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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.)

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.)

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.)

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.)

Cerebellar Purkinje cells arborize unique dendrites that exhibit a planar, fan shape. The dendritic branches fill the space of their receptive field with little overlap. This dendritic arrangement is well-suited to form numerous synapses with the afferent parallel fibers of the cerebellar granule cells in a non-redundant manner. Purkinje cell dendritic arbor morphology is achieved by a combination of dynamic local branch growth behaviors, including elongation, branching, and retraction. Impacting these behaviors, the self-avoidance of each branch terminal is essential to form the non-overlapping dendritic configuration. This review outlines recent advances in our understanding of the cellular and molecular mechanisms of dendrite formation during cerebellar Purkinje cell development.

Regional differences in dendritic architecture can influence connectivity and dendritic signal integration, with possible consequences for neuronal computation. In the cerebellum, analyses of Purkinje cells (PCs), which are functionally critical as they provide the sole output of the cerebellar cortex, have suggested that the cerebellar cortex is not uniform in structure as traditionally assumed. However, the limitations of traditional staining methods and microscopy capabilities have presented difficulties in investigating possible local variations in PC morphology. To address this question, we used male mice expressing green fluorescent protein selectively in PCs. Using Neurolucida 360 with confocal image stacks, we reconstructed dendritic arbors of PCs residing in lobule V (anterior) and lobule IX (posterior) of the vermis. We then analyzed morphologies of individual arbors and the structure of the assembled "jungle," comparing these features across anatomical locations and age groups. Strikingly, we found that in lobule IX, half of the reconstructed PCs had two primary dendrites emanating from their soma, whereas fewer than a quarter showed this characteristic in lobule V. Furthermore, PCs in lobule V showed more efficient spatial occupancy compared to lobule IX, as well as greater packing density and increased arbor overlap in the adult. When analyzing complete ensembles of PC arbors, we also observed "hot spots" of increased dendritic density in lobule V, whereas lobule IX showed a more homogeneous spread of dendrites. These differences suggest that input patterns and/or physiology of PCs could likewise differ along the vermis, with possible implications for cerebellar function., (© 2017 Wiley Periodicals, Inc.)

Background: Spinocerebellar ataxias (SCAs) are a group of cerebellar diseases characterized by progressive ataxia and cerebellar atrophy. Several forms of SCAs are caused by missense mutations or deletions in genes related to calcium signaling in Purkinje cells. Among them, spinocerebellar ataxia type 14 (SCA14) is caused by missense mutations in PRKCG gene which encodes protein kinase C gamma (PKCγ). It is remarkable that in several cases in which SCA is caused by point mutations in an individual gene, the affected genes are involved in the PKCγ signaling pathway and calcium signaling which is not only crucial for proper Purkinje cell function but is also involved in the control of Purkinje cell dendritic development. In this review, we will focus on the PKCγ signaling related genes and calcium signaling related genes then discuss their role for both Purkinje cell dendritic development and cerebellar ataxia., Methods: Research related to SCAs and Purkinje cell dendritic development is reviewed., Results: PKCγ dysregulation causes abnormal Purkinje cell dendritic development and SCA14. Carbonic anhydrase related protein 8 (Car8) encoding CAR8 and Itpr1 encoding IP3R1were identified as upregulated genes in one of SCA14 mouse model. IP3R1, CAR8 and PKCγ proteins are strongly and specifically expressed in Purkinje cells. The common function among them is that they are involved in the regulation of calcium homeostasis in Purkinje cells and their dysfunction causes ataxia in mouse and human. Furthermore, disruption of intracellular calcium homeostasis caused by mutations in some calcium channels in Purkinje cells links to abnormal Purkinje cell dendritic development and the pathogenesis of several SCAs., Conclusion: Once PKCγ signaling related genes and calcium signaling related genes are disturbed, the normal dendritic development of Purkinje cells is impaired as well as the integration of signals from other neurons, resulting in abnormal development, cerebellar dysfunction and eventually Purkinje cell loss., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Several studies have indicated that abnormal prenatal changes in the circulating glucocorticoids (GCs), induced by either maternal stress or exogenous GC administration, significantly alter the development of Purkinje cells (PCs). Among the suggested mechanisms that could mediate this GC-dependent PC susceptibility are changes in the expression of type-1 metabotropic glutamate receptors (mGluR1). In the current study, we analyzed whether a single course of prenatally administered betamethasone phosphate (BET) in pregnant rats increased the immunohistochemical expression of mGluR1 in PCs and decreased PC dendritic growth. The data obtained showed that in utero BET exposure resulted in a significant immunohistochemical overexpression of mGluR1 and a significant reduction in Purkinje cell dendritic outgrowth during postnatal life.

Neurensin1 (Nrsn1) gene, highly specific to neurons, has been considered to play a role in neurite growth during neuronal development and regeneration in mice. Intense expression of Nrsn1 was found particularly in projecting neurons like retinal ganglion cells and spinal motor neurons, suggesting that Neurensin1 is needed for active neurite growth. In the present study we cloned chick Nrsn1 gene and produced an antibody against cNrsn1 to examine Nrsn1 localization in the chick brain, since the chick is a suitable animal model for the study of developmental neurobiology. We found that there are neurons intensely stained for Nrsn1 antibody localized in the optic tectum, the cerebellum and the brain stem. These neurons are large in size and considered to be projecting neurons. In the cerebellum, Purkinje cells are the only one type of neurons stained for Nrsn1. During Purkinje cell development the arborized dendrites and axons become intensely stained at stages E17-18. A siRNA gene knock down was applied to the cultured embryonic cerebellar tissues and the result showed that Nrsn1 has an important role in dendrite formation of Purkinje cells. These findings suggest that Neurensin1 is also involved in neural development in the chick brain and that the embryonic chick brain is a good model to disclose the molecular and physiological functions of Nrsn1., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The cells that comprise the cerebellum perform a complex integration of neural inputs to influence motor control and coordination. The functioning of this circuit depends upon Purkinje cells and other cerebellar neurons forming in the precise place and time during development. Zebrafish provide a useful platform for modeling disease and studying gene function, thus a quantitative metric of normal zebrafish cerebellar development is key for understanding how gene mutations affect the cerebellum. To begin to quantitatively measure cerebellar development in zebrafish, we have characterized the spatial and temporal patterning of Purkinje cells during the first 2 weeks of development. Differentiated Purkinje cells first emerged by 2.8 days post fertilization and were spatially patterned into separate dorsomedial and ventrolateral clusters that merged at around 4 days. Quantification of the Purkinje cell layer revealed that there was a logarithmic increase in both Purkinje cell number as well as overall volume during the first 2 weeks, while the entire region curved forward in an anterior, then ventral direction. Purkinje cell dendrites were positioned next to parallel fibers as early as 3.3 days, and Purkinje cell diameter decreased significantly from 3.3 to 14 days, possibly due to cytoplasmic reappropriation into maturing dendritic arbors. A nearest neighbor analysis showed that Purkinje cells moved slightly apart from each other from 3 to 14 days, perhaps spreading as the organized monolayer forms. This study establishes a quantitative spatiotemporal map of Purkinje cell development in zebrafish that provides an important metric for studies of cerebellar development and disease., (© 2015 Wiley Periodicals, Inc.)

The establishment of cell-type-specific dendritic arbors is fundamental for proper neural circuit formation. Here, using temporal- and cell-specific knock-down, knock-out, and overexpression approaches, we show that multiple aspects of the dendritic organization of cerebellar Purkinje cells (PCs) are controlled by a single transcriptional factor, retinoic acid-related orphan receptor-alpha (RORα), a gene defective in staggerer mutant mice. As reported earlier, RORα was required for regression of primitive dendrites before postnatal day 4 (P4). RORα was also necessary for PCs to form a single Purkinje layer from P0 to P4. The knock-down of RORα from P4 impaired the elimination of perisomatic dendrites and maturation of single stem dendrites in PCs at P8. Filopodia and spines were also absent in these PCs. The knock-down of RORα from P8 impaired the formation and maintenance of terminal dendritic branches of PCs at P14. Finally, even after dendrite formation was completed at P21, RORα was required for PCs to maintain dendritic complexity and functional synapses, but their mature innervation pattern by single climbing fibers was unaffected. Interestingly, overexpression of RORα in PCs at various developmental stages did not facilitate dendrite development, but had specific detrimental effects on PCs. Because RORα deficiency during development is closely related to the severity of spinocerebellar ataxia type 1, delineating the specific roles of RORα in PCs in vivo at different time windows during development and throughout adulthood would facilitate our understanding of the pathogenesis of cerebellar disorders. Significance statement: The genetic programs by which each neuron subtype develops and maintains dendritic arbors have remained largely unclear. This is partly because dendrite development is modulated dynamically by neuronal activities and interactions with local environmental cues in vivo. In addition, dendrites are formed and maintained by the balance between their growth and regression; the effects caused by the disruption of transcription factors during the early developmental stages could be masked by dendritic growth or regression in the later stages. Here, using temporal- and cell-specific knock-down, knock-out, and overexpression approaches in vivo, we show that multiple aspects of the dendritic organization of cerebellar Purkinje cells are controlled by a single transcriptional factor, retinoic acid-related orphan receptor alpha., (Copyright © 2015 the authors 0270-6474/15/3512518-17$15.00/0.)

The spatiotemporal pattern of synaptic inputs to the dendritic tree is crucial for synaptic integration and plasticity. However, it is not known if input patterns driven by sensory stimuli are structured or random. Here we investigate the spatial patterning of synaptic inputs by directly monitoring presynaptic activity in the intact mouse brain on the micron scale. Using in vivo calcium imaging of multiple neighbouring cerebellar parallel fibre axons, we find evidence for clustered patterns of axonal activity during sensory processing. The clustered parallel fibre input we observe is ideally suited for driving dendritic spikes, postsynaptic calcium signalling, and synaptic plasticity in downstream Purkinje cells, and is thus likely to be a major feature of cerebellar function during sensory processing.

Neurotrophins regulate diverse aspects of neuronal development and plasticity, but their precise in vivo functions during neural circuit assembly in the central brain remain unclear. We show that the neurotrophin receptor tropomyosin-related kinase C (TrkC) is required for dendritic growth and branching of mouse cerebellar Purkinje cells. Sparse TrkC knockout reduced dendrite complexity, but global Purkinje cell knockout had no effect. Removal of the TrkC ligand neurotrophin-3 (NT-3) from cerebellar granule cells, which provide major afferent input to developing Purkinje cell dendrites, rescued the dendrite defects caused by sparse TrkC disruption in Purkinje cells. Our data demonstrate that NT-3 from presynaptic neurons (granule cells) is required for TrkC-dependent competitive dendrite morphogenesis in postsynaptic neurons (Purkinje cells)--a previously unknown mechanism of neural circuit development., (Copyright © 2014, American Association for the Advancement of Science.)

The climbing fiber input to Purkinje cells acts as a teaching signal by triggering a massive influx of dendritic calcium that marks the occurrence of instructive stimuli during cerebellar learning. Here, we challenge the view that these calcium spikes are all-or-none and only signal whether the instructive stimulus has occurred, without providing parametric information about its features. We imaged ensembles of Purkinje cell dendrites in awake mice and measured their calcium responses to periocular airpuffs that serve as instructive stimuli during cerebellar-dependent eyeblink conditioning. Information about airpuff duration and pressure was encoded probabilistically across repeated trials, and in two additional signals in single trials: the synchrony of calcium spikes in the Purkinje cell population, and the amplitude of the calcium spikes, which was modulated by a non-climbing fiber pathway. These results indicate that calcium-based teaching signals in Purkinje cells contain analog information that encodes the strength of instructive stimuli trial-by-trial., (Copyright © 2014, Najafi et al.)

Cerebellar Purkinje cells have the most elaborate dendritic trees among neurons in the brain. We examined the roles of ryanodine receptor (RyR), an intracellular Ca(2+) release channel, in the dendrite formation of Purkinje cells using cerebellar cell cultures. In the cerebellum, Purkinje cells express RyR1 and RyR2, whereas granule cells express RyR2. When ryanodine (10 µM), a blocker of RyR, was added to the culture medium, the elongation and branching of Purkinje cell dendrites were markedly inhibited. When we transferred small interfering RNA (siRNA) against RyR1 into Purkinje cells using single-cell electroporation, dendritic branching but not elongation of the electroporated Purkinje cells was inhibited. On the other hand, transfection of RyR2 siRNA into granule cells also inhibited dendritic branching of Purkinje cells. Furthermore, ryanodine reduced the levels of brain-derived neurotrophic factor (BDNF) in the culture medium. The ryanodine-induced inhibition of dendritic differentiation was partially rescued when BDNF was exogenously added to the culture medium in addition to ryanodine. Overall, these results suggest that RyRs expressed by both Purkinje and granule cells play important roles in promoting the dendritic differentiation of Purkinje cells and that RyR2 expressed by granule cells is involved in the secretion of BDNF from granule cells., (Copyright © 2013 Wiley Periodicals, Inc.)

The neurotransmitter GABA regulates many aspects of inhibitory synapse development. We tested the hypothesis that GABAA receptors (GABAARs) work together with the synaptic adhesion molecule neuroligin 2 (NL2) to regulate synapse formation in different subcellular compartments. We investigated mice ("γ2 knockdown mice") with an engineered allele of the GABAAR γ2 subunit gene which produced a mosaic expression of synaptic GABAARs in neighboring neurons, causing a strong imbalance in synaptic inhibition. Deletion of the γ2 subunit did not abolish synapse formation or the targeting of NL2 to distinct types of perisomatic and axo-dendritic contacts. Thus synaptic localization of NL2 does not require synaptic GABAARs. However, loss of the γ2 subunit caused a selective decrease in the number of axo-dendritic synapses on cerebellar Purkinje cells and cortical pyramidal neurons, whereas perisomatic synapses were not significantly affected. Notably, γ2-positive cells had increased axo-dendritic innervation compared with both γ2-negative and wild-type counterparts. Moreover heterologous synapses on spines, that are found after total deletion of GABAARs from all Purkinje cells, were rare in cerebella of γ2 knockdown mice. These findings reveal a selective role of γ2 subunit-containing GABAARs in regulating synapse development in distinct subcellular compartments, and support the hypothesis that the refinement of axo-dendritic synapses is regulated by activity-dependent competition between neighboring neurons.

DOC-2/DAB-2 interacting protein (Dab2IP) is a GTPase activating protein that binds to Disabled-1, a cytosolic adapter protein involved in Reelin signaling and brain development. Dab2IP regulates PI3K-AKT signaling and is associated with metastatic prostate cancer, abdominal aortic aneurysms and coronary heart disease. To date, the physiological function of Dab2IP in the nervous system, where it is highly expressed, is relatively unknown. In this study, we generated a mouse model with a targeted disruption of Dab2IP using a retrovirus gene trap strategy. Unlike reeler mice, Dab2IP knock-down mice did not exhibit severe ataxia or cerebellar hypoplasia. However, Dab2IP deficiency produced a number of cerebellar abnormalities such as a delay in the development of Purkinje cell (PC) dendrites, a decrease in the parallel fiber synaptic marker VGluT1, and an increase in the climbing fiber synaptic marker VGluT2. These findings demonstrate for the first time that Dab2IP plays an important role in dendrite development and regulates the number of synapses in the cerebellum.

In the present study, we analyzed the effects of enriched, social and isolated experiences on vermal Purkinje cell of the rat, together with anxiety-like behavior in the elevated-plus maze. Sprague-Dawley male rats were randomly submitted to either enriched, social, or isolated environments during the early postweaning period (postnatal days 22-32) and were then behaviorally evaluated in the elevated-plus maze and euthanized for histological analysis. Vermal Purkinje cells (sub-lobules VIa and VIb) were sampled, drawn under camera lucida and morphometrically assessed using the Sholl's concentric ring method. Data obtained indicate that environmental enrichment did not significantly modify the Purkinje cell dendritic branching. On the contrary, Purkinje cell of animals reared in social isolation exhibited a significant reduction in dendritic arborization, which was closely associated with anxiety-like behaviors. The data obtained indicate that, although environmental stimulation in normal animals does not produce significant changes in vermal Purkinje cell dendritic arborization, these cells are vulnerable to early stressful experiences, which is in close association with anxiety-like behaviors.

Neurons develop dendritic arbors in cell type-specific patterns. Using growing Purkinje cells in culture as a model, we performed a long-term time-lapse observation of dendrite branch dynamics to understand the rules that govern the characteristic space-filling dendrites. We found that dendrite architecture was sculpted by a combination of reproducible dynamic processes, including constant tip elongation, stochastic terminal branching, and retraction triggered by contacts between growing dendrites. Inhibition of protein kinase C/protein kinase D signaling prevented branch retraction and significantly altered the characteristic morphology of long proximal segments. A computer simulation of dendrite branch dynamics using simple parameters from experimental measurements reproduced the time-dependent changes in the dendrite configuration in live Purkinje cells. Furthermore, perturbation analysis to parameters in silico validated the important contribution of dendritic retraction in the formation of the characteristic morphology. We present an approach using live imaging and computer simulations to clarify the fundamental mechanisms of dendrite patterning in the developing brain.

Dendritic arborizations of many neurons are patterned by a process called self-avoidance, in which branches arising from a single neuron repel each other. By minimizing gaps and overlaps within the arborization, self-avoidance facilitates complete coverage of a neuron’s territory by its neurites. Remarkably, some neurons that display self-avoidance interact freely with other neurons of the same subtype, implying that they discriminate self from non-self. Here we demonstrate roles for the clustered protocadherins (Pcdhs) in dendritic self-avoidance and self/non-self discrimination. The Pcdh locus encodes 58 related cadherin-like transmembrane proteins, at least some of which exhibit isoform-specific homophilic adhesion in heterologous cells and are expressed stochastically and combinatorially in single neurons. Deletion of all 22 Pcdh genes in the mouse γ-subcluster (Pcdhg genes) disrupts self-avoidance of dendrites in retinal starburst amacrine cells (SACs) and cerebellar Purkinje cells. Further genetic analysis of SACs showed that Pcdhg proteins act cell-autonomously during development, and that replacement of the 22 Pcdhg proteins with a single isoform restores self-avoidance. Moreover, expression of the same single isoform in all SACs decreases interactions among dendrites of neighbouring SACs (heteroneuronal interactions). These results suggest that homophilic Pcdhg interactions between sibling neurites (isoneuronal interactions) generate a repulsive signal that leads to self-avoidance. In this model, heteroneuronal interactions are normally permitted because dendrites seldom encounter a matched set of Pcdhg proteins unless they emanate from the same soma. In many respects, our results mirror those reported for Dscam1 (Down syndrome cell adhesion molecule) in Drosophila: this complex gene encodes thousands of recognition molecules that exhibit stochastic expression and isoform-specific interactions, and mediate both self-avoidance and self/non-self discrimination. Thus, although insect Dscam and vertebrate Pcdh proteins share no sequence homology, they seem to underlie similar strategies for endowing neurons with distinct molecular identities and patterning their arborizations.

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents (3). Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells (11) are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period (4). We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

The development of a neuronal dendritic tree is modulated both by signals from afferent fibers and by an intrinsic program. We have previously shown that chronic activation of either type 1 metabotropic glutamate receptors (mGluR1s) or protein kinase C (PKC) in organotypic cerebellar slice cultures of mice and rats severely inhibits the growth and development of the Purkinje cell dendritic tree. The signaling events linking receptor activation to the regulation of dendritic growth remain largely unknown. We have studied whether channels allowing the entry of Ca(2+) into Purkinje cells, in particular the type 3 transient receptor potential cation channels (TRPC3s), P/Q-type Ca(2+) channels, and T-type Ca(2+) channels, might be involved in signaling after mGluR1 or PKC stimulation. We show that the inhibition of dendritic growth seen after mGluR1 or PKC stimulation is partially rescued by pharmacological blockade of P/Q-type and T-type Ca(2+) channels, indicating that activation of these channels mediating Ca(2+) influx contributes to the inhibition of dendritic growth. In contrast, the absence of Ca(2+) -permeable TRPC3s in TRPC3-deficient mice or pharmacological blockade had no effect on mGluR1-mediated and PKC-mediated inhibition of Purkinje cell dendritic growth. Similarly, blockade of Ca(2+) influx through glutamate receptor δ2 or R-type Ca(2+) channels or inhibition of release from intracellular stores did not influence mGluR1-mediated and PKC-mediated inhibition of Purkinje cell dendritic growth. These findings suggest that both T-type and P/Q-type Ca(2+) channels, but not TRPC3 or other Ca(2+) -permeable channels, are involved in mGluR1 and PKC signaling leading to the inhibition of dendritic growth in cerebellar Purkinje cells., (© 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Mutations in the gene encoding β-III spectrin give rise to spinocerebellar ataxia type 5, a neurodegenerative disease characterized by progressive thinning of the molecular layer, loss of Purkinje cells and increasing motor deficits. A mouse lacking full-length β-III spectrin (β-III⁻/⁻) displays a similar phenotype. In vitro and in vivo analyses of Purkinje cells lacking β-III spectrin, reveal a critical role for β-III spectrin in Purkinje cell morphological development. Disruption of the normally well ordered dendritic arborization occurs in Purkinje cells from β-III⁻/⁻ mice, specifically showing a loss of monoplanar organization, smaller average dendritic diameter and reduced densities of Purkinje cell spines and synapses. Early morphological defects appear to affect distribution of dendritic, but not axonal, proteins. This study confirms that thinning of the molecular layer associated with disease pathogenesis is a consequence of Purkinje cell dendritic degeneration, as Purkinje cells from 8-month-old β-III⁻/⁻ mice have drastically reduced dendritic volumes, surface areas and total dendritic lengths compared with 5- to 6-week-old β-III⁻/⁻ mice. These findings highlight a critical role of β-III spectrin in dendritic biology and are consistent with an early developmental defect in β-III⁻/⁻ mice, with abnormal Purkinje cell dendritic morphology potentially underlying disease pathogenesis.

Because of its highly branched dendrite, the Purkinje neuron requires significant computational resources if coupled electrical and biochemical activity are to be simulated. To address this challenge, we developed a scheme for reducing the geometric complexity; while preserving the essential features of activity in both the soma and a remote dendritic spine. We merged our previously published biochemical model of calcium dynamics and lipid signaling in the Purkinje neuron, developed in the Virtual Cell modeling and simulation environment, with an electrophysiological model based on a Purkinje neuron model available in NEURON. A novel reduction method was applied to the Purkinje neuron geometry to obtain a model with fewer compartments that is tractable in Virtual Cell. Most of the dendritic tree was subject to reduction, but we retained the neuron's explicit electrical and geometric features along a specified path from spine to soma. Further, unlike previous simplification methods, the dendrites that branch off along the preserved explicit path are retained as reduced branches. We conserved axial resistivity and adjusted passive properties and active channel conductances for the reduction in surface area, and cytosolic calcium for the reduction in volume. Rallpacks are used to validate the reduction algorithm and show that it can be generalized to other complex neuronal geometries. For the Purkinje cell, we found that current injections at the soma were able to produce similar trains of action potentials and membrane potential propagation in the full and reduced models in NEURON; the reduced model produces identical spiking patterns in NEURON and Virtual Cell. Importantly, our reduced model can simulate communication between the soma and a distal spine; an alpha function applied at the spine to represent synaptic stimulation gave similar results in the full and reduced models for potential changes associated with both the spine and the soma. Finally, we combined phosphoinositol signaling and electrophysiology in the reduced model in Virtual Cell. Thus, a strategy has been developed to combine electrophysiology and biochemistry as a step toward merging neuronal and systems biology modeling.

Cerebellar Purkinje cells have one of the most elaborate dendritic trees in the mammalian CNS, receiving excitatory synaptic input from a single climbing fiber (CF) and from ∼200,000 parallel fibers. The dendritic Ca(2+) signals triggered by activation of these inputs are crucial for the induction of synaptic plasticity at both of these synaptic connections. We have investigated Ca(2+) signaling in Purkinje cell dendrites in vivo by combining targeted somatic or dendritic patch-clamp recording with simultaneous two-photon microscopy. Both spontaneous and sensory-evoked CF inputs triggered widespread Ca(2+) signals throughout the dendritic tree that were detectable even in individual spines of the most distal spiny branchlets receiving parallel fiber input. The amplitude of these Ca(2+) signals depended on dendritic location and could be modulated by membrane potential, reflecting modulation of dendritic spikes triggered by the CF input. Furthermore, the variability of CF-triggered Ca(2+) signals was regulated by GABAergic synaptic input. These results indicate that dendritic Ca(2+) signals triggered by sensory-evoked CF input can act as associative signals for synaptic plasticity in Purkinje cells in vivo and may differentially modulate plasticity at parallel fiber synapses depending on the location of synapses, firing state of the Purkinje cell, and ongoing GABAergic synaptic input.

The ability of neurons to process synaptic inputs depends critically on their passive electrical properties. The intracellular resistivity, R(i), is one of the parameters that determine passive properties, yet few experiments have explored how changes in R(i) might affect synaptic integration. In this work, I addressed this issue by using targeted dendritic occlusion to locally increase R(i) in cerebellar Purkinje cells and examining the consequences of this manipulation for the summation of synaptic inputs. To achieve dendritic occlusion, I used two glass micropipettes to gently pinch the dendritic trunk close to the soma. This pinching produced stereotypical changes in the responses to test pulses applied at the soma under voltage and current clamp. A simple model confirmed that these changes were due to increases in R(i) in the dendritic trunk. These localized increases in R(i) produced striking alterations in the shapes of postsynaptic potentials at the soma, increasing their amplitude and accelerating their decay kinetics. As a consequence, dendritic occlusion sharpened temporal precision during the summation of synaptic inputs. These findings highlight the importance of local changes in intracellular resistivity for the passive electrical properties of neurons, with implications for their ability to process synaptic information., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Purkinje cells (PCs) are one of the principal neurons in the cerebellar cortex that play a central role in the coordination of fine-tuning body movement and balance. To acquire normal cerebellum function, PCs develop extensive dendritic arbors that establish synaptic connections with the parallel fibers of granule cells to form the proper neuronal circuitry. Therefore, dendritic arborization of PCs is an important developmental step to construct the mature neural network in the cerebellum. However, the genetic control of this process is not fully understood. In this study, Foxp4, a forkhead transcription factor that is expressed specifically in migrating and mature PCs of cerebellum from embryonic stages to adulthood, was knocked down by small interfering RNA (siRNA) in organotypic cerebellar slice culture. When Foxp4 expression was knocked down at postnatal day 5 (P5), no abnormalities for early dendritic remodeling of PCs were observed. However, when Foxp4 was knocked down in P10 cerebellar slices, the organization of PC dendritic arbors was highly impaired, leaving hypoplastic but non-apoptotic cell bodies. The radial alignment of Bergmann glial fibers that associated with PC dendrites was also lost. These results suggest that Foxp4 is dispensable for the early PC dendrite outgrowth, but is essential for the maintenance of PC dendritic arborization and subsequent association with Bergmann glial fibers., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Dendrite arborization patterns are critical determinants of neuronal connectivity and integration. Planar and highly branched dendrites of the cerebellar Purkinje cell receive specific topographical projections from two major afferent pathways; a single climbing fiber axon from the inferior olive that extend along Purkinje dendrites, and parallel fiber axons of granule cells that contact vertically to the plane of dendrites. It has been believed that murine Purkinje cell dendrites extend in a single parasagittal plane in the molecular layer after the cell polarity is determined during the early postnatal development. By three-dimensional confocal analysis of growing Purkinje cells, we observed that mouse Purkinje cells underwent dynamic dendritic remodeling during circuit maturation in the third postnatal week. After dendrites were polarized and flattened in the early second postnatal week, dendritic arbors gradually expanded in multiple sagittal planes in the molecular layer by intensive growth and branching by the third postnatal week. Dendrites then became confined to a single plane in the fourth postnatal week. Multiplanar Purkinje cells in the third week were often associated by ectopic climbing fibers innervating nearby Purkinje cells in distinct sagittal planes. The mature monoplanar arborization was disrupted in mutant mice with abnormal Purkinje cell connectivity and motor discoordination. The dendrite remodeling was also impaired by pharmacological disruption of normal afferent activity during the second or third postnatal week. Our results suggest that the monoplanar arborization of Purkinje cells is coupled with functional development of the cerebellar circuitry.

Dendritic spikes appear to be a ubiquitous feature of dendritic excitability. In cortical pyramidal neurons, dendritic spikes increase the efficacy of distal synapses, providing additional inward current to enhance axonal action potential (AP) output, thus increasing synaptic gain. In cerebellar Purkinje cells, dendritic spikes can trigger synaptic plasticity, but their influence on axonal output is not well understood. We have used simultaneous somatic and dendritic patch-clamp recordings to directly assess the impact of dendritic calcium spikes on axonal AP output of Purkinje cells. Dendritic spikes evoked by parallel fiber input triggered brief bursts of somatic APs, followed by pauses in spiking, which cancelled out the extra spikes in the burst. As a result, average output firing rates during trains of input remained independent of the input strength, thus flattening synaptic gain. We demonstrate that this "clamping" of AP output by the pause following dendritic spikes is due to activation of high conductance calcium-dependent potassium channels by dendritic spikes. Dendritic spikes in Purkinje cells, in contrast to pyramidal cells, thus have differential effects on temporally coded and rate coded information: increasing the impact of transient parallel fiber input, while depressing synaptic gain for sustained parallel fiber inputs.

Neurons have highly polarized structures that determine what parts of the soma elaborate the axon and dendrites. However, little is known about the mechanisms that establish neuronal polarity in vivo. Cerebellar Purkinje cells extend a single primary dendrite from the soma that ramifies into a highly branched dendritic arbor. We used the zebrafish cerebellum to investigate the mechanisms by which Purkinje cells acquire these characteristics. To examine dendritic morphogenesis in individual Purkinje cells, we marked the cell membrane using a Purkinje cell-specific promoter to drive membrane-targeted fluorescent proteins. We found that zebrafish Purkinje cells initially extend multiple neurites from the soma and subsequently retract all but one, which becomes the primary dendrite. In addition, the Golgi apparatus specifically locates to the root of the primary dendrite, and its localization is already established in immature Purkinje cells that have multiple neurites. Inhibiting secretory trafficking through the Golgi apparatus reduces dendritic growth, suggesting that the Golgi apparatus is involved in the dendritic morphogenesis. We also demonstrated that in a mutant of an atypical protein kinase C (aPKC), Prkci, Purkinje cells retain multiple primary dendrites and show disrupted localization of the Golgi apparatus. Furthermore, a mosaic inhibition of Prkci in Purkinje cells recapitulates the aPKC mutant phenotype. These results suggest that the aPKC cell autonomously controls the Golgi localization and thereby regulates the specification of the primary dendrite of Purkinje cells.

The molecular pathways controlling cerebellar Purkinje cell dendrite formation and maturation are poorly understood. The Purkinje cell degeneration (pcd) mutant mouse is characterized by mutations in Nna1, a gene discovered in an axonal regenerative context, but whose actual function in development and disease is unknown. We found abnormal development of Purkinje cell dendrites in postnatal pcd(Sid) mice and linked this deficit to a deletion mutation in exon 7 of Nna1. With single cell gene profiling and virus-based gene transfer, we analyzed a molecular pathway downstream to Nna1 underlying abnormal Purkinje cell dendritogenesis in pcd(Sid) mice. We discovered that mutant Nna1 dramatically increases intranuclear localization of lysyl oxidase propeptide, which interferes with NF-κB RelA signaling and microtubule-associated protein regulation of microtubule stability, leading to underdevelopment of Purkinje cell dendrites. These findings provide insight into Nna1's role in neuronal development and why its absence renders Purkinje cells more vulnerable., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: The active form (T3) of thyroid hormone (TH) controls critical aspects of cerebellar development, such as migration of postmitotic neurons and terminal dendritic differentiation of Purkinje cells. The effects of T3 on early dendritic differentiation are poorly understood., Results: In this study, we have analyzed the influence of T3 on the progression of the early steps of Purkinje cell dendritic differentiation in postnatal day 0 organotypic cerebellar cultures. These steps include, successively, regression of immature neuritic processes, a stellate cell stage, and the extension of several long and mature perisomatic protrusions before the growth of the ultimate dendritic tree. We also studied the involvement of RORalpha, a nuclear receptor controlling early Purkinje cell dendritic differentiation. We show that T3 treatment leads to an accelerated progression of the early steps of dendritic differentiation in culture, together with an increased expression of RORalpha (mRNA and protein) in both Purkinje cells and interneurons. Finally, we show that T3 failed to promote early dendritic differentiation in staggerer RORalpha-deficient Purkinje cells., Conclusions: Our results demonstrate that T3 action on the early Purkinje cell dendritic differentiation process is mediated by RORalpha.

Background: During cerebellar development, Purkinje cells (PCs) form the most elaborate dendritic trees among neurons in the brain, but the mechanism regulating PC arborization remains largely unknown. Geranylgeranyltransferase I (GGT) is a prenyltransferase that is responsible for lipid modification of several signaling proteins, such as Rho family small GTPase Rac1, which has been shown to be involved in neuronal morphogenesis. Here we show that GGT plays an important role in dendritic development of PCs., Results: We found that GGT was abundantly expressed in the developing rat cerebellum, in particular molecular layer (ML), the region enriched with PC dendrites. Inhibition or down-regulation of GGT using small interference RNA (siRNA) inhibited dendritic development of PCs. In contrast, up-regulation of GGT promoted dendritic arborization of PCs. Furthermore, neuronal depolarization induced by high K+ or treatment with brain-derived neurotrophic factor (BDNF) promoted membrane association of Rac1 and dendritic development of PCs in cultured cerebellar slices. The effect of BDNF or high K+ was inhibited by inhibition or down-regulation of GGT., Conclusion: Our results indicate that GGT plays an important role in Purkinje cell development, and suggest a novel role of GGT in neuronal morphogenesis in vivo.

Purkinje cell dendrites are excitable structures with intrinsic and synaptic conductances contributing to the generation and propagation of electrical activity. Voltage-gated potassium channel subunit Kv3.3 is expressed in the distal dendrites of Purkinje cells. However, the functional relevance of this dendritic distribution is not understood. Moreover, mutations in Kv3.3 cause movement disorders in mice and cerebellar atrophy and ataxia in humans, emphasizing the importance of understanding the role of these channels. In this study, we explore functional implications of this dendritic channel expression and compare Purkinje cell dendritic excitability in wild-type and Kv3.3 knockout mice. We demonstrate enhanced excitability of Purkinje cell dendrites in Kv3.3 knockout mice, despite normal resting membrane properties. Combined data from local application pharmacology, voltage clamp analysis of ionic currents, and assessment of dendritic Ca(2+) spike threshold in Purkinje cells suggest a role for Kv3.3 channels in opposing Ca(2+) spike initiation. To study the physiological relevance of altered dendritic excitability, we measured [Ca(2+)](i) changes throughout the dendritic tree in response to climbing fiber activation. Ca(2+) signals were specifically enhanced in distal dendrites of Kv3.3 knockout Purkinje cells, suggesting a role for dendritic Kv3.3 channels in regulating propagation of electrical activity and Ca(2+) influx in distal dendrites. These findings characterize unique roles of Kv3.3 channels in dendrites, with implications for synaptic integration, plasticity, and human disease.

Previous studies suggest that glial cells contribute to synaptogenesis in specific neurons from the postnatal CNS. Here, we studied whether this is true for Purkinje cells (PCs), which represent a unique neuronal cell type due to their large size, massive synaptic input, and high vulnerability. Using new glia-free cultures enriched in PCs from postnatal mice we show that these neurons survived and grew, but displayed only low levels of excitatory and inhibitory synaptic activity. Coculture with glial cells strongly enhanced the frequency and size of spontaneous and miniature excitatory synaptic currents as well as neurite growth and branching. Immunocytochemical staining for microtubule-associated protein 2- (MAP2-) positive neurites revealed impaired dendrite formation in PCs under glia-free conditions, which can explain the absence of synaptic activity. Glial signals strongly enhanced dendritogenesis in PCs and thus their ability to receive excitatory synaptic input from granule cells (GCs). The enhancement of dendrite formation was mimicked by glia-conditioned medium (GCM), whereas the increase in synaptic activity required physical presence of glia. This indicated that dendrite development is necessary but not sufficient for PCs to receive excitatory synaptic input and that synaptogenesis requires additional signals. The level of inhibitory synaptic activity was low even in cocultures due to a low incidence of inhibitory interneurons. Taken together, our results reinforce the idea that glial cells promote synaptogenesis in specific neuronal cell types.

Purkinje cell loss by excitotoxic damage is a typical finding in many cerebellar diseases. One important aspect of this high sensitivity of Purkinje cells to excitotoxic death might be the enormous size of their dendritic tree, with a high load of excitatory glutamate receptors. We have studied whether reduction in the size of the dendritic tree might confer resistance against excitotoxic death to Purkinje cells. We have grown Purkinje cells in organotypic cerebellar slice cultures under chronic activation of metabotropic glutamate receptors or of protein kinase C. Both treatments strongly reduced dendritic tree size. After this treatment, cells were exposed to the glutamate receptor agonist AMPA, which has a strong excitotoxic effect on Purkinje cells. We found that Purkinje cells with small dendritic trees were as sensitive to AMPA exposure as untreated control cells with large dendritic trees. Immunostaining against vesicular glutamate transporter 1 revealed that the small dendritic trees were densely covered by glutamatergic terminals. Our results indicate that the expansion of the dendritic tree and the total number of AMPA receptors per neuron do not play a major role in determining the susceptibility of Purkinje cells to excitotoxic death.

In 1909, Cajal published an up-dated version in French (Cajal, 1909-1911) of his main work Texture of the Nervous System of Man and Vertebrates (Cajal, 1899-1904), considered the most important book devoted to the nervous system. Owing that last year was the centenary of this publication, we decided to produce an article focused on Cajals description of the morphological changes that dendritic trees undergo during development. We will emphasize his brilliant hypotheses explaining the modelling of dendritic trees (the neurotropic hypothesis and the role of neuronal activity in the patterning of the dendritic trees), and the status of this topic in present day Neuroscience. Here, we will show original photographs taken from a selected collection of Cajals slides housed in the Cajal Museum (Instituto Cajal, CSIC, Madrid, Spain) illustrating the principal changes in neuronal morphology at different stages of development of the spinal cord, cerebellum and cerebral cortex. We will also discuss Cajals initial proposals regarding the influence of neurotropic substances (chemotactic hypothesis) and neural activity in the modelling of the dendritic tree, as well as the evidence that later confirmed these theories.

Neuronal dendrites are generated during development by a series of processes involving extension and retraction of dendritic branches in a first step, and subsequently stabilisation of existing dendrites through building of synaptic connections. These processes are tightly controlled at any of these time points and control of dendritic development follows individual differentiation stages. This review describes aspects of the maturation process in cerebellar Purkinje cells and spinal motoneurons. Although motoneurons are glutamatergic whereas Purkinje cells are GABAergic and thereby functionally very different, dendritic maturation processes appear to share common mechanisms and processes in both neuronal cell types. Genetically-regulated cell-intrinsic processes control dendritic outgrowth at an early stage, being thereafter supported by local growth factors. In contrast, increasing synaptic input promotes dendritic maturation by limiting overgrowth at a later stage, with Ca2+-dependent signalling involving PKC or CaMKII as the common mode of action. This series of events apparently is common for other neuronal cell types suggesting a generalised concept for intercellular control of neuronal connectivity.

Cerebellar Purkinje cells have the most elaborate dendritic trees among neurons in the brain. To date, the contributions of calcium-permeable AMPA receptors (CP-AMPARs) in calcium signaling and dendrite formation of Purkinje cells remain to be elucidated. In the present study, therefore, we examined the effects of 1-naphthyl acetyl spermine (NAS), a blocker of CP-AMPARs, on dendrite formation by cultured Purkinje cells. NAS markedly inhibited elongation and branching of Purkinje cell dendrites. Calcium imaging experiments using caged glutamate demonstrated that NAS inhibits the increase of intracellular calcium concentration in Purkinje cells after glutamate release. These results suggest that calcium signaling mediated through CP-AMPARs plays an important role in Purkinje cell dendrite formation.

The peculiar shape and disposition of Purkinje cell (PC) dendrites, planar and highly branched, offers an optimal model to analyze cellular and molecular regulators for the acquisition of neuronal dendritic trees. During the first 2 weeks after the end of the proliferation period, PCs undergo a 2-phase remodeling process of their dendrites. The first phase consists in the complete retraction of the primitive but extensive dendritic tree, together with the formation of multiple filopodia-like processes arising from the cell body. In the second phase, there is a progressive disappearance of the somatic processes along with rapid growth and branching of the mature dendrite. Mature Purkinje cell dendrites bear two types of spiny protrusions, named spine and thorn. The spines are numerous, elongated, located at the distal dendritic compartment and form synapses with parallel fibers, whereas the thorns are shorter, rounded, emerge from the proximal compartment and synapse with climbing fibers. Different culture models and mutant mice analyses suggest the identification of intrinsic versus extrinsic determinants of the Purkinje cell dendritic development. The early phase of dendritic remodeling might be cell autonomous and regulated by specific transcription factors such as retinoid-related orphan receptor alpha (RORalpha). Afferent fibers, trophic factors and hormones regulate the orientation and growth of the mature dendritic tree contributing, with still unknown intrinsic factors, to sculpt its general architecture. The formation of spines appears as an intrinsic phenomenon independent of their presynaptic partner, the parallel fibers, and confined to the distal compartment by inhibitory influences of the climbing fibers along the proximal compartment.

The shapes of dendritic arbors are fascinating and important, yet the principles underlying these complex and diverse structures remain unclear. Here, we analyzed basal dendritic arbors of 2,171 pyramidal neurons sampled from mammalian brains and discovered 3 statistical properties: the dendritic arbor size scales with the total dendritic length, the spatial correlation of dendritic branches within an arbor has a universal functional form, and small parts of an arbor are self-similar. We proposed that these properties result from maximizing the repertoire of possible connectivity patterns between dendrites and surrounding axons while keeping the cost of dendrites low. We solved this optimization problem by drawing an analogy with maximization of the entropy for a given energy in statistical physics. The solution is consistent with the above observations and predicts scaling relations that can be tested experimentally. In addition, our theory explains why dendritic branches of pyramidal cells are distributed more sparsely than those of Purkinje cells. Our results represent a step toward a unifying view of the relationship between neuronal morphology and function.

Functional neural circuits are formed by eliminating early-formed redundant synapses and strengthening necessary connections during development. In newborn mouse cerebellum, each Purkinje cell (PC) is innervated by multiple climbing fibers (CFs) with similar strengths. Subsequently, a single CF is selectively strengthened by postnatal day 7 (P7). We find that this competition among multiple CFs occurs on the soma before CFs form synapses along dendrites. Notably, in most PCs, the single CF that has been functionally strengthened (the "winner" CF) undergoes translocation to dendrites while keeping its synapses on the soma. Synapses of the weaker CFs (the "loser" CFs) remain around the soma and form "pericellular nests" with synapses of the winner CFs. Then most perisomatic synapses are eliminated nonselectively by P15. Thus, our results suggest that the selective translocation of the winner CF to dendrites in each PC determines the single CF that survives subsequent synapse elimination and persistently innervates the PC.

In mammals, climbing fiber axons compete for sole innervation at each Purkinje cell. At the same time, synapses disappear from Purkinje somata and appear in great numbers on the dendrites. In this issue of Neuron, Hashimoto et al. show that, by the time climbing fibers ascend the dendrites, the winner and losers are already decided.

Growing physiological evidence suggests that there are functional differences between synapses made by the ascending and parallel fiber segments of the granule axon on cerebellar Purkinje cells. Supporting this view, our previous electron microscopic studies suggested that these synapses also contacted different regions of the Purkinje cell dendrite, and in particular that ascending segment synapses are made exclusively on the smallest diameter Purkinje cell dendrites. In the current study we used serial electron microscopic techniques to reconstruct Purkinje cell dendritic segments up to almost 10 mum in length. Using a combination of anatomical and immunological labeling techniques we identified the ascending or parallel fiber origins of the excitatory synaptic inputs onto dendritic spines, as well as the location of inhibitory synapses made directly on the dendritic shaft. The results confirmed that there are regions of the Purkinje cell dendrite receiving exclusively ascending or parallel fiber synapses and that ascending segment synapses are only found on small-diameter dendrites. In addition, we describe for the first time small-diameter dendritic regions contacted by both types of excitatory synapses. While our data suggest that the majority of inhibitory inputs to the Purkinje cell tree are associated with parallel fiber synaptic inputs, we also found inhibitory inputs on dendritic regions with mixed ascending and parallel fiber inputs, or exclusively parallel fiber inputs. The finding that ascending and parallel fiber inputs can be segregated on the Purkinje cell dendritic tree provides further evidence that these excitatory granule cell synaptic inputs may be functionally distinct.

Long-term memory, a persistent form of synaptic plasticity, requires translation of a subset of mRNA present in neuronal dendrites during a short and critical period through a mechanism not yet fully elucidated. Western blotting analysis revealed a high content of eukaryotic translation initiation factor 5A (eIF5A) in the brain of neonatal rats, a period of intense neurogenesis rate, differentiation and synaptic establishment, when compared to adult rats. Immunohistochemistry analysis revealed that eIF5A is present in the whole brain of adult rats showing a variable content among the cells from different areas (e.g. cortex, hippocampus and cerebellum). A high content of eIF5A in the soma and dendrites of Purkinje cells, key neurons in the control of motor long-term memory in the cerebellum, was observed. Detection of high eIF5A content was revealed in dendritic varicosities of Purkinje cells. Evidence is presented herein that a reduction of eIF5A content is associated to brain aging.

Cerebellar Purkinje cells elaborate one of the most complex dendritic arbors among neurons to integrate the numerous signals they receive from the cerebellum circuitry. Their dendritic differentiation undergoes successive, tightly regulated phases of development involving both regressive and growth events. Although many players regulating the late phases of Purkinje cell dendritogenesis have been identified, intracellular factors controlling earlier phases of dendritic development remain mostly unknown. In this study, we explored the biological properties and functions of SCLIP, a protein of the stathmin family, in Purkinje cell dendritic differentiation and cerebellum development. Unlike the other stathmins, SCLIP is strongly expressed in Purkinje cells during cerebellar development and accumulates in their dendritic processes at a critical period of their formation and outgrowth. To reveal SCLIP functions, we developed a lentiviral-mediated approach on cerebellar organotypic cultures to inhibit or increase its expression in Purkinje cells in their tissue environment. Depletion of SCLIP promoted retraction of the Purkinje cell primitive process and then prevented the formation of new dendrites at early stages of postnatal development. It also prevented their elongation and branching at later phases of differentiation. Conversely, SCLIP overexpression promoted dendritic branching and development. Together, our results demonstrate for the first time that SCLIP is crucial for both the formation and proper development of Purkinje cell dendritic arbors. SCLIP appears thus as a novel and specific factor that controls the early phases of Purkinje cell dendritic differentiation during cerebellum development.

The small GTPase Rac controls cell morphology, gene expression, and reactive oxygen species formation. Manipulations of Rac activity levels in the cerebellum result in motor coordination defects, but activators of Rac in the cerebellum are unknown. P-Rex family guanine-nucleotide exchange factors activate Rac. We show here that, whereas P-Rex1 expression within the brain is widespread, P-Rex2 is specifically expressed in the Purkinje neurons of the cerebellum. We have generated P-Rex2(-/-) and P-Rex1(-/-)/P-Rex2(-/-) mice, analyzed their Purkinje cell morphology, and assessed their motor functions in behavior tests. The main dendrite is thinned in Purkinje cells of P-Rex2(-/-) pups and dendrite structure appears disordered in Purkinje cells of adult P-Rex2(-/-) and P-Rex1(-/-)/P-Rex2(-/-) mice. P-Rex2(-/-) mice show a mild motor coordination defect that progressively worsens with age and is more pronounced in females than in males. P-Rex1(-/-)/P-Rex2(-/-) mice are ataxic, with reduced basic motor activity and abnormal posture and gait, as well as impaired motor coordination even at a young age. We conclude that P-Rex1 and P-Rex2 are important regulators of Purkinje cell morphology and cerebellar function.

Despite the general uniformity in cellular composition of the adult cerebellar cortex, there is a complex underlying pattern of parasagittal stripes of Purkinje cells with characteristic molecular phenotypes and patterns of connectivity. It is not known whether interneuron processes are restricted at stripe boundaries. To begin to address the issue, three strategies were used to explore how cerebellar Golgi cell dendrites are organized with respect to parasagittal stripes: first, double immunofluorescence staining combining anti-neurogranin to identify Golgi cell dendrites with the Purkinje cell compartmentation antigens zebrin II/aldolase C, HNK-1, and phospholipase Cbeta4; second, zebrin II immunohistochemistry combined with a rapid Golgi-Cox impregnation procedure to reveal Golgi cell dendritic arbors; third, stripe antigen expression was used on sections of a GlyT2-EGFP transgenic mouse in which reporter expression is prominent in Golgi cell dendrites. In each case, the dendritic projections of Golgi cells were studied in the vicinity of Purkinje cell stripe boundaries. The data presented here show that the dendrites of a cerebellar interneuron, the Golgi cell, respect the fundamental cerebellar stripe cytoarchitecture.