Unit: Odor Perception and Memory
Director: LLEDO Pierre-Marie
The ability of adult brain to produce new neurons has excited much interest not only because of its fundamental significance, but also in part because of the potential to treat neurodegenerative diseases. While proliferation of neuronal precursors in the mature brain has been extensively studied in the past, very little was known about how neuronal progenitors migrate and integrate to the appropriate target area to become truly functional neurons and what are the factors controlling their recruitment into pre-existing neuronal networks.
We are currently investigating the mechanisms that regulate production of new neurons in the adult brain, their targeting to the appropriate regions, and their differentiation and survival in these areas. Understanding of these mechanisms will have profound advance in the cell replacement therapies by offering new strategies aimed to control not only the dispersal of grafted cells, but also the production and recruitment of endogenous precursor neurons.
The discovery of neuronal recruitment into adult brain microcircuits has been one of the major recent breakthroughs in developmental Neuroscience. Clearly, the next forthcoming steps are expected to bring new insights into the mechanisms that govern the production of new neurons, their targeting to appropriate regions, their differentiation into distinct neuronal subtypes and finally, their survival. In 2004, we have been investigating these fundamental issues by taking the olfactory bulb as a model. 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.
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 investigated (Alonso, de Chevigny, Gabellec, Saghatelyan, Violet).
We also have demonstrated that newly formed neurons become functional neurons in the adult brain that could be either dopaminergic or GABAergic (de Chevigny, Saghatelyan). A description of the progressive maturation of newly formed cells has also been provided in the adult brain (the lentiviral vector necessary for assessing the role of different factors influencing neurogenesis is provided by Pierre Charneau, Pasteur Institute). The production of adult-formed neurons has been compared with the one of early postnatal (Lemasson, Saghatelyan).
Tracking the migratoring cells in the adult forebrain in vivo (Davenne, Saghatelyan)
2) Understanding the molecular and environmental factors affecting neuronal proliferation, migration and differentiation of adult-generated neurons
We have analyzed the role of molecules from the extracellular matrix (tenascin-C and tenascin-R) that are either highly expressed along the migratory route of new neurons (tenascin-C) or in the target tissues (de Chevigny, Lemasson, Saghatelyan) .
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 (Alonso, Violet).
3) Identification of environmental factors affecting neuronal proliferation, migration and differentiation of adult-generated neurons
The role of sensory deprivation is particularly explored on the maturation of newly generated neurons in the adult olfactory bulb (Saghatelyan).
4) 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) (Gheusi, Lagier, Lemasson, Saghatelyan).
We compared the odor sensitivity (threshold tests), odor discrimination and odor memory in different types of mutant mice with altered neurogenesis pattern (Gheusi, Lagier, Lemasson, Saghatelyan).
5) 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. We are exploring different forms of learning and memory that could take place in newborn mice during odor-guided social transmission of offspring and mate recognition. This should undoubtedly constitute a critical step in understanding the role of bulbar neurogenesis in olfaction.
6) Seeking for the function of PrPC in adult neurogenesis
Based on the neuronal sub-cellular localization of PrPC, 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 PrPC might play a role in neuronal renewal. In adult wild-type mice, we found that PrPC is constitutively expressed in neuronal progenitors but not in neuroblasts migrating towards the olfactory bulb. Using "knocked-out" or overexpressing PrPC mice, we discovered that the expression level of PrPC influences the organization of chain migrating neuroblasts. Furthermore, using an in vitro neurosphere assay, we noted a strong correlation between the level of PrPC, the total number of SVZ neuronal stem cells and the proliferation rate of the stem cell precursors. The consequences of changing the level of PrPC 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 PrPC dramatically alters the rate of proliferation in the SVZ but not in the RMS. Taken together, these results show that PrPC 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 PrPC expression (de Chevigny, Gabellec).
Photo 1: In vivo forebrain imaging with fluorescence microendoscopy. A: Schematic representation of a sagittal view of the brain showing the site of labeling (the subventricular zone or SVZ), the adult-born neuroblasts (red dots) migrating from the SVZ to the olfactory bulb (OB) along the rostral migratory stream (RMS), and the site of imaging with the endoscope probe. B: GFP-labeled cells migrating along the RMS. All images were acquired at the same recording site and taken at different time points (indicated on top). Penetration depth: 3 mm. At time "0", stars indicate the initial positions. The arrowheads point to non-migrating cells and small arrows show moving cells. Scale bar = 60 µm.
Photo 2: Tenascin-R re-directs migrating neuroblasts. A: Schematic representation of mouse brain with tenascin-R (TN-R) rich areas shown in blue and migrating neuroblasts depicted in green. Note that ectopic expression of TN-R in the striatum re-routes migrating neuroblasts. B: TN-R-secreting grafted cells pre-labeled with a fluorescent dye (red) were placed into the striatum neighboring the SVZ. Migrating neuroblasts towards the TN-R immunopositive area (blue) are visualized by PSA-NCAM staining (green). C: A coronal section of the olfactory bulb showing the pattern of TN-R expression (red). Migrating neuroblasts are labeled with PSA-NCAM antibodies (green). Scale bars: b = 100 µm and c = 400 µm.
Keywords: Neural stem cells, Olfaction, Perception, Parkinson, Neuroscience