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Gastrointestinal stromal tumor (GIST), originating from the interstitial cells of Cajal (ICC), is characterized by frequent activating mutations of the KIT receptor tyrosine kinase. Despite the clinical success of imatinib, which targets KIT, most patients with advanced GIST develop resistance and eventually die of the disease. The ETS family transcription factor ETV1 is a master regulator of the ICC lineage. Using mouse models of Kit activation and Etv1 ablation, we demonstrate that ETV1 is required for GIST initiation and proliferation in vivo, validating it as a therapeutic target. We further uncover a positive feedback circuit where MAP kinase activation downstream of KIT stabilizes the ETV1 protein, and ETV1 positively regulates KIT expression. Combined targeting of ETV1 stability by imatinib and MEK162 resulted in increased growth suppression in vitro and complete tumor regression in vivo. The combination strategy to target ETV1 may provide an effective therapeutic strategy in GIST clinical management. Significance: ETV1 is a lineage-specific oncogenic transcription factor required for the growth and survival of GIST. We describe a novel strategy of targeting ETV1 protein stability by the combination of MEK and KIT inhibitors that synergistically suppress tumor growth. This strategy has the potential to change first-line therapy in GIST clinical management. Cancer Discov; 5(3); 304–15. ©2015 AACR. See related commentary by Duensing, p. 231 This article is highlighted in the In This Issue feature, p. 213

Rationale Prior research has accumulated a substantial amount of evidence on the ability of cocaine to produce short- and long-lasting molecular and structural plasticity in the corticostriatal-limbic circuitry. However, traditionally, the cerebellum has not been included in the addiction circuitry, even though growing evidence supports its involvement in the behavioural changes observed after repeated drug experiences. Objectives In the present study, we explored the ability of seven cocaine administrations to alter plasticity in the cerebellar vermis. Methods After six cocaine injections, one injection every 48 h, mice remained undisturbed for 1 month in their home cages. Following this withdrawal period, they received a new cocaine injection of a lower dose. Locomotion, behavioural stereotypes and several molecular and structural cerebellar parameters were evaluated. Results Cerebellar proBDNF and mature BDNF levels were both enhanced by cocaine. The high BDNF expression was associated with dendritic sprouting and increased terminal size in Purkinje neurons. Additionally, we found a reduction in extracellular matrix components that might facilitate the subsequent remodelling of Purkinje-nuclear neuron synapses. Conclusions Although speculative, it is possible that these cocaine-dependent cerebellar changes were incubated during withdrawal and manifested by the last drug injection. Importantly, the present findings indicate that cocaine is able to promote plasticity modifications in the cerebellum of sensitised animals similar to those in the basal ganglia. This work was supported by grants and fellowships: Ministerio de Economía y Competitividad [PSI2011- 29181], FPI-PREDOC2009/05, FPU12/04059, PPF 2013 (13I087.01/1) and UJI (P1.1B2011-42).

© 2015 the authors. Cerebellar GABAergic interneurons in mouse comprise multiple subsets of morphologically and neurochemically distinct phenotypes located at strategic nodes of cerebellar local circuits. These cells are produced by common progenitors deriving from the ventricular epithelium during embryogenesis and from the prospective white matter (PWM) during postnatal development. However, it is not clear whether these progenitors are also shared by other cerebellar lineages and whether germinative sites different from the PWM originate inhibitory interneurons. Indeed, the postnatal cerebellum hosts another germinal site along the Purkinje cell layer (PCL), in which Bergmann glia are generated up to first the postnatal weeks, which was proposed to be neurogenic. Both PCL and PWM comprise precursors displaying traits of juvenile astroglia and neural stem cell markers. First, we examine the proliferative and fate potential of these niches, showing that different proliferative dynamics regulate progenitor amplification at these sites. In addition, PCL and PWM differ in the generated progeny. GABAergic interneurons are produced exclusively by PWM astroglial-like progenitors, whereas PCL precursors produce only astrocytes. Finally, through in vitro, ex vivo, and in vivo clonal analyses we provide evidence that the postnatal PWM hosts a bipotent progenitor that gives rise to both interneurons and white matter astrocytes.

The different cerebellar phenotypes are generated according to a precise time schedule during embryonic and postnatal development. To assess whether the differentiative potential of cerebellar progenitors is progressively restricted in space and time we examined the fate of embryonic day 12 (E12) or postnatal day 4 (P4) cerebellar cells after heterotopic-heterochronic transplantation into the embryonic rat brain in utero or into organotypic CNS explants in vitro. Donor cells, isolated from transgenic mice overexpressing the enhanced-green fluorescent protein under the control of the beta-actin-promoter, engrafted throughout the host brainstem and diencephalon, whereas they rarely incorporated into specific telencephalic structures. In any recipient site, the vast majority of transplanted cells could be recognized as cerebellar phenotypes, and we did not obtain clear evidence that ectopically located cells adopted host-specific identities. Nevertheless, the two donor populations displayed different developmental potentialities. P4 progenitors exclusively generated granule cells and molecular layer interneurons, indicating that they are committed to late-generated cerebellar identities and not responsive to heterotopic-heterochronic environmental cues. In contrast, E12 precursors had the potential to produce all major cerebellar neurons, but the repertoire of adult phenotypes generated by these cells was different in distinct host regions, suggesting that they require instructive environmental information to acquire mature identities. Thus, cerebellar precursors are able to integrate into different foreign brain regions, where they develop mature phenotypes that survive long after transplantation, but they are committed to cerebellar fates at E12. Embryonic progenitors are initially capable, although likely not competent, to generate all cerebellar identities, but their potential is gradually restricted toward late-generated phenotypes.

There is a pressing need for treatments for neurodegenerative diseases. Hopes have been raised by the prospect of neural stem cell therapy; however, despite intense research activities and media attention, stem cell therapy for neurological disorders is still a distant goal. Effective strategies must be developed to isolate, enrich and propagate homogeneous populations of neural stem cells, and to identify the molecules and mechanisms that are required for their proper integration into the injured brain. This article examines these requirements, discusses the results obtained so far, and considers the steps that need to be taken to provide instruction to donor cells and to elucidate the neurogenic potential of the adult central nervous system environment.

The reestablishment of topographically organized connections is a necessary prerequisite to obtain a full anatomical repair following brain injury. One system where such an issue can be addressed is the olivocerebellar system, where, normally, clusters of inferior olive neurons project to neurochemically heterogeneous Purkinje cell compartments defined by the expression of cell-specific markers, such as zebrin II. To assess whether adult injured olivocerebellar axons that regenerate into cerebellar transplants are able to establish target-specific innervation of grafted Purkinje cells, we made surgical transections in the white matter of adult rat cerebella and placed solid grafts from the embryonic cerebellar anlage into the lesion site. The transplanted tissue developed highly organized minicerebella, in which Purkinje cells were distributed into distinct clusters of zebrin II-immunopositive or -immunonegative neurons, mimicking the cortical compartments present in the normal adult cerebellum. Olivocerebellar axons, labeled by biotinylated dextran amine tracing, regenerated into the transplants where they formed discrete patches made of several terminal arbors impinging upon Purkinje cell dendrites. Among 401 such climbing fiber patches, 96% exclusively innervated Purkinje cells of either phenotype and stopped at the border of the zebrin II+/− Purkinje cell clusters, whereas only 4% were extended across this boundary and innervated both zebrin II-positive and -negative Purkinje cells. The results obtained support the view that the embryonic cerebellar tissue provides target-specific information that can be decoded by ingrowing adult olivocerebellar axons in order to establish appropriate innervation patterns with zebrin II-positive or -negative Purkinje cell compartments.

The retinoblastoma family proteins pRB, p107, and p130 are phosphorylated and released from E2Fs in the late G(1) phase of the cell cycle. This phosphorylation is thought to contribute to the derepression of E2F-responsive genes and to be mediated, in part, by Cdk4 and Cdk6. Evidence that Cdk4/6 activity is inhibited by p16(INK4A) in most pRB(-) cells suggests that p107 and p130 may be underphosphorylated and remain associated with E2Fs during G(1)-S progression in cells that lack pRB. To examine this, we evaluated the cell cycle-dependent phosphorylation and E2F binding abilities of p107 and p130 in pRB(-), p16(+) Saos-2 osteosarcoma cells. p130, but not p107, was phosphorylated and released from E2F-4 in late G(1) and S phase cells, although p130 phosphorylation differed qualitatively in these and other pRB(-), p16(+) cells as compared with pRB(+), p16(-) cell types. p130 phosphorylation occurred in the absence of cyclin D-Cdk4/6 complexes, coincided with cyclin E- and Cdk2-associated kinase activity, and was prevented by expression of dominant negative Cdk2. Moreover, dominant negative Cdk2 prevented the dissociation of endogenous p130-E2F-4 complexes and inhibited E2F-4-dependent transcription. These findings show that p130 can be phosphorylated and functionally inactivated in a Cdk2-dependent process, and they highlight the involvement of distinct Cdks in the regulation of different pRB family proteins.

The myelin-associated proteins NI-35/250 exert a powerful inhibition on axon regeneration, but their function exerted on intact neurons is still unclear. In the adult CNS these proteins are thought to regulate axon growth processes to confine plasticity within restricted regions and to prevent the formation of aberrant connections. We have recently shown that application of neutralizing IN-1 antibody Fab fragment against NI-35/250 proteins to the adult cerebellum induces the expression of injury/growth-associated markers in intact Purkinje cells. Here, we asked whether these cellular modifications are accompanied by growth phenomena of Purkinje neurites. A single intraparenchymal application of IN-1 Fab fragment to the adult cerebellum induces a profuse sprouting of Purkinje axons along their intracortical course. The newly formed processes spread to cover most of the granular layer depth. A significant axon outgrowth is evident 2 d after injection; it tends to increase at 5 and 7 d, but it is almost completely reversed after 1 month. No axonal modifications occur in control Fab-treated cerebella. The IN-1 Fab fragment-induced cellular changes and axon remodeling are essentially reproduced by applying affinity-purified antibody 472 raised against a peptide sequence of the recombinant protein NI-220, thus confirming the specificity of the applied treatments on these myelin-associated molecules. Functional neutralization of NI-35/250 proteins induces outgrowth from uninjured Purkinje neurites in the adult cerebellum. Together with previous observations, this suggests that these molecules regulate axonal plasticity to maintain the proper targeting of terminal arbors within specific gray matter regions.

Inferior olive neurons are able to enlarge or retract their axonic terminal fields in response to changes in the extension of their target domain. Following Purkinje cell loss, the retraction of target-deprived climbing fibres is accompanied by a size reduction in the inferior olive neuron cell bodies. Here, we asked whether perikaryal modifications also occur when inferior olivary neurons enlarge their terminal fields to innervate supernumerary targets. To achieve this aim, we carried out a morphometric analysis on the somatic compartment of inferior olive neurons in two experimental conditions known to induce an expansion of their terminal field, i.e. a subtotal 3-acetylpyridine inferior olive lesion in the adult and a unilateral transection of the inferior cerebellar peduncle in newborn rats. In both experimental conditions, the inferior olive neurons that survived the lesion showed a remarkable increase in cell body and nuclear size, although the latter change was less pronounced in the 3-acetylpyridine-treated animals. These results show that both developing and mature inferior olive neurons are capable of adjusting their perikaryal phenotype to match the modifications of their target size.

Following injury to the adult mammalian cochlea, hair cells cannot be spontaneously replaced. Nonetheless, the postnatal cochlea contains progenitor cells, distinguished by the expression of nestin, which are able to proliferate and form neurospheres in vitro. Such resident progenitors might be endowed with reparative potential. However, to date little is known about their behaviour in situ following hair cell injury. Using adult mice and ex vivo cochlear cultures, we sought to determine whether: (i) resident cochlear progenitors respond to kanamycin ototoxicity and compensate for it; and (ii) the reparative potential of cochlear progenitors can be stimulated by the addition of growth factors. Morphological changes of cochlear tissue, expression of nestin mRNA and protein and cell proliferation were investigated in these models. Our observations show that ototoxic injury has modest effects on nestin expression and cell proliferation. On the other hand, the addition of growth factors to the injured cochlear explants induced the appearance of nestin-positive cells in the supporting cell area of the organ of Corti. The vast majority of nestin-expressing cells, however, were not proliferating. Growth factors also had a robust stimulatory effect on axonal sprouting and the proliferative response, which was more pronounced in injured cochleae. On the whole, our findings indicate that nestin expression after kanamycin ototoxicity is related to tissue reactivity rather than activation of resident progenitors attempting to replace the lost receptors. In addition, administration of growth factors significantly enhances tissue remodelling, suggesting that cochlear repair may be promoted by the exogenous application of regeneration-promoting substances.

The ability of Schwann cells to induce the regeneration of severed olivocerebellar and Purkinje cell axons across an injury up to their deafferented targets was tested by transplanting freshly dissociated cells from newborn rat sciatic nerves into surgically lesioned adult cerebella. The grafted glial cells consistently filled the lesion gap and migrated into the host parenchyma. Transected olivocerebellar axons vigorously regenerated into the graft, where their growth pattern and direction followed the arrangement of Schwann cell bundles. Although some of these axons terminated within the transplant, many of them rejoined the cerebellar parenchyma beyond the lesion. Here, their fate depended on the territory encountered. No growth occurred in the white matter. Numerous fibres penetrated into the granular layer and formed terminal branches that remained confined within this layer. A few of them, however, regenerated up to the molecular layer and formed climbing fibres on Purkinje cell dendrites. By contrast, the growth of transected Purkinje cell axons into the grafts was very poor. These results underscore the different intrinsic responsiveness of Purkinje cell and olivocerebellar axons to the growth-promoting action of Schwann cells, and show that the development and outcome of the regenerative phenomena is strongly conditioned by the spatial organization and specific features of the environmental cues encountered by the outgrowing axons along the course they follow. However, Schwann cells effectively bridge the lesion gap, induce the regeneration of olivocerebellar axons, and direct their growth up to the deafferented host cortex, where some of them succeed in reinnervating their natural targets.

It is unclear whether reparative processes in the injured mammalian brain are able to restore the topographic organisation of neuronal connections. To address this question, we have investigated the plasticity of the olivocerebellar system. This pathway has a precise topographic arrangement, in which subsets of inferior olivary neurons project to parasagittally oriented Purkinje cell compartments. Following unilateral transection of the inferior cerebellar peduncle in newborn rats, axons from the contralateral projection cross the cerebellar midline and reinnervate the deafferented hemicerebellum. By this experimental approach, we first analysed the behaviour of calcitonin gene-related peptide (CGRP)-immunoreactive climbing fibres. This marker is transiently expressed by a subset of developing inferior olivary axons, which terminate in the cerebellar cortex into several parasagittal strips. We show that transcommissural axons reestablish the original pattern of climbing fibre bands within a few days after lesion. Then, in adult animals injured at birth, we assessed whether the newly formed climbing fibre bands align with zebrin II+/- Purkinje cell compartments, as in normal conditions. The newly formed projection is organised in parasagittally oriented strips which mirror the distribution of their counterparts on the intact side and are precisely aligned to the heterogeneous Purkinje cell compartments. In addition, the patchy distribution of olivo-nuclear fibres suggests that specific reinnervation is also achieved in the deep nuclei. Thus, transcommissural olivocerebellar reinnervation is not random, but it is regulated by selective interactions between distinct subsets of olivocerebellar axons and target neurons aimed at reestablishing the correct projection map.

Neurons in mammals do not undergo replicative aging, and, in absence of pathologic conditions, their lifespan is limited only by the maximum lifespan of the organism. Whether neuronal lifespan is determined by the strain-specific lifetime or can be extended beyond this limit is unknown. Here, we transplanted embryonic mouse cerebellar precursors into the developing brain of the longer-living Wistar rats. The donor cells integrated into the rat cerebellum developing into mature neurons while retaining mouse-specific morphometric traits. In their new environment, the grafted mouse neurons did not die at or before the maximum lifespan of their strain of origin but survived as long as 36 mo, doubling the average lifespan of the donor mice. Thus, the lifespan of neurons is not limited by the maximum lifespan of the donor organism, but continues when transplanted in a longer-living host.

Stroke is a common and disabling global health-care problem, which is the third most common cause of death and one of the main causes of acquired adult disability in many countries. Rehabilitation interventions are a major component of patient care. In the last few years, brain stimulation, mirror therapy, action observation, or mental practice with motor imagery has emerged as interesting options as add-on interventions to standard physical therapies. The neural bases for poststroke recovery rely on the concept of plasticity, namely, the ability of central nervous system cells to modify their structure and function in response to external stimuli. In this review, we will discuss recent noninvasive strategies employed to enhance functional recovery in stroke patients and we will provide an overview of neural plastic events associated with rehabilitation in preclinical models of stroke.

This article reviews a series of experiments aimed at investigating the reciprocal trophic interactions which regulate the normal morphofunctional features and the plasticity of the adult rodent climbing fibre-Purkinje cell system. Climbing fibre deprivation induces profound functional and structural changes in the Purkinje cell. Among others, proximal Purkinje cells dendrites become studded with numerous newly formed spines some of which are innervated by parallel fibres. These structural modifications are reversed if the Purkinje cell is reinnervated by another climbing fibre. These results indicate that the olivocerebellar input inhibits spinogenesis on proximal Purkinje cell dendrites and prevents other afferents from invading its own target domain. It is proposed that the normal distribution of synapses on the Purkinje cell dendritic tree is controlled by the interplay between climbing and parallel fibre influences on Purkinje cell dendrites. Following Purkinje cell death, the distal climbing fibre branches are withdrawn. This atrophy progresses according to the time and mode of Purkinje cell degeneration and it is reversed if the climbing fibre is provided with a new target Purkinje cell. In addition, sprouting from intact climbing fibres and collateral reinnervation of Purkinje cells can be obtained by both subtotal inferior olive lesions and transplantation of embryonic cerebellar tissue on the surface of the adult cerebellum. These results indicate that specific signals produced by non-innervated Purkinje cells are responsible for inducing and guiding climbing fibre sprouting. By contrast, contact cues would be necessary for the formation and the maintenance of terminal arbour branches and synapses. It is suggested that these interactions which control the structural plasticity following lesion or transplantation also operate during the fine structural remodelling underlying the functional plasticity in the intact cerebellar cortex.

In the adult mammalian subventricular zone (SVZ), GFAP-positive neural stem cells (NSCs) generate neuroblasts that migrate tangentially along the rostral migratory stream (RMS) toward the olfactory bulb (OB). In the mouse brain, we found that the plasticity inhibitors Nogo-A and Nogo receptor 1 (NgR1) are differentially expressed in the SVZ–OB system, in which Nogo-A identifies immature neuroblasts and NgR1 germinal astrocytes. We therefore examined the role of Nogo-A and NgR1 in the regulation of neurogenesis. Pharmacological experiments show that Nogo-66/NgR1 interaction reduces the proliferation of NSCs. This is consistent with a negative-feedback loop, in which newly generated neurons modulate cell division of SVZ stem cells. Moreover, the Nogo-A–Δ20 domain promotes neuroblast migration toward the OB through activation of the Rho/ROCK (Rho-associated, coiled-coil containing protein kinase) pathway, without the participation of NgR1. Our findings reveal a new unprecedented function for Nogo-A and NgR1 in the homeostatic regulation of the pace of neurogenesis in the adult mouse SVZ and in the migration of neuroblasts along the RMS.

Background: Current tools for analgesia are often only partially successful, thus investigations of new targets for pain therapy stimulate great interest. Consequent to peripheral nerve injury, c-Jun N-terminal kinase (JNK) activity in cells of the dorsal root ganglia (DRGs) and spinal cord is involved in triggering neuropathic pain. However, the relative contribution of distinct JNK isoforms is unclear. Using knockout mice for single isoforms, and blockade of JNK activity by a peptide inhibitor, we have used behavioral tests to analyze the contribution of JNK in the development of neuropathic pain after unilateral sciatic nerve transection. In addition, immunohistochemical labelling for the growth associated protein (GAP)-43 and Calcitonin Gene Related Peptide (CGRP) in DRGs was used to relate injury related compensatory growth to altered sensory function. Results: Peripheral nerve injury produced pain–related behavior on the ipsilateral hindpaw, accompanied by an increase in the percentage of GAP43-immunoreactive (IR) neurons and a decrease in the percentage of CGRP-IR neurons in the lumbar DRGs. The JNK inhibitor, D-JNKI-1, successfully modulated the effects of the sciatic nerve transection. The onset of neuropathic pain was not prevented by the deletion of a single JNK isoform, leading us to conclude that all JNK isoforms collectively contribute to maintain neuropathy. Autotomy behavior, typically induced by sciatic nerve axotomy, was absent in both the JNK1 and JNK3 knockout mice. Conclusions: JNK signaling plays an important role in regulating pain threshold: the inhibition of all of the JNK isoforms prevents the onset of neuropathic pain, while the deletion of a single splice JNK isoform mitigates established sensory abnormalities. JNK inactivation also has an effect on axonal sprouting following peripheral nerve injury.

The correct function of the nervous system requires complex neural networks bearing precise connections. In principle, the high structural specificity of neural circuits could be achieved by genetically-determined processes, selected and refined during evolution. Highly conserved gene networks regulate some crucial steps of neural development, such as the regionalization of the neural tube and the initial phases of neurogenesis and synaptogenesis. A totally hardwired nervous system may meet the requirements of adaptation and natural selection at the population level. Nevertheless, it would be inadequate to allow individual organisms to cope with rapid changes of environmental conditions. Neural adaptation to external constraints can be partly achieved by introducing selective mechanisms in neural development. Accordingly, neurons are generated in excess and then partially eliminated to match the actual extension of innervation territories. Such mechanisms, however, are restricted to a set of potentialities, which must be predetermined in the ontogenetic program. On the other hand, constructive mechanisms, in which external stimuli directly influence structural modifications of neural circuits to produce adaptive responses, may allow individual organisms to cope with a wide variety of unprecedented situations. Thus, in the last ontogenetic period as well as in the adult, when the organism actively interacts with the external milieu, experience exerts a strong growth-promoting effect on neural circuits and connections inducing the emergence of specific functional properties. By this mechanism, which requires strict inhibitory control to prevent aberrant growth and dysfunction, the nervous system exploits external stimuli to create adaptive responses to unexpected situations.

We have investigated the long-term structural changes which affect Purkinje cells exposed to a single dose of kainic acid. Following intraparenchymal injection of the excitotoxin in the cerebellar cortex (1 microliter of a 1 mg/ml solution), Purkinje cells which survived within the lesioned area or close to its edges showed remarkable axonal abnormalities, involving the formation of torpedoes, hypertrophy of recurrent collaterals and atrophy of the corticofugal portion of the axon. In addition, their dendritic trees were often affected by conspicuous regressive alterations. The climbing fibres contacting these Purkinje cells were characterized by thick perisomatic plexuses, whereas their peridendritic branches were atrophic. The dendrites innervated by such atrophic olivary arbours were studded with huge numbers of newly formed spines. These alterations were already present a few days after kainic acid administration and persisted for the total period of observation of 6 months after the lesion. The remarkable similarity between the abnormalities of Purkinje cells exposed to kainic acid and those observed after axotomy indicates that in these two conditions common mechanisms determine analogous long-lasting modifications in the affected neurons. It is proposed that kainic acid-induced intracellular calcium overload disrupts cytoskeletal components and impairs axonal transport, thus depriving the affected Purkinje cells of retrograde trophic influences from their target neurons. As a consequence the affected neurons undergo long-lasting regressive modifications and compensatory remodelling phenomena.

By grafting solid pieces of cerebellar anlage onto the surface of the adult rat cerebellum, we have investigated the problem of the interactions between embryonic and adult neurons in an intact brain. A few days after grafting, embryonic astrocytic processes crossed the graft--host interface and radiated into the recipient molecular layer. Several grafted Purkinje cells also migrated into the host brain along such processes as well as adult Bergmann glia. Adult climbing fibres, labelled by means of Phaseolus vulgaris leucoagglutinin (PHA-L), sprouted new collateral branches which terminated on embryonic Purkinje cells at both extra- and intraparenchymal levels. No sign of activation of host astroglia or microglia was evident in the host cerebellum in relation to these processes. Embryonic Purkinje cells which migrated into the host cerebellum developed an adult-like morphology. Intraparenchymal grafts of neocortical embryonic tissue induced conspicuous growth of host olivary axons, characterized by a pattern which was different from that observed following cerebellar grafts. By contrast, when neocortical tissue was placed onto the surface of the recipient cerebellum, graft--host interactions were limited and climbing fibre sprouting was rarely seen. These results show that (i) supernumerary Purkinje cells can penetrate and settle in the adult intact cerebellar cortex, (ii) adult climbing fibres are able to innervate these new targets in the absence of any injury or activation of non-neuronal cells of the adult brain, and (iii) in the absence of damage to the adult brain, the plasticity of adult olivary axons is specifically elicited and controlled by embryonic Purkinje cells.

The progenitors of cerebellar GABAergic interneurons proliferate up to postnatal development in the prospective white matter, where they give rise to different neuronal subtypes, in defined quantities and according to precise spatiotemporal sequences. To investigate the mechanisms that regulate the specification of distinct interneuron phenotypes, we examined mice lacking the G1 phase-active cyclin D2. It has been reported that these mice show severe reduction of stellate cells, the last generated interneuron subtype. We found that loss of cyclin D2 actually impairs the whole process of interneuron genesis. In the mutant cerebella, progenitors of the prospective white matter show reduced proliferation rates and enhanced tendency to leave the cycle, whereas young postmitotic interneurons undergo severe delay of their maturation and migration. As a consequence, the progenitor pool is precociously exhausted and the number of interneurons is significantly reduced, although molecular layer interneurons are more affected than those of granular layer or deep nuclei. The characteristic inside-out sequence of interneuron placement in the cortical layers is also reversed, so that later born cells occupy deeper positions than earlier generated ones. Transplantation experiments show that the abnormalities of cyclin D2–/– interneurons are largely caused by cell-autonomous mechanisms. Therefore, cyclin D2 is not required for the specification of particular interneuron subtypes. Loss of this protein, however, disrupts regulatory mechanisms of cell cycle dynamics that are required to determine the numbers of interneurons of different types and impairs their rhythm of maturation and integration in the cerebellar circuitry.

The role of postsynaptic neurons in the maintenance of adult terminal axon arbours was investigated in the rat olivocerebellar system. The degeneration of Purkinje cells, the main target of olivary axons in the cerebellar cortex, was obtained by intraparenchymal application of kainate. The structural features of target-deprived climbing fibres, visualized by Phaseolus vulgaris leucoagglutinin tracing, were examined from two days to six months after the lesion. Following the degeneration of its Purkinje cell, the climbing fibre underwent remarkable regressive modifications involving the disappearance of most of the terminal arborization. Never the less, atrophie arbours still spanned through the molecular layer six months after the lesion. Morphometric evaluations showed that, one week after kainate application, total arbour length was already reduced to 52% of control, whereas the number of branches and of varicosities had both dropped around 40%. This retraction process progressed in the following stages to reach its maximum at about one month after the lesion, when total length was 30% of control and only 10% of branches and varicosities were still present. Only a slight tendency to a further decrease of the values could be detected at longer survival times. Branching pattern analysis revealed that such regressive phenomena mainly involved the distal compartment of the climbing fibres, the one made of fine varicose branchlets, while sparing the proximal thick branches. In addition, the whole process appeared to follow some rather strict guiding principles leading to an ordered branch retraction, from the periphery of the arbour inwards. Finally, in order to rule out the possibility that the observed changes could be due to a direct action of kainate on climbing fibres, we designed an alternative method of killing Purkinje cells by intraparenchymal injection of propidium iodide. The structural features of climbing fibres deprived of their target by such a procedure were very similar to those shown by arbours from time-matched kainate-lesioned animals at both qualitative and quantitative levels. Our results show that target deprivation induces remarkable structural modifications in the climbing fibre, leading to the retraction of most of the arbour. Never the less, the integrity of the Purkinje cell is not necessary for the maintenance of the whole arborization since its proximal compartment is maintained in the molecular layer for several months after target degeneration. It is proposed that the Purkinje cell, most likely by acting through a contact factor, directly controls the formation and the maintenance of the distal climbing fibre branches with their varicosities, which represent the presynaptic compartment of the axonal arbour.

Stem or progenitor cells acquire specific regional identities during early ontogenesis. Nonetheless, there is evidence that cells heterotopically transplanted to neurogenic regions of the developing or mature central nervous system may switch their fate to adopt host-specific phenotypes. Here, we isolated progenitor cells from different germinative sites along the neuraxis where GABAergic interneurons are produced (telencephalic subventricular zone, medial ganglionic eminence, ventral mesencephalon and dorsal spinal cord), and grafted them to the prospective white matter of the postnatal rat cerebellum, at the time when local interneurons are generated. The phenotype acquired by transplanted cells was assessed by different criteria, including expression of region-specific transcription factors, acquisition of morphological and neurochemical traits, and integration in the cerebellar cytoarchitecture. Regardless of their origin, all the different types of donor cells engrafted in the cerebellar parenchyma and developed mature neurons that shared some morphological and neurochemical features with local inhibitory interneurons, particularly in the deep nuclei. Nevertheless, transplanted cells failed to activate cerebellar-specific regulatory genes. In addition, their major structural features, the expression profiles of type-specific markers and the laminar placement in the recipient cortex did not match those of endogenous interneurons generated during the same developmental period. Therefore, although exogenous cells are influenced by the cerebellar milieu and show remarkable capabilities for adapting to the foreign environment, they essentially fail to switch their fate, integrate in the host neurogenic mechanisms and adopt clear-cut cerebellar identities.

Neurons with enhanced intrinsic growth capabilities can elongate their axons into non-permissive territories, but the mechanisms that enable the outgrowing processes to overcome environmental inhibition are largely unknown. To address this issue, we examined adult mouse Purkinje cells that overexpress the axonal growth-associated protein GAP-43. After injury, these neurons exhibit sprouting along the intracortical neuritic course and at the severed stump in the white matter. To determine whether GAP-43-overexpressing Purkinje cells are responsive to extrinsic inhibitory cues, we investigated the content and subcellular localization of major receptors for myelin-associated inhibitory proteins, PlexinB1 and the Nogo receptor (NgR) with the related co-receptors LINGO-1 and p75. Expression of these molecules, estimated by measuring perikaryal immunostaining intensity and Western blot, was not different in wild-type or transgenic mice, and it was not overtly modified after axotomy. Following injury, however, the content of PlexinB1 was significantly reduced in GAP-43-overexpressing neurites. Furthermore, in the same axons the distribution of both PlexinB1 and NgR was altered, being inverse to that of GAP-43. Labelling for the two receptors was conspicuously reduced on the axonal surface and it was almost undetectable in the outgrowing sprouts, which showed strong GAP-43 immunoreactivity. These observations indicate that although GAP-43 overexpression does not modify the expression of receptors for myelin-associated inhibitory factors, it interferes with their subcellular localization and exposure on the neuritic membrane. Therefore, GAP-43 promotes axon growth by multiple synergistic mechanisms that potentiate the intrinsic motility of the elongating processes, while reducing their sensitivity to environmental inhibition.

Background An autoimmune hypothesis has been suggested for a subtype of Obsessive–Compulsive Disorder (OCD) with childhood onset: obsessions, compulsions and/or tics would result from anti-streptococcal antibodies that cross-react with basal ganglia tissue based on molecular mimicry. Consistent with this hypothesis anti-brain antibodies were detected in sera of children with OCD and/or Tourette's syndrome. In the present study, we tested whether adults with OCD have anti-brain antibodies or other antibodies that serve as markers of autoimmunity. Methods Seventy-four DSM-IV OCD (YBOCS ≥ 16) subjects were recruited and compared to 44 controls with a current Major Depressive Episode for neurological symptoms, ALSO titres, anti-tissue and anti-thyroid antibodies. Anti-brain antibodies were tested by immunohistochemistry and Western blotting methods. Results The proportion of subjects with tic comorbidity or positive ASLO titre (> 200 IU/ml) was significantly greater in OCD than in MDE patients (21.6 vs. 2.3% and 16.3 vs. 2.3%, respectively). No other differences in antibody parameters were found. 4/74 OCD patients (5.4%) and none of the controls resulted positive for anti-brain antibodies, with a band around 50–60 kDa at the Western blot analysis. Limitations The methodology used to assess anti-brain antibodies. Conclusions The majority of adult OCD patients do not seem to have autoimmunity disturbances as compared to a control group. However, a greater percentage of subjects with positive ASLO titres were found among OCD patients. For a small proportion of OCD patients, moreover, autoimmune reactions towards neuronal structures are present although further investigations are needed to demonstrate its etiopathogenetic relevance.

A salient feature of the cerebellar Purkinje cells is the highly ordered distribution of their excitatory afferents on the dendritic tree. Climbing fibres synapse exclusively on the proximal dendrites, whereas parallel fibres articulate with the distal branches, the so-called spiny branchlets. This input organization is lost following the removal of climbing fibres. Such denervation results in the formation of a large number of new spines on the proximal dendrites, and these become contacted by sprouting parallel fibres, which thereby extend their domain of innervation. We have previously shown that the climbing fibres surviving a subtotal lesion of the inferior olive sprout and reinnervate neighbouring Purkinje cells. In the present ultrastructural study, we have investigated the features of Purkinje cells reinnervated by sprouting climbing fibres. The objectives were to examine the fine morphology of the newly formed synapses and to determine whether the modifications of Purkinje cell morphology and afferent organization are reversed by this reinnervation. Surviving climbing fibres were labelled by the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) and immunohistochemically visualized by means of the gold-substituted silver peroxidase technique, 2 and 6 months after 3-acetylpyridine lesions of the inferior olive in adult rats. Sprouting climbing fibres and newly formed arborizations were identified in the light microscope, isolated, and cut in serial ultrathin sections for electron microscopic analysis. The labelled boutons belonging to newly formed terminal plexuses exhibited the typical morphological features of climbing fibre terminals, i.e., a high number of round synaptic vesicles and a few small mitochondria. Most frequently they formed asymmetric synapses on stubby thorns protruding from the proximal Purkinje cell dendrites. In some instances, however, the postsynaptic element consisted of long slender spines or spines showing an atypical morphology. A number of labelled boutons was also in contact with the perikarya of reinnervated Purkinje cells, either articulating with spines or synapsing directly on the smooth somatic surface. The proximal dendrites of denervated Purkinje cells were characterized by large numbers of spines, which were frequently postsynaptic to parallel fibres. By contrast, Purkinje cells reinnervated by the sprouting climbing fibres generally showed a lower number of spines on their proximal dendrites, indicating a reversal of this morphological change. The aberrant parallel fibre input was also decreased on reinnervated dendrites or had completely disappeared. Nevertheless, some reinnervated Purkinje cells showed the persistence of some parallel fibre synapses on their proximal dendrites. On occasion, climbing fibre and parallel fibre boutons synapsed on the same spine. These results show that the sprouting of olivocerebellar axons leads to the formation of new contacts on Purkinje cell dendrites, which are almost identical in morphology and location to the normal climbing fibre input. Moreover, this collateral reinnervation substantially reverses the modifications which affect the denervated Purkinje cell dendrites and restores the normal afferent distribution. It is suggested that the interactions which influence the ontogenetic development of these connections are still active during regenerative phenomena in the adult animal.

The cerebellar circuits comprise a limited number of neuronal phenotypes embedded in a defined cytoarchitecture and generated according to specific spatio-temporal patterns. The local GABAergic network is composed of several interneuron phenotypes that play essential roles in information processing by modulating the activity of cerebellar cortical inputs and outputs. A major issue in the study of cerebellar development is to understand the mechanisms that underlie the generation of different interneuron classes and regulate their placement in the cerebellar architecture and integration in the cortico-nuclear network. Recent findings indicate that the variety of cerebellar interneurons derives from a single population of multipotent progenitors whose fate choices are determined by instructive environmental information. Such a strategy, which is unique for the cerebellum along the neuraxis, allows great flexibility in the control of the quality and quantity of GABAergic interneurons that are produced, thus facilitating the adaptive shaping of the cerebellar network to specific functional demands.

During development, Purkinje axons elongate along precise trajectories and acquire stereotypic branching patterns to innervate targets in the deep nuclei and cerebellar cortex. These processes are accomplished through cell-intrinsic mechanisms, whose operation is regulated by environmental signaling cues. Here, we show that Anosmin-1, the protein defective in the X-linked form of Kallmann syndrome, is one among such cues. Anosmin-1, that stimulates axon elongation and branching in the olfactory system, is expressed by Purkinje cells and deep nuclear neurons of the rat cerebellum during the ontogenetic period when Purkinje axons acquire their mature pattern. These neurons also express the putative Anosmin-1 receptor, fibroblast growth factor receptor 1. Application of Anosmin-1 to dissociated cultures of embryonic (embryonic day 17, E17) or postnatal (postnatal day 0, P0) rat cerebellar cells enhances neuritic elongation and exerts a strong promoting action on the budding of collateral branches and on the extension of terminal arbors. Opposite effects are observed when neutralizing anti-Anosmin-1 antibodies are applied to the same cultures. Comparable results are obtained by administering the protein or the blocking antibodies to organotypic cultures of postnatal (P0) rat cerebellum. In P10 cerebellar slices, Anosmin-1 does not enhance the spontaneous regenerative capabilities of severed Purkinje axons, but promotes the terminal outgrowth of injured neurites into embryonic neocortical explants apposed to the axotomy site. Although Anosmin-1 is unable to change the overall intrinsic growth competence of Purkinje cells, it exerts a powerful stimulatory action on the budding and extension of collateral branches and terminal plexus, contributing to the patterning of Purkinje axons.

Ever since the groundbreaking work of Ramon y Cajal, the cerebellar cortex has been recognized as one of the most regularly structured and wired parts of the brain formed by a rather limited set of distinct cells. Its rather protracted course of development, which persists well into postnatal life, the availability of multiple natural mutants, and, more recently, the availability of distinct molecular genetic tools to identify and manipulate discrete cell types have suggested the cerebellar cortex as an excellent model to understand the formation and working of the central nervous system. However, the formulation of a unifying model of cerebellar function has so far proven to be a most cantankerous problem, not least because our understanding of the internal cerebellar cortical circuitry is clearly spotty. Recent research has highlighted the fact that cerebellar cortical interneurons are a quite more diverse and heterogeneous class of cells than generally appreciated, and have provided novel insights into the mechanisms that underpin the development and histogenetic integration of these cells. Here, we provide a short overview of cerebellar cortical interneuron diversity, and we summarize some recent results that are hoped to provide a primer on current understanding of cerebellar biology.

To elucidate the mechanisms that regulate neuronal placement and integration in the cerebellar circuitry, we assessed the fate of Purkinje cells transplanted to embryonic, juvenile and adult hosts, asking how architectural changes of the developing cortex influence their anatomical incorporation. Donor Purkinje cells navigate through the host parenchyma either along their natural migratory pathway or following unusual routes. In the latter case, donor neurons fail to orientate correctly and to establish the cortico-nuclear projection. Purkinje cells that follow the physiological route achieve the typical orientation and connectivity, but end displaced in the molecular layer if their arrival in the recipient cortex is delayed. Navigation routes and final settling of donor neurons vary with host age, depending on the ontogenetic construction of cortical layering, and particularly on the maturation of granule cells. The migratory behavior and homing of transplanted Purkinje cells is modified after external granular layer ablation, or neutralization of reelin signaling produced by granule cells. Therefore, although the cerebellar milieu remains receptive for Purkinje cells even after the end of development, correct placement of donor neurons depends on the timing of their migration, related to cerebellar developmental dynamics and granule cell layering.

In the past few years, genetic fate mapping experiments have changed our vision of cerebellar development, particularly in redefining the origin of gabaergic and glutamatergic neurons of the cerebellar cortex and highlighting the precise spatio-temporal sequence of their generation. Here the authors review cerebellar neurogenesis and discuss the fate mapping studies with other new information stemming from transplantation experiments, in an effort to link the developmental potential of neural progenitor populations of the cerebellum with their spatio-temporal origin. NEUROSCIENTIST 14(1):91—100, 2008.

Fusion of bone marrow-derived cells with adult Purkinje cells in the cerebellum gives rise to binucleated Purkinje cells. Whether fusion can be modulated by epigenetic factors and whether fused neurons are stable has remained unclear. Here, we show that in mice and rats, partial ablation of Purkinje cells and local microglial activation in the absence of structural damage to the cerebellum increase the rate of fusion. Moreover, mouse Purkinje cells once fused with bone marrow-derived cells are viable for at least 7 months. We also show that cerebellar irradiation is unnecessary for the generation of binucleated Purkinje cells after bone marrow grafting. Moreover, binucleated Purkinje cells can be found in aged mice that did not receive any treatment, suggesting that fusion events occasionally occur throughout the whole lifespan of healthy, unmanipulated individuals. However, in aged chimeric mice that, after bone marrow transplant, have the majority of their nucleated blood cells fluorescent, the number of binucleated fluorescent Purkinje cells is two orders of magnitude less than the total number of binucleated Purkinje cells. This suggests that, in the majority of heterokaryons, either the incoming nucleus is quickly inactivated or fusion is not the only way to generate a binucleated Purkinje cell.

Background Plexins are a large family of transmembrane receptors for the Semaphorins, known for their role in the assembly of neural circuitry. More recently, Plexins have been implicated in diverse biological functions, including vascular growth, epithelial tissue morphogenesis and tumour development. In particular, PlexinB1, the receptor for Sema4D, has been suggested to play a role in neural development and in tumour angiogenesis, based on in vitro studies. However, the tissue distribution of PlexinB1 has not been extensively studied and the functional relevance of this receptor in vivo still awaits experimental testing. In order to shed light on PlexinB1 function in vivo, we therefore undertook the genomic targeting of the mouse gene to obtain loss of function mutants. Results This study shows that PlexinB1 receptor and its putative ligand, Sema4D, have a selective distribution in nervous and epithelial tissues during development and in the adult. PlexinB1 and Sema4D show largely complementary cell distribution in tissues, consistent with the idea that PlexinB1 acts as the receptor for Sema4D in vivo. Interestingly, PlexinB1 is also expressed in certain tissues in the absence of Sema4D, suggesting Sema4D independent activities. High expression of PlexinB1 was found in lung, kidney, liver and cerebellum. Mutant mice lacking expression of semaphorin receptor PlexinB1 are viable and fertile. Although the axon collapsing activity of Sema4D is impaired in PlexinB1 deficient neurons, we could not detect major defects in development, or in adult histology and basic functional parameters of tissues expressing PlexinB1. Moreover, in the absence of PlexinB1 the angiogenic response induced by orthotopically implanted tumours was not affected, suggesting that the expression of this semaphorin receptor in endothelial cells is redundant. Conclusion Our expression analysis suggests a multifaceted role of PlexinB1 during mouse development and tissue homeostasis in the adult. Nonetheless, the genetic deletion of PlexinB1 does not result in major developmental defects or clear functional abnormalities. We infer that PlexinB1 plays a redundant role in mouse development and it is not strictly required for tumour induced angiogenesis.

In the last few years Purkinje cells have become a most interesting model to investigate cellular/molecular mechanisms of axon regeneration and plasticity. Adult Purkinje cells are most peculiar for their weak cell body response to axotomy, which is accompanied by a strong resistance to injury and a virtually absolute inability to regenerate severed neurites, even in the presence of favourable environmental conditions. The same neurons show a vigorous intrinsic inclination toward axonal sprouting and structural plasticity, which can be elicited by removing extrinsic growth-inhibitory cues. These features gradually develop during early postnatal life, but the underlying mechanisms and biological significance remain unclear. This article reviews recent studies aimed at addressing these questions with respect to the general issue of brain repair. Indeed, understanding the reasons for the extremely poor regenerative capacity of Purkinje cells will be most important to elucidate basic biological mechanisms of axon regeneration and plasticity, and to promote circuit rewiring in the adult CNS.

Formation and maintenance of a neuronal network is based on a balance between plasticity and stability of synaptic connections. Several molecules have been found to regulate the maintenance of excitatory synapses but nothing is known about the molecular mechanisms involved in synaptic stabilization versus disassembly at inhibitory synapses. Here, we demonstrate that Nogo-A, which is well known to be present in myelin and inhibit growth in the adult CNS, is present in inhibitory presynaptic terminals in cerebellar Purkinje cells at the time of Purkinje cell-Deep Cerebellar Nuclei (DCN) inhibitory synapse formation and is then downregulated during synapse maturation. We addressed the role of neuronal Nogo-A in synapse maturation by generating several mouse lines overexpressing Nogo-A, starting at postnatal ages and throughout adult life, specifically in cerebellar Purkinje cells and their terminals. The overexpression of Nogo-A induced a progressive disassembly, retraction and loss of the inhibitory Purkinje cell terminals. This led to deficits in motor learning and coordination in the transgenic mice. Prior to synapse disassembly, the overexpression of neuronal Nogo-A led to the downregulation of the synaptic scaffold proteins spectrin, spectrin-E and beta-catenin in the postsynaptic neurons. Our data suggest that neuronal Nogo-A might play a role in the maintenance of inhibitory synapses by modulating the expression of synaptic anchoring molecules.

Purkinje cells, the first nerve cells to be described, are still one of the most interesting and useful experimental models to investigate different aspects of neural function. This special issue of The Cerebellum is dedicated to this very special neuron and contains a number of articles covering different topics of Purkinje cell research, from developmental neurobiology to neurophysiology and neuropathology. More than 150 years after their discovery, Purkinje cells remain one of the most popular neurons in the neuroscience community.

Although constitutive neurogenesis exclusively occurs in restricted regions of the adult mammalian brain, resident progenitors can be isolated from many different CNS sites, and neuronal neogeneration can be stimulated in vivo by injury or infusion of growth factors. To ask whether latent compensatory mechanisms, which may be exploited to promote repair processes, are present throughout the CNS, we examined the neurogenic potentialities of the adult rat cerebellum in normal conditions, following injury, and after infusion of growth factors. Degeneration of Purkinje cells was induced by intracerebroventricular administration of the toxin saporin, conjugated to anti-p75 antibodies. In addition, epidermal growth factor and basic fibroblast growth factor, or FGF8, were infused for 2 weeks to either intact or injured animals. In all conditions, proliferating cells were identified from bromodeoxyuridine (BrdU) incorporation. In the unmanipulated cerebellum there were rare dividing cells, mainly represented by NG2-positive presumptive oligodendrocyte precursors. Mitotic activity was strongly enhanced in cortical areas with Purkinje cell degeneration, being mostly sustained by microglia, plus minor fractions of NG2-expressing cells, astrocytes and oligodendrocytes. In contrast, growth factor infusion had a weak effect on both intact and injured cerebella. In all experimental conditions, we never found any BrdU-positive cells coexpressing distinctive markers for immature or differentiated cerebellar neurons. Therefore, although some progenitor cells reside in the adult cerebellum, the local environment, either intact or injured, does not provide efficient cues to direct their differentiation towards neuronal phenotypes. In addition, neurogenic potentialities cannot be induced or boosted by the application of growth factors which are effective in other CNS regions.

Different cerebellar phenotypes are generated according to a precise spatiotemporal schedule, in which projection neurons precede local interneurons. Glutamatergic neurons develop from the rhombic lip, whereas GABAergic neurons originate from the ventricular neuroepithelium. Progenitors in these germinal layers are committed toward specific phenotypes already at early ontogenetic stages. GABAergic interneurons are thought to derive from a subset of ventricular zone cells, which migrate in the white matter and proliferate up to postnatal life. During this period, different interneuron categories are produced according to an inside-out sequence, from the deep nuclei to the molecular layer (we show here that nuclear interneurons are also born during late embryonic and early postnatal days, after glutamatergic and GABAergic projection neurons). To ask whether distinct interneuron phenotypes share common precursors or derive from multiple fate-restricted progenitors, we examined the behavior of embryonic and postnatal rat cerebellar cells heterotopically/heterochronically transplanted to syngenic hosts. In all conditions, donor cells achieved a high degree of integration in the cerebellar cortex and deep nuclei and acquired GABAergic interneuron phenotypes appropriate for the host age and engraftment site. Therefore, contrary to other cerebellar types, which derive from dedicated precursors, GABAergic interneurons are produced by a common pool of progenitors, which maintain their full developmental potentialities up to late ontogenetic stages and adopt mature identities in response to local instructive cues. In this way, the numbers and types of inhibitory interneurons can be set by spatiotemporally patterned signals to match the functional requirements of developing cerebellar circuits.

The generation of cell diversity from undifferentiated progenitors is regulated by interdependent mechanisms, including cell intrinsic programs and environmental cues. This interaction can be investigated by means of heterochronic/heterotopic transplantation, which allows to examine the behaviour of precursor cells in an unusual environment. The cerebellum provides an ideal model to study cell specification, because its neurons originate according to a well-defined timetable and they can be are readily recognised by morphological features and specific markers. Cerebellar progenitors transplanted to the embryonic cerebellum develop fully mature cerebellar neurons, which often integrate in the host circuitry in a highly specific manner. In extracerebellar locations, cerebellar progenitors preferentially settle in caudal CNS regions where they exclusively acquire cerebellar identities. By contrast, neocortical precursors preferentially settle in rostral regions and fail to develop hindbrain phenotypes. The phenotypic repertoire generated by transplanted cerebellar progenitors is strictly dependent on their age. Embryonic progenitors originate all mature cerebellar cells, whereas postnatal ones exclusively generate later-born types, such as molecular layer interneurons and granule cells. Together, these observations foster the hypothesis that neural progenitors are first specified towards region-specific phenotypes along the rostro-caudal axis of the neural tube. Thereafter, the developmental potential of progenitor cells is progressively restricted towards later generated types. Such a progressive specification of precursor cells in space and time is stably transmitted to their progeny and it cannot be modified by local cues, when these cells are confronted with heterotopic and/or heterochronic environments.

The generation of cell diversity from undifferentiated progenitors is regulated by interdependent mechanisms, including cell intrinsic programs and environmental cues. This interaction can be investigated by means of heterochronic/heterotopic transplantation, which allows to examine the behaviour of precursor cells in an ususual environment. The cerebellum provides an ideal model to study cell specification, because its neurons originate according to a well-defined timetable and they can be are readily recognised by morphological features and specific markers. Cerebellar progenitors transplated to the embryonic cerebellum develop fully mature cerebellar neurons, which often integrate in the host circuitry in a highly specific manner. In extracerebellar locations, cerebellar progenitors preferentially settle in caudal CNS regions where they exclusively acquire cerebellar identities. By contrast, neocortical precursors preferentially settle in rostral regions and fail to develop hindbrain phenotypes. The phenotypic repertoire generated by transplanted cerebellar progenitors is strictly dependent on their age. Embryonic progenitors originate all mature cerebellar cells, whereas postnatal ones exclusively generate later-born types, such as molecular layer interneurons and granule cells. Together, these observations foster the hypothesis that neural progenitors are first specified towards region-specific phenotypes along the rostro-caudal axis of the neural tube. Thereafter, the developmental potential of progenitor cells is progressively restricted towrds later generated types. Such a progressive specification of precursor cells in space and time is stably transmitted to their progeny and it cannot be modified by local cues, when these cells are confronted with heterotopic and/or heterochronic environments.

Shc family of adaptor molecules has been demonstrated to play an important role during the transition from proliferating neural stem cells to postmitotic neurons. Previous studies from our group demonstrated a progressive decrease of ShcA levels occurring in coincidence with the end of embryonic neurogenesis and neuronal maturation, being ShcB and ShcC the major Shc molecules expressed in the mature brain. A growing body of evidence indicates that ShcB and ShcC are neuronal specific molecules exerting important roles in neuronal survival and phenotypic stability thus becoming potential attracting target molecules for development of drugs for interfering with brain demises. Here, we examine the expression pattern of ShcB and ShcC in neuronal populations composing the adult central and peripheral nervous system, in order to better elucidate their roles in vivo. We found a heterogeneous and peculiar presence and subcellular localization of ShcB and ShcC in specific neuronal populations, enlightening a potential specific requirement of these two molecules in the survival/maintenance of defined neuronal subtypes.

The last 30 years have seen major advancements in the field of neural transplantation, and several clinical trials have been prompted. In these procedures, fetal cells were grafted to the brain of patients with neurologic disorders, including Parkinson's or Huntington's disease. Despite some controversial results, there is a general agreement that this approach yields significant benefits. However, the application of fetal cell transplantation as a large-scale therapy is seriously hampered by ethical and methodological constraints related to the use of human abortive material. Recently, however, the discovery that stem or progenitor cells reside in adult central nervous system (CNS), and that neurogenic processes persist in some adult brain regions, has disclosed the possibility of promoting regeneration, by mobilizing endogenous elements. Both approaches assume that undifferentiated cells are able to acquire mature phenotypes and become specifically integrated in the adult CNS environment. Stem cells, defined as self-renewing multipotent cells are expected to be a potentially definitive solution to most of these difficulties. The conditions that have to be met to obtain successful cell replacement in the CNS are different in different types of diseases. It is emphasized that cell replacement may repair brain damage, but it does not counteract the underlying pathogenic mechanisms. Mobilization of endogenous progenitors might be a suitable alternative that would circumvent this problem. Embryonic stem cells, derived from the inner cell mass of preimplantation embryos, have the widest developmental potential.

The highly specific connection patterns of the mature CNS are shaped through finely regulated processes of axon growth and retraction. To investigate the relative contribution of cell-autonomous mechanisms and extrinsic cues in these events, we examined the development of Purkinje axon intracortical plexus in the rat cerebellum. During the first postnatal week, several new processes sprout from focal swellings along the initial portion of the Purkinje neurite and spread in the granular layer. Intense structural plasticity occurs during the following week, with pruning of collateral branches and remodeling of terminal arbors. The mature distribution of the Purkinje infraganglionic plexus, confined within the most superficial portion of the granular layer, is attained at approximately postnatal day 15. A similar neuritic branching pattern is also developed by Purkinje cells grown in cultures of dissociated cerebellar cells or transplanted to extracerebellar CNS regions, suggesting that cell-autonomous mechanisms contribute to determining the Purkinje axon phenotype.The structural remodeling of Purkinje intracortical plexus is concomitant with the development of cerebellar myelin. To ask whether myelin-associated factors contribute to the morphological maturation of Purkinje neurites, we prevented normal myelinogenesis by killing oligodendrocyte precursors with 5′-azacytidine or by applying neutralizing antibodies against the myelin-associated neurite growth inhibitor Nogo-A. In both conditions, Purkinje axons retained exuberant branches, and the terminal plexus spanned the entire extent of the granular layer. Thus, the formation of Purkinje axon collaterals is, in part, controlled by intrinsic determinants, but their growth and distribution are regulated by environmental signals, among which are myelin-derived cues.

Successful axon regeneration relies on the capability of the lesioned neurons to up-regulate a specific set of injury/growth-associated genes. In the adult central nervous system, the strength of the cell body response is generally related to the distance of the injury site from the perikaryon, being stronger for proximal lesions. Nevertheless, inferior olive (IO) cells react to injury and regenerate their axons even after distal transections. To investigate the mechanisms that regulate the IO growth properties, we examined the expression of injury/growth markers (nitric oxide synthase, growth-associated protein 43 and c-Jun) after target deletion or axotomy performed at different sites along the olivocerebellar pathway. Both axon injury and target loss disclose two subsets of IO neurons distributed within precise subnuclei: one subset up-regulates all markers in all conditions, whereas the other shows a mild c-Jun expression but remains unresponsive even after a very proximal axotomy. These observations indicate that distinct subpopulations of IO cells respond to different regulatory strategies. Unresponsive neurons appear insensitive to environmental positive or negative cues, suggesting that they are intrinsically unable to set up a cellular reaction to injury. In contrast, cell body changes in reactive neurons are elicited after the removal of retrogradely transported target-derived inhibitory signals. Target loss also induces degeneration of IO cells, whose survival remains partially dependent on Purkinje targets in adulthood. Thus, the intrinsic regenerative potential of a functionally homogeneous population is regulated by multiple mechanisms, specific for distinct neuronal subsets.