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Efferent and Afferent Connections of the Olfactory Bulb and Prepiriform cortex in the Pigeon (Columba livia) Yasuro Atoji,1* and J. Martin Wild2 1 2

Laboratory of Veterinary Anatomy, Faculty of Applied Biological Sciences, Gifu University, Gifu 501–1193, Japan Department of Anatomy, Faculty of Medical and Health Science, University of Auckland, Auckland 92019, New Zealand

ABSTRACT Although olfaction in birds is known to be involved in a variety of behaviors, there is comparatively little detailed information on the olfactory brain. In the pigeon brain, the olfactory bulb (OB) is known to project to the prepiriform cortex (CPP), piriform cortex (CPi), and dorsolateral corticoid area (CDL), which together are called the olfactory pallium, but centrifugal pathways to the OB have not been fully explored. Fiber connections of CPi and CDL have been reported, but those of other olfactory pallial nuclei remain unknown. The present study examines the fiber connections of OB and CPP in pigeons to provide a more detailed picture of their connections using tract-tracing methods. When anterograde and retrograde tracers were injected in OB, projections to a more extensive olfactory pallium were revealed, including the anterior olfactory nucleus,

CPP, densocellular part of the hyperpallium, tenia tecta, hippocampal continuation, CPi, and CDL. OB projected commissural fibers to the contralateral OB but did not receive afferents from the contralateral olfactory pallium. When tracers were injected in CPP, reciprocal ipsilateral connections with OB and nuclei of the olfactory pallium were observed, and CPP projected to the caudolateral nidopallium and the limbic system, including the hippocampal formation, septum, lateral hypothalamic nucleus, and lateral mammillary nucleus. These results show that the connections of OB have a wider distribution throughout the olfactory pallium than previously thought and that CPP provides a centrifugal projection to the OB and acts as a relay station to the limbic system. J. Comp. Neurol. 522:1728–1752, 2014. C 2013 Wiley Periodicals, Inc. V

INDEXING TERMS: fiber organization; olfactory pallium; limbic system; bird

In vertebrates olfactory stimuli in the form of chemical molecules interact with olfactory receptors on sensory cells in the olfactory epithelium and, in some species, also in the vomeronasal epithelium. Olfactory nerves then project to the main olfactory bulb (MOB) and, when a vomeronasal system is present, also to the accessory olfactory bulb (AOB; Baxi et al., 2006; Eisthen and Polese, 2007). The MOB exists in all vertebrates, but the AOB first appears in amphibians and is present in reptiles and mammals but is absent in birds. The size, localization, and lamination of the MOB and AOB vary among species and appear to be related to the behavioral patterns and living environment of each species (Eisthen and Polese, 2007). The oft-assumed equivalence of the AOB and pheromone detection and processing is not supported by the evidence (Baxi et al., 2006).

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In amniotes, the MOB projects to the olfactory cortex via the lateral, intermediate, or medial olfactory tracts. In reptiles, the MOB projects to the lateral cortex or piriform cortex, amygdala, olfactory tubercle, hippocampus, anterior olfactory nucleus, and nucleus of the lateral olfactory tract (Skeen et al., 1984; Reiner and Karten, 1985; Lohman and Smeets, 1993). The intermediate olfactory tract travels contralaterally to the lateral cortex. In mammals, the MOB projects to the lateral and medial olfactory cortices (Shipley et al., 2004). The

Grant sponsor: Ministry of Education, Culture, Sports, Science and Technology of Japan; Grant number: 21580360 (Y.A.). *CORRESPONDENCE TO: Yasuro Atoji, Laboratory of Veterinary Anatomy, Faculty of Applied Biological Sciences, Gifu University, Yanagido 1-1, Gifu 501–1193, Japan. E-mail: [email protected] Received April 16, 2013; Revised November 4, 2013; Accepted November 7, 2013. DOI 10.1002/cne.23504 Published online November 13, 2013 in Wiley (wileyonlinelibrary.com)

The Journal of Comparative Neurology | Research in Systems Neuroscience 522:1728–1752 (2014)

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Olfactory system in pigeons

lateral olfactory cortex consists of the anterior olfactory nucleus (AON), olfactory tubercle, piriform cortex (CPi), entorhinal cortex, nucleus of the lateral olfactory tract, periamygdaloid cortex, and cortical part of the amygdala (Broadwell and Jacobowitz, 1976; de Olmos et al., 1978; Luskin and Price, 1983; Shipley and Adamek, 1984; Carmichael et al., 1994). AON, CPi, and entorhinal cortex project back upon the main olfactory bulb. The MOB also receives commissural afferents from the contralateral AON. The medial olfactory cortex is composed of the indusium griseum, anterior hippocampal continuation, tenia tecta, infralimbic cortex, and olfactory tubercle (Shipley et al., 2004). Feedback to the main olfactory bulb from the olfactory cortex is essential for the control and integration of olfactory information. Olfaction in mammals is an important sense involved in emotional and neuroendocrine responses, species recognition, maternal functions, reproduction, and food selection and appreciation (Brown and Macdonald, 1985). For birds, recent evidence shows that, contrary to expectations, many species possess an extensive array of functional olfactory receptor genes, indicating that the sense of smell is well developed in birds generally (Steiger et al., 2008). Electrophysiological studies in chickens and pigeons show that odor stimuli evoke neuronal activity in the olfactory bulb (OB) or brain (Oosawa et al., 2000; McKeegan, 2002). In wild birds, odor recognition of partners or nests is reported for the blue tit and Antarctic prion (Bonadonna and Nevitt, 2004; Mennerat, 2008), and olfaction is used during foraging in the wandering albatross (Nevitt et al., 2008). Behavioral studies using nostril occlusion, olfactory nerve section, or lesions of the OB or CPi also indicate that, as in mammals, olfaction plays a critical role in reproduction and feeding (Balthazart and Schoffeniels, 1979; Jones and Roper, 1997; Hirano et al.,

2009; for reviews see Roper, 1999; Hagelin, 2006; Hagelin and Jones, 2007). Evidence accumulated over 40 years strongly supports the idea that olfaction is involved in navigation, especially in homing pigeons (Papi et al., 1971; Grubb, 1974; Papi, 1990; Wallraff, 2001, 2005; Gagliardo et al., 2006, 2008, 2009, 2011a,b; Ioale` et al., 2008). In general, however, neuronal circuits in the avian olfactory brain are less well understood than those in mammals. In pigeons, the OB projects to the prepiriform cortex (CPP), CPi, nucleus taeniae of the amygadala (TnA), and septum (Rieke and Wenzel, 1978; Reiner and Karten, 1985; Patzke et al., 2011). The first two, CPP and CPi, appear to be members of the lateral olfactory pallium in birds but it is not known whether these are the only members. The medial wall of the hemisphere and retrobulbar regions are assumed to connect with the OB, as in mammals and reptiles. In fact, injections of tritiated amino acids in the pigeon OB yield a distribution of silver grains over the medial wall and retrobulbar regions on autoradiograms, although the authors did not comment on labeling there (Reiner and Karten, 1985). On the basis of anterograde tracing, the OB receives input from CPi (Bingman et al., 1994), but other centrifugal projections from the lateral olfactory pallium have not been found. Furthermore, data on the medial olfactory pallium are not available. Experiments using retrograde tracer injections directly into the OB are needed to explore the full extent of the avian olfactory pallium and its centrifugal projections to the OB. The pigeon OB gives rise to heavy projections to CPP and CPi bilaterally (Bingman et al., 1994; Patzke et al., 2011). In the Karten and Hodos (1967) atlas of the pigeon brain, CPP appears to be a layered structure located in the rostral telencephalon, dorsal to the OB CPi is present on the lateral or ventrolateral surface of the caudal telencephalon, but CPP and CPi are

Abbreviations AD AI AON Bas CDL CH CPi CPP DMA DMP E HA HC HD HF IOT IR LHy LOT LR LSt

dorsal arcopallium intermediate arcopallium anterior olfactory nucleus basorostral pallial nucleus dorsolateral corticoid area habenular commissure piriform cortex prepiriform cortex dorsomedial anterior thalamic nucleus dorsomedial posterior thalamic nucleus entopallium apical part of the hyperpallium hippocampal continuation densocellular part of the hyperpallium hippocampal formation intermediate olfactory tract intermediate ridge lateral hypothalamic nucleus lateral olfactory tract lateral ridge lateral striatum

LVa M ML MOT MR MSt MVa N NC NCL OB OM PoA S SL SM TnA TT TuO V VTA

lateral vallecula mesopallium lateral mammillary nucleus medial olfactory tract medial ridge medial striatum medial vallecula nidopallium caudal nidopallium caudolateral nidopallium olfactory bulb occipitomesencephalic tract posterior pallial amygdala septum lateral septum medial septum nucleus taeniae of the amygadala tenia tecta olfactory tubercle lateral ventricle ventral tegmental area

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TABLE 1. Antibody Characterization Antigen Cholera toxin subunit B Parvalbumin

Immunogen

Manufacturer

Dilution

Purified CTB isolated from Vibria cholerae Purified parvalbumin from carp muscle

List Biological Laboratories (Campbell, CA), goat polyclonal, catalog No. 703 SWant (Bellinzona, Switzerrland), mouse monoclonal, catalog No. 235

1:40,000

separated rostrocaudally by some 7.5 mm, and no morphological relation between them has been noted. Fiber connections of CPi with olfactory-related or limbic areas in the telencephalon have been reported (Bingman et al., 1994; Patzke et al., 2011), but those of CPP remain unknown. In the present study, we used tracttracing methods to examine the efferents and afferents of OB and CPP in pigeons to provide more detailed knowledge of their connectivity.

MATERIALS AND METHODS Twenty-three pigeons of both sexes (weighing 249– 401 g) were used. Surgical procedures were approved by the committee for animal research and welfare of Gifu University.

Tract tracing Pigeons were anesthetized with a mixture of ketamine hydrochloride (50 mg/kg) and xylazine (20 mg/kg) and placed in a stereotaxic instrument (Narishige, Tokyo, Japan) such that the beak was parallel to the horizontal axis. Each animal received bilateral injections of two different tracers into the OB and prepiriform cortex, namely, biotinylated dextran amine (BDA; 10,000 molecular weight; Molecular Probes, Eugene, OR) on the right and cholera toxin B subunit (CTB; choleragenoid; List Biological Laboratories, Campbell, CA) on the left. BDA (10% in phosphate-buffered saline [PBS], pH 7.4) was used primarily for anterograde labeling, and CTB (1% in PBS) was used primarily for retrograde labeling. These were iontophoretically injected into the right and left sides, respectively, through glass micropipettes (outer diameter 10–15 lm, 4 lA positive current, 5 seconds on, 5 seconds off for 12–15 minutes). After a survival time of 7 days, the animals were anesthetized with sodium pentobarbital (50 mg/kg) and perfused with Ringer’s solution, followed by 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. Brains were removed and stored in the same fixative for 3–6 weeks until processing. They were then transferred to 30% sucrose in PBS at 4 C for 3 days and then cut transversely at 50 lm thickness on a cryostat. Both tracers were visualized in the same sections in sequential fashion: BDA first and CTB second. Sections were pretreated with 50% methanol in PBS containing 0.3% H2O2

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1:20,000

for 30 minutes. After being washed in PBS, they were incubated with streptavidin-peroxidase conjugate (Molecular Probes; 1:1,000) in PBS containing 0.3% Triton X-100 for 1 hour at room temperature, followed by 3,30 -diaminobenzidine tetrahydrochloride (DAB; 20 mg/100 ml) containing 0.0045% H2O2 and CoCl2 (15 mg/100 ml) in 0.1 M Tris-HCl buffer at pH 7.4. This resulted in a black reaction product. After washing thoroughly in PBS, the sections were incubated in PBS containing a goat anti-CTB antibody (List Biological Laboratories; lot No. 7032A3; 1:40,000), 0.3% Triton X-100, and 1% normal rabbit serum overnight at room temperature, followed by a biotinylated rabbit anti-goat IgG (Sigma, St. Louis, MO; 1:500) for 1 hour at room temperature, and finally incubated in streptavidin peroxidase conjugate for 1 hour at room temperature. The CTB antibody was raised against purified cholera toxin B subunit protein (choleragenoid) in goats (Table 1). CTB-peroxidase was visualized by DAB and H2O2, which resulted in a brown reaction product. Sections were then mounted, dehydrated, and coverslipped with DPX. All injection sites were confirmed in adjacent sections counterstained with 0.1% cresyl violet. Three immunohistochemical controls for CTB were carried out. First, nonimmune goat serum was incubated instead of the anti-CTB antibody. Second, sections without CTB injection were immunostained with the anti-CTB antibody. Third, sections were incubated with the primary antibody that had been preincubated with CTB (50 pg/1 ml). No specific reaction was observed in any of these three control sections.

Immunohistochemistry Two pigeons without tracer injections were perfused with 4% paraformaldehyde, and frozen brains were cut at 50 lm thickness as described above. Some floating sections were pretreated with 50% methanol containing 0.9% H2O2 for 30 minutes. After being washed in PBS, they were incubated with 1% normal horse serum and then incubated in PBS containing a mouse antiparvalbumin antibody (SWant, Bellinzona, Switzerland, lot No. 10–11F; Table 1) and 0.3% Triton X-100 at 4 C overnight. The sections were rinsed with PBS, incubated with a biotinylated horse anti-mouse IgG (Vector, Burlingame, CA; 1:500) for 1 hour at room temperature, and

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finally incubated in avidin-biotin-horseradish perioxidase complex (ABC Elite Kit; Vector) for 1 hour at room temperature. The peroxidase was visualized by DAB (20 mg/100 ml) containing 0.0045% H2O2 in 0.1 M Tris-HCl buffer at pH 7.4. Sections were then washed in PBS, mounted, dehydrated, and coverslipped with DPX. In controls, the primary antibody was replaced by nonimmune mouse antibody.

In situ hybridization Digoxigenin (DIG)-labeled antisense and sense RNA probes were made from purified vesicular glutamate transporter 2 (vGluT2) PCR product using a DIG RNA labeling kit (Roche, Mannheim, Germany) for nonradioisotope in situ hybridization. Briefly, forward primer (50 GGGGGACAAATTGCAGACTT-30 ; bases 1164–1183 of FJ_428226) attached T7 promoter sequence in 50 terminal (50 -TAATACGACTCACTATAGGG-30 ) and reverse primer (50 -TTCGCTTGTCTGTTCAGGGTCT-30 ; bases 1526–1547 of FJ_428226) attached Sp6 promoter sequence in 50 terminal (50 -ATTTAGGTGACACTATAGAA-30 ) were used for RT-PCR to obtain vGluT 2 PCR product (444 bp) from total RNA extracted from pigeon brains. The PCR product was purified using Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI), and 1 lg of purified PCR product was mixed with reagents of DIG RNA labeling kit; 2 ll of 103 NTP labeling mixture, 2 ll of 103 transcription buffer, 1 ll RNase inhibitor, 2 ll polymerase T7 for making sense or polymerase Sp6 for making antisense RNA probe, and RNase-free water to be a final volume as 20 ll. After incubation at 37 C for 4 hours, 1.25 ll of 8 M LiCl and 49 ll prechilled 100% ethanol were added, and the mixture was incubated again at 220 C overnight. Then, synthesized RNA transcript was collected by centrifugation at 13,000g for 15 minutes at 4 C, aliquoted into 100 ng/ll by RNase-free water, and stored at 260 C until use. For nonradioisotope in situ hybridization, two pigeons were anesthetized with sodium pentobarbital (50 mg/kg) and perfused with Ringer’s solution, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). Brains were removed and transferred to 30% sucrose in 0.1 M PB at 4 C for 3 days. The brains were cut transversely at 30 lm on a cryostat, mounted onto 3aminopropyltriethoxysilane-coated slides, and stored at 230 C. After being fixed again in 4% paraformaldehyde in 0.1 M PB for 20 minutes (all steps were performed at room temperature unless otherwise indicated) and rinsed in 0.1 M PB for 20 minutes, the sections were digested with proteinase K (Dako, Glostrup, Denmark) at the concentration of 15 lg/ml in PBS at 37 C for 30 minutes. After stopping the digestion by rinsing in cooled PBS, the sections were then acetylated in a solution com-

posed of 1.35% of trithanolamine, 0.25% of acetic anhydrite, and 0.058% of hydrochloric acid in RNase-free water for 10 minutes. Then, the sections dehydrated through an ethanol series and hybridized with antisense or sense DIG-labeled riboprobes (1.0 lg/ll) dissolved in a hybridization buffer composed of 20% dextran sulfate (Nacalai Tesque, Kyoto, Japan), 50% formamide (Nakalai Tesque), 2% blocking solution (pH 7.5; Roche), 0.01% N-lauroylsarcosine (NLS; Nacalai Tesque), and 0.01% of sodium lauryl sulfate (SDS; Nakalai Tesque) in 53 standard saline citrate (SSC; pH 7.4; 13 SSC contains 0.15 M sodium chloride and 0.015 M sodium citrate). The sections were first heated on a heat plate (95 C) for 4 minutes and then incubated at 55 C overnight. After the hybridization, the sections were rinsed in a solution composed of 50% formamide and 0.01% NLS in 23 SSC at 65 C for 30 minutes and incubated with RNase A (20lg/ml; Roche) in NTE buffer (500 mM NaCl, 10 mM Tris, and 1 mM EDTA, pH 8.0) at 37 C for 30 minutes. After being rinsed in Tris-buffered saline (TBS; pH 7.4; 25 mM Tris, 1.37 mM NaCl, and 0.27 mM KCl) containing 0.025% of Tween 20 (TBST), the sections were incubated with a blocking solution composed of 1% blocking reagent (Roche) and 2% normal sheep serum in TBS for 60 minutes. After rinsing in TBST, the sections were incubated with alkaline phosphatase-labeled anti-DIG antibody (Roche; 1:2,000) at 4 C overnight. After being rinsed in TBST, the sections were visualized by incubation with a mixture of nitroblue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate toluidine salt (BCIP; Roche) in a detection buffer (0.1 M NaCl, 50 mM MgCl2, and 0.1% Tween 20 in 0.1 M Tris, pH 9.5) overnight under the light-shielding condition. The sections were then rinsed in PBS and distilled water, dehydrated through a graded ethanol series, cleared with xylene, and coverslipped with Canada balsam. The nomenclature used in the present study is based on that of the Avian Brain Nomenclature Forum (Reiner et al., 2004) and Karten and Hodos (1967). Photomicrographs were taken with a digital camera (DS-Fi1; Nikon, Tokyo, Japan) mounted on a light microscope. Adjustment for contrast, brightness, and sharpness of microphotographs and layout and lettering were performed in Adobe Photoshop 7.0J (Adobe, Tokyo, Japan) and Adobe Illustrator 10.0J. Some photomicrographs at high magnification were superimposed using a software “Sensiv measure” (Mitani Co., Fukui, Japan) to superimpose a few microphotographs into one. Schematic illustrations of labeled neurons and fibers were drawn in Adobe Illustrator 10.0J. To describe additional results of labeling in CPP produced by injections of BDA and CTB in the hippocampal formation, dorsolateral corticoid area, and septum, sections from three cases (P-99, P-106, P-114), used in

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previous studies published in this journal (Atoji and Wild, 2004, 2005) were used.

RESULTS We found that the nomenclature identifying retrobulbar olfactory regions in the atlas of the pigeon brain by Karten and Hodos (1967) to be inadequate for our purposes, so we first describe additional structures at the macro- and microscopic levels. Anatomically, two furrows run rostrocaudally on the dorsal surface of the rostral telencephalon. The medial furrow is wider and longer than the lateral and we named it the medial vallecula. The latter we name the lateral vallecula (Fig. 1A,B); it houses a large vein, visible to the naked eye. Three ridges are thereby formed on the dorsal surface of the telencephalon: the medial ridge, which is the sagittal elevation or Wulst; intermediate ridge; and lateral ridge (Fig. 1A–C). Microscopically, certain nuclei or tracts were related to the olfactory system as a result of our tracing experiments, and their positions are described in Nisslstained sections: prepiriform cortex (CPP), anterior olfactory nucleus (AON), tenia tecta (TT), hippocampal continuation (HC), intermediate olfactory tract (IOT), lateral olfactory tract (LOT), and medial olfactory tract (MOT).

CPP The CPP is composed of three rostrocaudal parts. A rostral part is an obvious cell layer densely packed with basophillic neurons. The layer starts from the medial telencephalic wall at the OB peduncle, runs laterally in an arc over the lateral ventricle, curves dorsolaterally, and extends toward the lateral ridge (Fig. 1D–G). Here, an intermediate part of CPP is formed, which is wider than the layered part, and its cells are much more loosely packed, rendering it lighter than the surrounding nidopallium (Fig. 1B,C). Because it is deltoid in shape in coronal section, we call this intermediate part the sector part of CPP. As the sector part moves caudally, it becomes smaller and gradually approximates the ventral wall of the telencephalon at levels of A 13. More caudally still, the sector part is replaced by a small aggregate of neurons that extends caudally as far as the piriform cortex, approximately at levels of A 8 (Fig. 1H–M). This caudal part of CPP we call the cord. In situ hybridization showed that the most intense expression of vGluT2 mRNA was in CPP and OB (Fig. 2A–L). In CPP, vGluT2 expression was localized to the CPP layer (Fig. 2A) and sector (right pallium in Fig. 2A,B; see also Fig. 2C–H). In the CPP cord, vGluT2 expression was still found rostrally (Fig. 2I–L), but more caudally it became difficult to find. In controls, DIG-labeled sense probes did not yield any staining reaction.

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AON The AON is located between the layered part of CPP ventrally and the mesopallium dorsally. It appears lightly stained because of its low cell density (Figs. 1B,E, 3A,B).

TT The TT is located in the lower part of the medial wall of the telencephalon between A 14.00 and A 12.50 (Fig. 3A–C). Caudal to A 12.50, TT becomes thin and gradually disappears, to be replaced by the lateral septum. Neurons in TT are homogeneously distributed. Immunohistochemistry using an antiparvalbumin antibody showed a chemocytoarchitectural difference between TT and septum. Small numbers of stellate neurons were immunoreactive in TT (Fig. 3D,E), but no immunoreactivity was seen in the septum (Fig. 3F). In situ hybridization showed that vGluT2 mRNA was intensely expressed in TT neurons between A 14.00 and A12.50 but was found in very few neurons of the striatum and septum (Fig. 3G–I).

HC The HC appears approximately at A 13.00 as a thin layer of darkly stained, large neurons beginning immediately dorsal to TT in the medial wall. This layer becomes larger caudally, and continues with no discernible boundary to the V-shaped layer of the hippocampal formation (for subdivision of the hippocampal formation see Atoji and Wild, 2004; Fig. 3C). The rostral limit of the hippocampal formation in pigeons was estimated at A 10.25–10.75 by Atoji et al. (2002). Therefore, we call the medial wall structure dorsal to TT at A 13.00–11.00 the hippocampal continuation.

IOT The IOT is a thick fiber bundle located below the layered part of CPP near the OB peduncle. It extends approximately from A 14.50 to A 13.25 (Figs. 1A,B, 3A,B). Caudally, the bundle becomes flattened as it travels along the ventral surface of the telencephalon between the medial striatum and the nidopallium at rostral telencephalic levels. However, it is difficult to trace intermediate or caudal parts of IOT in Nisslstained sections, but the tract is revealed by BDA anterograde labeling as described below.

LOT The LOT is a thin layer located at the edge of the lateral hemisphere. It runs between CPP and the pia mater (Fig. 1L,M).

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Figure 1. Retrobulbar regions stained with cresyl violet. A: Transverse section at A 14.40. The CPP layer expands from the medial surface to the lateral surface. B: A section slightly caudal to A, at A 14.00, in which the CPP sector is shown at its largest in transverse section. C: Enlargement of the boxed area in B. The CPP sector in the lateral ridge appears to be lighter and less dense than the adjacent mesopallium. D–K: Rostrocaudal series of oblique sections through the hemisphere taken at the planes indicated by solid lines in D. The CPP layer (arrows) arcs between medial and lateral surfaces in E, shifts gradually to the dorsolateral wall in F–H, and moves onto the ventral surface in I,J. More caudally CPP disappears and is displaced by CPi (K; arrowheads). L: Low magnification of the ventrolateral telencephalon at A 10.50. M: Enlargement of the boxed area in L. The CPP cord is a cell aggregate separated from the nidopallium by a cell-poor layer (asterisk). The lateral olfactory tract (LOT) is a thin layer between CPP and the pia mater. Arrowheads in A and B indicate the medial olfactory tract in the medial wall. For abbreviations see list. Scale bars 5 1 mm in A,B,E–G,L; 500 lm in H–K; 250 lm in C,M. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Y. Atoji and J.M. Wild

Figure 2. A–L: In situ hybridization of vGluT2 mRNA. CPP is shown at low (A,G,I,K) and high (B,H,J,L) magnification in the rostrocaudal direction. Three parts of CPP layer (A,B), CPP sector (A,B,G,H), and CPP cord (I–L) express intense vGluT2 mRNA (arrows in B,H,J,L). Left hemisphere in A indicates that vGluT2 expression in the CPP layer continues in the CPP sector (arrow). Four consecutive sections (C–F) show transition of the layer (double-headed arrows) and sector (single-headed arrows) regions of the right CPP in A in the caudal–rostral direction. The circle in F indicates the point at which the two parts unite at the rostral pole of the telencephalon. For abbreviations see list. Scale bars 5 1 mm in A,C–G,I,K; 200 lm in B,H,J,L. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

MOT The MOT lies on the medial edge of the OB peduncle and TT (at arrowheads in Figs. 1A,B, 3A,B).

Injections into the OB Ten pigeons received injections of CTB and BDA into the OB. From these, the case with the best labeling

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(case P-228) was selected to depict and describe the results. CTB retrograde labeling. CTB was placed in a middle portion of the left OB. The distribution of CTB-labeled neurons is shown schematically in Figure 4. CTB spread within the OB but did not leak to retrobulbar regions (Figs. 4A, 5A). In the retrobulbar regions, heavy labeling was collectively seen in neurons of CPP, AON, TT, HC, and the densocellular part of the hyperpallium (HD;

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Figure 3. A–C: Position of AON, TT, and HC. Arrowheads in A and B indicate the medial olfactory tract in the medial wall. D: Parvalbumin immunostaining in a retrobulbar region. E: Enlargement of the boxed area in D. Several neurons (arrows) in TT are immunoreactive for parvalbumin. F: No parvalbumin immunoreactivity is seen in the lateral septum (SL). G–I: In situ hybridization of vGluT2 mRNA. Intense expression of vGluT2 is found in TT (arrows in G,H) but is nearly negative in the medial striatum (MSt) and lateral septum (SL) in H (A 13.00) and I (A 8.00). G: A 13.25. For abbreviations see list. Scale bars 5 1 mm in A–C; 500 lm in D,F–I; 50 lm in E. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figs. 4B–F, 5B). The HC contained CTB-labeled neurons at A 12.75–11.00 (Fig. 5C). Labeled neurons in HD were numerous ventromedially but became fewer dorsolaterally (Figs. 4B–D, 5B,D). Labeling in HD ended caudally at A 12.50–12.25. In CPP, the three parts of layer, sector, and cord contained CTB-labeled neurons (Figs. 4B–G, 5B,E,F, 6A,B). Labeled neurons in AON were homogeneously distributed (Fig. 5B). Sparse labeling was found in a ventromedial portion of the mesopallium, sandwiched between AON and HD (Figs. 5B, 6C). Labeling in TT expanded at A 13.00–12.75 (Fig. 6D). Caudally, a small area adjacent to IOT described below contained several labeled neurons, and the labeling increased approximately between A 12.50 and A 9.00. The olfactory tubercle and nucleus of the diagonal band contained several labeled neurons at the ventral edge of the telencephalon (Fig. 4G). In middle and caudal regions of the telenceph-

alon, small to moderate numbers of CTB-labeled neurons were found in CPi and the dorsolateral corticoid area (CDL). Labeled cells in the latter increased in number at A 5.00–3.00 (Figs. 4H–J, 6E). There were a few CTBlabeled cells in TnA and the medial arcopallium. In the contralateral OB, very few CTB-labeled neurons could be detected in the mitral cell or glomerular layers (Fig. 6F), because they were obscured by the BDA injection in the OB on that side. When a CTB injection was made in the OB that was not accompanied by a BDA injection in the contralateral OB, many retrogradely labeled neurons were found in the OB contralateral to the CTB injection (Fig. 6G,H). In the brainstem, a few neurons were labeled in the ventral tegmental area ipsilaterally (Fig. 6I). A few labeled neurons were also found in the periaqueductal gray at the levels of the caudal (A6) and rostral (A8) parts of the locus coeruleus. A6 and A8 themselves

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Figure 4. A–J: Schematic illustration of the rostrocaudal extent of labeling following injections of BDA in the right olfactory bulb and CTB in the left olfactory bulb. Case P-228. The core of the injection sites is marked in red (CTB) and in black (BDA), whereas the hatched area around the core indicates spread of the tracer. Dots represent neurons retrogradely labeled with CTB on the left side, and fibers and terminals anterogradely labeled with BDA are represented by short lines. One dot indicates 10 CTB-labeled neurons. BDA-labeled lines indicate the relative difference in distributions but do not represent real numbers. For abbreviations see list. Scale bar 5 1 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Figure 5. CTB and BDA labeling. A: Injection sites of CTB (left) and BDA (right) in the olfactory bulbs. B: Heavy CTB and BDA labeling in the retrobulbar region at A 14.25. C: CTB-labeled neurons (arrows) in the hippocampal continuation (HC) at A 12.00 (section turned 90 counterclockwise). D: Enlargement of the boxed area in B. CTB-labeled neurons in HD. Arrows show contralateral, BDA-labeled, varicose fibers passing through HD. E: Enlargement of the boxed area in B. Many CTB-labeled neurons are seen in the CPP sector and contralateral; BDA-labeled fiber bundles travel in the IOT. F: CTB-labeled neurons in the CPP sector and contralateral BDA-labeled fibers (arrowheads) at A 14.25. For abbreviations see list. Scale bars 5 1 mm in A,B; 500 lm in C; 50 lm in D; 100 lm in E,F. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

showed bilateral but predominantly ipsilateral labeling (Fig. 6J,K). BDA anterograde labeling. BDA was injected in a middle portion of the right OB. The distribution of BDAlabeled fibers is shown schematically in Figure 4. The tracer spread within the OB but did not extend to retrobulbar regions (Figs. 4A, 5A). The olfactory peduncle was crowded with a huge number of thick fibers and

thin, varicose fibers from the OB. Three main streams could be traced toward the olfactory pallium. In mediolateral order, the first stream took a medial course. It first traveled in the olfactory peduncle medially, reached the medial wall of the hemisphere, and formed a densely packed sheet of labeled fibers immediately under the pia mater (Figs. 4B–F, 6D, 7A). This is the MOT. Labeling in the MOT continued caudally until

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Figure 6. CTB and BDA labeling. Case P-228. A: Low magnification of a ventrolateral corner of the left rostral telencephalon stained with cresyl violet at A 11.00. B: Enlargement of an adjacent section corresponding to the boxed area in A. CTB-labeled neurons (arrowheads) in the CPP cord. Arrows indicate contralateral BDA-labeled fibers in the LOT. C: Enlargement of the boxed area in Figure 2B. CTB-labeled neurons in the mesopallium (M). Arrows show BDA-labeled fibers in the MOT. D: Numerous CTB-labeled neurons in the left (ipsilateral to the injection in OB) TT and numerous BDA-labeled fibers in the right (ipsilateral to the BDA injection) TT at A 3.00. Arrows show BDA-labeled fibers running in the ipsilateral MOT; arrowheads indicate BDA labeling in the contralateral MOT. E: CTB-labeled neurons in the ipsilateral CDL at A 3.75 together with superficially located contralateral BDA-labeled fibers (arrows). F: A CTB-labeled neuron (arrow) in the contralateral olfactory bulb, where BDA-labeled fibers formed dense networks. G: CTB injection site in the left olfactory bulb. Case P-231. H: Enlargement of the boxed area in G showing contralateral CTB-labeled neurons (arrows). I: Two CTB-labeled neurons (arrows) in the ventral tegmental area (VTA) at A 3.75. III, oculomotor nerve. J: Medium magnification of the dorsal pons at A 1.00. K: Enlargement of the boxed area in J showing several CTB-labeled neurons (arrows) in the caudal part of the locus coeruleus (A6). For other abbreviations see list. Scale bars 5 1 mm in A,G; 50 lm in B,F,H; 100 lm in C,E,I,K; 200 lm in D; 500 lm in J. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Olfactory system in pigeons

Figure 7. BDA labeling. Case P-228. A: Labeling in the first stream (I), second stream (II), and AON at A 14.00. Less labeling is seen in the CPP layer. B: Numerous labeled fibers in HD and the CPP sector at A 14.60. The second stream (II) lies between the two dashed lines. C: Low magnification of the rostral telencephalon at A 12.00. D: Enlargement of the boxed area marked D in C. Arrows show labeling in the LOT. E: Low magnification of the ventral telencephalon at A 7.90. A compact fiber bundle (arrowheads) from LOT joins IOT (shown in the box). F: Labeled fibers (arrow) in CPi at A 3.25. G: Enlargement of the boxed area marked G in C. Labeled fibers in IOT (arrow). H: Labeled fibers in the olfactory tubercle and nucleus of the diagonal band (arrows) at A 9.50. Small numbers of labeled fibers (arrowheads) are seen in the olfactory tubercle and nucleus of the diagonal band on the contralateral side. I: A 7.90. IOT traveling toward the stria medullaris (arrow) and a minor sheet of fibers heading for TnA and the ventral hippocampus (arrowheads). The solid line indicates a border between IOT and the minor sheet, and the dashed line shows a boundary between OM and TnA. J: A labeled fiber bundle passing through the habenular commissure at A 4.25. K: A few BDA-labeled fibers (arrows) in the contralateral olfactory bulb. For abbreviations see list. Scale bars 5 500 lm in A,B,J; 1 mm in C,E,F; 100 lm in D,G; 200 lm in H,I; 50 lm in K. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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approximately A 13.50, but more caudally still, the number of labeled fibers decreased markedly, finally disappearing into the neuropil medial to the septomesencephalic tract in the rostral septum. Along its course, MOT provided terminations in TT. Together with the MOT, a very large number of labeled fibers collected loosely in a medial part of the olfactory peduncle located between MOT and the olfactory and lateral ventricles. These fibers divided into a medial course to TT and a dorsolateral course to HD (Figs. 6D, 7A,B). Labeling in HD was moderately distributed, but only between A 14.50 and A 14.00. No labeling was seen in HC. The second stream traveled dorsolaterally toward the sector of CPP, and there formed a sheet of labeled fibers just under the pia mater. This is the lateral olfactory tract (LOT; Figs. 4B,C, 7B). LOT then coursed ventrally and caudally, becoming smaller as it did so (Figs. 4D–G, 7C,D). From A 8.50–7.75, LOT branched off a moderate number of fibers to IOT (described below; Figs. 4H, 7E). After branching, the LOT continued caudally to travel under the pia mater and terminated in CPi and more dorsally in the ventral part of CDL (Fig. 4I,J). BDA labeled fibers in CPi and CDL were located at the lateral surface of the hemisphere and extended to its caudal pole (Fig. 7F). In addition to LOT, the second stream contained fibers that ran dorsally to AON (Fig. 7A) and HD (Fig. 7B). Fibers in AON were numerous. The third stream made up the IOT. The IOT first formed a thick, compact layer near the lateral part of the olfactory peduncle (Fig. 7A) and ran caudally on the ventral surface of the telencephalon (Fig. 4B–E). The layered formation of the IOT gradually changed to a thick bundle between the striatum and the nidopallium, approximately at A 13.00, and gradually shifted its course laterally as the striatum expanded caudally (Figs. 4F, 7C,G). At A 10.00–9.00, IOT branched off a loose fiber bundle medially, which extended to the olfactory tubercle, nucleus of the diagonal band, and medial surface of the septum (Figs. 4G, 7H). The labeling in the septum extended to its caudal pole. At the ventral edge of the hemisphere at approximately A 8.50–7.75, IOT received a thick branch from LOT (Figs. 4H, 7E,I). At this juncture, IOT and a component of LOT separated into a major bundle heading contralaterally and a minor sheet heading ipsilaterally toward TnA and the ventral hippocampus. The former bundle was located medially (Fig. 7I). It entered the stria medullaris of the thalamus, crossed the habenular commissure, and reached the contralateral side (Figs. 4I, 7J). Before passing the habenular commissure, IOT branched off a few fibers to the medial habenular nucleus. On the contralateral side, IOT passed through the stria medullaris, traveled rostrally a short distance, and entered the ven-

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tral surface of the hemisphere under TnA. Here IOT divided into two bundles. One bundle turned caudally to travel along the ventral and ventrolateral surface of the telencephalon, finally to terminate in CPi and CDL (Figs. 4I,J, 6E). Some fibers entered TnA. Another bundle ran rostrally, taking a mirror-image course where LOT and IOT descended from the OB on the side of the injection (Fig. 4B–H). Thin, varicose fibers and thick fibers were traced to retrobulbar regions, including CPP, AON, TT, HD, IOT, and MOT (Figs. 5D–F, 6C,D), and a small number of labeled fibers reached the contralateral OB (Fig. 7K). In addition, a few fibers from this bundle were supplied to the medial edge of the septum via the nucleus of the diagonal band and olfactory tubercle (Fig. 7H).

Injection into the CPP sector Eleven pigeons received injections of CTB and BDA in the CPP sector. From these, two well-labeled cases (case P-241 for CTB and case P-243 for BDA) were selected as representative, and their results are described. CTB retrograde labeling. The injection of CTB into the left sector of CPP (case P-241) was placed at A 14.20, L 3.70, and V 0.50 below the pia mater. The distribution of CTB-labeled neurons is shown schematically in Figure 8. Diffusion of the tracer was mostly restricted to the CPP sector but slightly encroached on the dorsolateral edge of the mesopallium (Figs. 8B, 9A). In the telencephalon, CTB labeling in neuronal cell bodies was largely found in the rostral telencephalon ipsilaterally and in the arcopallium bilaterally. OB contained a few, weakly stained neurons in the mitral cell layer (Figs. 8A, 9B). In CPP, numerous neurons in the layered part were labeled, and those in the cord were also numerously labeled rostrally (Figs. 8B–G, 9C,D), but caudal to A 9.00 the labeling in the cord decreased to a low level. The olfactory pallium, including AON, TT, and HD, contained numerous CTB-labeled neurons (Figs. 8C–F, 9D,E), but HC was devoid of labeling. The labeling in HD ended caudally at A 11.75. The mesopallium contained a moderate number of labeled cells in its rostral pole near the injection site (Figs. 8C,D, 9E). In the nidopallium, labeled neurons were scattered around the injection site and the CPP cord. In addition, moderate numbers of labeled neurons was scattered throughout the medial quarter of the nidopallium at levels of the basorostral pallial nucleus. There were a few labeled neurons in the olfactory tubercle and the nucleus of the diagonal band but very few cells in the septum. Caudally, CPi and CDL showed low levels of labeling (Figs. 8I,J, 9F). Very few cells were labeled in the dorsolateral and dorsomedial subdivisions of the hippocampal formation (for subdivisions of the hippocampal formation

The Journal of Comparative Neurology | Research in Systems Neuroscience

Olfactory system in pigeons

Figure 8. A–J: Schematic illustration of the rostrocaudal extent of labeling following injections of BDA in the right CPP sector at A13.75 (case P-243) and CTB in the left CPP sector at A14.20 (case P-241); the figure is drawn from sections from two pigeons. The core of the injection site is marked in red (CTB) and in black (BDA), whereas the hatched area around the core indicates spread of tracer. Dots represent neurons retrogradely labeled with CTB on both sides; fibers and terminals anterogradely labeled with BDA, and confined to the ipsilateral side, are represented by short lines. One dot indicates 10 CTB-labeled neurons. BDA-labeled lines indicate the relative differences in distribution but do not represent real numbers. For abbreviations see list. Scale bar 5 1 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Figure 9. CTB labeling. Case P-241. A: Injection site of CTB in the left CPP sector at A 14.20. B: Weakly CTB-labeled neurons (arrows) in the mitral cell layer of the left OB. C: Labeled neurons in the CPP cord at A11.00. D: Many labeled neurons in the CPP layer and AON at A 14.25. E: Labeled neurons in HD (arrows), TT, and N at A 13.75. F–H: CTB labeled neurons in CDL at A 3.75 (F), ventral arcopallium at A 5.75 (G), and medial margin of the dorsomedial anterior thalamic nucleus (DMA) at A 6.50 (H). For abbreviations see list. Scale bars 5 1 mm in A; 100 lm in B,F,G; 200 lm in C,H; 500 lm in D,E. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

see Atoji and Wild, 2004). In the arcopallium, small numbers of labeled neurons were located mainly at the ventrolateral periphery of the intermediate arcopallium (Figs. 8H, 9G), i.e., the ventral arcopallium of Zeier and Karten (1971), and labeled neurons were less numerous at the ventrolateral surface of the intermediate arcopallium on the contralateral side. At the ventral surface between A 9.00 and A 7.75, a few labeled neurons were scattered near IOT, and the labeling continued into rostral TnA under the occipitomesencephalic tract. In the diencephalon, a rim of labeled neurons was seen at the medial margin of the dorsomedial anterior and posterior thalamic nuclei (Figs. 8I, 9H). Laterally, neurons were scat-

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tered in the dorsolateral anterior and posterior thalamic nuclei and in the subrotundal nucleus. In the brainstem, there were a few weakly labeled cells in the ventral tegmental area and A6 ipsilaterally. BDA anterograde labeling. The injection of BDA into the right CPP sector (case P-243) was placed at A 13.75, L 4.58, and V 0.28 below the pia mater. The distribution of BDA-labeled fibers is shown schematically in Figure 8. Diffusion of the tracer was restricted within the CPP sector (Figs. 8D, 10A). Three main streams of labeled fibers arose from the CPP sector, projecting to various parts of the brain. The streams were composed of thick fibers and thin, varicose fibers.

The Journal of Comparative Neurology | Research in Systems Neuroscience

Figure 10. BDA labeling. Case P-243. A: Injection site of BDA in the right CPP sector at A 13.75. B: Low magnification of the right (ipsilateral) olfactory bulb. C: Enlargement of the boxed area in B showing a BDA-labeled, varicose fiber (solid arrow). D: Two streams of labeled fibers at A 14.00. The second stream (II) bifurcates into ventromedial and dorsomedial courses, whereas the third stream (III) extends laterally. E,F: Labeled fibers in the tenia tecta (TT) at A 13.00 (E) and hippocampal continuation (HC) at A 11.25 (F). G: Numerous labeled fibers in the medial striatum (MSt; A 11.50). A dashed circle shows a fiber bundle that travels caudally through the ventromedial part of the pallial–subpallial lamina. H: Labeled fibers (solid arrows) in the septum at A 9.00. I: Low magnification of the caudal telencephalon at A 4.75. J: Enlargement of the boxed area marked J in I showing labeled fibers in the dorsolateral subdivision of the hippocampal formation (solid arrows). K: The third stream at the edge of the lateral ridge at A 12.50, where two fiber pathways are seen on the surface of the telencephalon. The upper pathway (arrowheads) travels caudally and dorsocaudally to temporo-parieto-occipital area and CDL. The lower pathway in the CPP cord gives rise to many fibers heading medially toward the nidopallium (N; open arrows). L: Enlargement of the box marked L in I, showing numerous fibers in a ventrolateral portion of the caudal nidopallium. M: Labeled fibers (solid arrows) in the ventral tegmental area (VTA) at A 4.00. III, oculomotor nerve. For abbreviations see list. Scale bars 5 1 mm in A,I; 500 lm in B,D,G; 200 lm in E,F,H,K; 100 lm in C,J,L,M. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Y. Atoji and J.M. Wild

The first stream was small and ran rostrally to the OB, in which it was distributed from the granular layer to the glomerular layer (Figs. 8A–C, 10B,C). The second stream projected mainly to the rostral telencephalon. It consisted of both compact and loose components (Fig. 10D). The compact component ran ventromedially to pass mainly through HD, with fewer fibers passing through adjacent parts of the mesopallium and intercalated part of the hyperpallium. The loose component commenced dorsomedially near a fan-like region ventral to the medial vallecula, then turned ventromedially to join the course of the compact component in HD (Fig. 8C–E). The labeling in HD of compact and loose components was distributed widely throughout the entire rostrocaudal extent of HD (Fig. 8B–G), in which thin, varicose fibers and thick fibers were mixed. In addition to HD, the stream extended to more ventromedial regions of the hemisphere and gave rise to numerous fibers that reached TT, HC, medial striatum, and the olfactory tubercle (Figs. 8B–F, 10E–G). It also sent a small number of fibers to the CPP layer and AON. Labeled fibers in TT continued to pass caudally and entered the septum (Fig. 10H). A few labeled fibers arose dorsocaudally from the second stream and traveled to the dorsolateral subdivision of the hippocampal formation (Figs. 8G–J, 10I,J). Also from the second stream, a loose, small bundle arose ventrally to head toward CPi via the ventral surface of the hemisphere. This bundle commenced at the ventromedial corner of the pallial–subpallial lamina between the medial striatum and the nidopallium and headed caudally to the ventral edge the hemisphere (Fig. 10G). Approximately at A 12.00, the bundle left the pallial–subpallial lamina, shifted medially under the olfactory tubercle, and became larger, gradually receiving fibers from the third stream described below. At levels of A 9.00–8.50, the bundle bifurcated into two pathways: one turned dorsolaterally, ascended along the ventrolateral wall of the hemisphere, and terminated in CPi; another coursed caudally to reach caudal regions of the arcopallium and nidopallium (Fig. 10K). The third stream was a large and long component, which projected as far as the caudal telencephalon. It first headed ventrolaterally toward the lateral ridge (Fig. 10D) and formed a continuous fiber sheet dorsally and then a fiber bundle in the CPP cord ventrally, on the edge of the lateral ridge (Figs. 8E,F, 10K). The dorsal sheet traveled caudally along the lateral surface of the hemisphere to reach CDL, the temporo-parieto-occipital area, and hippocampal formation (Fig. 8G–J). The bundle in the CPP cord traveled caudally on the ventrolateral surface of the telencephalon to reach CPi, branching off loose fibers toward the lateral nidopallium

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and arcopallium medially. The loose fibers reached lateral regions of the caudal nidopallium and arcopallium (Fig. 8G–J). Approximately at A 11.00–9.00, the bundle also gave rise to small numbers of fibers that ran along the ventromedial wall to reach the medial striatum and septum (Fig. 8G). Thus, the medial striatum and septum receive afferents from CPP via the second and third streams. In the caudal arcopallium, caudal nidopallium, and hippocampal formation, labeled fibers of the second and third streams appeared to be intermixed. BDA labeling was present in the ventrolateral part of the caudal nidopallium and sparse in the dorsolateral nidopallium (Figs. 8I,J, 10L). A few fibers in the caudal arcopallium and the nidopallium descended into the thalamus via the occipitomesencephalic tract. These fibers could be traced to the lateral hypothalamic area, lateral mammillary nucleus, and ventral tegmental area as far caudal as A 4.75 (Figs. 8H,I, 10M).

Injections in the limbic system The tracer injections into the CPP sector revealed wide projections to the limbic system. To confirm these projections, we re-examined tracer experiments using data from previously published studies (Atoji and Wild, 2004, 2005). In these two previous papers, the term CPP sector was not used, but it was described as part of the frontolateral nidopallium. When CTB was injected in a dorsolateral subdivision of the hippocampal formation (case P-106, A 5.75; Fig. 11A), CDL (case P-114, A 5.00; Fig. 11B), or septum (case P-99, A 7.75; Fig. 11C), neurons in the CPP sector were labeled in three cases (Fig. 11D–F). In a case of injection into CDL, neurons in the CPP cord were also labeled. TT was labeled by a CTB injection into the septum. However, no labeling was found in OB, AON, or CPP layer in either case. When BDA was injected on the right side of the dorsolateral subdivision of the hippocampal formation (case P-106, A 5.75; Fig. 11A), CDL (case P-114, A 4.75), or medial septum (case P99, A 7.85; Fig. 11C), thin and varicose fibers were numerous in the OB only in the case with a BDA injection in CDL (see Fig. 4L in Atoji and Wild, 2005). The course of label fibers to OB was through HD and the CPP sector at rostral telencephalic levels. A few fibers were seen in OB from the BDA injection in the medial septum. In this case, the labeled fibers appeared to reach OB via TT or the olfactory tubercle. In a case with a BDA injection in the dorsolateral subdivision of the hippocampal formation, a few labeled fibers were traced to the CPP sector, but such fibers were not found following a BDA injection in the medial septum. Labeled fibers in TT were found following injections in CDL and septum.

The Journal of Comparative Neurology | Research in Systems Neuroscience

Olfactory system in pigeons

Figure 11. A–C: Three different injection sites of BDA on the right side and CTB on the left side at the same rostrocaudal levels: dorsolateral subdivision of the hippocampal formation (P-106; A), CDL (P-114; B), and medial septal nucleus (P-99; C). D–F: CTB-labeled neurons in the CPP sector resulting from the injections shown in A (D), B (E), and C (F). These data are taken from Atoji and Wild (2004, 2005; see text). For abbreviations see list. Scale bars 5 1 mm in A–C; 200 lm in D–F. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

DISCUSSION The present study provides two major sets of evidence regarding the olfactory system of the pigeon. The first set suggests that the olfactory pallium is composed of more components than hitherto suspected. We should include the following retrobulbar regions: CPP layer and sector, AON, HD, TT, and HC as well as CPi and CDL of more caudal telencephalic regions. The retrobulbar and more caudal regions are in continuity via the CPP cord. Previous studies have shown that the olfactory pallium consists of CPi and CDL (Reiner and Karten, 1985; Bingman et al., 1994; Patzke et al., 2011). In contrast, the present study explored the whole olfactory pallium. The second set of evidence shows that the CPP sector connects with other nuclei of the olfactory pallium and suggests that it acts as a relay station by which olfactory information is distributed to the limbic system. The main fiber connections of the OB and CPP are summarized in Figure 12A,B. In Nissl-stained material, CPP can be distinguished from surrounding areas of the mesopallium and nidopallium on the basis of cell density and stain intensity (Karten and Hodos, 1969; present study). From previous in situ hybridization with 35S-labeled probes, we reported that vGluT2 mRNA, which is a selective marker for glutamatergic neurons, is strongly expressed in the whole pallium of pigeons but is partic-

ularly intense in OB and CPP (see Fig. 5A of Islam and Atoji, 2008). The present in situ hybridization with DIGlabeled probes confirmed that the most intense expression of vGluT2 mRNA is in the CPP sector and the rostral part of the CPP cord as well as in the CPP layer (Fig. 2A–L). More so than Nissl staining, vGluT2 expression clearly demarcates CPP from the surrounding areas. Furthermore, CPP is stained by Enc1 mRNA in chick embryos (Garcıa-Calero and Puelles, 2009). The fiber connections of CPP described below are different from those of visual, auditory or somatosensory nuclei and pathways in the nidopallium. Therefore, it appears that CPP is an independent nucleus in the avian pallium in terms of cytoarchitecture, gene expression, and hodology.

Olfactory system in birds OB connections. Rieke and Wenzel (1978) ablated the OB of the pigeon unilaterally and used the FinkHeimer technique to trace degenerating fibres. They found that the OB sends efferents to the CPP, mesopallium, nucleus accumbens, and medial striatum. Reiner and Karten (1985) injected tritiated amino acids in the pigeon OB, and on the basis of their results suggested that CPi and TnA made up the olfactory pallium. The projection that they found from OB to CPi was confirmed by wheat-germ agglutinin horseradish peroxide

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Figure 12. A,B: The main fiber connections of the olfactory bulb (A) and prepiriform cortex (B) are drawn schematically. Black arrows show efferent projections, and red arrows indicate afferent projections. A dashed line in A indicates the midline of the brain. C: Schematic drawing of the extent of the prepiriform cortex (CPP), piriform cortex (CPi), and dorsolateral coiticoid area (CDL) in a lateral view. A bandlike CPP (red) runs on the lateral aspect of the hemisphere and continues to CPi caudally. D–F: Comparative diagram of main projections of the intermediate olfactory tract (IOT) and lateral olfactory tract (LOT) in reptile (D), bird (E), and mammal (F). IOT and LOT are shown in red and black, respectively. The projection of the medial olfactory tract is not drawn, but see the text. D and F were drawn based on de Olmos et al. (1978), Martinez-Garcia et al. (1991), and Lohman and Smeets (1993). CA, anterior commissure; Cxl, lateral olfactory cortex; nLOT, nucleus of the lateral olfactory tract. For other abbreviations see list. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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(WGA-HRP) injections in CPi (Bingman et al., 1994). Later, Patzke et al. (2011) injected a more sensitive tracer, BDA, into the OB and found labeling in the layer part of CPP and in CDL, thereby adding two new members to the olfactory pallium. The present study with BDA injections in OB confirmed labeling in the CPP layer, CPi, and CDL, as reported by Patzke et al. (2011), and further found strong labeling in the retrobulbar regions of CPP sector and cord, AON, HD, and TT. CTB injections in OB revealed cell body labeling in these areas and in HC. Therefore, the olfactory pallium in pigeons is composed of CPP (layer, sector and cord), AON, HD, TT, HC, CPi, and CDL. These areas form a wide, continuous distribution. The OB projects to the olfactory pallium via olfactory tracts. Reiner and Karten (1985) described a route of the MOT toward the septum, but did not mention pathways toward CPi or TnA. Patzke et al. (2011) found two routes to the olfactory pallium. One (MOT) is the same as that of Reiner and Karten (1985), and another was described as a large fiber bundle, which arose from the ventral surface of the hemisphere near the olfactory peduncle. This latter bundle was seen to bifurcate into LOT toward CPi and IOT toward the contralateral side, crossing through the habenular commissure. The present study shows that IOT and LOT take separate routes. The LOT first runs dorsolaterally toward the lateral ridge and then turns ventrally or ventromedially. The IOT commences at the ventral surface near the olfactory peduncle. The IOT receives fiber components of LOT at the level of the septum and proceeds to the contralateral side. Patzke et al. (2011) reported that, after crossing, an ascending course to the contralateral OB passes through IOT. In the present study, we found that fiber components of IOT also pass through LOT and MOT and terminate in the contralateral OB. The olfactory system has a close relationship with the limbic system, in which the hippocampal formation is a pivotal area (Butler and Hodos, 2005), but no direct connections between the two structures have been shown (Atoji et al., 2002; Atoji and Wild, 2004). Bingman et al. (1994) reported a route by which the OB projects to the hippocampal formation via CPi in pigeons. The present results using BDA and CTB injections in OB or CPP revealed other pathways. HD, CDL, and TnA also have connections with the hippocampal formation (Atoji et al., 2002; Atoji and Wild, 2004, 2005, 2006). TT has reciprocal connections with the triangular subdivision of the hippocampal formation and with the septum (Atoji et al., 2002; Atoji and Wild, 2004; present study). CPP was shown to connect with the dorsolateral subdivision of the hippocampal formation in the present study. Therefore, relay stations from

OB to the hippocampal formation are CPP, HD, TT, CDL, and TnA, as well as CPi, and olfactory information is presumably conveyed to the hippocampal formation by these several stations. HC is also a prehippocampal area (Veenman et al., 1995) and expands at A 12.75–11.50, as described in Results. It continues without a visible boundary to the hippocampal formation caudally. In terms of fiber connections, the hippocampal formation has no direct connections with the OB (Atoji et al., 2002; Atoji and Wild, 2004; present study). When CTB is injected in the medial MSt, neuronal labeling is seen in HC but not in the hippocampal formation (Veenman et al., 1995). The present CTB injections in OB showed that HC, but not the hippocampal formation, projects to OB. The two retrograde studies indicate that HC is an area independent of the hippocampal formation. In a previous study, after CTB injections in the triangular subdivision of the hippocampal formation at A 9.50 or A 10.75, neuronal labeling was seen in HC at A 12.75 (Atoji et al., 2002). BDA injections in the same triangular subdivision at A 9.50 or A 10.30 produced fiber labeling in HC as far rostral as A 13.00. These tracer injections in the triangular subdivision indicate that HC is in communication with the hippocampal formation. Therefore, HC is probably an intermediary between the hippocampal formation and OB. TT forms a ventral part of the medial hemisphere wall between the OB and septum. It has reciprocal connections with the OB and CPP (present study) and also connects with the medial septum bidirectionally (Atoji and Wild, 2004). Parvalbumin-immunoreactive neurons were found in TT but not in the septum. In in situ hybridization studies, vGluT2 mRNA is expressed in the mammalian cortex and avian pallium (Hisano et al., 2000; Fremeau et al., 2001; Islam and Atoji, 2008). The present study demonstrated vGluT2 expression in TT, but not in the septum. This finding indicates that TT belongs to the pallium and is distinguishable from the subpallial septum. Therefore, the tripartite evidence of hodology, immunohistochemistry, and gene expression in the present study clearly shows that TT is an independent nucleus in the retrobulbar region of pigeons. TT plays the role of a ventral relay station to communicate between the OB and the subpallial septum, in parallel with direct communication via the MOT. HD has strong reciprocal connections with the OB and CPP. When CTB was injected in the triangular subdivision of the hippocampal formation, the number of labeled neurons in HD increased as the injection site was shifted rostrally from A 3.75, to A 6.50, to A 9.50 (Atoji et al., 2002). This pattern of labeling suggests a topographic innervation of the hippocampus by HD.

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Similarly, when CTB was injected in OB and CPP in the present study, neuronal labeling in HD was shown at A 14.25–12.25 and at A 14.25–11.75, respectively, suggesting a topographic olfactory innervation by HD, at least in its rostral part. In addition, HD of the pigeon projects to the visual part of the apical hyperpallium (Shimizu et al., 1995), and, in the zebra finch, lateral HD projects to area X and/or adjacent regions of the medial striatum (Wild and Williams, 1999). It thus appears that HD possesses both limbic and sensoryspecific components that are at least partly spatially separated. It can be noted that HD is one of the principal structures to undergo recategorization in the recent schema of pallial organization proposed by Jarvis et al. (2013). In that schema, HD is no longer considered hyperpallial but rather a dorsal part of the mesopallium (MD). Were this proposal to be supported, then the present results point to a very different hodology of the dorsal and ventral parts of the mesopallium. Bingman et al. (1994) reported that, on the basis of WGA-HRP injections in CPi, CPi projects to the contralateral OB. However, the present CTB injection in OB did not label neurons in the contralateral hemisphere, CPi included. This discrepancy could be explained by the fact that OB mitral cells project to the contralateral OB via collaterals of their CPi-projecting axons (Patzke et al., 2011, present study). Thus, when WGA-HRP is injected into CPi, it is retrogradely transported to OB mitral cell bodies and then is transferred to the contralateral OB via an axon collateral. CPP connections. The pigeon CPP has a layer or cord organization along the LOT in the rostral half of the hemisphere and continues to CPi in the caudal half (Fig. 12C). In the mallard, a well-developed layer is localized to the medial and mediodorsal surface of the rostral telencephalon (Ebinger et al., 1992). When Ebinger et al. injected 3H-proline into the mallard OB, silver grains were distributed over the layered part of CPP. However, the authors did not mention whether the labeling ends within the layer or continues caudally to CPi. Therefore, the similarity between the CPP layers in mallard and pigeon is unclear at present. The caudolateral nidopallium (NCL) has been suggested to be analogous to the prefrontal cortex of mammals. It gathers secondary inputs from auditory, visual, somatosensory, and trigeminal sensory systems (G€ unt€ urk€ un, 2005). Kr€oner and G€unt€urk€un (1999) reported that, when CTB was injected in caudal NCL at A 4.25, labeled neurons were seen in the frontolateral nidopallium at A 14.00 (see their Fig. 2), an area that seems to correspond to what we call the CPP sector. In the present study, a BDA injection in the CPP sector in case P-243 yielded sparse labeling in caudal NCL, but

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other cases showed more BDA labeling in caudal NCL at A 5.00–4.00 (data not shown). These results together suggest that caudal NCL could be an area receiving tertiary olfactory information, but, if so, it is positioned more caudally than the other sensoryrecipient areas in NCL. NCL projects to the medial MSt, which also receives afferents from wide areas of the telencephalic pallium, e.g., the ventral part of the apical hyperpallium, the medial part of HD, the prehippocampal area, and frontal and lateral parts of the frontal nidopallium, CDL, and arcopallium (Veenman et al., 1995; Kr€oner and G€unt€urk€un, 1999). The present BDA injections in CPP produced labeling in the medial MSt. Thus, the medial MSt receives higher-order inputs from the limbic and olfactory systems as well as from other sensory areas. The CPP sector sends descending projections to the lateral hypothalamic nucleus, lateral mammillary nucleus, and ventral tegmental area, which have fiber connections with the hippocampal formation and septum (Atoji et al., 2002; Atoji and Wild, 2004). CTB injections in the CPP sector did not label neurons in these nuclei. However, following 3H-leucine injections in the lateral mammillary nucleus, Berk and Hawkin (1985) showed silver grains over regions that we call the sector and cord of CPP (see their Fig. 3A–C). This suggests the possibility of reciprocal connections between the lateral mammillary nucleus and the CPP. The present CTB injection in CPP labeled many neurons at the medial margin of the dorsomedial anterior thalamic nucleus (DMA) and dorsomedial posterior thalamic nucleus (DMP). Veenman et al. (1997) suggested that the medial and dorsal parts of DMA and DMP are comparable to mammalian midline thalamic nuclei and confirmed their connections with visceral/limbic parts of the avian brain and, as we have shown previously, e.g., posterior pallial amygdala, lateral part of the bed nucleus of the stria terminalis, and septum (Atoji and Wild, 2004; Atoji et al., 2006). It seems that DMA and DMP have topographical projections to the limbic/olfactory system.

Comparative remarks The olfactory pallium in reptiles extends widely on the lateral surface of the telencephalon (Martinz-Garcia et al., 1991; Lanuza and Halpern, 1998). In mammals, the olfactory cortex spreads on the ventral surface of the hemisphere under the rhinal sulcus (de Olmos et al., 1978). In pigeons, the olfactory pallium is composed of a band-like CPP in the rostral telencephalon and a wide CPi and CDL in the caudal telencephalon (Fig. 12C).

The Journal of Comparative Neurology | Research in Systems Neuroscience

Olfactory system in pigeons

In the rat, layered extensions of the hippocampus dorsal and rostral to the corpus callosum are called the indusium griseum and anterior hippocampal continuation, respectively (Shipley et al., 2004). The anterior HC projects to the MOB, whereas the indusium griseum does not (Wyss and Sripanidkulchai, 1983; Adamek et al., 1984). Radtke-Schuller and K€unzle (2000) also observed projections from the anterior hippocampal formation to the MOB in the tenrec, although the projection was seen in only one of eight injections. The indusium griseum and anterior hippocampal continuation receive afferents from the MOB (Wyss and Spipanidkulchai, 1983; Adamek et al., 1984; Shipley and Adamek, 1984). Additionally, the MOB does not have direct connections with the hippocampus. The present CTB injections in OB revealed retrograde neuronal labeling in HC but not in the hippocampal formation. Therefore, HC, in which neurons were labeled by CTB injections, is probably comparable to the anterior HC in mammals. It remains to be determined whether the indusium griseum is present in birds. TT in mammals is a layered structure, which is located in the ventral part of the medial wall under the anterior hippocampal continuation and continues caudally to the lateral septum (Broadwell, 1975; Haberly and Price, 1978). The rat TT divides into dorsal and ventral parts, and the ventral TT has heavy reciprocal connections with the MOB (de Olmos et al., 1978; Haberly and Price, 1978; Wyss and Spipanidkulchai, 1983). The rabbit TT receives projections from the MOB (Broadwell, 1975). In the pigeon, TT is located in the ventral part of the medial wall and continues caudally to the lateral septum. BDA and CTB injections in OB demonstrated strong reciprocal connections with TT. The position and fiber connections of the pigeon TT are very similar to those of mammals, although the pigeon TT shows a homogenous appearance in Nissl stains rather than a layered structure. In reptiles, the MOB gives rise to the MOT, which emerges from the lateral aspect of the olfactory peduncle, passes caudally in a long olfactory peduncle, and splits into three tracts (MOT, IOT, and LOT) at a level corresponding to the beginning of the dorsal cortex (Martinz-Garcia et al., 1991; Lohman and Smeets, 1993, Lanuza and Halpern, 1998; Fig. 12D). The MOT projects to the medial wall of the hemisphere, including the retrobulbar formation and septum. The LOT runs on the lateral surface of the telencephalon and terminates in the lateral cortex and amygdala. The IOT commences at the ventral surface of the hemisphere and travels caudally to the stria medullaris in the thalamus, branching off fibers to the olfactory tubercle and nucleus of the diagonal band. It crosses the habenular commissure

and terminates in the contralateral lateral olfactory cortex but does not project to the contralateral MOB. The contralateral lateral olfactory cortex projects to the contralateral MOB. Before crossing, the IOT receives a contingent of fibers from LOT. In pigeons, the three tracts of MOT, IOT, and LOT emerge at the base of the OB and first run separately from their commencement (Fig. 12E). A later joining of fiber components of LOT and IOT agrees with the situation in reptiles. The main difference, however, is the termination of the contralateral IOT. IOT in reptiles terminates at the rostral part of the lateral cortex (Martinez-Garcia et al., 1991; Lohman and Smeets, 1993; Lanuza and Halpern, 1998), whereas IOT in pigeons reaches the OB (Patzke et al., 2011; present study). In mammals, only LOT from the MOB projects to the olfactory cortex (Scalia, 1966; Broadwell, 1975; Shipley et al., 2004; Fig. 12F). The MOB in the rat does not project to the contralateral OB directly, but AON and the nucleus of the LOT receive afferents from the MOB and project to the contralateral MOB via the anterior commissure (Broadwell and Jacobowitz, 1976; de Olmos et al., 1978; Haberly and Price, 1978; Luskin and Price, 1983; Santiago and Shammah-Lagnado, 2004). Under the cerebrum, the caudal part of the hypothalamic area in the rat receives descending projections from the olfactory cortex, including AON, CPi, periamygdaloid cortex, entorhinal cortex, and anterior cortical nucleus of the amygdala (Price et al., 1991). The mediodorsal nucleus in the thalamus receives similar projections from AON, CPi, anterior cortical nucleus of the amygdala, and periamygdaloid cortex (Price and Slotnick, 1983). In the present study, CPP gives rise to efferent fibers to the lateral hypothalamic nucleus, lateral mammillary nucleus, and ventral tegmental area but not to thalamic nuclei. Olfactory information in birds reaches the limbic nuclei in the hypothalamus and midbrain, as in mammals.

Functional implications Several lines of evidence implicate olfaction in avian behaviors. For example, blue tits perceive aromatic plant fragments inside the nest cavity (Mennerat, 2008). When aromatic plants (new moss collected outside territory areas) are added to the nests, the tits detect the change in nest odor from outside the nest. Antarctic prions recognize and discriminate individual odor in their burrows (Bonadonna and Nevitt, 2004). Wandering albatrosses are suggested to use the olfactory bulb for detecting the scent of prey (Nevitt et al., 2008). Zebra finch fledglings use olfactory cues to recognize their natal nest (Caspers and Krause, 2011). Sneddon et al. (1998) reported that chick embryos

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exposed to strawberry scent on incubation days 15–20 preferentially drank strawberry-flavored water after hatching, in contrast to embryos not so exposed. Jozsa et al. (2005) suggested that the embryonic olfactory memory of strawberry scent is mediated by pituitary adenyate cyclase activating polypeptide. Although ablation or lesion experiments in the olfactory system have not been performed in these behavioral studies, olfactory recognition or memory likely involves hippocampal function, as in pigeons (Colombo and Broadbent, 2000). It is possible that the olfactory inputs in the birds described above are conveyed indirectly to the hippocampal formation in ways similar to those described in the present article. With particular reference to pigeons, it is in homing behavior that olfaction seems to play such an important and critical role (Papi et al., 1971; Papi, 1990; Wallraff, 2005; Gagliardo et al., 2006, 2008, 2009, 2011a,b). In the brain, CPi seems to be a critical area involved in this mechanism, based on the deleterious effects of lesioning CPi on homing behavior from unfamiliar sites (Papi and Casini, 1990). The importance of CPi in olfactory navigation is confirmed by the expression of ZENK, an immediate early gene, after pigeon releases (Patzke et al., 2010). This gene expression study implicates the hippocampal formation as well as CPi. Olfactory input is transferred to the hippocampal formation, which is involved in learning, memory, or recognition (Colombo and Broadbent, 2000). In fact, although the hippocampus is not necessary for homing from unfamiliar locations in adult, experienced pigeons, it does appear to be important in learning a navigational map in young pigeons, although only if the lesion is of the left hippocampus (for review see Bingman et al., 2005). One projection route to the hippocampal formation involves OB projections to CPi, which in turn sends olfactory information to the hippocampal formation (Reiner and Karten, 1985; Bingman et al., 1994; Atoji et al., 2002; Atoji and Wild, 2004; present study). The pathway of OB!CPP!HF appears to involve glutamatergic transmission, because mitral cells in the OB and neurons in the CPP sector express mRNA of vGluT2 (Fig. 5A in Islam and Atoji, 2008; present study), CPP expresses mRNA of glutamate receptor 1 (Fig. 9A in Islam and Atoji, 2008), and the hippocampal formation expresses mRNA of glutamate receptor GluR1 and contains vGluT2 in axon terminals (Fig. 11A in Islam and Atoji, 2008; Fig. 12D–F in Atoji, 2011). In addition to this pathway, the present study revealed other routes to the hippocampal formation: 1) OB!CPP!HF, 2) OB!CDL!HF, 3) OB!HD!HF. Multiple circuits in the olfactory cortex indicate that they are more complex than previously understood and appear to work in

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parallel. The present study also demonstrated that the OB receives inputs from the olfactory cortex, including AON, CPP, CPi, HC, HD, and TT, inputs that no doubt mediate feedback to the OB. It will be important, therefore, in future studies of avian olfactory function to consider the multiple circuits of the olfactory cortex delineated in this and other studies.

ACKNOWLEDGMENTS We thank Dr. S. Saito for making DIG-labeled probes and M.R. Karim for assistance with in situ hybridization.

CONFLICT OF INTEREST STATEMENT The authors declare no conflicts of interest.

ROLE OF AUTHORS The authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: YA. Acquisition, analysis and interpretation of data: YA, JMW. Drafting and critical revision of manuscript: YA, JMW.

LITERATURE CITED Adamek GD, Shipley MT, Sanders MS. 1984. The indusium griseum in the mouse: architecture, Timm’s histochemistry and some afferent connections. Brain Res Bull 12: 657–668. Atoji Y. 2011. Immunohistochemical localization of vesicular glutamate transporter 2 (vGluT2) in the central nervous system of the pigeon (Columba livia). J Comp Neurol 519:2887–2905. Atoji Y, Wild JM. 2004. Fiber connections of the hippocampal formation and septum and subdivisions of the hippocampal formation in the pigeon as revealed by tract-tracing and kainic acid lesions. J Comp Neurol 475:426–461. Atoji Y, Wild JM. 2005. Afferent and efferent connections of the dorsolateral corticoid area and a comparison with connections of the temporo-parieto-occipital area in the pigeon (Columba livia). J Comp Neurol 485:165–182. Atoji Y, Wild JM. 2006. Anatomy of the avian hippocampal formation. Rev Neurosci 17:3–15. Atoji Y, Wild JM, Yamamoto Y, Suzuki Y. 2002. Intratelencephalic connections of the hippocampus in pigeons (Columba livia). J Comp Neurol 447:177–199. Atoji Y, Saito S, Wild JM. 2006. Fiber connections of the compact division of the posterior pallial amygdala and lateral part of the bed nucleus of the stria terminalis in the pigeon (Columba livia). J Comp Neurol 499:161–182. Balthazart J, Schoffeniels E. 1979. Pheromones are involved in the control of sexual behaviour of birds. Naturwissenschaften 66:55–56. Baxi KN, Dorries KM, Eisthen HL. 2006. Is the vomeronasal system really specialized for detecting pheromones? Trends Neurosci 29:1–7. Berk ML, Hawkin RF. 1985. Ascending projections of the mammillary region in the pigeon: emphasis on telencephalic connections. J Comp Neurol 239:330–340. Bingman VP, Casini G, Nocjar C, Jones T-J. 1994. Connections of the piriform cortex in homing pigeons (Columba livia)

The Journal of Comparative Neurology | Research in Systems Neuroscience

Olfactory system in pigeons

studied with fast blue and WGA-HRP. Brain Behav Evol 43:206–218. Bingman VP, Gagliardo A, Hough, GE II, Ioale` P, Kahn MC, Siegel JJ. 2005. The Avian hippocampus, homing in pigeons and the memory representation of large-scale space. Integr Comp Biol 45:555–564. Bonadonna F, Nevitt GA. 2004. Partner-specific odor recognition in an Antarctic seabird. Science 306:835. Broadwell RD. 1975. Olfactory relationships of the telencephalon and diencephalon in the rabbit. II. An autoradiographic study of the efferent connections of the main and accessory olfactory bulbs. J Comp Neurol 163:329– 346. Broadwell RD, Jacobowitz DM. 1976. Olfactory relationships of the telencephalon and diencephalon in the rabbit. III. The ipsilateral centrifugal fibers to the olfactory bulbar and retrobulbar formations. J Comp Neurol 170:321– 346. Brown RE, Macdonald DW. 1985. Social odours in mammals. Oxford: Clarendon Press. Butler AB, Hodos W. 2005. Comparative vertebrate neuroanatomy. Evolution and adaptation, 2nd ed. Hoboken, NJ: John Wiley & Sons. Carmichael ST, Clugnet MC, Price JL. 1994. Central olfactory connections in the macaque monkey. J Comp Neurol 346:403–434. Caspers BA, Krause ET. 2011. Odour-based natal nest recognition in the zebra finch (Taeniopygia guttata), a colonybreeding songbird. Biol Lett 7:184–186. Colombo M, Broadbent NJ. 2000. Is the avian hippocampus a functional homologue of the mammalian hippocampus? Neurosci Biobehav Rev 24:465–484. de Olmos J, Hardy H, Heimer L. 1978. The afferent connections of the main and accessory olfactory bulb formations in the rat: an experimental HRP-study. J Comp Neurol 181:213–244. Ebinger P, Rehk€amper G, Schr€oder H. 1992. Forebrain specialization and the olfactory system in anseriform birds. An architectonic and tracing study. Cell Tissue Res 268:81– 90. Eisthen HL, Polese G. 2007. Evolution of vertebrate olfactory subsystems. In Kaas JH, editor. Evolution of nervous systems. A comprehensive reference, vol 2. Amsterdam: Academic Press. p 355–406. Fremeau RT Jr, Troyer MD, Pahner I, Nygaard GO, Tran CH, Reimer RJ, Bellocchio EE, Fortin D, Storm-Mathisen J, Edwards RH. 2001. The expression of vesicular glutamate transporters defines two classes of excitatory synapse. Neuron 31:247–260. Gagliardo A, Ioale` P, Savini M, Wild JM. 2006. Having the nerve to home: trigeminal magnetoreceptor versus olfactory mediation of homing in pigeons. J Exp Biol 212: 4065–4071. Gagliardo A, Ioale` P, Savini M, Wild JM. 2008. Navigational abilities of homing pigeons deprived of olfactory or trigeminally mediated magnetic information when young. J Exp Biol 211:2046–2051. Gagliardo A, Ioale` P, Savini M, Wild JM. 2009. Navigational abilities of adult and experienced homing pigeons deprived of olfactory or trigeminally mediated magnetic information. J Exp Biol 212:3119–3124. Gagliardo A, Filannino C, Ioale` P, Pecchia T, Wikelski M, Vallortigara G. 2011a. Olfactory lateralization in homing pigeons: a GPS study on birds released with unilateral olfactory inputs. J Exp Biol 214:593–598. Gagliardo A, Ioale` P, Filannino C, Wikelski M 2011b. Homing pigeons only navigate in air with intact environmental

odours: a test of the olfactory activation hypothesis with GPS data loggers. PLoS ONE 6:e22385. Garcıa-Calero E, Puelles L. 2009. Enc1 expression in the chick telencephalon at intermediate and late stages of development. J Comp Neurol 517:564–580. Grubb TC. 1974. Olfactory navigation to the nesting burrow in Leach’s petrel Oceanodroma leucorrhoa. Anim Behav 22: 192–202. G€unt€urk€un O. 2005. The avian “prefrontal cortex” and cognition. Curr Opin Neurobiol 15:686–693. Haberly LB, Price JL. 1978. Association and commissural fiber systems of the olfactory cortex of the rat. II. Systems originating in the olfactory peduncle. J Comp Neurol 181: 781–808. Hagelin JC. 2006. Odors and chemical signaling. In: Jamieson BGM, editor. Reproductive behavior and phylogeny of aves, vol 6B. Enfield, NH: Science Publishers. p 76–119. Hagelin JC, Jones IL. 2007. Bird odors and other chemical substances: a defense mechanism or overlooked mode of intraspecific communication? Auk 124:1–21. Hirano A, Aoyama M, Sugita S. 2009. The role of uropygial gland in sexual behavior in domestic chicken Gallus gallus domesticus. Behav Processes 80:115–120. Hisano S, Hoshi K, Ikeda Y, Maruyama D, Kanemoto M, Ichijo H, Kojima I, Takeda J, Nogami H. 2000. Regional expression of a gene encoding a neuron-specific Na1-dependent inorganic phosphate cotransporter (DNPI) in the rat forebrain. Brain Res Mol Brain Res 83:34–43. Ioale` P, Savini M, Gagliardo A. 2008. Pigeon homing: the navigational map developed in adulthood is based on olfactory information. Ethology 114:95–102. Islam MR, Atoji Y. 2008. Distribution of vesicular glutamate transporter 2 and glutamate receptor 1 mRNA in the central nervous system of the pigeon (Columba livia). J Comp Neurol 511:658–677. Jarvis ED, Yu JY, Rivas MV, Horita H, Feenders G, Whitney O, Jarvis SC, Jarvis ER, Kubikova L, Puck AEP, Siang-Bakshi C, Martin S, McElroy M, Hara E, Howard J, Pfenning A, Mouritsen H, Chen C-C, Wada K. 2013. Global view of the functional molecular organization of the avian cerebrum: mirror images and functional columns. J Comp Neurol 521:3614–3665. Jones RB, Roper TJ. 1997. Olfaction in the domestic fowl: a critical review. Physiol Behav 62:1009–1018. Jozsa R, Hollosy T, Tamas A, Toth G, Lengvari I, Regl€odi D. 2005. Pituitary adenylate cyclase activating polypeptide plays a role in olfactory memory formation in chicken. Peptides 26:2344–2350. Karten HJ, Hodos W. 1967. A stereotaxic atlas of the brain of the pigeon (Columba livia). Baltimore: John Hopkins University Press. Kr€oner S, G€unt€urk€un O. 1999. Afferent and efferent connections of the caudolateral neostriatum in the pigeon (Columba livia): a retro- and anterograde pathway tracing study. J Comp Neurol 407:228–260. Lanuza E, Halpern M. 1998. Efferents and centrifugal afferents of the main and accessory olfactory bulbs in the snake Thamonphis sirtalis. Brain Behav Evol 51:1–22. Lohman AHM, Smeets WJAJ. 1993. Overview of the main and accessory olfactory bulb projections in reptiles. Brain Behav Evol 41:147–155. Luskin MB, Price JL. 1983. The topographic organization of associational fibers of the olfactory system in the rat, including centrifugal fibers to the olfactory bulb. J Comp Neurol 216:264–291. Martinez-Garcia F, Olucha FE, Teruel V, Lorente MJ, Schwerdtfeger WK. 1991. Afferent and efferent

The Journal of Comparative Neurology | Research in Systems Neuroscience

1751

Y. Atoji and J.M. Wild

connections of the olfactory bulbs in the lizard Podarcis hispanica. J Comp Neurol 305:337–347. McKeegan DEF. 2002. Spontaneous and odour evoked activity in single avian olfactory bulb neurons. Brain Res 929:48– 58. Mennerat A. 2008. Blue tits (Cyanistes caeruleus) respond to an experimental change in the aromatic plant odour composition of their nest. Behav Processes 79:189–191. Nevitt GA, Losekoot M, Weimerskirch H. 2008. Evidence for olfactory search in wandering albatross, Diomedea exulans. Proc Natl Acad Sci U S A 105:4576–4581. Oosawa T, Hirano Y, Tonosaki K. 2000. Electroencephalographic study of odor responses in the domestic fowl. Physiol Behav 71:203–205. Papi F. 1990. Olfactory navigation in birds. Experientia 46: 352–363. Papi F, Casini G. 1990. Pigeons with ablated pyriform cortex home from familiar but not from unfamiliar sites. Proc Natl Acd Sci U S A 87:3783–3787. Papi F, Fiore L, Fiaschi V, Benvenuti S. 1971. The influence of olfactory nerve section on the homing capacity of carrier pigeons. Monit Zool Ital 5:265–267. Patzke N, Manns M, G€unt€urk€un O, Ioale` P, Gagliardo A. 2010. Navigation-induced ZENK expression in the olfactory system of pigeons (Columba livia). Eur J Neurosci 31:2062– 2072. Patzke N, Manns M, G€unt€urk€un O. 2011. Telencephalic organization of the olfactory system in homing pigeons (Columba livia). Neuroscience 194:53–61. Price JL, Slotnick BM. 1983. Dual olfactory representation in the rat thalamus: an anatomical and electrophysiological study. J Comp Neurol 215:63–77. Price JL, Slotnick BM, Revial MF. 1991. Olfactory projections to the hypothalamus. J Comp Neurol 306:447–461. Radtke-Schuller S, K€unzle H. 2000. Olfactory bulb and retrobulbar regions in the hedgehog tenrec: organization and interconnections. J Comp Neurol 423:687–705. Reiner A, Karten HJ. 1985. Comparison of olfactory bulb projections in pigeons and turtles. Brain Behav Evol 27:11– 27. Reiner A, Perkel DJ, Bruce LL, Butler AB, Csillag A, Kuenzel W, Medina L, Paxinos G, Shimizu T, Striedter G, Wild JM, Ball GF, Durand S, G€unt€urk€un O, Lee DW, Mello CV, Powers A, White SA, Hough G, Kubikova L, Smulders TV, Wada K, Dugas-Ford J, Husband S, Yamamoto K, Yu J, Siang C, Jarvis ED. 2004. Revised nomenclature for avian telencephalon and some related brainstem nuclei. J Comp Neurol 473:377–414. Rieke GK, Wenzel BM. 1978. The forebrain projections of the pigeon olfactory bulb. J Morphol 158:41–56.

1752

Roper TJ. 1999. Olfaction in birds. In: Slater PJB, Rosenblat JS, Snowden CT, Roper TJ, editors. Advances in the study of behavior, vol 28. Boston: Academic Press. p 247–332. Santiago AC, Shammah-Lagnado SJ. 2004. Efferent connections of the nucleus of the lateral olfactory tract in the rat. J Comp Neurol 471:314–332. Scalia F. 1966. Some olfactory pathways in the rabbit brain. J Comp Neurol 126:285–310. Shimizu T, Cox K, Karten HJ. 1995. Intratelencephalic projections of the visual Wulst in pigeons (Columba livia). J Comp Neurol 359:551–572. Shipley MT, Adamek GD. 1984. The connections of the mouse olfactory bulb: a study using orhtograde and retrograde transport of wheat germ agglutinin conjugated to horseradish peroxidase. Brain Res Bull 12:669–688. Shipley MY, Ennis M, Puche A. 2004. Olfactory system. In: Paxinos G, editor. The rat nervous system, 3rd ed. Amsterdam: Elsevier. p 923–964. Skeen LC, Pindzola, RR, Schofield BR. 1984. Tangential organization of olfactory, association, and commissural projections to olfactory cortex in a species of reptile (Trionyx spiniferus), bird (Aix sponsa), and mammal (Tupaia glis). Brain Behav Evol 25:206–216. Sneddon H, Hadde R, Hepper PG. 1998. Chemosensory learning in the chicken embryo. Physiol Behav 64:133–139. Steiger SS, Fidler AE, Valcu M, Kempenaers B. 2008. Avian olfactory receptor gene repertoires: evidence for a welldeveloped sense of smell in birds? Proc R Soc Lond B Biol Sci 275:2309–2317. Veenman CL, Wild JM, Reiner A. 1995. Organization of the avian “corticostriatal” projection system: a retrograde and anterograde pathway tracing study in pigeons. J Comp Neurol 354:87–126. Veenman CL, Medina L, Reiner A. 1997. Avian homologues of mammalian intralaminar, mediodorsal and midline thalamic nuclei: immunohistochemical and hodological evidence. Brain Behav Evol 49:78–98. Wallraff HG. 2001. Navigation by homing pigeons: updated perspective. Ethol Ecol Evol 13:1–48. Wallraff HG. 2005. Avian navigation: pigeon homing as a paradigm. Berlin: Springer Verlag. Wild JM, Williams MN. 1999. Rostral Wulst of passerine birds: II. Intratelencephalic projections to nuclei associated with the auditory and song systems. J Comp Neurol 413: 520–534. Wyss JM, Sripanidkulchai K. 1983. The indusium griseum and anterior hippocampal continuation in the rat. J Comp Neurol 219:251–272. Zeier H, Karten HJ. 1971. The archistriatum of the pigeon: organization of afferent and efferent connections. Brain Res 31:313–326.

The Journal of Comparative Neurology | Research in Systems Neuroscience

Efferent and afferent connections of the olfactory bulb and prepiriform cortex in the pigeon (Columba livia).

Although olfaction in birds is known to be involved in a variety of behaviors, there is comparatively little detailed information on the olfactory bra...
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