Cellular development and evolution of the mammalian cerebellum.

The expansion of the neocortex, a hallmark of mammalian evolution1,2, was accompanied by an increase in cerebellar neuron numbers3. However, little is known about the evolution of the cellular programmes underlying the development of the cerebellum in mammals. In this study we generated single-nucleus RNA-sequencing data for around 400,000 cells to trace the development of the cerebellum from early neurogenesis to adulthood in human, mouse and the marsupial opossum. We established a consensus classification of the cellular diversity in the developing mammalian cerebellum and validated it by spatial mapping in the fetal human cerebellum. Our cross-species analyses revealed largely conserved developmental dynamics of cell-type generation, except for Purkinje cells, for which we observed an expansion of early-born subtypes in the human lineage. Global transcriptome profiles, conserved cell-state markers and gene-expression trajectories across neuronal differentiation show that cerebellar cell-type-defining programmes have been overall preserved for at least 160 million years. However, we also identified many orthologous genes that gained or lost expression in cerebellar neural cell types in one of the species or evolved new expression trajectories during neuronal differentiation, indicating widespread gene repurposing at the cell-type level. In sum, our study unveils shared and lineage-specific gene-expression programmes governing the development of cerebellar cells and expands our understanding of mammalian brain evolution.

Arc Oligomerization Is Regulated by CaMKII Phosphorylation of the GAG Domain: An Essential Mechanism for Plasticity and Memory Formation.

Arc is a synaptic protein essential for memory consolidation. Recent studies indicate that Arc originates in evolution from a Ty3-Gypsy retrotransposon GAG domain. The N-lobe of Arc GAG domain acquired a hydrophobic binding pocket in higher vertebrates that is essential for Arc's canonical function to weaken excitatory synapses. Here, we report that Arc GAG also acquired phosphorylation sites that can acutely regulate its synaptic function. CaMKII phosphorylates the N-lobe of the Arc GAG domain and disrupts an interaction surface essential for high-order oligomerization. In Purkinje neurons, CaMKII phosphorylation acutely reverses Arc's synaptic action. Mutant Arc that cannot be phosphorylated by CaMKII enhances metabotropic receptor-dependent depression in the hippocampus but does not alter baseline synaptic transmission or long-term potentiation. Behavioral studies indicate that hippocampus- and amygdala-dependent learning requires Arc GAG domain phosphorylation. These studies provide an atomic model for dynamic and local control of Arc function underlying synaptic plasticity and memory.

Novel genetic features of human and mouse Purkinje cell differentiation defined by comparative transcriptomics.

Comparative transcriptomics between differentiating human pluripotent stem cells (hPSCs) and developing mouse neurons offers a powerful approach to compare genetic and epigenetic pathways in human and mouse neurons. To analyze human Purkinje cell (PC) differentiation, we optimized a protocol to generate human pluripotent stem cell-derived Purkinje cells (hPSC-PCs) that formed synapses when cultured with mouse cerebellar glia and granule cells and fired large calcium currents, measured with the genetically encoded calcium indicator jRGECO1a. To directly compare global gene expression of hPSC-PCs with developing mouse PCs, we used translating ribosomal affinity purification (TRAP). As a first step, we used Tg(Pcp2-L10a-Egfp) TRAP mice to profile actively transcribed genes in developing postnatal mouse PCs and used metagene projection to identify the most salient patterns of PC gene expression over time. We then created a transgenic Pcp2-L10a-Egfp TRAP hPSC line to profile gene expression in differentiating hPSC-PCs, finding that the key gene expression pathways of differentiated hPSC-PCs most closely matched those of late juvenile mouse PCs (P21). Comparative bioinformatics identified classical PC gene signatures as well as novel mitochondrial and autophagy gene pathways during the differentiation of both mouse and human PCs. In addition, we identified genes expressed in hPSC-PCs but not mouse PCs and confirmed protein expression of a novel human PC gene, CD40LG, expressed in both hPSC-PCs and native human cerebellar tissue. This study therefore provides a direct comparison of hPSC-PC and mouse PC gene expression and a robust method for generating differentiated hPSC-PCs with human-specific gene expression for modeling developmental and degenerative cerebellar disorders.

Modulation of the dynamics of cerebellar Purkinje cells through the interaction of excitatory and inhibitory feedforward pathways.

The dynamics of cerebellar neuronal networks is controlled by the underlying building blocks of neurons and synapses between them. For which, the computation of Purkinje cells (PCs), the only output cells of the cerebellar cortex, is implemented through various types of neural pathways interactively routing excitation and inhibition converged to PCs. Such tuning of excitation and inhibition, coming from the gating of specific pathways as well as short-term plasticity (STP) of the synapses, plays a dominant role in controlling the PC dynamics in terms of firing rate and spike timing. PCs receive cascade feedforward inputs from two major neural pathways: the first one is the feedforward excitatory pathway from granule cells (GCs) to PCs; the second one is the feedforward inhibition pathway from GCs, via molecular layer interneurons (MLIs), to PCs. The GC-PC pathway, together with short-term dynamics of excitatory synapses, has been a focus over past decades, whereas recent experimental evidence shows that MLIs also greatly contribute to controlling PC activity. Therefore, it is expected that the diversity of excitation gated by STP of GC-PC synapses, modulated by strong inhibition from MLI-PC synapses, can promote the computation performed by PCs. However, it remains unclear how these two neural pathways are interacted to modulate PC dynamics. Here using a computational model of PC network installed with these two neural pathways, we addressed this question to investigate the change of PC firing dynamics at the level of single cell and network. We show that the nonlinear characteristics of excitatory STP dynamics can significantly modulate PC spiking dynamics mediated by inhibition. The changes in PC firing rate, firing phase, and temporal spike pattern, are strongly modulated by these two factors in different ways. MLIs mainly contribute to variable delays in the postsynaptic action potentials of PCs while modulated by excitation STP. Notably, the diversity of synchronization and pause response in the PC network is governed not only by the balance of excitation and inhibition, but also by the synaptic STP, depending on input burst patterns. Especially, the pause response shown in the PC network can only emerge with the interaction of both pathways. Together with other recent findings, our results show that the interaction of feedforward pathways of excitation and inhibition, incorporated with synaptic short-term dynamics, can dramatically regulate the PC activities that consequently change the network dynamics of the cerebellar circuit.

Programmed cell death in cerebellar Purkinje neurons.

Apoptosis, autophagy and necrosis are the three main types of programmed cell death. One or more of these types of programmed cell death may take place in neurons leading to their death in various neurodegenerative disorders in humans. Purkinje neurons (PNs) are among the most highly vulnerable population of neurons to cell death in response to intrinsic hereditary diseases or extrinsic toxic, hypoxic, ischemic, and traumatic injury. In this review, we will describe the three main types of programmed cell death, including the molecular mechanisms and the sequence of events in each of them, and thus illustrating the intracellular proteins that mediate and regulate each of these types. Then, we will discuss the role of Ca2+ in PN function and increased vulnerability to cell death. Additionally, PN death will be described in animal models, namely lurcher mutant mouse and shaker mutant rat, in order to illustrate the potential therapeutic implications of programmed cell death in PNs by reviewing the previous studies that were carried out to interfere with the programmed cell death in an attempt to rescue PNs from death.

PDK1 Regulates the Maintenance of Cell Body and the Development of Dendrites of Purkinje Cells by pS6 and PKCγ.

3-Phosphoinositide-dependent protein kinase-1 (PDK1) plays a critical role in the development of mammalian brain. Here, we investigated the role of PDK1 in Purkinje cells (PCs) by generating the PDK1-conditional knock-out mice (cKO) through crossing PV-cre or Pcp2-cre mice with Pdk1fl/fl mice. The male mice were used in the behavioral testing, and the other experiments were performed on mice of both sexes. These PDK1-cKO mice displayed decreased cerebellar size and impaired motor balance and coordination. By the electrophysiological recording, we observed the reduced spontaneous firing of PCs from the cerebellar slices of the PDK1-cKO mice. Moreover, the cell body size of PCs in the PDK1-cKO mice was time dependently reduced compared with that in the control mice. And the morphologic complexity of PCs was also decreased after PDK1 deletion. These effects may have contributed to the reduction of the rpS6 (reduced ribosomal protein S6) phosphorylation and the PKCγ expression in PDK1-cKO mice since the upregulation of pS6 by treatment of 3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-1, the agonist of mTOR1, partly rescued the reduction in the cell body size of the PCs, and the delivery of recombinant adeno-associated virus-PKCγ through cerebellar injection rescued the reduced complexity of the dendritic arbor in PDK1-cKO mice. Together, our data suggest that PDK1, by regulating rpS6 phosphorylation and PKCγ expression, controls the cell body maintenance and the dendritic development in PCs and is critical for cerebellar motor coordination.SIGNIFICANCE STATEMENT Here, we show the role of 3-phosphoinositide-dependent protein kinase-1 (PDK1) in Purkinje cells (PCs). The ablation of PDK1 in PCs resulted in a reduction of cell body size, and dendritic complexity and abnormal spontaneous firing, which attributes to the motor defects in PDK1-conditional knock-out (cKO) mice. Moreover, the ribosomal protein S6 (rpS6) phosphorylation and the expression of PKCγ are downregulated after the ablation of PDK1. Additionally, upregulation of rpS6 phosphorylation by3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-1 partly rescued the reduction in cell body size of PCs, and the overexpression of PKCγ in PDK1-KO PCs rescued the reduction in the dendritic complexity. These findings indicate that PDK1 contributes to the maintenance of the cell body and the dendritic development of PCs by regulating rpS6 phosphorylation and PKCγ expression.

Mosaic Analysis with Double Markers reveals IGF1R function in granule cell progenitors during cerebellar development.

During cerebellar development, granule cell progenitors (GCPs) proliferate exponentially for a fixed period, promoted by paracrine mitogenic factor Sonic Hedgehog (Shh) secreted from Purkinje cells (PCs). Dysregulation of Shh signaling leads to uncontrolled GCP proliferation and medulloblastoma. Serendipitously our previous work discovered insulin-like growth factor 1 (IGF1) as another key driver for medulloblastoma, which led to the current investigation into the role of IGF1 in GCPs during normal development. While the IGF1R conditional knockout model revealed GCP defects in anterior cerebellum, the posterior cerebellum was mostly intact, likely owing to incomplete excision of floxed alleles. To circumvent this hurdle, we enlisted a mouse genetic system called Mosaic Analysis of Double Markers (MADM), which sporadically generates homozygous null cells unequivocally labeled with GFP and their wildtype sibling cells labeled with RFP, enabling phenotypic analysis at single-cell resolution. Using MADM, we found that loss of IGF1R resulted in a 10-fold reduction of GCs in both anterior and posterior cerebellum; and that hindered S phase entry and increased cell cycle exit collectively led to this phenotype. Genetic interaction studies showed that IGF1 signaling prevents GCP cell cycle exit at least partially through suppressing the level of p27kip1, a negative regulator of cell cycle. Finally, we found that IGF1 is produced by PCs in a temporally regulated fashion: it is highly expressed early in development when GCPs proliferate exponentially, then gradually decline as GCPs commit to cell cycle exit. Taken together, our studies reveal IGF1 as a paracrine factor that positively regulates GCP cell cycle in cooperation with Shh, through dampening the level of p27 to prevent precocious cell cycle exit. Our work not only showcases the power of phenotypic analysis by the MADM system but also provides an excellent example of multi-factorial regulation of robust developmental programs.

Caspr2 interacts with type 1 inositol 1,4,5-trisphosphate receptor in the developing cerebellum and regulates Purkinje cell morphology.

Contactin-associated protein-like 2 (Caspr2) is a neurexin-like protein that has been associated with numerous neurological conditions. However, the specific functional roles that Caspr2 plays in the central nervous system and their underlying mechanisms remain incompletely understood. Here, we report on a functional role for Caspr2 in the developing cerebellum. Using a combination of confocal microscopy, biochemical analyses, and behavioral testing, we show that loss of Caspr2 in the Cntnap2-/- knockout mouse results in impaired Purkinje cell dendritic development, altered intracellular signaling, and motor coordination deficits. We also find that Caspr2 is highly enriched at synaptic specializations in the cerebellum. Using a proteomics approach, we identify type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) as a specific synaptic interaction partner of the Caspr2 extracellular domain in the molecular layer of the developing cerebellum. The interaction of the Caspr2 extracellular domain with IP3R1 inhibits IP3R1-mediated changes in cellular morphology. Together, our work defines a mechanism by which Caspr2 controls the development and function of the cerebellum and advances our understanding of how Caspr2 dysfunction might lead to specific brain disorders.

MTCL1 plays an essential role in maintaining Purkinje neuron axon initial segment.

The axon initial segment (AIS) is a specialized domain essential for neuronal function, the formation of which begins with localization of an ankyrin-G (AnkG) scaffold. However, the mechanism directing and maintaining AnkG localization is largely unknown. In this study, we demonstrate that in vivo knockdown of microtubule cross-linking factor 1 (MTCL1) in cerebellar Purkinje cells causes loss of axonal polarity coupled with AnkG mislocalization. MTCL1 lacking MT-stabilizing activity failed to restore these defects, and stable MT bundles spanning the AIS were disorganized in knockdown cells. Interestingly, during early postnatal development, colocalization of MTCL1 with these stable MT bundles was observed prominently in the axon hillock and proximal axon. These results indicate that MTCL1-mediated formation of stable MT bundles is crucial for maintenance of AnkG localization. We also demonstrate that Mtcl1 gene disruption results in abnormal motor coordination with Purkinje cell degeneration, and provide evidence suggesting possible involvement of MTCL1 dysfunction in the pathogenesis of spinocerebellar ataxia.

Learning from the past: A reverberation of past errors in the cerebellar climbing fiber signal.

The cerebellum allows us to rapidly adjust motor behavior to the needs of the situation. It is commonly assumed that cerebellum-based motor learning is guided by the difference between the desired and the actual behavior, i.e., by error information. Not only immediate but also future behavior will benefit from an error because it induces lasting changes of parallel fiber synapses on Purkinje cells (PCs), whose output mediates the behavioral adjustments. Olivary climbing fibers, likewise connecting with PCs, are thought to transport information on instant errors needed for the synaptic modification yet not to contribute to error memory. Here, we report work on monkeys tested in a saccadic learning paradigm that challenges this concept. We demonstrate not only a clear complex spikes (CS) signature of the error at the time of its occurrence but also a reverberation of this signature much later, before a new manifestation of the behavior, suitable to improve it.

Control of motor coordination by astrocytic tonic GABA release through modulation of excitation/inhibition balance in cerebellum.

Tonic inhibition in the brain is mediated through an activation of extrasynaptic GABAA receptors by the tonically released GABA, resulting in a persistent GABAergic inhibitory action. It is one of the key regulators for neuronal excitability, exerting a powerful action on excitation/inhibition balance. We have previously reported that astrocytic GABA, synthesized by monoamine oxidase B (MAOB), mediates tonic inhibition via GABA-permeable bestrophin 1 (Best1) channel in the cerebellum. However, the role of astrocytic GABA in regulating neuronal excitability, synaptic transmission, and cerebellar brain function has remained elusive. Here, we report that a reduction of tonic GABA release by genetic removal or pharmacological inhibition of Best1 or MAOB caused an enhanced neuronal excitability in cerebellar granule cells (GCs), synaptic transmission at the parallel fiber-Purkinje cell (PF-PC) synapses, and motor performance on the rotarod test, whereas an augmentation of tonic GABA release by astrocyte-specific overexpression of MAOB resulted in a reduced neuronal excitability, synaptic transmission, and motor performance. The bidirectional modulation of astrocytic GABA by genetic alteration of Best1 or MAOB was confirmed by immunostaining and in vivo microdialysis. These findings indicate that astrocytes are the key player in motor coordination through tonic GABA release by modulating neuronal excitability and could be a good therapeutic target for various movement and psychiatric disorders, which show a disturbed excitation/inhibition balance.

Role of Reelin in cell positioning in the cerebellum and the cerebellum-like structure in zebrafish.

The cerebellum and the cerebellum-like structure in the mesencephalic tectum in zebrafish contain multiple cell types, including principal cells (i.e., Purkinje cells and type I neurons) and granule cells, that form neural circuits in which the principal cells receive and integrate inputs from granule cells and other neurons. It is largely unknown how these cells are positioned and how neural circuits form. While Reelin signaling is known to play an important role in cell positioning in the mammalian brain, its role in the formation of other vertebrate brains remains elusive. Here we found that zebrafish with mutations in Reelin or in the Reelin-signaling molecules Vldlr or Dab1a exhibited ectopic Purkinje cells, eurydendroid cells (projection neurons), and Bergmann glial cells in the cerebellum, and ectopic type I neurons in the tectum. The ectopic Purkinje cells and type I neurons received aberrant afferent fibers in these mutants. In wild-type zebrafish, reelin transcripts were detected in the internal granule cell layer, while Reelin protein was localized to the superficial layer of the cerebellum and the tectum. Laser ablation of the granule cell axons perturbed the localization of Reelin, and the mutation of both kif5aa and kif5ba, which encode major kinesin I components in the granule cells, disrupted the elongation of granule cell axons and the Reelin distribution. Our findings suggest that in zebrafish, (1) Reelin is transported from the granule cell soma to the superficial layer by axonal transport; (2) Reelin controls the migration of neurons and glial cells from the ventricular zone; and (3) Purkinje cells and type I neurons attract afferent axons during the formation of the cerebellum and the cerebellum-like structure.

Loss of cerebellar glutamate transporters EAAT4 and GLAST differentially affects the spontaneous firing pattern and survival of Purkinje cells.

Loss of excitatory amino acid transporters (EAATs) has been implicated in a number of human diseases including spinocerebellar ataxias, Alzhiemer's disease and motor neuron disease. EAAT4 and GLAST/EAAT1 are the two predominant EAATs responsible for maintaining low extracellular glutamate levels and preventing neurotoxicity in the cerebellum, the brain region essential for motor control. Here using genetically modified mice we identify new critical roles for EAAT4 and GLAST/EAAT1 as modulators of Purkinje cell (PC) spontaneous firing patterns. We show high EAAT4 levels, by limiting mGluR1 signalling, are essential in constraining inherently heterogeneous firing of zebrin-positive PCs. Moreover mGluR1 antagonists were found to restore regular spontaneous PC activity and motor behaviour in EAAT4 knockout mice. In contrast, GLAST/EAAT1 expression is required to sustain normal spontaneous simple spike activity in low EAAT4 expressing (zebrin-negative) PCs by restricting NMDA receptor activation. Blockade of NMDA receptor activity restores spontaneous activity in zebrin-negative PCs of GLAST knockout mice and furthermore alleviates motor deficits. In addition both transporters have differential effects on PC survival, with zebrin-negative PCs more vulnerable to loss of GLAST/EAAT1 and zebrin-positive PCs more vulnerable to loss of EAAT4. These findings reveal that glutamate transporter dysfunction through elevated extracellular glutamate and the aberrant activation of extrasynaptic receptors can disrupt cerebellar output by altering spontaneous PC firing. This expands our understanding of disease mechanisms in cerebellar ataxias and establishes EAATs as targets for restoring homeostasis in a variety of neurological diseases where altered cerebellar output is now thought to play a key role in pathogenesis.

Zfp423/ZNF423 regulates cell cycle progression, the mode of cell division and the DNA-damage response in Purkinje neuron progenitors.

The Zfp423/ZNF423 gene encodes a 30-zinc-finger transcription factor involved in key developmental pathways. Although null Zfp423 mutants develop cerebellar malformations, the underlying mechanism remains unknown. ZNF423 mutations are associated with Joubert Syndrome, a ciliopathy causing cerebellar vermis hypoplasia and ataxia. ZNF423 participates in the DNA-damage response (DDR), raising questions regarding its role as a regulator of neural progenitor cell cycle progression in cerebellar development. To characterize in vivo the function of ZFP423 in neurogenesis, we analyzed allelic murine mutants in which distinct functional domains are deleted. One deletion impairs mitotic spindle orientation, leading to premature cell cycle exit and Purkinje cell (PC) progenitor pool deletion. The other deletion impairs PC differentiation. In both mutants, cell cycle progression is remarkably delayed and DDR markers are upregulated in cerebellar ventricular zone progenitors. Our in vivo evidence sheds light on the domain-specific roles played by ZFP423 in different aspects of PC progenitor development, and at the same time strengthens the emerging notion that an impaired DDR may be a key factor in the pathogenesis of JS and other ciliopathies.

DeTerm: Software for automatic detection of neuronal dendritic branch terminals via an artificial neural network.

Dendrites of neurons receive and process synaptic or sensory inputs. The Drosophila class IV dendritic arborization (da) neuron is an established model system to explore molecular mechanisms of dendrite morphogenesis. The total number of dendritic branch terminals is one of the frequently employed parameters to characterize dendritic arborization complexity of class IV neurons. This parameter gives a useful phenotypic readout of arborization during neurogenesis, and it is typically determined by laborious manual analyses of numerous images. Ideally, an automated analysis would greatly reduce the workload; however, it is challenging to automatically discriminate dendritic branch terminals from signals of surrounding tissues in whole-mount live larvae. Here, we describe our newly developed software, called DeTerm, which automatically recognizes and quantifies dendrite branch terminals via an artificial neural network. Once we input an image file of a neuronal dendritic arbor and its region of interest information, DeTerm is capable of labeling terminals of larval class IV neurons with high precision, and it also provides positional data of individual terminals. We further show that DeTerm is applicable to other types of neurons, including mouse cerebellar Purkinje cells. DeTerm is freely available on the web and was successfully tested on Mac, Windows and Linux.

Nitric Oxide Critically Regulates Purkinje Neuron Dendritic Development Through a Metabotropic Glutamate Receptor Type 1-Mediated Mechanism.

Nitric oxide (NO), specifically derived from neuronal nitric oxide synthase (nNOS), is a well-established regulator of synaptic transmission in Purkinje neurons (PNs), governing fundamental processes such as motor learning and coordination. Previous phenotypic analyses showed similar cerebellar structures between neuronal nitric oxide null (nNOS-/-) and wild-type (WT) adult male mice, despite prominent ataxic behavior within nNOS-/- mice. However, a study has yet to characterize PN molecular structure and their excitatory inputs during development in nNOS-/- mice. This study is the first to explore morphological abnormalities within the cerebellum of nNOS-/- mice, using immunohistochemistry and immunoblotting. This study sought to examine PN dendritic morphology and the expression of metabotropic glutamate receptor type 1 (mGluR1), vesicular glutamate transporter type 1 and 2 (vGluT1 and vGluT2), stromal interaction molecule 1 (STIM1), and calpain-1 within PNs of WT and nNOS-/- mice at postnatal day 7 (PD7), 2 weeks (2W), and 7 weeks (7W) of age. Results showed a decrease in PN dendritic branching at PD7 in nNOS-/- cerebella, while aberrant dendritic spine formation was noted in adult ages. Total protein expression of mGluR1 was decreased in nNOS-/- cerebella across development, while vGluT2, STIM1, and calpain-1 were significantly increased. Ex vivo treatment of WT slices with NOS inhibitor L-NAME increased calpain-1 expression, whereas treating nNOS-/- cerebellar slices with NO donor NOC-18 decreased calpain-1. Moreover, mGluR1 agonist DHPG increased calpain-1 in WT, but not in nNOS-/- slices. Together, these results indicate a novel role for nNOS/NO signaling in PN development, particularly by regulating an mGluR1-initiated calcium signaling mechanism.

Purkinje-cell plasticity and cerebellar motor learning are graded by complex-spike duration.

Behavioural learning is mediated by cellular plasticity, such as changes in the strength of synapses at specific sites in neural circuits. The theory of cerebellar motor learning relies on movement errors signalled by climbing-fibre inputs to cause long-term depression of synapses from parallel fibres to Purkinje cells. However, a recent review has called into question the widely held view that the climbing-fibre input is an 'all-or-none' event. In anaesthetized animals, there is wide variation in the duration of the complex spike (CS) caused in Purkinje cells by a climbing-fibre input. Furthermore, the amount of plasticity in Purkinje cells is graded according to the duration of electrically controlled bursts in climbing fibres. The duration of bursts depends on the 'state' of the inferior olive and therefore may be correlated across climbing fibres. Here we provide a potential functional context for these mechanisms during motor learning in behaving monkeys. The magnitudes of both plasticity and motor learning depend on the duration of the CS responses. Furthermore, the duration of CS responses seems to be a meaningful signal that is correlated across the Purkinje-cell population during motor learning. We suggest that during learning, longer bursts in climbing fibres lead to longer-duration CS responses in Purkinje cells, more calcium entry into Purkinje cells, larger synaptic depression, and stronger learning. The same graded impact of instructive signals for plasticity and learning might occur throughout the nervous system.

Selection for Divergent Reproductive Investment Affects Neuron Size and Foliation in the Cerebellum.

The cerebellum has a highly conserved internal circuitry, but varies greatly in size and morphology within and across species. Despite this variation, the underlying volumetric changes among the layers of the cerebellar cortex or their association with Purkinje cell numbers and sizes is poorly understood. Here, we examine intraspecific scaling relationships and variation in the quantitative neuroanatomy of the cerebellum in Japanese quail (Coturnix japonica) selected for high or low reproductive investment. As predicted by the circuitry of the cerebellum, the volumes of the constituent layers of the cerebellar cortex were strongly and positively correlated with one another and with total cerebellar volume. The number of Purkinje cells also significantly and positively co-varied with total cerebellar volume and the molecular layer, but not the granule cell layer or white matter volumes. Purkinje cell size and cerebellar foliation did not significantly covary with any cerebellar measures, but differed significantly between the selection lines. Males and females from the high-investment lines had smaller Purkinje cells than males and females from the low-investment lines and males from the high-investment lines had less folded cerebella than quail from the low-investment lines. These results suggest that within species, the layers of the cerebellum increase in a coordinated fashion, but Purkinje cell size and cerebellar foliation do not increase proportionally with overall cerebellum size. In contrast, selection for differential reproductive investment affects Purkinje cell size and cerebellar foliation, but not other quantitative measures of cerebellar anatomy.

Allometric Scaling Rules of the Cerebellum in Galliform Birds.

Although the internal circuitry of the cerebellum is highly conserved across vertebrate species, the size and shape of the cerebellum varies considerably. Recent comparative studies have examined the allometric rules between cerebellar mass and number of neurons, but data are lacking on the numbers and sizes of Purkinje and granule cells or scaling of cerebellar foliation. Here, we investigate the allometric rules that govern variation in the volumes of the layers of the cerebellum, the numbers and sizes of Purkinje cells and granule cells and the degree of the cerebellar foliation across 7 species of galliform birds. We selected Galliformes because they vary greatly in body and brain sizes. Our results show that the molecular, granule and white matter layers all increase in volume at the same rate relative to total cerebellum volume. Both numbers and sizes of Purkinje cells increased with cerebellar volume, but numbers of Purkinje cells increased at a much faster rate than size. Granule cell numbers increased with cerebellar volume, but size did not. Sizes and numbers of Purkinje cells as well as numbers of granule cells were positively correlated with the degree of cerebellar foliation, but granule cell size decreased with higher degrees of foliation. The concerted changes among the volumes of cerebellar layers likely reflects the conserved neural circuitry of the cerebellum. Also, our data indicate that the scaling of cell sizes can vary markedly across neuronal populations, suggesting that evolutionary changes in cell sizes might be more complex than what is often assumed.

Regulation of spinogenesis in mature Purkinje cells via mGluR/PKC-mediated phosphorylation of CaMKIIß.

Dendritic spines of Purkinje cells form excitatory synapses with parallel fiber terminals, which are the primary sites for cerebellar synaptic plasticity. Nevertheless, how density and morphology of these spines are properly maintained in mature Purkinje cells is not well understood. Here we show an activity-dependent mechanism that represses excessive spine development in mature Purkinje cells. We found that CaMKIIß promotes spine formation and elongation in Purkinje cells through its F-actin bundling activity. Importantly, activation of group I mGluR, but not AMPAR, triggers PKC-mediated phosphorylation of CaMKIIß, which results in dissociation of the CaMKIIß/F-actin complex. Defective function of the PKC-mediated CaMKIIß phosphorylation promotes excess F-actin bundling and leads to abnormally numerous and elongated spines in mature IP3R1-deficient Purkinje cells. Thus, our data suggest that phosphorylation of CaMKIIß through the mGluR/IP3R1/PKC signaling pathway represses excessive spine formation and elongation in mature Purkinje cells.