F. Jourdan

Identification and localization of dopamine receptor subtypes in rat olfactory mucosa and bulb: a combined in situ hybridization and ligand binding radioautographic approach.

Olfactory bulb (OB) of mammals contains a large population of dopaminergic interneurons within the glomerular layer. Dopamine has been shown in vivo to modulate several aspects of olfactory information processing. The dopamine receptors of olfactory bulb and mucosa are assessed here at the levels of mRNAs and radioligand binding sites with presently available tools. D1A mRNA was found in OB glomerular-, plexiform-, mitral-cell and granular layers, but not in olfactory mucosa. D1B mRNA was absent in olfactory bulb and mucosa. D1-like binding sites were detected with two distinct radioligands, in glomerular-, plexiform-, mitral cell- and granular layers of OB but not in olfactory mucosa. We thus demonstrate the previously doubtful presence of D1-like receptors in OB. D2 mRNAs were localized in the glomerular and granular layers of OB and in olfactory mucosa; lesser amounts of D3 mRNAs were found in OB glomerular and granular layer, but not in olfactory mucosa. No D4 mRNA was detected in either structure. High densities of D2-like, [125I]Iodosulpride-labelled binding sites, were revealed within lamina propria of olfactory mucosa, and confirmed in the olfactory nerve- and glomerular layers of OB. A faint but significant density of [3H]7-hydroxy-dipropyl-aminotetralin (OH-DPAT) labelled, D3 binding sites was detected in olfactory nerve- and glomerular layers of OB, but not in olfactory mucosa. Competition of [125I]Iodosulpride specific binding by three D2/D3 selective drugs yielded kinetics typical of the D2 receptor subtype in olfactory bulb and mucosa. Olfactory nerve- and glomerular layers of OB are proved thus to contain a predominant contingent of D2 receptors and a minor population of D3 receptors, while olfactory mucosa expresses only D2 receptors.

Specificity of spatial patterns of glomerular activation in the mouse olfactory bulb: computer-assisted image analysis of 2-deoxyglucose autoradiograms

We have developed a computer-assisted method for analyzing the 2-deoxyglucose (2-DG) autoradiograms of mice olfactory bulbs. The purpose of the study was to numerize the maps of glomerular activation in order to achieve a statistical comparison of the glomerular patterns evoked by different stimuli. The spatial distribution of glomerular activation was displayed on unfolded representations of the glomerular layer which were built up using glomerular optical densities (OD) measured systematically within 13 sections per bulb. Each bulbar sample was converted into an OD profile. A matrix composed of 18 OD profiles was submitted to a principal component analysis. The first factor which accounted for 28% of the variance separated unambiguously two clusters corresponding to the bulbs issued from animals stimulated with amylacetate and isovaleric acid, respectively. The second and third factors which accounted for 14% and 12% of the variance segregated the control group (animals exposed to pure air) from the odor-stimulated ones. It was demonstrated that the cluster separation was actually due to the specific spatial distribution of the most-labelled glomeruli. A particular attention was paid to the well-delineated glomerular activation evoked by isovaleric acid. The results demonstrate the specificity and reliability of the glomerular 2-DG patterns. The method should be useful for further comparisons of patterns elicited by larger sets of odorant compounds.

Acetylcholine induces neuritic outgrowth in rat primary olfactory bulb cultures

The rat olfactory bulb is innervated by basal forebrain cholinergic neurons and is endowed with both nicotinic and muscarinic receptors. The development of this centrifugal cholinergic innervation occurs mainly in early postnatal stages. This developmental time-course and the demonstration that acetylcholine can modulate some aspects of neuronal proliferation, differentiation or death, suggests the possible involvement of cholinergic afferents in the morphogenesis and/or plasticity of the olfactory bulb. The purpose of the present work was to assess whether acetylcholine could modulate neuronal morphogenesis in the olfactory bulb. Toward this aim, we developed a primary culture model of rat olfactory bulbs. Three major cell types were identified on the basis of their morphological and immunocytochemical phenotype: neuronal-shaped cells expressing the neuronal markers neuron specific enolase, microtubule associated protein 2, neural cell adhesion molecule and beta-tubulin III; glial-like cells immunoreactive for glial fibrillary acidic protein and flattened cells immunolabelled with antibodies against beta-tubulin III and nestin, most likely neuronal precursors. After three to six days of treatment with 100-microM carbachol, a cholinergic agonist, significant increase in neuritic length was observed in cultured olfactory bulb neurons. The neurite outgrowth effect of carbachol was abolished by co-treatment with 1 microM alpha-bungarotoxin, an alpha 7 subunit nicotinic receptor antagonist, but was not affected by the addition of 10 microM atropine, a general muscarinic antagonist. The effect of carbachol was also mimicked by the nicotinic agonists, nicotine (100 microM) and epibatidine (10 microM). This pharmacological profile suggested the involvement of nicotinic receptors of the alpha 7-like subtype as confirmed using 125I-alpha-bungarotoxin receptor autoradiography.Taken together, these data argue for a role for nicotinic receptors in neuritic outgrowth in the rat olfactory bulb and provide a cellular support to the previously described effects of acetylcholine on olfactory bulb plasticity in vivo.

Topography of centrifugal acetylcholinesterase-positive fibres in the olfactory bulb of the rat: Evidence for original projections in atypical glomeruli

An original pathway of centrifugal acetylcholinesterase-positive fibres is described in the olfactory bulb of the rat. A dense network of positive fibres spreads out superficially at the boundaries of the lateral olfactory tract and the glomerular layer. These labelled fibres converge towards atypical glomerular structures lying close to the classical olfactory glomeruli. The atypical glomeruli are located dorsally at the medial border of the accessory olfactory bulb, in the area previously described as the modified glomerular complex, and in the ventrolateral bulbar area. They structurally differ from typical glomeruli, as suggested by observations on semithin sections. The ultrastructural distribution of acetylcholinesterases into axonal and dendritic profiles, around and inside atypical glomeruli, is consistent with the hypothesis of centrifugal modulatory influences at this level. This study illustrates several new aspects of morphofunctional heterogeneity in the olfactory system. The glomerular layer of the main olfactory bulb can no longer be considered as morphologically and functionally uniform. Atypical glomeruli located in the mediodorsal and the ventrolateral boundaries of the glomerular layer are characterized by both structural features and an uncommonly high convergence of acetylcholinesterase-positive centrifugal fibres. Such areas might be involved in the processing of specific olfactory signals as demonstrated elsewhere for the modified glomerular complex.

Early postnatal Müller cell death leads to retinal but not optic nerve degeneration in NSE-Hu-Bcl-2 transgenic mice.

Topographically localized over-expression of the human Bcl-2 protein in retinal glial Müller cells of a transgenic mice (line 71) leads to early postnatal apoptotic Müller cell death and retinal degeneration. Morphological, immunohistological and confocal laser microscopic examination of transgenic and wild-type retinas were achieved on paraffin retinal sections, postnatally. Apoptosis occurs two to three days earlier in the internal nuclear layer of transgenic retinae, than in wild-type littermates. In parallel there was a progressive disappearance of transgenic Hu-Bcl-2 over-expression, as well as of the Müller cell markers, cellular retinaldehyde-binding protein and glutamine synthetase. This phenomenon led to retinal dysplasia, photoreceptor apoptosis and then retinal degeneration and proliferation of the retinal pigment epithelium. The optic nerve, however, remains intact. Two complementary observations confirm the pro-apoptotic action of Bcl-2 over-expression in Müller cells: (i) in the peri-papillary and peripheral regions where the transgene Bcl-2 is not expressed, cellular retinaldehyde-binding protein or glutamine synthetase immunostaining persist and Müller glia do not die; and (ii) the retina conserves a normal organisation in these two regions in spite of total retinal degeneration elsewhere. We conclude that retinal dysplasia and degeneration are linked to primary Müller cell disruption. Besides its generally accepted anti-apoptotic function, over-expression of Bcl-2 also exerts a pro-apoptotic action, at least in immature Müller glia. One may suppose that Bcl-2 translocation resulting in its over-expression in retinal Müller cells could be a putative mechanism for early retinal degeneration.

Apomorphine disrupts odour-induced patterns of glomerular activation in the olfactory bulb

The olfactory bulb of adult male rats stimulated with propionic acid vapours displays a characteristic focus of high metabolic activity in the dorso-medial glomeruli, as revealed by the 2-deoxyglucose (2DG) method. Injection of the dopaminergic agonist apomorphine (1.5 mg kg-1) prior to odour stimulation completely abolishes this selective pattern of glomerular activation, while the metabolic activity of other bulbar areas is not significantly altered. This effect is abolished by a previous injection of the dopamine antagonist haloperidol (0.1 mg kg-1). These observations emphasize the probable involvement of dopamine and dopamine receptors in the bulbar processing of olfactory information.

Spatial dimension in olfactory coding: a representation of the 2-deoxyglucose patterns of glomerular labeling in the olfactory bulb.

A method has been developed for visualizing the patterns of uptake of radioactive 2-deoxyglucose (2-DG) induced in the glomerular layer of rat olfactory bulbs by various olfactory stimuli. Some characteristics of these patterns such as shape, contrast, symmetry, specificity and variability, are discussed. This method is thought to be useful for understanding olfactory coding at the glomerular level.

Odour-induced c-fos expression in the rat olfactory bulb: involvement of centrifugal afferents

Expression of the proto-oncogene c-fos is known to increase in granule cells of the olfactory bulb following a sustained olfactory stimulation. Most granule cells displaying high levels of Fos accumulation are located in the bulbar columns defined by the odour-induced foci of high 2-deoxyglucose glomerular uptake. The present studies were undertaken in order to assess the possible involvement of centrifugal afferents in the modulation of odour-induced patterns of either 2-deoxyglucose accumulation or c-fos expression in the olfactory bulb. A unilateral olfactory peduncle section had no effect on the odour-induced 2-deoxyglucose foci but induced a significant decrease in the number of Fos-containing neurons in odour-selective areas of both olfactory bulbs, ipsilateral and contralateral to the lesion. This suppressive effect was much more pronounced in the side ipsilateral to the peduncle section. It is concluded that c-fos expression induced by a sustained stimulation with propionic acid vapours is not only determined by the olfactory peripheral input but also by afferents of central origin. In order to estimate the contingent involvements of the cholinergic and noradrenergic afferents in this control of c-fos expression, we attempted to mimic the effects of the surgical deafferentation on odour-induced c-fos expression by using a pharmacological approach with selective cholinergic and noradrenergic antagonists. The beta-adrenergic antagonist propanolol induced a suppression of the odour-related patterns of Fos accumulation similar to the one caused by the surgical deafferentation of the olfactory bulb. The muscarinic antagonist scopolamine did not alter c-fos expression in the odour-selective area but increased significantly Fos labelling in the other bulbar aspects. Pharmacological investigations indicate that the noradrenergic and cholinergic centrifugal systems are likely involved in the central modulation of c-fos expression in the OB. The Fos protein could be expressed as an early nuclear signal triggering further long-term modifications of the neuronal phenotype, in certain conditions of sensory stimulation involving the activation of centrifugal systems.

Accumulation of Ym1/2 protein in the mouse olfactory epithelium during regeneration and aging

A unique feature of the olfactory system is its efficiency to produce new neurons in the adult. Thus, destruction of the olfactory receptor neurons (ORNs) using chemical (intranasal perfusion with ZnSO4) or surgical (axotomy or bulbectomy) methods, leads to an enhanced rate of proliferation of their progenitors and to complete ORNs regeneration. The aim of our study was to identify new factors implied in this regenerative process. Using an electrophoretic method, we observed the accumulation of a 42 kDa protein after axotomy in the olfactory mucosa, but not in the olfactory bulb. Its expression started after a few days following injury and increased massively during the phase of ORN regeneration. The purification and the sequence characterization revealed that this protein was Ym1/2, recently identified in activated macrophages present in various tissues during inflammation. Western blotting analysis of Ym1/2 confirmed the accumulation of this protein in the regenerating olfactory mucosa consecutively to olfactory axotomy or bulbectomy but also after ZnSO4 irrigation of the nasal cavity. In the olfactory mucosa of control mice, Ym1/2 was hardly detectable in young animals and became more and more abundant with increasing age. In injured and aged mice, Ym1/2 mainly accumulates in the cytoplasm of supporting cells as well as in other cells located throughout the olfactory epithelium. Our results suggest that Ym1/2 is involved in olfactory epithelium remodeling following several kinds of lesions of the adult olfactory mucosa and support the view of a critical role of inflammatory cues in neurodegeneration and aging.

Immunocytochemical identification of luteinizing hormone-releasing hormone-positive fibres and terminals in the olfactory system of the rat

Luteinizing hormone-releasing hormone immunoreactivity was studied in the olfactory system of the rat in combination with acetylcholinesterase histochemistry. Neuronal perikarya containing luteinizing hormone-releasing hormone lie in the medial septal nucleus, the vertical limb of the diagonal band of Broca, the olfactory tubercule and the ganglionated plexus of the terminal nerve. Labelled fibres spread in the superficial layers of the main and accessory olfactory bulbs, some encompassing the strongly acetylcholinesterase-positive atypical glomeruli. Others are observed on the medial side of the bulb, running along the terminal nerve bundles and ganglia. These fibres join the vomeronasal nerve branches and proceed distally towards the nasal cavity. In the septal submucosa, immunoreactive fibres are partly associated with the terminal nerve network. Conspicuous endings filled with luteinizing hormone-releasing hormone are observed on blood vessels of the olfactory mucosa. Such well-differentiated terminals might be the neurosecretory afferents of a new neurohemal area. Immunoreactive terminals are also observed around the excretory ducts of the anterior medial glands. We have failed to observe any labelled fibres in the olfactory and vomeronasal epithelia. The results of the present study are discussed with respect to possible functional interpretations. It is suggested that significant amounts of luteinizing hormone-releasing hormone could be released in the submucosal capillaries in spite of the scarcity of immunoreactive fibres. Similar afferents could also modulate the secretory activity of some nasal glands. Synaptic events involving the neuropeptide might occur in the olfactory bulb, particularly in atypical glomerular areas previously characterized by their high acetylcholinesterase content. Finally, no anatomical support for a chemosensory function of fibres containing luteinizing hormone-releasing hormone has been brought out by our work.