Dendritic Arbor Development Research Articles

γ-TuRCs and the augmin complex are required for the development of highly branched dendritic arbors in Drosophila.

Microtubules are nucleated by γ-tubulin ring complexes (γ-TuRCs) and are essential for neuronal development. Nevertheless, γ-TuRC depletion has been reported to perturb only higher-order branching in elaborated Drosophila larval class IV dendritic arborization (da) neurons. This relatively mild phenotype has been attributed to defects in microtubule nucleation from Golgi outposts, yet most Golgi outposts lack associated γ-TuRCs. By analyzing dendritic arbor regrowth in pupae, we show that γ-TuRCs are also required for the growth and branching of primary and secondary dendrites, as well as for higher-order branching. Moreover, we identify the augmin complex (hereafter augmin), which recruits γ-TuRCs to the sides of pre-existing microtubules, as being required predominantly for higher-order branching. Augmin strongly promotes the anterograde growth of microtubules in terminal dendrites and thus terminal dendrite stability. Consistent with a specific role in higher-order branching, we find that augmin is expressed less strongly and is largely dispensable in larval class I da neurons, which exhibit few higher-order dendrites. Thus, γ-TuRCs are essential for various aspects of complex dendritic arbor development, and they appear to function in higher-order branching via the augmin pathway, which promotes the elaboration of dendritic arbors to help define neuronal morphology.

Dendritic arbor dynamics and stability in health and disease.

Dendritogenesis, a process of dendritic arbor development, is essential for the formation of functional neuronal networks, and in mammals, it begins in early life and continues into adulthood. It is a highly dynamic process in which dendritic branches form and regress until mature connectivity is achieved. Thereafter, dendritic branches are considered stable and do not undergo substantial rearrangements, although several exceptions have been described in the literature. After this long period of relative stability, significant changes in dendritic branching occur when the brain begins to age. Several neurological diseases, occurring both during development and in adulthood, have severe effects on the morphology of dendritic arbors, often associated with intellectual dysfunction. The molecular mechanisms of dendritogenesis are fairly well described. In contrast, knowledge of the molecular mechanisms of dendritic arbor stabilization and pathology‑induced instability is still quite incomplete, and several important questions remain unanswered. We describe the dynamic changes during development and adulthood and in different pathologies. Whenever possible, we also provide details on the molecular mechanisms behind dendritic dynamics and stability.

Convertase-dependent regulation of membrane-tethered and secreted ligands tunes dendrite adhesion.

During neural development, cellular adhesion is crucial for interactions among and between neurons and surrounding tissues. This function is mediated by conserved cell adhesion molecules, which are tightly regulated to allow for coordinated neuronal outgrowth. Here, we show that the proprotein convertase KPC-1 (homolog of mammalian furin) regulates the Menorin adhesion complex during development of PVD dendritic arbors in Caenorhabditis elegans. We found a finely regulated antagonistic balance between PVD-expressed KPC-1 and the epidermally expressed putative cell adhesion molecule MNR-1 (Menorin). Genetically, partial loss of mnr-1 suppressed partial loss of kpc-1, and both loss of kpc-1 and transgenic overexpression of mnr-1 resulted in indistinguishable phenotypes in PVD dendrites. This balance regulated cell-surface localization of the DMA-1 leucine-rich transmembrane receptor in PVD neurons. Lastly, kpc-1 mutants showed increased amounts of MNR-1 and decreased amounts of muscle-derived LECT-2 (Chondromodulin II), which is also part of the Menorin adhesion complex. These observations suggest that KPC-1 in PVD neurons directly or indirectly controls the abundance of proteins of the Menorin adhesion complex from adjacent tissues, thereby providing negative feedback from the dendrite to the instructive cues of surrounding tissues.

Dual Role of the P2X7 Receptor in Dendritic Outgrowth during Physiological and Pathological Brain Development.

At high levels, extracellular ATP operates as a "danger" molecule under pathologic conditions through purinergic receptors, including the ionotropic P2X7 receptor (P2X7R). Its endogenous activation is associated with neurodevelopmental disorders; however, its function during early embryonic stages remains largely unclear. Our objective was to determine the role of P2X7R in the regulation of neuronal outgrowth. For this purpose, we performed Sholl analysis of dendritic branches on primary hippocampal neurons and in acute hippocampal slices from WT mice and mice with genetic deficiency or pharmacological blockade of P2X7R. Because abnormal dendritic branching is a hallmark of certain neurodevelopmental disorders, such as schizophrenia, a model of maternal immune activation (MIA)-induced schizophrenia, was used for further morphologic investigations. Subsequently, we studied MIA-induced behavioral deficits in young adult mice females and males. Genetic deficiency or pharmacological blockade of P2X7R led to branching deficits under physiological conditions. Moreover, pathologic activation of the receptor led to deficits in dendritic outgrowth on primary neurons from WT mice but not those from P2X7R KO mice exposed to MIA. Likewise, only MIA-exposed WT mice displayed schizophrenia-like behavioral and cognitive deficits. Therefore, we conclude that P2X7R has different roles in the development of hippocampal dendritic arborization under physiological and pathologic conditions.SIGNIFICANCE STATEMENT Our main finding is a novel role for P2X7R in neuronal branching in the early stages of development under physiological conditions. We show how a decrease in the expression of P2X7R during brain development causes the receptor to play pathologic roles in adulthood. Moreover, we studied a neurodevelopmental model of schizophrenia and found that, at higher ATP concentrations, endogenous activation of P2X7R is necessary and sufficient for the development of positive and cognitive symptoms.

TRPC3 Channel Activity and Viability of Purkinje Neurons can be Regulated by a Local Signalosome.

Canonical transient receptor potential channels (TRPC3) may play a pivotal role in the development and viability of dendritic arbor in Purkinje neurons. This is a novel postsynaptic channel for glutamatergic synaptic transmission. In the cerebellum, TRPC3 appears to regulate functions relating to motor coordination in a highly specific manner. Gain of TRPC3 function is linked to significant alterations in the density and connectivity of dendritic arbor in Purkinje neurons. TRPC3 signals downstream of class I metabotropic glutamate receptors (mGluR1). Moreover, diacylglycerol (DAG) can directly bind and activate TRPC3 molecules. Here, we investigate a key question: How can the activity of the TRPC3 channel be regulated in Purkinje neurons? We also explore how mGluR1 activation, Ca2+ influx, and DAG homeostasis in Purkinje neurons can be linked to TRPC3 activity modulation. Through systems biology approach, we show that TRPC3 activity can be modulated by a Purkinje cell (PC)–specific local signalosome. The assembly of this signalosome is coordinated by DAG generation after mGluR1 activation. Our results also suggest that purinergic receptor activation leads to the spatial and temporal organization of the TRPC3 signaling module and integration of its key effector molecules such as DAG, PKCγ, DGKγ, and Ca2+ into an organized local signalosome. This signaling machine can regulate the TRPC3 cycling between active, inactive, and desensitized states. Precise activity of the TRPC3 channel is essential for tightly regulating the Ca2+ entry into PCs and thus the balance of lipid and Ca2+ signaling in Purkinje neurons and hence their viability. Cell-type–specific understanding of mechanisms regulating TRPC3 channel activity could be key in identifying therapeutic targeting opportunities.

Functional interdependence of the actin nucleator Cobl and Cobl-like in dendritic arbor development

Local actin filament formation is indispensable for development of the dendritic arbor of neurons. We show that, surprisingly, the action of single actin filament-promoting factors was insufficient for powering dendritogenesis. Instead, this required the actin nucleator Cobl and its only evolutionary distant ancestor Cobl-like acting interdependently. This coordination between Cobl-like and Cobl was achieved by physical linkage by syndapins. Syndapin I formed nanodomains at convex plasma membrane areas at the base of protrusive structures and interacted with three motifs in Cobl-like, one of which was Ca2+/calmodulin-regulated. Consistently, syndapin I, Cobl-like's newly identified N terminal calmodulin-binding site and the single Ca2+/calmodulin-responsive syndapin-binding motif all were critical for Cobl-like's functions. In dendritic arbor development, local Ca2+/CaM-controlled actin dynamics thus relies on regulated and physically coordinated interactions of different F-actin formation-promoting factors and only together they have the power to bring about the sophisticated neuronal morphologies required for neuronal network formation in mammals.

Characterization hiPSC-derived neural progenitor cells and neurons to investigate the role of NOS1AP isoforms in human neuron dendritogenesis

Abnormal dendritic arbor development has been implicated in a number of neurodevelopmental disorders, such as autism and Rett syndrome, and the neuropsychiatric disorder schizophrenia. Postmortem brain samples from subjects with schizophrenia show elevated levels of NOS1AP in the dorsolateral prefrontal cortex, a region of the brain associated with cognitive function. We previously reported that the long isoform of NOS1AP (NOS1AP-L), but not the short isoform (NOS1AP-S), negatively regulates dendrite branching in rat hippocampal neurons. To investigate the role that NOS1AP isoforms play in human dendritic arbor development, we adapted methods to generate human neural progenitor cells and neurons using induced pluripotent stem cell (iPSC) technology. We found that increased protein levels of either NOS1AP-L or NOS1AP-S decrease dendrite branching in human neurons at the developmental time point when primary and secondary branching actively occurs. Next, we tested whether pharmacological agents can decrease the expression of NOS1AP isoforms. Treatment of human iPSC-derived neurons with d-serine, but not clozapine, haloperidol, fluphenazine, or GLYX-13, results in a reduction in endogenous NOS1AP-L, but not NOS1AP-S, protein expression; however, d-serine treatment does not reverse decreases in dendrite number mediated by overexpression of NOS1AP isoforms. In summary, we demonstrate how an in vitro model of human neuronal development can help in understanding the etiology of schizophrenia and can also be used as a platform to screen drugs for patients.

Experience-Dependent Development of Dendritic Arbors in Mouse Visual Cortex.

The dendritic arbor of neurons constrains the pool of available synaptic partners and influences the electrical integration of synaptic currents. Despite these critical functions, our knowledge of the dendritic structure of cortical neurons during early postnatal development and how these dendritic structures are modified by visual experience is incomplete. Here, we present a large-scale dataset of 849 3D reconstructions of the basal arbor of pyramidal neurons collected across early postnatal development in visual cortex of mice of either sex. We found that the basal arbor grew substantially between postnatal day 7 (P7) and P30, undergoing a 45% increase in total length. However, the gross number of primary neurites and dendritic segments was largely determined by P7. Growth from P7 to P30 occurred primarily through extension of dendritic segments. Surprisingly, comparisons of dark-reared and typically reared mice revealed that a net gain of only 15% arbor length could be attributed to visual experience; most growth was independent of experience. To examine molecular contributions, we characterized the role of the activity-regulated small GTPase Rem2 in both arbor development and the maintenance of established basal arbors. We showed that Rem2 is an experience-dependent negative regulator of dendritic segment number during the visual critical period. Acute deletion of Rem2 reduced directionality of dendritic arbors. The data presented here establish a highly detailed, quantitative analysis of basal arbor development that we believe has high utility both in understanding circuit development as well as providing a framework for computationalists wishing to generate anatomically accurate neuronal models.SIGNIFICANCE STATEMENT Dendrites are the sites of the synaptic connections among neurons. Despite their importance for neural circuit function, only a little is known about the postnatal development of dendritic arbors of cortical pyramidal neurons and the influence of experience. Here we show that the number of primary basal dendritic arbors is already established before eye opening, and that these arbors primarily grow through lengthening of dendritic segments and not through addition of dendritic segments. Surprisingly, visual experience has a modest net impact on overall arbor length (15%). Experiments in KO animals revealed that the gene Rem2 is positive regulator of dendritic length and a negative regulator of dendritic segments.

Golgi Complex Dynamics and Its Implication in Prevalent Neurological Disorders.

Coupling of protein synthesis with protein delivery to distinct subcellular domains is essential for maintaining cellular homeostasis, and defects thereof have consistently been shown to be associated with several diseases. This function is particularly challenging for neurons given their polarized nature and differential protein requirements in synaptic boutons, dendrites, axons, and soma. Long-range trafficking is greatly enhanced in neurons by discrete mini-organelles resembling the Golgi complex (GC) referred to as Golgi outposts (GOPs) which play an essential role in the development of dendritic arborization. In this context, the morphology of the GC is highly plastic, and the polarized distribution of this organelle is necessary for neuronal migration and polarized growth. Furthermore, synaptic components are readily trafficked and modified at GOP suggesting a function for this organelle in synaptic plasticity. However, little is known about GOPs properties and biogenesis and the role of GOP dysregulation in pathology. In this review, we discuss current literature supporting a role for GC dynamics in prevalent neurological disorders such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and epilepsy, and examine the association of these disorders with the wide-ranging effects of GC function on common cellular pathways regulating neuronal excitability, polarity, migration, and organellar stress. First, we discuss the role of Golgins and Golgi-associated proteins in the regulation of GC morphology and dynamics. Then, we consider abnormal GC arrangements observed in neurological disorders and associations with common neuronal defects therein. Finally, we consider the cell signaling pathways involved in the modulation of GC dynamics and argue for a master regulatory role for Reelin signaling, a well-known regulator of neuronal polarity and migration. Determining the cellular pathways involved in shaping the Golgi network will have a direct and profound impact on our current understanding of neurodevelopment and neuropathology and aid the development of novel therapeutic strategies for improved patient care and prognosis.

Development of GABAergic Inputs Is Not Altered in Early Maturation of Adult Born Dentate Granule Neurons in Fragile X Mice.

Fragile X syndrome (FXS) is the most common form of inherited mental retardation and the most common known cause of autism. Loss of fragile X mental retardation protein (FMRP) in mice (Fmr1 KO) leads to altered synaptic and circuit maturation in the hippocampus that is correlated with alterations in hippocampal-dependent behaviors. Previous studies have demonstrated that loss of FMRP increased the rate of proliferation of progenitor cells and altered their fate specification in adult Fmr1 KO mice. While these studies clearly demonstrate a role for FMRP in adult neurogenesis in the hippocampus, it is not known whether the functional synaptic maturation and integration of adult-born dentate granule cells (abDGCs) into hippocampal circuits is affected in Fmr1 KO mice. Here, we used retroviral labeling to birthdate abDGCs in Fmr1 KO mice which allowed us to perform targeted patch clamp recording to measure the development of synaptic inputs to these neurons at precise time points after differentiation. The frequency and amplitude of spontaneous GABAergic events increased over the first three weeks after differentiation; however, this normal development of GABAergic synapses was not altered in Fmr1 KO mice. Furthermore, the relatively depolarized GABA reversal potential (EGABA) in immature abDGCs was unaffected by loss of FMRP as was the development of dendritic arbor of the adult generated neurons. These studies systematically characterized the functional development of abDGCs during the first four weeks after differentiation and demonstrate that the maturation of GABAergic synaptic inputs to these neurons is not grossly affected by the loss of FMRP.

Mapping the asynchrony of cortical maturation in the infant brain: A MRI multi-parametric clustering approach

While the main neural networks are in place at term birth, intense changes in cortical microstructure occur during early infancy with the development of dendritic arborization, synaptogenesis and fiber myelination. These maturational processes are thought to relate to behavioral acquisitions and the development of cognitive abilities. Nevertheless, in vivo investigations of such relationships are still lacking in healthy infants. To bridge this gap, we aimed to study the cortical maturation using non-invasive Magnetic Resonance Imaging, over a largely unexplored period (1–5 post-natal months). In a first univariate step, we focused on different quantitative parameters: longitudinal relaxation time (T1), transverse relaxation time (T2), and axial diffusivity from diffusion tensor imaging (λ//) These individual maps, acquired with echo-planar imaging to limit the acquisition time, showed spatial distortions that were first corrected to reliably match the thin cortical ribbon identified on high-resolution T2-weighted images. Averaged maps were also computed over the infants group to summarize the parameter characteristics during early infancy. In a second step, we considered a multi-parametric approach that leverages parameters complementarity, avoids reliance on pre-defined regions of interest, and does not require spatial constraints. Our clustering strategy allowed us to group cortical voxels over all infants in 5 clusters with distinct microstructural T1 and λ// properties The cluster maps over individual cortical surfaces and over the group were in sound agreement with benchmark post mortem studies of sub-cortical white matter myelination, showing a progressive maturation of 1) primary sensori-motor areas, 2) adjacent unimodal associative cortices, and 3) higher-order associative regions. This study thus opens a consistent approach to study cortical maturation in vivo.

Glial cell line-derived neurotrophic factor as a treatment after spinal cord injury.

Glial cell line-derived neurotrophic factor as a treatment after spinal cord injury.

Chronic pharmacological blockade of the Na+ /Ca2+ exchanger modulates the growth and development of the Purkinje cell dendritic arbor in mouse cerebellar slice cultures.

The Na+ /Ca2+ exchanger (NCX) is a bidirectional plasma membrane antiporter involved in Ca2+ homeostasis in eukaryotes. NCX has three isoforms, NCX1-3, and all of them are expressed in the cerebellum. Immunostaining on cerebellar slice cultures indicates that NCX is widely expressed in the cerebellum, including expression in Purkinje cells. The pharmacological blockade of the forward mode of NCX (Ca2+ efflux mode) by bepridil moderately inhibited growth and development of Purkinje cell dendritic arbor in cerebellar slice cultures. However, the blockade of the reverse mode (Ca2+ influx mode) by KB-R7943 severely reduced the dendritic arbor and induced a morphological change with thickened distal dendrites. The effect of KB-R7943 on dendritic growth was unrelated to the activity of voltage-gated calcium channels and was also apparent in the absence of bioelectrical activity indicating that it was mediated by NCX expressed in Purkinje cells. We have used additional NCX inhibitors including CB-DMB, ORM-10103, SEA0400, YM-244769, and SN-6 which have higher specificity for NCX isoforms and target either the forward, reverse, or both modes. These inhibitors caused a strong dendritic reduction similar to that seen with KB-R7943, but did not elicit thickening of distal dendrites. Our findings indicate that disturbance of the NCX-dependent calcium transport in Purkinje cells induces a reduction of dendritic arbor, which is presumably caused by changes in the calcium handling, and underline the importance of the calcium equilibrium for the dendritic development in cerebellar Purkinje cells.

Dendritic overgrowth and elevated ERK signaling during neonatal development in a mouse model of autism.

Autism spectrum disorder (hereafter referred to as “ASD”) is a heterogeneous neurodevelopmental condition characterized by impaired social communication and interactions, and restricted, repetitive activities or interests. Alterations in network connectivity and memory function are frequently observed in autism patients, often involving the hippocampus. However, specific changes during early brain development leading to disrupted functioning remain largely unclear. Here, we investigated the development of dendritic arbor of hippocampal CA1 pyramidal neurons in the BTBR T+tf/J (BTBR) mouse model of autism. BTBR mice display the defining behavioural features of autism, and also exhibit impaired learning and memory. We found that compared to control C57BL/6J (B6) animals, the lengths of both apical and basal dendrites were significantly greater in neonatal BTBR animals. Further, basal dendrites in the BTBR mice had higher branching complexity. In contrast, cross-sectional area of the soma was unchanged. In addition, we observed a similar density of CA1 pyramidal neurons and thickness of the neuronal layer between the two strains. Thus, there was a specific, compartmentalized overgrowth of dendrites during early development in the BTBR animals. Biochemical analysis further showed that the extracellular signal-regulated kinases (ERK) pathway was up-regulated in the hippocampus of neonatal BTBR animals. Since dendritic structure is critical for information integration and relay, our data suggest that altered development of dendrites could potentially contribute to impaired hippocampal function and behavior observed in the BTBR model, and that this might be related to increased activation of the ERK pathway.

Quantitative Map of Proteome Dynamics during Neuronal Differentiation

SummaryNeuronal differentiation is a multistep process that shapes and re-shapes neurons by progressing through several typical stages, including axon outgrowth, dendritogenesis, and synapse formation. To systematically profile proteome dynamics throughout neuronal differentiation, we took cultured rat hippocampal neurons at different developmental stages and monitored changes in protein abundance using a combination of stable isotope labeling and high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). Almost one third of all 4,500 proteins quantified underwent a more than 2-fold expression change during neuronal differentiation, indicating extensive remodeling of the neuron proteome. To highlight the strength of our resource, we studied the neural-cell-adhesion molecule 1 (NCAM1) and found that it stimulates dendritic arbor development by promoting actin filament growth at the dendritic growth cone. We anticipate that our quantitative map of neuronal proteome dynamics is a rich resource for further analyses of the many identified proteins in various neurodevelopmental processes.

Pea3 Transcription Factors, Etv4 and Etv5, Are Required for Proper Hippocampal Dendrite Development and Plasticity.

The proper formation and morphogenesis of dendrites is essential to the establishment of neuronal connectivity. We report that 2 members of the Pea3 family of transcription factors, Etv4 and Etv5, are expressed in hippocampal neurons during the main period of dendritogenesis, suggesting that they have a function in dendrite development. Here, we show that these transcription factors are physiological regulators of growth and arborization of pyramidal cell dendrites in the developing hippocampus. Gain and loss of function assays indicate that Etv4 and Etv5 are required for proper development of hippocampal dendritic arbors and spines. We have found that in vivo deletion of either Etv4 or Etv5 in hippocampal neurons causes deficits in dendrite size and complexity, which are associated with impaired cognitive function. Additionally, our data support the idea that Etv4 and Etv5 are part of a brain-derived neurotrophic factor-mediated transcriptional program required for proper hippocampal dendrite connectivity and plasticity.

Muscle- and Skin-Derived Cues Jointly Orchestrate Patterning of Somatosensory Dendrites

Sensory dendrite arbors are patterned through cell-autonomously and non-cell-autonomously functioning factors [1-3]. Yet, only a few non-cell-autonomously acting proteins have been identified, including semaphorins [4, 5], brain-derived neurotrophic factors (BDNFs) [6], UNC-6/Netrin [7], and theconserved MNR-1/Menorin-SAX-7/L1CAM cell adhesion complex [8, 9]. This complex acts from the skin to pattern the stereotypic dendritic arbors of PVD and FLP somatosensory neurons in Caenorhabditis elegans through the leucine-rich transmembrane receptor DMA-1/LRR-TM expressed on PVD neurons [8, 9]. Here we describe a role for thediffusible C.elegans protein LECT-2, which is homologous to vertebrate leukocyte cell-derived chemotaxin 2 (LECT2)/Chondromodulin II. LECT2/Chondromodulin II has been implicated in a variety of pathological conditions [10-13], but the developmental functions of LECT2 have remained elusive. We find that LECT-2/Chondromodulin II is required for development of PVD and FLP dendritic arbors and can act as a diffusible cue from a distance to shape dendritic arbors. Expressed in body-wall muscles, LECT-2 decorates neuronal processes and hypodermal cells in a pattern similar to the cell adhesion molecule SAX-7/L1CAM. LECT-2 functions genetically downstream of the MNR-1/Menorin-SAX-7/L1CAM adhesion complex and upstream of the DMA-1 receptor. LECT-2 localization is dependent on SAX-7/L1CAM, but not on MNR-1/Menorin or DMA-1/LRR-TM, suggesting that LECT-2 functions as part of the skin-derived MNR-1/Menorin-SAX-7/L1CAM adhesion complex. Collectively, our findings suggest that LECT-2/Chondromodulin II acts as a muscle-derived, diffusible cofactor together with a skin-derived cell adhesion complex to orchestrate the molecular interactions of three tissues during patterning of somatosensory dendrites.

MTOR kinase is needed for the development and stabilization of dendritic arbors in newly born olfactory bulb neurons.

ABSTRACTNeurogenesis is the process of neuron generation, which occurs not only during embryonic development but also in restricted niches postnatally. One such region is called the subventricular zone (SVZ), which gives rise to new neurons in the olfactory bulb (OB). Neurons that are born postnatally migrate through more complex territories and integrate into fully functional circuits. Therefore, differences in the differentiation of embryonic and postnatally born neurons may exist. Dendritogenesis is an important process for the proper formation of future neuronal circuits. Dendritogenesis in embryonic neurons cultured in vitro was shown to depend on the mammalian target of rapamycin (mTOR). Still unknown, however, is whether mTOR could regulate the dendritic arbor morphology of SVZ‐derived postnatal OB neurons under physiological conditions in vivo. The present study used in vitro cultured and differentiated SVZ‐derived neural progenitors and found that both mTOR complex 1 and mTOR complex 2 were required for the dendritogenesis of SVZ‐derived neurons. Furthermore, using a combination of in vivo electroporation of neural stem cells in the SVZ and genetic and pharmacological inhibition of mTOR, it was found that mTOR was crucial for the growth of basal and apical dendrites in postnatally born OB neurons under physiological conditions and contributed to the stabilization of their basal dendrites. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1308–1327, 2016

Hippocampal neuronal subtypes develop abnormal dendritic arbors in the presence of Fragile X astrocytes

Astrocytes are now recognized as key players in the neurobiology of neurodevelopmental disorders such as Fragile X syndrome. However, the nature of Fragile X astrocyte-mediated control of dendrite development in subtypes of hippocampal neurons is not yet known. We used a co-culture procedure in which wildtype primary hippocampal neurons were cultured with astrocytes from either a wildtype or Fragile X mouse, for either 7, 14 or 21days. The neurons were processed for immunocytochemistry with the dendritic marker MAP2, classified by morphological criteria into one of five neuronal subtypes, and subjected to Sholl analyses. Both linear and semi-log methods of Sholl analyses were applied to the neurons in order to provide an in depth analysis of the dendritic arborizations. We found that Fragile X astrocytes affect the development of dendritic arborization of all subtypes of wildtype hippocampal neurons. Furthermore, we show that hippocampal neurons with spiny stellate neuron morphology exhibit the most pervasive developmental delays, with significant dendritic arbor alterations persisting at 21days in culture. The results further dictate the critical role astrocytes play in governing neuronal morphology including altered dendrite development in Fragile X.

The GDNF-GFRα1 complex promotes the development of hippocampal dendritic arbors and spines via NCAM.

The formation of synaptic connections during nervous system development requires the precise control of dendrite growth and synapse formation. Although glial cell line-derived neurotrophic factor (GDNF) and its receptor GFRα1 are expressed in the forebrain, the role of this system in the hippocampus remains unclear. Here, we investigated the consequences of GFRα1 deficiency for the development of hippocampal connections. Analysis of conditional Gfra1 knockout mice shows a reduction in dendritic length and complexity, as well as a decrease in postsynaptic density specializations and in the synaptic localization of postsynaptic proteins in hippocampal neurons. Gain- and loss-of-function assays demonstrate that the GDNF-GFRα1 complex promotes dendritic growth and postsynaptic differentiation in cultured hippocampal neurons. Finally, in vitro assays revealed that GDNF-GFRα1-induced dendrite growth and spine formation are mediated by NCAM signaling. Taken together, our results indicate that the GDNF-GFRα1 complex is essential for proper hippocampal circuit development.