Disruption of Centrifugal Inhibition to Olfactory Bulb Granule Cells Impairs Olfactory Discrimination

Disruption of centrifugal inhibition to olfactory bulb granule cells impairs olfactory discrimination

Alexia Nunez-Parra1, Robert K. Maurer, Krista Krahe, Richard S. Smith, and Ricardo C. Araneda2

Department of Biology, University of Maryland, College Park, MD 20742

Edited* by Michael V. L. Bennett, Albert Einstein College of Medicine, Bronx, NY, and approved July 23, 2013 (received for review June 4, 2013) Granule cells (GCs) are the most abundant inhibitory neuronal type (23). Although mechanisms that promote excitation of GCs have in the olfactory bulb and play a critical role in olfactory processing. been studied extensively, the mechanisms that promote inhibition GCs regulate the activity of principal neurons, the mitral cells, of GCs have received less attention. The existence of a descending through dendrodendritic synapses, shaping the olfactory bulb out- inhibitory input suggests that regulation of GCs by afferent input to other brain regions. GC excitability is regulated precisely by hibition also can modulate olfactory processing. Previous studies intrinsic and extrinsic inputs, and this regulation is fundamental indicated that direct stimulation of HDB/MCPO can inhibit neu- for odor discrimination. Here, we used channelrhodopsin to stim- ronal activity in the OB (24); however, the presence of cholinergic ulate GABAergic axons from the basal forebrain selectively and projections from this region has confounded the interpretation of show that this stimulation generates reliable inhibitory responses these observations. Here, we expressed channelrhodopsin (ChR2) in GCs. Furthermore, selective in vivo inhibition of GABAergic neu- exclusively in inhibitory neurons of the HDB/MCPO to control rons in the basal forebrain by targeted expression of designer GABA release in the OB selectively and to determine its influence receptors exclusively activated by designer drugs produced a re- on GC function and olfactory discrimination. versible impairment in the discrimination of structurally similar odors, indicating an important role of these inhibitory afferents Results and Discussion in olfactory processing. Adenoviral injections of flexed ChR2 were made stereotactically in the HDB/MCPO of mice expressing Cre recombinase under habituation | uncaging the promoter of one of the isoforms of glutamate decarboxylase (GAD), the enzyme responsible for GABA synthesis (GAD65- NEUROSCIENCE Cre mice). Double immunofluorescence against the two GAD ensory information from the external world is integrated isoforms, GAD65 and GAD67, confirmed the presence of through a series of feed-forward stages toward higher cog- S GABAergic neurons across the HDB/MCPO axis (Fig. 1A and nitive cortical areas. At these different stages, sensory perception Fig. S1). The HDB/MCPO also is populated by cholinergic can be regulated by an individual’s internal state to enhance neurons that innervate the OB; 2 wk after injection, we found meaningful information relative to less valuable information + ChR2-positive (ChR2 ) neurons intermingled with neurons associated with different behavioral tasks (1, 2). Unlike other expressing choline acetyl transferase (ChAT), further corrobo- sensory systems, peripheral sensory input onto principal neurons rating that the virus injection is restricted to the HDB/MCPO. in the olfactory bulb (OB) is relayed directly to olfactory cortices + More importantly, ChR2 fibers were abundantly present in the and subcortical nuclei, bypassing the thalamus (3). The OB is the MOB and AOB at 6 wk after injection. (Fig. 1 B,1–3). In both first stage in which extensive fine-tuning and processing of ol- regions, HDB/MCPO GABAergic fibers targeted mostly the GC factory information occurs (4). This processing involves in- layer, where the soma and proximal dendrites of GCs are found; tegration of bottom-up and top-down information by the most very sparse labeling was observed in the external plexiform layer, abundant OB cell type, the granule cells (GCs). GCs establish – most of their connections with output neurons, the mitral and where most of GC MC synapses are found, or in the glomerular tufted cells (MCs herein), through the ubiquitous dendroden- fi dritic synapses (DDS) and with a few other subtypes of inter- Signi cance neurons (5–8). Importantly, the interaction between MCs and GCs is thought to give rise to network oscillations in the OB that Granule cells (GCs) are the most abundant neuronal type in the are associated with MC firing synchronization, adding an im- olfactory bulb (OB) and play a critical role in odor processing. portant time component to olfactory processing (9–13). GCs integrate bottom-up and top-down information and reg- In analogy with the regulation of thalamic neurons by cortical ulate the output of principal neurons to higher brain areas. inputs, GCs receive important feedback regulation from cortical Here, we provide direct evidence that GCs in the OB are regu- and subcortical projection areas of MCs and afferents from neu- lated by GABAergic neurons from the basal forebrain and that romodulatory systems (3, 14–16). Activation of these feedback disrupting this inhibition affects odor discrimination. Recent projections and neuromodulatory systems increases GC excit- work has highlighted the role of feedback excitatory cortical ability (14, 17, 18), thus increasing GABA-mediated inhibition at inputs to the OB. Like the excitatory cortical feedback, the in- DDS and regulating olfactory processing. For example, regula- hibitory input we describe could mediate fast changes in ol- tion of GC-mediated inhibition by noradrenaline in the main factory coding in the OB in response to rapid changes in olfactory bulbs (MOBs) and accessory olfactory bulbs (AOBs) environmental context. has been shown to affect a range of olfactory behaviors including Author contributions: A.N.-P., R.S.S., and R.C.A. designed research; A.N.-P., R.K.M., R.S.S., odor discrimination and more complex behaviors such as mem- and K.K. performed research; A.N.-P., R.K.M., R.S.S., and R.C.A. analyzed data; K.K. main- ory formation during mating (19). In addition, other studies have tained the animal colony; and A.N.-P. and R.C.A. wrote the paper. indicated an extensive innervation by GABAergic fibers origi- The authors declare no conflict of interest. nating in the horizontal limb of the diagonal band of Broca *This Direct Submission article had a prearranged editor. (HDB) and magnocellular preoptic area (MCPO) that prefer- 1Present address: Department of Cell and Developmental Biology, Rocky Mountain Taste entially targets the GC layer in the OB (20, 21). Interestingly, the and Smell Center, University of Colorado Medical School, Aurora, CO 80045. HDB/MCPO is also the origin of the cholinergic afferent fibers 2To whom correspondence should be addressed. E-mail: [email protected]. that target the OB (22), and this cholinergic input has been This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. shown to have an important influence in olfactory processing 1073/pnas.1310686110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1310686110 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 Fig. 1. Selective labeling of GABAergic neurons of the HDB/MCPO reveals a profuse projection into the OB. (A) Confocal imaging of HDB/MCPO sections of a GAD65-Cre mouse immunostained against GAD65 (green) and GAD67 (red). GABAergic neurons ex- press one of the markers exclusively (white arrow- head), but a few cells are positive for GAD65 and GAD67 (yellow, white arrow). (Right) After targeted injections, GABAergic neurons expressing ChR2 (red) are intermingled with cholinergic neurons stained for ChAT (green). (Scale bar: 25 μM.) (B) Confocal imaging of a sagittal section of the OB from a GAD65- Cre mouse showing extensive labeling of GABAergic ﬁbers expressing ChR2 (B2, red) throughout the inner layers of the OB after injection of ChR2 virus into the HDB/MCPO (B1). (Scale bar: 500 μM.) (B3)The afferents mostly innervate the GC layer with only sparse ﬁbers reaching beyond the MC layer to the external plexiform and glomerular layers (GL). (Scale bar: 100 μM.)

layer. The labeling pattern also indicates that inhibitory fibers (Fig. 2A). LightStim at 10 Hz produced a rapid and reversible target mostly GCs, but not MCs, in both the MOB and AOB decrease in spiking elicited by a depolarizing current stimulus (Fig. 1 B, 3). Similar to the excitatory feedback projections, this (MOB: 45.3 ± 9.9% reduction, n = 7, P < 0.004; AOB: 70.1 ± 12% inhibitory input could have a modulatory role on GCs through- reduction, n = 4, P < 0.01). Similar experiments under voltage- out the OB. clamp conditions at 0 mV (Fig. S2A) indicated the occurrence of + We next recorded from GCs surrounded by ChR2 GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs), which were fibers, which were recognized visually by the expression of tandem completely abolished by the GABAA blocker GABAzine (Fig. + dimer Tomato (tdTomato). Stimulation of these fibers with blue S2B). LightStim of ChR2 GABAergic fibers at 10 Hz evoked light (hereafter, LightStim) inhibited GCs in the MOB and AOB robust inhibitory postsynaptic currents (eIPSCs), synchronized

Fig. 2. Stimulation of ChR2 expressed in HDB/ MCPO GABAergic afferents inhibits GCs in the OB. (A)(Left) Diagram showing the recording array + used; we recorded from GCs surrounded by ChR2 GABAergic fibers and stimulated with blue light. (Center) Stimulation of GABAergic fibers in the vi- cinity of a recorded GC (50 pulses, 10 Hz) decreases the frequency of firing elicited by a depolarizing current stimulus in the MOB (16 pA, 10 s) (Upper) and in the AOB (24 pA, 15 s) (Lower). The raster plots represent the activity on three cells in each region, highlighting the reversible decrease in action potential frequency induced by LightStim. (Right) LightStim produced a frequency-dependent increase in inhibition of firing in GCs. Bar graph summarizing the reduction in GC firing produced by LightStim at 10 Hz (MOB, n = 7, *P < 0.004; AOB, n = 4, P < 0.02). (Scale bars: 1 s and 20 mV.) (B)(Upper Left) In voltage-clamp conditions, LightStim (10 Hz) elicited robust eIPSCs in a MOB GC. The Inset shows the expanded time axis; most light pulses produced a synchronized eIPSC. (Lower Left) In the same cell, the eIPSCs were not affected by the presence of blockers of glutamatergic transmission (CNQX, 10 μM; APV, 100 μM; LY367385 100 μM) but were completely abolished by the addition of TTX (0.5 μM). (Right) The integral under the area of the LightStim-induced eIPSCs at 10 Hz, or charge transfer (black), was not different in the presence of blockers (orange) but was completely abolished in the presence of TTX (gray bar; *P < 0.03).

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1310686110 Nunez-Parra et al. Downloaded by guest on September 24, 2021 with the LightStim pulses, in MOB GCs (20/22 cells; Fig. 2B). The diffuse projection pattern of GABAergic fibers from The eIPSCs, like the sIPSCs, were completely abolished in the the HDB/MCPO and the high frequency of occurrence of in- presence of GABAzine (Fig. S2C). Excitation of deep short hibitory responses elicited by activation of these axons suggest axon cells by cortical feedback projections (14) or direct excita- that inhibitory synapses occur throughout the soma and dendritic tion of another subtype of inhibitory neurons, Blanes cells, has tree of GCs. To explore this possibility further, we conducted been shown to inhibit MOB GCs (6). However, unlike these single-photon uncaging of GABA onto GCs using 1-(4-amino- studies, our observations suggest that inhibition of GCs by HDB/ butanoyl)-4-[1,3-bis(dihydroxyphosphoryloxy)propan-2-yloxy]-7- MCPO GABAergic neurons does not require the activation of nitroindoline (DNPI-GABA), while responses were recorded in local interneurons. Accordingly, blockers of excitatory gluta- GCs loaded with Alexa-594 to direct the uncaging spatially to the matergic transmission [100 μM (2R)-amino-5-phosphonopenta- recorded cell. As shown in Fig. 3, uncaging of GABA revealed noate (APV) and 10 μM 6-cyano-7-nitroquinoxaline-2,3-dione inhibitory responses throughout the soma and basal and apical (CNQX)] did not affect either the amplitude or the frequency of dendrites in GCs of the MOB and AOB (n = 4 slices each). the eIPSCs (Fig. 2B). In these experiments we also included the Larger current amplitudes were observed in the proximity of the metabotropic glutamate receptor blocker LY367385 (100 μM). soma (∼30 μm); however, the amplitude of the responses in Metabotropic glutamate receptors are found in MCs and GCs distal regions of the apical dendrites could be as large as the and have been shown to participate in GC→MC inhibition in the responses elicited in basal dendrites (Fig. 3D). The latency and MOB and AOB (25–27). To quantify the responses across dif- time course of the responses did not vary significantly across ferent cells better, we integrated the currents produced by the different regions, in agreement with the current kinetics observed LightStim sweep (SI Materials and Methods), which correspond in other inhibitory synapses stimulated using DNPI-GABA (29). to the total charge transfer. As shown in Fig. 2B, Right, the av- These results indicate that GABA inhibitory responses are very − erage charge at 10 Hz (19 ± 4 × 10 12 C, n = 11) was not reduced prominent throughout the GCs. To corroborate these findings − in the presence of blockers (16 ± 410 12 C, n = 9; P > 0.5). further, we determined the expression and location of postsynaptic However, the charge transfer elicited by LightStim was largely GABA receptors clusters using antibodies against gephyrin, an −12 abolished (0.6 ± 0.2 × 10 C, n = 2) by tetrodotoxin (TTX) (0.5 anchoring protein associated with postsynaptic clusters of GABAA μM), which is known to decrease responses to ChR2 activation receptors (30, 31). GCs were labeled by electroporation of GFP by inhibiting action potentials (14, 28). Similar results were in the subventricular zone. Confocal analysis of immunostaining obtained in recordings from AOB GCs, stimulated at 5 Hz against GFP and gephyrin indicated that GABA receptor clus- NEUROSCIENCE − − (control: 13 ± 3 × 10 12 C, n = 10; plus blockers: 9 ± 5 × 10 12 C, ters can be found throughout the GCs’ soma and proximal and − n = 3, P > 0.4; blockers plus TTX: 0.2 ± 0.2 × 10 12 C, n = 3, P < apical dendrites (Fig. S3). Importantly, the site of inhibition can 0.03). Previous work has indicated that local interneurons acti- have different consequences for GC excitability and information vated by excitatory afferent fibers are an important component processing. Spatially restricted excitation of GC dendrites can of GC inhibition (14). Our results support the notion that GCs produce localized inhibition of MCs through recurrent inhibition, receive an important direct inhibitory feedback projection from whereas global activation of GCs (i.e., somatic excitation) can affect the basal forebrain and, importantly, that olfactory behaviors a larger number of MCs through lateral inhibition (17, 32). Simi- mediated by the AOB and MOB could be regulated by HDB/ larly, we propose that GABAergic inhibitory inputs from the HDB/ MCPO GABAergic neurons (see Fig. 4). MCPO in the soma and proximal dendrites could exert a more

Fig. 3. GABA responses occur throughout the soma and dendritic tree of GCs. (A) Experimental setup used for GABA uncaging experiments. We recorded from the soma of GCs filled with Alexa 594 (red cell) while the slices were perfused with DPNI-GABA (2 mM) and photolysis was elicited by single-photon activation with a 405-nm laser. (B)GCfilled with Alexa 594 (20 μM). Colored circles show representative uncaging spots: The blue circle is 10 μm from soma; the red circle indicates a control uncaging event 4 μm from the blue spot; the orange circle indicates a basal dendrite 28 μm from soma; and the green circle indicates a distal dendrite 99 μm from soma. (Scale bars: 10 μm.) (C) Scatter plot of the amplitude (Upper) and rise times (10–90%) (Lower) of laser-evoked GABA IPSC on GC dendrites, as a function of distance from soma, for AOB (n = 4, orange) and MOB (n = 4, black) (107 spots, 1,070 events). Larger IPSC amplitudes were observed within the proximity of the soma (∼30 μm), but the rise time did not vary significantly. (D)(Upper) Representative laser-evoked IPSCs at the specified colored spots shown in B (average of 10 traces per spot). The purple asterisk indicates the time of photolysis. (Scale bars: 20 pA and 100 ms.) (Lower) Bar graph comparing amplitude and kinetics of the representative photolysis-evoked IPSCs shown above. Note that the response evoked at 4 μm from the dendrite (red) has a significantly lower amplitude and larger rise time.

Nunez-Parra et al. PNAS Early Edition | 3of6 Downloaded by guest on September 24, 2021 global effect on GCs’ activity, regulating not only recurrent in- the presence of CNO. Previous studies have shown that lesions in hibition of MCs, but lateral inhibition as well. the HDB, impair the animal’s ability to habituate to consecutive To determine the impact of HDB/MCPO inhibition in olfac- presentations of an odor (36); however, our behavioral test showed tory processing, we used designer receptors exclusively activated that these mice had no deficit in habituation to repetitive odor by designer drugs (DREADDs) technology to silence GABAergic presentations. Furthermore, the olfactory discrimination deficit neurons selectively. DREADDs are muscarinic receptors that observed is not caused by a change in the odor-detection threshold: have been mutated to respond selectively to the exogenous com- The detection threshold of the C7 ester was not different in mice pound clozapine-N-oxide (CNO) but not to the endogenous li- injected with PBS or CNO (Fig. S4A). Similarly, like control mice, gand acetylcholine (33). We injected a Cre-dependent expression hM4Di-injected mice learned to discriminate between stereo- virus encoding the inhibitory DREADD, AAV8/hSyn-DIO-HA- isomers of carvone in an associative learning paradigm, suggesting hM4D(Gi)-IRES-mCitrine (hM4Di), into the HDB/MCPO of that cortical processing is not affected (Fig. S4B). = GAD65-Cre mice. Animals injected with hM4Di (n 6) were Together our results indicate that afferent inhibition from the tested 4 wk after injection for olfactory discrimination, after PBS HDB/MPOC onto GCs is required for proper olfactory dis- (control mice) or CNO injections (treated mice) (Fig. 4). We crimination. We hypothesize that the inhibitory inputs, like ex- tested structurally similar odors (e.g., esters differing in one carbon citatory inputs, act to maintain a proper balance for the degree moiety) using the habituation/dishabituation paradigm. Animals of GC→MC inhibition in the OB, which is necessary for fine habituated to an odor (ethyl heptanoate; C7) show a reduction in discrimination. Future experiments are needed to determine the the sniffing time. Under control conditions (i.e., before the CNO impact of this inhibitory input in the processing of information injection), presentation of the novel odor (ethyl octanoate; C8) about social cues mediated by the AOB. Furthermore, proper fi resulted in a signi cant increase in investigation time, indicating olfactory coding depends on several properties such as local ± that the mice are able to discriminate between these odors (1.1 synaptic connectivity and also on network properties. Oscil- ± < 0.2 s vs. 4.1 0.4 s; P 0.01). More importantly, the same animals lations in the MOB are known to be particularly important for were unable to discriminate the odor pair 2 h after CNO injection information processing. There is evidence that γ oscillations arise ± ± < (0.5 mg/kg) (C7 0.7 0.3 s vs. C8 0.3 0.2 s; P 0.4) but had no from the interaction between cortical structures and bulbar fi dif culty discriminating these odors when tested 2 h after a PBS components (14) and that GABA inhibition onto GCs is required ± ± < injection (C7, 0.7 0.3 vs. C8, 0.3 0.2, P 0.02). Previous be- to maintain proper levels of γ oscillations (12). The HDB/MCPO havioral studies have shown that the maximal effect of CNO, receives inputs from several regions of the brain, including the acting on the DREADDs, occurs within a window of a few hours OB, amygdala, hypothalamus, and brainstem (37, 38). Therefore, (34, 35). Accordingly, 4 h after CNO injection, mice begin to re- the GABAergic inhibitory input from the basal forebrain could cover their ability to discriminate the C7/C8 pair, indicating that channel integrated information from other brain areas to the OB. the effect of CNO is reversible (C7 0.5 ± 0.2 s vs. C8 3.0 ± 0.7 s; < fi This information, like the excitatory cortical feedback, could P 0.02). The de cit in olfactory discrimination was limited to mediate fast changes in olfactory coding in the OB in response to closely related odors, and discrimination of esters differing by two rapid changes in environmental context. carbons, e.g., between ethyl hexanoate (C6) and C8, was normal 2 h after the CNO injection (1.25 ± 1.0 s vs. 4.6 ± 0.7 s; P < 0.01) Materials and Methods (Fig. 4). We note that these mice exhibited a significant increase in Animals. All experiments were conducted following the guidelines of the investigation time for the C8 odor after habituation to C6, even in Institutional Animal Care and Use Committee of the University of Maryland,

Fig. 4. Disruption of inhibition from the HDB/ MCPO affects odor discrimination. (Upper) A virus

encoding for hM4Di was injected into the HDB/ MCPO of GAD65-Cre mice. Four to five weeks later, animals received an i.p. injection of either PBS (control condition) or CNO (treated condition) and were tested for odor discrimination using the habituation/dishabituation paradigm or were tested for odor-detection threshold (SI Materials and Methods). (Lower) Before the CNO injection, mice habituated to C7 (grey bar) and showed a significant increase in investigation time in the presence of the dishabituated odor, C8 (red bar). Two hours after the CNO injection, mice habituated to C7 failed to show an increase in investigation time for C8. However, the investigation time was increased significantly for a pair of odors differing by two carbons (C6/C8). The inability to discriminate between C7 and C8 was recovered completely 4 h after CNO administration. *P < 0.05; **P < 0.02.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1310686110 Nunez-Parra et al. Downloaded by guest on September 24, 2021 College Park. Electrophysiological and behavioral experiments were per- detected using an amplitude threshold of 5–6 pA. Only events with fast ki- formed on C57/BL6 or GAD65-Cre 30- to 180-d-old female and male mice netics were considered for the analysis; for older ChR2-injected animals, the obtained from breeding pairs housed in our animal facility. Animals were cutoff was 4.5 ms. kept on a 12-h light/dark cycle with access to food and water ad libitum. Uncaging of GABA. We performed GABA uncaging as previously described Viral Injections. Expression of ChR2 in GAD65-Cre mice was achieved by (29). To correlate the physiological responses with the cell’s morphology, we targeted injection (1 μL) of the AAV2/5.CAGGS.flex.ChR2tdTomato adeno- included Alexa 594 (20 μM) in the recording pipette, so that we were able to virus into the HDB/MCPO (University of Pennsylvania vector core; SI Materials aim the uncaging laser spot to spatially restricted areas of the recorded GC. and Methods). Similarly, in GAD65-Cre mice used for behavior studies the The collimated output of a 405 nm laser (Coherent, LLC) was expanded μ HDB/MCPO was injected bilaterally with 0.5 L of the hM4Di adenovirus to 60% of the back aperture of a 63× Olympus objective. The spot has a (University of North Carolina vector core). Animals were allowed to recover Gaussian profile in the focal plane with a 1/e2 radius = 0.87 μm. Fluorescence and were used for electrophysiological recordings at least 6 wk after injection. illumination was achieved using a green LED (exciter 594 nm center wave – For behavioral studies, animals were used 4 5 wk after injection of hM4Di. length) (Chroma), and the emitted light was collected by a CCD camera (Hamamatsu). The concentration of DPNI-GABA was 2 mM (Tocris). Laser Confocal Imaging and Double-Labeling Immunofluorescence. To visualize ChR- flashes usually were of 100-μs duration with power intensities at the surface tdTomato expression, brains harvested from previously injected mice were of the slice up to 2 mW·μm−2. fixed with 4% (wt/vol) paraformaldehyde (SI Materials and Methods), and fi xed brain slices were mounted with Vectashield (Vector Laboratories) and Habituation/Dishabituation Test. Odor discrimination was assessed using the × visualized with a Leica SP5 confocal microscope. For double-labeling im- habituation/dishabituation test (SI Materials and Methods). Control mice and munofluorescence, we used free-floating sections incubated with 10% donkey animals injected 4–5 wk previously with hM4Di received a single i.p. injection serum (Sigma Aldrich) in PBS with 0.1% Triton X-100 (PBS-T) for 2 h at room of either PBS (control group) or 0.5 mg/kg CNO (treated group). Both groups temperature. Slices then were incubated overnight at room temperature with were tested for their ability to discriminate between C7 and C8 or between one or more of the following primary antibodies: mouse anti-GAD65 (1:300), C6 and C8. A clean standard mouse cage without bedding was used for the rabbit anti-GAD67 (1:100), and/or goat anti-ChAT (1:500). After the incubation tests, and mice were allowed to become familiar with the test environment with the primary antibodies, the samples were incubated for 2 h at room for 30 min. Each mouse was presented with three exposures to a wooden temperature with the secondary antibodies: donkey anti-mouse Alexa-488, block scented with 100 μL of the test odor at a 1:1,000 dilution. Exposures donkey anti-rabbit Alexa-594, and donkey anti-goat Alexa-488, all diluted at lasted 2 min with a 1-min intertrial interval. Each trial was videotaped, and 1:750 in PBS-T with 2.5% of donkey serum. The samples were mounted with the time the mouse spent investigating the block was quantified offline. Vectashield and visualized with a Leica SP5 × confocal microscope. Investigation was defined as the time during which the mouse’s nose was within a 2-cm radius from the block. Electrophysiological Recordings in OB Slices. Brain slice recordings were con- NEUROSCIENCE Data analysis was performed using the Igor Pro software (WaveMetrics, ducted as previously described (SI Materials and Methods) (39). We used Inc), and image analysis was performed using Image J software (National sagittal sections containing the MOB and AOB, respectively. After section- Institutes of Health). Data are shown as the mean ± SEM. Statistical signif- ing, the slices were placed in normal artificial cerebrospinal fluid [ACSF; in icance was determined using the Student t test. Statistical differences were mM, 125 NaCl, 25 NaHCO3, 1.25 NaH PO , 3 KCl, 2 CaCl2, 1 MgCl2, 3 myo- 2 4 considered significant at P < 0.05. inositol, 0.3 ascorbic acid, 2 Na-pyruvate and 15 glucose, continuously oxy-

genated (95% O2 and 5% CO2)] and were left to recuperate for 30–45 min. For LightStim at 473 nm, 5–50 pulses of 10 ms at 5 or 10 Hz were delivered ACKNOWLEDGMENTS. This work was supported by National Institutes of Health-National Institute on Deafness and Other Communication Disorders through the 40× objective, using a Lambda LS Xenon Lamp (Sutter) con- Grant DCR01-DC-009817 (to R.C.A.). A.N.-P. was supported by a Chilean gov- trolled by a Lambda SC Smart Shutter. The area of infection was assessed ernment fellowship (Becas Chile); R.K.M. was supported by an undergradu- ﬁ visually to con rm that the injection was restricted to the HDB/MCPO ate Howard Hughes Medical Institute research fellowship; R.S.S. was (Fig. 1 B, 1). eIPSCs were recorded at a holding potential of 0 mV and were supported by an National Science Foundation predoctoral fellowship and analyzed ofﬂine using the Synaptasoft Mini analysis program. Events were a Chateaubriand Fellowship from the French Embassy.

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