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

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

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

P53 inactivation leads to impaired motor synchronization in mice.

We have combined genetic and pharmacological approaches to investigate the behavioural consequences of inactivation of the murine p53 protein. Our behavioural analysis revealed that p53-null mice (p53KO) exhibit a very specific and significant motor deficit in rapid walking synchronization. This deficit, observed using the rotarod test, was the only behavioural defect of p53KO mice. We demonstrated that it was not due to an increase in neuronal number or abnormal connectivity in the olivo-cerebellar system, thought to control motor synchronization. In order to test the role of p53 in the central nervous system, we injected a pharmacological inhibitor of p53 activation, pifithrin-alpha, into the cerebellum of wild-type mice. This treatment mimicked the walking synchronization deficit of p53KO mice, suggesting that presence of p53 protein in the cerebellum is necessary to execute this synchronization of walking. Our investigation reveals a functional role of cerebellar p53 protein in adult walking synchronizatio

Mature Purkinje Cells Require the Retinoic Acid-Related Orphan Receptor-alpha (ROR alpha) to Maintain Climbing Fiber Mono-Innervation and Other Adult Characteristics

International audienceNeuronal maturation during development is a multistep process regulated by transcription factors. The transcription factor ROR alpha (retinoic acid-related orphan receptor alpha) is necessary for early Purkinje cell (PC) maturation but is also expressed throughout adulthood. To identify the role of ROR alpha in mature PCs, we used Cre-lox mouse genetic tools in vivo that delete it specifically from PCs between postnatal days 10 -21. Up to 14 d of age, differences between mutant and control PCs were not detectable: both were mono-innervated by climbing fibers (CFs) extending along their well-developed dendrites with spiny branchlets. By week 4, mutant mice were ataxic, some PCs had died, and remaining PC soma and dendrites were atrophic, with almost complete disappearance of spiny branchlets. The innervation pattern of surviving ROR alpha -deleted PCs was abnormal with several immature characteristics. Notably, multiple functional CF innervation was reestablished on these mature PCs, simultaneously with the relocation of CF contacts to the PC soma and their stem dendrite. This morphological modification of CF contacts could be induced even later, using lentivirus-mediated depletion of rora from adult PCs. These data show that the late postnatal expression of ROR alpha cell-autonomously regulates the maintenance of PC dendritic complexity, and the CF innervation status of the PC (dendritic vs somatic contacts, and mono-innervation vs multi-innervation). Thus, the differentiation state of adult neurons is under the control of transcription factors; and in their absence, adult neurons lose their mature characteristics and acquire some characteristics of an earlier developmental stage