Cerebellum Lecture: the Cerebellar Nuclei-Core of the Cerebellum.

The cerebellum is a key player in many brain functions and a major topic of neuroscience research. However, the cerebellar nuclei (CN), the main output structures of the cerebellum, are often overlooked. This neglect is because research on the cerebellum typically focuses on the cortex and tends to treat the CN as relatively simple output nuclei conveying an inverted signal from the cerebellar cortex to the rest of the brain. In this review, by adopting a nucleocentric perspective we aim to rectify this impression. First, we describe CN anatomy and modularity and comprehensively integrate CN architecture with its highly organized but complex afferent and efferent connectivity. This is followed by a novel classification of the specific neuronal classes the CN comprise and speculate on the implications of CN structure and physiology for our understanding of adult cerebellar function. Based on this thorough review of the adult literature we provide a comprehensive overview of CN embryonic development and, by comparing cerebellar structures in various chordate clades, propose an interpretation of CN evolution. Despite their critical importance in cerebellar function, from a clinical perspective intriguingly few, if any, neurological disorders appear to primarily affect the CN. To highlight this curious anomaly, and encourage future nucleocentric interpretations, we build on our review to provide a brief overview of the various syndromes in which the CN are currently implicated. Finally, we summarize the specific perspectives that a nucleocentric view of the cerebellum brings, move major outstanding issues in CN biology to the limelight, and provide a roadmap to the key questions that need to be answered in order to create a comprehensive integrated model of CN structure, function, development, and evolution.

Revisiting the development of cerebellar inhibitory interneurons in the light of single-cell genetic analyses.

The present review aims to provide a short update of our understanding of the inhibitory interneurons of the cerebellum. While these cells constitute but a minority of all cerebellar neurons, their functional significance is increasingly being recognized. For one, inhibitory interneurons of the cerebellar cortex are now known to constitute a clearly more diverse group than their traditional grouping as stellate, basket, and Golgi cells suggests, and this diversity is now substantiated by single-cell genetic data. The past decade or so has also provided important information about interneurons in cerebellar nuclei. Significantly, developmental studies have revealed that the specification and formation of cerebellar inhibitory interneurons fundamentally differ from, say, the cortical interneurons, and define a mode of diversification critically dependent on spatiotemporally patterned external signals. Last, but not least, in the past years, dysfunction of cerebellar inhibitory interneurons could also be linked with clinically defined deficits. I hope that this review, however fragmentary, may stimulate interest and help focus research towards understanding the cerebellum.

A CRISPR-Cas9-engineered mouse model for GPI-anchor deficiency mirrors human phenotypes and exhibits hippocampal synaptic dysfunctions.

Pathogenic germline mutations in PIGV lead to glycosylphosphatidylinositol biosynthesis deficiency (GPIBD). Individuals with pathogenic biallelic mutations in genes of the glycosylphosphatidylinositol (GPI)-anchor pathway exhibit cognitive impairments, motor delay, and often epilepsy. Thus far, the pathophysiology underlying the disease remains unclear, and suitable rodent models that mirror all symptoms observed in human patients have not been available. Therefore, we used CRISPR-Cas9 to introduce the most prevalent hypomorphic missense mutation in European patients, Pigv:c.1022C > A (p.A341E), at a site that is conserved in mice. Mirroring the human pathology, mutant Pigv341E mice exhibited deficits in motor coordination, cognitive impairments, and alterations in sociability and sleep patterns, as well as increased seizure susceptibility. Furthermore, immunohistochemistry revealed reduced synaptophysin immunoreactivity in Pigv341E mice, and electrophysiology recordings showed decreased hippocampal synaptic transmission that could underlie impaired memory formation. In single-cell RNA sequencing, Pigv341E-hippocampal cells exhibited changes in gene expression, most prominently in a subtype of microglia and subicular neurons. A significant reduction in Abl1 transcript levels in several cell clusters suggested a link to the signaling pathway of GPI-anchored ephrins. We also observed elevated levels of Hdc transcripts, which might affect histamine metabolism with consequences for circadian rhythm. This mouse model will not only open the doors to further investigation into the pathophysiology of GPIBD, but will also deepen our understanding of the role of GPI-anchor-related pathways in brain development.

Increased glutamate transporter-associated anion currents cause glial apoptosis in episodic ataxia 6.

Episodic ataxia type 6 is an inherited neurological condition characterized by combined ataxia and epilepsy. A severe form of this disease with episodes combining ataxia, epilepsy and hemiplegia was recently associated with a proline to arginine substitution at position 290 of the excitatory amino acid transporter 1 in a heterozygous patient. The excitatory amino acid transporter 1 is the predominant glial glutamate transporter in the cerebellum. However, this glutamate transporter also functions as an anion channel and earlier work in heterologous expression systems demonstrated that the mutation impairs the glutamate transport rate, while increasing channel activity. To understand how these changes cause ataxia, we developed a constitutive transgenic mouse model. Transgenic mice display epilepsy, ataxia and cerebellar atrophy and, thus, closely resemble the human disease. We observed increased glutamate-activated chloride efflux in Bergmann glia that triggers the apoptosis of these cells during infancy. The loss of Bergmann glia results in reduced glutamate uptake and impaired neural network formation in the cerebellar cortex. This study shows how gain-of-function of glutamate transporter-associated anion channels causes ataxia through modifying cerebellar development.

Developmental Maturation of the Cerebellar White Matter-an Instructive Environment for Cerebellar Inhibitory Interneurons.

In the developing cerebellum, the nascent white matter (WM) serves as an instructive niche for cerebellar cortical inhibitory interneurons. As their Pax2 expressing precursors transit the emerging WM, their laminar fate is programmed. The source(s) and nature of the signals involved remain unknown. Here, we used immunocytochemistry to follow the cellular maturation of the murine cerebellar WM during this critical period. During the first few days of postnatal development, when most Pax2 expressing cells are formed and many of them reach the cerebellar gray matter, only microglial cells can be identified in the territories through which Pax2 cells migrate. From p4 onward, cells expressing the oligodendrocytic or astrocyte markers, CNP-1, MBP or GFAP, started to appear in the nascent WM. Expression of macroglial markers increased with cerebellar differentiation, yet deep nuclei remained GFAP-negative at all ages. The progressive spread of maturing glia did not correlate with the exit of Pax2 cells from the WM, as indicated by the extensive mingling of these cells up to p15. Whereas sonic hedgehog-associated p75NTR expression could be verified in granule cell precursors, postmitotic Pax2 cells are p75NTR negative at all ages analyzed. Thus, if Pax2 cells, like their precursors, are sensitive to sonic hedgehog, this does not affect their expression of p75NTR. Our findings document that subsequently generated sets of Pax2 expressing precursors of inhibitory cerebellar interneurons are confronted with a dynamically changing complement of cerebellar glia. The eventual identification of fate-defining pathways should profit from the covariation with glial maturation predicted by the present findings.

Pharmacological Prevention of Neonatal Opioid Withdrawal in a Pregnant Guinea Pig Model.

Newborns exposed to prenatal opioids often experience intense postnatal withdrawal after cessation of the opioid, called neonatal opioid withdrawal syndrome (NOWS), with limited pre- and postnatal therapeutic options available. In a prior study in pregnant mice we demonstrated that the peripherally selective opioid antagonist, 6ß-naltrexol (6BN), is a promising drug candidate for preventive prenatal treatment of NOWS, and a therapeutic mechanism was proposed based on preferential delivery of 6BN to fetal brain with relative exclusion from maternal brain. Here, we have developed methadone (MTD) treated pregnant guinea pigs as a physiologically more suitable model, enabling detection of robust spontaneous neonatal withdrawal. Prenatal MTD significantly aggravates two classic maternal separation stress behaviors in newborn guinea pigs: calling (vocalizing) and searching (locomotion) - natural attachment behaviors thought to be controlled by the endogenous opioid system. In addition, prenatal MTD significantly increases the levels of plasma cortisol in newborns, showing that cessation of MTD at birth engages the hypothalamic-pituitary-adrenal (HPA) axis. We find that co-administration of 6BN with MTD prevents these withdrawal symptoms in newborn pups with extreme potency (ID50 ∼0.02 mg/kg), at doses unlikely to induce maternal or fetal withdrawal or to interfere with opioid antinociception based on many prior studies in rodents and non-human primates. Furthermore, we demonstrate a similarly high potency of 6BN in preventing opioid withdrawal in adult guinea pigs (ID50 = 0.01 mg/kg). This high potency appears to run counter to our pharmacokinetic studies showing slow 6BN transit of both the placenta and maternal blood brain barrier in guinea pigs, and calls into question the preferential delivery mechanism. Rather, it suggests a novel receptor mechanism to account for the selectively high potency of 6BN to suppress opioid dependence at all developmental stages, even in adults, as compared to its well-established low potency as a classical opioid antagonist. In conclusion, 6BN is an attractive compound for development of a preventive therapy for NOWS.

Moving into shape: cell migration during the development and histogenesis of the cerebellum.

The enormous expansion the vertebrate nervous system goes through from its first anlage to its adult shape and organization goes along with extensive rearrangements of its constituent cells and typical cellular migrations, often over long distances, and by convoluted pathways. Here, I try to summarize how the cells that form the cerebellum move and migrate during normal cerebellar histogenesis. The cerebellum is made up of a limited set of clearly distinguishable classes of cells, some of which are also readily accessible by genetic tools. Its structure and development have been the focus of studies dating back to at least Ramon y Cajal which have yielded fundamental insights into basic mechanisms of the development of the nervous. During cerebellar histogenesis, several distinct and well-discernable modes of migration may be recognized, some of which have been studied in considerable morphological and molecular detail. Still, often grace to the detail known, a wealth of open questions remains, and the cerebellar anlage remains a highly accessible and promising paradigm for those interested in nervous system development and cell migration in general. I also point out some of the issues that may warrant consideration when results from technically distinct studies are compared and integrated.

Migration of Interneuron Precursors in the Nascent Cerebellar Cortex.

The cerebellum arguably constitutes one of the best characterized central nervous circuits, and its structure, cellular function, and histogenesis have been described in exceptional quantitative detail. A notable exception to this is the development of its inhibitory interneurons, and in particular the extensive migrations of future basket and stellate cells. Here, we used acute slices from 8-day-old mice to assess the migration of Pax2-EGFP-tagged precursors of these cells en route to the molecular layer during their transit through the nascent cerebellar cortex. We document that movement of these cells is highly directed. Their speed and directional persistence are larger in the nascent granule cell layer than in the molecular layer. And they migrate periodically, with periods of effective, directed translocation separated by bouts of rather local movement. Finally, we document that the arrangement of these cells in the adult molecular layer is characterized by clustering. These data are discussed with a focus on potential generative mechanisms for the developmental pattern observed.

Synaptic input as a directional cue for migrating interneuron precursors.

During CNS development, interneuron precursors have to migrate extensively before they integrate in specific microcircuits. Known regulators of neuronal motility include classical neurotransmitters, yet the mechanisms that assure interneuron dispersal and interneuron/projection neuron matching during histogenesis remain largely elusive. We combined time-lapse video microscopy and electrophysiological analysis of the nascent cerebellum of transgenic Pax2-EGFP mice to address this issue. We found that cerebellar interneuronal precursors regularly show spontaneous postsynaptic currents, indicative of synaptic innervation, well before settling in the molecular layer. In keeping with the sensitivity of these cells to neurotransmitters, ablation of synaptic communication by blocking vesicular release in acute slices of developing cerebella slows migration. Significantly, abrogation of exocytosis primarily impedes the directional persistence of migratory interneuronal precursors. These results establish an unprecedented function of the early synaptic innervation of migrating neuronal precursors and demonstrate a role for synapses in the regulation of migration and pathfinding.

Mtss1 promotes maturation and maintenance of cerebellar neurons via splice variant-specific effects.

Efficient coupling of the actin cytoskeleton to the cell membrane is crucial for histogenesis and maintenance of the nervous system. At this critical interface, BAR (Bin-Amphiphysin-Rvs) proteins regulate membrane bending, shown to be instrumental for mobility and morphogenesis of individual cells. Yet, the systemic significance of these proteins remains largely unexplored. Here, we probe the role of a prominent member of this protein family, the inverse-BAR protein Mtss1, for the development and function of a paradigmatic neuronal circuit, the cerebellar cortex. Mtss1-null mice show granule cell ectopias, dysmorphic Purkinje cells, malformed axons, and a protracted neurodegeneration entailing age-dependent motor deficits. In postmitotic granule cells, which transiently express Mtss1 while they migrate and form neurites, Mtss1 impinges on directional persistence and neuritogenesis. The latter effect can be specifically attributed to its exon 12a splice variant. Targeted re-expression of Mtss1 in Mtss1-null animals indicated that these pathologies were largely due to cell type-specific and intrinsic effects. Together, our results provide a mechanistic perspective on Mtss1 function for brain development and degeneration and relate it to structural features of this protein.

Preferential Delivery of an Opioid Antagonist to the Fetal Brain in Pregnant Mice.

Prolonged fetal exposure to opioids results in neonatal abstinence syndrome (NAS), a major medical problem requiring intensive care and increased hospitalization times for newborns with NAS. Multiple strategies are currently available to alleviate withdrawal in infants with NAS. To prevent NAS caused by opioid maintenance programs in pregnant women, blocking fetal dependence without compromising the mother's opiate therapy is desirable. Here we tested in pregnant mice whether a peripherally selective opioid antagonist can preferentially enter the fetal brain and, thereby, in principle, selectively protect the fetus. We show using mass spectrometry that 6ß-naltrexol, a neutral opioid antagonist with very limited ability to cross the blood-brain barrier (BBB), readily crosses the placental barrier and enters the fetal brain at high levels, although it is relatively excluded from the maternal brain. Furthermore, owing to the late development of the BBB in postnatal mice, we show that 6ß-naltrexol can readily enter the juvenile mouse brain until at least postnatal day 14. Taking advantage of this observation, we show that long-term exposure to morphine starting in the second postnatal week causes robust and quantifiable dependence behaviors that are suppressed by concomitant administration of 6ß-naltrexol with much greater potency (ID50 0.022-0.044 mg/kg, or 1/500 the applied dose of morphine) than previously demonstrated for either the suppression of central nervous system opioid effects or the induction of withdrawal in adults. These results indicate that peripherally selective opioid antagonists capable of penetrating the placenta may be beneficial for preventing or reducing neonatal dependence and NAS in a dose range that should not interfere with maternal opioid maintenance.

Consensus Paper: Cerebellar Development.

The development of the mammalian cerebellum is orchestrated by both cell-autonomous programs and inductive environmental influences. Here, we describe the main processes of cerebellar ontogenesis, highlighting the neurogenic strategies used by developing progenitors, the genetic programs involved in cell fate specification, the progressive changes of structural organization, and some of the better-known abnormalities associated with developmental disorders of the cerebellum.

Paired box gene 8 (PAX8) expression is associated with sonic hedgehog (SHH)/wingless int (WNT) subtypes, desmoplastic histology and patient survival in human medulloblastomas.

AIMS: The paired box gene 8 (PAX8) plays crucial roles in organ patterning and cellular differentiation during development and tumorigenesis. Although its function is partly understood in vertebrate development, there is poor data concerning human central nervous system (CNS) development and brain tumours. METHODS: We investigated developing human (n = 19) and mouse (n = 3) brains as well as medulloblastomas (MBs) (n = 113) for PAX8 expression by immunohistochemistry. Human MB cell lines were assessed for PAX8 expression using polymerase chain reaction and immunoblotting and analysed for growth and migration following PAX8 knock-down by small interfering ribonucleic acid (siRNA). RESULTS: PAX8 protein expression was associated with germinal layers in human and murine forebrain and hindbrain development. PAX8 expression significantly decreased over time in the external granule cell layer but increased in the internal granule cell layer. In MB subtypes, we observed an association of PAX8 expression with sonic hedgehog (SHH) and wingless int subtypes but not with group 3 and 4 MBs. Beyond that, we detected high PAX8 levels in desmoplastic MB subtypes. Univariate analyses revealed high PAX8 levels as a prognostic factor associated with a significantly better patient prognosis in human MB (overall survival: Log-Rank P = 0.0404, Wilcoxon P = 0.0280; progression-free survival: Log-Rank P = 0.0225; Wilcoxon P = 0.0136). In vitro assays revealed increased proliferation and migration of MB cell lines after PAX8 siRNA knock-down. CONCLUSION: In summary, high PAX8 expression is linked to better prognosis in MBs potentially by suppressing both proliferative and migratory properties of MB cells. The distinct spatio-temporal expression pattern of PAX8 during brain development might contribute to the understanding of distinct MB subtype histogenesis.

The transcription factor Cux1 in cerebellar granule cell development and medulloblastoma pathogenesis.

Cux1, also known as Cutl1, CDP or Cut is a homeodomain transcription factor implicated in the regulation of normal and oncogenic development in diverse peripheral tissues and organs. We studied the expression and functional role of Cux1 in cerebellar granule cells and medulloblastoma. Cux1 is robustly expressed in proliferating granule cell precursors and in postmitotic, migrating granule cells. Expression is lost as postmigratory granule cells mature. Moreover, Cux1 is also strongly expressed in a well-established mouse model of medulloblastoma. In contrast, expression of CUX1 in human medulloblastoma tissue samples is lower than in normal fetal cerebellum. In these tumors, CUX1 expression tightly correlates with a set of genes which, when mapped on a global protein-protein interaction dataset, yields a tight network that constitutes a cell cycle control signature and may be related to p53 and the DNA damage response pathway. Antisense-mediated reduction of CUX1 levels in two human medulloblastoma cell lines led to a decrease in proliferation and altered motility. The developmental expression of Cux1 in the cerebellum and its action in cell lines support a role in granule cell and medulloblastoma proliferation. Its expression in human medulloblastoma shifts that perspective, suggesting that CUX1 is part of a network involved in cell cycle control and maintenance of DNA integrity. The constituents of this network may be rational targets to therapeutically approach medulloblastomas.

The molecular basis of the specificity and cross-reactivity of the NeuN epitope of the neuron-specific splicing regulator, Rbfox3.

Ever since its description and the generation of its defining antibody some 20 years ago, NeuN (Neural Nuclei) has been an invaluable tool for developmental neuroscientist sand neuropathologists to identify neurons and follow their normal or malignant development [corrected].The recent identification of the splicing factor Rbfox3 as the molecule constituting the genuine NeuN epitope has opened up a novel perspective on NeuN immunostaining and its interpretation. Here, we briefly review these recent developments, and we provide a series of data that allow to rationalize the specificity of the NeuN/A60 antibody on aldehyde-fixed tissues on the one hand, and its cross-reactivity with Synapsin I and R3hdm2 on Western blots on the other. We argue that rather than being considered as a mere marker for mature neurons, Rbfox3-mediated NeuN/A60 immunoreactivity may provide a window onto neuronal biology. Specifically, we hypothesize that the phosphorylation-dependent antigenicity of the Rbfox3/NeuN epitope should allow to visualize neuronal physiology realized through Rbfox3, including splicing, on the single-cell level.

Differential maturation of GIRK2-expressing neurons in the mouse cerebellum.

The cerebellar cortex is among the brain regions showing the highest expression levels of G-protein-gated inwardly-rectifying potassium (GIRK/Kir3) channels. Despite their critical contribution in modulating neuronal excitability during development and adult, the spatiotemporal expression of specific GIRK subunits in identified cerebellar neuron populations is unresolved. To characterize this onset of expression, we examined the GIRK2 protein expression in mouse cerebellum by western blot, light microscopy immunohistochemistry and immunofluorescence during perinatal development. Using western blots, GIRK2 expression was low at birth but reach its maximum at P5 before decreasing gradually to adult levels. Immunohistochemical localization indicated that GIRK2 is expressed in granule cells from early stages of development. At the embryonic stage, immunofluorescence techniques for the transcription factor Pax6 allowed to demonstrate that GIRK2 is expressed in granule cell precursors. This GIRK2 expression in granule cells continued throughout postnatal development and adulthood. In addition, the expression of Pax2-GFP allowed selective visualization of Golgi cells during pre- and postnatal development. We could not detect co-expression of Pax2-GFP and GIRK2 during prenatal and early postnatal development, but only at post-migratory stages of Golgi cells, once they are morphologically differentiated and located at the granule cell layer. In the adult cerebellum, we performed a detailed characterization on the expression of GIRK2 in different subpopulations of Golgi cells, using metabotropic glutamate receptor 2 (mGlu(2)) and neurogranin as markers, in GlyT2-GFP and GAD67-GFP mice, and showed that GIRK2 is present in at least four morphological and neurochemical non-overlapping populations of Golgi cells. Altogether, these findings shed new light on the developmental regulation of GIRK channels in the cerebellum, and the main expression in granule cells during perinatal development support the idea that GIRK2 may provide a significant route for modulating different aspects of cerebellar development.

Sex-dependent behavioral functions of the Purkinje cell-specific Gαi/o binding protein, Pcp2(L7).

We previously reported motor and non-motor enhancements in a mouse mutant with an inactivated Purkinje cell-specific gene, Pcp2(L7), that encodes a GoLoco domain-containing modulator of Gi/o protein-coupled receptors. Effects included elevated learning asymptote with repeated rotarod training, increased acquisition rate in tone-cued fear conditioning (FC), and subtle male-specific changes in both acoustic startle habituation and pre-pulse inhibition. We have further analyzed this mutant strain this time with a focus on male-female differences, and here we report a sex-dependent anxiety-like phenotype: male mutants are less anxious, and female mutants are more anxious, than same-sex wild types. Similarly, the fear responses measured during the tone in FC acquisition are decreased in male mutants and increased in female mutants relative to same-sex wild types. Overall, the dynamics of both acquisition and extinction of FC is affected in mutants but memory was not affected. In the social realm, compositional analysis of sociability and preference for social novelty data supports that both L7 genotype and sex contribute to these behaviors. These results provide direct evidence of emotional functions of the cerebellum due to the unambiguous cerebellar specificity of Pcp2(L7) expression and the lack of any confounding motor defects in the mutant. We attempt to synthesize these new data with what is previously known both about Pcp2(L7) and about the effects of sex and sex hormones on anxiety and fear behaviors: specifically, L7 is a bidirectional and sex-dependent damper that regulates the amplitude and/or rate of sensorimotor responses, potentially acting as a mood stabilizer.

Cell death as a regulator of cerebellar histogenesis and compartmentation.

Programmed cell death is essential for the homeostasis of tissues and organs. During the development of the central nervous system, programmed cell death is highly regulated and restricted to distinct developmental time points of histogenesis. In this review, we will summarize recent data on the temporal and spatial distribution of programmed Purkinje cell death within the cerebellar cortex. We point out that programmed cell death within distinct regions of the developing cerebellar cortex differs by type and its cellular consequences. We submit the concept that local Purkinje cell death is important for late compartmentation of the cerebellar cortex and its wiring. To support this hypothesis, we provide new data obtained from a cerebellar mutant with prolonged expression of Engrailed-2 specifically in cerebellar Purkinje cells which shows increased local physiological cell death and concomitant changes in the pattern of afferent innervation.