Reinnervation of late postnatal purkinje cells by climbing fibers: neosynaptogenesis without transient multi-innervation.

Synaptic partner selection and refinement of projections are important in the development of precise and functional neuronal connections. We investigated the formation of new synaptic connections in a relatively mature system to test whether developmental events can be recapitulated at later stages (i.e., after the mature synaptic organization has been established), using a model of postlesional reinnervation in the olivo-cerebellar pathway. During the development of this pathway, synaptic connections between climbing fibers (CFs) and Purkinje cells (PCs) are diffuse and redundant before synapse elimination refines the pattern. The regression of CFs during the first 2 postnatal weeks in the rat leads to mono-innervation of each PC. After unilateral transection of the rat olivo-cerebellar pathway and intracerebellar injection of BDNF 24 h after lesion, axons from the remaining inferior olive can sprout into the deafferented hemicerebellum and establish new contacts with denervated PCs at later developmental stages. We found that these contacts are first established on somatic thorns before the CFs translocate to the PC dendrites, recapitulating the morphological steps of normal CF-PC synaptogenesis, but on a relatively mature PC. However, electrophysiology of PC reinnervation by transcommissural CFs in these animals showed that each PC is reinnervated by only one CF. This mono-innervation contrasts with the reinnervation of grafted immature PCs in the same cerebellum. Our results provide evidence that relatively mature PCs do not receive several olivary afferents during late reinnervation, suggesting a critical role of the target cell state in the control of CF-PC synaptogenesis. Thus, synapse exuberance and subsequent elimination are not a prerequisite to reach a mature relationship between synaptic partners

Synaptogenèse et néosynaptogenèse dans le système olivo-cérébelleux des rongeurs

Pendant le développement de la voie olivo-cérébelleuse, la sélection des partenaires synaptiques repose sur une compétition synaptique qui se traduit par (1) la multi-innervation des cellules de Purkinje (CPs) puis par (2) la stabilisation synaptique sélective d une fibre grimpante (FG) unique. L élimination définitive des FGs surnuméraires permet la formation de couples CP-neurone olivaire spécifiques. Pendant ma thèse, je me suis posé la question de savoir si les partenaires synaptiques seraient capables de s engager de nouveau dans un processus de sélection lors d une réinnervation tardive et si la stabilisation des nouvelles connexions interviendrait après une phase de compétition synaptique. Cette question est importante pour déterminer si des neurones matures sont capables de reformer des réseaux synaptiques spécifiques après une lésion. Avec une approche in vivo chez le rat, nous avons montré que les neurones dans le système olivo-cérébelleux survivant à une lésion unilatérale des axones olivaires sont capables de créer de nouvelles connexions synaptiques après injection de BDNF exogène dans le cervelet. La stabilisation de nouvelles synapses pendant la réinnervation tardive se traduit par la mono-innervation d emblée des CPs et ne fait donc pas intervenir la compétition synaptique observée pendant le développement normal ou pendant la réinnervation de cellules immatures greffées dans le même cervelet. L établissement d une mono-innervation traduit l existence d une sélection des partenaires synaptiques mais l absence de multi-innervation indique que cette sélection ne résulte pas d une compétition synaptique. Cette étude montre que la maturation de la CP conditionne le processus de sélection des FGs in vivo en participant au contrôle du nombre d afférences pendant la réinnervation. Parallèlement, nous avons caractérisé un modèle de synaptogenèse dans des explants de rhombencéphale de souris. Nous avons montré que les axones olivaires multi-innervent les cellules de Purkinje dans les explants en développement puis rentrent en compétition jusqu à l établissement de la monoinnervation. En réalisant des expériences de cocultures, nous avons trouvé que la multi-innervation transitoire des CPs n est pas reproduite si les deux partenaires synaptiques sont matures et ont déjà vécu la période d élimination synaptique, confirmant les résultats obtenus in vivo. En revanche, des CPs matures peuvent être multi-innervées si elles n ont jamais été innervées auparavant et/ou si elles sont innervées par des FGs immatures ; mais elles sont alors incapables de s engager dans un processus de sélection. Ainsi, la compétition synaptique intervient pendant une période critique du développement et induit des changements à long-terme. La sélection des partenaires pendant une réinnervation post-lésionnelle intervient indépendamment de la formation de synapses.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Faire et refaire une synapse en choisissant un partenaire

Des études récentes réalisées in vivo et in vitro sur des neurones du cervelet de rongeur montrent que la formation des connexions synaptiques pendant une période critique du développement laisse une empreinte qui marque définitivement les neurones et les synapses qu’ils forment entre eux. Nous discutons la signification fonctionnelle et la nature de cette empreinte qui pourrait prendre la forme d’étiquettes moléculaires marquant et protégeant les connexions synaptiques sélectionnées pendant le développement. Ces étiquettes, à l’image d’un « plan », permettraient de maintenir ou de reformer des connexions spécifiques, garantissant une cohérence au niveau comportemental

Formation and Reformation of Climbing Fibre Synapses in the Cerebellum: a Similar Story?

International audienceThe assembly of neural circuits involves multiple sequential steps, in particular the formation and maturation of synaptic connections. This often prolonged process involves several stages including the appropriate morphological and physiological maturation of each synaptic partner as well as their mutual interaction in order to ensure correct cellular and subcellular targeting. Understanding the processes involved becomes critical if neural circuits are to be appropriately reassembled following lesion, atrophy or neurodegeneration. We study the climbing fibre to Purkinje cell synapse as an example of a neural circuit which undergoes initial synaptic formation, selective stabilisation and elimination of redundant connections, in order to better understand the relative roles of each synaptic partner in the process of synaptogenesis and post-lesion synapse reformation. In particular, we are interested in the molecules which may underlie these processes. Here, we present data showing that the maturational state of both the target Purkinje cell and the climbing fibre axon influence their capacity for synapse formation. The climbing fibre retains some ability to recapitulate developmental processes irrespective of its maturational state. In contrast, the experience of synaptic formation and selective stabilisation/elimination permanently changes the Purkinje cell so that it cannot be repeated. Thus, if the climbing fibre-Purkinje cell synapse is recreated after the period of normal maturation, the process of synaptic competition, involving the gradual weakening of one climbing fibre synapse and stabilisation of another, no longer takes place. Moreover, we show that these processes of synaptic competition can only proceed during a specific developmental phase. To understand why these changes occur, we have investigated the role of molecules involved in the development of the olivocerebellar path and show that brain-derived neurotrophic factor, through activation of its receptor TrkB, as well as polysialated neural cell adhesion molecule and the transcription factor ROR alpha regulate these processes

Maturation of GABAergic Transmission in Cerebellar Purkinje Cells Is Sex Dependent and Altered in the Valproate Model of Autism

Brain development is accompanied by a shift in gamma-aminobutyric acid (GABA) response from depolarizing-excitatory to hyperpolarizing-inhibitory, due to a reduction of intracellular chloride concentration. This sequence is delayed in Autism Spectrum Disorders (ASD). We now report a similar alteration of this shift in the cerebellum, a structure implicated in ASD. Using single GABAA receptor channel recordings in cerebellar Purkinje cells (PCs), we found two conductance levels (18 and 10 pS), the former being dominant in newborns and the latter in young-adults. This conductance shift and the depolarizing/excitatory to hyperpolarizing/inhibitory GABA shift occurred 4 days later in females than males. Our data support a sex-dependent developmental shift of GABA conductance and chloride gradient, leading to different developmental timing in males and females. Because these developmental sequences are altered in ASD, this study further stresses the importance of developmental timing in pathological neurodevelopment

Post-lesion transcommissural growth of olivary climbing fibres creates functional synaptic microzones

In the adult mammalian central nervous system, reinnervation and recovery from trauma is limited. During development, however, postlesion plasticity may generate alternate paths, providing models to investigate reinnervating axon–target interactions. After unilateral transection of the neonatal rat olivocerebellar path, axons from the ipsilateral inferior olive grow into the denervated hemicerebellum and develop climbing fibre (CF)-like arbors on Purkinje cells (PCs). However, the synaptic function and extent of PC reinnervation remain unknown. In adult rats pedunculotomized on postnatal day 3 the morphological and electrophysiological properties of reinnervating olivocerebellar axons were studied, using axonal reconstruction and patch-clamp PC recording of CF-induced synaptic currents. Reinnervated PCs displayed normal CF currents, and the frequency of PC reinnervation decreased with increasing laterality. Reinnervating CF arbors were predominantly normal but 6% branched within the molecular layer forming smaller secondary arbors. CFs arose from transcommissural olivary axons, which branched extensively near their target PCs to produce on average 36 CFs, which is six times more than normal. Axons terminating in the hemisphere developed more CFs than those terminating in the vermis. However, the precise parasagittal microzone organization was preserved. Transcommissural axons also branched, although to a lesser extent, to the deep cerebellar nuclei and terminated in a distribution indicative of the olivo-cortico-nuclear circuit. These results show that reinnervating olivocerebellar axons are highly plastic in the cerebellum, compensating anatomically and functionally for early postnatal denervation, and that this reparation obeys precise topographic constraints although axonal plasticity is modified by target (PC or deep nuclear neurons) interactions

Enhanced Survival of Wild-Type and Lurcher Purkinje Cells In Vitro Following Inhibition of Conventional PKCs or Stress-Activated MAP Kinase Pathways

International audienceRecent studies using both dissociated and organotypic cell cultures have shown that heterozygous Lurcher (Lc/+) Purkinje cells (PCs) grown in vitro share many of the same survival and morphological characteristics as Lc/+ PCs in vivo. We have used this established tissue culture system as a valuable model for studying cell death mechanisms in a relatively simple system where neurodegeneration is induced by a constitutive cation leak mediated by the Lurcher mutation in the delta 2 glutamate receptor (GluR delta 2). In this study, Ca++ imaging and immunocytochemistry studies indicate that intracellular levels of Ca++ are chronically increased in Lc/+ PCs and the concentration and/or distribution of the conventional PKC gamma isoform is altered in degenerating Lc/+ PCs. To begin to characterize the molecular mechanisms that regulate Lc/+ PC death, the contributions of conventional PKC pathways and of two MAP kinase family members, JNK and p38, were examined in slice cultures from wild-type and Lc/+ mutant mouse cerebellum. Cerebellar slice cultures from P0 pups were treated with either a conventional PKC inhibitor, a JNK inhibitor, or a p38 inhibitor either from 0 to 14 or 7 to 14 DIV. Treatment with either of the three inhibitors from 0 DIV significantly increased wild type and Lc/+ PC survival through 14 DIV, but only Lc/+ PC survival was significantly increased following treatments from 7 to 14 DIV. The results suggest that multiple PC death pathways are induced by the physical trauma of making organotypic slice cultures, naturally-occurring postnatal cell death, and the GluR delta 2 (Lc) mutation

Elimination of all redundant climbing fiber synapses requires granule cells in the postnatal cerebellum

Abstract Different afferent synapse populations interact to control the specificity of connections during neuronal circuit maturation. The elimination of all but one climbing-fiber onto each Purkinje cell during the development of the cerebellar cortex is a particularly well studied example of synaptic refinement. The suppression of granule cell precursors by X irradiation during postnatal days 4 to 7 prevents this synaptic refinement, indicating a critical role for granule cells. Several studies of cerebellar development have suggested that synapse elimination has a first phase which is granule cell-independent and a second phase which is granule cell-dependent. In this study, we show that sufficiently-strong irradiation restricted to postnatal days 5 or 6 completely abolishes climbing fiber synaptic refinement, leaving the olivo-cerebellar circuit in its immature configuration in the adult, with up to 5 climbing fibers innervating the Purkinje cell in some cases. This implies that the putative early phase of climbing fiber synapse elimination can be blocked by irradiation-induced granule cell loss if this loss is sufficiently large, and thus indicates that the entire process of climbing fiber synapse elimination requires the presence of an adequate number of granule cells. The specific critical period for this effect appears to be directly related to the timing of Purkinje cell and granule cell development in different cerebellar lobules, indicating a close, spatiotemporal synchrony between granule-cell development and olivo-cerebellar synaptic maturation