Amygdala Corticofugal Input Shapes Mitral Cell Responses in the Accessory Olfactory Bulb

The posteromedial cortical amygdala sends corticofugal afferents to the AOB

The AOB is densely innervated by cortical amygdala output neurons (Raisman, 1972; Gutiérrez-Castellanos et al., 2014; Oh et al., 2014; Allen Brain Mouse Connectivity Atlas, experiment #114249084). However, their precise target localization and identity are unknown. To precisely identify the source of neuronal projections to the AOB, we first locally injected the retrograde tracer Ct-b (Fig. 1A). Precise targeting of the AOB, with very limited spread to the MOB (4 of 10 subjects; Fig. 1B), consistently resulted in dense labeling of layers II and III in the PmCo (Fig. 1C,D).

Layer-specific Ct-b injections revealed that Ct-b injections in the GC alone are sufficient to retrogradely label PmCo neurons (data not shown). However, since the tracer can be taken up by passing axon terminals also directed to MCs, this method is not valid to assess the specificity of PmCo targets. To better determine the layer specificity of amygdala corticobulbar projection (herein referred to as ACPs) afferents to the AOB, we next used a pseudotyped rabies virus as a conditional retrograde tracer (PRV; Wickersham et al., 2007; Fig. 1E). In this experiment, PRV was injected into the AOB of a mouse line in which the expression of the protein rabies-G [RABVgp4 (which is required for viral amplification and retrograde PRV trans-synaptic transport)] and the avian receptor tumor virus receptor A [TVA; (required for the virus to access the host cells)] were under cre-dependent control (RABV mice; Takatoh et al., 2013). RABV mice were crossed either with mice expressing cre recombinase under the control of the MC-specific promoter Pcdh21 (MCs) or the nNOS (neuronal nitric oxide synthase) promoter, expressed by GC layer inhibitory neurons [GCs and main accessory cells (macs); Kosaka and Kosaka, 2007; Larriva-Sahd, 2008]. As neither Pcdh21 nor nNOS are expressed in the PmCo (Fig. 1F), both TVA and G expression were limited to the injection site (Fig. 1G1,H1). This allowed only monosynaptic retrograde tracing (e.g., no PRV expression was found in areas two synapses away from AOB starter neurons, such as the hypothalamus or the hippocampus). Although it is possible that TVA/G can be expressed elsewhere due to cre expression outside the AOB [e.g., Pcdh21 expression in the anterior piriform cortex (Nagai et al., 2005) and nNOS expression in the islands of Calleja, MeA, cerebellum, caudate putamen, cortex, hippocampus, hypothalamus], thus inducing neurons outside the AOB to be possible starters for PRV transport to the PmCo. However, this possibility could be ruled out as neither of these regions project to the AOB nor show PRV expression. Consistent with our Ct-b-tracing experiments, retrograde PRV labeling was found in several AOS regions, including the PmCo (Fig. 1G2,H2) and mainly from infection of nNOS^cre+ neurons (ratio of PRV⁺ cells PmCo/AOB: Pcdh21^cre+, 0.04 ± 0.02; nNOS^cre+, 0.54 ± 0.2; N = 4 brains for each strain, approximately five sections per animal, 1 section every 150 microns). These results confirmed that nNOS-expressing GC layer inhibitory neurons (GCs and macs), as opposed to MC neurons, are the major target of ACPs.

Sublaminar specificity of PmCo–AOB reciprocal connections

Although, PRV-RABV allows for layer-specific retrograde tracing, through this approach is not possible to quantify the relative contribution of different neuronal types (GCs or macs) to retrograde PRV infection. This limitation also prevents the calculation of relative amounts of input neurons reaching these neurons from any brain area. To estimate the number of starter neurons in the AOB and the relative contribution of GCs and macs to the retrograde PRV infection, we used a complementary viral approach (Watabe-Uchida et al., 2012; Menegas et al., 2015) to conditionally express rabies-G and TVA-mCherry in nNOS^cre-expressing neurons in the OB. This allowed for a more precise quantification of starter cells in the AOB, as those infected by PRV are GFP+ and those expressing the molecular component rabies-G and TVA are mCherry⁺ (Fig. 2A). Although double-labeled cells were found in both GCs and macs (Fig. 2A1), the majority were identified as GCs based on morphologic criteria (average percentage of total starters: GC 75,4%, macs 24,5%; N = 4; Fig. 2A3). Quantification of all PRV⁺ cells in the brains of infected animals showed consistent labeling in a restricted range of olfactory and limbic areas (Fig. 2A4). For each brain region, the relative percentage of traced neurons was calculated over the number of PRV cells collectively sampled in all brain areas (PRV-region/PRV-brain × 100; N = 4 brains, approximately four to five sections per animal, 1 tissue section every sixth animal). The amygdala alone gives rise to 9.7% of input neurons to AOB cre-expressing cells (Fig. 2A4). Of these, 83.5% are localized in the PmCo, with the majority arising from layer III (Fig. 2A4). These findings were consistent with our above Ct-b-tracing experiments (Fig. 1C,D). Overall, these results reveal that ACPs represent a major source of top-down feedback mainly targeting GCs in the AOB.

Molecular phenotype and connectivity of ACPs

To define the molecular phenotype of ACPs, we conducted immunohistochemistry on tissue sections from AOB Ct-b-injected brains. We found that almost all PmCo Ct-b⁺ neurons coexpressed the excitatory neuronal markers CaMKIIa and Tbr1 (∼90%; Fig. 3A), with no coexpression of markers of inhibitory neurons such as GAD or Dlx5/6 (Fig. 3B,C). Retrogradely traced ACPs also expressed Ctip2 and Cux1 (∼30% overlap; Fig. 3D), similar to other subpopulations of corticobulbar neurons in the piriform cortex (Diodato et al., 2016). A large majority (82.4%) of Ct-b⁺ ACPs also coexpressed Sim1, a limbic system marker (Semple and Hill, 2018; Fig. 3E).

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Figure 3.

Molecular phenotypes of PmCo–AOB projection neurons. A, PmCo corticobulbar projection neurons (ACPs) labeled with Ct-b after AOB retrograde tracing. Ct-b-labeled PmCo neurons (red) express the excitatory markers CaMKIIa (cyan, 92.2%) and Tbr1 (gray, 93.3%). B, C, Ct-b-labeled neurons in the PmCo do not express YFP in GAD^cre;Ryfp or Dlx5/6^cre;Ryfp mouse lines (cyan), confirming their excitatory phenotype. D, PmCo-Ct-b-labeled neurons express Cux1 (cyan, 37.1%) and Ctip2 (gray, 94.5%), typical cortical neuron markers in layer II–IV and V and VI, respectively. All Cux1⁺ PmCo neurons coexpressed Ctip2. E, Expression of the gene Sim1 reaches ca. 82.4% in PmCo Ct-b-positive cells. For each count, tissue collected (three to four sections) from three Ct-b-injected mice was used. Scale bars: left, 20 μm; right, 200 μm.

Interestingly, corticobulbar projection neurons in the piriform cortex have been shown to extend axon collaterals to other subcortical and cortical targets (Diodato et al., 2016). This implies the existence of top-down inputs from other high-order olfactory areas such as the PmCo. Specific gene expression patterns in piriform corticobulbar projections have been associated with this top-down cortical circuit (Diodato et al., 2016). In particular, Cux1/Ctip2-expressing piriform cortex neurons have been shown to project to both the OB and areas of the prefrontal cortex (PFC; Diodato et al., 2016). Thus, to evaluate the presence of ACP axon collaterals to other brain regions, we injected Ct-b coupled with different fluorophores into both the AOB (Ct-b 555) and other known targets of PmCo efferent projections (Ct-b 488; Gutiérrez-Castellanos et al., 2014; Fig. 4A). We found unbiased 555/488 dual labeling only when the MeA (7.6%), the medial prefrontal cortex (mPFC; 1.7%), and the entorhinal cortex (Ent; 2.8%) were targeted together with the AOB (N = 3; Fig. 4G,I,J). Since no detectable differences were found using either tracer in single-Ct-b injection experiments, we are confident that ACPs mainly target the AOB with very limited collateral axonal projections to the MeA, Ent, and mPFC. This result also reveals similarities between ACPs and the subpopulations of other corticobulbar projection neurons in the piriform cortex (Diodato et al., 2016).

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Figure 4.

Collateral projections of PmCo corticobulbar neurons. A, Dual Ct-b injections were used to identify possible additional target areas of the PmCo neurons retrogradely labeled from the AOB (Ct-b 555). Tracing was considered reliable in case of clear separation of the two injection sites and 555/488 colabeling of the same region (sr) or the same cells (sc). F–I, We considered nonspecific (ns) tracing experiments to be those in which the two tracers showed partial overlap near the two injection sites or in case of Ct-b 488 injections adjacent to the stria terminalis (for reference, see Allen Brain Connectivity Atlas, experiment #114249084) where AOB-directed ACPs course (e.g., G, I): in such cases, Ct-b 488 would be likely taken up by passing fibers and yield false-positive results (compare H to I and G to F to see how Ct-b overlap in the PmCo decreases as the injection site is moved either dorsally or rostrally, respectively). B–H, Injections of green Ct-b 488 (cyan) were targeted to different AOS regions, known main targets of the PmCo: the olfactory tubercle (Tu; B), the paraventricular nucleus (PVN; C, D), the bed nucleus of the stria terminalis (BNST; D), the ventromedial hypothalamic nucleus (VMH; E), the MeA (G; MePD, H), the basolateral amygdaloid nucleus (BLA, F), the endopiriform nuclei (K), and the medial prefrontal cortex were targeted (I, J). The pie charts in the panels showing the injections sites (left) indicate the coexpression of Ct-b 555 in Ct-b 488 fibers and therefore possible biases due to nonspecific tracing (tracing reliability is indicated according to the diagrams in A). Similarly, the coexpression of Ct-b 488 and Ct-b 555 in the PmCo is indicated in percentage in the panels on the right. In the regions showing higher coexpression, the absolute (averaged) values are indicated in the high-magnification insets. HDB, Nucleus of the diagonal band of Broca; ZI, zona incerta; BSTMP, bed nucleus stria terminalis medial division posterior part; PeF, perifornical nucleus; LH, lateral hypothalamic nucleus; opt, optic tract; M2, secondary motor cortex; Cg, cingulate cortex; IL, infralimbic cortex; DP, dorsal peduncular cortex; Den, dorsal endopiriform nucleus; VEn, ventral endopiriform nucleus. Scale bars: left panels, 20 μm; right panels, 200 μm. Data are the mean ± SEM.

To further validate these findings, we performed anterograde viral-tracing experiments using a CaMKIIa-specific adeno-associated virus (AAV), exploiting the high expression levels of CaMKIIa in ACPs. When viral injections were restricted to the PmCo (N = 6; Fig. 5A–D), there was negligible or no viral expression in any targets of PmCO efferent projections such as the BAOT, basolateral amygdala, olfactory tubercle, and mPFC (Fig. 5B–D). Negligible or no evidence of viral expression was detected in the MeA, Ent, and PFC (all receiving minimal ACP collateral input; Fig. 4F–K), with the strongest expression in the AOB GC and along the stria terminalis (Fig. 5A,B). Overall, as shown by different retrograde and anterograde tracing methods, these results confirm that the AOB is the major target of ACPs.

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Figure 5.

Anterograde tracing of PmCo projections using a CamKIIa-specific adenovirus. A, Z-projection of an image stack acquired from seriate tissue sections and showing the extent of viral labeling in the brain (mCherry) following AAV injection in the PmCo: the corticobulbar tract coursing through the stria terminalis (st) and reaching the AOB GC layer shows the most intense mCherry expression. In the lower panels: digitized version of the image stack showing from frontal and top views the course of PmCo fibers in the brain. The pattern of mCherry expression was reconstructed on each image by selecting pixels having values of hue/intensity/brightness equal or higher than those in the AOB GC layer. In the frontal view, the route of the injection is indicated by the white dotted line. In the top view, the sectioning planes relative to the images in B, C, and D are in indicated by the dashed yellow lines. B–D, Single images taken from the stack represented in A showing the extent of mCherry expression at the lateral levels indicated by the coordinates in the lower left corners of each panel. The AOB GC, the st, and the PmCo show the highest level of mCherry expression. Scale bars, 500 μm.

ACPs synapse onto AOB GABAergic interneurons

From our viral-tracing experiments, AOB GCs appear to be the main target of ACPs in the AOB. However, from this analysis it was not possible to evaluate the relative weight of ACP synaptic inputs onto either cell type or to assess the impact on the physiology of AOB circuits. To analyze these properties, we expressed ChR2 specifically in ACPs through conditional viral delivery in the PmCo of Sim1^cre mice (N = 28; Fig. 6A), as Sim1 is expressed by the majority of these neurons (Fig. 3E). Four to six weeks after viral injection, ChR2 was strongly expressed in the PmCo (Fig. 6A1), along the stria terminalis (Fig. 6A2) and in the AOB GC layer (Fig. 6A3). Perisomatic stimulation with blue light evoked excitatory responses in GCs with relatively fast kinetics and low onset variability (4.8 ± 0.2 ms, N = 23), which is consistent with a direct excitatory input from the PmCo (Fig. 6B). This was further confirmed by 4-AP-mediated rescue of evoked excitatory events, initially blocked with TTX (onset, 8.3 ± 0.7 ms; amplitude reduction, 77.9 ± 23.2 pA; N = 6; Fig. 6B; Petreanu et al., 2009). Excitatory input was instead completely eliminated by blockers of AMPA and NMDA glutamatergic transmission, DNQX and AP5, respectively (Fig. 6B). The absence of light-evoked IPSCs recorded at the reversal potential for excitation (0 mV) indicated a lack of indirect inhibitory transmission between the PmCo and AOB GCs (N = 23; Fig. 6B). MC layer or GL light stimulation did not result in any response (neither excitatory nor inhibitory) in either GCs (N = 23) or MCs (N = 28). Conversely, light activation of PmCo afferents to the GC layer evoked disynaptic IPSCs in MCs (Fig. 6C). These responses were approximately three times slower than those evoked in GCs (onset, 17.1 ± 0.3 ms; N = 28) and completely abolished by bicuculline, revealing their polysynaptic nature, likely resulting from GABA release from PmCo-activated GCs. Accordingly, trains of light pulses on GCs (4 ms at 20 Hz) induced distinct effects on MC firing (holding = −45 mV): either a sharp and transient decrease (compare 70% reduction: 1 s before LED vs 1 s after LED; found in N = 9 of 27 cells; paired t test before vs after, p = 0.019) or a gradual increase in the normalized spike frequency (∼30% increase: 1 s before LED vs 1 s after LED, found in N = 4/27 cells; paired t test before vs after, p = 0.002; Fig. 6C1).

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Figure 6.

A, ACPs establish direct synaptic contact with AOB GCs. A₁–A₃, Injection of double-floxed ChR2 mCherry-expressing adeno-associated virus in the PmCo (A₁) of Sim1^cre mice results in labeling (red) of the corticobulbar circuit coursing through the stria terminalis (st; A₂), and terminating in the AOB GC layer only (as shown by the color histogram on the side, A₃). B, Light stimulation of PmCo afferents to the GC layer induced TTX-sensitive excitatory responses in GCs at resting potential (−65 mV; onset, 4.8 ± 0.2 ms; N = 23). These are rescued by TTX-4AP bath application, indicating direct synaptic connectivity. C, No evoked post-synaptic current (EPSCs) is detected in MCs under the same conditions, while inhibitory currents (IPSCs; onset, 17.1 ± 0.3 ms; N = 28) are visualized using a high-chloride intracellular solution. C₁, Effects of repeated optogenetic stimulation of the GC layer on MC firing (five overlapped trials are shown for each effect): MC activity is either temporarily suppressed or facilitated (20 Hz light pulses, 4 ms each; frequency was compared 1 s before vs 1 s after LED stimulus onset). D, The macs receive direct PmCo inputs of lower amplitude compared to GCs. E, Circuit diagrams showing the putative effects of PmCo feedback on AOB MC firing: GC-mediated feedforward inhibition and mac–GC-mediated MC disinhibition. Above each trace, the recorded cell type and the site of LED stimulations are indicated in black and blue, respectively. a.u., Arbitrary units. Scale bars: A₃, 100 μm; A₁, A₂, 500 μm.

In the MOB, deep short axon cells (dSACs) are a type of inhibitory GABAergic neuron that provides feedforward inhibition to multiple GCs. dSACs are also the main recipient of Pir excitatory feedback, which in turn results in strong GC inhibition (Boyd et al., 2012; Markopoulos et al., 2012). Therefore, while direct GC-mediated inhibition can result in a reduction of MC firing rate, both dSAC-mediated disinhibition and GC-induced rebound firing (Desmaisons et al., 1999; Balu and Strowbridge, 2007) can explain the slow increase in MC firing we observed. In the AOB, macs have an analogous connectivity and function as dSACs (Larriva-Sahd, 2008). Surprisingly, the activation of PmCo afferents on macs (onset, 6.9 ± 0.6 ms; N = 10 cells) elicited excitatory events much lower in amplitude when compared to GCs (amplitude: GCs, 103.1 ± 21.9 pA, N = 23; macs, 20.9 ± 3.1, N = 10; t test, p = 0.005; Fig. 6B). The persistence of very low-magnitude responses detected in the presence of TTX and 4-AP (Fig. 6D) revealed the occurrence of direct synaptic connectivity with PmCo afferents; however, under these conditions (perisomatic or wide-field LED illumination), they were not sufficient to induce detectable light-evoked feedforward inhibition of GCs (Fig. 6B).

Given that our tracing experiments suggested a lower extent of PmCo–MC connectivity (Fig. 1G2), the lack of light-evoked EPSCs in MCs was unexpected. One possibility is that PmCo projections to MCs are either Sim1 ⁻ or simply too scarce to be detected. To rule out these possibilities, we used a CaMKIIa-specific viral vector as described above to target ChR2 expression to the highest possible number of corticobulbar projection neurons in the PmCo (CaMKIIa/Ct-b = 92.2%; Fig. 3A). In this case, during perisomatic LED stimulations, direct and fast excitatory responses were sometimes detectable in MCs (onset, 1.3 ± 0.2 ms; Fig. 7). However, by a thorough survey of all injected brains used in these experiments, we were able to rule out the origin of excitatory afferents to MCs in the PmCo: fast monosynaptic excitatory currents were detected only when CaMKIIa-expressing neurons in the BAOT—which also projects to the AOB (Fig. 1D)—were also infected (Fig. 7; Table 1). No other types of excitatory events (slower in onset) were detected on MCs. Collectively, these results conclusively validate the observation that ACPs innervate the AOB GC layer only and further confirm that this input is mainly directed to AOB GCs (Fig. 6E).

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Figure 7.

Optogenetically evoked MC excitatory responses are induced by the activation of BAOT afferents. A–B₂, Spread of CaMKIIa-ChR2 virus to the BAOT results in the expression of ChR2 not only in the PmCo projection neurons but also in those in the BAOT. Infection of this region results in mCherry labeling in the AOB GC and MC layers (see color histogram on the side, indicating the localization of mCherry labeling). C, Optogenetic stimulation of the MC layer evoked excitatory responses in MCs (onset, 1.3 ± 0.2 ms; 14 cells) only when the BAOT was infected. D, These direct responses (not blocked by TTX-4AP bath application) result in disynaptic excitatory events detectable in GCs, which are slower (onset, 7.7 ± 0.4 ms; 17 cells) compared with PmCo direct inputs elicited by GC stimulation (4.8 ± 0.2 ms; 23 cells; t test, p < 0.0005; Fig. 1B).

ACPs enhances AOB mitral cell excitatory output

In the MOB, GC-mediated inhibition has been proposed to be responsible for tuning MC responses to different odor inputs by sharpening their molecular receptive range through the suppression of nonspecific neuronal responses and facilitating relevant output (i.e., providing contrast enhancement; Yokoi et al., 1995; but, see also Fukunaga et al., 2014). In vivo experiments have shown that AOB MC firing can either increase or decrease in response to different odor stimuli (Luo et al., 2003), suggesting the presence of similar tuning mechanisms also in the AOB. In our experiments, corticofugal PmCo inputs induced either inhibitory or disinhibitory effects on AOB MCs (Fig. 6C1), which may potentially indicate a contribution to MC odor coding through contrast enhancement. To study this further, we conducted cell-attached recordings from AOB MCs during concurrent electrical stimulations of the VN and blue light excitation of PmCo afferents (Fig. 8A). VN stimulations consisted of a series of 4 × 100 Hz trains of 0.4 ms pulses delivered in 2 s trials (Schoppa, 2006). Light stimuli were partially interleaved with electrical pulses and consisted of 4 × 4/20 Hz trains of 4 ms light pulses delivered onto PmCo afferents in the AOB GC layer. Most cells (16 of 23 cells) were responsive to a single electrical pulse, as the current used for the stimulations was tuned each time to reach firing threshold [≥0.4 mA for both low (50 MΩ) and high (1 GΩ) resistance seals; two-way ANOVA, factors: current (levels: 0.04, 0.08, 0.4, 0.8, 4, and 8 pA); resistance (levels: 1 GΩ, 50 MΩ); current effect, p < 0.005; significant pairwise post hoc comparisons (p < 0.05): 0.04 and 0.08 pA vs 0.4, 0.8, 4, and 8 pA; Fig. 8B]. As expected, AOB MCs showed either excitatory or inhibitory responses to VN input stimulation alone (Fig. 8C,D). Comparing the firing rates during and after VN stimulations (40 ms ON vs 200 ms OFF), we selected excited (type I) and inhibited (type II) cells to further analyze the effect of PmCo feedback in relation to different VN inputs (paired t test, p ≤ 0.05; Fig. 8D). This categorization accounted for the relative changes in firing rates between baseline activity and evoked responses and was referred to a more restricted sample of cells (16 of 23 cells). Inhibited cells were the most represented [type II cells were ∼50% (10 of 23 cells) while type I cells only were ∼26% (6 of 23 cells)], probably due to the high number of inhibitory neurons recruited by electrode stimulations of the VN. Cells that had responses that did not fall into either of the two categories were considered not to have any statistically significant change in firing rate (“no change”; Fig. 8C). When current intensity was kept at subthreshold levels (0.08 mA), only type II MCs were observed (Fig. 8C). Together, these results suggest that the threshold for MC excitability is determined by both VN input and the extent of concurrent activation of local inhibitory circuits.

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Figure 8.

PmCo feedback enhances MC responses to vomeronasal inputs. A, Diagram showing the configuration used during recordings: electrical stimuli were targeted onto the VN, light stimuli were delivered onto the GC layer, and the firing rate of MCs was recorded in cell-attached mode. B, Tuning curves of MCs in response to current stimuli of different intensity were obtained at different seal resistance levels: 1 GΩ, black dots; 50 MΩ, circles. This preliminary test was made to define the threshold current for MC firing, used in the rest of the experiments (2-way ANOVA, p = 0.004). C, Percentage of the different types of MC responses following electrical VN stimulations above (0.8 mA) and below (0.08 mA) threshold. D, Raster plots of type I and type II MC activity during electrode stimulation (red dots). E, Average firing rate (across units and trials) of MCs divided by type (I or II) and stimulation. EO 4 Hz/EO 20 Hz (joint electrical and optical stimulation at 4 and 20 Hz, respectively. Red dots mark electrical stimuli; blue dots mark optical stimuli. F, Mean firing rate changes induced in MCs by optogenetic activation of PmCo feedback. The red lines represent the firing frequency at unstimulated baseline levels. G, Relative increase and decrease in firing rates during joint electrical and optical stimulation (y-axis) and electrical only stimulation (x-axis) both referred to baseline levels. Firing rate was computed during 40 ms of stimulation. H, Two LED stimulation protocols are color coded in darker (EO 20 Hz) or lighter (EO 4 Hz) red (for excitation) or blue (for inhibition). Percentage values refer to either values of E > 0 or E < 0). Scatter plot showing the lack of correlation between the relative effect of dual electrical and light stimulations on light-evoked responses alone. Data are the mean ± SEM.

When trains of light stimuli were delivered to PmCo afferents in the GC layer, the type I MC firing rate was increased on concurrent VN afferent stimulation, while type II MC responses remained low in frequency or were even slightly reduced (∼2 Hz; two-way ANOVA, factors: type (levels: I, II, no change); protocol (levels: E, EO 4 Hz, EO 20 Hz); interaction effect: F_(4,36) = 2.78, p = 0.04; type effect: p < 0.005; protocol effect: p = 0.03; post hoc comparisons: E vs EO 4 Hz, p = 0.014; E vs EO₂, 0 Hz, p = 0.018; type I, N = 4; type II, N = 10, no change, N = 7; Fig. 8E,F). In other words, considering only the two subsets of MCs characterized by significant VN-evoked firing rate changes (type I, II), the addition of optogenetically evoked PmCo input was mainly evident in the type I MCs. This implies that the predominant inhibitory effect of PmCo feedback on MCs observed during voltage-clamp experiments (Fig. 6C,C1 ) might be limited by the excitatory effects induced by the concurrent activation of VN afferents in both type I and II responses. Conversely, since both the activation of PmCo corticofugal afferents as well as electrical VN stimulation can induce GC-mediated rebound excitation (Schoppa, 2006), or other disinhibitory mechanisms enacted by local GABAergic circuits, these two effects might be additive in other cases, possibly explaining the more significant effect of dual stimulations on type I responses. Thus, in contrast to the generic and homogeneous impact of piriform afferents to MOB circuits (Boyd et al., 2012; but, see also Otazu et al., 2015), the effect of PmCo feedback depends on the polarity of VN-evoked responses in AOB MCs. Accordingly, photoactivation of PmCo afferents did not shift MC firing rates toward excitation during subthreshold VN stimulations (data not shown). However, this analysis was limited to the two extremes of the VN response range (type I and type II). To test whether the effect of dual PmCo/VN activation could be generalized to all VN-evoked responses, we compared the relative frequency changes [(F_evoked − F_baseline)/(F_evoked + F_baseline); see Materials and Methods] during VN stimulations (electrode-evoked vs baseline firing rates = E) to those evoked during dual VN/PmCo stimulations, in all recorded MCs (electrical + optical stimulation = EO). Firing rates during dual stimulations were either shifted toward excitation (in 62.5% of all cells for which F_EO > F_baseline, F_EO > F_E) in case of positive VN-evoked responses or inhibition (in 60.7% of all cells for which F_{EO <} F_baseline, F_EO < F_E) in case of negative ones (E < 0; χ² tests were run to compare the effect of stimulations to random data distributions yielded p values <0.0001). Thus, the effect of PmCo input on MC gain to VN-evoked activity appears to be conserved in most recorded neurons in our sample (Fig. 8G). Importantly, the differential effect of cortical input on MC VN-evoked firing does not depend on PmCo input alone since very low correlation was found (R ² = 0.2) comparing the effect of dual stimulations (EO) to the one of light stimulations alone (O; Fig. 8H). Light-evoked firing rate changes (in absence of paired electrical stimulations) were broadly inhibitory (F_O < F_baseline = 65.8%; similar rates were found in current-clamp experiments: N_inhibited/(N_inhibited + N_excited) × 100 = 69.2%). Together, these results indicate that PmCo feedback exerts differential and input-specific effects on MCs. This leads to an increase in the gain of MC responses to incoming stimuli, which is a typical functional requirement for odor discrimination by olfactory circuits. These data, together with our electrophysiological analysis, suggest that, similar to what occurs in piriform circuits, amygdala corticobulbar neurons might play a crucial role in shaping odor processing by the AOS through experience or brain state-dependent feedback.