Pax3 induces target-specific reinnervation through axon collateral expression of PSA-NCAM.

Damaged or dysfunctional neural circuits can be replaced after a lesion by axon sprouting and collateral growth from undamaged neurons. Unfortunately, these new connections are often disorganized and rarely produce clinical improvement. Here we investigate how to promote post-lesion axonal collateral growth, while retaining correct cellular targeting. In the mouse olivocerebellar path, brain-derived neurotrophic factor (BDNF) induces correctly-targeted post-lesion cerebellar reinnervation by remaining intact inferior olivary axons (climbing fibers). In this study we identified cellular processes through which BDNF induces this repair. BDNF injection into the denervated cerebellum upregulates the transcription factor Pax3 in inferior olivary neurons and induces rapid climbing fiber sprouting. Pax3 in turn increases polysialic acid-neural cell adhesion molecule (PSA-NCAM) in the sprouting climbing fiber path, facilitating collateral outgrowth and pathfinding to reinnervate the correct targets, cerebellar Purkinje cells. BDNF-induced reinnervation can be reproduced by olivary Pax3 overexpression, and abolished by olivary Pax3 knockdown, suggesting that Pax3 promotes axon growth and guidance through upregulating PSA-NCAM, probably on the axon's growth cone. These data indicate that restricting growth-promotion to potential reinnervating afferent neurons, as opposed to stimulating the whole circuit or the injury site, allows axon growth and appropriate guidance, thus accurately rebuilding a neural circuit.

Magnetic Stimulation as a Therapeutic Approach for Brain Modulation and Repair: Underlying Molecular and Cellular Mechanisms.

Neurological and psychiatric diseases generally have no cure, so innovative non-pharmacological treatments, including non-invasive brain stimulation, are interesting therapeutic tools as they aim to trigger intrinsic neural repair mechanisms. A common brain stimulation technique involves the application of pulsed magnetic fields to affected brain regions. However, investigations of magnetic brain stimulation are complicated by the use of many different stimulation parameters. Magnetic brain stimulation is usually divided into two poorly connected approaches: (1) clinically used high-intensity stimulation (0.5-2 Tesla, T) and (2) experimental or epidemiologically studied low-intensity stimulation (µT-mT). Human tests of both approaches are reported to have beneficial outcomes, but the underlying biology is unclear, and thus optimal stimulation parameters remain ill defined. Here, we aim to bring together what is known about the biology of magnetic brain stimulation from human, animal, and in vitro studies. We identify the common effects of different stimulation protocols; show how different types of pulsed magnetic fields interact with nervous tissue; and describe cellular mechanisms underlying their effects-from intracellular signalling cascades, through synaptic plasticity and the modulation of network activity, to long-term structural changes in neural circuits. Recent advances in magneto-biology show clear mechanisms that may explain low-intensity stimulation effects in the brain. With its large breadth of stimulation parameters, not available to high-intensity stimulation, low-intensity focal magnetic stimulation becomes a potentially powerful treatment tool for human application.

In vivo low-intensity magnetic pulses durably alter neocortical neuron excitability and spontaneous activity.

Magnetic brain stimulation is a promising treatment for neurological and psychiatric disorders. However, a better understanding of its effects at the individual neuron level is essential to improve its clinical application. We combined focal low-intensity repetitive transcranial magnetic stimulation (LI-rTMS) to the rat somatosensory cortex with intracellular recordings of subjacent pyramidal neurons in vivo. Continuous 10 Hz LI-rTMS reliably evoked firing at ∼4-5 Hz during the stimulation period and induced durable attenuation of synaptic activity and spontaneous firing in cortical neurons, through membrane hyperpolarization and a reduced intrinsic excitability. However, inducing firing in individual neurons by repeated intracellular current injection did not reproduce the effects of LI-rTMS on neuronal properties. These data provide a novel understanding of mechanisms underlying magnetic brain stimulation showing that, in addition to inducing biochemical plasticity, even weak magnetic fields can activate neurons and enduringly modulate their excitability. KEY POINTS: Repetitive transcranial magnetic stimulation (rTMS) is a promising technique to alleviate neurological and psychiatric disorders caused by alterations in cortical activity. Our knowledge of the cellular mechanisms underlying rTMS-based therapies remains limited. We combined in vivo focal application of low-intensity rTMS (LI-rTMS) to the rat somatosensory cortex with intracellular recordings of subjacent pyramidal neurons to characterize the effects of weak magnetic fields at single cell level. Ten minutes of LI-rTMS delivered at 10 Hz reliably evoked action potentials in cortical neurons during the stimulation period, and induced durable attenuation of their intrinsic excitability, synaptic activity and spontaneous firing. These results help us better understand the mechanisms of weak magnetic stimulation and should allow optimizing the effectiveness of stimulation protocols for clinical use.

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

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.

Mature Purkinje cells require the retinoic acid-related orphan receptor-α (RORα) to maintain climbing fiber mono-innervation and other adult characteristics.

Neuronal maturation during development is a multistep process regulated by transcription factors. The transcription factor RORα (retinoic acid-related orphan receptor α) is necessary for early Purkinje cell (PC) maturation but is also expressed throughout adulthood. To identify the role of RORα 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 RORα-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 RORα 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.

Formation and reformation of climbing fibre synapses in the cerebellum: a similar story?

The 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 RORα regulate these processes.

Enhanced survival of wild-type and Lurcher Purkinje cells in vitro following inhibition of conventional PKCs or stress-activated MAP kinase pathways.

Recent 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 δ2 glutamate receptor (GluRδ2). 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 PKCγ 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 GluRδ2 (Lc) mutation.

[Marked for life: synaptic stabilisation leaves an indelible trace on neuronal partners: a question of etiquette for neurons?]. / Faire et refaire une synapse en choisissant un partenaire : une question d'étiquette pour les neurones ?

Recent in vivo and in vitro studies have used the rodent olivocerebellar path to examine developmental synaptogenesis, in particular the mechanisms which permit the simultaneous stabilisation of some synapses and elimination of others. They reveal that while the formation of synapses can take place throughout life, elimination of supernumerary connections is limited to a critical period during development. Synapse elimination involves the strengthening of appropriate synapses and weakening of others, which are subsequently removed. This process is partly dependent on activity and post-synaptic signalling cascades and irreversibly changes each synaptic partner, leaving a trace of previous connectivity. Here we discuss the nature of this << trace >>, which may be a molecule that labels and protects those synapses to be retained, and its functional significance in maintaining the specificity of neuronal circuits needed for coordinate behaviour. double dagger.

Synapse elimination in olivo-cerebellar explants occurs during a critical period and leaves an indelible trace in Purkinje cells.

During developmental synaptogenesis, the pre- and postsynaptic cells undergo specific interactions that lead to the establishment of the mature circuit. We have studied the roles of the pre- and postsynaptic cells in establishing this mature innervation by using an in vitro model of synaptic development. We describe climbing fiber (CF)-Purkinje cell (PC) synaptogenesis in cultured mouse hindbrain explants and show that synaptic competition occurs during early development in vitro. By manipulating the maturation stage of each of the synaptic partners in a coculture experimental paradigm, we found that multi-innervation does not occur when both synaptic partners are mature and have already experienced synapse elimination; in contrast, mature PCs can be multi-innervated when they have never experienced synapse elimination and/or when CFs are immature. However in these cases, the normal process of synapse elimination is impaired. These results show that CF-synapse elimination occurs only during a PC-dependant critical period and triggers indelible signals that prevent synapse competition in the mature system.

Normal adult climbing fiber monoinnervation of cerebellar Purkinje cells in mice lacking MHC class I molecules.

Some immune system proteins have recently been implicated in the development and plasticity of neuronal connections. Notably, proteins of the major histocompatibility complex 1 (MHC class 1) have been shown to be involved in synaptic plasticity in the hippocampus and the development of projection patterns in the visual system. We examined the possible role for the MHC class 1 proteins in one well-characterized example of synaptic exuberance and subsequent refinement, the climbing fiber (CF) to Purkinje cell (PC) synapse. Cerebella from adult mice deficient for two MHC genes, H2-D1 and H2-K1, and for beta2-microglobulin gene were examined for evidence of deficient elimination of supernumerary CF synapses on their PCs. Electrophysiological and morphological evidence showed that, despite the absence of these MHC class 1 molecules, adult PCs in these transgenic mice are monoinnervated as in wild-type animals. These findings indicate that, at the level of restriction of afferent number at this synapse, functional MHC class 1 proteins are not required.

Participation of low-threshold Ca2+ spike in the Purkinje cells complex spike.

In Purkinje cells from cerebellar slice cultures, low-threshold Ca spike (LTS) gives rise to complex bursts in the soma that resemble the complex spike induced by climbing fibers stimulation. We show that LTS is reduced by T-type and R-type Ca channel blockers (SNX-482, nickel, or mibefradil). We propose that LTS is generated by openings of T-type Ca channels (alpha-1G and/or alpha-1I subunits) and R-type Ca channels (alpha-1E subunit isoforms with a weak sensitivity to SNX-482 and to nickel). Using mibefradil we show that climbing fiber stimulation activates LTS, which contributes to the shape of the response. This Ca entry may be involved in Ca-dependent synaptic plasticity of the parallel fiber input induced by climbing fiber activation.

BDNF increases homotypic olivocerebellar reinnervation and associated fine motor and cognitive skill.

Recovery of complex neural function after injury to the adult CNS is limited by minimal spontaneous axonal regeneration and/or sprouting from remaining pathways. In contrast, the developing CNS displays spontaneous reorganization following lesion, in which uninjured axons can develop new projections to appropriate target neurons and provide partial recovery of complex behaviours. Similar pathways can be induced in the mature CNS, providing models to optimize post-injury recovery of complex neural functions. After unilateral transection of a developing olivocerebellar path (pedunculotomy), remaining inferior olivary axons topographically reinnervate the denervated hemicerebellum and compensate functional deficits. Brain-derived neurotrophic factor (BDNF) partly recreates such reinnervation in the mature cerebellum. However the function of this incomplete reinnervation and any unwanted behavioural effects of BDNF remain unknown. We measured olivocerebellar reinnervation and tested rotarod and navigation skills in Wistar rats treated with BDNF/vehicle and pedunculotomized on day 3 (Px3; with reinnervation) or 11 (Px11; without spontaneous reinnervation). BDNF treatment did not affect motor or spatial behaviour in normal (control) animals. Px11-BDNF animals equalled controls on the rotarod, outperforming Px11-vehicle animals. Moreover, Px3-BDNF and Px11-BDNF animals achieved spatial learning and memory tasks as well as controls, with Px11-BDNF animals showing better spatial orientation than Px11-vehicle counterparts. BDNF slightly increased olivocerebellar reinnervation in Px3 animals and induced sparse (22% Purkinje cells) yet widespread reinnervation in Px11 animals. As reinnervation correlated with spatial function, these data imply that after injury even a small amount of reinnervation that is homotypic to correct target neurons compensates deficits in appropriate complex motor and spatial skills. As there was no effect in control animals, BDNF effectively induces this axon collateralisation without interfering with normal neuronal circuits.

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.

Afferent-target interactions during olivocerebellar development: transcommissural reinnervation indicates interdependence of Purkinje cell maturation and climbing fibre synapse elimination.

We have used a model of postlesional reinnervation to observe the interactions between synaptic partners during neosynaptogenesis to determine how the developmental states of the pre- and postsynaptic cells influence circuit maturation. After unilateral transection of the neonatal rat olivocerebellar pathway (pedunculotomy), axons from the remaining ipsilateral inferior olive grow into the denervated hemicerebellum and develop climbing fibre (CF) terminal arbors on Purkinje cells (PCs) at a later stage of development than normal. However, the significance of delayed CF-PC interactions on subsequent circuit maturation remains poorly defined. To examine this question, we recorded CF-induced currents in PCs and analysed PC morphology during the first two postnatal weeks in control animals and following left unilateral inferior cerebellar pedunculotomy on postnatal day (P)3. Our results show that transcommissural olivary axons multiply-reinnervate PCs in the denervated hemisphere over 4 days following pedunculotomy. Each PC received fewer CFs than did age-matched controls and the maximal multi-reinnervation was reached on P7, 2 days later than in controls. Consequently, the onset of CF synapse elimination in reinnervated PCs was delayed, but then proceeded in parallel with controls so that all PCs were monoinnervated by P15. Furthermore, reinnervated PCs had delayed dendritic maturation and subsequent dendritic abnormalities consistent with the role of CF innervation in PC dendritic growth. Thus, within the olivocerebellar system, our data suggest that target neurons depend upon sufficient afferent investment arriving at the correct time for their normal development, and maturation of the target neuron regulates afferent selection and therefore circuit maturation.

Developmental modifications of olivocerebellar topography: the granuloprival cerebellum reveals multiple routes from the inferior olive.

Correct function of neural circuits depends on highly organized neuronal connections, refined from less precise projections through synaptic elimination, collateral regression, or neuronal death. We examined regressive phenomena that define olivocerebellar topography during maturation from Purkinje cell polyinnervation to monoinnervation. We used bilateral retrograde tracing to determine the source of olivocerebellar afferents to posterior vermis lobules VII-VIII in a model of retained immature Purkinje cell polyinnervation, the granuloprival cerebellum. In controls, labelled neurons were found only in the contralateral inferior olive (ION) clustered in a small ventromedial locus that is congruent with known olivocerebellar topography. In granuloprival animals, olivary labelling appeared more dispersed and was present in homologous ipsilateral regions. Double-labelled neurons were never seen. Retrograde tracing following unilateral olivocerebellar transection in adult granuloprival rats revealed: 1) the origin of the normal (remaining) path projecting through the contralateral inferior peduncle was more localized than in irradiated nonpedunculotomized rats, 2) a small double-crossed path, and 3) a projection that ascends the peduncle ipsilateral to the ION of origin, part of which crosses the midline within the cerebellum. Electrophysiological and immunohistochemical assessment in the neonatal cerebellum revealed that transcommissural paths are not present during development but sprout within the irradiated cerebellum. Therefore, the olivocerebellar projection in the granuloprival rat, as a model of the immature path, shows parasagittal organization similar to that of controls in its normally crossed path but possesses additional abnormal projections. Thus, maturation of olivocerebellar topography involves removal of whole developmental paths to define laterality plus synapse elimination within largely predefined parasagittal zones.

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.

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 synchronization.

Lurcher GRID2-induced death and depolarization can be dissociated in cerebellar Purkinje cells.

The Lurcher mutation transforms the GRID2 receptor into a constitutively opened channel. In Lurcher heterozygous mice, cerebellar Purkinje cells are permanently depolarized, a characteristic that has been thought to be the primary cause of their death, which occurs from the second postnatal week onward. The more dramatic phenotype of Lurcher homozygotes is thought to be due to a simple gene dosage effect of the mutant allele. We have analyzed the phenotype of Lurcher/hotfoot heteroallelic mutants bearing only one copy of the Lurcher allele and no wild-type Grid2. Our results show that the absence of wild-type GRID2 receptors in these heteroallelic mutants induces an early and massive Purkinje cell death that is correlated with early signs of autophagy. This neuronal death is independent of depolarization and can be explained by the direct activation of autophagy by Lurcher GRID2 receptors through the recently discovered signaling pathway formed by GRID2, n-PIST, and Beclin1.