Joëlle Sacquet

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.

Neurogenic correlates of an olfactory discrimination task in the adult olfactory bulb

In the main olfactory bulb, stimuli are coded within the spatio‐temporal pattern of mitral cells activity. Granule cells are interneurons that shape the mitral cells activity, and are continuously generated in the adult main olfactory bulb. However, the role of granule cell renewal remains elusive. We show here that an associative olfactory discrimination task reduces the survival of newborn neurons. However, when the olfactory task involves perceptually related odorants, the learning process is slower and does not induce such a reduction in the number of new neurons. Mapping newborn cells within the granule cell layer of the main olfactory bulb reveals a clustered distribution that evolves with learning as a function of odorant similarity and partly overlaps with the immediate‐early gene Zif268 expression pattern. These data provide insight into the functional mechanisms underlying olfactory discrimination learning, and promote the importance of neurogenesis as a cellular basis for the restructuring of odor images in the main olfactory bulb.

Novelty determines the effects of olfactory enrichment on memory and neurogenesis through noradrenergic mechanisms.

Commonly used experimental paradigms of environmental enrichment combine increased social interactions and sensory inputs and renewal of the objects present in the environment. However, the specific contribution of novelty to the effects of enrichment is unclear. Here, we show that repeated daily exposure to single novel odorants and not to an enriched but stable olfactory environment improves short-term olfactory memory and neurogenesis in the mouse olfactory bulb. In addition, these positive effects are mediated by noradrenalin as they are blocked by a noradrenergic receptor antagonist. These data suggest that novelty recognition and noradrenergic mechanisms are crucial in mediating neural plasticity induced by olfactory enrichment.

Early locus coeruleus degeneration and olfactory dysfunctions in Tg2576 mice

Olfactory deficiency has been reported in the early stages of Alzheimers disease (AD) in humans but is very poorly understood due to the lack of investigations in animal models of AD. Recent studies point to the noradrenergic system as an important target of the AD pathological process. In addition, noradrenalin has been shown to influence adult neurogenesis which is implicated in cognitive functions. We have therefore investigated the olfactory neurogenesis and cognitive performances in young transgenic Tg2576 mice in relation with the status of the noradrenergic and the cholinergic systems. Tg2576 showed a deficit in neurogenesis in the olfactory bulb evidenced by an increased death of newborn cells and a reduced expression of PSA-NCAM. The locus coeruleus degenerated in Tg2576 between the age of 6.5 and 8 months. These changes were associated with olfactory memory impairments. Our findings indicate that a noradrenergic deficiency could play a role in the early stages of the pathological process in this transgenic model and induce olfactory cognitive impairments through an alteration of olfactory neurogenesis.

Neuronal expression of Nogo‐A mRNA and protein during neurite outgrowth in the developing rat olfactory system†

The major impediments to axonal regeneration in the central nervous system are growth‐inhibitory proteins present in the myelin sheath, and Nogo‐A is one of the most potent inhibitors synthesized by oligodendrocytes. However, neuronal expression of Nogo‐A during development suggests that it may have an additional role. The spatio‐temporal regulation of both Nogo‐A mRNA and protein expression was examined by in situ hybridization and immunohistochemistry in the developing rat olfactory system. During embryonic and postnatal development (from E13 to P6), Nogo‐A mRNA and protein were strongly expressed by differentiating neurons in the olfactory epithelium and in the olfactory bulb. From the second postnatal week, a progressive down‐regulation of both Nogo‐A mRNA and protein occurred, such that only a weak expression persisted in the adult olfactory system. Using double‐immunostainings in the adult olfactory epithelium, we determined that Nogo‐A was preferentially expressed by immature olfactory receptor neurons extending axonal processes toward the olfactory bulb. At all developmental stages, Nogo‐A protein was preferentially targeted in olfactory axons emerging from the olfactory epithelium. Using an in vitro model of olfactory axon growth, we demonstrated that, in addition to its presence along the entire axon length, Nogo‐A accumulated in axonal growth cone and at axonal branching points, with a distribution similar to that of microtubule‐associated proteins. Moreover, Nogo‐A was transiently expressed in dendritic processes in the postnatal olfactory bulb. Together, our data suggest that, in non‐pathological conditions, Nogo‐A may be involved in the processes of axonal growth and dendritic modeling through the regulation of microtubule dynamics.

CRMP5 regulates generation and survival of newborn neurons in olfactory and hippocampal neurogenic areas of the adult mouse brain.

The Collapsin Response Mediator Proteins (CRMPs) are highly expressed in the developing brain, and in adult brain areas that retain neurogenesis, ie: the olfactory bulb (OB) and the dentate gyrus (DG). During brain development, CRMPs are essentially involved in signaling of axon guidance and neurite outgrowth, but their functions in the adult brain remain largely unknown. CRMP5 has been initially identified as the target of auto-antibodies involved in paraneoplasic neurological diseases and further implicated in a neurite outgrowth inhibition mediated by tubulin binding. Interestingly, CRMP5 is also highly expressed in adult brain neurogenic areas where its functions have not yet been elucidated. Here we observed in both neurogenic areas of the adult mouse brain that CRMP5 was present in proliferating and post-mitotic neuroblasts, while they migrate and differentiate into mature neurons. In CRMP5−/− mice, the lack of CRMP5 resulted in a significant increase of proliferation and neurogenesis, but also in an excess of apoptotic death of granule cells in the OB and DG. These findings provide the first evidence that CRMP5 is involved in the generation and survival of newly generated neurons in areas of the adult brain with a high level of activity-dependent neuronal plasticity.

Expression of Zif268 in the granule cell layer of the adult mouse olfactory bulb is modulated by experience

Behavioral and physiological evidence indicates that odor processing in the main olfactory bulb is influenced by olfactory experience. At the cellular level, changes in inhibitory influence exerted by granular interneurons may contribute to restructuring odor representations. To assess experience‐dependent modulation in the responsiveness of granule cells, we measured the level and spatial distribution of odor‐induced expression of the immediate–early gene Zif268 in the granule cell layer of adult mice submitted or not to olfactory discrimination conditioning. We first show that stimulation by the reinforced odorant in conditioned animals did not induce any increase in Zif268 expression in contrast to stimulation with an unfamiliar odorant which induced an odor‐specific three‐fold increase in Zif268 expression. The same lack of Zif268 induction was observed in animals exposed to odorants without learning, indicating that familiarity to the odorant with or without conditioning similarly reduced responsiveness of granule cells to odorant stimulation. Second, conditioning induced a spatial reorganization of Zif268‐positive cells leading to higher contrast and significant enlargement of their distribution pattern. The latter effect was also present in animals exposed to the odorants without conditioning but was significantly weaker. Taken together, these data indicate that distinct populations of granule cells are solicited by odorant processing, depending on its familiarity or behavioral significance. Finally, we report that the expression pattern of Zif268 in the granule cell layer is constrained by anteroposterior and dorsolateral gradients in cell density, pointing to anatomical and possibly functional disparity within the layer.

Context-driven activation of odor representations in the absence of olfactory stimuli in the olfactory bulb and piriform cortex

Sensory neural activity is highly context dependent and shaped by experience and expectation. In the olfactory bulb (OB), the first cerebral relay of olfactory processing, responses to odorants are shaped by previous experiences including contextual information thanks to strong feedback connections. In the present experiment, mice were conditioned to associate an odorant with a visual context and were then exposed to the visual context alone. We found that the visual context alone elicited exploration of the odor port similar to that elicited by the stimulus when it was initially presented. In the OB, the visual context alone elicited a neural activation pattern, assessed by mapping the expression of the immediate early gene zif268 (egr-1) that was highly similar to that evoked by the conditioned odorant, but not other odorants. This OB activation was processed by olfactory network as it was transmitted to the piriform cortex. Interestingly, a novel context abolished neural and behavioral responses. In addition, the neural representation in response to the context was dependent on top-down inputs, suggesting that context-dependent representation is initiated in cortex. Modeling of the experimental data suggests that odor representations are stored in cortical networks, reactivated by the context and activate bulbar representations. Activation of the OB and the associated behavioral response in the absence of physical stimulus showed that mice are capable of internal representations of sensory stimuli. The similarity of activation patterns induced by imaged and the corresponding physical stimulus, triggered only by the relevant context provides evidence for an odor-specific internal representation.

STOP proteins contribute to the maturation of the olfactory system

Regulation of microtubule dynamics is crucial for axon growth and guidance as well as for the establishment of synaptic connections. STOPs (Stable Tubule Only Polypeptides) are microtubule-associated proteins that regulate microtubule stabilization but are also able to interact with actin or Golgi membranes. Here, we have investigated the involvement of STOPs during the development of the olfactory system. We first describe the spatio-temporal expression patterns of N- and E-STOP, the two neuronal-specific isoforms of STOP. E- and N-STOP are expressed in the axonal compartment of olfactory sensory neurons, but are differentially regulated during development. Interestingly, each neuronal isoform displays a specific gradient distribution within the olfactory nerve layer. Then, we have examined the development of the olfactory system in the absence of STOPs. Olfactory axons display a normal outgrowth and targeting in STOP-null mice, but maturation of the synapses in the glomerular neuropil is altered.

Spatio-temporal expression pattern of receptors for myelin-associated inhibitors in the developing rat olfactory system

The myelin-associated inhibitory proteins (Nogo-A, MAG and OMgp) that prevent axon regeneration in adult CNS, mediate their effects via a receptor referred as NgR1. Beside their inhibitory role in the adult CNS, Nogo-A and NgR1 might also be functionally involved in the developing nervous system. At the present time, no detailed study is available regarding either the onset of NgR1 expression during development or its spatio-temporal pattern of expression relative to the presence of Nogo-A. Two homologs of NgR1, NgR2 and NgR3, have been recently identified, but their function in the nervous system is still unknown in adult as well as during development. We have examined the spatio-temporal expression pattern of both NgR1, NgR2 and NgR3 mRNAs and corresponding proteins in the developing rat olfactory system using in situ hybridization and immunohistochemistry. From E15-E16 onwards, NgR1 mRNA was expressed by differentiating neurons in both the olfactory epithelium and the olfactory bulb. At all developmental stages, including adult animals, NgR1 protein was preferentially targeted to olfactory axons emerging from the olfactory epithelium. Using double-immunostainings in the postnatal olfactory mucosa, we confirm the neuronal localization of NgR1 and its preferential distribution along the olfactory axons. The NgR2 and NgR3 transcripts and their proteins display similar expression profiles in the olfactory system. Together, our data suggest that, in non-pathological conditions, NgR1 and its homologs may play a role in axon outgrowth in the rat olfactory system and may be relevant for the confinement of neural projections within the developing olfactory bulb.