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Alzheimer's disease (AD) is a severe neurodegenerative disorder and the most common form of dementia, causing the loss of cognitive function. Our previous study has shown, using a doubly mutated mouse model of AD (APP/PS1), that the neural adhesion molecule L1 directly binds amyloid peptides and decreases plaque load and gliosis when injected as an adeno-associated virus construct (AAV-L1) into APP/PS1 mice. In this study, we microinjected AAV-L1, using a Hamilton syringe, directly into the 3-month-old APP/PS1 mouse hippocampus and waited for a year until significant neurodegeneration developed. We stereologically counted the principal neurons and parvalbumin-positive interneurons in the hippocampus, estimated the density of inhibitory synapses around principal cells, and compared the AAV-L1 injection models with control injections of green fluorescent protein (AAV-GFP) and the wild-type hippocampus. Our results show that there is a significant loss of granule cells in the dentate gyrus of the APP/PS1 mice, which was improved by AAV-L1 injection, compared with the AAV-GFP controls (p < 0.05). There is also a generalized loss of parvalbumin-positive interneurons in the hippocampus of APP/PS1 mice, which is ameliorated by AAV-L1 injection, compared with the AAV-GFP controls (p < 0.05). Additionally, AAV-L1 injection promotes the survival of inhibitory synapses around the principal cells compared with AAV-GFP controls in all three hippocampal subfields (p < 0.01). Our results indicate that L1 promotes neuronal survival and protects the synapses in an AD mouse model, which could have therapeutic implications.
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Background: Familial hypercholesterolemia (FH) is an autosomal dominant disorder characterized by increased LDL-cholesterol levels. About 85% of FH cases are caused by LDLR mutations encoding the low-density lipoprotein receptor (LDLR). LDLR is synthesized in the endoplasmic reticulum (ER) where it undergoes post-translational modifications and then transported through Golgi apparatus to the plasma membrane. Over 2900 LDLR variants have been reported in FH patients with limited information on the pathogenicity and functionality of many of them. This study aims to elucidate the cellular trafficking and functional implications of LDLR missense variants identified in suspected FH patients using biochemical and functional methods. Methods: We used HeLa, HEK293T, and LDLR-deficient-CHO-ldlA7 cells to evaluate the subcellular localization and LDL internalization of ten LDLR missense variants (p.C167F, p.D178N, p.C243Y, p.E277K, p.G314R, p.H327Y, p.D477N, p.D622G, p.R744Q, and p.R814Q) reported in multiethnic suspected FH patients. We also analyzed the functional impact of three variants (p.D445E, p.D482H, and p.C677F), two of which previously shown to be retained in the ER. Results: We show that p.D622G, p.D482H, and p.C667F are largely retained in the ER whereas p.R744Q is partially retained. The other variants were predominantly localized to the plasma membrane. LDL internalization assays in CHO-ldlA7 cells indicate that p.D482H, p.C243Y, p.D622G, and p.C667F have quantitatively lost their ability to internalize Dil-LDL with the others (p.C167F, p.D178N, p.G314R, p.H327Y, p.D445E, p.D477N, p.R744Q and p.R814Q) showing significant losses except for p.E277K which retained full activity. However, the LDL internalization assay is only to able evaluate the impact of the variants on LDL internalization and not the exact functional defects such as failure to bind LDL. The data represented illustrate the hypomorphism nature of variants causing FH which may explain some of the variable expressivity of FH. Conclusion: Our combinatorial approach of in silico, cellular, and functional analysis is a powerful strategy to determine pathogenicity and FH disease mechanisms which may provide opportunitites for novel therapeutic strategies.
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The function of medial entorhinal cortex layer II (MECII) excitatory neurons has been recently explored. MECII dysfunction underlies deficits in spatial navigation and working memory. MECII neurons comprise two major excitatory neuronal populations, pyramidal island and stellate ocean cells, in addition to the inhibitory interneurons. Ocean cells express reelin and surround clusters of island cells that lack reelin expression. The influence of reelin expression by ocean cells and interneurons on their own morphological differentiation and that of MECII island cells has remained unknown. To address this, we used a conditional reelin knockout (RelncKO) mouse to induce reelin deficiency postnatally in vitro and in vivo. Reelin deficiency caused dendritic hypertrophy of ocean cells, interneurons and only proximal dendritic compartments of island cells. Ca2+ recording showed that both cell types exhibited an elevation of calcium frequencies in RelncKO, indicating that the hypertrophic effect is related to excessive Ca2+ signalling. Moreover, pharmacological receptor blockade in RelncKO mouse revealed malfunctioning of GABAB, NMDA and AMPA receptors. Collectively, this study emphasizes the significance of reelin in neuronal growth, and its absence results in dendrite hypertrophy of MECII neurons.
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Moléculas de Adhesión Celular Neuronal , Dendritas , Corteza Entorrinal , Proteínas de la Matriz Extracelular , Ratones Noqueados , Proteínas del Tejido Nervioso , Proteína Reelina , Serina Endopeptidasas , Animales , Corteza Entorrinal/metabolismo , Dendritas/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genética , Ratones , Interneuronas/metabolismo , Neuronas/metabolismo , Señalización del CalcioRESUMEN
During the first and second stages of postnatal development, neocortical neurons exhibit a wide range of spontaneous synchronous activity (SSA). Towards the end of the second postnatal week, the SSA is replaced by a more sparse and desynchronized firing pattern. The developmental desynchronization of neocortical spontaneous neuronal activity is thought to be intrinsically generated, since sensory deprivation from the periphery does not affect the time course of this transition. The extracellular protein reelin controls various aspects of neuronal development through multimodular signaling. However, so far it is unclear whether reelin contributes to the developmental desynchronization transition of neocortical neurons. The present study aims to investigate the role of reelin in postnatal cortical developmental desynchronization using a conditional reelin knockout (RelncKO) mouse model. Conditional reelin deficiency was induced during early postnatal development, and Ca2+ recordings were conducted from organotypic cultures (OTCs) of the somatosensory cortex. Our results show that both wild type (wt) and RelncKO exhibited an SSA pattern during the early postnatal week. However, at the end of the second postnatal week, wt OTCs underwent a transition to a desynchronized network activity pattern, while RelncKO activity remained synchronous. This changing activity pattern suggests that reelin is involved in regulating the developmental desynchronization of cortical neuronal network activity. Moreover, the developmental desynchronization impairment observed in RelncKO was rescued when RelncKO OTCs were co-cultured with wt OTCs. Finally, we show that the developmental transition to a desynchronized state at the end of the second postnatal week is not dependent on glutamatergic signaling. Instead, the transition is dependent on GABAAR and GABABR signaling. The results suggest that reelin controls developmental desynchronization through GABAAR and GABABR signaling.
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Proteínas de la Matriz Extracelular , Ratones Noqueados , Neocórtex , Proteínas del Tejido Nervioso , Proteína Reelina , Serina Endopeptidasas , Animales , Ratones , Neocórtex/metabolismo , Neocórtex/crecimiento & desarrollo , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Neuronas/metabolismo , Red Nerviosa/metabolismo , Red Nerviosa/crecimiento & desarrollo , Corteza Somatosensorial/metabolismo , Corteza Somatosensorial/crecimiento & desarrolloRESUMEN
Neuronal plasticity is a crucial mechanism for an adapting nervous system to change. It is shown to be regulated by perineuronal nets (PNNs), the condensed forms of the extracellular matrix (ECM) around neuronal bodies. By assessing the changes in the number, intensity, and structure of PNNs, the ultrastructure of the PNN mesh, and the expression of inhibitory and excitatory synaptic inputs on these neurons, we aimed to clarify the role of an ECM glycoprotein, tenascin-C (TnC), in the dorsal hippocampus. To enhance neuronal plasticity, TnC-deficient (TnC-/-) and wild-type (TnC+/+) young adult male mice were reared in an enriched environment (EE) for 8 weeks. Deletion of TnC in TnC-/- mice showed an ultrastructural reduction of the PNN mesh and an increased inhibitory input in the dentate gyrus (DG), and an increase in the number of PNNs with a rise in the inhibitory input in the CA2 region. EE induced an increased inhibitory input in the CA2, CA3, and DG regions; in DG, the change was also followed by an increased intensity of PNNs. No changes in PNNs or synaptic expression were found in the CA1 region. We conclude that the DG and CA2 regions emerged as focal points of alterations in PNNs and synaptogenesis with EE as mediated by TnC.
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Matriz Extracelular , Hipocampo , Plasticidad Neuronal , Sinapsis , Tenascina , Animales , Tenascina/metabolismo , Tenascina/genética , Masculino , Ratones , Hipocampo/metabolismo , Matriz Extracelular/metabolismo , Sinapsis/metabolismo , Ratones Noqueados , Neuronas/metabolismo , Ratones Endogámicos C57BL , Giro Dentado/metabolismoRESUMEN
Proper growth and branching of dendrites are crucial for adequate central nervous system (CNS) functioning. The neuronal dendritic geometry determines the mode and quality of information processing. Any defects in dendrite development will disrupt neuronal circuit formation, affecting brain function. Besides cell-intrinsic programmes, extrinsic factors regulate various aspects of dendritic development. Among these extrinsic factors are extracellular molecular signals which can shape the dendrite architecture during early development. This review will focus on extrinsic factors regulating dendritic growth during early neuronal development, including neurotransmitters, neurotrophins, extracellular matrix proteins, contact-mediated ligands, and secreted and diffusible cues. How these extracellular molecular signals contribute to dendritic growth has been investigated in developing nervous systems using different species, different areas within the CNS, and different neuronal types. The response of the dendritic tree to these extracellular molecular signals can result in growth-promoting or growth-limiting effects, and it depends on the receptor subtype, receptor quantity, receptor efficiency, the animal model used, the developmental time windows, and finally, the targeted signal cascade. This article reviews our current understanding of the role of various extracellular signals in the establishment of the architecture of the dendrites.
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Introduction: There is an increasing evidence supporting the hypothesis that traumatic experiences during early developmental periods might be associated with psychopathology later in life. Maternal deprivation (MD) in rodents has been proposed as an animal model for certain aspects of neuropsychiatric disorders. Methods: To determine whether early-life stress leads to changes in GABAergic, inhibitory interneurons in the limbic system structures, specifically the amygdala and nucleus accumbens, 9-day-old Wistar rats were exposed to a 24 h MD. On postnatal day 60 (P60), the rats were sacrificed for morphometric analysis and their brains were compared to the control group. Results: Results show that MD affect GABAergic interneurons, leading to the decrease in density and size of the calcium-binding proteins parvalbumin-, calbindin-, and calretinin-expressing interneurons in the amygdala and nucleus accumbens. Discussion: This study indicates that early stress in life leads to changes in the number and morphology of the GABAergic, inhibitory interneurons in the amygdala and nucleus accumbens, most probably due to the loss of neurons during postnatal development and it further contributes to understanding the effects of maternal deprivation on brain development.
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The extracellular matrix (ECM) of the nervous system can be considered as a dynamically adaptable compartment between neuronal cells, in particular neurons and glial cells, that participates in physiological functions of the nervous system. It is mainly composed of carbohydrates and proteins that are secreted by the different kinds of cell types found in the nervous system, in particular neurons and glial cells, but also other cell types, such as pericytes of capillaries, ependymocytes and meningeal cells. ECM molecules participate in developmental processes, synaptic plasticity, neurodegeneration and regenerative processes. As an example, the ECM of the hippocampal formation is involved in degenerative and adaptive processes related to epilepsy. The role of various components of the ECM has been explored extensively. In particular, the ECM protein reelin, well known for orchestrating the formation of neuronal layer formation in the cerebral cortex, is also considered as a player involved in the occurrence of postnatal granule cell dispersion (GCD), a morphologically peculiar feature frequently observed in hippocampal tissue from epileptic patients. Possible causes and consequences of GCD have been studied in various in vivo and in vitro models. The present review discusses different interpretations of GCD and different views on the role of ECM protein reelin in the formation of this morphological peculiarity.
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Reelin is a large secreted glycoprotein that regulates neuronal migration, lamination and establishment of dendritic architecture in the embryonic brain. Reelin expression switches postnatally from Cajal-Retzius cells to interneurons. However, reelin function in interneuron development is still poorly understood. Here, we have investigated the role of reelin in interneuron development in the postnatal neocortex. To preclude early cortical migration defects caused by reelin deficiency, we employed a conditional reelin knockout (RelncKO) mouse to induce postnatal reelin deficiency. Induced reelin deficiency caused dendritic hypertrophy in distal dendritic segments of neuropeptide Y-positive (NPY+) and calretinin-positive (Calr+) interneurons, and in proximal dendritic segments of parvalbumin-positive (Parv+) interneurons. Chronic recombinant Reelin treatment rescued dendritic hypertrophy in Relncko interneurons. Moreover, we provide evidence that RelncKO interneuron hypertrophy is due to presynaptic GABABR dysfunction. Thus, GABABRs in RelncKO interneurons were unable to block N-type (Cav2.2) Ca2+ channels that control neurotransmitter release. Consequently, the excessive Ca2+ influx through AMPA receptors, but not NMDA receptors, caused interneuron dendritic hypertrophy. These findings suggest that reelin acts as a 'stop-growth-signal' for postnatal interneuron maturation.
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Moléculas de Adhesión Celular Neuronal/metabolismo , Dendritas/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Interneuronas/citología , Neocórtex/crecimiento & desarrollo , Proteínas del Tejido Nervioso/metabolismo , Serina Endopeptidasas/metabolismo , Animales , Calbindina 2/metabolismo , Calcio/metabolismo , Moléculas de Adhesión Celular Neuronal/deficiencia , Moléculas de Adhesión Celular Neuronal/farmacología , Dendritas/efectos de los fármacos , Proteínas de la Matriz Extracelular/deficiencia , Proteínas de la Matriz Extracelular/farmacología , Hipertrofia , Interneuronas/efectos de los fármacos , Interneuronas/metabolismo , Ratones , Ratones Noqueados , Neocórtex/citología , Neocórtex/efectos de los fármacos , Neocórtex/patología , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/farmacología , Neuropéptido Y/metabolismo , Parvalbúminas/metabolismo , Receptores de GABA-B/metabolismo , Receptores de Glutamato/metabolismo , Proteína Reelina , Serina Endopeptidasas/deficiencia , Serina Endopeptidasas/farmacologíaRESUMEN
Reelin is a protein that is best known for its role in controlling neuronal layer formation in the developing cortex. Here, we studied its role for post-natal cortical network function, which is poorly explored. To preclude early cortical migration defects caused by Reelin deficiency, we used a conditional Reelin knock-out (RelncKO ) mouse, and induced Reelin deficiency post-natally. Induced Reelin deficiency caused hyperexcitability of the neocortical network in vitro and ex vivo. Blocking Reelin binding to its receptors ApoER2 and VLDLR resulted in a similar effect. Hyperexcitability in RelncKO organotypic slice cultures could be rescued by co-culture with wild-type organotypic slice cultures. Moreover, the GABAB receptor (GABAB R) agonist baclofen failed to activate and the antagonist CGP35348 failed to block GABAB Rs in RelncKO mice. Immunolabeling of RelncKO cortical slices revealed a reduction in GABAB R1 and GABAB R2 surface expression at the plasma membrane and western blot of RelncKO cortical tissue revealed decreased phosphorylation of the GABAB R2 subunit at serine 892 and increased phosphorylation at serine 783, reflecting receptor deactivation and proteolysis. These data show a role of Reelin in controlling early network activity, by modulating GABAB R function. Cover Image for this issue: https://doi.org/10.1111/jnc.15054.
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Moléculas de Adhesión Celular Neuronal/deficiencia , Proteínas de la Matriz Extracelular/deficiencia , Neocórtex/metabolismo , Proteínas del Tejido Nervioso/deficiencia , Receptores de GABA-B/fisiología , Serina Endopeptidasas/deficiencia , Transducción de Señal/fisiología , Animales , Animales Recién Nacidos , Moléculas de Adhesión Celular Neuronal/genética , Proteínas de la Matriz Extracelular/genética , Femenino , Agonistas de Receptores GABA-B/farmacología , Masculino , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Técnicas de Cultivo de Órganos , Proteína Reelina , Serina Endopeptidasas/genética , Transducción de Señal/efectos de los fármacosRESUMEN
NMDA receptors are important players for neuronal differentiation. We previously reported that antagonizing NMDA receptors with APV blocked the growth-promoting effects evoked by the overexpression of specific calcium-permeable or flip-spliced AMPA receptor subunits and of type I transmembrane AMPA receptor regulatory proteins which both exclusively modify apical dendritic length and branching of cortical pyramidal neurons. These findings led us to characterize the role of GluN2B and GluN2A for dendritogenesis using organotypic cultures of rat visual cortex. Antagonizing GluN2B with ifenprodil and Ro25-6981 strongly impaired basal dendritic growth of supra- and infragranular pyramidal cells at DIV 5-10, but no longer at DIV 15-20. Growth recovered after washout, and protein blots revealed an increase of synaptic GluN2B-containing receptors as indicated by a enhanced phosphorylation of the tyrosine 1472 residue. Antagonizing GluN2A with TCN201 and NVP-AAM077 was ineffective at both ages. Dendrite growth of non-pyramidal interneurons was not altered. We attempted to overexpress GluN2A and GluN2B. However, although the constructs delivered currents in HEK cells, there were neither effects on dendrite morphology nor an enhanced sensitivity to NMDA. Further, co-expressing GluN1-1a and GluN2B did not alter dendritic growth. Visualization of overexpressed, tagged GluN2 proteins was successful after immunofluorescence for the tag which delivered rather weak staining in HEK cells as well as in neurons. This suggested that the level of overexpression is too weak to modify dendrite growth. In summary, endogenous GluN2B, but not GluN2A is important for pyramidal cell basal dendritic growth during an early postnatal time window.
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BACKGROUND: Biolistic gene gun transfection has been used to transfect organotypic cultures (OTCs) or dissociated cultures in vitro. Here, we modified this technique to allow successful transfection of acute brain slices, followed by measurement of neuronal activity within a few hours. NEW METHOD: We established biolistic transfection of murine acute cortical slices to measure calcium signals. Acute slices are mounted on plasma/thrombin coagulate and transfected with a calcium sensor. Imaging can be performed within 4 h post transfection without affecting cell viability. RESULTS: Four hours after GCaMP6s transfection, acute slices display remarkable fluorescent protein expression level allowing to study spontaneous activity and receptor pharmacology. While optimal gas pressure (150 psi) and gold particle size used (1 µm) confirm previously published protocols, the amount of 5 µg DNA was found to be optimal for particle coating. COMPARISON WITH EXISTING METHODS: The major advantage of this technique is the rapid disposition of acute slices for calcium imaging. No transgenic GECI expressing animals or OTC for long periods are required. In acute slices, network interaction and connectivity are preserved. The method allows to obtain physiological readouts within 4 h, before functional tissue modifications might come into effect. Limitations of this technique are random transfection, low expression efficiency when using specific promotors, and preclusion or genetic manipulations that require a prolonged time before physiological changes become measurable, such as expression of recombinant proteins that require transport to distant subcellular localizations. CONCLUSION: The method is optimal for short-time investigation of calcium signals in acute slices.
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Biolística , Neuronas , Animales , ADN , Técnicas de Transferencia de Gen , Ratones , TransfecciónRESUMEN
The 30-amino acid peptide Y-P30 corresponds to the N-terminus of the primate-specific, sweat gland-derived dermcidin prepropeptide. Previous work has revealed that Y-P30 enhances the interaction of pleiotrophin and syndecans-2/3, and thus represents a natural ligand to study this signaling pathway. In immature neurons, Y-P30 activates the c-Src and p42/44 ERK kinase pathway, increases the amount of F-actin in axonal growth cones, and promotes neuronal survival, cell migration and axonal elongation. The action of Y-P30 on axonal growth requires syndecan-3 and heparan sulfate side chains. Whether Y-P30 has the potential to influence dendrites and dendritic protrusions has not been explored. The latter is suggested by the observations that syndecan-2 expression increases during postnatal development, that syndecan-2 becomes enriched in dendritic spines, and that overexpression of syndecan-2 in immature neurons results in a premature morphological maturation of dendritic spines. Here, analysing rat cortical pyramidal and non-pyramidal neurons in organotypic cultures, we show that Y-P30 does not alter the development of the dendritic arborization patterns. However, Y-P30 treatment decreases the density of apical, but not basal dendritic protrusions at the expense of the filopodia. Analysis of spine morphology revealed an unchanged mushroom/stubby-to-thin spine ratio and a shortening of the longest decile of dendritic protrusions. Whole-cell recordings from cortical principal neurons in dissociated cultures grown in the presence of Y-P30 demonstrated a decrease in the frequency of glutamatergic mEPSCs. Despite these differences in protrusion morphology and synaptic transmission, the latter likely attributable to presynaptic effects, calcium event rate and amplitude recorded in pyramidal neurons in organotypic cultures were not altered by Y-P30 treatment. Together, our data suggest that Y-P30 has the capacity to decelerate spinogenesis and to promote morphological, but not synaptic, maturation of dendritic protrusions.
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Espinas Dendríticas/metabolismo , Neocórtex/citología , Péptidos/metabolismo , Animales , Calcio/metabolismo , Diferenciación Celular , Células Cultivadas , Neocórtex/metabolismo , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Ratas , Sindecano-2/metabolismoRESUMEN
During neuronal development, AMPA receptors (AMPARs) and NMDA receptors (NMDARs) are important for neuronal differentiation. Kainate receptors (KARs) are closely related to AMPARs and involved in the regulation of cortical network activity. However, their role for neurite growth and differentiation of cortical neurons is unclear. Here, we used KAR agonists and overexpression of selected KAR subunits and their auxiliary neuropilin and tolloid-like proteins, NETOs, to investigate their influence on dendritic growth and network activity in organotypic cultures of rat visual cortex. Kainate at 500 nM enhanced network activity and promoted development of dendrites in layer II/III pyramidal cells, but not interneurons. GluK2 overexpression promoted dendritic growth in pyramidal cells and interneurons. GluK2 transfectants were highly active and acted as drivers for network activity. GluK1 and NETO1 specifically promoted dendritic growth of interneurons. Our study provides new insights for the roles of KARs and NETOs in the morphological and physiological development of the visual cortex.
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Dendritas/fisiología , Interneuronas/fisiología , Células Piramidales/fisiología , Receptores de Ácido Kaínico/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Animales , Animales Recién Nacidos , Dendritas/efectos de los fármacos , Interneuronas/efectos de los fármacos , Ácido Kaínico/farmacología , Técnicas de Cultivo de Órganos , Organogénesis/efectos de los fármacos , Organogénesis/fisiología , Subunidades de Proteína/agonistas , Subunidades de Proteína/fisiología , Células Piramidales/efectos de los fármacos , Ratas , Ratas Long-Evans , Receptores de Ácido Kaínico/agonistas , Corteza Visual/efectos de los fármacos , Corteza Visual/crecimiento & desarrollo , Receptor de Ácido Kaínico GluK2Asunto(s)
Interneuronas/citología , Interneuronas/efectos de los fármacos , Factor Inhibidor de Leucemia/farmacología , Sinapsis/efectos de los fármacos , Corteza Visual/citología , Animales , Animales Recién Nacidos , Axones/efectos de los fármacos , Factor Neurotrófico Derivado del Encéfalo/farmacología , Calcio/metabolismo , Dendritas/efectos de los fármacos , Técnicas In Vitro , Interneuronas/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuropéptido Y/genética , Neuropéptido Y/metabolismo , Técnicas de Cultivo de Órganos , Ratas , Ratas Long-Evans , Canales de Potasio Shaw/genética , Canales de Potasio Shaw/metabolismo , Sinaptotagmina II/genética , Sinaptotagmina II/metabolismo , Factores de TiempoRESUMEN
BACKGROUND: Calcium imaging has unraveled the calcium-dependent mechanisms underlying neuronal function. Acetoxymethyl ester (AM) dyes are widely employed for calcium imaging. Pluronic F127 (PF127) as a surfactant and dimethyl sulfoxide (DMSO) as a solvent are used to dissolve the dyes, but concentrations vary between protocols. How these substances affect loading efficiency is not well characterized. NEW METHOD: We aimed to characterize dye loading in slice cultures. We determined minimum concentrations of surfactant, solvent and dye. The current study shows that the efficiency of AM dye loading depends on the initial stock concentration of PF127. Lowering the PF127 and DMSO concentrations can improve the loading efficiency. RESULTS: Both, pluronic and DMSO are required for successful dye loading. However, dissolving the dyes in lower concentrations of PF127 yielded better staining efficiency. Moreover, lowering the DMSO concentration to â¼0.25% improves the efficiency. The strategy allows standard two-photon or confocal microscope monitoring of neuronal activity. The labeled cells display spontaneous and evoked calcium transients, and repetitive measurements for up to 24h after loading indicate that the method is not deleterious to neuronal function. COMPARISON WITH EXISTING METHOD(S): Dissolving the AM dyes in lower concentrations of PF127 combines the advantages of high loading efficiency, preserves cell viability and functional integrity, and allows repetitive measures over hours and days. Moreover, we found that the dye itself can be diluted to a final concentration of 1µM which reduces the experimental costs. CONCLUSION: The method is optimal for calcium imaging in slice cultures.
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Calcio/metabolismo , Colorantes , Ésteres , Microscopía/métodos , Neuronas/fisiología , Animales , Tamaño de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Colorantes/química , Dimetilsulfóxido/química , Ésteres/química , Indoles/química , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neuronas/efectos de los fármacos , Concentración Osmolar , Técnicas de Placa-Clamp , Poloxámero/química , Ratas , Solventes/química , Tensoactivos/química , Técnicas de Cultivo de Tejidos , Corteza Visual/fisiologíaRESUMEN
The 30-amino acid peptide Y-P30, generated from the N-terminus of the human dermcidin precursor protein, has been found to promote neuronal survival, cell migration and neurite outgrowth by enhancing the interaction of pleiotrophin and syndecan-3. We now show that Y-P30 activates Src kinase and extracellular signal-regulated kinase (ERK). Y-P30 promotes axonal growth of mouse embryonic stem cell-derived neurons, embryonic mouse spinal cord motoneurons, perinatal rat retinal neurons, and rat cortical neurons. Y-P30-mediated axon growth was dependent on heparan sulfate chains. Y-P30 decreased the proportion of collapsing/degenerating growth cones of cortical axons in an Src and ERK-dependent manner. Y-P30 increased for 90 min in axonal growth cones the level of Tyr418-phosphorylated Src kinase and the amount of F-actin, and transiently the level of Tyr-phosphorylated ERK. Levels of total Src kinase, actin, GAP-43, cortactin and the glutamate receptor subunit GluN2B were not altered. When exposed to semaphorin-3a, Y-P30 protected a significant fraction of growth cones of cortical neurons from collapse. These results suggest that Y-P30 promotes axonal growth via Src- and ERK-dependent mechanisms which stabilize growth cones and confer resistance to collapsing factors.
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Axones/efectos de los fármacos , Conos de Crecimiento/efectos de los fármacos , Células-Madre Neurales/efectos de los fármacos , Neuronas/citología , Péptidos/farmacología , Actinas/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos , Proteína GAP-43/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Imagen Molecular , Neuronas/efectos de los fármacos , Técnicas de Cultivo de Órganos , Ratas , Ratas Long-Evans , Retina/citología , Retina/efectos de los fármacos , Semaforina-3A/metabolismoRESUMEN
The ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazole propionate glutamate receptors (AMPARs) have been implicated in the establishment of dendritic architecture. The transmembrane AMPA receptor regulatory proteins (TARPs) regulate AMPAR function and trafficking into synaptic membranes. In the current study, we employ type I and type II TARPs to modulate expression levels and function of endogenous AMPARs and investigate in organotypic cultures (OTCs) of rat occipital cortex whether this influences neuronal differentiation. Our results show that in early development [5-10 days in vitro (DIV)] only the type I TARP γ-8 promotes pyramidal cell dendritic growth by increasing spontaneous calcium amplitude and GluA2/3 expression in soma and dendrites. Later in development (10-15 DIV), the type I TARPs γ-2, γ-3 and γ-8 promote dendritic growth, whereas γ-4 reduced dendritic growth. The type II TARPs failed to alter dendritic morphology. The TARP-induced dendritic growth was restricted to the apical dendrites of pyramidal cells and it did not affect interneurons. Moreover, we studied the effects of short hairpin RNA-induced knockdown of endogenous γ-8 and showed a reduction of dendritic complexity and amplitudes of spontaneous calcium transients. In addition, the cytoplasmic tail (CT) of γ-8 was required for dendritic growth. Single-cell calcium imaging showed that the γ-8 CT domain increases amplitude but not frequency of calcium transients, suggesting a regulatory mechanism involving the γ-8 CT domain in the postsynaptic compartment. Indeed, the effect of γ-8 overexpression was reversed by APV, indicating a contribution of NMDA receptors. Our results suggest that selected type I TARPs influence activity-dependent dendritogenesis of immature pyramidal neurons.
Asunto(s)
Canales de Calcio/metabolismo , Dendritas/metabolismo , Neocórtex/citología , Células Piramidales/metabolismo , Animales , Animales Recién Nacidos , Canales de Calcio/química , Señalización del Calcio/efectos de los fármacos , Dendritas/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Neocórtex/crecimiento & desarrollo , Neocórtex/metabolismo , Neurotoxinas/toxicidad , Lóbulo Occipital/efectos de los fármacos , Lóbulo Occipital/metabolismo , Técnicas de Cultivo de Órganos , Estructura Terciaria de Proteína , Células Piramidales/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Long-Evans , Receptores AMPA/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
Glutamatergic transmission converging on calcium signaling plays a key role in dendritic differentiation. In early development, AMPA receptor (AMPAR) transcripts are extensively spliced and edited to generate subunits that differ in their biophysical properties. Whether these subunits have specific roles in the context of structural differentiation is unclear. We have investigated the role of nine GluA variants and revealed a correlation between the expression of flip variants and the period of major dendritic growth. In interneurons, only GluA1(Q)-flip increased dendritic length and branching. In pyramidal cells, GluA2(Q)-flop, GluA2(Q)-flip, GluA3(Q)-flip and calcium-impermeable GluA2(R)-flip promoted dendritic growth, suggesting that flip variants with slower desensitization kinetics are more important than receptors with elevated calcium permeability. Imaging revealed significantly higher calcium signals in pyramidal cells transfected with GluA2(R)-flip as compared with GluA2(R)-flop, suggesting a contribution of voltage-activated calcium channels. Indeed, dendritic growth induced by GluA2(R)-flip in pyramidal cells was prevented by blocking NMDA receptors (NMDARs) or voltage-gated calcium channels (VGCCs), suggesting that they act downstream of AMPARs. Intriguingly, the action of GluA1(Q)-flip in interneurons was also dependent on NMDARs and VGCCs. Cell class-specific effects were not observed for spine formation, as GluA2(Q)-flip and GluA2(Q)-flop increased spine density in pyramidal cells as well as in interneurons. The results suggest that AMPAR variants expressed early in development are important determinants for activity-dependent dendritic growth in a cell type-specific and cell compartment-specific manner.