Errors in lamina growth of primary olfactory axons in the rat and mouse olfactory bulb

In the adult olfactory nerve pathway of rodents, each primary olfactory axon forms a terminal arbor in a single glomerulus in the olfactory bulb. During development, axons are believed to project directly to and terminate precisely within a glomerulus without any exuberant growth or mistargeting. To gain insight into mechanisms underlying this process, the trajectories of primary olfactory axons during glomerular formation were studied in the neonatal period. Histochemical staining of mouse olfactory bulb sections with the lectin Dolichos biflorus-agglutinin revealed that many olfactory axons overshoot the glomerular layer and course into the deeper laminae of the bulb in the early postnatal period. Single primary olfactory axons were anterogradely labelled either with the lipophilic carbocyanine dye, 1,1'-dioctodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), or with horseradish peroxidase (HRP) by localized microinjections into the nerve fiber layer of the rat olfactory bulb. Five distinct trajectories of primary olfactory axons were observed in DLI-labelled preparations at postnatal day 1.5 (P1.5). Axons either coursed directly to and terminated specifically within a glomerulus, branched before terminating in a glomerulus, bypassed glomeruli and entered the underlying external plexiform layer, passed through the glomerular layer with side branches into glomeruli, or branched into more than one glomerulus. HRP-labelled axon arbors from eight postnatal ages were reconstructed by camera lucida and were used to determine arbor length, arbor area, and arbor branch number. Whereas primary olfactory axons display errors in laminar targeting in the mammalian olfactory bulb, axon arbors typically achieve their adult morphology without exuberant growth. Many olfactory axons appear not to recognize appropriate cues to terminate within the glomerular layer during the early postnatal period. However, primary olfactory axons exhibit precise targeting in the glomerular layer after P5.5, indicating temporal differences in either the presence of guidance cues or the ability of axons to respond to these cues. (C) 1999 Wiley-Liss, Inc.

Shared and differential traits in the accessory olfactory bulb of caviomorph rodents with particular reference to the semiaquatic capybara

The vomeronasal system is crucial for social and sexual communication in mammals. Two populations of vomeronasal sensory neurons, each expressing G alpha i2 or G alpha o proteins, send projections to glomeruli of the rostral or caudal accessory olfactory bulb, rAOB and cAOB, respectively. In rodents, the G alpha i2- and G alpha o-expressing vomeronasal pathways have shown differential responses to small/volatile vs. large/non-volatile semiochemicals, respectively. Moreover, early gene expression suggests predominant activation of rAOB and cAOB neurons in sexual vs. aggressive contexts, respectively. We recently described the AOB of Octodon degus, a semiarid-inhabiting diurnal caviomorph. Their AOB has a cell indentation between subdomains and the rAOB is twice the size of the cAOB. Moreover, their AOB receives innervation from the lateral aspect, contrasting with the medial innervation of all other mammals examined to date. Aiming to relate AOB anatomy with lifestyle, we performed a morphometric study on the AOB of the capybara, a semiaquatic caviomorph whose lifestyle differs remarkably from that of O. degus. Capybaras mate in water and scent-mark their surroundings with oily deposits, mostly for male-male communication. We found that, similar to O. degus, the AOB of capybaras shows a lateral innervation of the vomeronasal nerve, a cell indentation between subdomains and heterogeneous subdomains, but in contrast to O. degus the caudal portion is larger than the rostral one. We also observed that four other caviomorph species present a lateral AOB innervation and a cell indentation between AOB subdomains, suggesting that those traits could represent apomorphies of the group. We propose that although some AOB traits may be phylogenetically conserved in caviomorphs, ecological specializations may play an important role in shaping the AOB.

Chemically and morphologically identifiable glomeruli in the rat olfactory bulb

Primary olfactory neurons that express the same odorant receptor are distributed mosaically throughout the olfactory neuroepithelium lining the nasal cavity, yet their axons converge and form discrete glomeruli in the olfactory bulb. We previously proposed that cell surface carbohydrates mediate the sorting out and selective fasciculation of primary olfactory axons en route to glomeruli. If this were the case, then axons that terminate in the same glomerulus would express the same complement of cell surface carbohydrates. In this study, we examined the expression of a novel carbohydrate (NOC-3) on neural cell adhesion molecule in the adult rat olfactory system. NOC-3 was expressed by a subset of neurons distributed throughout the olfactory neuroepithelium. The axons of these neurons entered the nerve fiber layer and terminated in a subset of glomeruli. It is interesting to note that we identified three unusually large glomeruli in the lateral, ventrolateral, and ventromedial olfactory bulb that were innervated by axons expressing NOC-3. NOC-3-expressing axons sorted out and fasciculated into discrete fascicles prior to entering these glomeruli. Each of these glomeruli was in a topographically fixed position in the olfactory bulbs of the same animal as well as in different animals, and their lengths were approximately 10% of the total length of the bulb. They could be identified reliably by both their topographical position and their unique morphology. These results reveal that axons expressing the same cell surface carbohydrates consistently target the same topographically fixed glomeruli, which supports a role for these molecules in axon navigation in the primary olfactory nerve pathway. J. Comp. Neurol. 436: 497-507, 2001. (C) 2001 Wiley-Liss, Inc.

Expression of neurexin ligands, the neuroligins and the neurexophilins, in the developing and adult rodent olfactory bulb

The neurexins are a large family of neuronal cell-surface proteins believed to be involved in intercellular signalling and the formation of intercellular junctions. To begin to assess the role of these proteins in the olfactory bulb, we describe here the expression patterns of their transmembrane and secreted ligands, the neuroligins and neurexophilins, during both embryonic and postnatal development. In situ hybridisation showed that neuroligin 1 and 2 were expressed by second order mitral cells during early postnatal development but not in adults. The secreted ligand for a-neurexin, neurexophilin 1, was also expressed in the postnatal olfactory bulb. Neurexophilin 1 was detected in only periglomerular cells during the early postnatal period of glomerular formation but later was also expressed in mitral cells. These results suggest that neurexin-ligand interactions may be important for development and/or maturation of synaptic connections in the primary olfactory pathway.

Laminar disorganisation of mitral cells in the olfactory bulb does not affect topographic targeting of primary olfactory axons

Primary olfactory neurons expressing the same odorant receptor protein typically project to topographically fixed olfactory bulb sites. While cell adhesion molecules and odorant receptors have been implicated in guidance of primary olfactory axons. the postsynaptic mitral cells may also have a role in final target selection. We have examined the effect of disorganisation of the mitral cell soma layer in mutant mice heterozygous for the beta-subunit of platelet activating factor acetylhydrolase (Lis1(-/+)) on the targeting of primary olfactory axons. Lis1(-/+) mice display abnormal lamination of neurons in the olfactory bulb. Lis1(-/+) mice were crossed with the P2-IRES-tau:LacZ line of transgenic mice that selectively expresses beta-galactosidase in primary olfactory neurons expressing the P2 odorant receptor. LacZ histochemistry revealed blue-stained P2 axons that targeted topographically fixed glomeruli in these mice in a manner similar to that observed in the parent P2-IRES-tau:LacZ line. Thus, despite the aberrant organisation of postsynaptic mitral cells in Lis1(-/+) mice, primary olfactory axons continued to converge and form glomeruli at correct sites in the olfactory bulb. Next we examined whether challenging primary olfactory axons in adult Lis(-/+) mice with regeneration would affect their ability to converge and form glomeruli. Following partial chemical ablation of the olfactory neuroepithelium with dichlobenil, primary olfactory neurons die and are replaced by newly differentiating neurons that project axons to the olfactory bulb where they converge and form glomeruli. Despite the aberrant mitral cell layer in Lis(-/+) mice. primary olfactory axons continued to converge and form glomeruli during regeneration. Together these results demonstrate that the convergence of primary olfactory axons during development and regeneration is not affected by gross perturbations to the lamination of the mitral cell layer. Thus, these results support evidence from other studies indicating that mitral cells do not play a major role in the convergence and targeting of primary olfactory axons in the olfactory bulb. (C) 2002 Elsevier Science B.V. All rights reserved.

Sorting and convergence of primary olfactory axons are independent of the olfactory bulb

Primary olfactory axons expressing the same odorant receptor gene sort out and converge to fixed sites in the olfactory bulb. We examined the guidance of axons expressing the P2 odorant receptor when they were challenged with different cellular environments in vivo. In the mutant extratoes mouse, the olfactory bulb is lacking and is replaced by a fibrocellular mass. In these animals, primary olfactory axons form glomerular-like loci despite the absence of normal postsynaptic targets. P2 axons are able to sort out from other axons in this fibrocellular mass and converge to form loci of like axons. The sites of these loci along mediolateral and ventrodorsal axes were highly variable. Similar convergence was observed for larger subpopulations of axons expressing the same cell surface carbohydrates. The sorting out and convergence of like axons also occurred during regeneration following bulbectomy. Olfactory axon behaviour in these models demonstrates that sorting and convergence of axons are independent of the target, which instead provides distinct topographic cues for guidance. (C) 2003 Wiley-Liss, Inc.

NEONATAL HANDLING AND THE MATERNAL ODOR PREFERENCE IN RAT PUPS: INVOLVEMENT OF MONOAMINES AND CYCLIC AMP RESPONSE ELEMENT-BINDING PROTEIN PATHWAY IN THE OLFACTORY BULB

Early-life environmental events, such as the handling procedure, can induce long-lasting alterations upon several behavioral and neuroendocrine systems. However, the changes within the pups that could be causally related to the effects in adulthood are still poorly understood. In the present study, we analyzed the effects of neonatal handling on behavioral (maternal odor preference) and biochemical (cyclic AMP response element-binding protein (CREB) phosphorylation, noradrenaline (NA), and serotonin (5-HT) levels in the olfactory bulb (OB)) parameters in 7-day-old male and female rat pups. Repeated handling (RH) abolished preference for the maternal odor in female pups compared with nonhandled (NH) and the single-handled (SH) ones, while in RH males the preference was not different than NH and SH groups. In both male and female pups, RH decreased NA activity in the OB, but 5-HT activity increased only in males. Since preference for the maternal odor involves the synergic action of NA and 5-HT in the OB, the maintenance of the behavior in RH males could be related to the increased 5-HT activity, in spite of reduction in the NA activity in the OB. RH did not alter CREB phosphorylation in the OB of both male and females compared with NH pups. The repeated handling procedure can affect the behavior of rat pups in response to the maternal odor and biochemical parameters related to the olfactory learning mechanism. Sex differences were already detected in 7-day-old pups. Although the responsiveness of the hypothalamic-pituitary-adrenal axis to stressors is reduced in the neonatal period, environmental interventions may impact behavioral and biochemical mechanisms relevant to the animal at that early age. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.

Astrocyte intermediate filaments are important markers in olfactory bulb of bovine herpesvirus type 5 natural infections

Astroglial cells are the most abundant cells in the mammalian central nervous system, yet our knowledge about their function in bovine Herpesvirus type 5 (BoHV-5) has been limited. The aim of this study was to detect by immunohistochemistry assay the reactive astrocytes for glial fibrilary acidic protein (GFAP) and vimentin (VIM), considered intermediate filaments of the cytoskeleton, localized in olfactory bulb from natural acute cases of BoHV-5 infection. All samples were submitted to virus isolation, real-time polymerase chain reaction (RT-PCR) and in situ hybridization (ISH) technique to confirm the virus transcription and respective genome. Samples were classified into four groups according to the severity of histological lesions. Groups III and IV, which histological lesions were classified as alacia, gliosis, satellitosis, neuronophagia and neuronal necrosis, 35% (± 1.8-2.1) of the inflammatory mononuclear cells, corresponded to CD3 positive lymphocytes. In the same group, 35% (± 1.8) of astrocytes were described as reactive to GFAP and VIM proteins. An agreement of r = 1.0 (P<0.0001) was found between histological lesions, intermediate filaments expression, viral DNA and transcription and CD3 lymphocytes. However, samples with mild histological lesions, 10.8 to 14.2% of astrocytes were classified as reactive to GFAP and VIM filaments. Our findings suggest that GFAP and VIM reactive astrocytes, in primary site of virus replication, seems to play an important role in neurovirulence, in spite of many questions concerning the virus immunopathology remains unclear.

Organisation and tyrosine hydroxylase and calretinin immunoreactivity in the main olfactory bulb of paca (cuniculus paca): a large caviomorph rodent

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Lateral inhibition and odor discrimination in a model of the olfactory bulb

DA is supported by a CAPES PhD grant and ACR is the recipient of research grants by CNPq and FAPESP.

Nitric oxide synthase protein levels, not the mRNA, are downregulated in olfactory bulb interneurons of reeler mice

Homozygous mutations in the Reelin gene result in severe disruption of brain development. The histogenesis of layered regions, like the neocortex, hippocampus and the cerebellum, is most notably affected in mouse reeler mutants and similar traits are also present in mice lacking molecular components of the Reelin signalling pathway. Moreover, there is evidence for an additional role of Reelin in sustaining synaptic plasticity in adult networks. Nitric oxide is an important gaseous messenger that can modulate neuronal plasticity both in developing and mature synaptic networks and has been shown to facilitate synaptic changes in the hippocampus, cerebellum and olfactory bulb. We studied the distribution and content of neuronal nitric oxide synthase in the olfactory bulbs of reeler and wildtype mice. Immunocytochemistry reveals that Reelin and neuronal nitric oxide synthase containing interneurons are two distinct, non overlapping cell populations of the olfactory bulb. We show by in situ hybridization that both nitrergic and Reelin expressing cells represent only a subset of olfactory bulb GABAergic neurons. Immunoblots show that neuronal nitric oxide synthase protein content is decreased by two thirds in reeler mice causing a detectable loss of immunolabelled cells throughout the olfactory bulb of this strain. However, neuronal nitric oxide synthase mRNA levels, essayed by quantitative real-time RT-PCR, are unaffected in the reeler olfactory bulb. Thus, disruption of the Reelin signalling pathway may modify the turnover of neuronal nitric oxide synthase in the olfactory bulb and possibly affects nitric oxide functions in reeler mice.

Olfactory bulb interneurons releasing NO exhibit the Reelin receptor ApoEr2 and part of those targeted by NO express Reelin

Nitric oxide (NO) and Reelin both modulate neuronal plasticity in developing and mature synaptic networks. We recently showed a loss of neuronal nitric oxide synthase (nNOS) protein in the olfactory bulb of reeler mutants and advanced the hypothesis that the Reelin and NO signalling pathways may influence each other. We now studied the distribution of NO sensitive guanylyl cyclase (NOsGC), Reelin and its receptor Apolipoprotein E2 (ApoEr2) in the olfactory bulb by multiple fluorescence labelling and tested whether nNOS and ApoEr2 colocalize in this area. We also essayed the protein content of NOsGC in the reeler olfactory bulb and tested whether there are any changes in nNOS and NOsGC protein in other reeler brain areas. Olfactory bulb interneurons expressing ApoEr2 and nNOS are only few in the glomerular layer but represent the large majority of granule cell layer interneurons. Conversely, NOsGC interneurons are rare in the granule cell layer and abundant as periglomerular cells. Reelin containing periglomerular cells almost entirely belong to the NOsGC subset. These data further support the hypothesis of a reciprocal signalling between Reelin/NOsGC and ApoEr2/nNOS expressing neurons to affect olfactory bulb activity. We also show that a significant rise in NOsGC content accompanies the decrease of nNOS protein in the reeler olfactory bulb. The same reciprocal changes present in the cortex/striatum and the hippocampus of reeler mice. Thus, the influence that the deficit of extracellular Reelin seems to exert on nNOS and its receptor is not limited to the olfactory bulb but is a general feature of the reeler brain.

Expression of adenosine A2a receptors gene in the olfactory bulb and spinal cord of rat and mouse

The expression of adenosine A2a receptors (A2aR) in the mammalian striatum is well known. In contrast the exact distribution of A2aR in other regions of the central nervous system remains unclear. The aim of this study was to investigate the A2aR gene expression in the rat olfactory bulb and spinal cord, two regions which are seldom included in mapping studies. Secondly, we compared the A2aR expression in the rat and in the mouse brain. Hybridization histochemistry was performed with an S35-labelled radioactive oligonucleotide probe. The results show strong expression of A2aR in the mouse and rat striatum in accordance with previous reports. In the olfactory bulb a weak but specific expression of A2aR was found in the granular cell layer in both species. In contrast, no significant expression of the A2aR gene was observed in other parts of the brain or the rat spinal cord. The presence of the A2aR in the mammalian olfactory bulb suggests a functional role for this receptor in olfaction.