Input dependent modulation of olfactory bulb activity by HDB GABAergic projections

Abstract

Basal forebrain modulation of central circuits is associated with active sensation, attention, and learning. While cholinergic modulations have been studied extensively the effect of non-cholinergic basal forebrain subpopulations on sensory processing remains largely unclear. Here, we directly compare optogenetic manipulation effects of two major basal forebrain subpopulations on principal neuron activity in an early sensory processing area, i.e. mitral/tufted cells (MTCs) in the olfactory bulb. In contrast to cholinergic projections, which consistently increased MTC firing, activation of GABAergic fibers from basal forebrain to the olfactory bulb leads to differential modulation effects: while spontaneous MTC activity is mainly inhibited, odor-evoked firing is predominantly enhanced. Moreover, sniff-triggered averages revealed an enhancement of maximal sniff evoked firing amplitude and an inhibition of firing rates outside the maximal sniff phase. These findings demonstrate that GABAergic neuromodulation affects MTC firing in a bimodal, sensory-input dependent way, suggesting that GABAergic basal forebrain modulation could be an important factor in attention mediated filtering of sensory information to the brain.

Figure 1

Selective targeting of cholinergic and GABAergic inputs from basal forebrain to the OB. (A) Left, Coronal section (Bregma 0.74) through BF in a ChAT-Cre mouse injected with a Cre-dependent AAV-ChR2 virus. White lines indicate the outline of HDB and VDB. Right, Magnification of HDB showing labeled ChAT+ neurons. (B) Left, Coronal section (Bregma 0.74) through BF in a GAD2-Cre mouse injected with a Cre-dependent AAV-ChR2 virus. White lines indicate the outline of HDB and VDB. Right, Magnification of HDB/VDB showing somata of GAD + neurons. (C) Immunohistochemical staining of coronal sections of GAD2-Cre:GCaMP6 reporter mice (Bregma 0.74). Double labeled neurons are marked with white arrows. (D) ChR2-EYFP-expressing axon terminals in different layers of the OB 4 weeks after AAV-ChR2-EYFP injection into BF of a ChAT-Cre and GAD-Cre mice. GL: glomerular layer, EPL: external plexiform layer, MCL: mitral cell layer, GCL: granule cell layer. (E) Normalized fluorescence intensity profiles show that axons reaching the external plexiform layer are less prominent in GAD-Cre mice. (F) Schematic of experimental approach. See Materials and Methods for details. (G) Data acquisition. In continuous recordings, action potential waveforms with a signal-to-noise ratio of at least 4 SD above baseline noise were saved to a disk (sample rate 24 kHz) and further isolated using off-line spike sorting. The vertical line indicates onset of odorant stimulation. Isolation of waveforms into three different units by principal components 1 and 2 (bottom, left). Spike waveforms of the isolated units (bottom, right). (H) Inter-spike-interval histograms for two units. One is a single unit (SU) and one a multi unit (MU). Only single units were analyzed further. For units to be classified as presumptive MTCs also a clear sniff-modulation in sniff-triggered spike averages had to be present (inset).

Figure 2

Optogenetic activation of cholinergic and GABAergic basal forebrain inputs to the OB modulates spontaneous as well as sensory-evoked MTC spiking. (A) Spike raster and rate histograms (bin width, 50 ms) from presumptive MTCs showing spontaneous spiking in the absence of inhalation (no sniff). Spike rate decreased during optical stimulation of the dorsal OB (“stim”, blue shaded area) in GAD-Cre mice and increased in ChAT-Cre animals. (B) Left, Plot of spontaneous firing rate in the 9 s before (no stim) and during (stim) optical stimulation for all tested units (ChAT-Cre, n = 27 units, purple; GAD-Cre, n = 44 units, orange). Squares indicated significantly modulated units. Right, Time course of changes in firing rate (mean ± SEM across all units) during optical stimulation (blue bar). The trace indicates changes in mean spike rate in 1 s bins relative to the mean rate before stimulation. The time axis is relative to the time of stimulation onset. (C) Spike raster and rate histogram of MTC spiking during inhalation of clean air and optical stimulation (blue shaded area). Inhalation-evoked spike rates decrease (top) or increase (bottom) during optical stimulation in GAD-Cre mice while only excitation in response to optical stimulation is observed in ChAT-Cre mice. The top trace (sniff) shows artificial inhalation as measured by a pressure sensor connected to the nasopharyngeal cannula. (D) Left, Plot of inhalation-evoked firing rates during clean air inhalation, averaged for the nine inhalations just before (no stim) and after (stim) optical stimulation (ChAT-Cre, n = 62 units, purple; GAD-Cre, n = 25 units, orange). Data were analyzed and plotted as in B. Squares indicate significantly modulated units. Right: Time course of changes in firing rate (mean ± SEM across all units) during optical stimulation (blue bar).

Figure 3

Optogenetic activation of cholinergic and GABAergic OB inputs modulates odor-evoked responses. (A) Odorant-evoked MTC spiking is enhanced by optical OB stimulation in ChAT-Cre mice in both odorant-activated (Unit1) and odorant-inhibited (Unit2) cells. (B) Odorant-evoked MTC spiking is increased (left) or decreased (right) by optical OB stimulation in GAD-Cre mice. (C) Plot of odorant-evoked changes in MTC spiking (∆ spikes/sniff) in the absence of (no stim) and during (stim) optogenetic stimulation of cholinergic (n = 56 units, purple) or GABAerig (n = 29, orange) afferents to the OB. (D) Time course of effects of optical stimulation on odorant-evoked spike rate, averaged across all units. The blue bar shows time of optical stimulation and simultaneous odorant presentation. Plotted is the mean change in odorant-evoked spike rate between trials with and without light stimulation, measured after each inhalation (at 1 Hz); the shaded area indicates variance (SEM) around mean. (E) Optical stimulation induced firing changes during the sniff cycle. Cholinergic modulation increased the firing rate across the sniff cycle. GABAergic modulation increased firing in peak and adjacent time bins while inhibiting firing outside the “preferred sniff phase”. Firing changes were calculated form sniff-triggered spike histograms as depicted in the inset. (F) Cumulative probability plots comparing changes in MTC firing rates caused by optical stimulation in the no sniff and odor condition. Plots reflect datasets plotted in Figs. 2B and 3C, respectively. Unlike the uniform suppression of spontaneous spiking observed in GAD-Cre mice, optical stimulation in the odor condition predominantly causes MTC excitation.

Figure 4

Switch in modulatory effect between conditions in GAD-Cre mice. (A) Spike raster and rate histogram (bin width, 50 ms) depicting a spike rate decrease for a MTC during optical stimulation of the dorsal OB (“stim”, blue shaded area). (B) The same unit plotted in A was also tested in the odor condition. Odorant-evoked spiking is enhanced by optical OB stimulation in this unit. (C) Plot of spontaneous firing rate in the 9 s before (no stim) and during (stim) optical stimulation for all units tested in both the no-sniff and the odor condition (n = 53 units). Squares indicated significantly modulated units. (D) Plot of odorant-evoked spiking changes (∆ spikes/sniff) in the absence of (no stim) and during (stim) optogenetic stimulation of the same units tested in C. (E) Quantitative comparison of stimulation-evoked spiking changes (∆ spikes/sec) in the no-sniff and odor condition. Circles, spiking changes for individual units; black bars, mean value. Lines connect the same unit across conditions. (F) Optical stimulation effects in the odor condition were positively correlated to baseline activity (Pearson’s r = 0.61; two-tailed, p = 1.47 ×10^-6). A negative correlation was observed in the no-sniff condition (Pearson’s r = −0.27; two-tailed, p = 0.05). (G) Effect of optical GABAergic OB fiber stimulation on odorant responses for MTCs tested with seven odorants. Blue, baseline response; red, response during optical stimulation. Odorants are ordered separately for each unit, with the strongest excitatory response in the baseline condition in the middle of the abscissa. Lines (±SEM) connect median responses across all tested trials. (H) Box plot comparing optically induced change in odorant-evoked spike rate (stim - no stim) for the strongest and weakest quartile of odorant-evoked responses taken from all cell–odorant pairs for excitatory as well as inhibitory responses. The box indicates median, 25–75th percentile ranges of the data, and whiskers indicate ±1 SD from the mean. (I) Representative PCA representation of OB response to odors with (red) and without (blue) OB HDB fiber activation. Symbols represent responses on individual trials to each odorant; ellipsoids are mean ± 1 SD. (J) Relative population response distances in neural activity space projected onto the first three principal components. Distances were normalized to the average ∆ odor distance. OB GABAergic fiber activation increases both variability in repeat and ∆ odor responses.