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Afferent and efferent connections of the cortical and medial nuclei of the amygdala in sheep
Journal of Chemical Neuroanatomy 37 (2009) 87–97
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Afferent and efferent connections of the cortical and medial nuclei of the amygdala in sheep M. Meurisse, E. Chaillou, F. Le´vy * INRA, UMR85 Physiologie de la Reproduction et des Comportements, CNRS, UMR6175, Universite´ de Tours, Haras Nationaux, F-37380 Nouzilly, France
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 June 2008 Received in revised form 4 September 2008 Accepted 4 September 2008 Available online 12 September 2008
The cortical (CoA) and the medial (MeA) nuclei of the amygdala are involved in the processing of olfactory information relevant to social recognition in the ewe. To better understand the neural pathways responsible for these effects, the connections of both CoA and MeA with the telencephalic and diencephalic regions were studied by injecting an anterograde (Biotin-Dextran-Amine, BDA) or a retrograde (Fluorogold, FG) neuronal tracer into either the CoA or the MeA. Concerning the primary olfactory structures, the CoA receives inputs from both the main olfactory bulb and the accessory olfactory bulb (AOB), while the MeA is innervated by cells only from the AOB. Among the other olfactory structures, only the entorhinal cortex and the tenia tecta are connected with both the CoA and the MeA. With respect to the other secondary olfactory structures, the connections with the CoA and the MeA show segregating neuronal routes. The CoA is connected with the accessory olfactory nucleus, the piriform, the endopiriform and the orbitofrontal cortices while the MeA exhibited connections with the nucleus of the lateral olfactory tract, the perirhinal and the insular cortices. Concerning the diencephalic structures, only the MeA receives projections from the PVN and the MBH. On the other hand, we showed that the BNST is the major site of connection with both the CoA and the MeA. Reciprocal projections were observed between the CoA and the MeA and between both nuclei and the basal or the lateral nuclei of the amygdala with the exception of the CoA which does not send inputs to the lateral nucleus. These data are discussed in relation with olfactory learning in the context of sexual and maternal behavior in sheep. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Fluorogold Dextran amine Neural tract-tracing Olfaction Social behavior
* Corresponding author at: INRA, UMR85 Physiologie de la Reproduction et des Comportements, F-37380 Nouzilly, France. Fax: +33 247427743. E-mail address: [email protected] (F. Le´vy). Abbreviations: 3V, third ventricle; AOB, accessory olfactory bulb; AON, anterior olfactory nucleus; AC, anterior commissure; B, basal amygdaloid nucleus; BNST, bed nucleus of the stria terminalis; C, caudate nucleus; CeA, central amygdaloid nucleus; CoA, cortical amygdaloid nucleus; E, endopiriform nucleus; EC, entorhinal cortex; Fx, fornix; Gl, glomerular layer of the olfactory bulb; Gr, granular cell layer of the olfactory bulb; HDB, horizontal limb of the diagonal band of Broca; IC, insular cortex; L, lateral amygdaloid nucleus; LHA, lateral hypothalamic area; LOT, lateral olfactory tract; LOTn, nucleus of the lateral olfactory tract; LV, lateral ventricle; MBH, mediobasal hypothalamus; MeA, medial amygdaloid nucleus; Mi, mitral cell layer of the olfactory bulb; MOB, main olfactory bulb; MS, medial septum; MT, mamillothalamic tract; OC, optic chiasma; OFC, orbitofrontal cortex; OT, optic tract; OV, olfactory ventricle; PC, piriform cortex; PM, premamillary nucleus; POA, preoptic area; POAdl, dorsolateral part of the POA; POAm, medial part of the POA; PRh, perirhinal cortex; Pu, putamen; PVN, paraventricular nucleus; RT, reticular nucleus of the thalamus; S, subiculum; SON, supraoptic nucleus; TT, tenia tecta; VA, ventral anterior nucleus of the thalamus; VDB, vertical limb of the diagonal band of Broca; VH, ventral hippocampus. 0891-0618/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jchemneu.2008.09.001
The sense of smell is of primary importance for social recognition among mammals. For instance, olfaction is involved in discrimination of familiar conspecifics in rats (Gheusi et al., 1994), in mate recognition in mice, voles, ferrets and sheep (Brennan and Keverne, 1997; Cohen-Tannoudji et al., 1989; Demas et al., 1997) and in the recognition of the offspring in ungulates (Kendrick et al., 1997a; Le´vy et al., 2004, 1996a; Poindron et al., 2007). More particularly, in sheep, exposure of ewes to a male or its odor triggers a release of luteinizing hormone (LH) and ovulation (Martin et al., 1986). Ewes form a selective memory for their lambs after 2 h of mother–young interaction following parturition (Kendrick et al., 1997a; Le´vy et al., 2004, 1996b). This memory is mediated by the main olfactory system, as its disruption through anosmia results in a loss of lamb recognition. As a consequence, both familiar and unfamiliar lambs are suckled (Ferreira et al., 2000; Le´vy et al., 1995; Poindron, 1976). Although these examples of olfactory recognition of conspecifics involve different behavioral contexts, they share common features, including a dependence on the main or the accessory olfactory bulbs (AOBs) which represent the first stage of sensory processing and on their associated functional changes (Brennan and Keverne, 1997; Cohen-Tannoudji
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et al., 1989; Dluzen et al., 1998a,b; Kendrick et al., 1997a,b, 1992). These various forms of olfactory learning depend also on the amygdala and in particular on the two nuclei that are directly associated with the two olfactory systems, the cortical nucleus (CoA) and the medial nucleus (MeA). For example, in sheep, the male odor increases Fos neural expression specifically in the CoA (Gelez and Fabre-Nys, 2006) and the effect of ram odor on the secretion of luteinizing hormone was completely blocked by inactivation of the CoA (Gelez et al., 2004). In parturient ewes exposed to lambs, primary olfactory projections, involving the CoA, show higher Fos expression than those of ewes that were not exposed to lambs (Keller et al., 2004a). Furthermore, inactivation of either the CoA or the MeA during the first 8 h post-partum impaired lamb olfactory recognition by the mother (Keller et al., 2004b). In sheep, the topography of connections of the CoA and the MeA with the rest of the brain has remained poorly understood. Tracttracing studies indicate interconnections between the anterior and posterolateral parts of the CoA and the main olfactory bulb (MOB; Jansen et al., 1998; Le´vy et al., 1999). MOB terminals were also observed within the MeA (Jansen et al., 1998). Connections between the AOB/anterior olfactory nucleus (AON) on the one hand, and the MeA and the posteromedial CoA on the other hand have been reported in one study (Jansen et al., 1998). However, how the two nuclei of the amygdala are connected with the rest of the brain is currently unknown and these data would help us to understand the possible neural networks involved in olfactoryguided reproductive behavior of sheep. In this context, the aim of the present work has been to investigate the afferent and efferent connections with both the CoA and the MeA by injecting either in the CoA and the MeA nuclei, a retrograde tracer, Fluorogold (FG), or an anterograde tracer, BiotinDextran-Amine (BDA), that were looked for in the body cells (FG) and in the fibers (BDA) of the telencephalic and diencephalic structures, known to be involved in social recognition. 1. Material and methods 1.1. Animals Fifteen adult intact Ile de France ewes, 2–5 years old, from the laboratory flock were studied. The majority of ewes (11/15) were used in the seasonal period of reproduction and all the ewes were multiparous. They were kept permanently indoors and fed ad libitum with dehydrated lucerne, maize and straw and had free access to water. All efforts were made to minimize the number of animals used and their suffering. Animal care and all procedures were in accordance with authorization A37801 of the French Ministry of Agriculture. Experimental procedures were approved by the local ethical authority (Comite´ Re´gional d’Ethique et d’Expe´rimentation Animale, Re´gion Centre-Limousin, procedure INRA 37-002). 1.2. Injection procedures Anesthesia was induced with a mixture of Pentothal (Rhoˆne-Poulenc, France) and atropine (20 mg; Lavoisier, Paris, France), and maintained with 4% halothane (Be´lamont, Neuilly, France) in oxygen. Full sterile procedures were used throughout surgery. The animal’s head was fixed in a stereotaxic frame adapted to sheep and modified for X-ray radiographic control (Blanc et al., 1992). Stereotaxic coordinates were calculated using lateral and dorsoventral X-rays of the skull after infusion of a radio-opaque agent (Iopamiron 300; Schering, Germany) in the lateral ventricle. For each ewe, horizontal and vertical tangents to the thalamic mass were used to calculate the coordinates of the implantation site in the rostrocaudal and in the dorsoventral axis. The middle of the third ventricle was used to calculate the lateral coordinates. The stereotaxic coordinates of the cortical and medial amygdala nuclei were as follows: CoA: rostrocaudal plane, 1.7 mm behind the vertical tangent; mediolateral plane, 14 mm from the middle of the third ventricle; depth, 7 mm above the horizontal tangent; MeA: rostrocaudal plane, 1.7 mm; mediolateral plane, 13.2 mm; depth, 1.5 mm. A 2% solution of FG (Interchim, Montluc¸on, France) in sterile distilled water was used for retrograde tracing and 5% solution of BDA (Sigma–Aldrich, L’Isle d’Abeau Chesnes, France) in sterile distilled water was used for anterograde tracing. A 1 ml Hamilton syringe filled with tracers was placed in a probe holder (Kopf) so that its tip was located either in the CoA or in the MeA. A
volume of 0.1 ml of FG alone or BDA alone was slowly injected unilaterally at the rate of 0.01 ml/min. The syringe was then left in place for 15 min after completion to avoid reflux of the tracer along the needle tract. FG was used in 11 ewes: six were injected in the CoA and five in the MeA. BDA was injected in four ewes: two received BDA in the CoA and two in the MeA. For the 5 days following surgery, animals received daily intramuscular injections of an antibiotic (5 ml of amoxicillin; Mixtencilline, Rhoˆne Me´rieux, Lyon, France) and an anti-inflammatory and analgesic preparation (Phe´nylarthrite, 5 ml of phenylbutazone, 0.02 g/ml; Ve´toquinol, Lure, France). 1.3. Histological procedures After a survival period of 15–21 days to allow for sufficient transport of tracers, the ewes received a lethal dose of thiopenthal and were sacrificed by decapitation by a licensed butcher in an official slaughterhouse (authorization No. A37801). Brains were immediately perfused via both carotid arteries with 2 l of 1% sodium nitrite followed by 4 l of ice-cold 4% paraformaldehyde in 0.1 M, pH 7.4, phosphate buffer. The brain was dissected out, cut into blocks and postfixed overnight in the same fixative. The tissues were then stored in 30% sucrose until sectioned. Frontal free-floating sections were cut with a freezing microtome at a thickness of 40 mm and stored at 4 8C in phosphate-buffered saline (PBS) containing 0.1% sodium azide, until processing for FG or BDA visualization. A 1:30 series of sections were stained with cresyl violet in order to identify the appropriate neuroanatomical structures. 1.4. Immunohistochemistry FG was revealed by an antibody providing a permanent image and an excellent resolution (Chang et al., 1990). Sensitivity was increased by detecting the antibody with a nickel (Ni)-enhanced diaminobenzidine (DAB) tetrahydrochloride peroxidase antiperoxidase (PAP) technique. BDA histochemistry yields extensive and detailed labeling of axons and terminals (Kobbert et al., 2000). The tracer was revealed using a standard streptavidin-biotinylated horseradish peroxidase (HRP) procedure to produce a permanent reaction product. 1.4.1. FG staining Endogenous peroxidase activity was first inhibited by incubating the sections for 30 min at 4 8C in PBS containing 1% H2O2. Sections were then incubated for 1 h at room temperature in PBS containing 0.3% Triton X-100, 0.1% sodium azide and 1% BSA (PBST). Afterwards, they were incubated with the 1:15,000 primary antiserum (anti-FG Chemicon, Euromedex, Souffelweyersheim, France) and gently shaken for 48 h at 4 8C. Sections were washed in PBS and incubated 3:30 h at 4 8C in a 1:400 sheep anti-rabbit antibody diluted in PBS–BSA 1%. They were rinsed repeatedly in PBS and incubated overnight at 4 8C in 1:80,000 rabbit PAP complex (Dakocytomation, Trappes, France). Sections were rinsed twice in PBS and twice in Tris–HCl. Immunoreactivity was visualized after incubation in a solution of 0.02% DAB (Sigma–Aldrich, L’Isle d’Abeau Chesnes, France), 0.3% Ni and 0.003% H2O2 in Tris– HCl (50 mM, pH 7.6). The specificity of the immunoreactive reaction was tested by preincubating the anti-FG antibody with an excess of 1 mg/ml of FG (Le´vy et al., 1999). 1.4.2. BDA staining After endogenous peroxidase inhibition, sections were incubated for 1 h at room temperature in PBS containing 0.3% Triton X-100 and 1% BSA. Streptavidin-HRP (Abcys, Paris, France) was diluted 1:500 in the same solution, and sections were left inside overnight at 4 8C. Sections were then rinsed twice in PBS and twice in Tris– HCl. Sections were incubated in a solution of 0.02% DAB (Sigma–Aldrich, L’Isle d’Abeau Chesnes, France), 0.3% Ni and 0.003% H2O2 in Tris–HCl (50 mM, pH 7.6) until a black reaction product was visible. Finally, they were counterstained with neutral red for a better visualization of the neurons and fibers. All sections were then washed, mounted on gelatin-coated slides, dried, and dehydrated through graded alcohol, cleared in toluene and coverslipped with Depex1. 1.5. Analysis of sections Sections were examined under a Zeiss Axiophoto microscope (Zeiss, St. Cloud, France) with bright-field illumination. Across telencephalic and diencephalic structures, 1 section out of 15 was examined; for the olfactory bulb, one section out of three was visualized. Neuroanatomical sites were based on the rat stereotaxic atlas of Paxinos and Watson (1986), on the description of Breder et al. (1992) and on the cresyl violet staining. In order to better locate the FG and BDA injection sites within amygdala nuclei, they were drawn with the help of a video camera fixed to the microscope, and were superimposed onto the drawings of adjacent Nisslstained sections. For FG-retrograde tracing, immunoreactive cells were plotted by capturing images onto a computer monitor. Through a video camera fixed to a microscope, 10 cells by structure were processed with a video analysis software (Mercator, Explora Nova, la Rochelle, France), yielding information on the level of
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Fig. 1. Successive schematic drawings of coronal sections through the sheep brain from rostral (a) to caudal (n) showing the distribution of Fluorogold labeled neurons (*) when the tracer was injected in the medial nucleus of the amygdala (left panel) or the cortical nucleus of the amygdala (right panel).
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Fig. 1. (Continued )
grey of each cell. Three classes of staining intensity were defined according to a grey scale (GS) which extend from 0 (black) to 256 (white): GS < 80, high intensity; 81 < GS < 135, moderate intensity; GS > 136, light intensity. This video analysis software was also used to determine the density of BDA-labeled terminals: dense labeling (+++) represents more than 20 fibers/mm2, moderate labeling (++) represents between 10 and 20 fibers/mm2, light labeling (+) represents less than 10 fibers/mm2; () represents a lack of labeling.
2. Results The distribution of labeled structures is shown on schematic drawings of frontal sections (Fig. 1). As the results were similar in all animals, the schematic drawings represent the mean distribution of labeled neurons of the 11 sheep. For each animal, the FG
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Fig. 1. (Continued ).
injection site appeared as an intensely stained area of 1.5 0.2 mm wide (Fig. 2A, A0 , B and B0 ). Within labeled neurons, FG was located in small granules inside the perikaria as well in dendritic processes. Cell nuclei were never stained. According to the subdivisions of amygdala used by Jansen et al. (1998), diffusion of FG from the injection sites was mainly located in the anterior cortical nucleus (C1) in one ewe and in the posterolateral cortical nucleus (C2) in five ewes. In the MeA, FG sites were mainly located in the anterior division of the MeA in two ewes and in the posterior division in four ewes. Diffusion of BDA from the injection sites was mainly located in the anterior cortical nucleus (C1) in the two ewes, in the anterior subdivision of
the MeA in one ewe and in the posterior subdivision in the other animal (Fig. 2C, C0 , D and D0 ). The distribution of FG-labeled neurons was predominantly ipsilateral. The BDA injection sites were similar to FG injection sites (Fig. 2C and D). The results are summarized in Table 1. FG and BDA labeling in the central nucleus of the amygdala was not taken into account because either the injection in the MeA was very close to this nucleus or the guide cannula went through this nucleus in the case of injection into the CoA. Immunolabeled fibers were considered to be a site of projections if fibers were intermixed and presented varicosities. Straight and thick labeled fibers with no varicosities were
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Fig. 2. Low power bright-field photomicrographs illustrating FG and BDA injection sites in the cortical (A and C) or medial (B and D) nuclei of the amygdala. Drawings of the different nuclei were made from adjacent sections stained with cresyl violet (not shown). (A0 )–(D0 ): high power bright-field photomicrographs of injection sites shown in (A)– (D). B, basal amygdaloid nucleus; C1, anterior cortical amygdaloid nucleus; C2, posterolateral cortical amygdaloid nucleus; CoA, cortical amygdaloid nucleus; L, lateral amygdaloid nucleus; MeA, medial amygdaloid nucleus; OT, optic tract. Scale bar = 0.5 mm.
considered to be fibers of passage. Although BDA is an anterograde tracer, a little retrograde labeling (1–5 neurons) can be seen in the following structures: the lateral olfactory tractus nucleus (LOTn; in the two ewes injected in the MeA), the basal and lateral amygdala (in one ewe injected in the MeA) and the subiculum (in one ewe injected in the CoA). 2.1. Connections with the medial amygdala 2.1.1. Telencephalic structures Mitral/tufted (M/T) cells of the AOB showed an important retrograde staining (30–70 neurons per section) (Figs. 1b, 5C and 5D). They were multipolar, distributed throughout the anterior-
posterior extent of the AOB, arranged parallel to the lateral olfactory tract. Their staining intensity varied between intermediate and high. The MOB was never stained (Fig. 1a). The tenia tecta (TT) presented few retrogradely labeled cells (less than 5 neurons per section), multipolar, with intermediate intensity, distributed along the border of section (Fig. 1d). In this system, only LOTn showed an important retrograde and anterograde labeling (Fig. 1j). There were numerous high intensive FG immunoreactive neurons (FG-IR; 30 neurons per section), loosely packed, and a heavy number of BDA-labeled fibers. Reciprocal projections between the MeA and the insular cortex (IC) were observed. FG-IR bipolar neurons (between 40 and 50 neurons per section) were mainly present in the ventral part of IC
M. Meurisse et al. / Journal of Chemical Neuroanatomy 37 (2009) 87–97 Table 1 Distribution of labeled fibers in telencephalic and diencephalic structures following an injection of BDA in the medial (MeA) or cortical nuclei of the amygdala (CoA) Neuroanatomical locus
Injection of BDA in the MeA
Injection of BDA in the CoA
Telencephalic structures MOB AOB AON TT LOTn OFC IC PC E PRh HDB VDB CoA B L MeA VH (CA1) S EC
– – – – +++ – ++ – – + + – ++ + + SITE – – +
– + + + – + – – – – + + SITE ++ – + + ++ +
Diencephalic structures BNST POAdl POAm PVN LHA MBH
++ + – – – –
+++ + + – + –
Symbols indicate the density of labeling, varying from light to heavy (+ to +++).
(Fig. 1f–j). They were sparse and presented a weak intensity. Moderate number of short BDA-labeled fibers was seen in the dorsal and in the ventral part of this area. The vertical limb (VDB; five cells per section) and the horizontal limb (HDB; 10–50 FG-IR neurons per section) of the diagonal band of Broca (DBB) projected onto the MeA (Fig. 1f–i). Intermediate labeled multipolar neurons were sparse and distributed parallel to the border of the section. They included both large multipolar (30 mm) and bipolar types. The HDB, but not the VDB, received light projections from the MeA. Within the ventral hippocampus (VH), there was an important projection to the MeA (50–100 cells per section) originated from the subiculum and CA1 subfield (Fig. 1m). High intense retrogradely labeled neurons were closely packed and perpendicular to the border of the section. They were multipolar and presented numerous processes. No BDA-labeled fibers were observed in these two regions. Reciprocal connections were observed between the
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entorhinal cortex (EC) and the MeA. Intermediate to high FG-IR neurons (100 neurons per section) were seen. They were multipolar, perpendicular to the border of the section, and located preferentially in the ventral part of the area (Fig. 1n). Few, short and sparse BDA fibers were observed in the ventral part of the EC. A weak density of FG-IR neurons (1–5 neurons per section) was noted in the perirhinal cortex (PRh) at the level of the perirhinal scissure (Fig. 1k–n). MeA provided light inputs to the PRh. 2.1.2. Diencephalic structures The bed nucleus of the stria terminalis (BNST) projected onto the MeA (Fig. 1i and j). Some variations were observed in the density of retrogradely labeled perikaria since the majority of the ewes presented 1–20 neurons per section while one ewe presented a higher density of neurons (50–100 neurons per section; Fig. 3A). The BNST contained bipolar FG-IR neurons, strongly labeled in the cytoplasm and processes. Parallel to the border of the lateral ventricle, they formed a continuum with the preoptic area (POA). The BNST received heavy projections from the MeA (Fig. 3B). In the POA, the neurons which project to the MeA (30 neurons per section) were distributed in the whole structure. They were multipolar (15–20 mm), with no specific orientation. Few BDAlabeled fibers were only detected in the dorsolateral part of the POA (POAdl). The paraventricular nucleus (PVN) of the hypothalamus presented few FG-IR cells (2 neurons per section), mainly located in its dorsal part whereas in the close vicinity of the PVN more retrogradely labeled neurons were observed (5–10 neurons per section, Fig. 1l). Only BDA immunostained fibers of passage were seen in the PVN. A weak density of retrogradely labeled neurons was found in the lateral (LHA) and mediobasal hypothalamic (MBH) areas (5–10 neurons per section), sparsely distributed along the third ventricle (Fig. 1k–m). These two areas were devoid of BDA-labeled fibers. 2.1.3. Intra-amygdaloid connections The connections between the MeA and the CoA are reciprocal. The CoA, specifically its posterior part, projected substantially to the MeA (30–100 FG-IR neurons per section, Figs. 1l and 4A). The FG-IR neurons were multipolar and oriented perpendicularly to the border of the section. The MeA send moderate projections to the CoA in which sparse BDA-IR fibers were detected (Fig. 4D). The parvicellular and intermediate part of the basal amygdala (B) presented multipolar, weakly labeled neurons (5–50 neurons per section, Fig. 1l and m), without particular orientation. Numerous FG-IR neurons showing an intermediate to weak intensity were found in the lateral amygdala (L; 50–100 neurons per section, Fig. 1m). The MeA send light projections to the B and the lateral nuclei.
Fig. 3. FG-labeled neurons (A) and Biotin-Dextran-Amine (BDA) fibers (B) observed in the bed nucleus of the stria terminalis after injection in the ipsilateral medial nucleus of the amygdala. Note the abundance of the fibers coming from the medial amygdala to the bed nucleus of the stria terminalis. Scale bar = 100 mm.
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Fig. 4. Connections between the medial (MeA) and the cortical (CoA) nuclei of the amygdala. (A) FG-labeled neurons in the CoA following an injection into the MeA; (B) BDAlabeled terminals in the MeA following an injection into the CoA; (C) FG-labeled neurons in the MeA following an injection into the CoA; (D) BDA-labeled terminals in the CoA following an injection into the MeA. Note the relative abundance of inputs from the CoA to the MeA (A and B) in comparison to the inputs from the MeA to the CoA (C and D). Scale bar = 100 mm.
2.2. Connections with cortical amygdala 2.2.1. Telencephalic structures A moderate retrograde labeling (5–30 neurons per section) was observed in the mitral cells through the rostrocaudal axis of the MOB (Figs. 1a, 5A and 5B) whereas light retrograde labeling was observed in the M/T cells of the AOB (1–20 neurons per section, Figs. 1b and 5F). In the AOB, FG-IR neurons were multipolar, distributed throughout its rostrocaudal extent and parallel to the LOT. Light projections from the CoA were seen in the AOB (Fig. 5E). In the AON, few weak labeled neurons were found (5 neurons per section, Fig. 1c). The TT showed a substantial number of FG-IR neurons (50–100 neurons per section, Fig. 1d and e). The neurons were multipolar, intensely labeled and were located mainly in the ventral part of the structure, perpendicularly to the border of the section. The AON and the TT received light projections from the CoA. In the TT, short dispersed BDA positive fibers were confined to its ventral part, perpendicular to the border of the section. The piriform cortex (PC) sent moderate inputs to the CoA (Fig. 1d– m). FG-IR neurons were detected in both anterior and posterior divisions of the PC. In its posterior division, they were more numerous in the ventral part (more than 50 neurons per section) than in the dorsal part of the area. The endopiriform nucleus (E) showed dense projections to the CoA (50–100 neurons per section, Fig. 1f–j). FG-IR multipolar neurons were present both in the ventral and in the dorsal part of the nucleus and showed an intermediate to high intensity of staining. No BDA fiber was seen in this nucleus. The CoA received light inputs from the orbitofrontal cortex (OFC; 5–30 neurons per section, Fig. 1d). Neurons showed weak intensity of staining and were sparse and oriented perpendicular to the border of the section. Light BDA-labeled fibers were found in the OFC. We noted a light projection from the HDB and the VDB to the CoA (5–10 neurons per section, Fig. 1f–i). Large multipolar neurons were observed, sparsely distributed along the border of the section.
They showed a weak intensity of staining. Light BDA-labeled fibers were found in the HDB and the VDB. The CoA received numerous inputs from the subiculum (more than 100 neurons per section) and from the ventral part of the EC (50–100 neurons per section, Fig. 1m and n). Moderate projections from the CoA to the hippocampus terminated in the subiculum. Light projections were also detected in the CA1 subfield and in the ventral part of the EC. 2.2.2. Diencephalic structures The CoA received few inputs from the BNST (1–5 neurons per section, Fig. 1i and j) whereas it projected heavily on the BNST as numerous BDA-labeled axons showed. In the POA, no FG-IR neuron was found but light stained short BDA-labeled fibers with varicosities were observed in the POAdl and in the medial nucleus of the POA. A light anterograde labeling was seen throughout the LHA. 2.2.3. Intra-amygdaloid connections The MeA contained few FG-IR neurons showing a low labeling intensity (1–5 neurons per section, Figs. 1k, 1l and 4C). FG-IR neurons were sparse, with weak staining. Numerous fibers of BDA were seen in the MeA (Fig. 4B). The B and the posterior part of the lateral amygdala contained numerous FG-IR neurons (30–50 neurons per section, Fig. 1l and m). Only the B received moderate projections from the CoA. 3. Discussion The present report describes, for the first time in the sheep, the neural connections of the CoA and the MeA with the telencephalic and diencephalic regions. The findings are summarized in Fig. 6. Concerning the primary olfactory structures (AOB and MOB), we found that the CoA receives inputs from both the MOB and the
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Fig. 5. Connections between the medial (MeA) and the cortical (CoA) nuclei of the amygdala and the main (MOB) or the accessory (AOB) olfactory bulb. Low magnification (A, scale bar = 200 mm) and high magnification (B, scale bar = 40 mm) of FG-labeled neurons in the MOB following an injection into the CoA. Low magnification (C, scale bar = 100 mm) and high magnification (D, scale bar = 100 mm) of FG-labeled neurons in the AOB following an injection into the MeA. BDA-labeled terminals in the AOB following an injection into the CoA (E, scale bar = 40 mm) and FG-labeled neurons in the AOB following an injection into the CoA (F, scale bar = 100 mm).
AOB, while the MeA is innervated by cells only in the AOB. Among the other olfactory structures, only the TT and the EC are connected with both the CoA and the MeA. With respect to the other secondary olfactory structures, the connections with the CoA and the MeA show segregating neuronal routes. The CoA is connected with the AON, the piriform, the endopiriform and the orbitofrontal cortices while the MeA exhibited connections with the LOTn, the insular and the perirhinal cortices. Concerning the diencephalic structures, only the MeA receives projections from the PVN and the MBH. On the other hand, we showed that the BNST is the major site of connection with both the CoA and the MeA. This study confirmed that the CoA is the major nucleus of the amygdala innervated by the MOB and that it receives inputs from the AOB and the AON (Jansen et al., 1998). However, we did not observe reciprocal inputs from the CoA to the MOB, as previously shown (Jansen et al., 1998; Le´vy et al., 1999). The MeA received afferents only from the AOB, but not from the MOB and any efferent to the AOB and the AON were visualized as previously reported (Jansen et al., 1998). While we used the anterograde tracer BDA, Jansen et al. (1998) injected the retrograde and anterograde tracer wheat germ agglutinin-conjugated horseradish peroxidase (WGA-
HRP) and Le´vy et al. (1999) injected the retrograde tracer, FG. Differences of transport and/or incorporation of these tracers could account for some of these differences. It could also be possible that the volume of BDA injected (0.1 ml) into the CoA or the MeA was too small with regard to a scattered distribution of the neurons and consequently terminals were insufficiently labeled. However, pilot studies showed that an injection of a 0.2 ml of tracer in the MeA extends beyond the central and basal nuclei of the amygdala. Similarly to other mammals studied so far, the CoA and the MeA could be characterized as olfactory-related amygdala. Projections from the MOB to the CoA were also reported in the macaque monkey (Carmichael et al., 1994), in the cat (Russchen, 1982), in the mouse (Shipley and Adamek, 1984) and in the rat (McDonald, 1998; Swanson, 2003). Inputs from the AOB to the MeA were also observed in the cat (Russchen, 1982), as well as in the rat (Swanson, 2003) but were not reported in the monkey. Connections from the AOB to the CoA were only reported in the rat (Swanson, 2003). Concerning the secondary olfactory structures, we did not observe connections between the CoA and the insular cortex, and between the MeA and the orbitofrontal, the piriform, the endopiriform cortices, as it was described in the rat and in the
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Fig. 6. Schematic drawing that shows a summary of the afferents and efferent connections of the cortical and medial nuclei of the amygdala. ( moderate projections; ( ) heavy projections.
hamster (Kevetter and Winans, 1981a,b; Pitkanen et al., 2000, for review). In addition, as in the cat but contrary to the rat, no projection from the piriform cortex to the MeA was observed in the sheep (McDonald, 1998; Pitkanen et al., 2000). This lack of connections could reflect species differences between sheep and rats. Therefore, connections between the MeA or the CoA and the majority of secondary olfactory structures follow two segregated routes. On the one hand, the CoA is connected with the orbitofrontal, the piriform and the endopiriform cortices and on the other hand, the MeA is linked to the insular cortex and the perirhinal cortex. These species differences also could be due to projections too weak to detect and multiple injections of tracers would reveal them. As for the connections between the CoA or the MeA and the other telencephalic (DBB, subiculum, CA1) and the diencephalic regions, their organization is quite similar to the one observed in the rat (Pitkanen et al., 2000). Specifically, the CoA is interconnected with the BNST and sends inputs to the POA while the MeA in addition receives projections from the PVN, the LHA and the MBH. Thus, it appears that the MeA is the site of connections with hypothalamic structures as in the rat (Pitkanen et al., 2000). In sheep, olfaction is essential for the control of reproductive behavior. Exposure of ewes to male odor triggered ovulation (Martin et al., 1986) and mothers formed an olfactory memory for their lambs at parturition (Le´vy et al., 1996b). Processing of these odors depends on the main olfactory system and the CoA. Destruction of the neuroreceptors of the olfactory epithelium or inactivation of the CoA impaired the ability of ewes to respond to the ram or the lamb odor (Gelez et al., 2004; Gelez and Fabre-Nys, 2004; Keller et al., 2004b; Le´vy et al., 1995). In this regard, the present study provides evidence for a substantial innervation from the MOB to the CoA. In addition, direct projections from the CoA to the POA suggest that the male odor could directly reach the GnRH
) light projections; (
)
neurons located in the POA which control the LH response (Gelez and Fabre-Nys, 2006). Contrary to male odor, processing of individual lamb odor also depends on the MeA since inactivation of this nucleus blocked lamb olfactory memory (Keller et al., 2004b). Interconnections between the MeA and the CoA and strong inputs from the BNST/POA and from hypothalamic structures to the MeA allow us to propose a hypothetical pathway supporting the formation of lamb olfactory memory. Parturition activates neurons of the PVN and of the POAm/BNST and this activation is necessary for the normal development of maternal responsiveness (Da Costa et al., 1996; Perrin et al., 2007). Consecutively, these diencephalic regions would activate the MeA through their efferents to this amygdala nucleus. On the other hand, the lamb odor would activate the MeA through the projection from the CoA to the MeA. Thus, the MeA would be a major site in which lamb odor and PVN/POAm/BNST activation induced by parturition would be processed. Investigations of social recognition in rodents show that the MeA is essential for olfactory recognition of conspecifics (Binns and Brennan, 2005; Ferguson et al., 2002). Taken together, these data favour the hypothesis that the MeA forms a hub in the neural network controlling mammalian social behavior (Brennan and Kendrick, 2006). In summary, the CoA and the MeA connections of the ewe appear for the most part to resemble those described for other species. A possible exception concerns the connections with secondary olfactory structures for which two separate pathways were observed. Previous anatomical studies reveal that there are a scant population of scattered mitral/tufted cells in the AOB (Salazar et al., 2007) and a paucity of AOB input from the amygdala (Jansen et al., 1998). In addition, preventing the vomeronasal system from functioning does not affect the LH response to ram odor (CohenTannoudji et al., 1989) or the recognition of lamb olfactory cues (Le´vy et al., 1995). However, our data reveal the existence of a substantial projection from the AOB to the MeA, and to a lesser
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extent to the CoA, which is not in favour of a reduced role of this accessory olfactory system in sheep. Acknowledgements We thank the whole staff of the hospital for the care they provided to the animals during the experiment. The authors are grateful to N. Jouaneau and G. Venier for their expert technical assistance and to M.Keller and Y. Tillet for their helpful comments. This research was supported by a grant A2430 of Re´gion Centre. References Binns, K.E., Brennan, P.A., 2005. Changes in electrophysiological activity in the accessory olfactory bulb and medial amygdala associated with mate recognition in mice. European Journal of Neuroscience 21 (9), 2529–2537. Blanc, M.R., Locatelli, A., Malpaux, B., Le´vy, F., Tillet, Y., 1992. Technique for permanent implantation of cannulae into the sheep brain. In: Dziuk, P.J., Wheeler, M.B. (Eds.), Handbook of Techniques for Reproduction Studies on Pigs, Sheep, Cows, Horses, Chickens, Goats and Rabbits. University of Illinois, Urbana. Breder, C.D., Smith, W.L., Raz, A., Masferrer, J., Seibert, K., Needleman, P., Saper, C.B., 1992. Distribution and characterization of cyclooxygenase immunoreactivity in the ovine brain. Journal of Comparative Neurology 322 (3), 409–438. Brennan, P.A., Kendrick, K.M., 2006. Mammalian social odours: attraction and individual recognition. Philosophical Transactions of the Royal Society B: Biological Sciences 361 (1476), 2061–2078. Brennan, P.A., Keverne, E.B., 1997. Neural mechanisms of mammalian olfactory learning. Progress in Neurobiology 51 (4), 457–481. Carmichael, S.T., Clugnet, M.C., Price, J.L., 1994. Central olfactory connections in the macaque monkey. Journal of Comparative Neurology 346 (3), 403–434. Chang, H.T., Kuo, H., Whittaker, J.A., Cooper, N.G.F., 1990. Light and electron microscopic analysis of projection neurons retrogradely labeled with FluoroGold: notes on the application of antibodies to Fluoro-Gold. Journal of Neuroscience Methods 35, 31–37. Cohen-Tannoudji, J., Lavenet, C., Locatelli, A., Tillet, Y., Signoret, J.P., 1989. Noninvolvement of the accessory olfactory system in the LH response of anoestrous ewes to male odour. Journal of Reproduction and Fertility 86 (1), 135–144. Da Costa, A.P., Guevara-Guzman, R.G., Ohkura, S., Goode, J.A., Kendrick, K.M., 1996. The role of oxytocin release in the paraventricular nucleus in the control of maternal behaviour in the sheep. Journal of Neuroendocrinology 8 (3), 163–177. Demas, G.E., Williams, J.M., Nelson, R.J., 1997. Amygdala but not hippocampal lesions impair olfactory memory for mate in prairie voles (Microtus ochrogaster). American Journal of Physiology 273 (5 (Pt 2)), R1683–R1689. Dluzen, D.E., Muraoka, S., Engelmann, M., Landgraf, R., 1998a. The effects of infusion of arginine vasopressin, oxytocin, or their antagonists into the olfactory bulb upon social recognition responses in male rats. Peptides 19 (6), 999–1005. Dluzen, D.E., Muraoka, S., Landgraf, R., 1998b. Olfactory bulb norepinephrine depletion abolishes vasopressin and oxytocin preservation of social recognition responses in rats. Neuroscience Letters 254 (3), 161–164. Ferguson, J.N., Young, L.J., Insel, T.R., 2002. The neuroendocrine basis of social recognition. Frontiers in Neuroendocrinology 23 (2), 200–224. Ferreira, G., Terrazas, A., Poindron, P., Nowak, R., Orgeur, P., Levy, F., 2000. Learning of olfactory cues is not necessary for early lamb recognition by the mother. Physiology and Behavior 69, 405–412. Gelez, H., Archer, E., Chesneau, D., Magallon, T., Fabre-Nys, C., 2004. Inactivation of the olfactory amygdala prevents the endocrine response to male odour in anoestrus ewes. European Journal of Neuroscience 19 (6), 1581– 1590. Gelez, H., Fabre-Nys, C., 2004. The ‘‘male effect’’ in sheep and goats: a review of the respective roles of the two olfactory systems. Hormones and Behaviour 46 (3), 257–271. Gelez, H., Fabre-Nys, C., 2006. Neural pathways involved in the endocrine response of anestrous ewes to the male or its odor. Neuroscience 140 (3), 791–800. Gheusi, G., Bluthe, R.M., Goodall, G., Dantzer, R., 1994. Social and individual recognition in rodents: methodological aspects and neurobiological bases. Behavioural Processes 33, 59–88.
97
Jansen, H.T., Iwamoto, G.A., Jackson, G.L., 1998. Central connections of the ovine olfactory bulb formation identified using wheat germ agglutinin-conjugated horseradish peroxidase. Brain Research Bulletin 45 (1), 27–39. Keller, M., Meurisse, M., Le´vy, F., 2004a. Mapping the neural substrates involved in maternal responsiveness and lamb olfactory memory in parturient ewes using Fos imaging. Behavioral Neuroscience 118 (6), 1274–1284. Keller, M., Perrin, G., Meurisse, M., Ferreira, G., Levy, F., 2004b. Cortical and medial amygdala are both involved in the formation of olfactory offspring memory in sheep. European Journal of Neuroscience 20 (12), 3433–3441. Kendrick, K.M., Da Costa, A.P., Broad, K.D., Ohkura, S., Guevara, R., Le´vy, F., Keverne, E.B., 1997a. Neural control of maternal behaviour and olfactory recognition of offspring. Brain Research Bulletin 44 (4), 383–395. Kendrick, K.M., Guevara-Guzman, R., Zorrilla, J., Hinton, M.R., Broad, K.D., Mimmack, M., Ohkura, S., 1997b. Formation of olfactory memories mediated by nitric oxide. Nature 388 (6643), 670–674. Kendrick, K.M., Le´vy, F., Keverne, E.B., 1992. Changes in the sensory processing of olfactory signals induced by birth in sheep. Science 256 (5058), 833–836. Kevetter, G.A., Winans, S.S., 1981a. Connections of the corticomedial amygdala of the golden hamster. I. Efferents of the vomeronasal amygdale. Journal of Comparative Neurology 197, 81–98. Kevetter, G.A., Winans, S.S., 1981b. Connections of the corticomedial amygdala of the golden hamster. II. Efferents of the olfactory amygdale. Journal of Comparative Neurology 179, 99–111. Kobbert, C., Apps, R., Bechmann, I., Lanciego, J.L., Mey, J., Thanos, S., 2000. Current concepts in neuroanatomical tracing. Progress in Neurobiology 62 (4), 327–351. Le´vy, F., Keller, M., Poindron, P., 2004. Olfactory regulation of maternal behavior in mammals. Hormones and Behaviour 46 (3), 284–302. Le´vy, F., Kendrick, K., Keverne, E.B., Porter, R.H., Romeyer, A., 1996a. Physiological, sensory and experiential factors of parental care in sheep. Advances in the Study of Behavior 25, 385–473. Le´vy, F., Kendrick, K.M., Keverne, E.B., Proter, R.H., Romeyer, A., 1996b. Physiological, sensory, and experiential factors of prenatal care in sheep. In: Rosenblatt, J.S., Snowdon, C.T. (Eds.), Advances in the Study of Behavior. Academic Press, San Diego, pp. 385–416. Le´vy, F., Locatelli, A., Piketty, V., Tillet, Y., Poindron, P., 1995. Involvement of the main but not the accessory olfactory system in maternal behavior of primiparous and multiparous ewes. Physiology and Behavior 57 (1), 97–104. Le´vy, F., Meurisse, M., Ferreira, G., Thibault, J., Tillet, Y., 1999. Afferents to the rostral olfactory bulb in sheep with special emphasis on the cholinergic, noradrenergic and serotonergic connections. Journal of Chemical Neuroanatomy 16 (4), 245–263. Martin, G., Oldham, C., Cognie´, Y., Pearce, D., 1986. The physiological responses of anovulatory ewes to the introduction of rams: a review. Livestock Production Science 15, 219–247. McDonald, A.J., 1998. Cortical pathways to the mammalian amygdala. Progress in Neurobiology 55 (3), 257–332. Paxinos, G., Watson, C., 1986. The Rat Brain Stereotaxic Coordinates, 2nd ed. Academic Press, San Diego, CA. Perrin, G., Meurisse, M., Levy, F., 2007. Inactivation of the medial preoptic area or the bed nucleus of the stria terminalis differentially disrupts maternal behavior in sheep. Hormones and Behaviour 52 (4), 461–473. Pitkanen, A., Pikkarainen, M., Nurminen, N., Ylinen, A., 2000. Reciprocal connections between the amygdala and the hippocampal formation, perirhinal cortex, and postrhinal cortex in rat. A review. Annals of New York Academy of Sciences 911, 369–391. Poindron, P., 1976. Mother–young relationships in intact or anosmic ewes at the time of suckling. Biology of Behaviour 2, 161–177. Poindron, P., Le´vy, F., Keller, M., 2007. Maternal responsiveness and maternal selectivity in domestic sheep and goats: the two facets of maternal attachment. Developmental Psychobiology 49 (1), 54–70. Russchen, F.T., 1982. Amygdalopetal projections in the cat. I. Cortical afferent connections. A study with retrograde and anterograde tracing techniques. Journal of Comparative Neurology 206 (2), 159–179. Salazar, I., Quinteiro, P.S., Aleman, N., Cifuentes, J.M., Troconiz, P.F., 2007. Diversity of the vomeronasal system in mammals: the singularities of the sheep model. Microscopy Research and Technique 70 (8), 752–762. Shipley, M.T., Adamek, G.D., 1984. The connections of the mouse olfactory bulb: a study using orthograde and retrograde transport of wheat germ agglutinin conjugated to horseradish peroxidase. Brain Research Bulletin 12 (6), 669–688. Swanson, L.W., 2003. The amygdala and its place in the cerebral hemisphere. Annals of New York Academy of Sciences 985, 174–184.
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Journal of Chemical Neuroanatomy journal homepage: www.elsevier.com/locate/jchemneu
Afferent and efferent connections of the cortical and medial nuclei of the amygdala in sheep M. Meurisse, E. Chaillou, F. Le´vy * INRA, UMR85 Physiologie de la Reproduction et des Comportements, CNRS, UMR6175, Universite´ de Tours, Haras Nationaux, F-37380 Nouzilly, France
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 June 2008 Received in revised form 4 September 2008 Accepted 4 September 2008 Available online 12 September 2008
The cortical (CoA) and the medial (MeA) nuclei of the amygdala are involved in the processing of olfactory information relevant to social recognition in the ewe. To better understand the neural pathways responsible for these effects, the connections of both CoA and MeA with the telencephalic and diencephalic regions were studied by injecting an anterograde (Biotin-Dextran-Amine, BDA) or a retrograde (Fluorogold, FG) neuronal tracer into either the CoA or the MeA. Concerning the primary olfactory structures, the CoA receives inputs from both the main olfactory bulb and the accessory olfactory bulb (AOB), while the MeA is innervated by cells only from the AOB. Among the other olfactory structures, only the entorhinal cortex and the tenia tecta are connected with both the CoA and the MeA. With respect to the other secondary olfactory structures, the connections with the CoA and the MeA show segregating neuronal routes. The CoA is connected with the accessory olfactory nucleus, the piriform, the endopiriform and the orbitofrontal cortices while the MeA exhibited connections with the nucleus of the lateral olfactory tract, the perirhinal and the insular cortices. Concerning the diencephalic structures, only the MeA receives projections from the PVN and the MBH. On the other hand, we showed that the BNST is the major site of connection with both the CoA and the MeA. Reciprocal projections were observed between the CoA and the MeA and between both nuclei and the basal or the lateral nuclei of the amygdala with the exception of the CoA which does not send inputs to the lateral nucleus. These data are discussed in relation with olfactory learning in the context of sexual and maternal behavior in sheep. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Fluorogold Dextran amine Neural tract-tracing Olfaction Social behavior
* Corresponding author at: INRA, UMR85 Physiologie de la Reproduction et des Comportements, F-37380 Nouzilly, France. Fax: +33 247427743. E-mail address: [email protected] (F. Le´vy). Abbreviations: 3V, third ventricle; AOB, accessory olfactory bulb; AON, anterior olfactory nucleus; AC, anterior commissure; B, basal amygdaloid nucleus; BNST, bed nucleus of the stria terminalis; C, caudate nucleus; CeA, central amygdaloid nucleus; CoA, cortical amygdaloid nucleus; E, endopiriform nucleus; EC, entorhinal cortex; Fx, fornix; Gl, glomerular layer of the olfactory bulb; Gr, granular cell layer of the olfactory bulb; HDB, horizontal limb of the diagonal band of Broca; IC, insular cortex; L, lateral amygdaloid nucleus; LHA, lateral hypothalamic area; LOT, lateral olfactory tract; LOTn, nucleus of the lateral olfactory tract; LV, lateral ventricle; MBH, mediobasal hypothalamus; MeA, medial amygdaloid nucleus; Mi, mitral cell layer of the olfactory bulb; MOB, main olfactory bulb; MS, medial septum; MT, mamillothalamic tract; OC, optic chiasma; OFC, orbitofrontal cortex; OT, optic tract; OV, olfactory ventricle; PC, piriform cortex; PM, premamillary nucleus; POA, preoptic area; POAdl, dorsolateral part of the POA; POAm, medial part of the POA; PRh, perirhinal cortex; Pu, putamen; PVN, paraventricular nucleus; RT, reticular nucleus of the thalamus; S, subiculum; SON, supraoptic nucleus; TT, tenia tecta; VA, ventral anterior nucleus of the thalamus; VDB, vertical limb of the diagonal band of Broca; VH, ventral hippocampus. 0891-0618/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jchemneu.2008.09.001
The sense of smell is of primary importance for social recognition among mammals. For instance, olfaction is involved in discrimination of familiar conspecifics in rats (Gheusi et al., 1994), in mate recognition in mice, voles, ferrets and sheep (Brennan and Keverne, 1997; Cohen-Tannoudji et al., 1989; Demas et al., 1997) and in the recognition of the offspring in ungulates (Kendrick et al., 1997a; Le´vy et al., 2004, 1996a; Poindron et al., 2007). More particularly, in sheep, exposure of ewes to a male or its odor triggers a release of luteinizing hormone (LH) and ovulation (Martin et al., 1986). Ewes form a selective memory for their lambs after 2 h of mother–young interaction following parturition (Kendrick et al., 1997a; Le´vy et al., 2004, 1996b). This memory is mediated by the main olfactory system, as its disruption through anosmia results in a loss of lamb recognition. As a consequence, both familiar and unfamiliar lambs are suckled (Ferreira et al., 2000; Le´vy et al., 1995; Poindron, 1976). Although these examples of olfactory recognition of conspecifics involve different behavioral contexts, they share common features, including a dependence on the main or the accessory olfactory bulbs (AOBs) which represent the first stage of sensory processing and on their associated functional changes (Brennan and Keverne, 1997; Cohen-Tannoudji
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et al., 1989; Dluzen et al., 1998a,b; Kendrick et al., 1997a,b, 1992). These various forms of olfactory learning depend also on the amygdala and in particular on the two nuclei that are directly associated with the two olfactory systems, the cortical nucleus (CoA) and the medial nucleus (MeA). For example, in sheep, the male odor increases Fos neural expression specifically in the CoA (Gelez and Fabre-Nys, 2006) and the effect of ram odor on the secretion of luteinizing hormone was completely blocked by inactivation of the CoA (Gelez et al., 2004). In parturient ewes exposed to lambs, primary olfactory projections, involving the CoA, show higher Fos expression than those of ewes that were not exposed to lambs (Keller et al., 2004a). Furthermore, inactivation of either the CoA or the MeA during the first 8 h post-partum impaired lamb olfactory recognition by the mother (Keller et al., 2004b). In sheep, the topography of connections of the CoA and the MeA with the rest of the brain has remained poorly understood. Tracttracing studies indicate interconnections between the anterior and posterolateral parts of the CoA and the main olfactory bulb (MOB; Jansen et al., 1998; Le´vy et al., 1999). MOB terminals were also observed within the MeA (Jansen et al., 1998). Connections between the AOB/anterior olfactory nucleus (AON) on the one hand, and the MeA and the posteromedial CoA on the other hand have been reported in one study (Jansen et al., 1998). However, how the two nuclei of the amygdala are connected with the rest of the brain is currently unknown and these data would help us to understand the possible neural networks involved in olfactoryguided reproductive behavior of sheep. In this context, the aim of the present work has been to investigate the afferent and efferent connections with both the CoA and the MeA by injecting either in the CoA and the MeA nuclei, a retrograde tracer, Fluorogold (FG), or an anterograde tracer, BiotinDextran-Amine (BDA), that were looked for in the body cells (FG) and in the fibers (BDA) of the telencephalic and diencephalic structures, known to be involved in social recognition. 1. Material and methods 1.1. Animals Fifteen adult intact Ile de France ewes, 2–5 years old, from the laboratory flock were studied. The majority of ewes (11/15) were used in the seasonal period of reproduction and all the ewes were multiparous. They were kept permanently indoors and fed ad libitum with dehydrated lucerne, maize and straw and had free access to water. All efforts were made to minimize the number of animals used and their suffering. Animal care and all procedures were in accordance with authorization A37801 of the French Ministry of Agriculture. Experimental procedures were approved by the local ethical authority (Comite´ Re´gional d’Ethique et d’Expe´rimentation Animale, Re´gion Centre-Limousin, procedure INRA 37-002). 1.2. Injection procedures Anesthesia was induced with a mixture of Pentothal (Rhoˆne-Poulenc, France) and atropine (20 mg; Lavoisier, Paris, France), and maintained with 4% halothane (Be´lamont, Neuilly, France) in oxygen. Full sterile procedures were used throughout surgery. The animal’s head was fixed in a stereotaxic frame adapted to sheep and modified for X-ray radiographic control (Blanc et al., 1992). Stereotaxic coordinates were calculated using lateral and dorsoventral X-rays of the skull after infusion of a radio-opaque agent (Iopamiron 300; Schering, Germany) in the lateral ventricle. For each ewe, horizontal and vertical tangents to the thalamic mass were used to calculate the coordinates of the implantation site in the rostrocaudal and in the dorsoventral axis. The middle of the third ventricle was used to calculate the lateral coordinates. The stereotaxic coordinates of the cortical and medial amygdala nuclei were as follows: CoA: rostrocaudal plane, 1.7 mm behind the vertical tangent; mediolateral plane, 14 mm from the middle of the third ventricle; depth, 7 mm above the horizontal tangent; MeA: rostrocaudal plane, 1.7 mm; mediolateral plane, 13.2 mm; depth, 1.5 mm. A 2% solution of FG (Interchim, Montluc¸on, France) in sterile distilled water was used for retrograde tracing and 5% solution of BDA (Sigma–Aldrich, L’Isle d’Abeau Chesnes, France) in sterile distilled water was used for anterograde tracing. A 1 ml Hamilton syringe filled with tracers was placed in a probe holder (Kopf) so that its tip was located either in the CoA or in the MeA. A
volume of 0.1 ml of FG alone or BDA alone was slowly injected unilaterally at the rate of 0.01 ml/min. The syringe was then left in place for 15 min after completion to avoid reflux of the tracer along the needle tract. FG was used in 11 ewes: six were injected in the CoA and five in the MeA. BDA was injected in four ewes: two received BDA in the CoA and two in the MeA. For the 5 days following surgery, animals received daily intramuscular injections of an antibiotic (5 ml of amoxicillin; Mixtencilline, Rhoˆne Me´rieux, Lyon, France) and an anti-inflammatory and analgesic preparation (Phe´nylarthrite, 5 ml of phenylbutazone, 0.02 g/ml; Ve´toquinol, Lure, France). 1.3. Histological procedures After a survival period of 15–21 days to allow for sufficient transport of tracers, the ewes received a lethal dose of thiopenthal and were sacrificed by decapitation by a licensed butcher in an official slaughterhouse (authorization No. A37801). Brains were immediately perfused via both carotid arteries with 2 l of 1% sodium nitrite followed by 4 l of ice-cold 4% paraformaldehyde in 0.1 M, pH 7.4, phosphate buffer. The brain was dissected out, cut into blocks and postfixed overnight in the same fixative. The tissues were then stored in 30% sucrose until sectioned. Frontal free-floating sections were cut with a freezing microtome at a thickness of 40 mm and stored at 4 8C in phosphate-buffered saline (PBS) containing 0.1% sodium azide, until processing for FG or BDA visualization. A 1:30 series of sections were stained with cresyl violet in order to identify the appropriate neuroanatomical structures. 1.4. Immunohistochemistry FG was revealed by an antibody providing a permanent image and an excellent resolution (Chang et al., 1990). Sensitivity was increased by detecting the antibody with a nickel (Ni)-enhanced diaminobenzidine (DAB) tetrahydrochloride peroxidase antiperoxidase (PAP) technique. BDA histochemistry yields extensive and detailed labeling of axons and terminals (Kobbert et al., 2000). The tracer was revealed using a standard streptavidin-biotinylated horseradish peroxidase (HRP) procedure to produce a permanent reaction product. 1.4.1. FG staining Endogenous peroxidase activity was first inhibited by incubating the sections for 30 min at 4 8C in PBS containing 1% H2O2. Sections were then incubated for 1 h at room temperature in PBS containing 0.3% Triton X-100, 0.1% sodium azide and 1% BSA (PBST). Afterwards, they were incubated with the 1:15,000 primary antiserum (anti-FG Chemicon, Euromedex, Souffelweyersheim, France) and gently shaken for 48 h at 4 8C. Sections were washed in PBS and incubated 3:30 h at 4 8C in a 1:400 sheep anti-rabbit antibody diluted in PBS–BSA 1%. They were rinsed repeatedly in PBS and incubated overnight at 4 8C in 1:80,000 rabbit PAP complex (Dakocytomation, Trappes, France). Sections were rinsed twice in PBS and twice in Tris–HCl. Immunoreactivity was visualized after incubation in a solution of 0.02% DAB (Sigma–Aldrich, L’Isle d’Abeau Chesnes, France), 0.3% Ni and 0.003% H2O2 in Tris– HCl (50 mM, pH 7.6). The specificity of the immunoreactive reaction was tested by preincubating the anti-FG antibody with an excess of 1 mg/ml of FG (Le´vy et al., 1999). 1.4.2. BDA staining After endogenous peroxidase inhibition, sections were incubated for 1 h at room temperature in PBS containing 0.3% Triton X-100 and 1% BSA. Streptavidin-HRP (Abcys, Paris, France) was diluted 1:500 in the same solution, and sections were left inside overnight at 4 8C. Sections were then rinsed twice in PBS and twice in Tris– HCl. Sections were incubated in a solution of 0.02% DAB (Sigma–Aldrich, L’Isle d’Abeau Chesnes, France), 0.3% Ni and 0.003% H2O2 in Tris–HCl (50 mM, pH 7.6) until a black reaction product was visible. Finally, they were counterstained with neutral red for a better visualization of the neurons and fibers. All sections were then washed, mounted on gelatin-coated slides, dried, and dehydrated through graded alcohol, cleared in toluene and coverslipped with Depex1. 1.5. Analysis of sections Sections were examined under a Zeiss Axiophoto microscope (Zeiss, St. Cloud, France) with bright-field illumination. Across telencephalic and diencephalic structures, 1 section out of 15 was examined; for the olfactory bulb, one section out of three was visualized. Neuroanatomical sites were based on the rat stereotaxic atlas of Paxinos and Watson (1986), on the description of Breder et al. (1992) and on the cresyl violet staining. In order to better locate the FG and BDA injection sites within amygdala nuclei, they were drawn with the help of a video camera fixed to the microscope, and were superimposed onto the drawings of adjacent Nisslstained sections. For FG-retrograde tracing, immunoreactive cells were plotted by capturing images onto a computer monitor. Through a video camera fixed to a microscope, 10 cells by structure were processed with a video analysis software (Mercator, Explora Nova, la Rochelle, France), yielding information on the level of
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Fig. 1. Successive schematic drawings of coronal sections through the sheep brain from rostral (a) to caudal (n) showing the distribution of Fluorogold labeled neurons (*) when the tracer was injected in the medial nucleus of the amygdala (left panel) or the cortical nucleus of the amygdala (right panel).
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Fig. 1. (Continued )
grey of each cell. Three classes of staining intensity were defined according to a grey scale (GS) which extend from 0 (black) to 256 (white): GS < 80, high intensity; 81 < GS < 135, moderate intensity; GS > 136, light intensity. This video analysis software was also used to determine the density of BDA-labeled terminals: dense labeling (+++) represents more than 20 fibers/mm2, moderate labeling (++) represents between 10 and 20 fibers/mm2, light labeling (+) represents less than 10 fibers/mm2; () represents a lack of labeling.
2. Results The distribution of labeled structures is shown on schematic drawings of frontal sections (Fig. 1). As the results were similar in all animals, the schematic drawings represent the mean distribution of labeled neurons of the 11 sheep. For each animal, the FG
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Fig. 1. (Continued ).
injection site appeared as an intensely stained area of 1.5 0.2 mm wide (Fig. 2A, A0 , B and B0 ). Within labeled neurons, FG was located in small granules inside the perikaria as well in dendritic processes. Cell nuclei were never stained. According to the subdivisions of amygdala used by Jansen et al. (1998), diffusion of FG from the injection sites was mainly located in the anterior cortical nucleus (C1) in one ewe and in the posterolateral cortical nucleus (C2) in five ewes. In the MeA, FG sites were mainly located in the anterior division of the MeA in two ewes and in the posterior division in four ewes. Diffusion of BDA from the injection sites was mainly located in the anterior cortical nucleus (C1) in the two ewes, in the anterior subdivision of
the MeA in one ewe and in the posterior subdivision in the other animal (Fig. 2C, C0 , D and D0 ). The distribution of FG-labeled neurons was predominantly ipsilateral. The BDA injection sites were similar to FG injection sites (Fig. 2C and D). The results are summarized in Table 1. FG and BDA labeling in the central nucleus of the amygdala was not taken into account because either the injection in the MeA was very close to this nucleus or the guide cannula went through this nucleus in the case of injection into the CoA. Immunolabeled fibers were considered to be a site of projections if fibers were intermixed and presented varicosities. Straight and thick labeled fibers with no varicosities were
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Fig. 2. Low power bright-field photomicrographs illustrating FG and BDA injection sites in the cortical (A and C) or medial (B and D) nuclei of the amygdala. Drawings of the different nuclei were made from adjacent sections stained with cresyl violet (not shown). (A0 )–(D0 ): high power bright-field photomicrographs of injection sites shown in (A)– (D). B, basal amygdaloid nucleus; C1, anterior cortical amygdaloid nucleus; C2, posterolateral cortical amygdaloid nucleus; CoA, cortical amygdaloid nucleus; L, lateral amygdaloid nucleus; MeA, medial amygdaloid nucleus; OT, optic tract. Scale bar = 0.5 mm.
considered to be fibers of passage. Although BDA is an anterograde tracer, a little retrograde labeling (1–5 neurons) can be seen in the following structures: the lateral olfactory tractus nucleus (LOTn; in the two ewes injected in the MeA), the basal and lateral amygdala (in one ewe injected in the MeA) and the subiculum (in one ewe injected in the CoA). 2.1. Connections with the medial amygdala 2.1.1. Telencephalic structures Mitral/tufted (M/T) cells of the AOB showed an important retrograde staining (30–70 neurons per section) (Figs. 1b, 5C and 5D). They were multipolar, distributed throughout the anterior-
posterior extent of the AOB, arranged parallel to the lateral olfactory tract. Their staining intensity varied between intermediate and high. The MOB was never stained (Fig. 1a). The tenia tecta (TT) presented few retrogradely labeled cells (less than 5 neurons per section), multipolar, with intermediate intensity, distributed along the border of section (Fig. 1d). In this system, only LOTn showed an important retrograde and anterograde labeling (Fig. 1j). There were numerous high intensive FG immunoreactive neurons (FG-IR; 30 neurons per section), loosely packed, and a heavy number of BDA-labeled fibers. Reciprocal projections between the MeA and the insular cortex (IC) were observed. FG-IR bipolar neurons (between 40 and 50 neurons per section) were mainly present in the ventral part of IC
M. Meurisse et al. / Journal of Chemical Neuroanatomy 37 (2009) 87–97 Table 1 Distribution of labeled fibers in telencephalic and diencephalic structures following an injection of BDA in the medial (MeA) or cortical nuclei of the amygdala (CoA) Neuroanatomical locus
Injection of BDA in the MeA
Injection of BDA in the CoA
Telencephalic structures MOB AOB AON TT LOTn OFC IC PC E PRh HDB VDB CoA B L MeA VH (CA1) S EC
– – – – +++ – ++ – – + + – ++ + + SITE – – +
– + + + – + – – – – + + SITE ++ – + + ++ +
Diencephalic structures BNST POAdl POAm PVN LHA MBH
++ + – – – –
+++ + + – + –
Symbols indicate the density of labeling, varying from light to heavy (+ to +++).
(Fig. 1f–j). They were sparse and presented a weak intensity. Moderate number of short BDA-labeled fibers was seen in the dorsal and in the ventral part of this area. The vertical limb (VDB; five cells per section) and the horizontal limb (HDB; 10–50 FG-IR neurons per section) of the diagonal band of Broca (DBB) projected onto the MeA (Fig. 1f–i). Intermediate labeled multipolar neurons were sparse and distributed parallel to the border of the section. They included both large multipolar (30 mm) and bipolar types. The HDB, but not the VDB, received light projections from the MeA. Within the ventral hippocampus (VH), there was an important projection to the MeA (50–100 cells per section) originated from the subiculum and CA1 subfield (Fig. 1m). High intense retrogradely labeled neurons were closely packed and perpendicular to the border of the section. They were multipolar and presented numerous processes. No BDA-labeled fibers were observed in these two regions. Reciprocal connections were observed between the
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entorhinal cortex (EC) and the MeA. Intermediate to high FG-IR neurons (100 neurons per section) were seen. They were multipolar, perpendicular to the border of the section, and located preferentially in the ventral part of the area (Fig. 1n). Few, short and sparse BDA fibers were observed in the ventral part of the EC. A weak density of FG-IR neurons (1–5 neurons per section) was noted in the perirhinal cortex (PRh) at the level of the perirhinal scissure (Fig. 1k–n). MeA provided light inputs to the PRh. 2.1.2. Diencephalic structures The bed nucleus of the stria terminalis (BNST) projected onto the MeA (Fig. 1i and j). Some variations were observed in the density of retrogradely labeled perikaria since the majority of the ewes presented 1–20 neurons per section while one ewe presented a higher density of neurons (50–100 neurons per section; Fig. 3A). The BNST contained bipolar FG-IR neurons, strongly labeled in the cytoplasm and processes. Parallel to the border of the lateral ventricle, they formed a continuum with the preoptic area (POA). The BNST received heavy projections from the MeA (Fig. 3B). In the POA, the neurons which project to the MeA (30 neurons per section) were distributed in the whole structure. They were multipolar (15–20 mm), with no specific orientation. Few BDAlabeled fibers were only detected in the dorsolateral part of the POA (POAdl). The paraventricular nucleus (PVN) of the hypothalamus presented few FG-IR cells (2 neurons per section), mainly located in its dorsal part whereas in the close vicinity of the PVN more retrogradely labeled neurons were observed (5–10 neurons per section, Fig. 1l). Only BDA immunostained fibers of passage were seen in the PVN. A weak density of retrogradely labeled neurons was found in the lateral (LHA) and mediobasal hypothalamic (MBH) areas (5–10 neurons per section), sparsely distributed along the third ventricle (Fig. 1k–m). These two areas were devoid of BDA-labeled fibers. 2.1.3. Intra-amygdaloid connections The connections between the MeA and the CoA are reciprocal. The CoA, specifically its posterior part, projected substantially to the MeA (30–100 FG-IR neurons per section, Figs. 1l and 4A). The FG-IR neurons were multipolar and oriented perpendicularly to the border of the section. The MeA send moderate projections to the CoA in which sparse BDA-IR fibers were detected (Fig. 4D). The parvicellular and intermediate part of the basal amygdala (B) presented multipolar, weakly labeled neurons (5–50 neurons per section, Fig. 1l and m), without particular orientation. Numerous FG-IR neurons showing an intermediate to weak intensity were found in the lateral amygdala (L; 50–100 neurons per section, Fig. 1m). The MeA send light projections to the B and the lateral nuclei.
Fig. 3. FG-labeled neurons (A) and Biotin-Dextran-Amine (BDA) fibers (B) observed in the bed nucleus of the stria terminalis after injection in the ipsilateral medial nucleus of the amygdala. Note the abundance of the fibers coming from the medial amygdala to the bed nucleus of the stria terminalis. Scale bar = 100 mm.
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Fig. 4. Connections between the medial (MeA) and the cortical (CoA) nuclei of the amygdala. (A) FG-labeled neurons in the CoA following an injection into the MeA; (B) BDAlabeled terminals in the MeA following an injection into the CoA; (C) FG-labeled neurons in the MeA following an injection into the CoA; (D) BDA-labeled terminals in the CoA following an injection into the MeA. Note the relative abundance of inputs from the CoA to the MeA (A and B) in comparison to the inputs from the MeA to the CoA (C and D). Scale bar = 100 mm.
2.2. Connections with cortical amygdala 2.2.1. Telencephalic structures A moderate retrograde labeling (5–30 neurons per section) was observed in the mitral cells through the rostrocaudal axis of the MOB (Figs. 1a, 5A and 5B) whereas light retrograde labeling was observed in the M/T cells of the AOB (1–20 neurons per section, Figs. 1b and 5F). In the AOB, FG-IR neurons were multipolar, distributed throughout its rostrocaudal extent and parallel to the LOT. Light projections from the CoA were seen in the AOB (Fig. 5E). In the AON, few weak labeled neurons were found (5 neurons per section, Fig. 1c). The TT showed a substantial number of FG-IR neurons (50–100 neurons per section, Fig. 1d and e). The neurons were multipolar, intensely labeled and were located mainly in the ventral part of the structure, perpendicularly to the border of the section. The AON and the TT received light projections from the CoA. In the TT, short dispersed BDA positive fibers were confined to its ventral part, perpendicular to the border of the section. The piriform cortex (PC) sent moderate inputs to the CoA (Fig. 1d– m). FG-IR neurons were detected in both anterior and posterior divisions of the PC. In its posterior division, they were more numerous in the ventral part (more than 50 neurons per section) than in the dorsal part of the area. The endopiriform nucleus (E) showed dense projections to the CoA (50–100 neurons per section, Fig. 1f–j). FG-IR multipolar neurons were present both in the ventral and in the dorsal part of the nucleus and showed an intermediate to high intensity of staining. No BDA fiber was seen in this nucleus. The CoA received light inputs from the orbitofrontal cortex (OFC; 5–30 neurons per section, Fig. 1d). Neurons showed weak intensity of staining and were sparse and oriented perpendicular to the border of the section. Light BDA-labeled fibers were found in the OFC. We noted a light projection from the HDB and the VDB to the CoA (5–10 neurons per section, Fig. 1f–i). Large multipolar neurons were observed, sparsely distributed along the border of the section.
They showed a weak intensity of staining. Light BDA-labeled fibers were found in the HDB and the VDB. The CoA received numerous inputs from the subiculum (more than 100 neurons per section) and from the ventral part of the EC (50–100 neurons per section, Fig. 1m and n). Moderate projections from the CoA to the hippocampus terminated in the subiculum. Light projections were also detected in the CA1 subfield and in the ventral part of the EC. 2.2.2. Diencephalic structures The CoA received few inputs from the BNST (1–5 neurons per section, Fig. 1i and j) whereas it projected heavily on the BNST as numerous BDA-labeled axons showed. In the POA, no FG-IR neuron was found but light stained short BDA-labeled fibers with varicosities were observed in the POAdl and in the medial nucleus of the POA. A light anterograde labeling was seen throughout the LHA. 2.2.3. Intra-amygdaloid connections The MeA contained few FG-IR neurons showing a low labeling intensity (1–5 neurons per section, Figs. 1k, 1l and 4C). FG-IR neurons were sparse, with weak staining. Numerous fibers of BDA were seen in the MeA (Fig. 4B). The B and the posterior part of the lateral amygdala contained numerous FG-IR neurons (30–50 neurons per section, Fig. 1l and m). Only the B received moderate projections from the CoA. 3. Discussion The present report describes, for the first time in the sheep, the neural connections of the CoA and the MeA with the telencephalic and diencephalic regions. The findings are summarized in Fig. 6. Concerning the primary olfactory structures (AOB and MOB), we found that the CoA receives inputs from both the MOB and the
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Fig. 5. Connections between the medial (MeA) and the cortical (CoA) nuclei of the amygdala and the main (MOB) or the accessory (AOB) olfactory bulb. Low magnification (A, scale bar = 200 mm) and high magnification (B, scale bar = 40 mm) of FG-labeled neurons in the MOB following an injection into the CoA. Low magnification (C, scale bar = 100 mm) and high magnification (D, scale bar = 100 mm) of FG-labeled neurons in the AOB following an injection into the MeA. BDA-labeled terminals in the AOB following an injection into the CoA (E, scale bar = 40 mm) and FG-labeled neurons in the AOB following an injection into the CoA (F, scale bar = 100 mm).
AOB, while the MeA is innervated by cells only in the AOB. Among the other olfactory structures, only the TT and the EC are connected with both the CoA and the MeA. With respect to the other secondary olfactory structures, the connections with the CoA and the MeA show segregating neuronal routes. The CoA is connected with the AON, the piriform, the endopiriform and the orbitofrontal cortices while the MeA exhibited connections with the LOTn, the insular and the perirhinal cortices. Concerning the diencephalic structures, only the MeA receives projections from the PVN and the MBH. On the other hand, we showed that the BNST is the major site of connection with both the CoA and the MeA. This study confirmed that the CoA is the major nucleus of the amygdala innervated by the MOB and that it receives inputs from the AOB and the AON (Jansen et al., 1998). However, we did not observe reciprocal inputs from the CoA to the MOB, as previously shown (Jansen et al., 1998; Le´vy et al., 1999). The MeA received afferents only from the AOB, but not from the MOB and any efferent to the AOB and the AON were visualized as previously reported (Jansen et al., 1998). While we used the anterograde tracer BDA, Jansen et al. (1998) injected the retrograde and anterograde tracer wheat germ agglutinin-conjugated horseradish peroxidase (WGA-
HRP) and Le´vy et al. (1999) injected the retrograde tracer, FG. Differences of transport and/or incorporation of these tracers could account for some of these differences. It could also be possible that the volume of BDA injected (0.1 ml) into the CoA or the MeA was too small with regard to a scattered distribution of the neurons and consequently terminals were insufficiently labeled. However, pilot studies showed that an injection of a 0.2 ml of tracer in the MeA extends beyond the central and basal nuclei of the amygdala. Similarly to other mammals studied so far, the CoA and the MeA could be characterized as olfactory-related amygdala. Projections from the MOB to the CoA were also reported in the macaque monkey (Carmichael et al., 1994), in the cat (Russchen, 1982), in the mouse (Shipley and Adamek, 1984) and in the rat (McDonald, 1998; Swanson, 2003). Inputs from the AOB to the MeA were also observed in the cat (Russchen, 1982), as well as in the rat (Swanson, 2003) but were not reported in the monkey. Connections from the AOB to the CoA were only reported in the rat (Swanson, 2003). Concerning the secondary olfactory structures, we did not observe connections between the CoA and the insular cortex, and between the MeA and the orbitofrontal, the piriform, the endopiriform cortices, as it was described in the rat and in the
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Fig. 6. Schematic drawing that shows a summary of the afferents and efferent connections of the cortical and medial nuclei of the amygdala. ( moderate projections; ( ) heavy projections.
hamster (Kevetter and Winans, 1981a,b; Pitkanen et al., 2000, for review). In addition, as in the cat but contrary to the rat, no projection from the piriform cortex to the MeA was observed in the sheep (McDonald, 1998; Pitkanen et al., 2000). This lack of connections could reflect species differences between sheep and rats. Therefore, connections between the MeA or the CoA and the majority of secondary olfactory structures follow two segregated routes. On the one hand, the CoA is connected with the orbitofrontal, the piriform and the endopiriform cortices and on the other hand, the MeA is linked to the insular cortex and the perirhinal cortex. These species differences also could be due to projections too weak to detect and multiple injections of tracers would reveal them. As for the connections between the CoA or the MeA and the other telencephalic (DBB, subiculum, CA1) and the diencephalic regions, their organization is quite similar to the one observed in the rat (Pitkanen et al., 2000). Specifically, the CoA is interconnected with the BNST and sends inputs to the POA while the MeA in addition receives projections from the PVN, the LHA and the MBH. Thus, it appears that the MeA is the site of connections with hypothalamic structures as in the rat (Pitkanen et al., 2000). In sheep, olfaction is essential for the control of reproductive behavior. Exposure of ewes to male odor triggered ovulation (Martin et al., 1986) and mothers formed an olfactory memory for their lambs at parturition (Le´vy et al., 1996b). Processing of these odors depends on the main olfactory system and the CoA. Destruction of the neuroreceptors of the olfactory epithelium or inactivation of the CoA impaired the ability of ewes to respond to the ram or the lamb odor (Gelez et al., 2004; Gelez and Fabre-Nys, 2004; Keller et al., 2004b; Le´vy et al., 1995). In this regard, the present study provides evidence for a substantial innervation from the MOB to the CoA. In addition, direct projections from the CoA to the POA suggest that the male odor could directly reach the GnRH
) light projections; (
)
neurons located in the POA which control the LH response (Gelez and Fabre-Nys, 2006). Contrary to male odor, processing of individual lamb odor also depends on the MeA since inactivation of this nucleus blocked lamb olfactory memory (Keller et al., 2004b). Interconnections between the MeA and the CoA and strong inputs from the BNST/POA and from hypothalamic structures to the MeA allow us to propose a hypothetical pathway supporting the formation of lamb olfactory memory. Parturition activates neurons of the PVN and of the POAm/BNST and this activation is necessary for the normal development of maternal responsiveness (Da Costa et al., 1996; Perrin et al., 2007). Consecutively, these diencephalic regions would activate the MeA through their efferents to this amygdala nucleus. On the other hand, the lamb odor would activate the MeA through the projection from the CoA to the MeA. Thus, the MeA would be a major site in which lamb odor and PVN/POAm/BNST activation induced by parturition would be processed. Investigations of social recognition in rodents show that the MeA is essential for olfactory recognition of conspecifics (Binns and Brennan, 2005; Ferguson et al., 2002). Taken together, these data favour the hypothesis that the MeA forms a hub in the neural network controlling mammalian social behavior (Brennan and Kendrick, 2006). In summary, the CoA and the MeA connections of the ewe appear for the most part to resemble those described for other species. A possible exception concerns the connections with secondary olfactory structures for which two separate pathways were observed. Previous anatomical studies reveal that there are a scant population of scattered mitral/tufted cells in the AOB (Salazar et al., 2007) and a paucity of AOB input from the amygdala (Jansen et al., 1998). In addition, preventing the vomeronasal system from functioning does not affect the LH response to ram odor (CohenTannoudji et al., 1989) or the recognition of lamb olfactory cues (Le´vy et al., 1995). However, our data reveal the existence of a substantial projection from the AOB to the MeA, and to a lesser
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extent to the CoA, which is not in favour of a reduced role of this accessory olfactory system in sheep. Acknowledgements We thank the whole staff of the hospital for the care they provided to the animals during the experiment. The authors are grateful to N. Jouaneau and G. Venier for their expert technical assistance and to M.Keller and Y. Tillet for their helpful comments. This research was supported by a grant A2430 of Re´gion Centre. References Binns, K.E., Brennan, P.A., 2005. Changes in electrophysiological activity in the accessory olfactory bulb and medial amygdala associated with mate recognition in mice. European Journal of Neuroscience 21 (9), 2529–2537. Blanc, M.R., Locatelli, A., Malpaux, B., Le´vy, F., Tillet, Y., 1992. Technique for permanent implantation of cannulae into the sheep brain. In: Dziuk, P.J., Wheeler, M.B. (Eds.), Handbook of Techniques for Reproduction Studies on Pigs, Sheep, Cows, Horses, Chickens, Goats and Rabbits. University of Illinois, Urbana. Breder, C.D., Smith, W.L., Raz, A., Masferrer, J., Seibert, K., Needleman, P., Saper, C.B., 1992. Distribution and characterization of cyclooxygenase immunoreactivity in the ovine brain. Journal of Comparative Neurology 322 (3), 409–438. Brennan, P.A., Kendrick, K.M., 2006. Mammalian social odours: attraction and individual recognition. Philosophical Transactions of the Royal Society B: Biological Sciences 361 (1476), 2061–2078. Brennan, P.A., Keverne, E.B., 1997. Neural mechanisms of mammalian olfactory learning. Progress in Neurobiology 51 (4), 457–481. Carmichael, S.T., Clugnet, M.C., Price, J.L., 1994. Central olfactory connections in the macaque monkey. Journal of Comparative Neurology 346 (3), 403–434. Chang, H.T., Kuo, H., Whittaker, J.A., Cooper, N.G.F., 1990. Light and electron microscopic analysis of projection neurons retrogradely labeled with FluoroGold: notes on the application of antibodies to Fluoro-Gold. Journal of Neuroscience Methods 35, 31–37. Cohen-Tannoudji, J., Lavenet, C., Locatelli, A., Tillet, Y., Signoret, J.P., 1989. Noninvolvement of the accessory olfactory system in the LH response of anoestrous ewes to male odour. Journal of Reproduction and Fertility 86 (1), 135–144. Da Costa, A.P., Guevara-Guzman, R.G., Ohkura, S., Goode, J.A., Kendrick, K.M., 1996. The role of oxytocin release in the paraventricular nucleus in the control of maternal behaviour in the sheep. Journal of Neuroendocrinology 8 (3), 163–177. Demas, G.E., Williams, J.M., Nelson, R.J., 1997. Amygdala but not hippocampal lesions impair olfactory memory for mate in prairie voles (Microtus ochrogaster). American Journal of Physiology 273 (5 (Pt 2)), R1683–R1689. Dluzen, D.E., Muraoka, S., Engelmann, M., Landgraf, R., 1998a. The effects of infusion of arginine vasopressin, oxytocin, or their antagonists into the olfactory bulb upon social recognition responses in male rats. Peptides 19 (6), 999–1005. Dluzen, D.E., Muraoka, S., Landgraf, R., 1998b. Olfactory bulb norepinephrine depletion abolishes vasopressin and oxytocin preservation of social recognition responses in rats. Neuroscience Letters 254 (3), 161–164. Ferguson, J.N., Young, L.J., Insel, T.R., 2002. The neuroendocrine basis of social recognition. Frontiers in Neuroendocrinology 23 (2), 200–224. Ferreira, G., Terrazas, A., Poindron, P., Nowak, R., Orgeur, P., Levy, F., 2000. Learning of olfactory cues is not necessary for early lamb recognition by the mother. Physiology and Behavior 69, 405–412. Gelez, H., Archer, E., Chesneau, D., Magallon, T., Fabre-Nys, C., 2004. Inactivation of the olfactory amygdala prevents the endocrine response to male odour in anoestrus ewes. European Journal of Neuroscience 19 (6), 1581– 1590. Gelez, H., Fabre-Nys, C., 2004. The ‘‘male effect’’ in sheep and goats: a review of the respective roles of the two olfactory systems. Hormones and Behaviour 46 (3), 257–271. Gelez, H., Fabre-Nys, C., 2006. Neural pathways involved in the endocrine response of anestrous ewes to the male or its odor. Neuroscience 140 (3), 791–800. Gheusi, G., Bluthe, R.M., Goodall, G., Dantzer, R., 1994. Social and individual recognition in rodents: methodological aspects and neurobiological bases. Behavioural Processes 33, 59–88.
97
Jansen, H.T., Iwamoto, G.A., Jackson, G.L., 1998. Central connections of the ovine olfactory bulb formation identified using wheat germ agglutinin-conjugated horseradish peroxidase. Brain Research Bulletin 45 (1), 27–39. Keller, M., Meurisse, M., Le´vy, F., 2004a. Mapping the neural substrates involved in maternal responsiveness and lamb olfactory memory in parturient ewes using Fos imaging. Behavioral Neuroscience 118 (6), 1274–1284. Keller, M., Perrin, G., Meurisse, M., Ferreira, G., Levy, F., 2004b. Cortical and medial amygdala are both involved in the formation of olfactory offspring memory in sheep. European Journal of Neuroscience 20 (12), 3433–3441. Kendrick, K.M., Da Costa, A.P., Broad, K.D., Ohkura, S., Guevara, R., Le´vy, F., Keverne, E.B., 1997a. Neural control of maternal behaviour and olfactory recognition of offspring. Brain Research Bulletin 44 (4), 383–395. Kendrick, K.M., Guevara-Guzman, R., Zorrilla, J., Hinton, M.R., Broad, K.D., Mimmack, M., Ohkura, S., 1997b. Formation of olfactory memories mediated by nitric oxide. Nature 388 (6643), 670–674. Kendrick, K.M., Le´vy, F., Keverne, E.B., 1992. Changes in the sensory processing of olfactory signals induced by birth in sheep. Science 256 (5058), 833–836. Kevetter, G.A., Winans, S.S., 1981a. Connections of the corticomedial amygdala of the golden hamster. I. Efferents of the vomeronasal amygdale. Journal of Comparative Neurology 197, 81–98. Kevetter, G.A., Winans, S.S., 1981b. Connections of the corticomedial amygdala of the golden hamster. II. Efferents of the olfactory amygdale. Journal of Comparative Neurology 179, 99–111. Kobbert, C., Apps, R., Bechmann, I., Lanciego, J.L., Mey, J., Thanos, S., 2000. Current concepts in neuroanatomical tracing. Progress in Neurobiology 62 (4), 327–351. Le´vy, F., Keller, M., Poindron, P., 2004. Olfactory regulation of maternal behavior in mammals. Hormones and Behaviour 46 (3), 284–302. Le´vy, F., Kendrick, K., Keverne, E.B., Porter, R.H., Romeyer, A., 1996a. Physiological, sensory and experiential factors of parental care in sheep. Advances in the Study of Behavior 25, 385–473. Le´vy, F., Kendrick, K.M., Keverne, E.B., Proter, R.H., Romeyer, A., 1996b. Physiological, sensory, and experiential factors of prenatal care in sheep. In: Rosenblatt, J.S., Snowdon, C.T. (Eds.), Advances in the Study of Behavior. Academic Press, San Diego, pp. 385–416. Le´vy, F., Locatelli, A., Piketty, V., Tillet, Y., Poindron, P., 1995. Involvement of the main but not the accessory olfactory system in maternal behavior of primiparous and multiparous ewes. Physiology and Behavior 57 (1), 97–104. Le´vy, F., Meurisse, M., Ferreira, G., Thibault, J., Tillet, Y., 1999. Afferents to the rostral olfactory bulb in sheep with special emphasis on the cholinergic, noradrenergic and serotonergic connections. Journal of Chemical Neuroanatomy 16 (4), 245–263. Martin, G., Oldham, C., Cognie´, Y., Pearce, D., 1986. The physiological responses of anovulatory ewes to the introduction of rams: a review. Livestock Production Science 15, 219–247. McDonald, A.J., 1998. Cortical pathways to the mammalian amygdala. Progress in Neurobiology 55 (3), 257–332. Paxinos, G., Watson, C., 1986. The Rat Brain Stereotaxic Coordinates, 2nd ed. Academic Press, San Diego, CA. Perrin, G., Meurisse, M., Levy, F., 2007. Inactivation of the medial preoptic area or the bed nucleus of the stria terminalis differentially disrupts maternal behavior in sheep. Hormones and Behaviour 52 (4), 461–473. Pitkanen, A., Pikkarainen, M., Nurminen, N., Ylinen, A., 2000. Reciprocal connections between the amygdala and the hippocampal formation, perirhinal cortex, and postrhinal cortex in rat. A review. Annals of New York Academy of Sciences 911, 369–391. Poindron, P., 1976. Mother–young relationships in intact or anosmic ewes at the time of suckling. Biology of Behaviour 2, 161–177. Poindron, P., Le´vy, F., Keller, M., 2007. Maternal responsiveness and maternal selectivity in domestic sheep and goats: the two facets of maternal attachment. Developmental Psychobiology 49 (1), 54–70. Russchen, F.T., 1982. Amygdalopetal projections in the cat. I. Cortical afferent connections. A study with retrograde and anterograde tracing techniques. Journal of Comparative Neurology 206 (2), 159–179. Salazar, I., Quinteiro, P.S., Aleman, N., Cifuentes, J.M., Troconiz, P.F., 2007. Diversity of the vomeronasal system in mammals: the singularities of the sheep model. Microscopy Research and Technique 70 (8), 752–762. Shipley, M.T., Adamek, G.D., 1984. The connections of the mouse olfactory bulb: a study using orthograde and retrograde transport of wheat germ agglutinin conjugated to horseradish peroxidase. Brain Research Bulletin 12 (6), 669–688. Swanson, L.W., 2003. The amygdala and its place in the cerebral hemisphere. Annals of New York Academy of Sciences 985, 174–184.