Between Order and Chaos: Understanding the Mechanism and Pathology of RAN Translation.

Repeat-associated non-AUG (RAN) translation is a pathogenic mechanism in which repetitive sequences are translated into aggregation-prone proteins from multiple reading frames, even without a canonical AUG start codon. Since its discovery in spinocerebellar ataxia type 8 (SCA8) and myotonic dystrophy type 1 (DM1), RAN translation is now known to occur in the context of 12 disease-linked repeat expansions. This review discusses recent advances in understanding the regulatory mechanisms controlling RAN translation and its contribution to the pathophysiology of repeat expansion diseases. We discuss the key findings in the context of Fragile X Tremor Ataxia Syndrome (FXTAS), a neurodegenerative disorder caused by a CGG repeat expansion in the 5' untranslated region of FMR1.

In vivo and in vitro properties of STX2484: a novel non-steroidal anti-cancer compound active in taxane-resistant breast cancer.

BACKGROUND: STX2484 is a novel non-steroidal compound with potent anti-proliferative activity. These studies aimed to identify STX2484's mechanism of action, in vivo efficacy and activity in taxane-resistant breast cancer models. METHODS: Effects of STX2484 and paclitaxel on proliferation, cell cycle and apoptosis were assessed in vitro in drug-resistant (MCF-7(DOX)) and non-resistant cells (MCF-7(WT)). STX2484 efficacy in ßIII tubulin overexpression in MCF-7 cells was also determined. Anti-angiogenic activity was quantified in vitro by a co-culture model and in vivo using a Matrigel plug assay. An MDA-MB-231 xenograft model was used to determine STX2484 efficacy in vivo. RESULTS: STX2484 is a tubulin disruptor, which induces p53 expression, Bcl2 phosphorylation, caspase-3 cleavage, cell cycle arrest and apoptosis. In addition, STX2484 is a potent anti-angiogenic agent in vitro and in vivo. In breast cancer xenografts, STX2484 (20 mg kg(-1) p.o.) suppressed tumour growth by 84% after 35 days of daily dosing, with limited toxicity. In contrast to paclitaxel, STX2484 efficacy was unchanged in two clinically relevant drug-resistant models. CONCLUSIONS: STX2484 is an orally bioavailable microtubule-disrupting agent with in vivo anti-angiogenic activity and excellent in vivo efficacy with no apparent toxicity. Crucially, STX2484 has superior efficacy to paclitaxel in models of clinical drug resistance.

Genetic Studies of Hypertrophic Cardiomyopathy in Singaporeans Identify Variants in TNNI3 and TNNT2 That Are Common in Chinese Patients.

BACKGROUND: To assess the genetic architecture of hypertrophic cardiomyopathy (HCM) in patients of predominantly Chinese ancestry. METHODS: We sequenced HCM disease genes in Singaporean patients (n=224) and Singaporean controls (n=3634), compared findings with additional populations and White HCM cohorts (n=6179), and performed in vitro functional studies. RESULTS: Singaporean HCM patients had significantly fewer confidently interpreted HCM disease variants (pathogenic/likely pathogenic: 18%, P<0.0001) but an excess of variants of uncertain significance (24%, P<0.0001), as compared to Whites (pathogenic/likely pathogenic: 31%, excess of variants of uncertain significance: 7%). Two missense variants in thin filament encoding genes were commonly seen in Singaporean HCM (TNNI3:p.R79C, disease allele frequency [AF]=0.018; TNNT2:p.R286H, disease AF=0.022) and are enriched in Singaporean HCM when compared with Asian controls (TNNI3:p.R79C, Singaporean controls AF=0.0055, P=0.0057, genome aggregation database-East Asian AF=0.0062, P=0.0086; TNNT2:p.R286H, Singaporean controls AF=0.0017, P<0.0001, genome aggregation database-East Asian AF=0.0009, P<0.0001). Both these variants have conflicting annotations in ClinVar and are of low penetrance (TNNI3:p.R79C, 0.7%; TNNT2:p.R286H, 2.7%) but are predicted to be deleterious by computational tools. In population controls, TNNI3:p.R79C carriers had significantly thicker left ventricular walls compared with noncarriers while its etiological fraction is limited (0.70 [95% CI, 0.35-0.86]) and thus TNNI3:p.R79C is considered variant of uncertain significance. Mutant TNNT2:p.R286H iPSC-CMs (induced pluripotent stem cells derived cardiomyocytes) show hypercontractility, increased metabolic requirements, and cellular hypertrophy and the etiological fraction (0.93 [95% CI, 0.83-0.97]) support the likely pathogenicity of TNNT2:p.R286H. CONCLUSIONS: As compared with Whites, Chinese HCM patients commonly have low penetrance risk alleles in TNNT2 or TNNI3 but exhibit few clinically actionable HCM variants overall. This highlights the need for greater study of HCM genetics in non-White populations.

Rupture limit evaluation of human cerebral aneurysms wall: Experimental study.

BACKGROUND AND PURPOSE: Rupture risk of intracranial aneurysms is a major issue for public healthcare. A way to obtain an individual rupture risk assessment is a main objective of many research teams in the world. For many years, we have investigated the relationship between the mechanical properties of aneurysm wall tissues and the rupture risk. In this work, we try to go further and investigate rupture limit values. METHODS: Following surgical clipping, a specific conservation protocol was applied to aneurysmal tissues in order to preserve their mechanical properties. Thirty-nine intracranial aneurysms (27 females, 12 males) were tested using a uniaxial tensile test machine under physiological conditions, temperature, and saline isotonic solution. These represented 24 unruptured and 15 ruptured aneurysms. Stress/strain curves were then obtained for each sample, and a fitting algorithm was applied following a Yeoh hyperelastic model with 2 parameters. Moreover, uniaxial tensile tests were conducted until rupture of samples to obtain values of stress and strain rupture limit. RESULTS: The significant parameter a C2 of the hyperelastic Yeoh model, allowed us to classify samples' rigidity following the terminology we adopted in previous papers (Costalat et al., 2011; Sanchez et al., 2013): Soft, Stiff and Intermediate. Moreover, strain/stress rupture limit values were gathered and analyzed thanks to the tissue rigidity, the status of the aneurysm (initially ruptured or unruptured) and the gender of the patient. CONCLUSION: Strain rupture limit was found quite stable around 20% and seems not to be correlated with the status of the aneurysm (initially ruptured or unruptured), neither with the gender of the patient. However, stretch and stress rupture limit seems not to be independent on the rigidity. The study confirms that ruptured aneurysms mainly present a soft tissue and unruptured aneurysms present a stiff material.

MetaNetter 2: A Cytoscape plugin for ab initio network analysis and metabolite feature classification.

Metabolomics frequently relies on the use of high resolution mass spectrometry data. Classification and filtering of this data remain a challenging task due to the plethora of complex mass spectral artefacts, chemical noise, adducts and fragmentation that occur during ionisation and analysis. Additionally, the relationships between detected compounds can provide a wealth of information about the nature of the samples and the biochemistry that gave rise to them. We present a biochemical networking tool: MetaNetter 2 that is based on the original MetaNetter, a Cytoscape plugin that creates ab initio networks. The new version supports two major improvements: the generation of adduct networks and the creation of tables that map adduct or transformation patterns across multiple samples, providing a readout of compound relationships. We have applied this tool to the analysis of adduct patterns in the same sample separated under two different chromatographies, allowing inferences to be made about the effect of different buffer conditions on adduct detection, and the application of the chemical transformation analysis to both a single fragmentation analysis and an all-ions fragmentation dataset. Finally, we present an analysis of a dataset derived from anaerobic and aerobic growth of the organism Staphylococcus aureus demonstrating the utility of the tool for biological analysis.

Long-term fate and distribution of newborn cells in the adult mouse olfactory bulb: Influences of olfactory deprivation.

The adult subventricular zone produces neuroblasts that migrate to the main olfactory bulb, where they differentiate into interneurons in the glomerular and granular layers. Using bromodeoxyuridine labeling, the survival of newborn cells was assessed in these two layers of the MOB in control mice and in mice unilaterally deprived from sensory input by naris occlusion. In control main olfactory bulbs, bromodeoxyuridine-positive cell density decreased about 70% between 15 and 180 days post-bromodeoxyuridine administration but earlier in the glomerular layer than in the granular layer. At all time points examined, newborn cell density was higher in the deep granular layer than in the superficial granular layer. Occlusion started at the age of 2 months and lasted for 15, 30, 45, 60 or 180 days. The newborn cell survival was similarly reduced in both layers by occlusion, during a critical period 15 and 45 days post-occlusion. Interestingly, olfactory deprivation decreased bromodeoxyuridine-positive cell density in the deep granular layer only, indicating a greater dependence of cell fate on sensory input in this sub-layer. Neuronal differentiation was assessed in the granular layer and glomerular layer by multiple double-labeling 45 days post-bromodeoxyuridine-injections, the time point at which the proportion of bromodeoxyuridine-positive cells expressing a neuronal marker reached approximately 85% in the granular layer and approximately 50% in the glomerular layer. Naris occlusion did not significantly affect these proportions. Taken together, our results reveal that the survival of newborn cells has a different time course in the glomerular layer and in the granular layer, but is similarly decreased in each layer by olfactory deprivation. In addition, our data suggest a functional heterogeneity of neurogenesis within the granular layer.

Leukemia inhibitory factor is a key signal for injury-induced neurogenesis in the adult mouse olfactory epithelium.

The mammalian olfactory epithelium (OE) is composed of primary olfactory sensory neurons (OSNs) that are renewed throughout adulthood by local, restricted neuronal progenitor cells. The molecular signals that control this neurogenesis in vivo are unknown. Using olfactory bulb ablation (OBX) in adult mice to trigger synchronous mitotic stimulation of neuronal progenitors in the OE, we show the in vivo involvement of a cytokine in the cellular events leading to the regeneration of the OE. We find that, of many potential mitogenic signals, only leukemia inhibitory factor (LIF) is induced before the onset of neuronal progenitor proliferation. The rise in LIF mRNA expression peaks at 8 hr after OBX, and in situ RT-PCR and immunocytochemistry indicate that LIF is upregulated, in part, in the injured neurons themselves. This rise in LIF is necessary for injury-induced neurogenesis, as OBX in the LIF knock-out mouse fails to stimulate cell proliferation in the OE. Moreover, delivery of exogenous LIF to the intact adult OE using an adenoviral vector stimulates BrdU labeling in the apical OE. Taken together, these results suggest that injured OSNs release LIF as a stimulus to initiate their own replacement.

Serine protease inhibitor Spi2 mediated apoptosis of olfactory neurons.

The olfactory epithelium of adult mouse, where primary sensory neurons are massively committed to apoptosis by removal of their synaptic target, was used as a model to determine in vivo mechanisms for neuronal cell death induction. A macro-array assay revealed that the death of olfactory neurons is accompanied with over-expression of the serine protease inhibitor Spi2. This over-expression is associated with decreased serine protease activity in the olfactory mucosa. Moreover, in vitro or in vivo inhibition of serine proteases induced apoptotic death of olfactory neuronal cells. Interestingly, Spi2 over-expression is not occurring in olfactory neurons but in cells of the lamina propria, suggesting that Spi2 may act extracellularly as a cell death inducer. In that sense, we present evidence that in vitro Spi2 overexpression generates a secreted signal for olfactory neuron death. Hence, taken together these results document a possible novel mechanism for apoptosis induction that might occur in response to neurodegenerative insults.

Small stress protein Hsp27 accumulation during dopamine-mediated differentiation of rat olfactory neurons counteracts apoptosis.

The small stress protein Hsp27 is expressed during mammalian neural development. We have analyzed the role of this protein in immortalized rat olfactory neuroblasts. In the presence of dopamine a fraction of these cells differentiate into neurons while the remaining cells undergo apoptosis. We report here that the dopamine induced differentiation and apoptosis are associated with a transient and specific accumulation of Hsp27. Moreover, transfection experiments have shown that Hsp27 overexpression drastically decreases the fraction of cells undergoing apoptosis. In contrast, reduction of the endogenous level of Hsp27 led to abortion of differentiation and, therefore, drastically increased the number of apoptotic cells. Furthermore, in the normal cell population we show that Hsp27 accumulation takes place only in differentiating cells that were not undergoing apoptosis. We therefore conclude that Hsp27 may represent a key protein that controls the decision of olfactory precursor cells to undergo either differentiation or cell death.

The Projections of Mitral Cells from Small Local Regions of the Olfactory Bulb: An Anterograde Tracing Study Using PHA-L (Phaseolus vulgaris Leucoagglutinin).

Numerous anatomical and electrophysiological studies have demonstrated a lack of simple point-to-point topographical relationships between the olfactory bulb and primary olfactory projection areas. They reveal instead, a complex pattern of divergence and convergence. Furthermore, several authors reported that a single mitral cell could project onto different widely spaced cortical regions of the olfactory cortex. In the present study, we attempted to label the projections of a few mitral cells so close together so that they might be assumed to be connected to the same glomerulus, and to determine if these cells had similar patterns of axonal projections. For this purpose small Phaseolus vulgaris leucoagglutinin (PHA-L) injections were performed in the olfactory bulb of adult rats. We found that labelling two to five mitral cells, lying close together in the mitral cell layer, resulted in well-delineated patches of labelled fibres in the cortex. The number of patches was not related to the number of labelled mitral cells but the fibre density in each patch increased with the number of PHA-L filled somata in the olfactory bulb. We conclude that mitral cells lying close together in the mitral cell layer have similar patterns of axonal projections. Functional implications of such an organization in olfactory coding is discussed.

Cholinergic innervation of olfactory glomeruli in the rat: an ultrastructural immunocytochemical study.

The ultrastructural organization of cholinergic afferents to the rat olfactory bulb (OB) was studied with the aid of choline acetyltransferase (ChAT) immunocytochemistry in electron microscopy. Particular attention has been paid to a subset of glomeruli characterized by a remarkably high density of cholinergic afferents. Numerous cholinergic terminals making symmetric or asymmetric synaptic contacts were observed in the periglomerular area. ChAT-labelled terminals have a diameter ranging from 0.3 to 1.5 micron and contain numerous clear agranular vesicles. Axo-somatic and axo-dendritic contacts were both observed in contact with several types of target neurons. Three types of cholinoceptive, noncholinergic neurons could be identified: periglomerular cells, superficial short-axon cells, and external tufted cells. Our results provide an anatomical substrate for the hypotheses concerning the complex effects of acetylcholine in the processing of sensory information in the olfactory bulb.

Postnatal development of cholinergic markers in the rat olfactory bulb: a histochemical and immunocytochemical study.

The present study has defined the developmental time course and the distribution patterns of neuronal fibers and cell bodies displaying acetylcholinesterase (AChE) activity or choline acetyltransferase (ChAT) immunoreactivity in the rat olfactory bulb. The results indicate that the deployment of centrifugal AChE-containing fibers is essentially postnatal. The subset of atypical glomeruli, including the modified glomerular complex, is innervated as early as the first postnatal day while the normal ones are not reached by this type of afferent before postnatal day 6. The comparison of AChE labelling with ChAT immunoreactivity strongly supports the assumption that AChE-containing fibers represent mainly, if not exclusively, the cholinergic bulbopetal innervation emanating from the basal forebrain. A quantitative study has confirmed that the density of labelled fibers increases gradually in the postnatal period and spreads heterogeneously among the bulbar layers. The selective precocious innervation of atypical glomeruli is in favor of their involvement in the early processing of olfactory information in the olfactory bulb. Acetylcholinesterase is also expressed within a subset of ChAT-negative interneurons of the developing olfactory bulb. The number of neurons expressing AChE increases from birth to postnatal day 15 and then decreases to reach the adult value on about postnatal day 30. This neuronal population could constitute a cholinoceptive subset mediating the effects of cholinergic afferents on the bulbar neuronal network.

Morphometric modifications associated with early sensory experience in the rat olfactory bulb: I. Volumetric study of the bulbar layers.

Current models of sensory coding in the olfactory bulb are based on the notion of topographical specificity in the processing of stimuli. Part of this hypothesis comes from studies of patterns of radiolabelled 2-deoxyglucose uptake, and local morphometric variations of the mitral cell size observed following prolonged exposure to an odor. The present study explored the possibility that exposing young rats to a long-term stimulation with an odor would induce spatially distributed volumetric variations of the bulbar layers. Three groups of 5 rats have been studied: (1) stimulated with ethyl acetoacetate from birth to 1 month of age, (2) unilaterally deprived following early cauterization of one nostril, and (3) normal animals of same age. Fourteen frontal histological sections uniformly distributed along the rostrocaudal axis of the olfactory bulb were used for this study. Areas of the bulbar layers were measured with the aid of an image analyser and the volume of the corresponding layers deduced by computation. Following complete sensory deprivation, the volume of all bulbar layers, except the periventricular core, was homogeneously reduced along the rostrocaudal axis of the olfactory bulb. Following long-term stimulation with ethyl acetoacetate, volume reduction was significantly higher in anterior and middle regions than in the posterior part of the olfactory bulb. It is assumed that neuronal pathways activated by ethyl acetoacetate stimulation are mainly located in the posterior part of the olfactory bulb. Functional interpretations of these results are discussed with respect to the spatial dimension in olfactory coding.

Morphometric study of the glomerular population in the mouse olfactory bulb: numerical density and size distribution along the rostrocaudal axis.

A morphometric study of the glomerular population in the olfactory bulb of the mouse has been carried out by using stereological methods. On the basis of the assumption that the glomerular population is a polydispersed system of spheres, glomerular profile distributions obtained from profile measurements were subjected to a mathematical unfolding procedure to obtain the actual glomerular size distribution. We used a distribution-free method to account for the combined effects of overprojection due to section thickness and truncation (two missing profile mechanisms). Results proved better than those obtained directly from profile measurements without stereological analysis. Several new findings were obtained. First, significant variations of the glomerulus sizes were found along the rostrocaudal axis. The glomeruli are larger in the middle region of the olfactory bulb, whereas their numerical density decreases in the same region. Moreover, the profile density is homogeneous along the rostrocaudal axis. In other words, the relative surface occupied by the periglomerular cells in the glomerular layer is invariant. As a consequence, it may be concluded that the variations in size and numerical density are inversely correlated. Thus, since the glomeruli are larger in the middle region, their number per unit volume is logically smaller in this same area. Finally, the computerization of all these data has led us to estimate the number of glomeruli (1,810 +/- 41) in the olfactory bulb of the mouse. In order to get a comparative idea of their advantages and disadvantages, other standard stereological methods were used in the present study to determine this number. Functional interpretations of the variations of the size and numerical density along the rostrocaudal axis of the olfactory bulb are discussed with respect to ontogenetic and morphofunctional data obtained elsewhere.

Morphometric modifications associated with early sensory experience in the rat olfactory bulb: II. Stereological study of the population of olfactory glomeruli.

The present study explores the local variations of size and number of olfactory glomeruli induced by the exposure of young rats to long-term stimulation with a single odor. Three groups of 5 rats were used that were either: (1) stimulated with ethyl acetoacetate from birth to 1 month of age, (2) unilaterally deprived following early occlusion of one nare, or (3) normal animals of the same age. Areas and coordinates of all glomerular profiles were measured in 14 coronal sections uniformly distributed along the rostrocaudal axis of the olfactory bulb. A distribution-free stereological method was applied to compute the size and number of glomeruli either along the bulbar rostrocaudal extent or in the bulbar coronal plane. Following complete sensory deprivation or long-term stimulation with ethyl acetoacetate, the mean diameter of glomeruli was significantly reduced everywhere, except in the ventrolateral and ventromedial regions of the posterior olfactory bulb in rats reared with a single odor. In both of these areas, the number of glomeruli was either significantly increased following long duration exposure or significantly reduced following unilateral deprivation. Thus these results show that selective modifications of the olfactory environment during postnatal maturation induce morphometric variations in specific areas of the glomerular layer. These data are discussed with respect to the concept of the topographical coding of odor quality at the level of the glomeruli and plasticity of the olfactory system during postnatal development.

Developmental profiles of various cholinergic markers in the rat main olfactory bulb using quantitative autoradiography.

The existence of possible relationships among the developmental profile of various cholinergic markers in the main olfactory bulb (OB) was assessed by using in vitro quantitative autoradiography. Muscarinic receptors were visualized with [3H]pirenzepine (muscarinic M1-like sites) and [3H]AF-DX 384 (muscarinic M2-like sites); nicotinic receptors by using [3H]cytisine (nicotinic 42-like subtype) and [125I] alpha-bungarotoxin (nicotinic 7-like subtype); cholinergic nerve terminals by using [3H]vesamicol (vesicular acetylcholine transport sites) and [3H]hemicholinium-3 (high-affinity choline uptake sites). These various cholinergic markers exhibited their lowest levels at birth and reached adult values by the end of the 4-5 postnatal weeks. However, the density of presynaptic cholinergic markers and nicotinic receptors at postnatal day 2 represented a large proportion of the levels observed in adulthood, and displays a transient overexpression around postnatal day 20. In contrast, the postnatal development of cholinergic muscarinic M1-like and M2-like receptors is apparently regulated independently of the presynaptic cholinergic markers and nicotinic receptors. Two neurochemically and anatomically separate olfactory glomeruli subsets were observed in the posterior OB of the developing rat. These atypical glomeruli expressed large amounts of [3H]vesamicol-and [3H]hemicholinium binding sites without significant amounts of muscarinic M1, M2, or nicotinic alpha 4 beta 2 receptor binding sites. A significant density of [125I] alpha-bungarotoxin binding sites could be detected only at early postnatal ages. A few olfactory glomeruli specifically restricted to the dorsal posterior OB expressed a high density of [3H]cytisine binding sites but lacked significant binding of the two presynaptic cholinergic markers used here, suggesting their noncholinergic but cholinoceptive nature.

Atypical olfactory glomeruli contain original olfactory axon terminals: an ultrastructural horseradish peroxidase study in the rat.

The labelling of olfactory bulb glomeruli following horseradish peroxidase lavage of the nasal cavity has been studied in the rat. In such conditions, atypical glomeruli, previously described according to their high acetylcholinesterase content, display a strong tracer accumulation. The course of afferent olfactory fibres could be followed along the lateral and dorsal surface of the olfactory bulbs. The primary olfactory axons ending in atypical glomeruli have been identified with horseradish peroxidase in electron microscopy. They differ significantly from classical olfactory terminals owing to the presence of large dense-cored vesicles accompanying small clear ones. Moreover, the olfactory terminals do not gather in dark nodules as they do classically in olfactory glomeruli. The study demonstrates that a subset of olfactory neuroreceptors displaying original ultrastructural characteristics projects selectively into atypical olfactory glomeruli. Ultrastructural features indicate that olfactory information processing taking place in the neuropil might be similar to that which occurs in typical glomeruli. Considered together, the atypical olfactory neuroreceptors, glomeruli and acetylcholinesterase-containing centrifugal fibres could constitute a new olfactory subsystem. This hypothesis is discussed by taking into account previous demonstration of other olfactory subsystems devoted to the processing of olfactory cues of fundamental biological importance.

Deprivation of sensory inputs to the olfactory bulb up-regulates cell death and proliferation in the subventricular zone of adult mice.

The main olfactory bulb (MOB) is the first relay on the olfactory sensory pathway and the target of the neural progenitor cells generated in the subventricular zone (SVZ) lining the lateral ventricles and which migrate along the rostral extension of the SVZ, also called the rostral migratory stream (RMS). Within the MOB, the neuroblasts differentiate into granular and periglomerular interneurons. A reduction in the number of granule cells during sensory deprivation suggests that neurogenesis may be influenced by afferent activity. Here, we show that unilateral sensory deafferentation of the MOB by axotomy of the olfactory receptor neurons increases apoptotic cell death in the SVZ and along the rostro-caudal extent of the RMS. The vast majority of dying cells in the RMS are migrating neuroblasts as indicated by double Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling/PSA-NCAM labeling. Counting bromodeoxyuridine-labeled cells in animals killed immediately or 4 days after tracer administration showed a bilateral increase in proliferation in the SVZ and RMS which was balanced by cell death on the operated side. These data suggest that olfactory inputs are required for the survival of newborn neural progenitors. The greatest enhancement in proliferation occurred in the extension of the RMS located in the MOB, revealing a population of local precursors mitotically stimulated following axotomy. Together, these findings indicate that olfactory inputs may strongly modulate the balance between neurogenesis and apoptosis in the SVZ and RMS and provide a model for further investigation of the underlying molecular mechanisms of this activity-dependent neuronal plasticity.

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.

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".