Development and evolution of cerebellar neural circuits

The cerebellum controls smooth and skillful movements and it is also involved in higher cognitive and emotional functions. The cerebellum is derived from the dorsal part of the anterior hindbrain and contains two groups of cerebellar neurons: glutamatergic and gamma-aminobutyric acid (GABA)ergic neurons. Purkinje cells are GABAergic and granule cells are glutamatergic. Granule and Purkinje cells receive input from outside of the cerebellum from mossy and climbing fibers. Genetic analysis of mice and zebrafish has revealed genetic cascades that control the development of the cerebellum and cerebellar neural circuits. During early neurogenesis, rostrocaudal patterning by intrinsic and extrinsic factors, such as Otx2, Gbx2 and Fgf8, plays an important role in the positioning and formation of the cerebellar primordium. The cerebellar glutamatergic neurons are derived from progenitors in the cerebellar rhombic lip, which express the proneural gene Atoh1. The GABAergic neurons are derived from progenitors in the ventricular zone, which express the proneural gene Ptf1a. The mossy and climbing fiber neurons originate from progenitors in the hindbrain rhombic lip that express Atoh1 or Ptf1a. Purkinje cells exhibit mediolateral compartmentalization determined on the birthdate of Purkinje cells, and linked to the precise neural circuitry formation. Recent studies have shown that anatomy and development of the cerebellum is conserved between mammals and bony fish (teleost species). In this review, we describe the development of cerebellar neurons and neural circuitry, and discuss their evolution by comparing developmental processes of mammalian and teleost cerebellum.     

Anatomy of zebrafish cerebellum and screen for mutations affecting its development

The cerebellum is important for the integration of sensory perception and motor control, but its structure has mostly been studied in mammals. Here, we describe the cell types and neural tracts of the adult zebrafish cerebellum using molecular markers and transgenic lines. Cerebellar neurons are categorized to two major groups: GABAergic and glutamatergic neurons. The Purkinje cells, which are GABAergic neurons, express parvalbumin7, carbonic anhydrase 8, and aldolase C like (zebrin II). The glutamatergic neurons are vglut1⁺ granule cells and vglut2^high cells, which receive Purkinje cell inputs; some vglut2^high cells are eurydendroid cells, which are equivalent to the mammalian deep cerebellar nuclei. We found olig2⁺ neurons in the adult cerebellum and ascertained that at least some of them are eurydendroid cells. We identified markers for climbing and mossy afferent fibers, efferent fibers, and parallel fibers from granule cells. Furthermore, we found that the cerebellum-like structures in the optic tectum and antero-dorsal hindbrain show similar Parvalbumin7 and Vglut1 expression profiles as the cerebellum. The differentiation of GABAergic and glutamatergic neurons begins 3 days post-fertilization (dpf), and layers are first detectable 5 dpf. Using anti-Parvalbumin7 and Vglut1 antibodies to label Purkinje cells and granule cell axons, respectively, we screened for mutations affecting cerebellar neuronal development and the formation of neural tracts. Our data provide a platform for future studies of zebrafish cerebellar development.     

Induction of excitatory and inhibitory presynaptic differentiation by GluD1

The δ subfamily of ionotropic glutamate receptor subunits consists of GluD1 and GluD2. GluD2, which is selectively expressed in cerebellar Purkinje neurons, has been shown to contribute to the formation of synapses between granule neurons and Purkinje neurons through interaction with Cbln1 (cerebellin precursor protein1) and presynaptic Neurexin. On the other hand, the synaptogenic activity of GluD1, which is expressed not in the cerebellum but in the hippocampus, remains to be characterized. Here, we report that GluD1 expressed in non-neuronal HEK cells, induced presynaptic differentiation of granule neurons through its N-terminal domain in co-cultures with cerebellar neurons, similarly to GluD2. We also show that GluD1 rescued the defect of synapse formation in GluD2-knockout Purkinje neurons, indicating the functional similarity of GluD1 and GluD2. In contrast, GluD1 expression alone did not induce presynaptic differentiation in co-cultures of HEK cells with hippocampal neurons. However, when Cbln1 was exogenously added to the culture medium, GluD1 induced presynaptic differentiation of not only glutamatergic presynaptic terminals but also GABAergic ones. Cbln1 is not expressed in hippocampal neurons but is expressed in entorhinal cortical neurons projecting to the hippocampus. In co-cultures of HEK cells expressing GluD1 and entorhinal cortical neurons, both glutamatergic and GABAergic presynaptic terminals were formed on the HEK cells without exogenous application of Cbln1. These results suggest that GluD1 might contribute to the formation of specific synapses in the hippocampus such as those formed by the projecting neurons of the entorhinal cortex.     

An in vitro model of human neocortical development using pluripotent stem cells: cocaine-induced cytoarchitectural alterations

Neocortical development involves ordered specification of forebrain cortical progenitors to various neuronal subtypes, ultimately forming the layered cortical structure. Modeling of this process using human pluripotent stem cells (hPSCs) would enable mechanistic studies of human neocortical development, while providing new avenues for exploration of developmental neocortical abnormalities. Here, we show that preserving hPSCs aggregates – allowing embryoid body formation – while adding basic fibroblast growth factor (bFGF) during neuroepithelial development generates neural rosettes showing dorsal forebrain identity, including Mash1⁺ dorsal telencephalic GABAergic progenitors. Structures that mirrored the organization of the cerebral cortex formed after rosettes were seeded and cultured for 3 weeks in the presence of FGF18, BDNF and NT3. Neurons migrated along radial glia scaffolding, with deep-layer CTIP2⁺ cortical neurons appearing after 1 week and upper-layer SATB2⁺ cortical neurons forming during the second and third weeks. At the end of differentiation, these structures contained both glutamatergic and GABAergic neurons, with glutamatergic neurons being most abundant. Thus, this differentiation protocol generated an hPSC-based model that exhibits temporal patterning and a neuronal subtype ratio similar to that of the developing human neocortex. This model was used to examine the effects of cocaine during neocorticogenesis. Cocaine caused premature neuronal differentiation and enhanced neurogenesis of various cortical neuronal subtypes. These cocaine-induced changes were inhibited by the cytochrome P450 inhibitor cimetidine. This in vitro model enables mechanistic studies of neocorticogenesis, and can be used to examine the mechanisms through which cocaine alters the development of the human neocortex.KEY WORDS: Neocortical development, Dorsal forebrain model, hPSCs, Cocaine, Premature neuronal differentiation     

SMAD4 is essential for generating subtypes of neurons during cerebellar development

Cerebellum development involves the coordinated production of multiple neuronal cell types. The cerebellar primordium contains two germinative zones, the rhombic lip (RL) and the ventricular zone (VZ), which generate different types of glutamatergic and GABAergic neurons, respectively. What regulates the specification and production of glutamatergic and GABAergic neurons as well as the subtypes for each of these two broad classes remains largely unknown. Here we demonstrate with conditional genetic approaches in mice that SMAD4, a major mediator of BMP and TGFβ signaling, is required early in cerebellar development for maintaining the RL and generating subsets of RL-derived glutamatergic neurons, namely neurons of the deep cerebellar nuclei, unipolar brush cells, and the late cohort of granule cell precursors (GCPs). The early cohort of GCPs, despite being deficient for SMAD4, is still generated. In addition, the numbers of GABAergic neurons are reduced in the mutant and the distribution of Purkinje cells becomes abnormal. These studies demonstrate a temporally and spatially restricted requirement for SMAD4 in generating subtypes of cerebellar neurons.     

Fibroblast growth factor 2 regulates dopaminergic neuron development in vivo

Fibroblast growth factor 2 (FGF-2) is a neurotrophic factor participating in regulation of proliferation, differentiation, apoptosis and neuroprotection in the central nervous system. With regard to dopaminergic (DA) neurons of substantia nigra pars compacta (SNpc), which degenerate in Parkinson's disease, FGF-2 improves survival of mature DA neurons in vivo and regulates expansion of DA progenitors in vitro. To address the physiological role of FGF-2 in SNpc development, embryonic (E14.5), newborn (P0) and juvenile (P28) FGF-2-deficient mice were investigated. Stereological quantification of DA neurons identified normal numbers in the ventral tegmental area, whereas the SNpc of FGF-2-deficient mice displayed a 35% increase of DA neurons at P0 and P28, but not at earlier stage E14.5. Examination of DA marker gene expression by quantitative RT-PCR and in situ hybridization revealed a normal patterning of embryonic ventral mesencephalon. However, an increase of proliferating Lmx1a DA progenitors in the subventricular zone of the ventral mesencephalon of FGF-2-deficient embryos indicated altered cell cycle progression of neuronal progenitors. Increased levels of nuclear FgfR1 in E14.5 FGF-2-deficient mice suggest alterations of integrative nuclear FgfR1 signaling (INFS). In summary, FGF-2 restricts SNpc DA neurogenesis in vivo during late stages of embryonic development.     

Effect of K+- and kainate-mediated depolarization on survival and functional maturation of GABAergic and glutamatergic neurons in cultures of dissociated mouse cerebellum

The effect of the depolarizing agents, an elevated potassium concentration (25 mM) or kainic acid (50 μM) on neuronal survival and differentiation was investigated in cultures of dissociated neurons from cerebella of 7-day-old mice. When maintained in the presence of an antimitotic agent such cultures consist primarily of glutamatergic and GABAergic neurons. Cell survival was monitored by measurement of DNA, and differentiation by determining uptake and depolarization coupled release of glutamate (D-aspartate as label) and GABA. The depolarizing agents were added separately or together either from the start of the culture period (7–8 days) or at day 5 in culture. The main findings are that K⁺ depolarization is important for differentiation of glutamatergic neurons but not for GABAergic neurons. This depolarizing signal is important during the early phase of development in culture. For glutamatergic neurons, kainate may replace K⁺ as a depolarizing signal whereas in case of the GABAergic neurons, kainate was toxic particularly during the late phase of development. It was further observed that the glutamatergic neurons when maintained in a medium with 5 mM K⁺ during the first 5 days in culture became sensitive to kainate toxicity when this amino acid was added at day 5. This was not the case when the medium contained 25 mM K⁺ from the start of the culture period. Special issue dedicated to Dr. Kinya Kuriyama.     

Synaptic communication between neurons and NG2+ cells

Chemical synaptic transmission provides the basis for much of the rapid signaling that occurs within neuronal networks. However, recent studies have provided compelling evidence that synapses are not used exclusively for communication between neurons. Physiological and anatomical studies indicate that a distinct class of glia known as NG2(+) cells also forms direct synaptic junctions with both glutamatergic and GABAergic neurons. Glutamatergic signaling can influence intracellular Ca(2+) levels in NG2(+) cells by activating Ca(2+) permeable AMPA receptors, and these inputs can be potentiated through high frequency stimulation. Although the significance of this highly differentiated form of communication remains to be established, these neuro-glia synapses might enable neurons to influence rapidly the behavior of this ubiquitous class of glial progenitors.