 ± 3 pA (thick black line). Right, The amplitude histogram of the min-eIPSCs. Amplitudes show a bimodal distribution with a small peak centered at 48 pA and a higher peak at 100 pA. Black lines indicate the fitting of two Gaussian distributions to the amplitude distribution. B, Probability distribution histograms for the rise time (left, 10%-90% of the peak) and decay time (right, τ_w) of the min-eIPSC events shown in A. An equivalent number of events were taken from each cell (median 26). Bottom, Ticks represent the values for each event. Thick black lines indicate average rise time (1.6 ± 0.1 ms) and decay time (50.6 ± 1.8 ms). C, eIPSCs recorded in GCs, using a CsCl-based internal solution, in response to LED stimulation (5 ms, 10 Hz). At this frequency of stimulation, the peak amplitude decreases with time, but each eIPSC appears synchronous throughout the train (black traces). The eIPSCs are unaffected by the perfusion of a mixture of the cholinergic blockers mecamylamine (MM, 10 μm) and atropine (Atrp, 3 μm) (purple trace; n = 5, p = 0.25), but completely blocked by the GABA_AR blocker gabazine (Gbz, 10 μm) (gray trace; n = 3, p = 0.04). D, Left, Light-evoked IPSCs in GCs are desynchronized by the equimolar replacement of calcium by strontium (Sr²⁺, 2 mm). Right, Overlay of peak normalized IPSCs for control (black) and strontium (pink) showing similar kinetics. C, D, The holding potential is −70 mV. E, Histogram overlay of the eIPSC amplitudes during control (gray) and strontium (pink) application (n = 3 cells).

Figure 4.

Activation of BF GABAergic inputs disinhibits MCs and reduces DDI. A, Left, Diagram of the experimental configuration. MCs were recorded either in current or voltage clamp while the sensory axons in the ON were activated by electrical stimulation. BF GABAergic axons expressing ChR2 were activated by blue light in the GL. Right, Responses in a representative MC recorded while stimulating the ON with a glass electrode (100 µA, 100 µs, 4 Hz, top black ticks) in the presence (left) or absence (right) of LED stimulation (at 10 Hz, blue ticks). The stimulus intensity was adjusted to elicit firing in the MC. Bottom, Spike raster plots for 10 trials in the cell shown above. The membrane potential was −57 mV (with zero current injection). B, Summary bar graphs for spike frequency showing a significant increase in the firing rate during the LED stimulation compared with control (n = 4, p = 0.01). C, Synaptic currents evoked in an MC by electrical stimulation of the ON (100 µA, 100 µs, arrow). Recordings were performed in symmetrical chloride, in which excitatory and inhibitory currents are seen as inward deflections. A single ON stimulation produced a long-lasting inward current, which was reduced in the presence of LED stimulation in the GL (blue ticks). The holding potential is −60 mV. D, The large barrage of evoked synaptic activity by the ON stimulation is greatly suppressed by LED stimulation (n = 10, p < 0.001), and completely abolished by blockers of GABA_A and glutamate receptors (gabazine [Gbz], 10 μm; kynurenic acid [KA], 1 mm, respectively; n = 6, p = 0.01). E, Left, Diagram of the experimental arrangement. MCs were recorded either in current or voltage clamp while LED stimulation was directed to the GCL. Right, Voltage traces of a representative MC held at peri-threshold membrane potential in control and in the presence of LED stimulation (4 Hz). Spike raster plots for 20 trials are shown in the traces below. F, Summary bar graphs for spike frequency showing a significant increase in the firing rate during the LED stimulation compared with control (n = 6, p = 0.004). Light directed to the GL did not significantly change the firing rate of MCs. Results are shown in Extended Data Figure 4-2A, B. G, Overlay of average current traces showing DDI on a MC evoked by a short depolarization (0 mV, 50 ms) in control and in the presence of LED stimulation (10 Hz). The holding potential is −60 mV. H, Summary bar plot showing a significant difference in the synaptic charge transferred in control versus during LED stimulation (n = 8, p = 0.003). Consistently, Gbz (10 μm) completely blocked the evoked dendrodendritic current in MCs (n = 10, p = 0.0002). The reduction of the ON-evoked response and depolarization induced inhibition in MCs by light stimulation of BF-LRGNs axons persisted in the presence of dopamine receptor antagonists. These results are shown in Extended Data Figure 4-1. In addition, light stimulation of the GL significantly reduced DDI in MCs, shown in Extended Data Figure 4-2C, D. *p < 0.05. **p < 0.01.

Figure 5.

Layer-specific modulation of LFP oscillations by activation of BF GABAergic inputs. A, Left, Image of a recorded section of the OB showing expression of ChR2-tdTomato achieved by an injection of AAV5-Flex-ChR2-tdTomato virus in the MCPO. Right, Diagram of the experimental configuration. A low-resistance patch electrode was placed in the EPL to record the LFP in OB slices containing BF GABAergic axons expressing ChR2. Oscillatory activity was elicited by stimulating the ON with a brief high-frequency stimulus (100 µA, 100 Hz for 50 ms). In alternate trials, we stimulated the BF GABAergic axons with a blue LED (5 ms, 10 Hz for 2 s) directed to the GCL or the GL using a 40× objective focused ∼400 μm apart. B, Raw traces of LFP recordings in the EPL during electrical stimulation of the ON (black ticks) in control (top), with LED stimulation over the GCL (blue ticks, middle) or the GL (bottom), and in the presence of the synaptic blockers kynurenic acid (KA, 1 mm) and gabazine, 10 μm). C, Band pass filtered LFP traces for the different conditions; low frequency, 2-12 Hz (θ, gray), and high frequency, 25-85 Hz (γ, black). D, Mean normalized 300 Hz low pass power spectra for a 1 s window of LFP recording during GL (left) and GCL (right) LED stimulation. Power was normalized with respect to the pre-ON stimulation period. The power spectra show significant activity <20 Hz, as well as a shoulder at higher frequency. E, Pair comparison of the normalized power of the θ (top plots) and γ frequency bands (bottom plots) in the absence (control) and presence of light stimulation (LED). Light stimulation in the GL significantly reduced the power of the θ band (n = 6, p = 0.001), but not the γ band (n = 6, p = 0.31), whereas LED stimulation in the GCL significantly reduced the power of the γ band (n = 5, p = 0.05), but not the θ band (n = 5, p = 0.11). Representative raw power spectrograms before and after ON stimulation, as well as normalized power spectrograms in the presence of synaptic blockers are shown in Extended Data Figure 5-1. *p < 0.05.

Figure 6.

Activation of BF GABAergic inputs desynchronizes MCs. A, Left top, Diagram of the experimental configuration. MCs were recorded in current clamp while axons of BF GABAergic neurons expressing ChR2 were locally activated in the GCL by blue light (5 ms, 10 Hz). MCs were stimulated with fluctuating currents that simulate sensory input of increasing synchrony on a 4 Hz sine wave (top). The simulated current injection had low (first 4 current bursts) and high synchrony (last 4 current bursts). Overlaid voltage traces from an MC held at −60 mV in response to the current stimuli, during control (top traces) and blue light stimulation (LED 5 ms, 10 Hz, bottom traces). Raster plots underneath show single-cell responses in 40 trials. B, The overall firing rate of MCs was significantly increased in the presence of blue light stimulation (n = 7, p = 0.02). C, The spike jitter was significantly increased during low synchrony and high synchrony simulated sensory inputs in the presence of blue light stimulation (low synchrony: p = 0.02; high synchrony: p < 0.001). D, Spike-triggered average during low synchrony (left) and high synchrony (right) in the presence (red) or absence (blue) of blue light stimulation. The peak current needed to elicit spikes was smaller in the presence of blue light stimulation (low synchrony peak: p < 0.001; high synchrony peak: p < 0.001). *p < 0.05. **p < 0.01.