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Stem cell lines that faithfully maintain the regional identity and developmental potency of progenitors in the human brain would create new opportunities in developmental neurobiology and provide a resource for generating specialized human neurons. However, to date, neural progenitor cultures derived from the human brain have either been short-lived or exhibit restricted, predominantly glial, differentiation capacity. Pluripotent stem cells are an alternative source, but to ascertain definitively the identity and fidelity of cell types generated solely in vitro is problematic. Here, we show that hindbrain neuroepithelial stem (hbNES) cells can be derived and massively expanded from early human embryos (week 5-7, Carnegie stage 15-17). These cell lines are propagated in adherent culture in the presence of EGF and FGF2 and retain progenitor characteristics, including SOX1 expression, formation of rosette-like structures, and high neurogenic capacity. They generate GABAergic, glutamatergic and, at lower frequency, serotonergic neurons. Importantly, hbNES cells stably maintain hindbrain specification and generate upper rhombic lip derivatives on exposure to bone morphogenetic protein (BMP). When grafted into neonatal rat brain, they show potential for integration into cerebellar development and produce cerebellar granule-like cells, albeit at low frequency. hbNES cells offer a new system to study human cerebellar specification and development and to model diseases of the hindbrain. They also provide a benchmark for the production of similar long-term neuroepithelial-like stem cells (lt-NES) from pluripotent cell lines. To our knowledge, hbNES cells are the first demonstration of highly expandable neuroepithelial stem cells derived from the human embryo without genetic immortalization.

By serving as the sole output of the cerebellar cortex, integrating a myriad of afferent stimuli, Purkinje cells (PCs) constitute the principal neuron in cerebellar circuits. Several neurodegenerative cerebellar ataxias feature a selective cell-autonomous loss of PCs, warranting the development of regenerative strategies. To date, very little is known as to the regulatory cascades controlling PC development. During central nervous system development, the proneural gene neurogenin 2 (Neurog2) contributes to many distinct neuronal types by specifying their fate and/or dictating development of their morphological features. By analyzing a mouse knock-in line expressing Cre recombinase under the control of Neurog2 cis-acting sequences we show that, in the cerebellar primordium, Neurog2 is expressed by cycling progenitors cell-autonomously fated to become PCs, even when transplanted heterochronically. During cerebellar development, Neurog2 is expressed in G1 phase by progenitors poised to exit the cell cycle. We demonstrate that, in the absence of Neurog2, both cell-cycle progression and neuronal output are significantly affected, leading to an overall reduction of the mature cerebellar volume. Although PC fate identity is correctly specified, the maturation of their dendritic arbor is severely affected in the absence of Neurog2, as null PCs develop stunted and poorly branched dendrites, a defect evident from the early stages of dendritogenesis. Thus, Neurog2 represents a key regulator of PC development and maturation.

In most CNS regions, the variety of inhibitory interneurons originates from separate pools of progenitors residing in discrete germinal domains, where they become committed to specific phenotypes and positions during their last mitosis. We show here that GABAergic interneurons of the rodent cerebellum are generated through a different mechanism. Progenitors for these interneurons delaminate from the ventricular neuroepithelium of the embryonic cerebellar primordium and continue to proliferate in the prospective white matter during late embryonic and postnatal development. Young postmitotic interneurons do not migrate immediately to their final destination, but remain in the prospective white matter for several days. The different interneuron categories are produced according to a continuous inside-out positional sequence, and cell identity and laminar placement in the cerebellar cortex are temporally related to birth date. However, terminal commitment does not occur while precursors are still proliferating, and postmitotic cells heterochronically transplanted to developing cerebella consistently adopt host-specific phenotypes and positions. However, solid grafts of prospective white matter implanted into the adult cerebellum, when interneuron genesis has ceased, produce interneuron types characteristic of the donor age. Therefore, specification of cerebellar GABAergic interneurons occurs through a hitherto unknown process, in which postmitotic neurons maintain broad developmental potentialities and their phenotypic choices are dictated by instructive cues provided by the microenvironment of the prospective white matter. Whereas in most CNS regions the repertoire of inhibitory interneurons is produced by recruiting precursors from different origins, in the cerebellum it is achieved by creating phenotypic diversity from a single source.

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.

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.

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.

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.

Structural remodeling or repair of neural circuits depends on the balance between intrinsic neuronal properties and regulatory cues present in the surrounding microenvironment. These processes are also influenced by experience, but it is still unclear how external stimuli modulate growth-regulatory mechanisms in the central nervous system. We asked whether environmental stimulation promotes neuronal plasticity by modifying the expression of growth-inhibitory molecules, specifically those of the extracellular matrix. We examined the effects of an enriched environment on neuritic remodeling and modulation of perineuronal nets in the deep cerebellar nuclei of adult mice. Perineuronal nets are meshworks of extracellular matrix that enwrap the neuronal perikaryon and restrict plasticity in the adult CNS. We found that exposure to an enriched environment induces significant morphological changes of Purkinje and precerebellar axon terminals in the cerebellar nuclei, accompanied by a conspicuous reduction of perineuronal nets. In the animals reared in an enriched environment, cerebellar nuclear neurons show decreased expression of mRNAs coding for key matrix components (as shown by real time PCR experiments), and enhanced activity of matrix degrading enzymes (matrix metalloproteinases 2 and 9), which was assessed by in situ zymography. Accordingly, we found that in mutant mice lacking a crucial perineuronal net component, cartilage link protein 1, perineuronal nets around cerebellar neurons are disrupted and plasticity of Purkinje cell terminal is enhanced. Moreover, all the effects of environmental stimulation are amplified if the afferent Purkinje axons are endowed with enhanced intrinsic growth capabilities, induced by overexpression of GAP-43. Our observations show that the maintenance and growth-inhibitory function of perineuronal nets are regulated by a dynamic interplay between pre- and postsynaptic neurons. External stimuli act on this interaction and shift the balance between synthesis and removal of matrix components in order to facilitate neuritic growth by locally dampening the activity of inhibitory cues.