Hypoxic Preconditioning Differentially Affects GABAergic and Glutamatergic Neuronal Cells in the Injured Cerebellum of the Neonatal Rat

In this study we examined cerebellar alterations in a neonatal rat model of hypoxic-ischemic brain injury with or without hypoxic preconditioning (Pc). Between postnatal days 7 and 15, the cerebellum is still undergoing intense cellular proliferation, differentiation and migration, dendritogenesis and synaptogenesis. The expression of glutamate decarboxylase 1 (GAD67) and the differentiation factor NeuroD1 were examined as markers of Purkinje and granule cells, respectively. We applied quantitative immunohistochemistry to sagittal cerebellar slices, and Western blot analysis of whole cerebella obtained from control (C) rats and rats submitted to Pc, hypoxia-ischemia (L) and a combination of both treatments (PcL). We found that either hypoxia-ischemia or Pc perturbed the granule cells in the posterior lobes, affecting their migration and final placement in the internal granular layer. These effects were partially attenuated when the Pc was delivered prior to the hypoxia-ischemia. Interestingly, whole nuclear NeuroD1 levels in Pc animals were comparable to those in the C rats. However, a subset of Purkinje cells that were severely affected by the hypoxic-ischemic insult—showing signs of neuronal distress at the levels of the nucleus, cytoplasm and dendritic arborization—were not protected by Pc. A monoclonal antibody specific for GAD67 revealed a three-band pattern in cytoplasmic extracts from whole P15 cerebella. A ∼110 kDa band, interpreted as a potential homodimer of a truncated form of GAD67, was reduced in Pc and L groups while its levels were close to the control animals in PcL rats. Additionally we demonstrated differential glial responses depending on the treatment, including astrogliosis in hypoxiated cerebella and a selective effect of hypoxia-ischemia on the vimentin-immunolabeled intermediate filaments of the Bergmann glia. Thus, while both glutamatergic and GABAergic cerebellar neurons are compromised by the hypoxic-ischemic insult, the former are protected by a preconditioning hypoxia while the latter are not.     

Cellular and molecular control of dendritic growth and development of cerebellar Purkinje cells

Purkinje cells are the principal neurons of the cerebellar cortex and are characterized by a large and highly branched dendritic tree. For this reason, they have for a long time been an attractive model system to study the regulation of dendritic growth and differentiation. In this article, I will first review studies on different aspects of Purkinje cell dendritic development and then go on to present studies which have aimed at experimentally altering Purkinje cell dendritic development. Some of the cellular and molecular mechanisms which have been shown by these studies to be important determinants of Purkinje cell dendritic development will be discussed, in particular the role of the parallel fiber input, of hormones, and of neuronal growth factors. The organotypic slice culture method will be introduced as an important experimental tool to study Purkinje cell dendritic development under controlled conditions. Using cerebellar slice cultures, protein kinase C (PKC) has been identified as a major determinant of Purkinje cell dendritic development and the contribution of specific isoforms of PKC will be discussed. Finally, it will be shown that Purkinje cell dendritic development in slice cultures does not depend on the activation of glutamate receptors and appears to be independent of the presence of the neurotrophin BDNF. These studies indicate that the initial outgrowth of the Purkinje cell dendritic tree can occur in the absence of signals derived from afferent fibers, but is under control of PKC signaling.     

Different regulation of Purkinje cell dendritic development in cerebellar slice cultures by protein kinase Calpha and -beta

Activity of protein kinase C (PKC), and in particular the PKCgamma-isoform, has been shown to strongly affect and regulate Purkinje cell dendritic development, suggesting an important role for PKC in activity-dependent Purkinje cell maturation. In this study we have analyzed the role of two additional Ca(2+)-dependent PKC isoforms, PKCalpha and -beta, in Purkinje cell survival and dendritic morphology in slice cultures using mice deficient in the respective enzymes. Pharmacological PKC activation strongly reduced basal Purkinje cell dendritic growth in wild-type mice whereas PKC inhibition promoted branching. Purkinje cells from mice deficient in PKCbeta, which is expressed in two splice forms by granule but not Purkinje cells, did not yield measurable morphological differences compared to respective wild-type cells under either experimental condition. In contrast, Purkinje cell dendrites in cultures from PKCalpha-deficient mice were clearly protected from the negative effects on dendritic growth of pharmacological PKC activation and showed an increased branching response to PKC inhibition as compared to wild-type cells. Together with our previous work on the role of PKCgamma, these data support a model predicting that normal Purkinje cell dendritic growth is mainly regulated by the PKCgamma-isoform, which is highly activated by developmental processes. The PKCalpha isoform in this model forms a reserve pool, which only becomes activated upon strong stimulation and then contributes to the limitation of dendritic growth. The PKCbeta isoform appears to not be involved in the signaling cascades regulating Purkinje cell dendritic maturation in cerebellar slice cultures.     

Different regulation of Purkinje cell dendritic development in cerebellar slice cultures by protein kinase Cα and ‐β

Activity of protein kinase C (PKC), and in particular the PKCγ‐isoform, has been shown to strongly affect and regulate Purkinje cell dendritic development, suggesting an important role for PKC in activity‐dependent Purkinje cell maturation. In this study we have analyzed the role of two additional Ca²⁺‐dependent PKC isoforms, PKCα and ‐β, in Purkinje cell survival and dendritic morphology in slice cultures using mice deficient in the respective enzymes. Pharmacological PKC activation strongly reduced basal Purkinje cell dendritic growth in wild‐type mice whereas PKC inhibition promoted branching. Purkinje cells from mice deficient in PKCβ, which is expressed in two splice forms by granule but not Purkinje cells, did not yield measurable morphological differences compared to respective wild‐type cells under either experimental condition. In contrast, Purkinje cell dendrites in cultures from PKCα‐deficient mice were clearly protected from the negative effects on dendritic growth of pharmacological PKC activation and showed an increased branching response to PKC inhibition as compared to wild‐type cells. Together with our previous work on the role of PKCγ, these data support a model predicting that normal Purkinje cell dendritic growth is mainly regulated by the PKCγ‐isoform, which is highly activated by developmental processes. The PKCα isoform in this model forms a reserve pool, which only becomes activated upon strong stimulation and then contributes to the limitation of dendritic growth. The PKCβ isoform appears to not be involved in the signaling cascades regulating Purkinje cell dendritic maturation in cerebellar slice cultures. © 2003 Wiley Periodicals, Inc. J Neurobiol 57: 95–109, 2003     

Ceramide and Its Interconvertible Metabolite Sphingosine Function as Indispensable Lipid Factors Involved in Survival and Dendritic Differentiation of Cerebellar Purkinje Cells

Abstract: Ceramide generated from sphingomyelin has emerged as a new but conserved type of biologically active lipid. We previously found that endogenous sphingolipids are required for the normal growth of cultured cerebellar Purkinje neurons and that sphingomyelin is present abundantly in the somatodendritic region of these cells. To gain further insight into a potential role of the sphingomyelin/ceramide pathway, we investigated the effects of depletion of sphingolipids on the phenotypic growth and survival of immature Purkinje cells and the ability of ceramide or other sphingolipids to antagonize these effects. Inhibition of ceramide synthesis by ISP-1, a specific inhibitor of serine palmitoyltransferase, decreased cellular levels of sphingolipids. This treatment resulted in a decrease in cell survival accompanied by an induction of apoptotic cell death and aberrant dendritic differentiation of Purkinje cells with no detectable changes in other cerebellar neurons. Cell-permeable ceramides, sphingosine, or sphingomyelin overcame these abnormalities more effectively than other sphingolipids when added simultaneously with ISP-1. Exposure to bacterial sphingomyelinase in turn enhanced cell survival and dendritic branching complexity of Purkinje cells at different optimal concentrations. Furthermore, cell-permeable ceramide acted synergistically with the neurotrophin family, which has been previously shown to support Purkinje cell survival. These observations suggest that ceramide is a requisite for the survival and the dendritic differentiation of Purkinje cells.     

Very-KIND, a KIND domain containing RasGEF, controls dendrite growth by linking Ras small GTPases and MAP2

The regulation of cytoskeletal components in the dendritic shaft core is critical for dendrite elongation and branching. Here, we report that a brain-specific Ras guanine nucleotide exchange factor (RasGEF) carrying two kinase non-catalytic C-lobe domains (KINDs), very-KIND (v-KIND), regulates microtubule-associated protein 2 (MAP2). v-KIND is expressed in developing mouse brain, predominantly in the cerebellar granule cells. v-KIND not only activates Ras small GTPases via the C-terminal RasGEF domain, but also specifically binds to MAP2 via the second KIND domain (KIND2), leading to threonine phosphorylation of MAP2. v-KIND overexpression suppresses dendritic extension and branching of hippocampal neurons and cerebellar granule cells, whereas knockdown of endogenous v-KIND expression promotes dendrite growth. These findings suggest that v-KIND mediates a signaling pathway that links Ras and MAP2 to control dendrite growth.     

The role of Notch and Rho GTPase signaling in the control of dendritic development

Dendritic patterning exerts a profound influence on neuronal connectivity. Recent studies indicate that mammalian Notch receptors are expressed by postmitotic neurons and that Notch signaling has a considerable influence on dendritic growth and branching. Investigations into the intracellular effectors of dendritic development have revealed that dendritic growth and branching are differentially affected by activation of the Rho-family GTPases, RhoA, Rac1, and Cdc42. These observations suggest that the differential activation of Notch receptors and Rho-family GTPases by extracellular signals may be important in the generation of morphological diversity in the developing nervous system.     

Redox- and pH-dependent association of plastocyanin with lipid bilayers: effect on protein conformation and thermal stability

The effect of electrostatic interactions on the conformation and thermal stability of plastocyanin (Pc) was studied by infrared spectroscopy. Association of any of the two redox states of the protein with positively charged membranes at neutral pH does not significantly change the secondary structure of Pc. However, upon membrane binding, the denaturation temperature decreases, regardless of the protein redox state. The extent of destabilization depends on the proportion of positively charged lipid headgroups in the membrane, becoming greater as the surface density of basic phospholipids increases. In contrast, at pH 4.8 the membrane binding-dependent conformational change becomes redox-sensitive. While the secondary structures and thermal stabilities of free and membrane-bound oxidized Pc are similar under acidic conditions, the conformation of the reduced form of the protein drastically rearranges upon membrane association. This rearrangement does not depend on electrostatic interactions to occur, since it is also observed in the presence of uncharged lipid bilayers. The conformational transition, only observed for reduced Pc, involves the exposure of hydrophobic regions that leads to intermolecular interactions at the membrane surface. Membrane-mediated partial unfolding of reduced Pc can be reversed by readjusting the pH to neutrality, in the absence of electrostatic interactions. This redox-dependent behavior might reflect specific structural requirements for the interaction of Pc with its redox partners.     

The Morphological Identity of Insect Dendrites

Dendrite morphology, a neuron's anatomical fingerprint, is a neuroscientist's asset in unveiling organizational principles in the brain. However, the genetic program encoding the morphological identity of a single dendrite remains a mystery. In order to obtain a formal understanding of dendritic branching, we studied distributions of morphological parameters in a group of four individually identifiable neurons of the fly visual system. We found that parameters relating to the branching topology were similar throughout all cells. Only parameters relating to the area covered by the dendrite were cell type specific. With these areas, artificial dendrites were grown based on optimization principles minimizing the amount of wiring and maximizing synaptic democracy. Although the same branching rule was used for all cells, this yielded dendritic structures virtually indistinguishable from their real counterparts. From these principles we derived a fully-automated model-based neuron reconstruction procedure validating the artificial branching rule. In conclusion, we suggest that the genetic program implementing neuronal branching could be constant in all cells whereas the one responsible for the dendrite spanning field should be cell specific.     

Dependence of Transformation of Intrinsic Rhythmic Impulse Activity of Neurons on Spatio-Temporal Organization of Synaptic Actions on Dendrites: A Simulation Study

The aim of the study to elucidate the biophysical mechanisms able to determine specific transformations of the patterns of output signals of neurons (neuronal impulse codes) depending on the spatio-temporal organization of synaptic actions coming to the dendrites. We studied mathematical models of the neocortical layer 5 pyramidal neurons built according to the results of computer reconstruction of their dendritic arborizations and experimental data on the voltage-dependent conductivities of their dendritic membrane. This work is a continuation of our previous studies that showed the existence of certain relations between the complexity of neural impulse codes, on the one hand, and the complexity, size, metrical asymmetry of branching, and nonlinear membrane properties of the dendrites, on the other hand. This relation determines synchronous (with some phase shifts) or asynchronous transitions of asymmetrical dendritic subtrees between high and low depolarization states during the generation of output impulse patterns in response to distributed tonic activation of dendritic inputs. In this work we demonstrate the first time that the appearance and pattern of transformations of complex periodical impulse trains at the neuron’s output associated with receiving a short series of presynaptic action potentials are determined not only by the time of arrival of such a series, but also by their spatial addressing to asymmetric dendritic subtrees; the latter, in this case, may be in the same (synchronous transitions) or different (asynchronous transitions) electrical states. Biophysically, this phenomenon is based on a significant excess of the driving potential for a synaptic excitatory current in low-depolarization regions, as compared with that in high-depolarization dendritic regions receiving phasic synaptic stimuli. These findings open a novel aspect of the functioning of neurons and neuronal networks.     

One Rule to Grow Them All: A General Theory of Neuronal Branching and Its Practical Application

Understanding the principles governing axonal and dendritic branching is essential for unravelling the functionality of single neurons and the way in which they connect. Nevertheless, no formalism has yet been described which can capture the general features of neuronal branching. Here we propose such a formalism, which is derived from the expression of dendritic arborizations as locally optimized graphs. Inspired by Ramón y Cajal''s laws of conservation of cytoplasm and conduction time in neural circuitry, we show that this graphical representation can be used to optimize these variables. This approach allows us to generate synthetic branching geometries which replicate morphological features of any tested neuron. The essential structure of a neuronal tree is thereby captured by the density profile of its spanning field and by a single parameter, a balancing factor weighing the costs for material and conduction time. This balancing factor determines a neuron''s electrotonic compartmentalization. Additions to this rule, when required in the construction process, can be directly attributed to developmental processes or a neuron''s computational role within its neural circuit. The simulations presented here are implemented in an open-source software package, the “TREES toolbox,” which provides a general set of tools for analyzing, manipulating, and generating dendritic structure, including a tool to create synthetic members of any particular cell group and an approach for a model-based supervised automatic morphological reconstruction from fluorescent image stacks. These approaches provide new insights into the constraints governing dendritic architectures. They also provide a novel framework for modelling and analyzing neuronal branching structures and for constructing realistic synthetic neural networks.     

The immunoglobulin‐like superfamily member IGSF3 is a developmentally regulated protein that controls neuronal morphogenesis

The establishment of a functional brain depends on the fine regulation and coordination of many processes, including neurogenesis, differentiation, dendritogenesis, axonogenesis, and synaptogenesis. Proteins of the immunoglobulin‐like superfamily (IGSF) are major regulators during this sequence of events. Different members of this class of proteins play nonoverlapping functions at specific developmental time‐points, as shown in particular by studies of the cerebellum. We have identified a member of the little studied EWI subfamily of IGSF, the protein IGSF3, as a membrane protein expressed in a neuron specific‐ and time‐dependent manner during brain development. In the cerebellum, it is transiently found in membranes of differentiating granule cells, and is particularly concentrated at axon terminals. There it co‐localizes with other IGSF proteins with well‐known functions in cerebellar development: TAG‐1 and L1. Functional analysis shows that IGSF3 controls the differentiation of granule cells, more precisely axonal growth and branching. Biochemical experiments demonstrate that, in the developing brain, IGSF3 is in a complex with the tetraspanin TSPAN7, a membrane protein mutated in several forms of X‐linked intellectual disabilities. In cerebellar granule cells, TSPAN7 promotes axonal branching and the size of TSPAN7 clusters is increased by downregulation of IGSF3. Thus IGSF3 is a novel regulator of neuronal morphogenesis that might function through interactions with multiple partners including the tetraspanin TSPAN7. This developmentally regulated protein might thus be at the center of a new signaling pathway controlling brain development. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 75–92, 2017     

Genetic manipulation of single neurons in vivo reveals specific roles of flamingo in neuronal morphogenesis

To study the roles of intracellular factors in neuronal morphogenesis, we used the mosaic analysis with a repressible cell marker (MARCM) technique to visualize identifiable single multiple dendritic (MD) neurons in living Drosophila larvae. We found that individual neurons in the peripheral nervous system (PNS) developed clear morphological polarity and diverse dendritic branching patterns in larval stages. Each MD neuron in the same dorsal cluster developed a unique dendritic field, suggesting that they have specific physiological functions. Single-neuron analysis revealed that Flamingo did not affect the general dendritic branching patterns in postmitotic neurons. Instead, Flamingo limited the extension of one or more dorsal dendrites without grossly affecting lateral branches. The dendritic overextension phenotype was partially conferred by the precocious initiation of dorsal dendrites in flamingo mutant embryos. In addition, Flamingo is required cell autonomously to promote axonal growth and to prevent premature axonal branching of PNS neurons. Our molecular analysis also indicated that the amino acid sequence near the first EGF motif is important for the proper localization and function of Flamingo. These results demonstrate that Flamingo plays a role in early neuronal differentiation and exerts specific effects on dendrites and axons.     

Control of dendritic development by the Drosophila fragile X-related gene involves the small GTPase Rac1

Fragile X syndrome is caused by loss-of-function mutations in the fragile X mental retardation 1 gene. How these mutations affect neuronal development and function remains largely elusive. We generated specific point mutations or small deletions in the Drosophila fragile X-related (Fmr1) gene and examined the roles of Fmr1 in dendritic development of dendritic arborization (DA) neurons in Drosophila larvae. We found that Fmr1 could be detected in the cell bodies and proximal dendrites of DA neurons and that Fmr1 loss-of-function mutations increased the number of higher-order dendritic branches. Conversely, overexpression of Fmr1 in DA neurons dramatically decreased dendritic branching. In dissecting the mechanisms underlying Fmr1 function in dendrite development, we found that the mRNA encoding small GTPase Rac1 was present in the Fmr1-messenger ribonucleoprotein complexes in vivo. Mosaic analysis with a repressor cell marker (MARCM) and overexpression studies revealed that Rac1 has a cell-autonomous function in promoting dendritic branching of DA neurons. Furthermore, Fmr1 and Rac1 genetically interact with each other in controlling the formation of fine dendritic branches. These findings demonstrate that Fmr1 affects dendritic development and that Rac1 is partially responsible for mediating this effect.     

Dendritic development: lessons from Drosophila and related branches

Dendrites show remarkable diversity in morphology and function, but the mechanisms that produce the characteristic forms is poorly understood. Insect systems offer a unique opportunity to manipulate and study identified neurons in otherwise undisturbed environments. Recent studies in Drosophila show that dendritic targeting, branching patterns, territories, and metamorphic remodeling are controlled in specific ways, by intrinsic genetic programs and extrinsic cues, with important implications for function. Here, we review some recent advances in our understanding of dendritic development in insects, focusing primarily on insights that have been gained from studies of Drosophila.     

The development of neuronal morphology in insects

Neurons are highly polarized cells with some regions specified for information input--typically the dendrites--and others specialized for information output--the axons. By extending to a specific location and branching in a specific manner, the processes of neurons determine at a fundamental level how the nervous system is wired to produce behavior. Recent studies suggest that relatively small changes in neuronal morphology could conceivably contribute to striking behavioral distinctions between invertebrate species. We review recent data that begin to shed light on how neurons extend dendrites to their targets and acquire their particular branching morphologies, drawing primarily on data from genetic model organisms. We speculate about how and why the actions of these genes might facilitate the diversification of dendritic morphology.     

The neural EGF family member CALEB/NGC mediates dendritic tree and spine complexity

The development of dendritic arborizations and spines is essential for neuronal information processing, and abnormal dendritic structures and/or alterations in spine morphology are consistent features of neurons in patients with mental retardation. We identify the neural EGF family member CALEB/NGC as a critical mediator of dendritic tree complexity and spine formation. Overexpression of CALEB/NGC enhances dendritic branching and increases the complexity of dendritic spines and filopodia. Genetic and functional inactivation of CALEB/NGC impairs dendritic arborization and spine formation. Genetic manipulations of individual neurons in an otherwise unaffected microenvironment in the intact mouse cortex by in utero electroporation confirm these results. The EGF-like domain of CALEB/NGC drives both dendritic branching and spine morphogenesis. The phosphatidylinositide 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling pathway and protein kinase C (PKC) are important for CALEB/NGC-induced stimulation of dendritic branching. In contrast, CALEB/NGC-induced spine morphogenesis is independent of PI3K but depends on PKC. Thus, our findings reveal a novel switch of specificity in signaling leading to neuronal process differentiation in consecutive developmental events.