Cortical Neurons Require Otx1 for the Refinement of Exuberant Axonal Projections to Subcortical Targets

AbstractInformation processing in the nervous system depends on the creation of specific synaptic connections between neurons and targets during development. The homeodomain transcription factor Otx1 is expressed in early-generated neurons of the developing cerebral cortex. Within layer 5, Otx1 is expressed by neurons with subcortical axonal projections to the midbrain and spinal cord. Otx1 is also expressed in the precursors of these neurons, but is localized to the cytoplasm. Nuclear translocation of Otx1 occurs when layer 5 neurons enter a period of axonal refinement and eliminate a subset of their long-distance projections. Otx1 mutant mice are defective in the refinement of these exuberant projections, suggesting that Otx1 is required for the development of normal axonal connectivity and the generation of coordinated motor behavior

In vivo Bioluminescence Imaging of Ca(2+) Signalling in the Brain of Drosophila

Many different cells' signalling pathways are universally regulated by Ca(2+) concentration [Ca(2+)] rises that have highly variable amplitudes and kinetic properties. Optical imaging can provide the means to characterise both the temporal and spatial aspects of Ca(2+) signals involved in neurophysiological functions. New methods for in vivo imaging of Ca(2+) signalling in the brain of Drosophila are required for probing the different dynamic aspects of this system. In studies here, whole brain Ca(2+) imaging was performed on transgenic flies with targeted expression of the bioluminescent Ca(2+) reporter GFP-aequorin (GA) in different neural structures. A photon counting based technique was used to undertake continuous recordings of cytosolic [Ca(2+)] over hours. Time integrals for reconstructing images and analysis of the data were selected offline according to the signal intensity. This approach allowed a unique Ca(2+) response associated with cholinergic transmission to be identified by whole brain imaging of specific neural structures. Notably, [Ca(2+)] transients in the Mushroom Bodies (MBs) following nicotine stimulation were accompanied by a delayed secondary [Ca(2+)] rise (up to 15 min. later) in the MB lobes. The delayed response was sensitive to thapsigargin, suggesting a role for intra-cellular Ca(2+) stores. Moreover, it was reduced in dunce mutant flies, which are impaired in learning and memory. Bioluminescence imaging is therefore useful for studying Ca(2+) signalling pathways and for functional mapping of neurophysiological processes in the fly brain

Non-Invasive In Vivo Imaging of Calcium Signaling in Mice

Rapid and transient elevations of Ca2+ within cellular microdomains play a critical role in the regulation of many signal transduction pathways. Described here is a genetic approach for non-invasive detection of localized Ca2+ concentration ([Ca2+]) rises in live animals using bioluminescence imaging (BLI). Transgenic mice conditionally expressing the Ca2+-sensitive bioluminescent reporter GFP-aequorin targeted to the mitochondrial matrix were studied in several experimental paradigms. Rapid [Ca2+] rises inside the mitochondrial matrix could be readily detected during single-twitch muscle contractions. Whole body patterns of [Ca2+] were monitored in freely moving mice and during epileptic seizures. Furthermore, variations in mitochondrial [Ca2+] correlated to behavioral components of the sleep/wake cycle were observed during prolonged whole body recordings of newborn mice. This non-invasive imaging technique opens new avenues for the analysis of Ca2+ signaling whenever whole body information in freely moving animals is desired, in particular during behavioral and developmental studies

Study of Fgf15 gene expression in developing mouse brain

Fibroblast growth factor 15 (Fgf15) is a gene regulated by the expression of Otx2 in developing mouse brain (Proc Natl Acad Sci USA 97 (2000) 14388). Otx2 gene codes for a transcription factor and is fundamental for the regionalisation and development of the anterior neural plate and cephalic region of the vertebrate embryo (Development 124 (1997) 3639). In addition, the thalamic expression of Fgf15 has been recently reported under the control of Shh signalling gene, expressed in the diencephalic basal plate (Development 129 (2002) 4807). In the present work, we have analysed Fgf15 expression pattern during mouse neural development. Fgf15 appeared early in the developing neural epithelium, in domains where Fgf8 gene is also expressed and, at later stages, in specific groups of neural cells. Fgf8 is an important signalling protein with demonstrated morphogenetic activity in several embryonic regions. Fgf15 expression is localized, like Fgf8, in secondary neural tube organizers: the isthmic organizer (IsO) and the anterior neural ridge (ANR).L.G. was supported by Seneca Foundation Fellowship BP 00396/CV/00. This work was supported by the Seneca Foundation 00708/CV/99, European Union grants (UE QLG2-CT-1999-00793; UE QLRT-1999-31556; UE QLRT-1999-31625; QLRT-2000-02310) and Spanish grants (DIGESIC-MEC PM98-0056 and FEDEN) to S.M.Peer reviewe

Retrograde trans-synaptic transfer of green fluorescent protein allows the genetic mapping of neuronal circuits in transgenic mice

The function of the nervous system is a consequence of the intricate synaptic connectivity of its neurons. Our understanding of these highly complex networks has profited enormously from methods used over the past two decades that are based on the mechanical injection of tracer molecules into brain regions. We have developed a genetic system for the mapping of synaptic connections during development of the mammalian central nervous system and in the mature brain. It is based on the transsynaptic transfer of green fluorescent protein (GFP) in the brains of mice using a fusion protein with a nontoxic fragment of tetanus toxin (TTC) expressed in defined neurons. These transgenic mice allowed us to visualize neurons, at single-cell resolution, that are in synaptic contact by the detection of GFP in interconnected circuits. Targeted genetic expression with a specific promoter permitted us to transfer GFP to defined subsets of neurons and brain regions. GFP–TTC is coexpressed with a lacZ reporter gene to discriminate neurons that produce the tracer from cells that have acquired it transneuronally. The marker shows selective transfer in the retrograde direction. We have used electron microscopic detection of GFP to define the ultrastructural features of the system. Our work opens up a range of possibilities for brain slice and in vivo studies taking advantage of the fluorescence of GFP. We point the way toward the use of powerful multiphoton technology and set the stage for the transsynaptic transfer of other proteins in the brains of mice

Chimeric green fluorescent protein-aequorin as bioluminescent Ca2+ reporters at the single-cell level.

Monitoring calcium fluxes in real time could help to understand the development, the plasticity, and the functioning of the central nervous system. In jellyfish, the chemiluminescent calcium binding aequorin protein is associated with the green fluorescent protein and a green bioluminescent signal is emitted upon Ca(2+) stimulation. We decided to use this chemiluminescence resonance energy transfer between the two molecules. Calcium-sensitive bioluminescent reporter genes have been constructed by fusing green fluorescent protein and aequorin, resulting in much more light being emitted. Chemiluminescent and fluorescent activities of these fusion proteins have been assessed in mammalian cells. Cytosolic Ca(2+) increases were imaged at the single-cell level with a cooled intensified charge-coupled device camera. This bifunctional reporter gene should allow the investigation of calcium activities in neuronal networks and in specific subcellular compartments in transgenic animals

Utilisation du fragment C-terminal de la neurotoxine tétanique pour visualiser et analyser des connexions neuronales et pour le transfert d'une activité biologique associée

Tout en étant dépourvu de toxicité, le fragment Cterminal de la neurotoxine tétanique ou fragment TTC conserve les propriétés de transport rétrograde et trans-synaptique de la toxine native. L'association du fragment TTC avec un marqueur fluorescent ou avec la β-galactosidase facilite sa détection. Ces marqueurs ont donc logiquement été utilisés pour visualiser et étudier les connexions neuronales établies. Dans cette revue de synthèse, nous développerons les potentialités des différentes approches utilisées, à savoir l'injection de protéine purifiée, l'utilisation d'adénovirus et la transgenèse. Comme l'activité nerveuse joue un rôle essentiel dans l'internalisation du fragment TTC, la fonctionnalité des connexions ainsi visualisées peut être également appréhendée. Des modifications quantitatives du transport rétrograde neuronal peuvent être également détectées. Le fragment TTC représente donc un excellent outil pour analyser la connectivité et la fonctionnalité d'un réseau neuronal. D'autre part, le fragment TTC a été très rapidement pressenti comme un vecteur particulièrement intéressant pour le transport et la libération d'activité biologique ou encore de gènes au sein d'un réseau neuronal. Dans cette optique, des constructions contenant un domaine de translocation membranaire permettant la libération cytosolique de l'activité biologique associée ont été étudiées. Enfin, nous rapporterons les premiers résultats, particulièrement encourageants, obtenus avec le fragment TTC pour cibler exclusivement vers les neurones l'action d'un facteur neurotrophique. Cette spécificité d'action permettrait alors d'éviter des effets secondaires dus à une action de ces facteurs sur d'autres cellules que les neurones