Wild-type Hippocampal Neurons Research Articles

Targeting Tiam1 enhances hippocampal-dependent learning and memory in the adult brain and promotes NMDA receptor-mediated synaptic plasticity and function.

Excitatory synapses and the actin-rich dendritic spines on which they reside are indispensable for information processing and storage in the brain. In the adult hippocampus, excitatory synapses must balance plasticity and stability to support learning and memory. However, the mechanisms governing this balance remain poorly understood. Tiam1 is an actin cytoskeleton regulator prominently expressed in the dentate gyrus (DG) throughout life. Previously, we showed that Tiam1 promotes dentate granule cell synapse and spine stabilization during development, but its role in the adult hippocampus remains unclear. Here, we deleted Tiam1 from adult forebrain excitatory neurons (Tiam1fKO ) and assessed the effects on hippocampal-dependent behaviors. Adult male and female Tiam1fKO mice displayed enhanced contextual fear memory, fear extinction, and spatial discrimination. Investigation into underlying mechanisms revealed that dentate granule cells from Tiam1fKO brain slices exhibited augmented synaptic plasticity and NMDA-type glutamate receptor (NMDAR) function. Additionally, Tiam1 loss in primary hippocampal neurons blocked agonist-induced NMDAR internalization, reduced filamentous actin levels, and promoted activity-dependent spine remodeling. Notably, strong NMDAR activation in wild-type hippocampal neurons triggered Tiam1 loss from spines. Our results suggest that Tiam1 normally constrains hippocampal-dependent learning and memory in the adult brain by restricting NMDAR-mediated synaptic plasticity in the DG. We propose that Tiam1 achieves this by limiting NMDAR availability at synaptic membranes and stabilizing spine actin cytoskeleton, and that these constraints can be alleviated by activity-dependent degradation of Tiam1. These findings reveal a previously unknown mechanism restricting hippocampal synaptic plasticity and highlight Tiam1 as a therapeutic target for enhancing cognitive function.Significance statement The precise and dynamic regulation of excitatory synapses within hippocampal circuits is indispensable for learning and memory. These specialized connections must remain malleable to support the acquisition of relevant new information, but stable enough to protect the loss of previously stored memories. Dysregulation of this intricate balance drives cognitive decline following central nervous system injury and disease. Here, we establish the Rac1 guanine nucleotide exchange factor Tiam1 as an essential regulator of this balance that functions in the adult hippocampus to limit learning and memory. Our findings identify a previously unknown mechanism for restricting hippocampal synaptic plasticity that represents a potential therapeutic target for improving cognitive function.

Neuron-restrictive silencer factor/repressor element 1-silencing transcription factor (NRSF/REST) controls spatial K+ buffering in primary cortical astrocytes.

Neuron-restrictive silencer factor/repressor element 1 (RE1)-silencing transcription factor (NRSF/REST) is a transcriptional repressor of a large cluster of neural genes containing RE1 motifs in their promoter region. NRSF/REST is ubiquitously expressed in non-neuronal cells, including astrocytes, while it is down-regulated during neuronal differentiation. While neuronal NRSF/REST homeostatically regulates intrinsic excitability and synaptic transmission, the role of the high NRSF/REST expression levels in the homeostatic functions of astrocytes is poorly understood. Here, we investigated the functional consequences of NRSF/REST deletion in primary cortical astrocytes derived from NRSF/REST conditional knockout mice (KO). We found that NRSF/REST KO astrocyte displayed a markedly reduced activity of inward rectifying K+ channels subtype 4.1 (Kir4.1) underlying spatial K+ buffering that was associated with a decreased expression and activity of the glutamate transporter-1 (GLT-1) responsible for glutamate uptake by astrocytes. The effects of the impaired astrocyte homeostatic functions on neuronal activity were investigated by co-culturing wild-type hippocampal neurons with NRSF/REST KO astrocytes. Interestingly, neurons experienced increased neuronal excitability at high firing rates associated with decrease after hyperpolarization and increased amplitude of excitatory postsynaptic currents. The data indicate that astrocytic NRSF/REST directly participates in neural circuit homeostasis by regulating intrinsic excitability and excitatory transmission and that dysfunctions of NRSF/REST expression in astrocytes may contribute to the pathogenesis of neurological disorders.

RNA editing-mediated regulation of calcium-dependent activator protein for secretion (CAPS1) localization and its impact on synaptic transmission.

Calcium-dependent activator protein for secretion 1 (CAPS1) is a SNARE accessory protein that facilitates formation of the SNARE complex to enable neurotransmitter release. Messenger RNAs encoding CAPS1 are subject to a site-specific adenosine-to-inosine (A-to-I) editing event resulting in a glutamate-to-glycine (E-to-G) substitution in the C-terminal domain of the encoded protein product. The C-terminal domain of CAPS1 is necessary for its synaptic enrichment and Cadps RNA editing has been shown previously to enhance the release of neuromodulatory transmitters. Using mutant mouse lines engineered to solely express CAPS1 protein isoforms encoded by either the non-edited or edited Cadps transcript, primary neuronal cultures from mouse hippocampus were used to explore the effect of Cadps editing on neurotransmission and CAPS1 synaptic localization at both glutamatergic and GABAergic synapses. While the editing of Cadps does not alter baseline evoked neurotransmission, it enhances short-term synaptic plasticity, specifically short-term depression, at inhibitory synapses. Cadps editing also alters spontaneous inhibitory neurotransmission. Neurons that solely express edited Cadps have a greater proportion of synapses that contain CAPS1 than neurons that solely express non-edited Cadps for both glutamatergic and GABAergic synapses. Editing of Cadps transcripts is regulated by neuronal activity, as global network stimulation increases the extent of transcripts edited in wild-type hippocampal neurons, whereas chronic network silencing decreases the level of Cadps editing. Taken together, these results provide key insights into the importance of Cadps editing in modulating its own synaptic localization, as well as the modulation of neurotransmission at inhibitory synapses in hippocampal neurons.

Involvement of p75NTR in the effects of Aβ on L-type Ca2+ channel in cultured neuronal networks

Ca2+ overload in neurons has been implicated in Alzheimer's Disease (AD). Upregulation of Ca2+ through L-type Ca2+ channels was known to be involved in the neurodegeneration induced by amyloid-β (Aβ) peptides in AD. However, little is known about the mechanism by which upregulation of L-type Ca2+ channel currents is linked to Aβ-induced neuronal toxicity. In the present study, we found that the L-type Ca2+ current in transgenic AD mice (Tg2576) neurons is greater than in wild-type (WT) neurons, and this Ca2+ channel current change were rescued in Tg2576/p75NTR+/− (p75 neurotrophin receptor) neurons. We further examined the changes in the gating of L-type Ca2+ channels following Aβ42 treatment, and the results showed that the L-type Ca2+ channel current was significantly increased by Aβ42 treatment in WT hippocampal neurons. Blocking or decreasing the expression of p75NTR eliminated the influence of Aβ42 on the L-type Ca2+ channel current in WT hippocampal neurons. We also evaluated how Aβ42 affected the voltage-dependent activation and inactivation of L-type Ca2+ channels in cultured WT neurons. The results indicated that the half-maximal activation voltage (V1/2) was left shifted, and the half-inactivation voltage (V1/2) displayed a right shift in neuron treated by Aβ42. Decreasing the expression of p75NTR eliminated the effect of Aβ42 on voltage-dependent activation and inactivation of the L-type Ca2+ channel. These results indicate that Aβ42 changes L-type Ca2+ channel currents by modulating the channel's activation and inactivation dynamics, while decreasing p75NTR expression can remove this effect.

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.

TNF-Α Protection of Hippocampal Neurons from Kainic Acid Induced Neurodegeneration is Associated with Up-Regulation of Nfκb and AKT as well as Down-Regulation of P38 MAPK Signaling Molecules

Background: Previously, our study showed that TNF-α plays a protective role in kainic acid (KA) induced neurodegeneration in vivo in mice. Here, we further clarified the protective role of TNF-α in KA-induced hippocampal neuronal damage in vitro and elucidated the potential signaling pathways. Methods: Hippocampal neurons were isolated from embryonic 16 days (E16) TNF-α knockout (KO) and wild-type (WT) mice with C57BL/6 background, respectively, and exposed to KA with and without recombinant TNF-α, as well as anti-TNF-α antibody. After 24-hours exposure to KA, lactate dehydrogenase (LDH) production and neurotoxicity were detected for evaluating neuronal survival rates. The production of nitric oxide (NO) from supernatants and the expression of nuclear factor kappa B (NFкB), inhibitor of NFкB alpha (IκBα), p38 mitogen-activated protein kinase (MAPK) and AKT in hippocampal neurons were measured. Results: Comparing with WT mice, the neurons of TNF-α KO mice showed more susceptibility to KA-induced neurotoxicity, as demonstrated by higher production of LDH and lower neuronal survival rates, as well as elevated NO production. It is also evidenced that pre-blocked TNF-α molecule in the neuron cultures of WT mice with anti-TNF-α antibody significantly enhanced the production of LDH and NO, and decreased the neuronal survival rate. In contrast, the neurons from WT mice pre-exposed to recombinant TNF-α were more resistant to KA induced neurotoxicity. TNF-α deficiency down-regulated phospho-IκBα, NFкB, total AKT and phospho-AKT, as well as up-regulated phospho-p38 MAPK expressions after KA insult. The reverse results were achieved in WT hippocampal neurons with TNF-α pre-administration. Conclusion: Our findings demonstrated an association between the protective effect of TNF-α on neurons as assessed following KA insult, with the up-regulation of NFκB and AKT, as well as down-regulation of p38 MAPK signaling molecules.

Presenilins regulate calcium homeostasis and presynaptic function via ryanodine receptors in hippocampal neurons

Presenilin (PS) plays a central role in the pathogenesis of Alzheimer's disease, and loss of PS causes progressive memory impairment and age-related neurodegeneration in the mouse cerebral cortex. In hippocampal neurons, PS is essential for neurotransmitter release, NMDA receptor-mediated responses, and long-term potentiation. PS is also involved in the regulation of calcium homeostasis, although the precise site of its action is less clear. Here we investigate the mechanism by which PS regulates synaptic function and calcium homeostasis using acute hippocampal slices from PS conditional knockout mice and primary cultured postnatal hippocampal neurons, in which PS is inducibly inactivated. Using two different calcium probes, Fura-2 and Mag-Fura-2, we found that inactivation of PS in primary hippocampal neurons does not affect calcium concentration in the endoplasmic reticulum. Rather, in the absence of PS, levels of ryanodine receptor (RyR) are reduced in the hippocampus, measured by Western analysis and radioligand binding assay, although the mRNA expression is unaffected. RyR-mediated function is also impaired, as indicated by reduced RyR agonist-induced calcium release from the ER and RyR-mediated synaptic responses in the absence of PS. Furthermore, knockdown of RyR expression in wild-type hippocampal neurons by two independent shRNAs to levels comparable with the RyR protein reduction in PS-deficient hippocampal neurons mimics the defects exhibited in calcium homeostasis and presynaptic function. Collectively, our findings show that PS regulates calcium homeostasis and synaptic function via RyR and suggest that disruption of intracellular calcium homeostasis may be an early pathogenic event leading to presynaptic dysfunction in Alzheimer's disease.

Phosphorylation of CRMP2 is involved in proper bifurcation of the apical dendrite of hippocampal CA1 pyramidal neurons

The neural circuit in the hippocampus is important for higher brain functions. Dendrites of CA1 pyramidal neurons mainly receive input from the axons of CA3 pyramidal neurons in this neural circuit. A CA1 pyramidal neuron has a single apical dendrite and multiple basal dendrites. In wild-type mice, most of CA1 pyramidal neurons extend a single trunk, or alternatively, the apical dendrite bifurcates into two daughter trunks at the stratum radiatum layer. We previously reported the proximal bifurcation phenotype in Sema3A-/-, p35-/-, and CRMP4-/- mice. Cdk5/p35 phosphorylates CRMP2 at Ser522, and inhibition of this phosphorylation suppressed Sema3A-induced growth cone collapse. In this study, we analyzed the bifurcation points of the apical dendrites of hippocampal CA1 pyramidal neurons in CRMP2KI/KI mice in which the Cdk5/p35-phosphorylation site Ser522 was mutated into an Ala residue. The proximal bifurcation phenotype was not observed in CRMP2KI/KI mice; however, severe proximal bifurcation of apical dendrites was found in CRMP2KI/KI;CRMP4-/- mice. Cultured hippocampal neurons from CRMP2KI/KI and CRMP2KI/KI;CRMP4-/- embryos showed an increased number of dendritic branching points compared to those from wild-type embryos. Sema3A increased the number of branching points and the total length of dendrites in wild-type hippocampal neurons, but these effects of Sema3A for dendrites were not observed in CRMP2KI/KI and CRMP2KI/KI;CRMP4-/-hippocampal neurons. Binding of CRMP2 to tubulin increased in both CRMP2KI/KI and CRMP2KI/KI:CRMP4-/- brain lysates. These results suggest that CRMP2 and CRMP4 synergistically regulate dendritic development, and CRMP2 phosphorylation is critical for proper bifurcation of apical dendrite of CA1 pyramidal neurons.

Immobilized Pool of NCAM180 in the Postsynaptic Membrane Is Homeostatically Replenished by the Flux of NCAM180 from Extrasynaptic Regions

Homeostatic mechanisms maintaining high levels of adhesion molecules in synapses over prolonged periods of time remain incompletely understood. We used fluorescence recovery after photobleaching experiments to analyze the steady state turnover of the immobile pool of green fluorescent protein-labeled NCAM180, the largest postsynaptically accumulating isoform of the neural cell adhesion molecule (NCAM). We show that there is a continuous flux of NCAM180 to the postsynaptic membrane from nonsynaptic regions of dendrites by diffusion. In the postsynaptic membrane, the newly delivered NCAM180 slowly intermixes with the immobilized pool of NCAM180. Preferential immobilization and accumulation of NCAM180 in the postsynaptic membrane is reduced after disruption of the association of NCAM180 with the spectrin cytoskeleton and in the absence of the homophilic interactions of NCAM180 in synapses. Our observations indicate that the homophilic interactions and binding to the cytoskeleton promote immobilization of NCAM180 and its accumulation in the postsynaptic membrane. Flux of NCAM180 from extrasynaptic regions and its slow intermixture with the immobile pool of NCAM180 in the postsynaptic membrane may be important for the continuous homeostatic replenishment of NCAM180 protein at synaptic contacts without compromising the long term synaptic contact stability.

Synapsin I Is an Oligomannose-Carrying Glycoprotein, Acts As an Oligomannose-Binding Lectin, and Promotes Neurite Outgrowth and Neuronal Survival When Released via Glia-Derived Exosomes

Oligomannosidic glycans play important roles in nervous system development and function. By performing a phage display screening with oligomannose-specific antibodies, we identified an oligomannose-mimicking peptide that was functionally active in modulating neurite outgrowth and neuron-astrocyte adhesion. Using the oligomannose-mimicking peptide in crosslinking experiments, synapsin I was identified as a novel oligomannose-binding protein in mouse brain. Further analyses not only verified that synapsin I is an oligomannose-binding lectin, but also indicated that it is a glycoprotein carrying oligomannose and Lewis(x). We also found that synapsin I is expressed in glia-enriched cultures and is released from glial cells via exosomes. Incubation of glial-derived exosomes in the presence of high KCl concentrations or subjecting glial cell cultures to either oxygen/glucose deprivation or hydrogen peroxide resulted in release of synapsin I from exosomes. Application of synapsin I promoted neurite outgrowth from hippocampal neurons and increased survival of cortical neurons upon hydrogen peroxide treatment or oxygen/glucose deprivation. Coculture experiments using wild-type hippocampal neurons and wild-type or synapsin-deficient glial cells showed enhanced neurite outgrowth when synapsin was expressed by glial cells. Synapsin-induced neurite outgrowth was dependent on oligomannose on synapsin I and the neural cell adhesion molecule NCAM at the neuronal cell surface. The data indicate that, under conditions of high neuronal activity and/or oxidative stress, synapsin can be released from glial-derived exosomes and promotes neurite outgrowth and neuronal survival by modulating the interactions between glia and neurons.

Non–cell autonomous influence of MeCP2-deficient glia on neuronal dendritic morphology

The neurodevelopmental disorder Rett syndrome (RTT) is caused by sporadic mutations in the transcriptional factor methyl-CpG-binding protein 2 (MeCP2). Although it is thought that the primary cause of RTT is cell autonomous, resulting from a lack of functional MeCP2 in neurons, whether non-cell autonomous factors contribute to the disease is unknown. We found that the loss of MeCP2 occurs not only in neurons but also in glial cells of RTT brains. Using an in vitro co-culture system, we found that mutant astrocytes from a RTT mouse model, and their conditioned medium, failed to support normal dendritic morphology of either wild-type or mutant hippocampal neurons. Our studies suggest that astrocytes in the RTT brain carrying MeCP2 mutations have a non-cell autonomous effect on neuronal properties, probably as a result of aberrant secretion of soluble factor(s).

Synaptic Scaffolding Molecule α Is a Scaffold To Mediate N-Methyl-d-Aspartate Receptor-Dependent RhoA Activation in Dendrites

Synaptic scaffolding molecule (S-SCAM) interacts with a wide variety of molecules at excitatory and inhibitory synapses. It comprises three alternative splicing variants, S-SCAMalpha, -beta, and -gamma. We generated mutant mice lacking specifically S-SCAMalpha. S-SCAMalpha-deficient mice breathe and feed normally but die within 24 h after birth. Primary cultured hippocampal neurons from mutant mice have abnormally elongated dendritic spines. Exogenously expressed S-SCAMalpha corrects this abnormal morphology, while S-SCAMbeta and -gamma have no effect. Active RhoA decreases in cortical neurons from mutant mice. Constitutively active RhoA and ROCKII shift the length of dendritic spines toward the normal level, whereas ROCK inhibitor (Y27632) blocks the effect by S-SCAMalpha. S-SCAMalpha fails to correct the abnormal spine morphology under the treatment of N-methyl-d-aspartate (NMDA) receptor inhibitor (AP-5), Ca(2+)/calmodulin kinase inhibitor (KN-62), or tyrosine kinase inhibitor (PP2). NMDA treatment increases active RhoA in dendrites in wild-type hippocampal neurons, but not in mutant neurons. The ectopic expression of S-SCAMalpha, but not -beta, recovers the NMDA-responsive accumulation of active RhoA in dendrites. Phosphorylation of extracellular signal-regulated kinase 1/2 and Akt and calcium influx in response to NMDA are not impaired in mutant neurons. These data indicate that S-SCAMalpha is a scaffold required to activate RhoA protein in response to NMDA receptor signaling in dendrites.

Mice with GFAP‐targeted loss of neurofibromin demonstrate increased axonal MET expression with aging

Neurofibromatosis 1 (NF1) is a common genetic disease that predisposes patients to peripheral nerve tumors and central nervous system (CNS) abnormalities including low-grade astrocytomas and cognitive disabilities. Using mice with glial fibrillary acidic protein (GFAP)-targeted Nf1 loss (Nf1(GFAP)CKO mice), we found that Nf1(-/-) astrocytes proliferate faster and are more invasive than wild-type astrocytes. In light of our previous finding that aberrant expression of the MET receptor tyrosine kinase contributes to the invasiveness of human NF1-associated malignant peripheral nerve sheath tumors, we sought to determine whether MET expression is aberrant in the brains of Nf1 mutant mice. We found that Nf1(-/-) astrocytes express slightly more MET than wild-type cells in vitro, but do not express elevated MET in situ. However, fiber tracts containing myelinated axons in the hippocampus, midbrain, cerebral cortex, and cerebellum express higher than normal levels of MET in older (> or =6 months) Nf1(GFAP)CKO mice. Both Nf1(GFAP)CKO and wild-type astrocytes induced MET expression in neurites of wild-type hippocampal neurons in vitro, suggesting that astrocyte-derived signals may induce MET in Nf1 mutant mice. Because the Nf1 gene product functions as a RAS GTPase, we examined MET expression in the brains of mice with GFAP-targeted constitutively active forms of RAS. MET was elevated in axonal fiber tracts in mice with active K-RAS but not H-RAS. Collectively, these data suggest that loss of Nf1 in either astrocytes or GFAP(+) neural progenitor cells results in increased axonal MET expression, which may contribute to the CNS abnormalities in children and adults with NF1.

A role for myosin VI in postsynaptic structure and glutamate receptor endocytosis

Myosin VI (Myo6) is an actin-based motor protein implicated in clathrin-mediated endocytosis in nonneuronal cells, though little is known about its function in the nervous system. Here, we find that Myo6 is highly expressed throughout the brain, localized to synapses, and enriched at the postsynaptic density. Myo6-deficient (Snell's waltzer; sv/sv) hippocampus exhibits a decrease in synapse number, abnormally short dendritic spines, and profound astrogliosis. Similarly, cultured sv/sv hippocampal neurons display decreased numbers of synapses and dendritic spines, and dominant-negative disruption of Myo6 in wild-type hippocampal neurons induces synapse loss. Importantly, we find that sv/sv hippocampal neurons display a significant deficit in the stimulation-induced internalization of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid–type glutamate receptors (AMPARs), and that Myo6 exists in a complex with the AMPAR, AP-2, and SAP97 in brain. These results suggest that Myo6 plays a role in the clathrin-mediated endocytosis of AMPARs, and that its loss leads to alterations in synaptic structure and astrogliosis.

Acid-sensing Ion Channel 2 (ASIC2) Modulates ASIC1 H+-activated Currents in Hippocampal Neurons

Hippocampal neurons express subunits of the acid-sensing ion channel (ASIC1 and ASIC2) and exhibit large cation currents that are transiently activated by acidic extracellular solutions. Earlier work indicated that ASIC1 contributed to the current in these neurons and suggested its importance for normal behavior. However, the specific contribution of ASIC1 and ASIC2 subunits to acid-evoked currents in hippocampal neurons remained uncertain. To decipher the individual role of the ASIC subunits, we studied H(+)-gated currents in neurons from both ASIC1 and ASIC2 null mice. We found that much of the current was produced by ASIC1a/2a heteromultimeric channels, and individual subunits made distinct contributions. The ASIC1a subunit was key in establishing current amplitude. The ASIC2a subunit had little effect on amplitude but influenced desensitization, recovery from desensitization, pH sensitivity, and the response to modulatory agents. We also found heterogeneity in the contribution of ASIC2 throughout the neuronal population, with individual neurons expressing both ASIC1a homomultimeric and ASIC1a/2a heteromultimeric channels. Studies of neurons heterozygous for disrupted ASIC alleles indicated that the properties of H(+)-gated currents are dependent on the proportion of the individual subunits. These findings indicate that the absolute and relative amounts of ASIC subunits determine the amplitude and properties of hippocampal H(+)-gated currents and therefore may contribute to normal physiology and pathophysiology.

1610 Novel cerebellar afferents to the cerebellar nuclei from the brain stem

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 21 days. 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 21 days in culture. The results further dictate the critical role astrocytes play in governing neuronal morphology including altered dendrite development in Fragile X.