The olfactory system has become an important model for investigating sensory processing as well as the cellular basis of learning and memory. We are investigating how the external word of scents may sculpt the activity of the first central relay of the olfactory system, i.e., the main olfactory bulb (OB). This relay is also one of the few regions of the brain to continuously replace one of its neuronal populations: the local GABAergic interneurons. How the newly formed neurons integrate into a pre-existing neural network and how basic olfactory functions are maintained when a large percentage of neurons are subjected to continuous renewal, are important questions that we are currently exploring. In particular, we are analyzing the sensitivity of the bulbar neurogenesis on the level of sensory inputs and, in turn, how the neurogenesis may adjust the neural network functioning to optimize odor information processing. Our results, though correlative so far, suggested that adult-born interneurons integrate functionally into the adult OB and bring specific functions to the pre-existing neuronal circuitry. We are now exploring the functional consequences of the adult neurogenesis and how it may be appropriate for the sense of smell. It is worth mentioning that these investigations not only provide new fuel for the molecular and cellular bases of sensory perception but should also shed light onto the cellular bases of learning and memory.

In 2002, our activity was aimed at understanding adult neurogenesis: the factors controlling generation, migration and integration of new neurons in adult brains and the functional consequences of this phenomenon. We have achieved this through a multidisciplinary, fully integrated approach. Methodologically this had included morphological, electrophysiological, and molecular characterization of new neurons in vitro and in vivo as well as behavior approaches to investigate the functional contribution of new neurons to pre-existing brain circuitry. Special emphasis was given to investigate the relative contribution of intrinsic (cell-autonomous) and extrinsic (environmental) cues regulating this process. Using the adult OB as a model, we investigated how neurons proliferate, differentiate, migrate and populate the different layers of the adult OB. We also explored the possible consequences of the bulbar plasticity for olfactory performances.

1) Characterization of adult-generated neurons (cellular physiology, morphology, immuno-histochemical profiles)

• The cellular targets and the way by which newly generated cells incorporate into the existing neuronal circuitry according to some molecular cues (extracellular matrix components and neurotransmitters) have been described using electrophysiological and immuno-histochemical approaches.

• We also have demonstrated that newly formed neurons become functional neurons in the adult brain. To show that, a number of different lines of evidence have been provided, including morphological, molecular and electrophysiological characterization of in vivo labeled cells showing neuronal activity. A description of the progressive maturation of newly formed cells has also been provided in the adult brain (the retroviral vector necessary for assessing the role of different factors influencing neurogenesis is provided by Pierre Charneau, Pasteur Institute). The properties of adult-formed neurons have been compared with those found in embryonic and early postnatal neurons.

2) Understanding the molecular and environmental factors affecting neuronal proliferation, migration and differentiation of adult-generated neurons

• We have analyzed the role of neuronal cell adhesion molecules such as PSA-NCAM and the extracellular matrix proteins that are highly expressed along the migratory route of new neurons in rodents.

• The role of ionotropic neurotransmitter receptors in regulating neuroblast proliferation/migration are currently investigated both in vivo and in vitro experiments on explants and on neurospheres.

3) Identification of environmental factors affecting neuronal proliferation, migration and differentiation of adult-generated neurons

• The role of sensory deprivation, enriched environment, learning and social cues on adult neurogenesis are currently explored.

4) The contribution of the medial ganglionic eminence during brain development

• We have identified two populations of early-generated neurons that control corticogenesis. By combining immunocytochemistry with elecrophysiology, we brought new insights into the cellular identity of neurons that play crucial developmental roles.

5) Understanding the functional consequences of adult neurogenesis at the behavioral level

• We have performed electrophysiological analysis of neural synchronization in the olfactory bulb after modification of neuroblast proliferation, migration and/or survival (using mutant mice and sensory deprived rats).

• We compared the odor sensitivity (threshold tests), odor discrimination and odor memory in different types of mutant mice with altered neurogenesis pattern.

6) Behavioral relevance of bulbar neurogenesis during brain development and adulthood

• In order to fully appreciate the relevance of neurogenesis, it is important to take advantage of naturally occurring situations in which the production of neurons could play a role. For example, the exploitation of different forms of learning and memory which could take place in newborn mice during odor-guided social transmission of offspring and mate recognition have been partially investigated and should undoubtedly constitute a critical step in understanding the role of bulbar neurogenesis in olfaction.

7) Seeking for the function of PrP^C in adult neurogenesis

• Based on the neuronal sub-cellular localization of PrP^C, on its high levels of expression in a region of intense neuronal renewal in the adult and on its coupling with some signal transduction pathways, we hypothesized that PrP^C might play a role in neuronal renewal. In adult wild-type mice, we found that PrP^C is constitutively expressed in neuronal progenitors but not in neuroblasts migrating towards the olfactory bulb. Using "knocked-out" or overexpressing PrP^C mice, we discovered that the expression level of PrP^C influences the organization of chain migrating neuroblasts. Furthermore, using an in vitro neurosphere assay, we noted a strong correlation between the level of PrP^C, the total number of SVZ neuronal stem cells and the proliferation rate of the stem cell precursors. The consequences of changing the level of PrP^C expression on adult neurogenesis was further investigated in vivo using intraperitoneal injections of 5-bromo-2-deoxyuridine, a marker of dividing cells. We found that changing the level of PrP^C dramatically alters the rate of proliferation in the SVZ but not in the RMS. Taken together, these results show that PrP^C plays a role in adult neurogenesis and suggest that it may negatively regulate cellular proliferation and/or survival. We will now use a transgenic line in which PrP gene expression is induced on demand by oral doxycline in an endogenous PrP deleted context. This will allow us to investigate more precisely the temporal relationship between adult neurogenesis and the regulation of PrP^C expression.

Photo 1 : Trophic factors in the main olfactory bulb.

Photo 2 : Odor-induced oscillations in the main olfactory bulb.

Keywords: Development, Interneuron, Oscillations, Prion, Stem cells