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Dendritic spines are considered a morphological proxy for excitatory synapses, rendering them a target of many different lines of research. Over recent years, it has become possible to image simultaneously large numbers of dendritic spines in 3D volumes of neural tissue. In contrast, currently no automated method for spine detection exists that comes close to the detection performance reached by human experts. However, exploiting such datasets requires new tools for the fully automated detection and analysis of large numbers of spines. Here, we developed an efficient analysis pipeline to detect large numbers of dendritic spines in volumetric fluorescence imaging data. The core of our pipeline is a deep convolutional neural network, which was pretrained on a general-purpose image library, and then optimized on the spine detection task. This transfer learning approach is data efficient while achieving a high detection precision. To train and validate the model we generated a labelled dataset using five human expert annotators to account for the variability in human spine detection. The pipeline enables fully automated dendritic spine detection and reaches a near human-level detection performance. Our method for spine detection is fast, accurate and robust, and thus well suited for large-scale datasets with thousands of spines. The code is easily applicable to new datasets, achieving high detection performance, even without any retraining or adjustment of model parameters.
Highlights
• Enables immunostaining and visualization of epitopes deep within brain slices
• Utilizes expansion microscopy to increase imaging resolution
• Optimized for brain organotypic slice cultures and tested in acute brain slices
• Analysis workflow for protein distribution (surface vs. intracellular pool) using Imaris
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Summary
Assessing protein distribution with super-resolution in tissue is often complicated and restrictive. Here, we describe a protocol for immunostaining and expansion microscopy imaging of mouse brain organotypic slice cultures. We detail an Imaris analysis workflow to analyze the surface vs intracellular distribution of AMPA receptors at super-resolution during homeostatic plasticity. We have optimized the protocol for brain organotypic slice culture and tested in acute brain slices. This protocol is suitable to study protein distribution under multiple plasticity paradigms.
Neuro-vascular communication is essential to synchronize central nervous system development. Here, we identify angiopoietin/Tie2 as a neuro-vascular signaling axis involved in regulating dendritic morphogenesis of Purkinje cells (PCs). We show that in the developing cerebellum Tie2 expression is not restricted to blood vessels, but it is also present in PCs. Its ligands angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) are expressed in neural cells and endothelial cells (ECs), respectively. PC-specific deletion of Tie2 results in reduced dendritic arborization, which is recapitulated in neural-specific Ang1-knockout and Ang2 full-knockout mice. Mechanistically, RNA sequencing reveals that Tie2-deficient PCs present alterations in gene expression of multiple genes involved in cytoskeleton organization, dendritic formation, growth, and branching. Functionally, mice with deletion of Tie2 in PCs present alterations in PC network functionality. Altogether, our data propose Ang/Tie2 signaling as a mediator of intercellular communication between neural cells, ECs, and PCs, required for proper PC dendritic morphogenesis and function.
Endothelial tip cells are essential for VEGF-induced angiogenesis, but underlying mechanisms are elusive. The Ena/VASP protein family, consisting of EVL, VASP, and Mena, plays a pivotal role in axon guidance. Given that axonal growth cones and endothelial tip cells share many common features, from the morphological to the molecular level, we investigated the role of Ena/VASP proteins in angiogenesis. EVL and VASP, but not Mena, are expressed in endothelial cells of the postnatal mouse retina. Global deletion of EVL (but not VASP) compromises the radial sprouting of the vascular plexus in mice. Similarly, endothelial-specific EVL deletion compromises the radial sprouting of the vascular plexus and reduces the endothelial tip cell density and filopodia formation. Gene sets involved in blood vessel development and angiogenesis are down-regulated in EVL-deficient P5-retinal endothelial cells. Consistently, EVL deletion impairs VEGF-induced endothelial cell proliferation and sprouting, and reduces the internalization and phosphorylation of VEGF receptor 2 and its downstream signaling via the MAPK/ERK pathway. Together, we show that endothelial EVL regulates sprouting angiogenesis via VEGF receptor-2 internalization and signaling.
EphrinB2 and GRIP1 stabilize mushroom spines during denervation-induced homeostatic plasticity
(2021)
Highlights
• Denervation induces mushroom spine loss and AMPAR redistribution to the surface
• GRIP1 and ephrinB2 mediate homeostatic mechanisms after lesion
• Stimulation with the ephrinB2 receptor EphB4 promotes a surface shift of AMPARs
• AMPARs surface shift restores impaired spine recovery after lesion in GRIP1 mutants
Summary
Despite decades of work, much remains elusive about molecular events at the interplay between physiological and structural changes underlying neuronal plasticity. Here, we combined repetitive live imaging and expansion microscopy in organotypic brain slice cultures to quantitatively characterize the dynamic changes of the intracellular versus surface pools of GluA2-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) across the different dendritic spine types and the shaft during hippocampal homeostatic plasticity. Mechanistically, we identify ephrinB2 and glutamate receptor interacting protein (GRIP) 1 as mediating AMPAR relocation to the mushroom spine surface following lesion-induced denervation. Moreover, stimulation with the ephrinB2 specific receptor EphB4 not only prevents the lesion-induced disappearance of mushroom spines but is also sufficient to shift AMPARs to the surface and rescue spine recovery in a GRIP1 dominant-negative background. Thus, our results unravel a crucial role for ephrinB2 during homeostatic plasticity and identify a potential pharmacological target to improve dendritic spine plasticity upon injury.
In mammalian species, including humans, the hippocampal dentate gyrus (DG) is a primary region of adult neurogenesis. Aberrant adult hippocampal neurogenesis is associated with neurological pathologies. Understanding the cellular mechanisms controlling adult hippocampal neurogenesis is expected to open new therapeutic strategies for mental disorders. Microglia is intimately associated with neural progenitor cells in the hippocampal DG and has been implicated, under varying experimental conditions, in the control of the proliferation, differentiation and survival of neural precursor cells. But the underlying mechanisms remain poorly defined. Using fluorescent in situ hybridization we show that microglia in brain express the ADP-activated P2Y13 receptor under basal conditions and that P2ry13 mRNA is absent from neurons, astrocytes, and neural progenitor cells. Disrupting P2ry13 decreases structural complexity of microglia in the hippocampal subgranular zone (SGZ). But it increases progenitor cell proliferation and new neuron formation. Our data suggest that P2Y13 receptor-activated microglia constitutively attenuate hippocampal neurogenesis. This identifies a signaling pathway whereby microglia, via a nucleotide-mediated mechanism, contribute to the homeostatic control of adult hippocampal neurogenesis. Selective P2Y13R antagonists could boost neurogenesis in pathological conditions associated with impaired hippocampal neurogenesis.
Background: Altered neuronal development is discussed as the underlying pathogenic mechanism of autism spectrum disorders (ASD). Copy number variations of 16p11.2 have recurrently been identified in individuals with ASD. Of the 29 genes within this region, quinolinate phosphoribosyltransferase (QPRT) showed the strongest regulation during neuronal differentiation of SH-SY5Y neuroblastoma cells. We hypothesized a causal relation between this tryptophan metabolism-related enzyme and neuronal differentiation. We thus analyzed the effect of QPRT on the differentiation of SH-SY5Y and specifically focused on neuronal morphology, metabolites of the tryptophan pathway, and the neurodevelopmental transcriptome.
Methods: The gene dosage-dependent change of QPRT expression following Chr16p11.2 deletion was investigated in a lymphoblastoid cell line (LCL) of a deletion carrier and compared to his non-carrier parents. Expression of QPRT was tested for correlation with neuromorphology in SH-SY5Y cells. QPRT function was inhibited in SH-SY5Y neuroblastoma cells using (i) siRNA knockdown (KD), (ii) chemical mimicking of loss of QPRT, and (iii) complete CRISPR/Cas9-mediated knock out (KO). QPRT-KD cells underwent morphological analysis. Chemically inhibited and QPRT-KO cells were characterized using viability assays. Additionally, QPRT-KO cells underwent metabolite and whole transcriptome analyses. Genes differentially expressed upon KO of QPRT were tested for enrichment in biological processes and co-regulated gene-networks of the human brain.
Results: QPRT expression was reduced in the LCL of the deletion carrier and significantly correlated with the neuritic complexity of SH-SY5Y. The reduction of QPRT altered neuronal morphology of differentiated SH-SY5Y cells. Chemical inhibition as well as complete KO of the gene were lethal upon induction of neuronal differentiation, but not proliferation. The QPRT-associated tryptophan pathway was not affected by KO. At the transcriptome level, genes linked to neurodevelopmental processes and synaptic structures were affected. Differentially regulated genes were enriched for ASD candidates, and co-regulated gene networks were implicated in the development of the dorsolateral prefrontal cortex, the hippocampus, and the amygdala.
Conclusions: In this study, QPRT was causally related to in vitro neuronal differentiation of SH-SY5Y cells and affected the regulation of genes and gene networks previously implicated in ASD. Thus, our data suggest that QPRT may play an important role in the pathogenesis of ASD in Chr16p11.2 deletion carriers.
Regulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking in response to neuronal activity is critical for synaptic function and plasticity. Here, we show that neuronal activity induces the binding of ephrinB2 and ApoER2 receptors at the postsynapse to regulate de novo insertion of AMPA receptors. Mechanistically, the multi-PDZ adaptor glutamate-receptor-interacting protein 1 (GRIP1) binds ApoER2 and bridges a complex including ApoER2, ephrinB2, and AMPA receptors. Phosphorylation of ephrinB2 in a serine residue (Ser-9) is essential for the stability of such a complex. In vivo, a mutation on ephrinB2 Ser-9 in mice results in a complete disruption of the complex, absence of ApoER2 downstream signaling, and impaired activity-induced and ApoER2-mediated AMPA receptor insertion. Using compound genetics, we show the requirement of this complex for long-term potentiation (LTP). Together, our findings uncover a cooperative ephrinB2 and ApoER2 signaling at the synapse, which serves to modulate activity-dependent AMPA receptor dynamic changes during synaptic plasticity.
Synaptic release sites are characterized by exocytosis-competent synaptic vesicles tightly anchored to the presynaptic active zone (PAZ) whose proteome orchestrates the fast signaling events involved in synaptic vesicle cycle and plasticity. Allocation of the amyloid precursor protein (APP) to the PAZ proteome implicated a functional impact of APP in neuronal communication. In this study, we combined state-of-the-art proteomics, electrophysiology and bioinformatics to address protein abundance and functional changes at the native hippocampal PAZ in young and old APP-KO mice. We evaluated if APP deletion has an impact on the metabolic activity of presynaptic mitochondria. Furthermore, we quantified differences in the phosphorylation status after long-term-potentiation (LTP) induction at the purified native PAZ. We observed an increase in the phosphorylation of the signaling enzyme calmodulin-dependent kinase II (CaMKII) only in old APP-KO mice. During aging APP deletion is accompanied by a severe decrease in metabolic activity and hyperphosphorylation of CaMKII. This attributes an essential functional role to APP at hippocampal PAZ and putative molecular mechanisms underlying the age-dependent impairments in learning and memory in APP-KO mice.
A critical role for VEGF and VEGFR2 in NMDA receptor synaptic function and fear-related behavior
(2016)
Vascular endothelial growth factor (VEGF) is known to be required for the action of antidepressant therapies but its impact on brain synaptic function is poorly characterized. Using a combination of electrophysiological, single-molecule imaging and conditional transgenic approaches, we identified the molecular basis of the VEGF effect on synaptic transmission and plasticity. VEGF increases the postsynaptic responses mediated by the N-methyl-d-aspartate type of glutamate receptors (GluNRs) in hippocampal neurons. This is concurrent with the formation of new synapses and with the synaptic recruitment of GluNR expressing the GluN2B subunit (GluNR-2B). VEGF induces a rapid redistribution of GluNR-2B at synaptic sites by increasing the surface dynamics of these receptors within the membrane. Consistently, silencing the expression of the VEGF receptor 2 (VEGFR2) in neural cells impairs hippocampal-dependent synaptic plasticity and consolidation of emotional memory. These findings demonstrated the direct implication of VEGF signaling in neurons via VEGFR2 in proper synaptic function. They highlight the potential of VEGF as a key regulator of GluNR synaptic function and suggest a role for VEGF in new therapeutic approaches targeting GluNR in depression.