Retrograde labelling of mitral/tufted cells in the mouse accessory olfactory bulb following local injections of the lipophilic tracer DiI into the vomeronasal amygdala

It has recently become apparent that there are two classes of vomeronasal receptor neurons that project to functionally separate anterior and posterior sub-regions of the mammalian accessory olfactory bulb. However, anterograde tracing of the projections from these sub-regions, in the mouse, has revealed that the processing pathways are not segregated at the level of the vomeronasal amygdala. Both sub-regions have overlapping projections to the superficial lamina of the medial and posterior medial cortical nuclei of the amygdala. However, differential projections have been found in the opossum, in which only the posterior sub-region projects to the deeper laminae of the medial amygdala. Therefore, there may be species differences in these projections that are important for the control of reproductive behaviour. This study used an alternative approach of retrogradely tracing mitral/tufted cell projections from different nuclei of the vomeronasal amygdala back to the accessory olfactory bulb of mice. Local injections of the lipophilic tracer DiI were made into the antero-dorsal and postero-ventral divisions of the medial amygdala, and into the postero-medial cortical amygdala. In each case, provided the DiI affected the superficial lamina Ia, labelled mitral/tufted cells were found distributed throughout the anterior-posterior extent of the accessory olfactory bulb. These results confirm that mitral/tufted cells of the anterior and posterior sub-regions of the accessory olfactory bulb project to both the medial and postero-medial cortical nuclei of the amygdala. There was no evidence for differential projections from the anterior and posterior sub-regions accessory olfactory bulb in mice, as has been reported to occur in other species.     

Differential projections from the anterior and posterior divisions of the accessory olfactory bulb to the medial amygdala in the opossum, Monodelphis domestica

The vomeronasal sensory epithelium of mammals contains apical and basal cell populations expressing different G proteins and putative pheromone receptors, which project, respectively, to the anterior and posterior divisions of the accessory olfactory bulb (AOB). In order to analyse whether these segregated pathways are preserved in the connections between the AOB and the amygdala, conjugated dextran-amines were iontophoretically injected into the anterior and posterior divisions of the AOB. We found that efferent projections from both divisions essentially overlap throughout the vomeronasal recipient amygdala. In the medial amygdaloid complex, both divisions project to lamina 1A of layer 1 of the anterodorsal, anteroventral, posterodorsal and posteroventral nuclei. The posterior division alone, however, projects to lamina 1B and layers 2 and 3 of the anterodorsal, anteroventral and posteroventral nuclei. These results constitute a link between molecular, anatomical and functional approaches on the study of the vomeronasal system. Molecular and functional studies support that the two segregated pathways between the vomeronasal organ and the AOB are functionally different. Similarly, the anatomical approaches to the further connections of this system indicate that the medial amygdala possesses ventral and dorsal divisions that are hodologically and functionally different. The present results demonstrate a differential projection from the posterior AOB to the ventral division of the medial amygdala. These findings indicate that the segregated pathways of the vomeronasal system continue to the level of the amygdala, and they provide some clues about the functional implications.     

Olfactory bulbectomy increases food intake and hypothalamic neuropeptide Y in obesity-prone but not obesity-resistant rats

Obese individuals often suffer from depression. The olfactory bulbectomy (OBX) model is an animal model of depression that produces behavioral, physiological, and neurochemical alterations resembling clinical depression. The OBX model was employed to assess depression-related changes in food intake in obesity-prone, Osborne–Mendel (OM) rats and obesity-resistant, S5B/Pl rats. OBX increased food intake in OM rats beginning 7 days following surgery, however, OBX did not alter food intake in S5B/Pl rats at any time point. Fourteen days following surgery, OBX significantly increased locomotor activity (total lines crossed and rears) in the openfield test in OM and S5B/Pl rats. Fifteen days following surgery, prepro-neuropeptide Y (NPY) mRNA levels were significantly increased in the hypothalamus of bulbectomized OM rats and in the medial nucleus of the amygdala of bulbectomized OM and S5B/Pl rats. OBX decreased NPY Y2 receptor mRNA levels in the hypothalamus and medial nucleus of the amygdala in OM rats, while increasing NPY Y2 receptor mRNA levels in the medial nucleus of the amygdala of S5B/Pl rats. These data indicate that though both obesity-prone and obesity-resistant strains were susceptible to the locomotor effects of OBX, food intake and hypothalamic prepro-NPY mRNA were only increased in OM rats. Therefore, strain specific alterations in hypothalamic NPY may account for increased food intake in the obesity-prone rats following OBX, and suggests a potential mechanism to explain the comorbidity of obesity and depression.     

A horseradish peroxidase study of the olfactory system of the frog, Rana esculenta

The olfactory system of the frog Rana esculenta was studied by using horseradish peroxidase (HRP) tracing of axonal pathways. Injections of HRP were made in the main olfactory bulb (MOB), accessory olfactory bulb (AOB), anterior olfactory nucleus (AON), the amygdala (AMY), and in a zone of the leteral wall of the telencephalic hemisphere immediately posterior to the AOB. Projections from these sites are described and are generally similar to those obtained by degeneration methods. However, HRP reveals more extensive olfactory connections than previously reported. Ipsilateral, contralateral, and bilateral projections are described. The MOB, AOB, and AON have ipsilateral connections to each other. The MOB and AOB have very different projections. The MOB and AON project via the habenular commissure (HC) to the contralateral medial wall of the telencephalon. Ipsilateral MOB fibers also terminate in this cell-free zone where the medial forebrain bundle (MFB) originates. The AOB projects to the lateral cortex of the contralateral telencephalic hemisphere via the HC and also to the ipsilateral AMY and lateral forebrain bundle (LFB) from where some fibers project contralaterally. HRP injections in the AMY retrogradely fill cells in the ipsilateral AOB, two nuclei of the ipsilateral hypothalamus and a nucleus of cells caudal to the ipsilateral nucleus isthmi. Fibers are also labeled that project to the contralateral AMY. Few fibers were observed to decussate in the interpeduncular nucleus or optic chiasma. No olfactory fibers were found to project to the habenular nuclei, and no labeled neurons were found to project to the olfactory bulbs. No morphological asymmetry was observed qualitatively in the distribution of olfactory fibers in the two halves of the brain.     

Efferents and centrifugal afferents of the main and accessory olfactory bulbs in the hamster

The efferents and centrifugal afferents of the hamster olfactory bulbs were studied using orthograde and retrograde tracing techniques. Following injections of tritiated amino acids which were restricted to the main olfactory bulb (MOB), autoradiographic grains were observed ipsilaterally over layer IA of the entire anterior olfactory nucleus (AON), the ventral portion of the hippocampal rudiment (HR), the entire prepyriform cortex and olfactory tubercle, the anterior and posterolateral cortical amygdaloid nuclei and the lateral entorhinal cortex. An ipsilateral projection to the nucleus of the lateral olfactory tract (nLOT) was also indicated. No subcortical or contralateral projections were observed. Amino acid injections into the accessory olfactory bulb (AOB) revealed ipsilateral projections to the superficial plexiform layer of the medial and posteromedial cortical amygdaloid nuclei and to the bed nucleus of the accessory olfactory tract (nAOT) and the bed nucleus of the stria terminalis (nST). Following injections of HRP which were restricted to the MOB, contralateral HRP-positive neurons were found predominantly in pars externa and to a lesser extent in the other subdivisions of the AON. Centrifugal projections to the MOB were identified ipsilaterally from the entire AON, the ventral portion of the HR, the anterior portion of the prepyriform cortex, and the nLOT. No labelled neurons were found in the olfactory tubercle, the anterior and posterolateral cortical amygdaloid nuclei or the entorhinal cortex. Centrifugal projections to the MOB were also identified from subcortical structures of the ipsilateral basal forebrain and from midline structures of the midbrain. Labelling occurred in the fusiform neurons of the diagonal band near the medial base of the forebrain at the level of caudal olfactory tubercle. Heavy labelling was seen in a distinct group of large, predominantly multipolar neurons (magnocellular preoptic area) that continued from the level of caudal olfactory tubercle to the level of the nLOT. This band of HRP-positive neurons could be followed more caudally to a position dorsal and medial to the nLOT near the lateral margin of the lateral anterior hypothalamic area. The midbrain projections to the MOB originated in the dorsal and median raphe nuclei. After injections of HRP into the AOB, centrifugal projections were identified from the nAOT and the posteromedial cortical amygdaloid nucleus. In addition, isolated neurons were labelled in the medial cortical amygdaloid nucleus but no labelled neurons were found in the nST. These results support the notion of two anatomically distinct olfactory systems and demonstrate two previously unreported pathways through which the limbic system may modulate sensory processing in the olfactory bulb.     

Connections of the corticomedial amygdala in the golden hamster. I. Efferents of the "vomeronasal amygdala"

The medial (M) an posteromedial cortical (C3) amygdaloid nuclei and the nucleus of the accessory olfactory tract (NAOT) are designated the "vomeronasal amygdala" because they are the only components of the amygdala to receive a direct projection from the accessory olfactory bulb (AOB). The efferents of M and C3 were traced after injections of 3H-proline into the amygdala in male golden hamsters. Frozen sections of the brains were processed for autoradiography. The efferents of the "vomeronasal amygdala" are largely to areas which are primary and secondary terminal areas along the vomeronasal pathway, although the efferents from C3 and M terminate in different layers in these areas than do the projections from the vomeronasal nerve or the AOB. Specifically, C3 projects ipsilaterally to the internal granule cell layer of the AOB, the cellular layer of NAOT, and layer Ib of M. Additional fibers from C3 terminate in a retrocommissural component of the bed nucleus of the strain terminalis (BNST) bilaterally, and in the cellular layers of the contralateral C3. The medial nucleus projects to the cellular layer of the ipsilateral NAOT, layer Ib of C3, and bilaterally to the medial component of BNST. Projections from M to non-vomeronasal areas terminate in the medial preoptic area-anterior hypothalamic junction, ventromedial nucleus of the hypothalamus, ventral premammillary nucleus and possibly in the ventral subiculum. These results demonstrate reciprocal connections between primary and secondary vomeronasal areas between the secondary areas themselves. They suggest that M, but not C3, projects to areas outside this vomeronasal network. The medial amygdaloid nucleus is therefore an important link between the vomeronasal organ and areas of the brain not receiving direct vomeronasal input.     

The vomeronasal cortex – afferent and efferent projections of the posteromedial cortical nucleus of the amygdala in mice

Most mammals possess a vomeronasal system that detects predominantly chemical signals of biological relevance. Vomeronasal information is relayed to the accessory olfactory bulb (AOB), whose unique cortical target is the posteromedial cortical nucleus of the amygdala. This cortical structure should therefore be considered the primary vomeronasal cortex. In the present work, we describe the afferent and efferent connections of the posteromedial cortical nucleus of the amygdala in female mice, using anterograde (biotinylated dextranamines) and retrograde (Fluorogold) tracers, and zinc selenite as a tracer specific for zinc‐enriched (putative glutamatergic) projections. The results show that the posteromedial cortical nucleus of the amygdala is strongly interconnected not only with the rest of the vomeronasal system (AOB and its target structures in the amygdala), but also with the olfactory system (piriform cortex, olfactory‐recipient nuclei of the amygdala and entorhinal cortex). Therefore, the posteromedial cortical nucleus of the amygdala probably integrates olfactory and vomeronasal information. In addition, the posteromedial cortical nucleus of the amygdala shows moderate interconnections with the associative (basomedial) amygdala and with the ventral hippocampus, which may be involved in emotional and spatial learning (respectively) induced by chemical signals. Finally, the posteromedial cortical nucleus of the amygdala gives rise to zinc‐enriched projections to the ventrolateral septum and the ventromedial striatum (including the medial islands of Calleja). This pattern of intracortical connections (with the olfactory cortex and hippocampus, mainly) and cortico‐striatal excitatory projections (with the olfactory tubercle and septum) is consistent with its proposed nature as the primary vomeronasal cortex.     

Differential projections of the main and accessory olfactory bulb in the frog.

The central projections of the main olfactory bulb and the accessory olfactory bulb of the adult leopard frog (Rana pipiens) were reexamined, by using a horseradish peroxidase anterograde tracing method that fills axons with a continuous deposit of reaction product. The fine morphology preserved by this method allowed the terminal fields of the projection tracts to be delineated reliably, and for the first time. Herrick's amygdala has been newly subdivided into cortical and medial nuclei on the basis of cytoarchitecture, dendritic morphology, and the differential projections of the main and accessory olfactory tracts. The main olfactory bulb projects through the medial and lateral olfactory tracts to the postolfactory eminence, the rostral end of the medial cortex, the rostral end of the medial septal nucleus, the cortical amygdaloid nucleus, the nucleus of the hemispheric sulcus, and both the dorsal and ventral divisions of the lateral cortex, including its retrobulbar fringe. The lateral olfactory tract overlaps the dorsal edge of the striatal plate along the ventral border of the lateral cortex, but it is not certain whether any striatal cells are postsynaptic to the tract fibers. The lateral cortex is the largest of these territories, and receives the terminals of the main olfactory projection throughout its extent. It extends from the olfactory bulb to the posterior pole, and from the striatum to the summit of the hemisphere, where it borders the dorsal cortex. The medial and lateral olfactory tracts combine in the region of the amygdala to form a part of the stria medullaris thalami. These fibers cross in the habenular commissure and terminate in the contralateral cortical amygdaloid nucleus and periamygdaloid part of the lateral cortex. Cells projecting to the main olfactory bulb are found in the diagonal band and adjacent cell groups, but there is no evidence of an interbulbar projection arising from either the olfactory bulb proper or a putative anterior olfactory nucleus. The accessory olfactory bulb projects through the accessory olfactory tract to the medial and cortical amygdaloid nuclei. A fascicle of the tract crosses in the anterior commissure to terminate in the contralateral amygdala. While the main and accessory olfactory projections may converge in the cortical amygdaloid nucleus, the medial amygdaloid nucleus is connected exclusively with the accessory olfactory bulb.     

Afferent and efferent connections of the olfactory bulbs in the lizard Podarcis hispanica.

The connections of the olfactory bulbs of Podarcis hispanica were studied by tract-tracing of injected horseradish peroxidase. Restricted injections into the main olfactory bulb (MOB) resulted in bilateral terminallike labeling in the medial part of the anterior olfactory nucleus (AON) and in the rostral septum, lateral cortex, nucleus of the lateral olfactory tract, and ventrolateral amygdaloid nucleus. Bilateral retrograde labeling was found in the rostral lateral cortex and in the medial and dorsolateral AON. Ipsilaterally the dorsal cortex, nucleus of the diagonal band, lateral preoptic area, and dorsolateral amygdala showed labeled cell bodies. Retrogradely labeled cells were also found in the midbrain raphe nucleus. Results from injections into the rostral lateral cortex and lateral olfactory tract indicate that the mitral cells are the origin of the centripetal projections of the MOB. Injections in the accessory olfactory bulb (AOB) produced ipsilateral terminallike labeling of the ventral AON, bed nucleus of the accessory olfactory tract, central and ventromedial amygdaloid nuclei, medial part of the bed nucleus of the stria terminalis, and nucleus sphericus. Retrograde labeling of neurons was observed ipsilaterally in the bed nucleus of the accessory olfactory tract and stria terminalis, in the central amygdaloid nucleus, dorsal cortex, and nucleus of the diagonal band. Bilateral labeling of somata was found in the ventral AON, the nucleus sphericus (hilus), and in the mesencephalic raphe nucleus and locus coeruleus. Injections into the dorsal amygdala showed that the mitral neurons are the cells of origin of the AOB centripetal projections. Reciprocal connections are present between AOB and MOB. To our knowledge, this is the first study to address the afferent connections of the olfactory bulbs in a reptile. On the basis of the available data, a discussion is provided of the similarities and differences between the reptilian and mammalian olfactory systems, as well as of the possible functional role of the main olfactory connections in reptiles.     

Lateral and medial amygdala of anuran amphibians and their relation to olfactory and vomeronasal information

The amygdala of anurans is currently considered as a complex of nuclei that share many features with their counterparts in amniotes. In the present study, the subdivisions of the amygdala that are directly related to olfactory and vomeronasal information, were investigated in the anurans Rana perezi and Xenopus laevis. In particular, the connectivity of the main and accessory olfactory bulbs and their related amygdaloid nuclei was studied by means of in vivo and in vitro tract-tracing with dextran amines. The projections observed from the main olfactory bulb clearly innervate the newly redefined lateral amygdala within the ventral pallium and, to a lesser extent, the rostral portion of the medial amygdala. Injections into the accessory olfactory bulb exclusively revealed projections to the medial amygdala. Tracer applications into the lateral and medial nuclei revealed abundant intra-amygdaloid connections. The dual flow of olfactory and vomeronasal projections throughout the telencephalon was not strictly segregated since the lateral pallium and the lateral amygdala, both receiving olfactory information, were found to project to the medial amygdala (the only target of vomeronasal information), which in turn projects to the lateral amygdala. Additionally, both the lateral and the medial amygdala strongly project to the hypothalamus through the anuran equivalent of the stria terminalis. The main hodological features found in the present study suggest that forerunners of the olfactory and vomeronasal amygdaloid nuclei can be distinguished in anurans. This supports the notion that all tetrapods share a common pattern of organization of the amygdaloid complex, which links environmental (olfactory/vomeronasal) information and the behavioural response of the animal.     

Cortical projection patterns of magnocellular basal nucleus subdivisions as revealed by anterogradely transportedPhaseolus vulgaris leucoagglutinin

The present paper deals with a detailed analysis of cortical projections from the magnocellular basal nucleus (MBN) and horizontal limb of the diagonal band of Broca (HDB) in the rat. The MBN and HDB were injected iontophoretically with the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L). After immunocytochemical visualization of labeled efferents, the distribution of projections over the cortical mantle, olfactory regions and amygdala were studied by light microscopy. Based on differences in cortical projection patterns, the MBN was subdivided in anterior, intermediate and posterior portions (MBNa, MBNi and MBNp). All subdivisions maintain neocortical projections and are subject to an anterior to posterior topographic arrangement. In the overall pattern, however, the frontal cortex is the chief target. Furthermore, all MBN parts project to various regions of meso- and allocortex, which are progressively more dense when the tracer injection is more anteriorly placed. The most conspicuous finding, however, was a ventrolateral to dorsomedial cortical projection pattern as the PHA-L injection site moved from posterior to anterior. Thus, the posterior MBN projects predominantly to lateral neo- and mesocortex while the anterior MBN sends more fibers to the medial cortical regions. Furthermore, the MBNa is a source of considerable afferent input to the olfactory nuclei and as such should be regarded as a transition to the HDB. The HDB, apart from projecting densely to olfactory bulb and related nuclei, maintains a substantial output to the medial prefrontal cortical regions and entorhinal cortex, as well. Comparison of young vs aged cases indicate that aging does not appear to have a profound influence on cortical innervation patterns, at least as studied with the PHA-L method.     

Segregated pathways to the vomeronasal amygdala: differential projections from the anterior and posterior divisions of the accessory olfactory bulb

Apically and basally located receptor neurons in the vomeronasal sensory epithelium express G(i2 alpha)- and G(o alpha)-proteins, V1R and V2R vomeronasal receptors, project to the anterior and posterior accessory olfactory bulb and respond to different stimuli, respectively. The extent to which secondary projections from the two portions of the accessory olfactory bulb are convergent in the vomeronasal amygdala is controversial. This issue is addressed by using anterograde and retrograde tract-tracing methods in rats including electron microscopy. Injections of dextran-amines, Fluoro Gold, cholera toxin-B subunit and Fast Blue were delivered to the anterior and posterior accessory olfactory bulb, bed nucleus of the stria terminalis, dorsal anterior amygdala and bed nucleus of the accessory olfactory tract/anteroventral medial amygdaloid nucleus. We have demonstrated that, apart from common vomeronasal-recipient areas, only the anterior accessory olfactory bulb projects to the bed nucleus of the stria terminalis, medial division, posteromedial part, and only the posterior accessory olfactory bulb projects to the dorsal anterior amygdala and deep cell layers of the bed nucleus of the accessory olfactory tract and the anteroventral medial amygdaloid nucleus. These results provide evidence that, excluding areas of convergence, the V1R and V2R vomeronasal pathways project to specific areas of the amygdala. These two vomeronasal subsystems are therefore anatomically and functionally separated in the telencephalon.     

Characterization of neuropeptide Y2 receptor protein expression in the mouse brain. I. Distribution in cell bodies and nerve terminals

Neuropeptide Y (NPY), a 36-amino-acid peptide, mediates biological effects by activating Y1, Y2, Y5, and y6 receptors. NPY neurons innervate many brain regions, including the hypothalamus, where NPY is involved in regulation of a broad range of homeostatic functions. We examined, by immunohistochemistry with tyramide signal amplification, the expression of the NPY Y2 receptor (Y2R) in the mouse brain with a newly developed rabbit polyclonal antibody. Y2R immunoreactivity was specific with its absence in Y2R knockout (KO) mice and in adjacent sections following preadsorption with the immunogenic peptide (10(-5) M). Y2R-positive processes were located in many brain regions, including the olfactory bulb, some cortical areas, septum, basal forebrain, nucleus accumbens, amygdala, hippocampus, hypothalamus, substantia nigra compacta, locus coeruleus, and solitary tract nucleus. However, colchicine treatment was needed to detect Y2R-like immunoreactivity in cell bodies in many, but not all, areas. The densest distributions of cell bodies were located in the septum basal forebrain, including the bed nucleus, and amygdala, with lower density in the anterior olfactory nucleus, nucleus accumbens, caudal striatum, CA1, CA2, and CA3 hippocampal fields, preoptic nuclei lateral hypothalamus, and A13 DA cells. The widespread distribution of Y2R-positive cell bodies and fibers suggests that NPY signaling through the Y2R is common in the mouse brain. Localization of the Y2R suggests that it is mostly presynaptic, a view supported by its frequent absence in cell bodies in the normal mouse and its dramatic increase in cell bodies of colchicine-treated mice.     

Differential centrifugal afferents to the anterior and posterior accessory olfactory bulb.

The centrifugal afferents to the anterior and posterior divisions of the accessory olfactory bulb (AOB) were investigated after iontophoretic injections of dextranamines. Injections affecting the anterior and posterior or just the posterior division of the AOB gave rise to retrogradely labeled cells in the bed nuclei of the accessory olfactory tract and stria terminalis and in the anterodorsal medial and posteromedial cortical (PMCo) amygdaloid nuclei. Injections restricted to the anterior division of the AOB yielded similar results, although no cells were observed in the PMCo. These results demonstrate differential centrifugal inputs to the anterior and posterior divisions of the AOB, probably to the granular layer, and provide further support for the hypothesis of a functionally segregated vomeronasal system.     

A direct main olfactory bulb projection to the 'vomeronasal' amygdala in female mice selectively responds to volatile pheromones from males

The main olfactory system, like the accessory olfactory system, responds to pheromones involved in social communication. Whereas pheromones detected by the accessory system are transmitted to the hypothalamus via the medial ('vomeronasal') amygdala, the pathway by which pheromones are detected and transmitted by the main system is not well understood. We examined in female mice whether a direct projection from mitral/tufted (M/T) cells in the main olfactory bulb (MOB) to the medial amygdala exists, and whether medial amygdala-projecting M/T cells are activated by volatile urinary odors from conspecifics or a predator (cat). Simultaneous anterograde tracing using Phaseolus vulgaris leucoagglutinin and Fluoro-Ruby placed in the MOB and accessory olfactory bulb (AOB), respectively, revealed that axons of MOB M/T cells projected to superficial laminae of layer Ia in anterior and posterodorsal subdivisions of the medial amygdala, whereas projection neurons from the AOB sent axons to non-overlapping, deeper layer Ia laminae of the same subdivisions. Placement of the retrograde tracer cholera toxin B into the medial amygdala labeled M/T cells that were concentrated in the ventral MOB. Urinary volatiles from male mice, but not from female conspecifics or cat, induced Fos in medial amygdala-projecting MOB M/T cells of female subjects, suggesting that information about male odors is transmitted directly from the MOB to the 'vomeronasal' amygdala. The presence of a direct MOB-to-medial amygdala pathway in mice and other mammals could enable volatile, opposite-sex pheromones to gain privileged access to diencephalic structures that control mate recognition and reproduction.     

Central Connections of the Ovine Olfactory Bulb Formation Identified Using Wheat Germ Agglutinin-Conjugated Horseradish Peroxidase

Pheromonal stimuli elicit rapid behavioral and reproductive endocrine changes in the ewe. The neural pathways responsible for these effects in sheep are unknown, in part, because the olfactory bulb projections have not been examined in this species. Using the anterograde and retrograde neuronal tracer, wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP), we describe the afferent and efferent olfactory bulb connections of the Suffolk ewe. Injections of WGA-HRP limited to the main olfactory bulb resulted in retrograde labeling of cells in numerous telencephalic, diencephalic, and metencephalic regions. Terminal labeling was limited to layer Ia of ipsilateral cortical structures extending rostrally from the anterior olfactory nucleus (AON), piriform cortex, anterior-, and posterolateral-cortical amygdaloid nuclei to lateral entorhinal cortex caudally. Injections involving the accessory olfactory bulb and AON produced additional labeling of cells within the bed nucleus of the stria terminalis (BNST), medial nucleus of the amygdala, and a few cells in the posteromedial cortical nucleus of the amygdala. Terminal labeling included a small dorsomedial quadrant of BNST and also extended to the far lateral portions of the supraoptic nucleus. A clearly defined accessory olfactory tract and nucleus was not evident, perhaps due to limitations in the sensitivity of the method. With this possible exception, the afferent and efferent olfactory connections in the sheep appear similar to those reported for other species.     

Immunohistochemical localization of an inositol 1,4,5-trisphosphate receptor, P400, in neural tissue: studies in developing and adult mouse brain.

The immunohistochemical localization of P400/inositol 1,4,5-trisphosphate (InsP3) receptor protein was studied in developing and adult mouse brain by using monoclonal antibodies. The developmental expression pattern of P400/InsP3 receptor protein differed among different classes of neurons. It was first detected in the somata of immature Purkinje cells at embryonic day 17, in the ventrolateral region of the posterior vermis in the cerebellum. Axonal immunoreactivity within the cerebellar nuclei was first present at postnatal day 3. Neurons in the retrosplenial cortex, the anterior olfactory nucleus, and the CA1 region of the hippocampus expressed immunoreactivity earlier than other regions of the brain. In the adult brain, not only the Purkinje cell but also many other types of cells in many areas of the brain expressed P400/InsP3 receptor, though to a lesser extent. These included the neurons in the striatum, globus pallidus, nucleus accumbens septi, anterior olfactory nucleus, olfactory tubercle, precommissural hippocampus, hippocampus, substantia nigra, cerebral cortex, pons, and certain hypothalamic nuclei. Forebrain cortical regions that receive afferents from the olfactory bulb, such as the anterior olfactory nucleus, olfactory tubercle, prepiriform cortex, entorhinal cortex, and amygdala, exhibited distinct immunoreactivity, while olfactory bulb was almost devoid of staining. Immunoreactivity in the axonal pathways was also found in the limbic-hypothalamic pathways, strionigral projection, and part of the corpus callosum. Results of Western blot analysis and 3H-InsP3 binding assay were consistent with the qualitative regional differences of immunoreactivity demonstrated by immunohistochemical study. The location of InsP3 receptor in the brain correlates well with the InsP3 binding sites demonstrated by an autoradiographic study.     

Hodological characterization of the septum in anuran amphibians: I. Afferent connections

On the basis of Nissl-stained sections, we subdivided the septum of the gray treefrog Hyla versicolor in the lateral, central, and medial septal complex. The afferent projections of the different septal nuclei were studied by combined retrograde and anterograde tracing with biotin ethylendiamine (Neurobiotin). The central and medial septal complex receives direct input from regions of the olfactory bulb and from all other limbic structures of the telencephalon (e.g., amygdalar regions, nucleus accumbens), whereas projections to the lateral septal complex are absent or less extensive. The medial pallium projects to all septal nuclei. In the diencephalon, the anterior thalamic nucleus provides the main ascending input to all subnuclei of the anuran septum, which can be interpreted as a limbic/associative pathway. The ventromedial thalamic nucleus projects to the medial and lateral septal complex and may thereby transmit multisensory information to the limbic system. Anterior preoptic nucleus, suprachiasmatic nucleus, and hypothalamic nuclei innervate the central and lateral septal complex. Only the nuclei of the central septal complex receive input from the brainstem. Noteworthy is the relatively strong projection from the nucleus raphe to the central septal complex, but not to the other septal nuclei.     

Olfactory projections in the lepidosirenid lungfishes

Olfactory nerve and olfactory bulb projections in lepidosirenid lungfishes were experimentally determined with neural tracers. Unilateral injections of DiI into the olfactory nerve labeled the accessory and main olfactory bulbs as well as fibers of the anterior root of the terminal nerve, which terminates extensively in cell groups of the medial hemispheric wall, the dorsal and lateral pallia, and the preoptic nuclei and posterior tubercle. Lepidosirenid lungfishes do not exhibit separate vomeronasal nerves, but previous data indicate that calbindin-positive receptors within basal crypts of the olfactory epithelium are homologous to the vomeronasal organ of tetrapods. Unilateral injections of DiI into the accessory olfactory bulb reveal an accessory olfactory tract which terminates primarily if not solely in the ipsilateral medial amygdalar nucleus as in amphibians. Unilateral injections of tracers into the main olfactory bulb reveal extensive projections to all cell groups in the ipsilateral telencephalic hemisphere, except for the medial amygdalar nucleus, as well as secondary olfactory projections (decussating in the habenular commissure) to the contralateral dorsal pallium and main olfactory bulb. Secondary olfactory projections also terminate bilaterally in diencephalic and midbrain centers after partial decussation in the anterior and postoptic commissures, as well as in the ventral hypothalamus and posterior tubercle. Cladistic analysis of the extensive secondary olfactory projections indicates that this pattern is primitive for all bony fishes whereas the reduction in secondary olfactory projections in amphibians, particularly anurans, is a derived, simplified pattern.