Search

We studied morphological features of the cerebellum in 14-day-old Wistar rats from reduced litters (the number of pups was reduced from 10-12 to 6 on the next day after birth). The control group comprised 14-day-old animals from litters of medium size (10-12 rat pups). Rats from reduced litters had greater body weight and brain weight. The weight of the cerebellum, together with the weight of the adjacent part of the brain stem and the thickness of cerebellar cortex also significantly exceeded the corresponding parameters in control animals. The thickness of the molecular layer of the cerebellar cortex and the numerical density of Purkinje cells in these rats did not differ significantly from the control. The thickness of the external granular (neurogenic) layer of the cerebellar cortex in rats from reduced litters was smaller. This can indicate accelerated reduction of this layer that persists in rats until days 20-22 of age. Numerical density of cells in the external granular layer of control and experimental animals was similar. Numerical density Ki-67 + cells in this layer, as well as GFAP + glial cells in the granular layer of the cerebellar cortex in rats from reduced litters significantly exceeded the corresponding parameters in control animals. The cerebellum of rats from litters reduced 1 day after birth had a number of differences in important indicators reflecting the rate of its development during the neonatal and suckling periods of ontogeny., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

This review article will cover the recent developments in the new evolving field of Purkinje bioengineering and the development of human Purkinje networks. Recent work has progressed to the point of a methodological and systematic process to bioengineer Purkinje networks. This involves the development of 3D models based on human anatomy, followed by the development of tunable biomaterials, and strategies to reprogram stem cells to Purkinje cells. Subsequently, the reprogrammed cells and the biomaterials are coupled to bioengineer Purkinje networks, which are then tested using a small animal injury model. In this article, we discuss this process as a whole and then each step separately. We then describe potential applications of bioengineered Purkinje networks and challenges in the field that need to be overcome to move this field forward. Although the field of Purkinje bioengineering is new and in a state of infancy, it holds tremendous potential, both for therapeutic applications and to develop tools that can be used for disease modeling., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The expansion of the neocortex, a hallmark of mammalian evolution 1,2 , was accompanied by an increase in cerebellar neuron numbers 3 . 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., (© 2023. The Author(s).)

Although circadian and sleep disorders are frequently associated with autism spectrum disorders (ASD), it remains elusive whether clock gene disruption can lead to autistic-like phenotypes in animals. The essential clock gene Bmal1 has been associated with human sociability and its missense mutations are identified in ASD. Here we report that global Bmal1 deletion led to significant social impairments, excessive stereotyped and repetitive behaviors, as well as motor learning disabilities in mice, all of which resemble core behavioral deficits in ASD. Furthermore, aberrant cell density and immature morphology of dendritic spines were identified in the cerebellar Purkinje cells (PCs) of Bmal1 knockout (KO) mice. Electrophysiological recordings uncovered enhanced excitatory and inhibitory synaptic transmission and reduced firing rates in the PCs of Bmal1 KO mice. Differential expression of ASD- and ataxia-associated genes (Ntng2, Mfrp, Nr4a2, Thbs1, Atxn1, and Atxn3) and dysregulated pathways of translational control, including hyperactivated mammalian target of rapamycin complex 1 (mTORC1) signaling, were identified in the cerebellum of Bmal1 KO mice. Interestingly, the antidiabetic drug metformin reversed mTORC1 hyperactivation and alleviated major behavioral and PC deficits in Bmal1 KO mice. Importantly, conditional Bmal1 deletion only in cerebellar PCs was sufficient to recapitulate autistic-like behavioral and cellular changes akin to those identified in Bmal1 KO mice. Together, these results unveil a previously unidentified role for Bmal1 disruption in cerebellar dysfunction and autistic-like behaviors. Our findings provide experimental evidence supporting a putative role for dysregulation of circadian clock gene expression in the pathogenesis of ASD., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

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., Competing Interests: The author declares no conflict of interest., (© 2022 The Author(s). Published by IMR Press.)

The brain selectively allocates attention from a continuous stream of sensory input. This process is typically attributed to computations in distinct regions of the forebrain and midbrain. Here, we explore whether cerebellar Purkinje cells encode information about the selection of sensory inputs and could thereby contribute to non-motor forms of learning. We show that complex spikes of individual Purkinje cells change the sensory modality they encode to reflect changes in the perceived salience of sensory input. Comparisons with mouse models deficient in cerebellar plasticity suggest that changes in complex spike activity instruct potentiation of Purkinje cells simple spike firing, which is required for efficient learning. Our findings suggest that during learning, climbing fibers do not directly guide motor output, but rather contribute to a general readiness to act via changes in simple spike activity, thereby bridging the sequence from non-motor to motor functions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Sensory processing is essential for motor control. Climbing fibers from the inferior olive transmit sensory signals to Purkinje cells, but how the signals are represented in the cerebellar cortex remains elusive. To examine the olivocerebellar organization of the mouse brain, we perform quantitative Ca 2+ imaging to measure complex spikes (CSs) evoked by climbing fiber inputs over the entire dorsal surface of the cerebellum simultaneously. The surface is divided into approximately 200 segments, each composed of ∼100 Purkinje cells that fire CSs synchronously. Our in vivo imaging reveals that, although stimulation of four limb muscles individually elicits similar global CS responses across nearly all segments, the timing and location of a stimulus are derived by Bayesian inference from coordinated activation and inactivation of multiple segments on a single trial basis. We propose that the cerebellum performs segment-based, distributed-population coding that represents the conditional probability of sensory events., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Synaptotagmin 7 (Syt7) is a high-affinity calcium sensor that is implicated in multiple aspects of synaptic transmission. Here, we study the influence of Syt7 on the climbing fiber (CF) to Purkinje cell (PC) synapse. We find that small facilitation and prominent calcium-dependent recovery from depression at this synapse do not rely on Syt7 and that Syt7 is not normally present in CFs. We expressed Syt7 in CFs to assess the consequences of introducing Syt7 to a synapse that normally lacks Syt7. Syt7 expression does not promote asynchronous release or accelerate recovery from depression. Syt7 decreases the excitatory postsynaptic current (EPSC) magnitude, consistent with a decrease in the initial probability of release (P R ). Syt7 also increases synaptic facilitation to such a large extent that it could not arise solely as an indirect consequence of decreased P R . Thus, the primary consequence of Syt7 expression in CFs, which normally lack Syt7, is to promote synaptic facilitation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purkinje cells are critically involved in processing the cerebellar functions by shaping and coordinating commands that they receive. Here, we demonstrate experimentally that in the adult zebrafish valvular part of the cerebellum, the Purkinje cells exhibited variable firing and functional responses and allowed the categorization into three firing classes. Compared with the Purkinje cells in the corpus cerebelli, the valvular Purkinje cells receive weak and occasional input from the inferior olive and are not active during locomotion. Together, our findings expand further the regional functional differences of the Purkinje cell population and expose their non-locomotor functionality., (© 2021. The Author(s).)

Histone variants are crucial regulators of chromatin structure and gene transcription, yet their functions within the brain remain largely unexplored. Here, we show that the H2A histone variant H2A.Z is essential for neuronal survival. Mice lacking H2A.Z in GABAergic neurons or Purkinje cells (PCs) present with a progressive cerebellar ataxia accompanied by widespread degeneration of PCs. Ablation of H2A.Z in other neuronal subtypes also triggers cell death. H2A.Z binds to the promoters of key nuclear-encoded mitochondrial genes to regulate their expression and promote organelle function. Bolstering mitochondrial activity genetically or by organelle transplant enhances the survival of H2A.Z-ablated neurons. Changes in bioenergetic status alter H2A.Z occupancy at the promoters of nuclear-encoded mitochondrial genes, an adaptive response essential for cell survival. Our results highlight that H2A.Z fulfills a key, conserved role in neuronal survival by acting as a transcriptional rheostat to regulate the expression of genes critical to mitochondrial function., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purkinje cells of the cerebellum have a complex arborized arrangement of dendrites and are among the most distinctive cell types of the nervous system. Although the neuromorphology of Purkinje cells has been well described for some mammals and teleost fish, for most vertebrates less is known. Here we used a modified Golgi-Cox method to investigate the neuromorphology of Purkinje cells from the lizard Eublepharis macularius, the leopard gecko. Using Sholl and Branch Structure Analyses, we sought to investigate whether the neuromorphology of gecko Purkinje cells was altered in response to tail loss and regeneration. Tail loss is an evolved mechanism commonly used by geckos to escape predation. Loss of the tail represents a significant and sudden change in body length and mass, which is only partially recovered as the tail is regenerated. We predicted that tail loss and regeneration would induce a quantifiable change in Purkinje cell dendrite arborization. Post hoc comparisons of Sholl analyses data showed that geckos with regenerated tails have significant changes in dendrite diameter and the number of dendrite intersections in regions corresponding to the position of parallel fiber synapses. We propose that the neuromorphological alterations observed in gecko Purkinje cells represent a compensatory response to tail regrowth, and perhaps a role in motor learning., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

T-type Ca 2+ channels, generating low threshold calcium influx in neurons, play a crucial role in the function of neuronal networks and their plasticity. To further investigate their role in the complex field of research in plasticity of neurons on a molecular level, this study aimed to analyse the impact of the vascular endothelial growth factor (VEGF) on these channels. VEGF, known as a player in vasculogenesis, also shows potent influence in the central nervous system, where it elicits neuronal growth. To investigate the influence of VEGF on the three T-type Ca 2+ channel isoforms, Cav3.1 (encoded by Cacna1g ), Cav3.2 (encoded by Cacna1h ), and Cav3.3 (encoded by Cacna1i ), lasermicrodissection of in vivo-grown Purkinje cells (PCs) was performed, gene expression was analysed via qPCR and compared to in vitro-grown PCs. We investigated the VEGF receptor composition of in vivo- and in vitro-grown PCs and underlined the importance of VEGF receptor 2 for PCs. Furthermore, we performed immunostaining of T-type Ca 2+ channels with in vivo- and in vitro-grown PCs and showed the distribution of T-type Ca 2+ channel expression during PC development. Overall, our findings provide the first evidence that the mRNA expression of Cav3.1, Cav3.2, and Cav3.3 increases due to VEGF stimulation, which indicates an impact of VEGF on neuronal plasticity.

The architecturally stereotypical structure of cerebellum is ideal for investigating the generation of neuronal diversity, but in vitro models for assessing early cerebellar progenitor differentiation were lacking. Here, we report a detailed protocol for long-term in vitro generation of Pax6 + granule cells and Calbindin + Purkinje cells from common Sox2 + embryonic cerebellar progenitors. We describe the procedure for dissecting mouse cerebellar anlage, cell seeding, and tamoxifen-induced labeling of progenitor cells, followed by time-lapse video recording of clonal expansion and neuronal differentiation. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Our previous studies on cannabinoid type1 receptor (CB1R) activation on Methamphetamine (METH)-induced neurodegeneration and locomotion impairments in male rats suggest an interaction between CB1Rs and METH. However, the role of these receptors in METH-neurotoxicity has not been fully identified. Therefore, the purpose of the present study is to investigate the involvement of CB1Rs in these effects. We conducted an electrophysiological study to evaluate functional interactions between METH and CB1Rs using whole-cell patch current clamp recording. Furthermore, we designed the Nissl staining protocol to assess the effect of METH on the basic cerebellar Purkinje cell structure. Our findings revealed that METH significantly increased the action potential half-width, spontaneous interspike intervals, first spike latency, and decreased the rebound action potential and spontaneous firing frequency. Using CB1R agonist and antagonist, our results showed a significant interaction with some of the electrophysiological alterations induced by METH. Further, Nissl staining revealed that the exposure to the combination of METH and SR141716A resulted in the necrotic cell death. Results of the current study raises the possibility that METH consumption profoundly affect the intrinsic membrane properties of cerebellar Purkinje neurons and cannabinoid system manipulations may counteract some of these effects. In summary, our findings provide further insights into the modulatory role of the endocannabinoid system in METH-induced neurologic changes, which can be used in the development of potential therapeutic interventions for METH dependence.

Reduced cerebellar volume and motor dysfunction have previously been observed in the GFAP-IL6 murine model of chronic neuroinflammation. This study aims to extend these findings by investigating the effect of microglial activation and ageing on the total number of Purkinje cells and the morphology of their dendritic arborization. Through comparison of transgenic GFAP-IL6 mice and their wild-type counterparts at the ages of 12 and 24-months, we were able to investigate the effects of ageing and chronic microglial activation on Purkinje cells. Unbiased stereology was used to estimate the number of microglia in Iba1 + stained tissue and Purkinje cells in calbindin stained tissue. Morphological analyses were made using 3D reconstructions of images acquired from the Golgi-stained cerebellar tissue. We found that the total number of microglia increased by approximately 5 times in the cerebellum of GFAP-IL6 mice compared to their WT littermates. The number of Purkinje cells decreased by as much as 50 % in aged wild type mice and 83 % in aged GFAP-IL6 mice. The remaining Purkinje cells in these cohorts were found to have significant reductions in their total dendritic length and number of branching points, indicating how the complexity of the Purkinje cell dendritic arbor reduces through age and inflammation. GFAP-IL6 mice, when compared to WT mice, had higher levels of microglial activation and more profound neurodegenerative changes in the cerebellum. The presence of constitutive IL6 production, driving chronic neuroinflammation, may account for these neurodegenerative changes in GFAP-IL6 mice., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist.

Hodgkin and Huxley's seminal neuron model describes the propagation of voltage spikes in axons, but it cannot explain certain full-neuron features crucial for understanding the neural code. We consider channel current fluctuations in a trisection of the Hodgkin-Huxley model, allowing an analytic-mechanistic explanation of these features and yielding consistently excellent matches with in vivo recordings of cerebellar Purkinje neurons, which we use as model systems. This shows that the neuronal encoding is described conclusively by a soft-thresholding function having just three parameters.

The dimorphism among male, female and freemartin intersex bovines, focusing on the vermal lobules VIII and IX, was analyzed using a novel data analytics approach to quantify morphometric differences in the cytoarchitecture of digitalized sections of the cerebellum. This methodology consists of multivariate and multi-aspect testing for cytoarchitecture-ranking, based on neuronal cell complexity among populations defined by factors, such as sex, age or pathology. In this context, we computed a set of shape descriptors of the neural cell morphology, categorized them into three domains named size, regularity and density, respectively. The output and results of our methodology are multivariate in nature, allowing an in-depth analysis of the cytoarchitectonic organization and morphology of cells. Interestingly, the Purkinje neurons and the underlying granule cells revealed the same morphological pattern: female possessed larger, denser and more irregular neurons than males. In the Freemartin, Purkinje neurons showed an intermediate setting between males and females, while the granule cells were the largest, most regular and dense. This methodology could be a powerful instrument to carry out morphometric analysis providing robust bases for objective tissue screening, especially in the field of neurodegenerative pathologies.

The objective of the present study was to investigate the morphometrical features of the cat cerebellum using design-based stereology. Cerebellar hemispheres from four male cats were examined. Isotropic, uniform random sections were obtained and processed for light microscopy. Cerebellar total volume (V), white (WM) and grey matter (GM) volume fractions, and the volumes of the molecular and granular layers were measured using the Cavalieri's estimator and the point counting system. Cerebellar surface area was estimated using test lines, and Purkinje cellular and nuclear volumes were analysed using the nucleator technique. The volume of the cat cerebellar hemispheres was 2.06 ± 0.29 cm 3 . The relative volume fractions of the GM and WM were 70.6 ± 2.6% and 29.3 ± 2.6%, respectively. The surface area of the cerebellar hemisphere was 68.2 ± 17.8 cm 2 . The volumes of the molecular and granular layers were estimated at 0.89 ± 0.16 cm 3 and 0.56 ± 0.1 cm 3 , respectively. The Purkinje cell volumes were found to be ranging from 1,717 to 28,489 µm 3 , of which cells with a perikaryon volume of 6,994 µm 3 had a higher incidence. The Purkinje nuclear volume was estimated at 440-3,561 µm 3 , and nuclei with a volume of 1,252 µm 3 were the most frequently occurring ones. Our data might contribute to the veterinary comparative neuroanatomy knowledge, help develop experimental studies in this field, and possibly lead to advancement in the diagnosis and treatment of nervous diseases in the cat., (© 2020 Wiley-VCH GmbH.)

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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 (IP 3 R1) 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 IP 3 R1 inhibits IP 3 R1-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., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Argent et al.)

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.

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 Pdk1 fl/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., (Copyright © 2020 the authors.)

Competing Interests: Declaration of competing interest The authors declare no competing interests.

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., Competing Interests: The authors declare no competing interest.

Calcium-calmodulin dependent protein kinase II (CaMKII) regulates many forms of synaptic plasticity, but little is known about its functional role during plasticity induction in the cerebellum. Experiments have indicated that the β isoform of CaMKII controls the bidirectional inversion of plasticity at parallel fibre (PF)-Purkinje cell (PC) synapses in cerebellar cortex. Because the cellular events that underlie these experimental findings are still poorly understood, we developed a simple computational model to investigate how β CaMKII regulates the direction of plasticity in cerebellar PCs. We present the first model of AMPA receptor phosphorylation that simulates the induction of long-term depression (LTD) and potentiation (LTP) at the PF-PC synapse. Our simulation results suggest that the balance of CaMKII-mediated phosphorylation and protein phosphatase 2B (PP2B)-mediated dephosphorylation of AMPA receptors can determine whether LTD or LTP occurs in cerebellar PCs. The model replicates experimental observations that indicate that β CaMKII controls the direction of plasticity at PF-PC synapses, and demonstrates that the binding of filamentous actin to CaMKII can enable the β isoform of the kinase to regulate bidirectional plasticity at these synapses.

Most cerebellar neurons arise from two embryonic stem niches: a rhombic lip niche, which generates all the cerebellar excitatory glutamatergic neurons, and a ventricular zone niche, which generates the inhibitory GABAergic Purkinje cells, which are neurons that constitute the deep cerebellar nuclei and Bergman glia. Recently, a third stem cell niche has been described that arises as a secondary germinal zone from the ventricular zone niche. The cells of this niche are defined by the cell surface marker prominin-1 and are localized to the developing white matter of the postnatal cerebellum. This niche accounts for the late born molecular layer GABAergic interneurons along with postnatally generated cerebellar astrocytes. In addition to their developmental role, this niche is gaining translational importance in regards to its involvement in neurodegeneration and tumorigenesis. The biology of these cells has been difficult to decipher because of a lack of efficient techniques for their purification. Demonstrated here are efficient methods to purify, culture, and differentiate these postnatal cerebellar stem cells.

Introduction: The application of brief high voltage electrical pulses to tissue can lead to an irreversible or reversible electroporation effect in a cell-specific manner. In the management of ventricular arrhythmias, the ability to target different tissue types, specifically cardiac conduction tissue (His-Purkinje System) vs. cardiac myocardium would be advantageous. We hypothesize that pulsed electric fields (PEFs) can be applied safely to the beating heart through a catheter-based approach, and we tested whether the superficial Purkinje cells can be targeted with PEFs without injury to underlying myocardial tissue., Methods: In an acute (n = 5) and chronic canine model (n = 6), detailed electroanatomical mapping of the left ventricle identified electrical signals from myocardial and overlying Purkinje tissue. Electroporation was effected via percutaneous catheter-based Intracardiac bipolar current delivery in the anesthetized animal. Repeat Intracardiac electrical mapping of the heart was performed at acute and chronic time points; followed by histological analysis to assess effects., Results: PEF demonstrated an acute dose-dependent functional effect on Purkinje, with titration of pulse duration and/or voltage associated with successful acute Purkinje damage. Electrical conduction in the insulated bundle of His (n = 2) and anterior fascicle bundle (n = 2), was not affected. At 30 days repeat cardiac mapping demonstrated resilient, normal electrical conduction throughout the targeted area with no significant change in myocardial amplitude (pre 5.9 ± 1.8 mV, 30 days 5.4 ± 1.2 mV, p = 0.92). Histopathological analysis confirmed acute Purkinje fiber targeting, with chronic studies showing normal Purkinje fibers, with minimal subendocardial myocardial fibrosis., Conclusion: PEF provides a novel, safe method for non-thermal acute modulation of the Purkinje fibers without significant injury to the underlying myocardium. Future optimization of this energy delivery is required to optimize conditions so that selective electroporation can be utilized in humans the treatment of cardiac disease., Competing Interests: The authors have declared that no competing interests exist.

Cerebellar granule cells (GCs) make up the majority of all neurons in the vertebrate brain, but heterogeneities among GCs and potential functional consequences are poorly understood. Here, we identified unexpected gradients in the biophysical properties of GCs in mice. GCs closer to the white matter (inner-zone GCs) had higher firing thresholds and could sustain firing with larger current inputs than GCs closer to the Purkinje cell layer (outer-zone GCs). Dynamic Clamp experiments showed that inner- and outer-zone GCs preferentially respond to high- and low-frequency mossy fiber inputs, respectively, enabling dispersion of the mossy fiber input into its frequency components as performed by a Fourier transformation. Furthermore, inner-zone GCs have faster axonal conduction velocity and elicit faster synaptic potentials in Purkinje cells. Neuronal network modeling revealed that these gradients improve spike-timing precision of Purkinje cells and decrease the number of GCs required to learn spike-sequences. Thus, our study uncovers biophysical gradients in the cerebellar cortex enabling a Fourier-like transformation of mossy fiber inputs., Competing Interests: IS, LW, AE, MH, NB, SM, ID, KD, ES, IB, MK, PI, SH No competing interests declared, (© 2020, Straub et al.)

Whole-cell patch-clamp technique was employed to record chloride ionic current I GABA evoked by fast (600 msec) application of GABA to hippocampal pyramidal neurons and cerebellar Purkinje cells isolated from rat brain. GABA solution in the application pipette was either neutral (pH 7.4) or acidic (pH 7.0 or 6.0). Application of protons to neurons causes a rapid, reversible, and dose-dependent decrease in the amplitude of I GABA ; the effect was more pronounced on hippocampal neurons (carrying both synaptic and extrasynaptic GABA A receptors) than in cerebellar Purkinje cells (predominantly equipped with synaptic GABA A receptors). In hippocampal neurons, pharmacological isolation of extrasynaptic component from total I GABA was performed with GABA A receptor antagonist gabazine (50 nM). The extrasynaptic component of I GABA was stronger blocked by protons than total I GABA . It was concluded that acidic medium produced more potent blocking effect on extrasynaptic GABA A receptors than on synaptic ones.

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., (© 2020 S. Karger AG, Basel.)

In humans, pyruvate dehydrogenase complex (PDC) deficiency impairs brain energy metabolism by reducing the availability of the functional acetyl‑CoA pool. This "hypometabolic defect" results in congenital lactic acidosis and abnormalities of brain morphology and function, ranging from mild ataxia to profound psychomotor retardation. Our previous study showed reduction in total cell number and dendritic arbors in the cerebellar Purkinje cells in systemic PDC‑deficient mice. Phenylbutyrate has been shown to increase PDC activity in cultured fibroblasts from PDC‑deficient patients. Hence, we investigated the effects of postnatal (days 2‑35) phenylbutyrate administration on the cerebellar Purkinje cell population in PDC‑deficient female mice. Histological analyses of different regions of cerebellar cortex from the brain‑specific PDC‑deficient saline‑injected mice revealed statistically significant reduction in the Purkinje cell density and increased cell size of the individual Purkinje cell soma compared to control PDC‑normal, saline‑injected group. Administration of phenylbutyrate to control mice did not cause significant changes in the Purkinje cell density and cell size in the studied regions. In contrast, administration of phenylbutyrate variably lessened the ill effects of PDC deficiency on Purkinje cell populations in different areas of the cerebellum. Our results lend further support for the possible use of phenylbutyrate as a potential treatment for PDC deficiency.

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., (© 2020 S. Karger AG, Basel.)

Ecomorphological studies evaluating the impact of environmental and biological factors on the brain have so far focused on morphology or size measurements, and the ecological relevance of potential multi-level variations in brain architecture remains unclear in vertebrates. Here, we exploit the extraordinary ecomorphological diversity of squamates to assess brain phenotypic diversification with respect to locomotor specialization, by integrating single-cell distribution and transcriptomic data along with geometric morphometric, phylogenetic, and volumetric analysis of high-definition 3D models. We reveal significant changes in cerebellar shape and size as well as alternative spatial layouts of cortical neurons and dynamic gene expression that all correlate with locomotor behaviours. These findings show that locomotor mode is a strong predictor of cerebellar structure and pattern, suggesting that major behavioural transitions in squamates are evolutionarily correlated with mosaic brain changes. Furthermore, our study amplifies the concept of 'cerebrotype', initially proposed for vertebrate brain proportions, towards additional shape characters.

The brain has traditionally been considered to be a target site of peripheral steroid hormones. On the other hand, extensive studies over the past thirty years have demonstrated that the brain is a site of biosynthesis of several steroids. Such steroids synthesized de novo from cholesterol in the brain are called neurosteroids. To investigate the biosynthesis and biological actions of neurosteroids in the brain, data on the regio- and temporal-specific synthesis of neurosteroids are needed. In the mid 1990s, the Purkinje cell, an important cerebellar neuron, was discovered as a major cell producing neurosteroids in the brain of vertebrates. It was the first demonstration of de novo neuronal biosynthesis of neurosteroids in the brain. Subsequently, neuronal biosynthesis of neurosteroids and biological actions of neurosteroids have become clear by the follow-up studies using the Purkinje cell as an excellent cellular model. Progesterone and estradiol, which are known as sex steroid hormones, are actively synthesized de novo from cholesterol in the Purkinje cell during development, when cerebellar neuronal circuit formation occurs. Importantly, progesterone and estradiol synthesized in the Purkinje cell promote dendritic growth, spinogenesis and synaptogenesis via their cognate nuclear receptors in the Purkinje cell. Neurotrophic factors may mediate these neurosteroid actions. Futhermore, allopregnanolone (3α,5α-tetrahydroprogesterone), a progesterone metabolite, is also synthesized in the cerebellum and acts on the survival of Purkinje cells. On the other hand, at the beginning of 2010s, the pineal gland, an endocrine organ located close to the cerebellum, was discovered as an important site of the biosynthesis of neurosteroids. Allopregnanolone, a major pineal neurosteroid, acts on the Purkinje cell for the survival of Purkinje cells by suppressing the expression of caspase-3, a crucial mediator of apoptosis. I as a recipient of Kobayashi Award from the Japan Society for Comparative Endocrinology in 2016 summarize the discovery of cerebellar and pineal neurosteroids and their biological actions on the growth and survival of Purkinje cells during development., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Syntaphilin (SNPH) is a major mitochondrial anchoring protein targeted to axons and excluded from dendrites. In this study, we provide in vivo evidence that this spatial specificity is lost in Shiverer (Shi) mice, a model for progressive multiple sclerosis (MS), resulting in inappropriate intrusion of SNPH into dendrites of cerebellar Purkinje cells with neurodegenerative consequences. Thus, reconstituting dendritic SNPH intrusion in SNPH-KO mice by viral transduction greatly sensitizes Purkinje cells to excitotoxicity when the glutamatergic climbing fibers are stimulated. Finally, we demonstrate in vitro that overexpression of SNPH in dendrites compromises neuronal viability by inducing N-methyl-D-aspartate (NMDA) excitotoxicity, reducing mitochondrial calcium uptake, and interfering with quality control of mitochondria by blocking somal mitophagy. Collectively, we propose that inappropriate immobilization of dendritic mitochondria by SNPH intrusion produces excitotoxicity and suggest that interception of dendritic SNPH intrusion is a therapeutic strategy to combat neurodegeneration., (Published by Elsevier Inc.)

Microglia, the brain resident macrophages, critically shape forebrain neuronal circuits. However, their precise function in the cerebellum is unknown. Here we show that human and mouse cerebellar microglia express a unique molecular program distinct from forebrain microglia. Cerebellar microglial identity was driven by the CSF-1R ligand CSF-1, independently of the alternate CSF-1R ligand, IL-34. Accordingly, CSF-1 depletion from Nestin + cells led to severe depletion and transcriptional alterations of cerebellar microglia, while microglia in the forebrain remained intact. Strikingly, CSF-1 deficiency and alteration of cerebellar microglia were associated with reduced Purkinje cells, altered neuronal function, and defects in motor learning and social novelty interactions. These findings reveal a novel CSF-1-CSF-1R signaling-mediated mechanism that contributes to motor function and social behavior., (© 2019 Kana et al.)

The molecular mechanisms by which environmental light conditions affect cerebellar development are incompletely understood. We showed that circadian disruption by light-at-night induced Purkinje cell death through pineal allopregnanolone (ALLO) activity during early life in chicks. Light-at-night caused the loss of diurnal variation of pineal ALLO synthesis during early life and led to cerebellar Purkinje cell death, which was suppressed by a daily injection of ALLO. The loss of diurnal variation of pineal ALLO synthesis induced not only reduction in pituitary adenylate cyclase-activating polypeptide (PACAP), a neuroprotective hormone, but also transcriptional repression of the cerebellar Adcyap1 gene that produces PACAP, with subsequent Purkinje cell death. Taken together, pineal ALLO mediated the effect of light on early cerebellar development in chicks., Competing Interests: SH, MK, TT, KT, MS, MN, MO, IS, GH, VC, XL, TM, JK, TN, KM, HO, TT, TT, AM, KT No competing interests declared, (© 2019, Haraguchi et al.)

Neuronal activity is believed to be important for brain development; however, it remains unclear as to how spatiotemporal distributions of synaptic excitation contribute to neural network formation. Bifurcated axons of cerebellar granule cells, parallel fibers (PFs), are made in an orderly inside-out manner during postnatal development. In this study, we induced a blockade of neurotransmitter release from specific bundles of developing PFs and tested the effects of biased PF inputs on cerebellar development. The blockade of different layers of PFs at different developmental times results in varying degrees of abnormal cerebellar development. Furthermore, cerebellar network abnormalities are not restored when PF inputs are restored in adulthood and, hence, result in motor dysfunction. We thus conclude that spatiotemporally unbiased synaptic transmission from sequentially developed PFs is crucial for cerebellar network formation and motor function, supporting the idea that unbiased excitatory synaptic transmission is crucial for network formation., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

The cerebellum is critical for controlling motor and non-motor functions via cerebellar circuit that is composed of defined cell types, which approximately account for more than half of neurons in mammals. The molecular mechanisms controlling developmental progression and maturation processes of various cerebellar cell types need systematic investigation. Here, we analyzed transcriptome profiles of 21119 single cells of the postnatal mouse cerebellum and identified eight main cell clusters. Functional annotation of differentially expressed genes revealed trajectory hierarchies of granule cells (GCs) at various states and implied roles of mitochondrion and ATPases in the maturation of Purkinje cells (PCs), the sole output cells of the cerebellar cortex. Furthermore, we analyzed gene expression patterns and co-expression networks of 28 ataxia risk genes, and found that most of them are related with biological process of mitochondrion and around half of them are enriched in PCs. Our results also suggested core transcription factors that are correlated with interneuron differentiation and characteristics for the expression of secretory proteins in glia cells, which may participate in neuronal modulation. Thus, this study presents a systematic landscape of cerebellar gene expression in defined cell types and a general gene expression framework for cerebellar development and dysfunction., (© The Author(s) (2019). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., (© 2019 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

While mixed primary cerebellar cultures prepared from embryonic tissue have proven valuable for dissecting structure-function relationships in cerebellar Purkinje neurons (PNs), this technique is technically challenging and often yields few cells. Recently, mouse embryonic stem cells (mESCs) have been successfully differentiated into PNs, although the published methods are very challenging as well. The focus of this study was to simplify the differentiation of mESCs into PNs. Using a recently described neural differentiation media, we generate monolayers of neural progenitor cells from mESCs and differentiate them into PN precursors using specific extrinsic factors. These PN precursors are then differentiated into mature PNs by co-culturing them with granule neuron (GN) precursors also derived from neural progenitors using different extrinsic factors. The morphology of mESC-derived PNs is indistinguishable from PNs grown in primary culture in terms of gross morphology, spine length, and spine density. Furthermore, mESC-derived PNs express Calbindin D28K, IP3R1, IRBIT, PLCβ4, PSD93, and myosin IIB-B2, all of which are either PN-specific or highly expressed in PNs. Moreover, we show that mESC-derived PNs form synapses with GN-like cells as in primary culture, express proteins driven by the PN-specific promoter Pcp2/L7, and exhibit the defect in spine ER inheritance seen in PNs isolated from dilute-lethal (myosin Va-null) mice when expressing a Pcp2/L7-driven miRNA directed against myosin Va. Finally, we define a novel extracellular matrix formulation that reproducibly yields monolayer cultures conducive for high-resolution imaging. Our improved method for differentiating mESCs into PNs should facilitate the dissection of molecular mechanisms and disease phenotypes in PNs.

Sparse labeling of neurons contributes to uncovering their morphology, and rapid expression of a fluorescent protein reduces the experiment range. To achieve the goal of rapid and sparse labeling of neurons in vivo, we established a rapid method for depicting the fine structure of neurons at 24 h post-infection based on a mutant virus-like particle of Semliki Forest virus. Approximately 0.014 fluorescent focus-forming units of the mutant virus-like particle transferred enhanced green fluorescent protein into neurons in vivo, and its affinity for neurons in vivo was stronger than for neurons in vitro and BHK21 (baby hamster kidney) cells. Collectively, the mutant virus-like particle provides a robust and convenient way to reveal the fine structure of neurons and is expected to be a helper virus for combining with other tools to determine their connectivity. Our work adds a new tool to the approaches for rapid and sparse labeling of neurons in vivo.

An 8-year-old female Magellanic penguin (Spheniscus magellanicus) started to show epilepsy-like seizures. Subsequent magnetic resonance imaging (MRI) examinations did not reveal any responsible lesions. The neurological symptoms worsened at the age of 10. This penguin became recumbent and died 6 months later after the apparition of the recumbency. At necropsy, only multiple yellowish necrotic lesions in the air sacs and lungs were found. Histopathological evaluation of the brain showed a marked loss of Purkinje cells and many hypertrophied parvalbumin-positive basket/stellate cells were seen in the cerebellar cortex. Calbindin immunohistochemistry demonstrated disrupted arrangement of dendrites in the Purkinje cells. This case was diagnosed as cerebellar cortical degeneration with a very late onset and a slow progression in a Magellanic penguin.

ADP ribosylation factor 6 (Arf6) is a small GTPase that regulates various neuronal events including formation of the axon, dendrites and dendritic spines, and synaptic plasticity through actin cytoskeleton remodeling and endosomal trafficking. EFA6C, also known as Psd2, is a guanine nucleotide exchange factor for Arf6 that is preferentially expressed in the cerebellar cortex of adult mice, particularly in Purkinje cells. However, the roles of EFA6C in cerebellar development and functions remain unknown. In this study, we generated global EFA6C knockout (KO) mice using the CRISPR/Cas9 system and investigated their cerebellar phenotypes by histological and behavioral analyses. Histological analyses revealed that EFA6C KO mice exhibited normal gross anatomy of the cerebellar cortex, in terms of the thickness and cellularity of each layer, morphology of Purkinje cells, and distribution patterns of parallel fibers, climbing fibers, and inhibitory synapses. Electron microscopic observation of the cerebellar molecular layer revealed that the density of asymmetric synapses of Purkinje cells was significantly lower in EFA6C KO mice compared with wild-type control mice. However, behavioral analyses using accelerating rotarod and horizontal optokinetic response tests failed to detect any differences in motor coordination, learning or adaptation between the control and EFA6C KO mice. These results suggest that EFA6C plays ancillary roles in cerebellar development and motor functions., Competing Interests: The authors have declared that no competing interests exist.

Modern electron microscopy offers a wide variety of tools to investigate the ultrastructural organization of cells and tissues and to accurately pinpoint intracellular localizations of macromolecules of interest. New volumetric electron microscopy techniques and new instrumentation provide unique opportunities for high-throughput analysis of comparatively large volumes of tissue and their complete reconstitution in three-dimensional (3D) electron microscopy. However, due to a variety of technical issues such as the limited penetration of label into the tissue, low antigen preservation, substantial electron density of secondary detection reagents, and many others, the adaptation of immuno-detection techniques for use with such 3D imaging methods as focused ion beam-scanning electron microscopy (FIB-SEM) has been challenging. Here, we describe a sample preparation method for 3D FIB-SEM, which results in an optimal preservation and staining of ultrastructural details at a resolution necessary for tracing immunolabeled neuronal structures and detailed reconstruction of synapses. This technique is applicable to neuronal and non-neuronal cells, tissues, and a wide variety of antigens.

Staufen2 (Stau2) is an RNA-binding protein that is involved in dendritic spine morphogenesis and function. Several studies have recently investigated the role of Stau2 in the regulation of its neuronal target mRNAs, with particular focus on the hippocampus. Here, we provide evidence for Stau2 expression and function in cerebellar Purkinje cells. We show that Stau2 downregulation (Stau2 GT ) led to an increase of glutamate receptor ionotropic delta subunit 2 (GluD2) in Purkinje cells when animals performed physical activity by voluntary wheel running compared with the age-matched wildtype (WT) mice (C57Bl/6J). Furthermore, Stau2 GT mice showed lower performance in motor coordination assays but enhanced motor learning abilities than did WT mice, concomitantly with an increase in dendritic GluD2 expression. Together, our results suggest the novel role of Stau2 in Purkinje cell synaptogenesis in the mouse cerebellum.

Mammalian target of rapamycin (mTOR) is a central regulator of cellular metabolism. The importance of mTORC1 signaling in neuronal development and functions has been highlighted by its strong relationship with many neurological and neuropsychiatric diseases. Previous studies demonstrated that hyperactivation of mTORC1 in forebrain recapitulates tuberous sclerosis and neurodegeneration. In the mouse cerebellum, Purkinje cell-specific knockout of Tsc1/2 has been implicated in autistic-like behaviors. However, since TSC1/2 activity does not always correlate with clinical manifestations as evident in some cases of tuberous sclerosis, the intriguing possibility is raised that phenotypes observed in Tsc1/2 knockout mice cannot be attributable solely to mTORC1 hyperactivation. Here we generated transgenic mice in which mTORC1 signaling is directly hyperactivated in Purkinje cells. The transgenic mice exhibited impaired synapse elimination of climbing fibers and motor discoordination without affecting social behaviors. Furthermore, mTORC1 hyperactivation induced prominent apoptosis of Purkinje cells, accompanied with dysregulated cellular homeostasis including cell enlargement, increased mitochondrial respiratory activity, and activation of pseudohypoxic response. These findings suggest the different contributions between hyperactivated mTORC1 and Tsc1/2 knockout in social behaviors, and reveal the perturbations of cellular homeostasis by hyperactivated mTORC1 as possible underlying mechanisms of neuronal dysfunctions and death in tuberous sclerosis and neurodegenerative diseases.