François Jourdan

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.

BDNF/ TrkB interaction regulates migration of SVZ precursor cells via PI3‐K and MAP‐K signalling pathways

Neuroblasts born in the subventricular zone (SVZ) migrate along the rostral migratory stream, reaching the olfactory bulb (OB) where they differentiate into local interneurons. Several extracellular factors have been suggested to control specific steps of this process. The brain‐derived neurotrophic factor (BDNF) has been demonstrated to promote morphological differentiation and survival of OB interneurons. Here we show that BDNF and its receptor TrkB are expressed in vivo throughout the migratory pathway, implying that BDNF might also mediate migratory signals. By using in vitro models we demonstrate that BDNF promotes migration of SVZ neuroblasts, acting both as inducer and attractant through TrkB activation. We show that BDNF induces cAMP response element‐binding protein (CREB) activation in migrating neuroblasts via phosphatidylinositol 3‐kinase (PI3‐K) and mitogen‐activated protein kinase (MAP‐K) signalling. Pharmacological blockade of these pathways on SVZ explants significantly reduces CREB activation and impairs neuronal migration. This study identifies a function of BDNF in the SVZ system, which involves multiple protein kinase pathways leading to neuroblast migration.

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.

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.

Induction of apoptosis in rat olfactory neuroepithelium by synaptic target ablation

The olfactory system provides a useful in vivo model for studying the influence of synaptic targets on the survival of relay neurones. The bipolar sensory neurones located in the olfactory mucosa project synaptically onto the ipsilateral olfactory bulb, and their survival depends on the integrity of this connection. We demonstrate here that the retrograde neuronal degeneration induced by olfactory bulb removal involves apoptosis. As revealed by typical nucleosome-sized fragmentations of the genomic DNA, the apoptosis rate reaches a maximum 32 h after bulbectomy. A transient c-fos mRNA accumulation was detected, peaking 16 h after bulbectomy, suggesting that c-fos is involved in the early steps of programmed cell death.

In vitro induction of apoptosis or differentiation by dopamine in an immortalized olfactory neuronal cell line

Abstract: A new neuronal cell line was generated by transfection of rat olfactory epithelium with immortalizing recombinant oncogene E1A of adenovirus‐2. The resulting 13.S.1.24 line of transformed cells expressed an antigenic phenotype of olfactory neuronal progenitors. Addition of dopamine to 13.S.1.24 cultures induced reduction of cell number within 2 days. Two hallmarks of apoptosis were detected in dopamine‐treated cultures: internucleosomal DNA fragmentation and nuclear condensation. Dopamine did not alter the cell proliferation rate, as assessed by [3H]thymidine incorporation. Dopamine also stimulated differentiation of surviving 13.S.1.24 cells into bipolar olfactory marker protein‐immunoreactive neurons. Time‐dependency assessments over 1 week of treatment indicated that apoptosis and differentiation induced by dopamine were concomitant. Both apoptosis and differentiation triggered by dopamine were dose‐dependent, half‐maximal effects being obtained with ∼10 µM dopamine. Mediation of both effects by dopaminergic D2 receptors was supported by several observations: active dopamine doses in micromolar ranges, quinpirole agonism and eticlopride antagonism, D2‐characteristic rank order of potency among the three agonists tested, and specific binding of a selective D2‐like radioligand to 13.S.1.24 cells. The present data altogether indicated that dopamine commits immortalized olfactory neuronal cells in vitro either to apoptosis or to olfactory‐like differentiation via D2 dopaminergic receptors.

Comparative laminar distribution of various autoradiographic cholinergic markers in adult rat main olfactory bulb.

To provide anatomical information on the complex effects of acetylcholine (ACh) in the olfactory bulb (OB), the distribution of different cholinergic muscarinic and nicotinic receptor sub-types was studied by quantitative in vitro autoradiography. The muscarinic M1-like and M2-like sub-types, as well as the nicotinic bungarotoxin-insensitive (alpha 4 beta 2-like) and bungarotoxin-sensitive (alpha 7-like) receptors were visualized using [3H]pirenzepine, [3H]AF-DX 384, [3H]cytisine and [125I] alpha-bungarotoxin (BTX), respectively. In parallel, labelling patterns of [3H]vesamicol (vesicular acetylcholine transport sites) and [3H]hemicholinium-3 (high-affinity choline uptake sites), two putative markers of cholinergic nerve terminals, were investigated. Specific labelling for each cholinergic radioligand is distributed according to a characteristic laminar and regional pattern within the OB revealing the lack of a clear overlap between cholinergic afferents and receptors. The presynaptic markers, [3H]vesamicol and [3H]hemicholinium-3, demonstrated similar laminar pattern of distribution with two strongly labelled bands corresponding to the glomerular layer and the area around the mitral cell layer. Muscarinic M1-like and M2-like receptor sub-types exhibited unique distribution with their highest levels seen in the external plexiform layer (EPL). Intermediate M1-like and M2-like binding densities were found throughout the deeper bulbar layers. In the glomerular layer, the levels of muscarinic receptor subtypes were low, the level of M2-like sites being higher than M1. Both types of nicotinic receptor sub-types displayed distinct distribution pattern. Whereas [125I] alpha-BTX binding sites were mostly concentrated in the superficial bulbar layers, [3H]cytisine binding was found in the glomerular layers, as well as the mitral cell layer and the underlying laminae. An interesting feature of the present study is the visualization of two distinct cholinoceptive glomerular subsets in the posterior OB. The first one exhibited high levels of both [3H]vesamicol and [3H]hemicholinium-3 sites. It corresponds to the previously identified atypical glomeruli and apparently failed to express any of the cholinergic receptors under study. In contrast, the second subset of glomeruli is not enriched with cholinergic nerve terminal markers but displayed high amounts of [3H]cytisine/nicotinic binding sites. Taken together, these results suggest that although muscarinic receptors have been hypothesized to be mostly involved in cholinergic olfactory processing and short-term memory in the OB, nicotinic receptors, especially of the cytisine/ alpha 4 beta 2 sub-type, may have important roles in mediating olfactory transmission of efferent neurons as well as in a subset of olfactory glomeruli.

Expression of collapsin response mediator proteins 1, 2 and 5 is differentially regulated in newly generated and mature neurons of the adult olfactory system.

Collapsin‐response mediator proteins (CRMPs) are highly expressed in the developing brain where they take part in several aspects of neuronal differentiation. CRMPs are still present postnatally, but their function remains speculative in the adult brain. We studied the expression and localization of CRMP1, CRMP2 and CRMP5 in two areas of the nervous system with persistent neurogenesis in adult mice, the olfactory mucosa and the olfactory bulb. In the olfactory mucosa, we have established that CRMP expression is restricted to postmitotic cells of the olfactory neurons lineage. CRMP5 is coexpressed with growth associated protein of 43 kDa (GAP43) in immature olfactory neurons and is down‐regulated in olfactory marker protein‐positive mature neurons. In contrast, CRMP1 and CRMP2 persist at all stages of differentiation from immature GAP43‐positive to fully mature olfactory neurons. In the olfactory bulb, CRMP1, CRMP2 and CRMP5 are abundant in neuronal progenitors of the subependymal layer and in differentiating interneurons. In both areas, the subcellular distribution of CRMP1 or CRMP2 is different in mature vs. immature neurons, suggesting that these proteins are sequentially involved in various cellular events during neuronal lifetime. The variations of CRMP expression following axotomy are consistent with their differential localization and functional involvement in immature vs. mature neurons of the olfactory system. Our data bring new insight to the putative functions of CRMPs within areas of the adult nervous system with permanent neurogenesis, some related to differentiation of newly generated neurons but others occurring in mature neurons with a limited lifespan.

Activation of noradrenergic transmission by α2-adrenoceptor antagonists counteracts deafferentation-induced neuronal death and cell proliferation in the adult mouse olfactory bulb

The olfactory bulb is the target of neural progenitor cells that are generated in the subventricular zone of the lateral ventricle in the adult brain. This permanent neurogenesis is likely influenced by olfactory input to the bulb since previous studies have shown that cell proliferation and/or apoptotic death are stimulated by naris closure or surgical transection of the olfactory nerve. Since the olfactory bulb is densely innervated by noradrenergic afferents originating in the locus coeruleus, we have studied the impact of pharmacologically activating this noradrenergic system on cell death and proliferation following unilateral olfactory axotomy in the adult mouse olfactory bulb. We found that noradrenaline release in the olfactory bulb was significantly increased by intraperitoneal injections of the selective alpha(2)-adrenoceptor antagonists, dexefaroxan (0.63 mg/kg) and 5-fluoro-methoxyidazoxan (F 14413; 0.16 mg/kg). A chronic treatment with either compound for 7 days following olfactory axotomy significantly reduced neuronal death, glial activation and cell proliferation in the deafferented olfactory bulb. These data (1) confirm that alpha(2)-adrenoceptor antagonists, presumably by facilitating central noradrenergic transmission, afford neuroprotection in vivo, as previously shown in models of cerebral ischemia, excitotoxicity and devascularization-induced neurodegeneration, and (2) support a role of the locus coeruleus noradrenergic system in promoting survival of neurons in areas of the brain where neurogenesis persists in the adult.

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]α‐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).