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HE ORGANIZATION OF FEEDBACK PROJECTIONS IN A PATHWAY
MPORTANT FOR PROCESSING PHEROMONAL SIGNALS
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Institute of Biophysics, Chinese Academy of Sciences, Beijing,00101, China

National Institute of Biological Sciences, #7 Science Park Road,hongguancun Life Science Park, Beijing, 102206, China

bstract—In most of the mammalian sensory systems therere massive cortical feedback projections to early processingtations. The mammalian accessory olfactory system is con-idered unique in several aspects. It is specialized for pro-essing pheromonal signals and plays a critical role in reg-lating sociosexual behaviors. Furthermore, pheromonalignals are believed to bypass cortex and reach the hypo-halamic behavioral centers after merely three forward pro-ections. Because the organization of the feedback projec-ions in the accessory olfactory system remains largely un-lear, the importance of the feedback projections in therocessing of pheromonal signals has been ignored. Here wehow that in mice the feedback projections from the beducleus of stria terminalis (BST) and the vomeronasal amyg-ala to the accessory olfactory bulb (AOB) are topographi-ally organized and use different neurotransmitters. By ret-ograde and anterograde tracing, we find that the feedbackrojection from the BST terminates in the AOB mitral cell

ayer, whereas that from the amygdala terminates in the AOBranule cell layer. By combining tracing, genetic labeling ofABAergic neurons, and immunostaining against a markerf glutamatergic synapses, we observe that the BST-to-AOBrojection is GABAergic whereas the amygdala-to-AOB pro-

ection is glutamatergic. In addition, a substantial number ofeedback neurons in the amygdala and BST express estro-en receptors. Thus, the accessory olfactory system, likether sensory systems, possesses extensive feedbackrojections. Moreover, our results suggest that centralormonal cues may modulate the processing of phero-onal signals at early stations through the precisely orga-ized feedback projections. © 2009 IBRO. Published bylsevier Ltd. All rights reserved.

ey words: amygdala, olfactory, GABA, glutamate, estrogen,eproduction.

Corresponding author. Tel: �86-10-80726688-8320; fax: �86-10-0723342.-mail address: [email protected] (M. Luo).bbreviations: AOB, accessory olfactory bulb; BDA, biotinylated-dex-

ran amine; BST, bed nucleus of stria terminalis; BSTif, bed nucleus oftria terminalis, interfascicular nucleus; BSTmp, bed nucleus of striaerminalis, posteriormedial division; DAPI, 4=,6-diamidino-2-phenylin-ole; dLOT, dorsal lateral olfactory tract; ER-�, estrogen receptorlpha; GAD, glutamate decarboxylase; GCL, granule cell layer; GFP,reen fluorescent protein; GL, glomerular layer; MCL, mitral cell layer;eA, medial amygdala; MePD, medial amygdala, posterior dorsalivision; MePV, medial amygdala, posterior ventral division; MOB,ain olfactory bulb; PBS, phosphate-buffered saline; PMCo, posterior

cedial cortical amygdala; TRDA, Texas Red conjugated dextran-mine; VGLUT, vesicular glutamate transporter.

306-4522/09 $ - see front matter © 2009 IBRO. Published by Elsevier Ltd. All rightoi:10.1016/j.neuroscience.2009.03.065

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prominent feature of the circuit organization of mamma-ian sensory systems lies in the massive feedback fromensory cortical areas to early sensory stations (Alitto andsrey, 2003). For example, the fibers projecting back from

he visual cortex to the lateral geniculate nucleus far out-umber those from the retina to the lateral geniculateucleus (Van Horn et al., 2000). In the main olfactoryystem, the main olfactory bulb (MOB) relays input fromhe olfactory epithelium to the piriform cortex by bypassinghe thalamus (Neville and Haberly, 2004). However, theOB also receives massive feedback projections from theiriform cortex (Broadwell and Jacobowitz, 1976; Willhitet al., 2006). The sheer size of the feedback projectionsoints to the importance of top-down signals in recon-tructing sensory percepts by modulating signal process-

ng at early stations.The accessory olfactory system is specialized for pro-

essing pheromonal signals for intra-species social com-unication (Powers and Winans, 1975; Stowers et al.,002; Dulac and Torello, 2003; Halpern and Martinez-arcos, 2003; Kimchi et al., 2007). Pheromonal signalsre relayed to hypothalamic behavioral centers via merelyhree synapses (Fig. 1A). Sensory neurons in the vomer-nasal organ (VNO) project to the accessory olfactory bulbAOB), which in turn projects to the bed nucleus of striaerminalis (BST) and the vomeronasal amygdala, includinghe medial amygdala (MeA) and posteromedial corticalmygdala (PMCo) (Scalia and Winans, 1975; Kevetter andinans, 1981; Martinez-Marcos, 2008). These areas then

roject to various hypothalamic nuclei to regulate innateociosexual behaviors (Canteras et al., 1995; Dong andwanson, 2004; Choi et al., 2005). Because of thischeme of forward projections to hypothalamic behavioralenters and lack of well-described feedback projections,he potential role of feedback projections in the accessorylfactory system has been largely ignored.

Sociosexual behaviors are highly adaptive behaviorshat are not only triggered by pheromonal signals but alsoeavily influenced by an animal’s internal state and sen-ory signals from other modalities, including those from theain olfactory system (Wood and Newman, 1995a; Wood,997; Holy and Guo, 2005; Martel and Baum, 2009). Thus,he processing of pheromonal signals at early stations maye modulated by feedback projections. The AOB is knowno receive centrifugal projections from the amygdala andST that richly express sex steroid hormone receptors

Newman, 1999). However, we still lack fundamental infor-ation about the organization of these feedback projec-

ions. For example, do these projections terminate in spe-
ific layers in the AOB? What are their basic neurotrans-
s reserved.

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ig. 1. Tracer injection into the AOB MCL retrogradely labeled neurons in the BST. (A) A highly simplified schematic illustrating the typical view of projectionsithin the vomeronasal system. (B) A schematic showing the experimental design and results following tracer injection into the AOB MCL. After the depositf tract tracers (BDA, MW�3 KD) into the AOB MCL, retrogradely labeled somata (red circles) were observed only in the ipsilateral BST. (C) Drawing of two serialoronal sections showing the distribution pattern of retrogradely labeled somata (red circles) in the BST. Each circle indicates a cluster of labeled somata. Theosition of the sections in mm, from front to back: Bregma, AP �0.34; AP �0.46. (D) Three consecutive parasagittal sections showing one injection sitearrows) in the AOB MCL. Labeling in the GCL (arrowheads) likely resulted from tracer pickup by granule cell dendrites in the MCL. Red, BDA tracers; blue,API labeling for marking the layer organization in the AOB. (E) Three coronal sections showing the retrogradely labeled somata (red) in BSTmp and BSTif,

wo subnuclei of the BST, following tracer injection shown in (D). The number of labeled cells tended to be low because each panel shows the labeling withinsingle optical section. Background grayscale pictures were bright-field images to illustrate the locations of brain nuclei. Scale bars in (C, D)�100 �m. A,

nterior; D, dorsal; L, lateral. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.


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S. Fan and M. Luo / Neuroscience xx (2009) xxx 3

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itters? And do feedback neurons in these areas alsoxpress hormone receptors that could modulate their out-ut to align with internal hormonal states? In this study weddressed these questions by combining precise tract

racing, genetic labeling and immunohistochemistry.

EXPERIMENTAL PROCEDURES

ll procedures described in this study followed institutional guide-ines for animal care and use of the National Institute of Biologicalciences, Beijing, China. They were also in accordance with theational Institutes of Health Guide for the Care and Use of Lab-ratory Animals. All efforts were made to minimize the numbernd suffering of animals used.

ract tracing

ale mice (C57BL/6 or glutamate decarboxylase (GAD)67-GFP,–8 weeks, �25 g in weight) were anesthetized with sodiumentobarbital (80 mg/kg i.p.) and then mounted and kept warm incustom-designed stereotaxic apparatus. Various forms of flu-

rophore-conjugated dextran amines (all from Invitrogen, Carls-ad, CA, USA) were injected either iontophoretically or by pres-ure. Biotinylated-dextran amine (BDA; MW�3 KD) and Texased conjugated dextran amine (TRDA; MW�3 KD) were used for

etrograde tracing, and BDA and TRDA of higher moleculareight (MW�10 KD) were selected for anterograde tracing. Fornterograde tracing, tracers were dissolved in physiological salinedye concentration�10%). For retrograde tracing, they were dis-olved in sodium citric solution (pH�3.0) to enhance the efficiencyf retrograde labeling (Reiner et al., 2000). For tracer delivery tohe vomeronasal amygdala or the BST, tracers were injected byontophoresis (5–10 �A, 1-s on/1-s off, 10 min) into these twouclei based on stereotaxic coordinates provided by an estab-

ished mouse brain atlas (Paxinos and Franklin, 2000). Tracernjection into the AOB was carried out by extending injectionipettes 2.4–2.8 mm forward from a cortical area (2 mm anterior-osterior and 0.95 mm medial-lateral from the bregma) to the AOBith a 45° angle. After a survival time of 10–15 days, mice wereilled by an overdose pentobarbital injection and perfused tran-cardially with pre-cooled physiological saline and 4% paraformal-ehyde solution in 0.1 M phosphate-buffered saline (PBS). Brainsere cryoprotected with 30% sucrose in 0.1 M PBS for 24–36 hnd sectioned in coronal or parasagittal planes (20–60 �m thick)sing a cryostat (Leica CM1900; Leica, Wetzlar, Germany). Toisualize BDA signals, brain slices were first washed in PBS with.5% Triton X-100 and then stained with cy3-conjugated strepta-idin (1:500; Invitrogen).

abeling of GAD67, vesicular glutamate transporterVGLUT) 2 and estrogen receptor alpha (ER-�)

ABA, the major inhibitory neurotransmitter in the brain, is syn-hesized by two enzymes, GAD67 and GAD65 (Dupuy and Houser,996). Because these two enzymes are typically co-expressed,AD67 has been used as a reliable marker of GABAergic neurons

DeDiego et al., 1994). We identified GABAergic neurons by theresence of green fluorescent protein (GFP) in GAD67-GFP�neo) knock-in mice. In these mice, GFP is selectively expressednder the control of the endogenous GAD67 gene promoterTamamaki et al., 2003). These GFP-knock-in mice have beenxtensively characterized and GFP has been found to be a reli-ble marker of GABAergic neurons. Henceforth we refer to theseice as GAD67-GFP mice for simplicity. For immunostaininggainst VGLUT2, we used anti-VGLUT2 primary antibody made inuinea pig (1:2500, 96 h at 4 °C; Chemicon, Temecula, CA, USA),ollowed by biotin-conjugated secondary antibody generated from
onkey (1:500, overnight at 4 °C; Jackson ImmunoResearch, a
Please cite this article in press as: Fan S, Luo M, The organization of fpheromonal signals, Neuroscience (2009), doi: 10.1016/j.neuroscience.

est Grove, PA, USA), and finally stained by cy2-conjugatedtreptavidin (1:500, overnight at 4 °C; Invitrogen). For immunostain-ng against ER-�, we used an anti-ER-� primary antibody made inabbit (1:1000; overnight at 4 °C; Upstate Biochemical, Lake Placid,Y, USA), followed by a cy2-conjugated secondary antibody made inoat (1:500; overnight at 4 °C; Jackson ImmunoResearch). Negativeontrols were incubated without primary antibodies.

Brain sections were mounted in medium containing 50%lycerol and DAPI (4=,6-diamidino-2-phenylindole), cover-lipped, and sealed with nail polish. DAPI is a fluorescent DNAounterstain that serves to mark cell density. Fluorescent con-ocal images were acquired with a laser scanning confocalicroscope (Zeiss LSM 510; Carl Zeiss, Gottingen, Germany). For

resentation purposes, multiple optical sections (Z-interval 2–5 �m)ere projected into single frames. Images were further processedsing image analysis software to enhance contrast and brightnessAdobe Photoshop; Adobe, San Diego, CA, USA).

Delineation of brain nuclei was based on an establishedouse brain atlas (Paxinos and Franklin, 2000).

RESULTS

imilar to the MOB, the AOB is organized into distinctayers, including the glomerular layer (GL), the mitral cellayer (MCL), and the granule cell layer (GCL) (Shepherd etl., 2004). Unlike those in the MOB, mitral cells in the AOBo not form a thin layer but occupy a wide area thatorresponds to the external plexiform layer and MCL in theOB. Mitral cells are the projection neurons of the AOB,nd granule cells in the GCL are GABAergic interneurons.arlier studies have shown that the AOB receives feed-ack projections from the BST and vomeronasal amygdalaRaisman, 1972; Broadwell and Jacobowitz, 1976; Davist al., 1978; de Olmos et al., 1978; Kevetter and Winans,981; Barber, 1982). However, the MCL had not beennown to receive any major feedback projections fromither the BST or the vomeronasal amygdala. We firstested whether the MCL receives any feedback projec-ions, and if so, whether the source of feedback projectioniffers from that to the GCL. We then examined neuro-ransmitter phenotypes of the feedback projections to theCL and GCL. Finally, we examined the presence of ER-�

n the feedback neurons to the AOB.

he BST projects to the AOB MCL

o test the presence of feedback projections to the AOBCL, we first injected BDA precisely into the AOB MCL by

ontophoresis (Fig. 1B). Since the vomeronasal amygdaland the BST are two major areas that receive ascendingrojections from the AOB (Scalia and Winans, 1975), wepecifically examined the presence of retrogradely labeledomata in these two areas (Fig. 1B, C). Out of a total of 42njections into the AOB, eight of them were restricted to theOB MCL. As shown by a representative example in Fig.D, a precise MCL injection had several characteristics.irstly, we could observe a small area of intensive labelingithin the MCL only. Because mitral cells extend theirpical dendrites into the GL, labeling in glomeruli wasxpected for these injections. But we did not observe any

abeling in the somata of the periglomerular cells in the GL.ranule cells in the GCL extend their dendrites into the MCL
nd form dendro-dendritic synapses with the dendrites of
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itral cells (Taniguchi and Kaba, 2001; Ma and Lowe, 2004),hus the labeling in the somata of granule cells in the GCLas also expected. However, the injections appeared tovoid the dorsal lateral olfactory tract (dLOT), which sepa-ates the MCL and GCL. Additionally the labeling density inhe GCL was much lower than that in the MCL, suggestinghat tracers did not spill over into the GCL.

Following restricted tracer injection into the MCL, weeliably observed retrogradely labeled somata in the ipsilat-ral BST but not in the amygdala (Fig. 1C, E; n�8 out of eightice). By examining a series of coronal sections, we found

hat the retrograde labeling was mostly distributed in twoubnuclei in the ipsilateral BST: bed nucleus of stria termina-is, posteriormedial division (BSTmp) and bed nucleus of striaerminalis, interfascicular nucleus (BSTif) (Fig. 1E). Thesewo subnuclei are known to receive direct input from thepsilateral AOB (Scalia and Winans, 1975; Wood and Swann,005), suggesting that their projections to the AOB are in-eed “feedback” projections. The number of the retrogradely

abeled cells in the BST tended to be small. This may indicatehat only a small number of BST neurons provide feedbackrojections. Alternatively, it could be the result of the smallize of our injections in the AOB. Nevertheless, these resultstrongly suggest that the AOB MCL receives feedback pro-ections from the ipsilateral BST but not the amygdala.

To further confirm that that the AOB MCL layer re-eives feedback projection from the ipsilateral BST, weade tracer injection into the BST and then examined theistribution pattern of anterogradely labeled terminals in

he olfactory bulb (Fig. 2A–C; n�5 mice). We sectioned thelfactory bulb parasagittally so that the AOB MCL and theCL were present on the same sections and could beasily distinguished. Consistent with our results from ret-ograde tracing (Fig. 1), tracer deposit into the BST antero-radely labeled axonal terminals that were distributed inhe ipsilateral AOB MCL but not in the GL or GCL (Fig. 2D).ometimes we found that a few fibers ran parallel to theorder between the GL and MCL but never crossed intohe GL. Some fibers were occasionally observed in theCL. For two reasons we believe that these fibers in

he GCL represent the fibers of passage from the BST tohe AOB MCL. First, anterograde labeling was observednly in the posterior end of the GCL through which thebers from the BST traverse. Second, we did not observeny varicosities or branching from these fibers, furtheruggesting that these fibers passed from the GCL to theCL and did not form synapses within the GCL. In con-

rast, closer examination of these projection fibers re-ealed many varicosities in the MCL (Fig. 2E), suggestingynaptic formations there. Despite the fact that there wasome variability across the injection sites in the BST, weid not observe any clear difference in the distributionattern of anterograde labeling, suggesting a lack of moreubtle topography within the feedback projection from theST to the AOB MCL. We did not observe any anterograde

abeling in the MOB following BST injection (data nothown), suggesting that the feedback projections from theST selectively target the AOB. Thus, our results from
oth retrograde tracing (Fig. 1) and anterograde tracing i

Fig. 2) strongly suggest that the BST provides feedbackrojection to the AOB MCL.

he vomeronasal amygdala projects to theOB GCL

arlier studies have revealed that the vomeronasal amyg-ala provides a feedback projection to the AOB GCL (Ra-

sman, 1972; Kevetter and Winans, 1981; Barber, 1982).owever, these studies did not combine anterograde or

etrograde tracing, thus their results are subjected to theonfounding factor of fibers of passage. In addition, thisonclusion had been derived mostly by less sensitive trac-

ng techniques and large injection sites. We decided toeexamine this issue by improving our approaches in threespects. First, we made small injection sites by preciselyargeting either AOB GCL or the vomeronasal amygdala.econd, we combined both anterograde and retrograde

racing. Lastly, the tracers we used (dextran amines) wereeveloped more recently and are more sensitive than trac-rs used in earlier studies.

To examine the source of feedback projections to theOB GCL, we carried out retrograde tracing by makingmall tracer injections into the GCL and then examined theistribution pattern of retrogradely labeled somata in themygdala and BST (Fig. 3A, B). Out of a total of 42 injectionites, six were restricted to the AOB GCL as determined byhe fact that tracer-labeled somata were observed only in theCL (Fig. 3C). Because granule cells extend their dendrites

nto the MCL, we observed labeling of their dendrites in theCL but did not observe any labeling of somata there. Fol-

owing tracer injections into the AOB GCL, we observedetrogradely labeled somata only within the ipsilateral vome-onasal amygdala but not in the BST. Within the amygdala,etrograde labeling was observed within the medial amygdalaosteroventral subnucleus (MePV) and the PMCo (Fig. 3D).oth of these two subnuclei receive forward input from theOB (Scalia and Winans, 1975), suggesting that the signalsent from these two nuclei back to the AOB are indeedeedback signals.

To directly test whether the vomeronasal amygdalarojects to the AOB GCL, we injected anterograde trac-rs (BDA, MW�10 KD) into vomeronasal amygdala cov-ring mostly the MePV and PMCo and then examined

he distribution pattern of anterogradely labeled fibers inhe AOB (Fig. 4A, B). Special care was taken so that thenjection sites largely overlapped with the MePV andMCo (Figs. 3 and 4C). In the AOB, we observed inten-ive anterograde labeling in the ipsilateral GCL but no

abeling in the MCL (Fig. 4D). Many varicosities can bebserved along these fibers (Fig. 4E), suggesting syn-ptic formation in the GCL.

One early study suggests that the PMCo provides aeak projection to the contralateral AOB (Barber, 1982).e failed to observe any obvious retrograde labeling in the

ontralateral PMCo following tracer injections into the AOBCL. Similarly, we did not observe any obvious antero-rade labeling in the contralateral AOB following PMCo

njections. This contralateral projection might be too weak


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ig. 2. Tracer injection into the BST anterogradely labeled centrifugal fibers in the AOB MCL. (A) A schematic showing the experimental design andesults following tracer injection into the BST. After deposit of tract tracers (BDA, MW�10 KD) into the BST, anterogradely labeled terminals (crossedines) were observed only in the ipsilateral AOB MCL. (B) Drawing of three coronal sections showing the extent of injection sites (red) in the BST ofhree mice. The position of the sections in mm, from front to back: Bregma, AP �0.22; AP �0.34; AP �0.46. (C) Pictures showing the extent ofnjection site in the BST of one mouse along the rostral–caudal axis. In this mouse, tracers were injected into both the BSTmp and BSTif. (D) 1–3:hree consecutive parasagittal sections showing that the presence of anterogradely labeled fibers (red) was restricted to the AOB MCL. (4) A zoom-iniew of one area in (3) showing that the centrifugal fiber from the BST did not cross into the GL. (E) A zoom-in view showing the presence of manyaricosities along one anterogradely labeled fiber. Arrows point to three of the varicosities as examples. Scale bars�200 �m in (C), (D 1–3)�100 �m,
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o be detected with small injection sites. Two studies indi-ate that the anterior MeA and the bed nucleus of theccessory olfactory tract provide feedback projection to theOB (Barber, 1982; Martel and Baum, 2009). We occa-ionally observed retrograde labeling in these two nucleiollowing tracer injection into the AOB. However, the label-ng tended to be inconsistent and weak, likely because our

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ig. 3. Tracer injection into the AOB GCL retrogradely labeled neuroesign and results following tracer injection into the AOB GCL. Retromygdala of the ipsilateral side after tract tracer (BDA, MW�3 KD) injhe distribution pattern of retrogradely labeled somata (red circles) inregma, AP �1.82; AP �1.94; AP �2.06. (C) Three consecutive para

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ig. 4. Tracer injection into the vomeronasal amygdala anterogradely labeled axonal fibers in the AOB GCL. (A) A schematic showing thexperimental design and results following tracer injection into the vomeronasal amygdala. After deposit of tract tracers (BDA, MW�10 KD) into theePV and PMCo, anterogradely labeled terminals (crossed lines) were observed only in the ipsilateral AOB GCL. (B) Drawing of three coronal

ections showing the extent of injection sites (red) in the vomeronasal amygdala. The position of the sections, from front to back: Bregma, AP �1.94;P �2.06; AP �2.18. (C) Raw pictures showing the extent of injection sites in the amygdala along the rostral–caudal axis. Tracers were injected intooth the MePV and PMCo. (D) 1–3: Three consecutive parasagittal sections showing that the presence of anterogradely labeled fibers (red) wasestricted to the AOB GCL. (4) A zoom-in view of one area in (3) showing that the feedback fibers from the amygdala did not cross the dLOT. (E) Aoom-in view showing the presence of many varicosities along the anterogradely labeled fibers. Arrows point to four of the varicosities as examples.cale bars�200 �m in (C), (D 1–3)�100 �m, (D4 and E)�20 �m. For interpretation of the references to color in this figure legend, the reader is

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anglia is that the projection neurons of these two structurese different neurotransmitters: the cortical pyramidal neu-ons are glutamatergic and excitatory whereas basal gan-lion projection neurons are GABAergic and inhibitorySwanson, 2000). Whether the feedback projections are glu-amatergic or GABAergic will thus determine the fundamentalign of their computation (excitation or inhibition) in the AOB.

To test whether the feedback projections from the BSTnd vomeronasal amygdala were GABAergic, we injected

racers into either the MCL or the GCL of the AOB fromAD67-GFP knock-in mice. In these mice, GFP expres-

ion is driven by the endogenous GAD67 promoter andhus provides a reliable marker for GABAergic neuronsTamamaki et al., 2003; Bian et al., 2008). Following tracernjections, we examined the presence of GFP in the retro-radely labeled somata in both the BST and the amygdala.ut of five injections into the AOB GCL of GAD67-GFPice, we found that all of the retrogradely labeled somata

n the BST expressed GFP (Fig. 5A; n�54 cells in five micexamined). In contrast, we did not observe any GFP ex-ression in any of the retrogradely labeled somata in theomeronasal amygdala following tracer injections into theOB GCL (Fig. 5B; n�123 cells in two mice examined).hese results thus suggest that the feedback neurons of

he BST are GABAergic, whereas those of the vomerona-al amygdala are not.

We next examined whether the vomeronasal amyg-ala provides a glutamatergic feedback projection to theOB GCL. Following tracer injection into the vomeronasalmygdala (Fig. 5C), we carried out immunostaininggainst VGLUT2, a marker of glutamatergic neurons in theomeronasal amygdala (Fremeau et al., 2001). SinceGLUT2 is a VGLUT that is mainly expressed in axonal

erminals (Herzog et al., 2001), we examined whether thenterogradely labeled terminals in the AOB GCL were

mmunopositive to VGLUT2. Dense VGLUT2 immunore-ctivity was observed in the AOB GCL (Fig. 5D). More

mportantly, numerous varicosities of these anterogradelyabeled terminals were strongly VGLUT2� (Fig. 5D). Thus,he feedback projection from the vomeronasal amygdala tohe AOB GCL is most likely glutamatergic.

any feedback neurons in the BST and vomeronasalmygdala express ER-�

ex steroid hormones play a very important role in mam-alian sociosexual behavior (Remage-Healey and Bass,006). Both the BST and the vomeronasal amygdalaensely express � and � types of nuclear estrogen recep-

ors (Mitra et al., 2003; Merchenthaler et al., 2004). Knock-ng out the ER-� gene results in dramatic deficits in socio-exual behavior in both male and female mice (Ogawa etl., 1997; Wersinger et al., 1997; Couse and Korach,999). Since both the BST and vomeronasal amygdalarovide feedback projections to the AOB, we askedhether these feedback neurons express ER-�. Following

racer injections into the AOB, we examined whether thereas ER-� immunoreactivity in the retrogradely labeledomata in these two areas. We found that in both the BST
nd vomeronasal amygdala, more than half of the retro- v

radely labeled neurons expressed ER-� (Fig. 6; n�50/96ells and three mice for BST and 66/104 cells and two miceor vomeronasal amygdala). Thus, central hormonal cuesay have an effect on modulating the processing of phero-onal signal at an early station such as the AOB by acting

ia the feedback pathways described here.

DISCUSSION

t is a common theme in the sensory systems that cortexrovides heavy feedback projections to early stations suchs the thalamus to modulate signal processing (Alitto andsrey, 2003). The accessory olfactory system is critical forrocessing pheromonal signals that regulate social andeproductive behavior. This system is considered uniqueecause it relays pheromonal signals to hypothalamic be-avioral centers after just three synapses. It also bypassesonventional cortical areas and thus the pheromonal sig-als are thought to mediate behaviors directly and imme-iately. Our results in this study reveal that both the AOBCL and GCL receive feedback projections that are orga-ized in a layer- and neurotransmitter-specific way. Inddition, many feedback neurons in these two areas ex-ress ER-�. These findings have several implications forur understanding of the processing of social and repro-uctive olfactory signals.

ew findings of this study

y combining precise tract tracing, immunohistochemistrynd genetic labeling of GABAergic neurons in this study,e reveal several new features of the organization of these

wo feedback projections (Fig. 7). First we show that therojection from the BST terminates only in the AOB MCL.y making large injections into the olfactory bulb one earlytudy suggested that the AOB receives input from the BST,ut it did not specify the specific layer that receives thisentrifugal input (de Olmos et al., 1978). Our results alsohow that the projection from the vomeronasal amygdalaerminates in the AOB GCL. Using HRP as a retrograderacer, Broadwell and Jacobowitz found that the AOB re-eives centrifugal inputs from the amygdala, locus coer-leus, and raphe nucleus in rabbits (Broadwell and Jaco-owitz, 1976). Several additional studies show that theOB receives input from the vomeronasal amygdala and

ndicate this centrifugal projection likely terminates in theOB GCL (Raisman, 1972; Kevetter and Winans, 1981;arber, 1982). These early observations on the projection

rom the vomeronasal amygdala are now confirmed by ussing more modern techniques and systematic tract trac-

ng. In addition to tract tracing, our results show that theST feedback to the AOB MCL is GABAergic and themygdalar feedback to AOB GCL is glutamatergic. Lastly,e show that more than half of the feedback neurons inoth the BST and the amygdala express ER-�.

unctional implications of our results

eurons in the AOB respond to pheromonal signals (Luot al., 2003; Hendrickson et al., 2008) and project to the
omeronasal amygdala and BST (Scalia and Winans,

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ig. 5. The feedback neurons from the BST to the AOB are GABAergic, while those from the vomeronasal amygdala are likely glutamatergic.A) BST feedback neurons were retrogradely labeled by tracer injection into the AOB (red in left-hand panel). GABAergic neurons were markedy the presence of GFP in the GAD67-GFP knock-in mice. We observed the presence of GFP in all of these retrogradely labeled neurons in

he BST (green in the middle panel). Right hand panel shows the overlay of the tracer labeling and GFP labeling. Insets show the zoom-in viewf the same neuron pointed by arrows. (B) In contrast, none of the retrogradely labeled neurons in the vomeronasal amygdala wereFP-positive. (C) Three coronal sections showing one injection site of anterograde tracers (TRDA, MW�10 KD) in both the MePV and theMCo. (D) The anterogradely labeled fibers in the AOB GCL (red, left-hand panel) were positive for VGLUT2 (green, middle panel). Panel at

ight shows the overlay of anterograde labeling and VGLUT2 staining, demonstrating the colocalization of the tracer and VGLUT2 protein. Insetshow the zoom-in view of the fibers pointed by arrows in the AOB GCL. Scale bars�50 �m in (A and B) and in insets�10 �m, in (C)�200 �m,n (D)�20 �m and in insets�10 �m. For interpretation of the references to color in this figure legend, the reader is referred to the Web version
f this article.

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975). It is interesting to note that although the entireomeronasal amygdala receives input from the AOB, onlyome subnuclei project back to the AOB. The source ofeedback in the vomeronasal amygdala includes the MePVnd PMCo but not the anterior subnucleus and the medial

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ig. 6. Many feedback neurons in the vomeronasal amygdala and BSany retrogradely labeled somata (red, left-hand panel) were positive

or retrograde labeling and ER-� immunostaining. Insets show the zooeedback neurons in the vomeronasal amygdala were immunopositivehe references to color in this figure legend, the reader is referred to

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ig. 7. A schematic diagram summarizing the major findings of thistudy. The feedback neurons in the BST project specifically to the AOBCL and are GABAergic while those in the vomeronasal amygdalaroject only to the AOB GCL and are glutamatergic. Moreover, many

If the feedback neurons in both the BST and vomeronasal amygdalaxpress ER-� (white diamonds).


mygdala, posteriodorsal subnucleus (MePD). Some stud-es suggest functional specialization for these subnuclei.or example, the MePV is thought to be more involved inefensive behavior and mainly receives input from theosterior AOB that is associated with V2R family of theomeronasal receptor genes (Belluscio et al., 1999; Delunta et al., 2002; Choi et al., 2005; Martinez-Marcos,008). In contrast, the MePD is believed to be more in-olved in mating behavior and mainly receives input fromhe anterior AOB that is associated with the V1R geneamily (Belluscio et al., 1999; Del Punta et al., 2002; Mar-inez-Marcos, 2008). It is thus possible that signals impor-ant for defensive behavior are sent back to the AOB toodulate the processing of all pheromonal signals. We

how that the vomeronasal amygdala provides a glutama-ergic and most likely excitatory projections to the GCL.ranule cells in the GCL are in turn GABAergic and inhibitory

Shepherd et al., 2004). A key feature of AOB mitral cells liesn their lack of extensive basal dendrites (Del Punta et al.,002; Ma and Lowe, 2004), and the GABAergic synapses

ormed between granule cells and mitral cells are mainlyocated on the apical dendrites. Thus the GABAergic granuleells in the AOB could gate the sensory input to mitral cells by

nhibition on apical dendrites. A recent study shows thatABAergic circuits in the AOB sharpen the response selec-

ivity of mitral cells to pheromones (Hendrickson et al., 2008).

pha Merge

mmunopositive for ER-�. (A) Following tracer injection into the AOB,(green, middle panel). Right hand panel shows the overlay of images

of a small area pointed by arrows in the panels. (B) Similarly, many. Scale bars�50 �m in (B) and 10 �m in insets. For interpretation ofversion of this article.

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nasal amygdala can have a critical control on the sensoryesponses of AOB mitral cells.

The BST is another brain center that receives directnput from the AOB and contributes to regulating sociosex-al behavior in mammals (Wood and Swann, 2005). It also

nteracts heavily with the vomeronasal amygdala (Dong etl., 2001; Wood and Swann, 2005). Our results demon-trate that it provides a GABAergic output to the AOB MCL.ome studies indicate that the posterior division of BST is

mportant for defensive behavior (Dong and Swanson,004). In contrast, tracing studies suggest that the BSTeceives input from anterior AOB, which also projects tohe MePD that is believed to be involved in mating behav-or (Choi et al., 2005; Martinez-Marcos, 2008). No matterhat the nature of the feedback signals is, our data show

hat this projection is GABAergic and thus most likelynhibitory. It is possible that BST feedback projection in-ibits mitral cells by directly acting on the dendrites andomata of these cells. Alternatively, it can synapse on theendrites of granule cells and thus inhibits GABAergic gran-le cells and then disinhibits mitral cells. Several recent stud-

es suggest that the BST provides feedback projections to thehalamus in other sensory systems, suggesting that the BSTontributes to the modulation of sensory signals by animal’snternal states, such as energy balance (Dong and Swanson,006; Shin et al., 2008). It will be interesting to test whetherhe BST feedback projections to thalamus in other sensoryystems are also GABAergic.

The feedback neurons may be activated by signalsrom other sensory modalities. Although the accessorylfactory system is specialized for processing pheromonalignals, mammalian sociosexual behaviors are known toe regulated by other sensory signals, such as the phero-onal signals detected by the main olfactory system, theltrasonic calls made by other conspecific individuals, andomatosensory signals (Stern, 1990; Holy and Guo, 2005;artel and Baum, 2009). The vomeronasal amygdala is

nown to receive volatile pheromonal signals from theOB (Schaefer et al., 2001; Pro-Sistiaga et al., 2007;ang et al., 2009). One recent study using c-Fos mappinghows that the feedback neurons to the AOB can bectivated by pheromonal signals processed by the mainlfactory system (Martel and Baum, 2009). It will be inter-sting to test whether the feedback neurons in the BSTnd the amygdala can respond to social and reproductiveignals from auditory, somatosensory, and visual systems.n addition, it will be interesting to test the physiologicalffects of the sensory signals from other modalities on therocessing of pheromonal signals in the AOB.

Sociosexual behaviors require proper integration ofheromonal signals and hormonal cues (Wood and New-an, 1995a; Remage-Healey and Bass, 2006). In rodents,

he BST and vomeronasal amygdala are considered to beotential sites for this integration because both of thesewo nuclei receive pheromonal input from the AOB andxpress high density of receptors for sex steroid hormonesKrieger et al., 1976; Wood and Newman, 1995a,b). Ouresults show that a substantial number of feedback neu-
ons express ER-�, which is extremely important for reg-

lating the reproductive behavior of both males and fe-ales (Wersinger et al., 1997). Some of the testosteroneffects are believed to reflect the action of estrogen formed

n the amygdala via the aromatization of testosteroneSimpson et al., 1994). Thus, these feedback projectionsrovide a potential mechanism by which central hormonalues can modulate the processing of pheromonal signalst stage as early as the AOB.

cknowledgments—We thank Yuchio Yanagawa for GAD67-GFPice and A. L. Person for critical reading of the manuscript. This

tudy was supported by grants from China Ministry of Science andechnology, Natural Science Foundation of China, and Humanrontier Science Program to M.L.

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(Accepted 23 March 2009)


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ARTICLE IN PRESS - NIBS · 2009-05-07 · Please cite this article in press as: Fan S, Luo M, The organization of feedback projections in a pathway important for processing pheromonal