Automated Analysis of Neuronal Morphology in 2D Fluorescence Micrographs through an Unsupervised Semantic Segmentation of Neurons.

Brain function emerges from a highly complex network of specialized cells that are interlinked by billions of synapses. The synaptic connectivity between neurons is established between the elongated processes of their axons and dendrites or, together, neurites. To establish these connections, cellular neurites have to grow in highly specialized, cell-type dependent patterns covering extensive distances and connecting with thousands of other neurons. The outgrowth and branching of neurites are tightly controlled during development and are a commonly used functional readout of imaging in the neurosciences. Manual analysis of neuronal morphology from microscopy images, however, is very time intensive and prone to bias. Most automated analyses of neurons rely on reconstruction of the neuron as a whole without a semantic analysis of each neurite. A fully-automated classification of all neurites still remains unavailable in open-source software. Here we present a standalone, GUI-based software for batch-quantification of neuronal morphology in two-dimensional fluorescence micrographs of cultured neurons with minimal requirements for user interaction. Single neurons are first reconstructed into binarized images using a Hessian-based segmentation algorithm to detect thin neurite structures combined with intensity- and shape-based reconstruction of the cell body. Neurites are then classified into axon, dendrites and their branches of increasing order using a geodesic distance transform of the cell skeleton. The software was benchmarked against a published dataset and reproduced the phenotype observed after manual annotation. Our tool promises accelerated and improved morphometric studies of neuronal morphology by allowing for consistent and automated analysis of large datasets.

Layer 6b controls brain state via apical dendrites and the higher-order thalamocortical system.

The deepest layer of the cortex (layer 6b [L6b]) contains relatively few neurons, but it is the only cortical layer responsive to the potent wake-promoting neuropeptide orexin/hypocretin. Can these few neurons significantly influence brain state? Here, we show that L6b-photoactivation causes a surprisingly robust enhancement of attention-associated high-gamma oscillations and population spiking while abolishing slow waves in sleep-deprived mice. To explain this powerful impact on brain state, we investigated L6b's synaptic output using optogenetics, electrophysiology, and monoCaTChR ex vivo. We found powerful output in the higher-order thalamus and apical dendrites of L5 pyramidal neurons, via L1a and L5a, as well as in superior colliculus and L6 interneurons. L6b subpopulations with distinct morphologies and short- and long-term plasticities project to these diverse targets. The L1a-targeting subpopulation triggered powerful NMDA-receptor-dependent spikes that elicited burst firing in L5. We conclude that orexin/hypocretin-activated cortical neurons form a multifaceted, fine-tuned circuit for the sustained control of the higher-order thalamocortical system.

Layer 1 of somatosensory cortex: an important site for input to a tiny cortical compartment.

Neocortical layer 1 has been proposed to be at the center for top-down and bottom-up integration. It is a locus for interactions between long-range inputs, layer 1 interneurons, and apical tuft dendrites of pyramidal neurons. While input to layer 1 has been studied intensively, the level and effect of input to this layer has still not been completely characterized. Here we examined the input to layer 1 of mouse somatosensory cortex with retrograde tracing and optogenetics. Our assays reveal that local input to layer 1 is predominantly from layers 2/3 and 5 pyramidal neurons and interneurons, and that subtypes of local layers 5 and 6b neurons project to layer 1 with different probabilities. Long-range input from sensory-motor cortices to layer 1 of somatosensory cortex arose predominantly from layers 2/3 neurons. Our optogenetic experiments showed that intra-telencephalic layer 5 pyramidal neurons drive layer 1 interneurons but have no effect locally on layer 5 apical tuft dendrites. Dual retrograde tracing revealed that a fraction of local and long-range neurons was both presynaptic to layer 5 neurons and projected to layer 1. Our work highlights the prominent role of local inputs to layer 1 and shows the potential for complex interactions between long-range and local inputs, which are both in position to modify the output of somatosensory cortex.

In-ovo echocardiography for application in cardiovascular research.

Preclinical cardiovascular research relies heavily on non-invasive in-vivo echocardiography in mice and rats to assess cardiac function and morphology, since the complex interaction of heart, circulation, and peripheral organs are challenging to mimic ex-vivo. While n-numbers of annually used laboratory animals worldwide approach 200 million, increasing efforts are made by basic scientists aiming to reduce animal numbers in cardiovascular research according to the 3R's principle. The chicken egg is well-established as a physiological correlate and model for angiogenesis research but has barely been used to assess cardiac (patho-) physiology. Here, we tested whether the established in-ovo system of incubated chicken eggs interfaced with commercially available small animal echocardiography would be a suitable alternative test system in experimental cardiology. To this end, we defined a workflow to assess cardiac function in 8-13-day-old chicken embryos using a commercially available high resolution ultrasound system for small animals (Vevo 3100, Fujifilm Visualsonics Inc.) equipped with a high frequency probe (MX700; centre transmit: 50 MHz). We provide detailed standard operating procedures for sample preparation, image acquisition, data analysis, reference values for left and right ventricular function and dimensions, and inter-observer variabilities. Finally, we challenged incubated chicken eggs with two interventions well-known to affect cardiac physiology-metoprolol treatment and hypoxic exposure-to demonstrate the sensitivity of in-ovo echocardiography. In conclusion, in-ovo echocardiography is a feasible alternative tool for basic cardiovascular research, which can easily be implemented into the small animal research environment using existing infrastructure to replace mice and rat experiments, and thus, reduce use of laboratory animals according to the 3R principle.

Plasma membrane phospholipid phosphatase-related proteins as pleiotropic regulators of neuron growth and excitability.

Neuronal plasma membrane proteins are essential for integrating cell extrinsic and cell intrinsic signals to orchestrate neuronal differentiation, growth and plasticity in the developing and adult nervous system. Here, we shed light on the family of plasma membrane proteins phospholipid phosphatase-related proteins (PLPPRs) (alternative name, PRGs; plasticity-related genes) that fine-tune neuronal growth and synaptic transmission in the central nervous system. Several studies uncovered essential functions of PLPPRs in filopodia formation, axon guidance and branching during nervous system development and regeneration, as well as in the control of dendritic spine number and excitability. Loss of PLPPR expression in knockout mice increases susceptibility to seizures, and results in defects in sensory information processing, development of psychiatric disorders, stress-related behaviors and abnormal social interaction. However, the exact function of PLPPRs in the context of neurological diseases is largely unclear. Although initially described as active lysophosphatidic acid (LPA) ecto-phosphatases that regulate the levels of this extracellular bioactive lipid, PLPPRs lack catalytic activity against LPA. Nevertheless, they emerge as atypical LPA modulators, by regulating LPA mediated signaling processes. In this review, we summarize the effects of this protein family on cellular morphology, generation and maintenance of cellular protrusions as well as highlight their known neuronal functions and phenotypes of KO mice. We discuss the molecular mechanisms of PLPPRs including the deployment of phospholipids, actin-cytoskeleton and small GTPase signaling pathways, with a focus on identifying gaps in our knowledge to stimulate interest in this understudied protein family.

The impact of phosphorylated PTEN at threonine 366 on cortical connectivity and behaviour.

The lipid phosphatase PTEN (phosphatase and tensin homologue on chromosome 10) is a key tumour suppressor gene and an important regulator of neuronal signalling. PTEN mutations have been identified in patients with autism spectrum disorders, characterized by macrocephaly, impaired social interactions and communication, repetitive behaviour, intellectual disability, and epilepsy. PTEN enzymatic activity is regulated by a cluster of phosphorylation sites at the C-terminus of the protein. Here, we focused on the role of PTEN T366 phosphorylation and generated a knock-in mouse line in which Pten T366 was substituted with alanine (PtenT366A/T366A). We identify that phosphorylation of PTEN at T366 controls neuron size and connectivity of brain circuits involved in sensory processing. We show in behavioural tests that PtenT366/T366A mice exhibit cognitive deficits and selective sensory impairments, with significant differences in male individuals. We identify restricted cellular overgrowth of cortical neurons in PtenT366A/T366A brains, linked to increases in both dendritic arborization and soma size. In a combinatorial approach of anterograde and retrograde monosynaptic tracing using rabies virus, we characterize differences in connectivity to the primary somatosensory cortex of PtenT366A/T366A brains, with imbalances in long-range cortico-cortical input to neurons. We conclude that phosphorylation of PTEN at T366 controls neuron size and connectivity of brain circuits involved in sensory processing and propose that PTEN T366 signalling may account for a subset of autism-related functions of PTEN.

Precursor types predict the stability of neuronal branches.

Branches are critical for neuron function, generating the morphological complexity required for functional networks. They emerge from different, well-described, cytoskeletal precursor structures that elongate to branches. While branches are thought to be maintained by shared cytoskeletal regulators, our data from mouse hippocampal neurons indicate that the precursor structures trigger alternative branch maintenance mechanisms with differing stabilities. Whereas branches originating from lamellipodia or growth cone splitting events collapse soon after formation, branches emerging from filopodia persist. Furthermore, compared to other developing neurites, axons stabilise all branches and preferentially initiate branches from filopodia. These differences explain the altered stability of branches we observe in neurons lacking the plasma membrane protein phospholipid phosphatase-related protein 3 (PLPPR3, also known as PRG2) and in neurons treated with netrin-1. Rather than altering branch stability directly, PLPPR3 and netrin-1 boost a 'filopodia branch programme' on axons, thereby indirectly initiating more long-lived branches. In summary, we propose that studies on branching should distinguish overall stabilising effects from effects on precursor types, ideally using multifactorial statistical models, as exemplified in this study.

Dichloroacetate and PX-478 exhibit strong synergistic effects in a various number of cancer cell lines.

BACKGROUND: One key approach for anticancer therapy is drug combination. Drug combinations can help reduce doses and thereby decrease side effects. Furthermore, the likelihood of drug resistance is reduced. Distinct alterations in tumor metabolism have been described in past decades, but metabolism has yet to be targeted in clinical cancer therapy. Recently, we found evidence for synergism between dichloroacetate (DCA), a pyruvate dehydrogenase kinase inhibitor, and the HIF-1α inhibitor PX-478. In this study, we aimed to analyse this synergism in cell lines of different cancer types and to identify the underlying biochemical mechanisms. METHODS: The dose-dependent antiproliferative effects of the single drugs and their combination were assessed using SRB assays. FACS, Western blot and HPLC analyses were performed to investigate changes in reactive oxygen species levels, apoptosis and the cell cycle. Additionally, real-time metabolic analyses (Seahorse) were performed with DCA-treated MCF-7 cells. RESULTS: The combination of DCA and PX-478 produced synergistic effects in all eight cancer cell lines tested, including colorectal, lung, breast, cervical, liver and brain cancer. Reactive oxygen species generation and apoptosis played important roles in this synergism. Furthermore, cell proliferation was inhibited by the combination treatment. CONCLUSIONS: Here, we found that these tumor metabolism-targeting compounds exhibited a potent synergism across all tested cancer cell lines. Thus, we highly recommend the combination of these two compounds for progression to in vivo translational and clinical trials.

The actin binding protein drebrin helps to protect against the development of seizure-like events in the entorhinal cortex.

The actin binding protein drebrin plays a key role in dendritic spine formation and synaptic plasticity. Decreased drebrin protein levels have been observed in temporal lobe epilepsy, suggesting the involvement of drebrin in the disease. Here we investigated the effect of drebrin knockout on physiological and pathophysiological neuronal network activities in mice by inducing gamma oscillations, involved in higher cognitive functions, and by analyzing pathophysiological epileptiform activity. We found that loss of drebrin increased the emergence of spontaneous gamma oscillations suggesting an increase in neuronal excitability when drebrin is absent. Further analysis showed that although the kainate-induced hippocampal gamma oscillations were unchanged in drebrin deficient mice, seizure like events measured in the entorhinal cortex appeared earlier and more frequently. The results suggest that while drebrin is not essential for normal physiological network activity, it helps to protect against the formation of seizure like activities during pathological conditions. The data indicate that targeting drebrin function could potentially be a preventive or therapeutic strategy for epilepsy treatment.

Drebrin controls scar formation and astrocyte reactivity upon traumatic brain injury by regulating membrane trafficking.

The brain of mammals lacks a significant ability to regenerate neurons and is thus particularly vulnerable. To protect the brain from injury and disease, damage control by astrocytes through astrogliosis and scar formation is vital. Here, we show that brain injury in mice triggers an immediate upregulation of the actin-binding protein Drebrin (DBN) in astrocytes, which is essential for scar formation and maintenance of astrocyte reactivity. In turn, DBN loss leads to defective astrocyte scar formation and excessive neurodegeneration following brain injuries. At the cellular level, we show that DBN switches actin homeostasis from ARP2/3-dependent arrays to microtubule-compatible scaffolds, facilitating the formation of RAB8-positive membrane tubules. This injury-specific RAB8 membrane compartment serves as hub for the trafficking of surface proteins involved in astrogliosis and adhesion mediators, such as ß1-integrin. Our work shows that DBN-mediated membrane trafficking in astrocytes is an important neuroprotective mechanism following traumatic brain injury in mice.

Harnessing PTEN's Growth Potential in Neuronal Development and Disease.

PTEN is a powerful regulator of neuronal growth. It globally suppresses axon extension and branching during both nervous system development and regeneration, by antagonizing growth-promoting PI3K/PI(3,4,5)P3 signaling. We recently identified that the transmembrane protein PRG2/LPPR3 functions as a modulator of PTEN function during axon morphogenesis. Our work demonstrates that through inhibition of PTEN activity, PRG2 stabilizes membrane PI(3,4,5)P3. In turn, PRG2 deficiency attenuates the formation of branches in a PTEN-dependent manner, albeit without affecting the overall growth capacity of extending axons. Thus, PRG2 is poised to temporally and locally relieve growth suppression mediated by PTEN in neurons and, in effect, to redirect growth specifically to axonal branches. In this commentary, we discuss potential implications and unresolved questions regarding the regulation of axonal PTEN in neurons. Given their widespread implication during neuronal development and regeneration, identification of mechanisms that confer spatiotemporal control of PTEN may unveil new approaches to reprogram PI3K signaling in neurodevelopmental disorders and regeneration research.

Synergisms of genome and metabolism stabilizing antitumor therapy (GMSAT) in human breast and colon cancer cell lines: a novel approach to screen for synergism.

BACKGROUND: Despite an improvement of prognosis in breast and colon cancer, the outcome of the metastatic disease is still severe. Microevolution of cancer cells often leads to drug resistance and tumor-recurrence. To target the driving forces of the tumor microevolution, we focused on synergistic drug combinations of selected compounds. The aim is to prevent the tumor from evolving in order to stabilize disease remission. To identify synergisms in a high number of compounds, we propose here a three-step concept that is cost efficient, independent of high-throughput machines and reliable in its predictions. METHODS: We created dose response curves using MTT- and SRB-assays with 14 different compounds in MCF-7, HT-29 and MDA-MB-231 cells. In order to efficiently screen for synergies, we developed a screening tool in which 14 drugs were combined (91 combinations) in MCF-7 and HT-29 using EC25 or less. The most promising combinations were verified by the method of Chou and Talalay. RESULTS: All 14 compounds exhibit antitumor effects on each of the three cell lines. The screening tool resulted in 19 potential synergisms detected in HT-29 (20.9%) and 27 in MCF-7 (29.7%). Seven of the top combinations were further verified over the whole dose response curve, and for five combinations a significant synergy could be confirmed. The combination Nutlin-3 (inhibition of MDM2) and PX-478 (inhibition of HIF-1α) could be confirmed for all three cell lines. The same accounts for the combination of Dichloroacetate (PDH activation) and NHI-2 (LDH-A inhibition). Our screening method proved to be an efficient tool that is reliable in its projections. CONCLUSIONS: The presented three-step concept proved to be cost- and time-efficient with respect to the resulting data. The newly found combinations show promising results in MCF-7, HT-29 and MDA-MB231 cancer cells.

PI 3-kinase delta enhances axonal PIP3 to support axon regeneration in the adult CNS.

Peripheral nervous system (PNS) neurons support axon regeneration into adulthood, whereas central nervous system (CNS) neurons lose regenerative ability after development. To better understand this decline whilst aiming to improve regeneration, we focused on phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol (3,4,5)-trisphosphate (PIP3 ). We demonstrate that adult PNS neurons utilise two catalytic subunits of PI3K for axon regeneration: p110α and p110δ. However, in the CNS, axonal PIP3 decreases with development at the time when axon transport declines and regenerative competence is lost. Overexpressing p110α in CNS neurons had no effect; however, expression of p110δ restored axonal PIP3 and increased regenerative axon transport. p110δ expression enhanced CNS regeneration in both rat and human neurons and in transgenic mice, functioning in the same way as the hyperactivating H1047R mutation of p110α. Furthermore, viral delivery of p110δ promoted robust regeneration after optic nerve injury. These findings establish a deficit of axonal PIP3 as a key reason for intrinsic regeneration failure and demonstrate that native p110δ facilitates axon regeneration by functioning in a hyperactive fashion.

Layer 6b Is Driven by Intracortical Long-Range Projection Neurons.

Layer 6b (L6b), the deepest neocortical layer, projects to cortical targets and higher-order thalamus and is the only layer responsive to the wake-promoting neuropeptide orexin/hypocretin. These characteristics suggest that L6b can strongly modulate brain state, but projections to L6b and their influence remain unknown. Here, we examine the inputs to L6b ex vivo in the mouse primary somatosensory cortex with rabies-based retrograde tracing and channelrhodopsin-assisted circuit mapping in brain slices. We find that L6b receives its strongest excitatory input from intracortical long-range projection neurons, including those in the contralateral hemisphere. In contrast, local intracortical input and thalamocortical input were significantly weaker. Moreover, our data suggest that L6b receives far less thalamocortical input than other cortical layers. L6b was most strongly inhibited by PV and SST interneurons. This study shows that L6b integrates long-range intracortical information and is not part of the traditional thalamocortical loop.

The Axonal Membrane Protein PRG2 Inhibits PTEN and Directs Growth to Branches.

In developing neurons, phosphoinositide 3-kinases (PI3Ks) control axon growth and branching by positively regulating PI3K/PI(3,4,5)P3, but how neurons are able to generate sufficient PI(3,4,5)P3 in the presence of high levels of the antagonizing phosphatase PTEN is difficult to reconcile. We find that normal axon morphogenesis involves homeostasis of elongation and branch growth controlled by accumulation of PI(3,4,5)P3 through PTEN inhibition. We identify a plasma membrane-localized protein-protein interaction of PTEN with plasticity-related gene 2 (PRG2). PRG2 stabilizes membrane PI(3,4,5)P3 by inhibiting PTEN and localizes in nanoclusters along axon membranes when neurons initiate their complex branching behavior. We demonstrate that PRG2 is both sufficient and necessary to account for the ability of neurons to generate axon filopodia and branches in dependence on PI3K/PI(3,4,5)P3 and PTEN. Our data indicate that PRG2 is part of a neuronal growth program that induces collateral branch growth in axons by conferring local inhibition of PTEN.

PTEN in Autism and Neurodevelopmental Disorders.

Phosphatase and tensin homolog (PTEN) is a classical tumor suppressor that antagonizes phosphatidylinositol 3-phosphate kinase (PI3K)/AKT signaling. Although there is a strong association of PTEN germline mutations with cancer syndromes, they have also been described in a subset of patients with autism spectrum disorders with macrocephaly characterized by impairments in social interactions and communication, repetitive behavior and, occasionally, epilepsy. To investigate PTEN's role during neurodevelopment and its implication for autism, several conditional Pten knockout mouse models have been generated. These models are valuable tools to understand PTEN's spatiotemporal roles during neurodevelopment. In this review, we will highlight the anatomical and phenotypic results from animal studies and link them to cellular and molecular findings.

Optically Induced Calcium-Dependent Gene Activation and Labeling of Active Neurons Using CaMPARI and Cal-Light.

The advent of optogenetic methods has made it possible to use endogeneously produced molecules to image and manipulate cellular, subcellular, and synaptic activity. It has also led to the development of photoactivatable calcium-dependent indicators that mark active synapses, neurons, and circuits. Furthermore, calcium-dependent photoactivation can be used to trigger gene expression in active neurons. Here we describe two sets of protocols, one using CaMPARI and a second one using Cal-Light. CaMPARI, a calcium-modulated photoactivatable ratiometric integrator, enables rapid network-wide, tunable, all-optical functional circuit mapping. Cal-Light, a photoactivatable calcium sensor, while slower to respond than CaMPARI, has the capacity to trigger the expression of genes, including effectors, activators, indicators, or other constructs. Here we describe the rationale and provide procedures for using these two calcium-dependent constructs (1) in vitro in dissociated primary neuronal cell cultures (CaMPARI & Cal-Light); (2) in vitro in acute brain slices for circuit mapping (CaMPARI); (3) in vivo for triggering photoconversion or gene expression (CaMPARI & Cal-Light); and finally, (4) for recovering photoconverted neurons post-fixation with immunocytochemistry (CaMPARI). The approaches and protocols we describe are examples of the potential uses of both CaMPARI & Cal-Light. The ability to mark and manipulate neurons that are active during specific epochs of behavior has a vast unexplored experimental potential.

Unique properties of PTEN-L contribute to neuroprotection in response to ischemic-like stress.

Phosphatase and tensin homolog (PTEN) signalling might influence neuronal survival after brain ischemia. However, the influence of the less studied longer variant termed PTEN-L (or PTENα) has not been studied to date. Therefore, we examined the translational variant PTEN-L in the context of neuronal survival. We identified PTEN-L by proteomics in murine neuronal cultures and brain lysates and established a novel model to analyse PTEN or PTEN-L variants independently in vitro while avoiding overexpression. We found that PTEN-L, unlike PTEN, localises predominantly in the cytosol and translocates to the nucleus 10-20 minutes after glutamate stress. Genomic ablation of PTEN and PTEN-L increased neuronal susceptibility to oxygen-glucose deprivation. This effect was rescued by expression of either PTEN-L indicating that both PTEN isoforms might contribute to a neuroprotective response. However, in direct comparison, PTEN-L replaced neurons were protected against ischemic-like stress compared to neurons expressing PTEN. Neurons expressing strictly nuclear PTEN-L NLS showed increased vulnerability, indicating that nuclear PTEN-L alone is not sufficient in protecting against stress. We identified mutually exclusive binding partners of PTEN-L or PTEN in cytosolic or nuclear fractions, which were regulated after ischemic-like stress. GRB2-associated-binding protein 2, which is known to interact with phosphoinositol-3-kinase, was enriched specifically with PTEN-L in the cytosol in proximity to the plasma membrane and their interaction was lost after glutamate exposure. The present study revealed that PTEN and PTEN-L have distinct functions in response to stress and might be involved in different mechanisms of neuroprotection.