This Research Topic is devoted to update the knowledge of the cerebellum covering: (i) development of the cerebellum (ii) anatomy of cerebellar and related nuclei projections (iii) role of the cerebellum in motor and non-motor learning

Universidad Pablo de Olavide

Miguel Hernández University of Elche

Frontiers in Neuroanatomy

The Cerebellum: From Development to Learning

Over the last 60 years, the spotlight of research has periodically returned to the cerebellum as new techniques and insights have emerged. Because of its simple homogeneous structure, limited diversity of cell types and characteristic behavioral pathologies, the cerebellum is a natural home for studies of cell specification, patterning, and neuronal migration. However, recent evidence has extended the traditional range of perceived cerebellar function to include modulation of cognitive processes and implicated cerebellar hypoplasia and Purkinje neuron hypo-cellularity with autistic spectrum disorder. In the light of this emerging frontier, we review the key stages and genetic mechanisms behind cerebellum development. In particular, we discuss the role of the midbrain hindbrain isthmic organizer in the development of the cerebellar vermis and the specification and differentiation of Purkinje cells and granule neurons. These developmental processes are then considered in relation to recent insights into selected human developmental cerebellar defects: Joubert syndrome, Dandy–Walker malformation, and pontocerebellar hypoplasia. Finally, we review current research that opens up the possibility of using the mouse as a genetic model to study the role of the cerebellum in cognitive function.

Over the last 60 years, the spotlight of research has periodically returned to the cerebellum as new techniques and insights have emerged. Because of its simple homogeneous structure, limited diversity of cell types and characteristic behavioral pathologies, the cerebellum is a natural home for studies of cell specification, patterning, and neuronal migration. However, recent evidence has extended the traditional range of perceived cerebellar function to include modulation of cognitive processes and implicated cerebellar hypoplasia and Purkinje neuron hypo-cellularity with autistic spectrum disorder. In the light of this emerging frontier, we review the key stages and genetic mechanisms behind cerebellum development. In particular, we discuss the role of the midbrain hindbrain isthmic organizer in the development of the cerebellar vermis and the specification and differentiation of Purkinje cells and granule neurons. These developmental processes are then considered in relation to recent insights into selected human developmental cerebellar defects: Joubert syndrome, Dandy&#x2013;Walker malformation, and pontocerebellar hypoplasia. Finally, we review current research that opens up the possibility of using the mouse as a genetic model to study the role of the cerebellum in cognitive function.

Congenital hypoplasia of the cerebellum: developmental causes and behavioral consequences

Historically, the molecular and cellular mechanisms of cerebellar development were investigated through structural descriptions and studying spontaneous mutations in animal models and humans. Advances in experimental embryology, genetic engineering, and neuroimaging techniques render today the possibility to approach the analysis of molecular mechanisms underlying histogenesis and morphogenesis of the cerebellum by experimental designs. Several genes and molecules were identified to be involved in the cerebellar plate regionalization, specification, and differentiation of cerebellar neurons, as well as the establishment of cellular migratory routes and the subsequent neuronal connectivity. Indeed, pattern formation of the cerebellum requires the adequate orchestration of both key morphogenetic signals, arising from distinct brain regions, and local expression of specific transcription factors. Thus, the present review wants to revisit and discuss these morphogenetic and molecular mechanisms taking place during cerebellar development in order to understand causal processes regulating cerebellar cytoarchitecture, its highly topographically ordered circuitry and its role in brain function.

Historically, the molecular and cellular mechanisms of cerebellar development were investigated through structural descriptions and studying spontaneous mutations in animal models and humans. Advances in experimental embryology, genetic engineering, and neuroimaging techniques render today the possibility to approach the analysis of molecular mechanisms underlying histogenesis and morphogenesis of the cerebellum by experimental designs. Several genes and molecules were identified to be involved in the cerebellar plate regionalization, specification, and differentiation of cerebellar neurons, as well as the establishment of cellular migratory routes and the subsequent neuronal connectivity. Indeed, pattern formation of the cerebellum requires the adequate orchestration of both key morphogenetic signals, arising from distinct brain regions, and local expression of specific transcription factors. Thus, the present review wants to revisit and discuss these morphogenetic and molecular mechanisms taking place during cerebellar development in order to understand causal processes regulating cerebellar cytoarchitecture, its highly topographically ordered circuitry and its role in brain function.

Cellular and molecular basis of cerebellar development

The cerebellum plays a key role in the acquisition and execution of motor tasks whose physiological foundations were postulated on Purkinje cells' long-term depression (LTD). Numerous research efforts have been focused on understanding the cerebellum as a site of learning and/or memory storage. However, the controversy on which part of the cerebellum participates in motor learning, and how the process takes place, remains unsolved. In fact, it has been suggested that cerebellar cortex, deep cerebellar nuclei, and/or their combination with some brain structures other than the cerebellum are responsible for motor learning. Different experimental approaches have been used to tackle this question (cerebellar lesions, pharmacological agonist and/or antagonist of cerebellar neurotransmitters, virus tract tracings, etc.). One of these approaches is the study of spontaneous mutations affecting the cerebellar cortex and depriving it of its main input–output organizer (i.e., the Purkinje cell). In this review, we discuss the results obtained in our laboratory in motor learning of both Lurcher (Lc/+) and tambaleante (tbl/tbl) mice as models of Purkinje-cell-devoid cerebellum.

The cerebellum plays a key role in the acquisition and execution of motor tasks whose physiological foundations were postulated on Purkinje cells' long-term depression (LTD). Numerous research efforts have been focused on understanding the cerebellum as a site of learning and/or memory storage. However, the controversy on which part of the cerebellum participates in motor learning, and how the process takes place, remains unsolved. In fact, it has been suggested that cerebellar cortex, deep cerebellar nuclei, and/or their combination with some brain structures other than the cerebellum are responsible for motor learning. Different experimental approaches have been used to tackle this question (cerebellar lesions, pharmacological agonist and/or antagonist of cerebellar neurotransmitters, virus tract tracings, etc.). One of these approaches is the study of spontaneous mutations affecting the cerebellar cortex and depriving it of its main input–output organizer (i.e., the Purkinje cell). In this review, we discuss the results obtained in our laboratory in motor learning of both Lurcher (Lc/+) and tambaleante (tbl/tbl) mice as models of Purkinje-cell-devoid cerebellum.

Motor learning of mice lacking cerebellar Purkinje cells

Between the first and the second postnatal week, the development of rodent Purkinje cells is characterized by several profound transitions. Purkinje cells acquire their typical dendritic “espalier” tree morphology and form distal spines. During the first postnatal week, they are multi-innervated by climbing fibers and numerous collateral branches sprout from their axons, whereas from the second postnatal week, the regression of climbing fiber multi-innervation begins, and Purkinje cells become innervated by parallel fibers and inhibitory molecular layer interneurons. Furthermore, their periods of developmental cell death and ability to regenerate their axon stop and their axons become myelinated. Thus a Purkinje cell during the first postnatal week looks and functions differently from a Purkinje cell during the second postnatal week. These fundamental changes occur in parallel with a peak of circulating thyroid hormone in the mouse. All these features suggest to some extent an interesting analogy with amphibian metamorphosis.

Between the first and the second postnatal week, the development of rodent Purkinje cells is characterized by several profound transitions. Purkinje cells acquire their typical dendritic &#x201c;espalier&#x201d; tree morphology and form distal spines. During the first postnatal week, they are multi-innervated by climbing fibers and numerous collateral branches sprout from their axons, whereas from the second postnatal week, the regression of climbing fiber multi-innervation begins, and Purkinje cells become innervated by parallel fibers and inhibitory molecular layer interneurons. Furthermore, their periods of developmental cell death and ability to regenerate their axon stop and their axons become myelinated. Thus a Purkinje cell during the first postnatal week looks and functions differently from a Purkinje cell during the second postnatal week. These fundamental changes occur in parallel with a peak of circulating thyroid hormone in the mouse. All these features suggest to some extent an interesting analogy with amphibian metamorphosis.

Profound morphological and functional changes of rodent Purkinje cells between the first and the second postnatal weeks: a metamorphosis?

The patterning of the embryonic cerebellum is vital to establish the elaborate zone and stripe architecture of the adult. This review considers early stages in cerebellar Purkinje cell patterning, from the organization of the ventricular zone to the development of Purkinje cell clusters—the precursors of the adult stripes.

The patterning of the embryonic cerebellum is vital to establish the elaborate zone and stripe architecture of the adult. This review considers early stages in cerebellar Purkinje cell patterning, from the organization of the ventricular zone to the development of Purkinje cell clusters&#x2014;the precursors of the adult stripes.

PATTERN FORMATION DURING DEVELOPMENT OF THE EMBRYONIC CEREBELLUM

The presence of two antagonistic groups of deep cerebellar nuclei neurons has been reported as necessary for a proper dynamic control of learned motor responses. Most models of cerebellar function seem to ignore the biomechanical need for a double activation–deactivation system controlling eyelid kinematics, since most of them accept that, for closing the eyelid, only the activation of the orbicularis oculi (OO) muscle (via the red nucleus to the facial motor nucleus) is necessary, without a simultaneous deactivation of levator palpebrae motoneurons (via unknown pathways projecting to the perioculomotor area). We have analyzed the kinetic neural commands of two antagonistic types of cerebellar posterior interpositus neuron (IPn) (types A and B), the electromyographic (EMG) activity of the OO muscle, and eyelid kinematic variables in alert behaving cats during classical eyeblink conditioning, using a delay paradigm. We addressed the hypothesis that the interpositus nucleus can be considered an agonist–antagonist system controlling eyelid kinematics during motor learning. To carry out a comparative study of the kinetic–kinematic relationships, we applied timing and dispersion pattern analyses. We concluded that, in accordance with a dominant role of cerebellar circuits for the facilitation of flexor responses, type A neurons fire during active eyelid downward displacements—i.e., during the active contraction of the OO muscle. In contrast, type B neurons present a high tonic rate when the eyelids are wide open, and stop firing during any active downward displacement of the upper eyelid. From a functional point of view, it could be suggested that type B neurons play a facilitative role for the antagonistic action of the levator palpebrae muscle. From an anatomical point of view, the possibility that cerebellar nuclear type B neurons project to the perioculomotor area—i.e., more or less directly onto levator palpebrae motoneurons—is highly appealing.

The presence of two antagonistic groups of deep cerebellar nuclei neurons has been reported as necessary for a proper dynamic control of learned motor responses. Most models of cerebellar function seem to ignore the biomechanical need for a double activation&#x2013;deactivation system controlling eyelid kinematics, since most of them accept that, for closing the eyelid, only the activation of the orbicularis oculi (OO) muscle (via the red nucleus to the facial motor nucleus) is necessary, without a simultaneous deactivation of levator palpebrae motoneurons (via unknown pathways projecting to the perioculomotor area). We have analyzed the kinetic neural commands of two antagonistic types of cerebellar posterior interpositus neuron (IPn) (types A and B), the electromyographic (EMG) activity of the OO muscle, and eyelid kinematic variables in alert behaving cats during classical eyeblink conditioning, using a delay paradigm. We addressed the hypothesis that the interpositus nucleus can be considered an agonist&#x2013;antagonist system controlling eyelid kinematics during motor learning. To carry out a comparative study of the kinetic&#x2013;kinematic relationships, we applied timing and dispersion pattern analyses. We concluded that, in accordance with a dominant role of cerebellar circuits for the facilitation of flexor responses, type A neurons fire during active eyelid downward displacements&#x2014;i.e., during the active contraction of the OO muscle. In contrast, type B neurons present a high tonic rate when the eyelids are wide open, and stop firing during any active downward displacement of the upper eyelid. From a functional point of view, it could be suggested that type B neurons play a facilitative role for the antagonistic action of the levator palpebrae muscle. From an anatomical point of view, the possibility that cerebellar nuclear type B neurons project to the perioculomotor area&#x2014;i.e., more or less directly onto levator palpebrae motoneurons&#x2014;is highly appealing.

An agonist–antagonist cerebellar nuclear system controlling eyelid kinematics during motor learning

Malformations of the midbrain (MB) and hindbrain (HB) have become topics of considerable interest in the neurology and neuroscience literature in recent years. The combined advances of imaging and molecular biology have improved analyses of structures in these areas of the central nervous system, while advances in genetics have made it clear that malformations of these structures are often associated with dysfunction or malformation of other organ systems. This review focuses upon the importance of communication between clinical researchers and basic scientists in the advancement of knowledge of this group of disorders. Disorders of anteroposterior (AP) patterning, cerebellar hypoplasias, disorders associated with defects of the pial limiting membrane (cobblestone cortex), disorders of the Reelin pathway, and disorders of the primary cilium/basal body organelle (molar tooth malformations) are the main focus of the review.

Malformations of the midbrain (MB) and hindbrain (HB) have become topics of considerable interest in the neurology and neuroscience literature in recent years. The combined advances of imaging and molecular biology have improved analyses of structures in these areas of the central nervous system, while advances in genetics have made it clear that malformations of these structures are often associated with dysfunction or malformation of other organ systems. This review focuses upon the importance of communication between clinical researchers and basic scientists in the advancement of knowledge of this group of disorders. Disorders of anteroposterior (AP) patterning, cerebellar hypoplasias, disorders associated with defects of the pial limiting membrane (cobblestone cortex), disorders of the Reelin pathway, and disorders of the primary cilium/basal body organelle (molar tooth malformations) are the main focus of the review.

Developmental disorders of the midbrain and hindbrain

All cerebellar neurons derive from progenitors that proliferate in two germinal neuroepithelia: the ventricular zone (VZ) generates GABAergic neurons, whereas the rhombic lip is the origin of glutamatergic types. Among VZ-derivatives, GABAergic projection neurons, and interneurons are generated according to distinct strategies. Projection neurons (Purkinje cells and nucleo-olivary neurons) are produced at the onset of cerebellar neurogenesis by discrete progenitor pools located in distinct VZ microdomains. These cells are specified within the VZ and acquire mature phenotypes according to cell-autonomous developmental programs. On the other hand, the different categories of inhibitory interneurons derive from a single population of Pax-2-positive precursors that delaminate into the prospective white matter (PWM), where they continue to divide up to postnatal development. Heterotopic/heterochronic transplantation experiments indicate that interneuron progenitors maintain full developmental potentialities up to the end of cerebellar development and acquire mature phenotypes under the influence of environmental cues present in the PWM. Furthermore, the final fate choice occurs in postmitotic cells, rather than dividing progenitors. Extracerebellar cells grafted to the prospective cerebellar white matter are not responsive to local neurogenic cues and fail to adopt clear cerebellar identities. Conversely, cerebellar cells grafted to extracerebellar regions retain typical phenotypes of cerebellar GABAergic interneurons, but acquire type-specific traits under the influence of local cues. These findings indicate that interneuron progenitors are multipotent and sensitive to spatio-temporally patterned environmental signals that regulate the genesis of different categories of interneurons, in precise quantities and at defined times and places.

All cerebellar neurons derive from progenitors that proliferate in two germinal neuroepithelia: the ventricular zone (VZ) generates GABAergic neurons, whereas the rhombic lip is the origin of glutamatergic types. Among VZ-derivatives, GABAergic projection neurons, and interneurons are generated according to distinct strategies. Projection neurons (Purkinje cells and nucleo-olivary neurons) are produced at the onset of cerebellar neurogenesis by discrete progenitor pools located in distinct VZ microdomains. These cells are specified within the VZ and acquire mature phenotypes according to cell-autonomous developmental programs. On the other hand, the different categories of inhibitory interneurons derive from a single population of Pax-2-positive precursors that delaminate into the prospective white matter (PWM), where they continue to divide up to postnatal development. Heterotopic/heterochronic transplantation experiments indicate that interneuron progenitors maintain full developmental potentialities up to the end of cerebellar development and acquire mature phenotypes under the influence of environmental cues present in the PWM. Furthermore, the final fate choice occurs in postmitotic cells, rather than dividing progenitors. Extracerebellar cells grafted to the prospective cerebellar white matter are not responsive to local neurogenic cues and fail to adopt clear cerebellar identities. Conversely, cerebellar cells grafted to extracerebellar regions retain typical phenotypes of cerebellar GABAergic interneurons, but acquire type-specific traits under the influence of local cues. These findings indicate that interneuron progenitors are multipotent and sensitive to spatio-temporally patterned environmental signals that regulate the genesis of different categories of interneurons, in precise quantities and at defined times and places.

The Genesis of Cerebellar GABAergic Neurons: Fate Potential and Specification Mechanisms

The study of the links and interactions between development and motor learning has noticeable implications for the understanding and management of neurodevelopmental disorders. This is particularly relevant for the cerebellum which is critical for sensorimotor learning. The olivocerebellar pathway is a key pathway contributing to learning of motor skills. Its developmental maturation and remodeling are being unraveled. Advances in genetics have led to major improvements in our appraisal of the genes involved in cerebellar development, especially studies in mutant mice. Cerebellar neurogenesis is compartmentalized in relationship with neurotransmitter fate. The Engrailed-2 gene is a major actor of the specification of cerebellar cell types and late embryogenic morphogenesis. Math1, expressed by the rhombic lip, is required for the genesis of glutamatergic neurons. Mutants deficient for the transcription factor Ptf1a display a lack of Purkinje cells and gabaergic interneurons. Rora gene contributes to the developmental signaling between granule cells and Purkinje neurons. The expression profile of sonic hedgehog in postnatal stages determines the final size/shape of the cerebellum. Genes affecting the development impact upon the physiological properties of the cerebellar circuits. For instance, receptors are developmentally regulated and their action interferes directly with developmental processes. Another field of research which is expanding relates to very preterm neonates. They are at risk for cerebellar lesions, which may themselves impair the developmental events. Very preterm neonates often show sensori-motor deficits, highlighting another major link between impaired developments and learning deficiencies. Pathways playing a critical role in cerebellar development are likely to become therapeutical targets for several neurodevelopmental disorders.

The study of the links and interactions between development and motor learning has noticeable implications for the understanding and management of neurodevelopmental disorders. This is particularly relevant for the cerebellum which is critical for sensorimotor learning. The olivocerebellar pathway is a key pathway contributing to learning of motor skills. Its developmental maturation and remodeling are being unraveled. Advances in genetics have led to major improvements in our appraisal of the genes involved in cerebellar development, especially studies in mutant mice. Cerebellar neurogenesis is compartmentalized in relationship with neurotransmitter fate. The Engrailed-2 gene is a major actor of the specification of cerebellar cell types and late embryogenic morphogenesis. Math1, expressed by the rhombic lip, is required for the genesis of glutamatergic neurons. Mutants deficient for the transcription factor Ptf1a display a lack of Purkinje cells and gabaergic interneurons. Rora gene contributes to the developmental signaling between granule cells and Purkinje neurons. The expression profile of sonic hedgehog in postnatal stages determines the final size/shape of the cerebellum. Genes affecting the development impact upon the physiological properties of the cerebellar circuits. For instance, receptors are developmentally regulated and their action interferes directly with developmental processes. Another field of research which is expanding relates to very preterm neonates. They are at risk for cerebellar lesions, which may themselves impair the developmental events. Very preterm neonates often show sensori-motor deficits, highlighting another major link between impaired developments and learning deficiencies. Pathways playing a critical role in cerebellar development are likely to become therapeutical targets for several neurodevelopmental disorders.