Systematic Quantification of Synapses in Primary Neuronal Culture.

Most neurological disorders display impaired synaptic connectivity. Hence, modulation of synapse formation may have therapeutic relevance. However, the high density and small size of synapses complicate their quantification. To improve synapse-oriented screens, we analyzed the labeling performance of synapse-targeting antibodies on neuronal cell cultures using segmentation-independent image analysis based on sliding window correlation. When assessing pairwise colocalization, a common readout for mature synapses, overlap was incomplete and confounded by spurious signals. To circumvent this, we implemented a proximity ligation-based approach that only leads to a signal when two markers are sufficiently close. We applied this approach to different marker combinations and demonstrate its utility for detecting synapse density changes in healthy and compromised cultures. Thus, segmentation-independent analysis and exploitation of resident protein proximity increases the sensitivity of synapse quantifications in neuronal cultures and represents a valuable extension to the analytical toolset for in vitro synapse screens.

Progressive tau aggregation does not alter functional brain network connectivity in seeded hTau.P301L mice.

Progressive accumulation of hyperphosphorylated tau is a hallmark of various neurodegenerative disorders including Alzheimer's disease. However, to date, the functional effects of tau pathology on brain network connectivity remain poorly understood. To directly interrogate the impact of tau pathology on functional brain connectivity, we conducted a longitudinal experiment in which we monitored a fibril-seeded hTau.P301L mouse model using correlative whole-brain microscopy and resting-state functional MRI. Despite a progressive aggravation of tau pathology across the brain, the major resting-state networks appeared unaffected up to 15 weeks after seeding. Targeted analyses also showed that the connectivity of regions with high levels of hyperphosphorylated tau was comparable to that observed in controls. In line with the ostensible retention of connectivity, no behavioural changes were detected between seeded and control hTau.P301L mice as determined by three different paradigms. Our data indicate that seeded tau pathology, with accumulation of tau aggregates throughout different regions of the brain, does not alter functional connectivity or behaviour in this mouse model. Additional correlative functional studies on different mouse models should help determine whether this is a generalizable trait of tauopathies.

High-throughput microscopy exposes a pharmacological window in which dual leucine zipper kinase inhibition preserves neuronal network connectivity.

Therapeutic developments for neurodegenerative disorders are redirecting their focus to the mechanisms that contribute to neuronal connectivity and the loss thereof. Using a high-throughput microscopy pipeline that integrates morphological and functional measurements, we found that inhibition of dual leucine zipper kinase (DLK) increased neuronal connectivity in primary cortical cultures. This neuroprotective effect was not only observed in basal conditions but also in cultures depleted from antioxidants and in cultures in which microtubule stability was genetically perturbed. Based on the morphofunctional connectivity signature, we further showed that the effects were limited to a specific dose and time range. Thus, our results illustrate that profiling microscopy images with deep coverage enables sensitive interrogation of neuronal connectivity and allows exposing a pharmacological window for targeted treatments. In doing so, we revealed a broad-spectrum neuroprotective effect of DLK inhibition, which may have relevance to pathological conditions that ar.e associated with compromised neuronal connectivity.

Regional vulnerability and spreading of hyperphosphorylated tau in seeded mouse brain.

We have exploited whole brain microscopy to map the progressive deposition of hyperphosphorylated tau in intact, cleared mouse brain. We found that the three-dimensional spreading pattern of hyperphosphorylated tau in the brain of an aging Tau.P301L mouse model did not resemble that observed in AD patients. Injection of synthetic or patient-derived tau fibrils in the CA1 region resulted in a more faithful spreading pattern. Atlas-guided volumetric analysis showed a connectome-dependent spreading from the injection site and also revealed hyperphosphorylated tau deposits beyond the direct anatomical connections. In fibril-injected brains, we also detected a persistent subpopulation of rod-like and swollen microglia. Furthermore, we showed that the hyperphosphorylated tau load could be reduced by intracranial co-administration of, and to a lesser extent, by repeated systemic dosing with an antibody targeting the microtubule-binding domain of tau. Thus, the combination of targeted seeding and in toto staging of tau pathology allowed assessing regional vulnerability in a comprehensive manner, and holds potential as a preclinical drug validation tool.

Image-Based Profiling of Synaptic Connectivity in Primary Neuronal Cell Culture.

Neurological disorders display a broad spectrum of clinical manifestations. Yet, at the cellular level, virtually all these diseases converge into a common phenotype of dysregulated synaptic connectivity. In dementia, synapse dysfunction precedes neurodegeneration and cognitive impairment by several years, making the synapse a crucial entry point for the development of diagnostic and therapeutic strategies. Whereas high-resolution imaging and biochemical fractionations yield detailed insight into the molecular composition of the synapse, standardized assays are required to quickly gauge synaptic connectivity across large populations of cells under a variety of experimental conditions. Such screening capabilities have now become widely accessible with the advent of high-throughput, high-content microscopy. In this review, we discuss how microscopy-based approaches can be used to extract quantitative information about synaptic connectivity in primary neurons with deep coverage. We elaborate on microscopic readouts that may serve as a proxy for morphofunctional connectivity and we critically analyze their merits and limitations. Finally, we allude to the potential of alternative culture paradigms and integrative approaches to enable comprehensive profiling of synaptic connectivity.

SliceMap: an algorithm for automated brain region annotation.

Summary: Many neurodegenerative disorders, such as Alzheimer's Disease, pertain to or spread from specific sites of the brain. Hence, accurate disease staging or therapy assessment in transgenic model mice demands automated analysis of selected brain regions. To address this need, we have developed an algorithm, termed SliceMap, that enables contextual quantification by mapping anatomical information onto microtome-cut brain slices. For every newly acquired high-resolution image of a brain slice, the algorithm performs a coarse congealing-based registration to a library of pre-annotated reference slices. A subset of optimally matching reference slices is then used for refined, elastic registration. Morphotextural metrics are used to measure registration performance and to automatically detect poorly cut slices. We have implemented our method as a plugin for FIJI image analysis freeware, and we have used it to regionally quantify tau pathology in brain slices from a tauopathy (P301S) mouse model. By enabling region-based quantification, our method contributes to a more accurate assessment of neurodegenerative disease development. Availability and implementation: The method is available as a plugin for FIJI from https://github.com/mbarbie1/SliceMap/, along with an example dataset and user instructions. Contact: winnok.devos@uantwerpen.be. Supplementary information: Supplementary data are available at Bioinformatics online.

Dysregulation of Microtubule Stability Impairs Morphofunctional Connectivity in Primary Neuronal Networks.

Functionally related neurons assemble into connected networks that process and transmit electrochemical information. To do this in a coordinated manner, the number and strength of synaptic connections is tightly regulated. Synapse function relies on the microtubule (MT) cytoskeleton, the dynamics of which are in turn controlled by a plethora of MT-associated proteins, including the MT-stabilizing protein Tau. Although mutations in the Tau-encoding MAPT gene underlie a set of neurodegenerative disorders, termed tauopathies, the exact contribution of MT dynamics and the perturbation thereof to neuronal network connectivity has not yet been scrutinized. Therefore, we investigated the impact of targeted perturbations of MT stability on morphological (e.g., neurite- and synapse density) and functional (e.g., synchronous calcium bursting) correlates of connectivity in networks of primary hippocampal neurons. We found that treatment with MT-stabilizing or -destabilizing compounds impaired morphofunctional connectivity in a reversible manner. We also discovered that overexpression of MAPT induced significant connectivity defects, which were accompanied by alterations in MT dynamics and increased resistance to pharmacological MT depolymerization. Overexpression of a MAPT variant harboring the P301L point mutation in the MT-binding domain did far less, directly linking neuronal connectivity with Tau's MT binding affinity. Our results show that MT stability is a vulnerable node in tauopathies and that its precise pharmacological tuning may positively affect neuronal network connectivity. However, a critical balance in MT turnover causes it to be a difficult therapeutic target with a narrow operating window.

Sustained synchronized neuronal network activity in a human astrocyte co-culture system.

Impaired neuronal network function is a hallmark of neurodevelopmental and neurodegenerative disorders such as autism, schizophrenia, and Alzheimer's disease and is typically studied using genetically modified cellular and animal models. Weak predictive capacity and poor translational value of these models urge for better human derived in vitro models. The implementation of human induced pluripotent stem cells (hiPSCs) allows studying pathologies in differentiated disease-relevant and patient-derived neuronal cells. However, the differentiation process and growth conditions of hiPSC-derived neurons are non-trivial. In order to study neuronal network formation and (mal)function in a fully humanized system, we have established an in vitro co-culture model of hiPSC-derived cortical neurons and human primary astrocytes that recapitulates neuronal network synchronization and connectivity within three to four weeks after final plating. Live cell calcium imaging, electrophysiology and high content image analyses revealed an increased maturation of network functionality and synchronicity over time for co-cultures compared to neuronal monocultures. The cells express GABAergic and glutamatergic markers and respond to inhibitors of both neurotransmitter pathways in a functional assay. The combination of this co-culture model with quantitative imaging of network morphofunction is amenable to high throughput screening for lead discovery and drug optimization for neurological diseases.

NRP-1 Receptor Expression Mismatch in Skin of Subjects with Experimental and Diabetic Small Fiber Neuropathy.

The in vivo cutaneous nerve regeneration model using capsaicin is applied extensively to study the regenerative mechanisms and therapeutic efficacy of disease modifying molecules for small fiber neuropathy (SFN). Since mismatches between functional and morphological nerve fiber recovery are described for this model, we aimed at determining the capability of the capsaicin model to truly mimic the morphological manifestations of SFN in diabetes. As nerve and blood vessel growth and regenerative capacities are defective in diabetes, we focused on studying the key regulator of these processes, the neuropilin-1 (NRP-1)/semaphorin pathway. This led us to the evaluation of NRP-1 receptor expression in epidermis and dermis of subjects presenting experimentally induced small fiber neuropathy, diabetic polyneuropathy and of diabetic subjects without clinical signs of small fiber neuropathy. The NRP-1 receptor was co-stained with CD31 vessel-marker using immunofluorescence and analyzed with Definiens® technology. This study indicates that capsaicin application results in significant loss of epidermal NRP-1 receptor expression, whereas diabetic subjects presenting small fiber neuropathy show full epidermal NRP-1 expression in contrast to the basal expression pattern seen in healthy controls. Capsaicin induced a decrease in dermal non-vascular NRP-1 receptor expression which did not appear in diabetic polyneuropathy. We can conclude that the capsaicin model does not mimic diabetic neuropathy related changes for cutaneous NRP-1 receptor expression. In addition, our data suggest that NRP-1 might play an important role in epidermal nerve fiber loss and/or defective regeneration and that NRP-1 receptor could change the epidermal environment to a nerve fiber repellant bed possibly through Sem3A in diabetes.

Systematic Analysis of the Cytokine and Anhedonia Response to Peripheral Lipopolysaccharide Administration in Rats.

Inflammatory processes may cause depression in subsets of vulnerable individuals. Inflammation-associated behavioral changes are commonly modelled in rodents by administration of bacterial lipopolysaccharide (LPS). However, the time frame in which immune activation and depressive-like behavior occur is not very clear. In this study, we showed that systemic administration of LPS robustly increased circulating levels of corticosterone, leptin, pro- and anti-inflammatory cytokines, and chemokines. Serum concentrations of most analytes peaked within the first 6 h after LPS injection and returned to baseline values by 24 h. Chemokine levels, however, remained elevated for up to 96 h. Using an optimized sucrose preference test (SPT) we showed that sickness behavior was present from 2 to 24 h. LPS-induced anhedonia, as measured by decreased sucrose preference, lasted up to 96 h. To mimic the human situation, where depression develops after chronic inflammation, rats were preexposed to repeated LPS administration or subchronic restraint stress and subsequently challenged with LPS. While these procedures did not increase the duration of anhedonia, our results do indicate that inflammation may cause depressive symptoms such as anhedonia. Using our SPT protocol, more elaborate rodent models can be developed to study the mechanisms underlying inflammation-associated depression in humans.

BiDiFuse: a FIJI plugin for fusing bi-directionally recorded microscopic image volumes.

Deep tissue imaging is increasingly used for non-destructive interrogation of intact organs and small model organisms. An intuitive approach to increase the imaging depth by almost a factor of 2 is to record a sample from two sides and fuse both image stacks. However, imperfect three-dimensional alignment of both stacks presents a computational challenge. We have developed a FIJI plugin, called BiDiFuse, which merges bi-directionally recorded image stacks via 3D rigid transformations. The method is broadly applicable, considering it is compatible with all optical sectioning microscopes and it does not rely on fiducial markers for image registration. AVAILABILITY AND IMPLEMENTATION: The method is freely available as a plugin for FIJI from https://github.com/JanDetrez/BiDiFuse/ CONTACT: winnok.devos@uantwerpen.be.

Image Informatics Strategies for Deciphering Neuronal Network Connectivity.

Brain function relies on an intricate network of highly dynamic neuronal connections that rewires dramatically under the impulse of various external cues and pathological conditions. Amongst the neuronal structures that show morphological plasticity are neurites, synapses, dendritic spines and even nuclei. This structural remodelling is directly connected with functional changes such as intercellular communication and the associated calcium bursting behaviour. In vitro cultured neuronal networks are valuable models for studying these morpho-functional changes. Owing to the automation and standardization of both image acquisition and image analysis, it has become possible to extract statistically relevant readouts from such networks. Here, we focus on the current state-of-the-art in image informatics that enables quantitative microscopic interrogation of neuronal networks. We describe the major correlates of neuronal connectivity and present workflows for analysing them. Finally, we provide an outlook on the challenges that remain to be addressed, and discuss how imaging algorithms can be extended beyond in vitro imaging studies.

Peripheral Administration of Tumor Necrosis Factor-Alpha Induces Neuroinflammation and Sickness but Not Depressive-Like Behavior in Mice.

Clinical observations indicate that activation of the TNF-α system may contribute to the development of inflammation-associated depression. Here, we tested the hypothesis that systemic upregulation of TNF-α induces neuroinflammation and behavioral changes relevant to depression. We report that a single intraperitoneal injection of TNF-α in mice increased serum and brain levels of the proinflammatory mediators TNF-α, IL-6, and MCP-1, in a dose- and time-dependent manner, but not IL-1ß. Protein levels of the anti-inflammatory cytokine IL-10 increased in serum but not in the brain. The transient release of immune molecules was followed by glial cell activation as indicated by increased astrocyte activation in bioluminescent Gfap-luc mice and elevated immunoreactivity against the microglial marker Iba1 in the dentate gyrus of TNF-α-challenged mice. Additionally, TNF-α-injected mice were evaluated in a panel of behavioral tests commonly used to study sickness and depressive-like behavior in rodents. Our behavioral data imply that systemic administration of TNF-α induces a strong sickness response characterized by reduced locomotor activity, decreased fluid intake, and body weight loss. Depressive-like behavior could not be separated from sickness at any of the time points studied. Together, these results demonstrate that peripheral TNF-α affects the central nervous system at a neuroimmune and behavioral level.

Effect of stress and peripheral immune activation on astrocyte activation in transgenic bioluminescent Gfap-luc mice.

Neuroinflammation and the accompanying activation of glial cells is an important feature of many neurodegenerative conditions. It is known that factors such as peripheral infections and stress can influence immune processes in the brain. However, the effect of these stressors on astrocyte activation in vivo remains elusive. In this study, transgenic Gfap-luc mice expressing the luciferase gene under the transcriptional control of the glial fibrillary acidic protein promoter were used to quantify the kinetics of in vivo astrocyte activation following immune challenges relevant to clinical inflammation. It was found that astrocytes respond rapidly to peripheral immune activation elicited by either bacterial lipopolysaccharide (LPS) or the viral mimetic polyinosinic:polycytidylic acid (poly(I:C)). By measuring bioluminescence and 18-kDa translocator protein radioligand binding in the same animal it was observed that LPS induces both astrocyte as well as microglial activation at 6 h post-administration. Furthermore, the astrocyte response decreased upon repeated systemic LPS injections, indicating development of tolerance to the LPS challenge. Finally, restraining Gfap-luc mice for 1 h daily on 5 consecutive days did not affect brain bioluminescence, thereby indicating that sub-chronic stress does not influence astrocyte activation under unchallenged conditions. However, stressed animals showed a reduced response to a subsequent systemic LPS injection, suggesting that the immune system is compromised in these animals. Here, we demonstrate that Gfap-luc mice can be used to study astrocyte activation in response to stimuli relevant for clinical inflammation and that this approach may provide a more complete characterization of existing and novel models of neuroinflammation

Pharmacological characterization of cultivated neuronal networks: relevance to synaptogenesis and synaptic connectivity.

Mental disorders, such as schizophrenia or Alzheimer's disease, are associated with impaired synaptogenesis and/or synaptic communication. During development, neurons assemble into neuronal networks, the primary supracellular mediators of information processing. In addition to the orchestrated activation of genetic programs, spontaneous electrical activity and associated calcium signaling have been shown to be critically involved in the maturation of such neuronal networks. We established an in vitro model that recapitulates the maturation of neuronal networks, including spontaneous electrical activity. Upon plating, mouse primary hippocampal neurons grow neurites and interconnect via synapses to form a dish-wide neuronal network. Via live cell calcium imaging, we identified a limited period of time in which the spontaneous activity synchronizes across neurons, indicative of the formation of a functional network. After establishment of network activity, the neurons grow dendritic spines, the density of which was used as a morphological readout for neuronal maturity and connectivity. Hence, quantification of neurite outgrowth, synapse density, spontaneous neuronal activity, and dendritic spine density allowed to study neuronal network maturation from the day of plating until the presence of mature neuronal networks. Via acute pharmacological intervention, we show that synchronized network activity is mediated by the NMDA-R. The balance between kynurenic and quinolinic acid, both neuro-active intermediates in the tryptophan/kynurenine pathway, was shown to be decisive for the maintenance of network activity. Chronic modulation of the neurotrophic support influenced the network formation and revealed the extreme sensitivity of calcium imaging to detect subtle alterations in neuronal physiology. Given the reproducible cultivation in a 96-well setup in combination with fully automated analysis of the calcium recordings, this approach can be used to build a high-content screening assay usable for neurotoxicity screening, target identification/validation, or phenotypic drug screening.

Systemic immune activation leads to neuroinflammation and sickness behavior in mice.

Substantial evidence indicates an association between clinical depression and altered immune function. Systemic administration of bacterial lipopolysaccharide (LPS) is commonly used to study inflammation-associated behavioral changes in rodents. In these experiments, we tested the hypothesis that peripheral immune activation leads to neuroinflammation and depressive-like behavior in mice. We report that systemic administration of LPS induced astrocyte activation in transgenic GFAP-luc mice and increased immunoreactivity against the microglial marker ionized calcium-binding adapter molecule 1 in the dentate gyrus of wild-type mice. Furthermore, LPS treatment caused a strong but transient increase in cytokine levels in the serum and brain. In addition to studying LPS-induced neuroinflammation, we tested whether sickness could be separated from depressive-like behavior by evaluating LPS-treated mice in a panel of behavioral paradigms. Our behavioral data indicate that systemic LPS administration caused sickness and mild depressive-like behavior. However, due to the overlapping time course and mild effects on depression-related behavior per se, it was not possible to separate sickness from depressive-like behavior in the present rodent model.

Quantitation of chronic and acute treatment effects on neuronal network activity using image and signal analysis: toward a high-content assay.

Upon maturation, primary neuronal cultures form an interconnected network based on neurite outgrowth and synaptogenesis in which spontaneous electrical activity arises. Measurement of network activity allows quantification of neuronal health and maturation. A fluorescent indicator was used to monitor secondary calcium influxes after the occurrence of action potentials, allowing us to examine activity of hippocampal cultures via confocal live cell imaging. Subsequently, nuclear staining with DAPI allows accurate cell segmentation. To analyze the calcium recording in a robust, observer-independent manner, we implemented an automated image- and signal-processing algorithm and validated it against a visual, interactive procedure. Both methods yielded similar results on the emergence of synchronized activity and allowed robust quantitative measurement of acute and chronic modulation of drugs on network activity. Both the number of days in vitro (DIV) and neutralization of nerve growth factor (NGF) have a significant effect on synchronous burst frequency and correlation. Acute effects are demonstrated using 5-HT (serotonin) and ethylene glycol tetra-acetic acid. Automated analysis allowed measuring additional features, such as peak decay times and bursting frequency of individual neurons. Based on neuronal cell cultures in 96-well plates and accurate calcium recordings, the analysis method allows development of an integrated high-content screening assay. Because molecular biological techniques can be applied to assess the influence of genes on network activity, it is applicable for neurotoxicity or neurotrophics screening as well as development of in vitro disease models via, for example, pharmacologic manipulation or RNAi.

Systemic anti-vascular endothelial growth factor therapies induce a painful sensory neuropathy.

Systemic vascular endothelial growth factor inhibition, in combination with chemotherapy, improves the outcome of patients with metastatic cancer. Peripheral sensory neuropathies occurring in patients receiving both drugs are attributed to the chemotherapy. Here, we provide unprecedented evidence that vascular endothelial growth factor receptor inhibitors trigger a painful neuropathy and aggravate paclitaxel-induced neuropathies in mice. By using transgenic mice with altered neuronal vascular endothelial growth factor receptor expression, systemic inhibition of vascular endothelial growth factor receptors was shown to interfere with the endogenous neuroprotective activities of vascular endothelial growth factor on sensory neurons. In vitro, vascular endothelial growth factor prevented primary dorsal root ganglion cultures from paclitaxel-induced neuronal stress and cell death by counteracting mitochondrial membrane potential decreases and normalizing hyperacetylation of α-tubulin. In contrast, vascular endothelial growth factor receptor inhibitors exerted opposite effects. Intriguingly, vascular endothelial growth factor or vascular endothelial growth factor receptor inhibitors exerted their effects through a mechanism whereby Hdac6, through Hsp90, controls vascular endothelial growth factor receptor-2-mediated expression of the anti-apoptotic Bcl2. Our observations that systemic anti-vascular endothelial growth factor therapies interfere with the neuroprotective activities of vascular endothelial growth factor may have important implications for the application of anti-vascular endothelial growth factor therapies in cancer patients.

Pharmacological evaluation of rat dorsal root ganglion neurons as an in vitro model for diabetic neuropathy.

BACKGROUND: Diabetic neuropathy is a complication of diabetes mellitus that develops in about 50% of people with diabetes. Despite its widespread occurrence and devastating effects, this complication is still not fully understood, and there is no treatment available to prevent its development. METHODS: In this study, immunocytochemistry for activating transcription factor 3, a marker for cell injury, was used to investigate the stress response in dorsal root ganglion neurons in both in vitro and ex vivo models of diabetic neuropathy. RESULTS: Our findings showed increased activating transcription factor 3 expression in hyperglycemic culture conditions and in dorsal root ganglion neurons isolated from diabetic rats. Glial cell line-derived neurotrophic factor, a substance with known neuroprotective properties, was able to reduce diabetes mellitus-induced neuronal stress in vitro, while gabapentin and carbamazepine, currently used to treat neuropathic pain, showed only limited effects. CONCLUSION: Growth factors may have a therapeutic benefit as neurotrophic agents in the treatment of diabetic peripheral neuropathy, but gabapentin and carbamazepine have no direct protective effect on sensory neurons. This research also indicates that immunocytochemistry for activating transcription factor 3 is a valuable tool for evaluation of pharmacological substances in dorsal root ganglion cultures.

Neuronal FLT1 receptor and its selective ligand VEGF-B protect against retrograde degeneration of sensory neurons.

Even though VEGF-B is a homologue of the potent angiogenic factor VEGF, its angiogenic activities have been controversial. Intrigued by findings that VEGF-B may also affect neuronal cells, we assessed the neuroprotective and vasculoprotective effects of VEGF-B in the skin, in which vessels and nerves are functionally intertwined. Although VEGF-B and its FLT1 receptor were prominently expressed in dorsal root ganglion (DRG) neurons innervating the hindlimb skin, they were not essential for nerve function or vascularization of the skin. However, primary DRG cultures lacking VEGF-B or FLT1 exhibited increased neuronal stress and were more susceptible to paclitaxel-induced cell death. Concomitantly, mice lacking VEGF-B or a functional FLT1 developed more retrograde degeneration of sensory neurons in a model of distal neuropathy. On the other hand, the addition of the VEGF-B isoform, VEGF-B(186), to DRG cultures antagonized neuronal stress, maintained the mitochondrial membrane potential and stimulated neuronal survival. Mice overexpressing VEGF-B(186) or FLT1 selectively in neurons were protected against the distal neuropathy, whereas exogenous VEGF-B(186), either delivered by gene transfer or as a recombinant factor, was protective by directly affecting sensory neurons and not the surrounding vasculature. Overall, this indicates that VEGF-B, instead of acting as an angiogenic factor, exerts direct neuroprotective effects through FLT1. These findings also suggest a clinically relevant role for VEGF-B in preventing distal neuropathies.