Input dependent modulation of olfactory bulb activity by HDB GABAergic projections

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.

To assess the impact of cholinergic and GABAergic fiber stimulation on MTC excitability in the absence of sensory input, we optically activated BF axons without ongoing inhalation ( Fig. 2A). In this condition, MTC display an irregular firing pattern 28,57,58 . As shown previously 28 , optogenetic activation of cholinergic fibers in ChAT-Cre mice leads to a significant increase of spontaneous MTC spiking from 2.05 ± 2.34 Hz (mean ± SD) before stimulation to 2.40 ± 2.24 Hz during stimulation (n = 27 units from 5 mice; p = 0.0157 Wilcoxon signed rank test). 8 of these units (30%) showed a significant stimulation-evoked increase in firing activity when tested on a unit-by-unit basis (Mann-Whitney U test); none showed a decrease (Fig. 2B, left).
In contrast, optical stimulation in GAD2-Cre mice led to a significant decrease in MTC spontaneous spiking, from 5.43 ± 4.07 Hz (mean ± SD) before stimulation to 3.45 ± 3.54 Hz during stimulation (n = 44 units from 5 mice; p = 1.07 ×10 −8 , Wilcoxon signed rank test, Fig. 2A,B). When tested on a unit-by-unit basis 20 of the 44 recorded units showed a significant reduction in firing activity while none showed a significant increase (Fig. 2B, left). The median reduction in spike rate across these cells was 1.78 ± 1.36 Hz. Across the population of all recorded units, the decrease in spontaneous firing rate persisted for the duration of the 10 s optical stimulation (Fig. 2B, right). Following the stimulation, an increase in spiking was observed that returned to prestimulation levels within 20 s after stimulation ceased. Since population time course onsets appeared different for GABAergic and cholinergic modulation of spontaneous MTC firing we calculated onset times and found that cholinergic fiber stimulation derived effects were indeed significantly slower compared to GABAergic fiber derived effects (GAD-Cre 0.29 ± 0.27 s vs. ChAT-Cre 1.33 ± 1.43 s, p = 9.19 ×10 −5 , Mann-Whitney U test).
Thus, optogenetic activation of GABAergic BF fibers at the level of the OB leads to a fast reduction of MTC spontaneous activity while activating cholinergic fibers causes output neuron excitation in a slower manner, demonstrating that these subpopulations exert opposing effects with distinct time courses on spontaneous MTC firing.
To rule out optical activation artifacts, we stimulated the OB of uninjected control mice with the same parameters as before during the no sniff condition since in this condition even small changes could have been detected (Supp. Fig. 2). We found that optical stimulation led to no significant change in spontaneous firing rate (n = 19 units from three mice; 6.10 ± 4.15 Hz before stimulation, 6.20 ± 4.25 Hz during stimulation (mean ± SD); p = 0.365, Wilcoxon signed rank test). Thus, the light-evoked modulation of spontaneous MTC firing in ChR2-injected ChAT-Cre and GAD-Cre mice was attributable to cholinergic/GABAergic signaling mediated by the optogenetic activation of cholinergic/GABAergic axons in the OB.
www.nature.com/scientificreports www.nature.com/scientificreports/ 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). www.nature.com/scientificreports www.nature.com/scientificreports/ Optogenetic activation of cholinergic and GABAergic axons in the OB modulates inhalationevoked Mtc spiking. Next, we investigated the effect of cholinergic and GABAergic axon activation on MTC responses during artificial inhalation of clean air (Fig. 2C,D). Inhalation-linked spiking patterns could be observed in 62 units (4 mice) in ChAT-Cre and 25 units (6 mice) in GAD-Cre mice, most likely reflecting weak sensory-evoked responses 28,59,60 .
Optical stimulation of cholinergic axons in ChAT-Cre mice significantly increased inhalation-linked spiking of MTCs (Fig. 2C), with the median spike rate increasing from 2.86 ± 1.72 Hz to 4.03 ± 2.45 Hz during optical stimulation (p = 2.99 ×10 −11 , Wilcoxon signed rank test). When tested on a unit-by-unit basis, 37 of 62 recorded units (60%) showed a significant optical stimulation evoked increase in spiking (Fig. 2D); none showed a decrease. Inhalation evoked spiking of these 37 cells increased by 1.33 ± 1.29 spikes/sniff/s. In GAD2-Cre mice, optical stimulation led to mixed effects on inhalation-linked spiking that were not significant across the population of MTCs (1.8 ± 1.66 Hz before stimulation, 2.26 ± 2.13 Hz during stimulation; n = 25 units from 6 mice; p = 0.58 Wilcoxon signed rank test, Fig. 2C,D). When tested on a unit-by-unit basis three of the 25 (12%) units showed a significant decrease and two (8%) a significant increase in firing activity. Inhalation evoked spiking decreased by 0.67 ± 0.14 spikes/ sniff/s and increased by 2.35 ± 0.71 spikes/ sniff/s for www.nature.com/scientificreports www.nature.com/scientificreports/ the significantly inhibited and excited units, respectively. The averaged time course depicted an initial decrease in MTC firing rate that, in contrast to the time course in the no sniff condition, returned to prestimulation levels already during the stimulation period (Fig. 2D). Following stimulation, spike rate increased above baseline levels for approx. 20 s before returning to baseline. Taken together, while optogenetic stimulation of cholinergic fibers in both conditions was qualitatively similar, activating GABAergic fibers leads to mixed effects of MTC spiking in the sniff condition that was not observed during spontaneous spiking.

Optogenetic activation of cholinergic and GABAergic axons in the OB enhances odorant-evoked
Mtc spiking. Since optical activation of BF inputs to the bulb modulates inhalation-linked MTC spiking consistent with modulating weak sensory-evoked responses, we next evaluated the impact of bulbar cholinergic and GABAergic modulation on odorant responses. We compared MTC responses to odorant stimulation applied with and without optogenetic activation of cholinergic or GABAergic BF axons (Fig. 3A,B). In line with the findings from the previously tested conditions light activation of cholinergic fibers in ChAT-Cre mice increased MTC spiking (Fig. 3A,C). Across the population of recorded presumptive MTCs (n = 56 units, 5 mice), BF axon stimulation significantly increased MTC odor activity, with an increase from 6.49 ± 3.47 spikes/sniff/s (median ± SD) during odorant presentation alone to 8.98 ± 4.22 spikes/sniff/s during odorant paired with light (p = 3.93 ×10 −10 , Wilcoxon signed rank test). 25 out of the 56 recorded cells (45%) showed a significant increase in odor-evoked spiking when tested on a unit-by-unit basis; none showed a decrease (Fig. 3C). 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.
www.nature.com/scientificreports www.nature.com/scientificreports/ Optogenetic activation of GABAergic fibers in the OB during odor presentation elicited heterogeneous effects that, across the population of recorded cells (n = 29 units, 4 mice), were not significant (5.44 ± 3.12 spikes/sniff/s during odorant presentation alone, 4.93 ± 4.30 spikes/sniff/s during odorant paired with light, p = 0.52, Wilcoxon signed rank test). However, tested on a unit-by-unit basis eight out of 29 recorded cells (28%) showed a significant increase and three (10%) a significant decrease in odor-evoked firing activity (Fig. 3C). The modulation in spike rate across the cells showing a significant increase/decrease was 2.17/2.48 spikes/sniff/s (from 7.53 ± 4.1 to 9.7 ± 5.32/from 5.44 ± 0.57 to 2.96 ± 0.39), respectively. The averaged activity time course of the recorded units displayed an initial brief reduction of firing rated that switched to excitation during the stimulation period (Fig. 3D).
We also examined optical stimulation-induced changes in odor-evoked MTC spiking pattern by generating sniff-triggered spike histograms aligned to the start of inhalation for ChAT-cre and GAD-cre mice (Fig. 3E inset, see 28 . Neither cholinergic nor GABAergic basal forebrain derived modulation showed a significant change of the time bin of peak firing across the population of recorded units (p = 0.63 and 1 for ChAT-cre and GAD-cre mice, respectively, paired t test). However, comparing the optical stimulation-induced firing change per bin revealed a profound difference between cholinergic and GABAergic modulation (Fig. 3E): while 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".
The BF receives input from different olfactory areas 45 and it has been shown that even during sleep and anesthesia, cholinergic and GABAergic BF neurons are rhythmically discharging [61][62][63][64] . We therefore tested the effect of inhibiting cholinergic and GABAergic BF projections to the OB using the light-gated chloride pump Halorhodopsin as an optogenetic silencer (Supp. Fig. 3). Despite robust, yet sparser expression of Halo-YFP, labeling could be observed in the OB for both ChAT-Cre and GAD-Cre mice four weeks after viral injection (Supp. Fig. 3A). Optogenetic stimulation during recording of presumptive MTCs (Supp. Fig. 3B) showed, when tested on a unit-by-unit basis, no significant effects in ChAT-Cre animals (8 units, 2 mice) while only two out of 28 recorded cells showed a weak but significant decrease in odor-evoked spiking (0.47 spikes/sniff/s) in GAD-Cre animals (Supp. Fig. 3C). No significant modulation effects were observed in the other tested conditions (no sniff and sniff, data not shown). This was also true for units showing strong sensory-evoked spiking, rendering it unlikely that effects went undetected due to low spike counts. The surprisingly weak effects of optogenetic inhibition might be the result of only weak spontaneous cholinergic and GABAergic HDB activity in the anesthetized animal.
GABAergic modulation of oB output is sensory input dependent. Unlike spontaneous spiking, which got suppressed by optical stimulation in GAD-Cre mice, the same optical stimulation during odor stimulation predominantly caused MTC excitation (Fig. 3F). In a separate set of experiments, we therefore investigated if this suppression to excitation transition can also be observed on a single unit basis, or might be caused by a recording bias e.g. through different populations of output neurons being detectable in the different conditions. We recorded GABAergic modulation effects in individual MTC tested in both the spontaneous as well as the odorant evoked condition in one continuous session (Fig. 4A,B depict recordings from the same unit).
As shown previously (Fig. 2), optical stimulation in the no sniff condition led to a significant reduction in MTC spontaneous spiking across all recorded units (3.61 ± 3.43 Hz before stimulation; 3.14 ± 3.37 Hz during stimulation; n = 53 units from 3 mice; p = 0.014 Wilcoxon signed rank test). When tested on a unit-by-unit basis 5 of the 53 units recorded showed a significant reduction in firing activity and only two units showed an increase (Fig. 4C). Similar to the previous findings (Fig. 3), GABAergic axon activation during odor presentation had a predominantly excitatory effect on MTC activity (7.35 ± 8.12 spikes/sniff/s during odorant presentation alone, 8.13 ± 9.47 spikes/sniff/s during odorant paired with light, p = 9.86 ×10 −10 , Wilcoxon signed rank test). Moreover, all significantly modulated units (22 out of 53 cells (42%)) showed an optical stimulation-induced increase in odor-evoked spiking, none showed a decrease. Quantitative comparison of the optogenetic stimulation effect in both conditions showed a significantly more positive modulation in the odor compared to the spontaneous condition ( Fig. 4E; spontaneous spiking −0.31 ± 1.61 vs. odor-evoked spiking 1.98 ± 2.01 (median ± SD); measured as ∆ spikes/s relative to non-stimulation condition; p = 6.34 ×10 −9 , Wilcoxon signed rank test). Comparing the firing rate of individual units between the conditions revealed that almost all recorded units (48 of 53 units) showed a more positive, optically evoked modulation in spike rate in the odor compared to the no sniff condition.
To investigate whether the observed modulation was dependent on baseline firing rates we plotted the optical stimulation effect as a function of baseline activity. As expected, baseline firing activity was lower in the no-sniff compared to the odor condition (3.61 ± 3.43 (median ± SD) spikes/sec compared to 7.35 ± 8.11 spikes/ sec; p = 2.53 ×10 −6 , Wilcoxon signed rank test). Interestingly, while 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). These results suggest that the stimulation effect most likely depends on the amount of sensory input rather than solely on the absolute firing rate prior to optical stimulation. The results furthermore indicate that the inhibition to excitation transition across conditions can also be observed on a single unit basis and thus that GABAergic fiber derived modulation can act in a bimodal way on single OB output neurons.
Previous studies have suggested a sharpening of MTC odorant responses by cholinergic modulation through preferentially suppressing weak MTC responses and enhancing inhibitory responses 27,33,43 . Since we only observed additive cholinergic modulation effects on MTC activity, suggesting no change of tuning in accordance with a previous report 28 , we asked if the GABAergic basal forebrain subsystem might be capable of sharpening odor responses in the OB. In a separate set of experiments (36 units, from 3 animals), we therefore tested the response www.nature.com/scientificreports www.nature.com/scientificreports/ www.nature.com/scientificreports www.nature.com/scientificreports/ of the same MTC to multiple odorants with and without optical GABAergic OB fiber stimulation. Consistent with our previous findings, we found that odorant responses were mostly enhanced (0.39 ± 3.22 spikes/sniff/s during odorant presentation alone, 2.41 ± 5.01 spikes/sniff/s during odorant paired with light, p = 1.79 ×10 −40 , Wilcoxon signed rank test). Stimulation effects on tuning curves varied between units. An average tuning curve, calculated across the population of all recorded units (Fig. 4G) displayed a uniform, odor strength independent increase in firing rather than a change in tuning. However, when odorant-cell responses were collapsed across units creating a large dataset of 252 cell odor pairs (Fig. 4H), a comparison of the optically induced change in odorant-evoked spike rate (stim-no stim) for the strongest and weakest quartile of odorant-evoked responses revealed weak but significant differences for excitatory (unpaired Wilcoxon rank-sum test, p = 0.03; n = 37 cell-odor pairs) as well as inhibitory (unpaired Wilcoxon rank-sum test, p = 0.01; n = 24 cell-odor pairs) responses. These results might point to strong odorant responses receiving more excitatory modulation from GABAergic basal forebrain derived fibers no matter if their particular odorant response is excitatory or inhibitory. Based on this result we asked whether MTC responses to different odorants become more dissimilar with optogenetic stimulation of GABAergic fibers, potentially enhancing odor classification in the respective cells.
We therefore characterized the OB HDB stimulation effect by constructing trial-by-trial response vectors composed of spike counts for each cell and projected these high-dimensional responses onto their three principal components (Fig. 4I). We quantified the variance for repeated odor presentations of each odor (repeat) and responses to different odors (∆ odor), with and without stimulation (Fig. 4J). OB HDB fiber activation led to an increased Euclidian distance in repeat and ∆ odor responses pointing to more dissimilar odor responses, potentially supporting enhanced odor classification.
Here, we examine the effect of GABAergic long-range projections in the olfactory bulb (OB) and provide for the first time a direct comparison of cholinergic and GABAergic modulation effects on early sensory processing under the same experimental conditions. expression and activation of channelrhodopsin in basal forebrain projections. Differential expression of channelrhodopsin in either GABAergic or cholinergic long-range projections was achieved by injection of AAV-ChR2 into the BF of GAD2 or ChAT-cre mice, respectively. Expression patterns showed a relatively homogenous distribution of cholinergic fibers in the OB in accordance with previous publications. Distribution of GABAergic fibers only differed slightly from a previous publication that used the same transgenic mouse line with a similar expression strategy 47 . Similar to their results we saw dense labeling in the granule cell layer, but equally strongly labeled glomerular layer and a slightly weaker labeled mitral cell layer. This slight difference could be the result of viral injection sites since Nunez-Parra et al. targeted more posterior parts of BF.
A recent report points to a potential coexpression of cholinergic and GABAergic markers at the level of the OB 83 . Different from this study we found that less than 10% of GAD expressing cells were also positive for a cholinergic marker. In combination with the distinct cholinergic and GABAergic expression patterns and photostimulation effects observed here, our findings suggest that these projections are mainly separated and ACh and GABA are most likely not being predominantly released from the same fibers.
Activation of long-range projections was performed by photostimulating axonal fibers at the dorsal surface of the OB. While known to be less effective compared to somatic stimulation 84 , optogenetic fiber stimulation at the level of the OB was necessary to obviate potential indirect BF modulation effects of OB activity: First, optogenetically activating BF might cause a stimulation of brain areas also targeted by BF fibers that in turn project to the OB 25,76,77,85-91 . Second, a direct BF stimulation in GAD-Cre mice would cause activation of a large amount of GAD positive inhibitory BF interneurons 92,93 . Third, cholinergic BF neurons were shown to excite GABAergic projection neurons [94][95][96] , thereby also rendering a direct BF stimulation problematic. Axonal fibers at the dorsal surface of the OB were activated using a continuous stimulation paradigm, which has been shown to be most effective in activating cholinergic axonal fibers 28,97 . BF fiber silencing with inhibitory opsins hardly showed any MTC activity modulation in the different conditions (sniff, no sniff, odor). The surprisingly weak effects of optogenetic inhibition might be the result of only weak spontaneous cholinergic and GABAergic HDB activity in the anesthetized animal.

Effects of basal forebrain projections on OB output cell activity.
Recording from olfactory bulb output neurons, we show that, in contrast to a local and specific activation of cholinergic fibers, that add an excitatory bias to mitral/tufted cell firing, a selective activation of GABAergic BF fibers leads to bimodal, sensory 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.
www.nature.com/scientificreports www.nature.com/scientificreports/ input dependent effect on OB output: whereas optogenetic stimulation mainly inhibited spontaneous MTC firing, odor-evoked MTC cell spiking was predominantly enhanced; an effect that could also be observed on a single neuron level. Though we cannot exclude that the subpopulation of GABAergic cells showing cholinergic markers is partially or completely overlapping with GABAergic bulbopetal neurons in the HDB, this strong functional divergence, renders this possibility, in our opinion, unlikely.
Onset time, at least for modulation of spontaneous firing was slower for cholinergic modulation compared to GABAergic modulation. This is in agreement with a previous publication using that same stimulation technique, showing that cholinergic effects of spontaneous activity appear slower compared to modulations on sensory-evoked activity ( 28 Fig. 3H). The underlying causes of this slow onset remain to be elucidated, but it might be that in the absence of sensory input the effect of "direct" cholinergic MTC modulation is especially prominent. So far there are no reports demonstrating synaptic contacts of cholinergic fibers with MTCs, despite the presence of nAChRs on MTCs 98 . Thus, at least part of cholinergic transmission could happen extrasynaptically, a process that operates on a slower time scale than synaptic transmission 99 .
Additionally, MTCs showed a reduction of firing outside and an increase of firing within the preferred sniff phase. This modulation is strongly reminiscent of a model of a bimodal gain change evoked by attention 9 also referred to as filtering. These filter processes are reported to dampen activity to non-attended or background stimuli while enhancing relevant sensory input. Indeed, in the odor condition, the size of the GABAergic modulation effect was dependent on the odor-evoked firing activity for both excitatory and inhibitory odor responses, pointing to a multiplicative population firing change for relevant olfactory stimuli while decreasing background activity. Additionally, our data seem to show that odor responses within cells become more dissimilar with GABAergic fiber stimulation, raising the possibility of enhancing odor classification of MTCs.
Our direct comparison of cholinergic and GABAergic OB fiber activation suggest that both BF derived fiber systems might have a role in gain modulation of OB output, but in a very distinct way: while cholinergic modulation seems to act in a fashion rather similar to baseline control 46 , GABAergic modulation seems to lead to a filtering of weak signals in the OB. possible circuit mechanisms underlying bulbar GABAergic modulation. The circuit mechanisms underlying basal forebrain derived modulation effects remain to be elucidated since a comprehensive list of targets for BF fibers in the OB is missing especially for GABAergic projections. Cholinergic and GABAergic systems most likely exert multiple effects within the OB with different receptors, cell types, and modes of transmission (volume vs. strict synaptic transmission) involved 98 .
Though cholinergic modulation of OB circuits has been described in numerous publications (see for review 98 ), the circuit mechanisms underlying this modulation are quite complex and not yet described in their entirety. Cholinergic fibers from the HDB project to all layers of the OB 28,55,56 and there is evidence for the presence of both nicotinic (nAChRs 100,101 ,) as well as muscarinic ACh receptors 29,102 within the OB. It is so far unknown how the different players of cholinergic modulation in the OB interact to create an effect on olfactory bulb output. The here described excitatory effect on MTCs is in agreement with a previous publication 28 and might be derived from activation of nAChRs on mitral cells and/or external tufted cells which provide MTC feed-forward excitation [103][104][105] .
Just like in several other brain areas 106 , GABAergic BF axons in the OB are so far only reported to synapse on inhibitory interneurons 23,47 : centrifugal GABAergic afferents were shown to inhibit granule cells 47 deep short axon cells 48,83 as well as different types of periglomerular cells 48 . Inhibition of those inhibitory interneuron subtypes would, in the simplest case, result in a disinhibition of mitral and tufted cells and could explain the here observed MTC excitation. The sensory input dependent increase in excitatory modulation between conditions and the correlation of effects size with odorant evoked responses could be the result of an increase in sensory dependent activity in OB interneurons 107 leading to a further increase in disinhibition (Suppl. Fig. 4). The observed inhibition in MTCs could be elicited by a multisynaptic effect via several types of inhibitory interneurons: neurons like deep short axon cells and PG cells (which receive GABAergic BF input 48 ) were shown to inhibit other inhibitory interneurons like periglomerular and granule cells, respectively (see 108 ). However, the fast, strong, and persistent nature of this inhibition, rather argues for a direct inhibitory input to MTCs (Suppl. Fig. 4) similar to that observed in the basolateral amygdala where BF GABAergic fibers provide indirect disinhibition, as well as direct inhibition of pyramidal neurons 109 . Since recording interneuron activity in the OB in anesthetized animals is challenging due to their diminished activity [110][111][112] , future experiments in awake animals will test the relative contribution of different types of neurons on the observed effects.
It will be interesting to see how the, here described, dual modulation effect of GABAergic basal forebrain fibers translates to olfactory sensory responses in the awake animal. During wakefulness, the proposed direct inhibitory effect on MTCs could be diminished due to the sparsening of MTC responses 110 or contribute itself to sparser MTC responses. At the same time, the excitatory effect via inhibitory interneurons might be vastly increased due to the increased activity in those neurons 111 . In general, while it is hard to estimate the net modulation effect, it is likely that the increase in interneuron and basal forebrain neuron activity in awake animals 64,113 will lead to a stronger effect of BF modulation in this condition.

conclusion
Our recordings provide, for the first time, a detailed comparison of BF cholinergic and GABAergic influence on early sensory processing and highlight the potential of the noncholinergic BF population to modulate perception. By inhibiting weak and facilitating strong inputs, GABAergic BF fibers in the OB likely increase MTCs signal-to-noise ratio, a hallmark of attentional processes that have been previously attributed mainly to cholinergic processes [2][3][4]9,10 . Our findings are in line with recent data indicating that the classical view on the (cholinergic) BF system might be oversimplified: activity of non-cholinergic BF neurons was more strongly correlated www.nature.com/scientificreports www.nature.com/scientificreports/ with arousal and attention [11][12][13][14][15] , whereas cholinergic neuron activity was correlated with body movements, pupil dilations, licking, punishment 75,114 as well as primary reinforcers and outcome expectations 11 . The distinct early sensory modulation effects of cholinergic and GABAergic BF neurons observed in this study might therefore be owed to the different functions of these two basal forebrain systems. Here we targeted cholinergic and GABAergic projections neurons located primarily in the same area (HDB) since previous publications have shown that populations from different basal forebrain centers can have heterogeneous effects 81 . However, we cannot exclude that the effects observed during GABAergic fiber stimulation were, at least partially, derived from GABAergic fibers originating in the MCPO, a nucleus containing a majority of GABAergic bulbopetal neurons 23,25,47,115 . Future studies, examining the influence of GABAergic projections from different basal forebrain centers will hopefully be able to clarify this issue. Addressing the exact interplay between ACh and GABA in olfactory bulb sensory processing in the awake animal will be critical to fully understand the relative contribution of each system.

Materials and Methods
Animal strains and care. We  olfactometry. Odorants were presented as dilutions from saturated vapor in cleaned, humidified air using a custom olfactometer under computer control 28,117,118 . Odorants were typically presented for 10 seconds. All odorants were obtained at 95-99% purity from Sigma-Alrich and stored under nitrogen/argon. The following odorants were used: ethyl butyrate, 2-hexanone, methyl valerate, valeraldehyde, methyl hexanoate, isoamyl acetate, sec-butyl acetate, vinyl butyrate, and ethyl tiglate. Odorants were presented at 1% saturated vapor (s.v.). extracellular recordings and optical stimulation. MTC unit recordings and optical OB stimulation were performed as described previously 28 with several modifications. Briefly, mice were anesthetized with pentobarbital (50 mg/kg) and placed in a stereotaxic device. Mice were double tracheotomized and an artificial inhalation paradigm was used to control air and odorant inhalation independent of respiration [119][120][121] . Extracellular recordings were obtained from OB units using sixteen channel electrodes (NeuroNexus, A1x16-5mm50-413-A16, Atlas Neuro, E16 + R-100-S1-L6 NT) and an RZ5 digital acquisition system (TDT, Tucker Davis Technologies). Recording sites were confined to the dorsal OB. Action potential waveforms with a signal-to-noise ratio of at least 4 SD above baseline noise were saved to a disk and further isolated using off-line spike sorting (Open-Sorter; TDT, Fig. 1F). Sorting was done using the Bayesian or (in fewer cases) K-Means cluster cutting algorithms in OpenSorter. Units were defined as "single units" if they fell within discrete clusters in a space made up of principal components 1 and 2. Units with interspike intervals lower than the absolute refractory period (<2.5 ms) were excluded from further analysis 122 (Fig. 1G). For units to be classified as presumptive MTCs, units additionally had to be located in the vicinity of the mitral cell layer, show spiking activity in the absence of odorants, and a clear sniff-modulation (the maximum spike rate in a 100 ms bin had to be at least two times the minimum spike rate for the PSTH (Fig. 1G insets)); similar as described in 57 . Subsequent analyses were performed using custom scripts in Matlab. Odorant alone ('baseline') and odorant plus optical stimulation trials (at least 3 trials each) were interleaved for all odorants (inter-stimulus interval 40-50 s). Recordings were subject to unit-by-unit statistical analysis as described below.
For optical OB stimulation, light was presented as a single 10 sec pulse either alone or simultaneous with odorant presentation using a 470 or 565 nm LED and controller (LEDD1B, Thorlabs) and a 1 mm optical fiber positioned within 3 mm of the dorsal OB surface as in earlier studies 28 . The light power at the tip of the fiber was maximal 3 and 10 mW for the 565 nm and 470 nm LED, respectively. extracellular Data analysis. Basic processing and analysis of extracellular data followed protocols previously described for multichannel MTC recordings 28 . Responses to optical or odorant stimulation were analyzed differently depending on the experimental paradigm. Stimulation effects on spontaneous spike rate (no artificial inhalation, "no-sniff " condition) were measured by calculating spikes/second (Hz) for the 9 sec before or during www.nature.com/scientificreports www.nature.com/scientificreports/ stimulation. Selection of 'sniff modulated' units was performed as described previously 28 . Response latency was defined as the time difference between the stimulus onset and the response onset. For determining response onset we calculated the response latency of single units from averaged conditions by taking the first bin where the averaged response intersects a horizontal line indicating two standard deviations (SD) above the baseline activity within the evaluation window. The bin number was then converted to and reported in milliseconds (ms). Inhalation-evoked responses during inhalation of clean air ("sniff " condition) were measured by averaging the number of spikes per 1-sec period following each inhalation in the 9 inhalations pre-stimulation or during stimulation and across multiple trials (minimum of 3 trials in each condition for all units). Odorant-evoked responses were measured as changes in the mean number of spikes evoked per 1-sec inhalation cycle (Δ spikes/sniff) during odorant presentation, relative to the same number of inhalations just prior to odorant presentation. For statistical analysis, significance for changes in firing rate for baseline versus optical stimulation was tested on a unit-by-unit basis using the Mann-Whitney U test on units recorded in 5 or more trials per condition. principal component analysis. Principal components were computed from population response vectors using the Matlab statistical toolbox. Responses are odor presentation spike counts per trial for each cell. To compute PC distance, 3-dimensional Euclidean distances were computed for stimulation and no stimulation trials for each cell before averaging. Summary statistics were computed on these average trial-pair distances.
Statistical analysis. Significance was determined using paired Student's t-test, Wilcoxon signed rank test and Mann-Whitney U test, where appropriate. Significance was defined as *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001. All tests are clearly stated in the main text.
Histology and immunohistochemistry. Viral (AAV.FLEX.ChR2.YFP, AAV.FLEX.HR.YFP) expression in BF cells/axonal projections was evaluated with post hoc histology in all experiments to confirm accurate targeting of BF neurons and a lack of expression in OB neurons as described in 28,111 . Briefly, mice were deeply anesthetized with an overdose of sodium pentobarbital and perfused with 4% paraformaldehyde in PBS. Tissue sections were evaluated from native fluorescence without immunohistochemical amplification with a Leica TCS SP2 confocal laser scanning microscope at 10x or 20x magnification. The fluorescence intensity of confocal images was analyzed and plotted in ImageJ. Only mice showing a solid OB fiber expression were used for analysis.
For immunohistochemistry animals were perfused as described above. Brains were cryoprotected in 30% sucrose in PBS followed by embedding in optimal cutting temperature compound (Tissue Freezing Medium; Leica). Coronal slices (10-14 μm) were cut on a cryostat (CM 3050 S, Leica). Slices were first rinsed with PBS and nonspecific binding was blocked with 10% normal goat serum diluted in PBS with 0.2% Triton X-100 for 1 h at room temperature before incubation with primary antibody (rabbit anti-VAChT, 1:300; Synaptic Systems) in PBS with 0.02% Triton-X overnight at 4 °C. As a secondary antibody goat anti-rabbit Alexa Fluor 546 (Invitrogen) was used. The secondary antibody was incubated for 1 h at room temperature before labeling was evaluated as reported above. Colocalization of protein labeling was quantified in ImageJ by manually counting cells from confocal stacks of representative sections at ~Bregma 0.7 in each animal.

Data availability
Data supporting this article have been uploaded as electronic supplementary material.