This essay is adapted from the thesis “Relational analyses of the connectome”. It combines sections from both the introduction and discussion chapters without any significant modifications to the content.
Relational neuroscience is a rapidly growing field in neuroscience that emphasizes the study of relationships between brain components to gain a better understanding of the biological mechanisms underlying healthy and disrupted brain function. It can be viewed as a complementary approach to previous specialist approaches, as it connects data and ideas obtained from single-scale, single-modality, or single-domain studies. The overview provided in this text demonstrates how relational studies across four major research themes - cross-element, cross-scale, cross-modal, and cross-domain - can offer new insights into the healthy and diseased brain. Recognizing relational neuroscience as a distinct field provides a framework for understanding current research practices and advancing relational research methodologies.
The human brain is a marvel of complexity, with a vast number of neurons and other types of brain cells and a wide range of functional dynamics, making its examination a challenging task (D. S. Bassett and Gazzaniga 2011). Neuroscience has traditionally addressed this challenge by specializing into distinct research areas, investigating specific sub-hypotheses. Pragmatic divisions such as between neuroanatomy and brain function, cellular and systems-level neuroimaging, and neurological and psychiatric conditions, and so on, are often made to provide concepts that are accurate and comprehensible enough for practical application in theory generation and testing (Casadevall and Fang 2015; Leahey, Beckman, and Stanko 2016). However, studying components ‘on their own’, apart from other components of different specializations or scales, ignores the biologically relevant relationships across components.
A first often overlooked aspect in brain research is the relationship between descriptions of brain processes used at different spatial scales. The workings of the brain are typically described and studied by means of components defined at specific scales. Examples of commonly studied components are ‘the synapse’, ‘the neuron’ or ‘a brain region’, but such divisions ignore that the functions of a component are interlinked between different scales. Processes on a small scale affect processes on larger scales and vice versa. For instance, the chemical architecture of brain regions, including the presence of receptors, directly influences macroscale functional dynamics (Kringelbach et al. 2020). Moreover, large-scale processes, such as behavioral changes, can modify microscopic properties such as synapse density (Svatkova et al. 2015). These examples illustrate how examining a component in isolation on a single spatial scale may limit the extent to which the full dynamics of a component (e.g., a receptor, the synapse or behavior) can be understood.
Second, methodological specialization around specific imaging or measurement methods has resulted in studies that investigate brain processes through observations from a single imaging modality. However, brain processes are transmodal and require the acquisition of multiple complementary modalities at the same time to provide a comprehensive description of a process (Sander, Hansen, and Wey 2020). For example, sensory stimuli processing can be measured on the macroscale by means of positron emission tomography (PET) quantifying the involved neurotransmission activity, as well as by means of functional magnetic resonance imaging (MRI) to obtain temporal information on sensory processing (Sander, Hansen, and Wey 2020). Linking findings from different imaging methodologies can help to provide a better representation of examined brain mechanisms.
A third class of often overlooked relationships are those between neuroscientific concepts, such as between specific cognitive functions, brain circuits or disorders. While it might be pragmatic to divide these concepts into discrete categories, it is insufficient for understanding the underlying brain mechanisms. In practice, these neuroscientific components frequently overlap, as they represent items that exist on continuous gradients and dividing these gradients into discrete components results in heterogeneous and overlapping categories. For example, the classification of neurons was traditionally based on distinct classes. However, recent RNA-seq studies have revealed that these classes are not homogeneous but rather display continuous variation in transcriptomics within cell types (Cembrowski and Menon 2018). The classical division of the cortex into cortical areas is another example, as the cortex exhibits continuous gene-expression patterns, rather than solely discrete boundaries between cortical areas (Huntenburg, Bazin, and Margulies 2018). This issue extends to the diagnostic nosology of psychiatric disorders. Individuals who receive the same diagnosis, such as major depressive disorder, may not share most of their symptoms. On the other hand, individuals with a different diagnosis may show indistinguishable symptoms (D. Borsboom and Cramer 2013). Moreover, it is all too common for different diagnostic labels to fit individuals with mental distress almost equally well. The most notable example is the triad of the most prevalent psychiatric disorders: major depressive disorder, anxiety disorders and insomnia disorder (Wittchen et al. 2011). These disorders encompass two-thirds of all psychiatric disorders. Their symptoms overlap and vulnerable people frequently experience a combination or sequence of all three throughout their lifetime [Ohayon and Roth (2003)](Soehner and Harvey 2012). This is a most important epidemiological observation if one aims to find neural correlates specific to the vulnerability for a psychiatric disorder.
Empowered by methodological advances and availability of data, an increasing number of studies in the field now recognize the importance of the relationships between different components and incorporate the investigation of neuroscience relations in their study designs. In parallel to developments in other fields of science (see Box Relational sciences), this trend can be described as ‘Relational Neuroscience’: the research practice focused on interactions and dependencies between supposedly discrete and self-contained neurobiological elements. Relational neuroscience is positioned at the intersection of multiple multidisciplinary neuroscience fields (see Box Related neuroscience fields for an overview) and its establishment as a distinct field provides a framework for understanding current research practices and developing relational research methods. By integrating the relationships across levels, between modalities and across domains, relational neuroscience studies have the potential to provide more biologically realistic descriptions of brain processes and provide fundamental new insights into the workings of the healthy brain and disease mechanisms.
In the following sections, we provide a more detailed overview of this growing trend in neuroscience of studying relationships. We outline the methodology used and contributions of relational studies across four major core themes: studies that incorporate relationships between computational elements, between scales, between modalities and between domains (see Figure 1). This overview highlights how relational neuroscience can complement within-field studies to achieve a more comprehensive understanding of the biological mechanisms underlying healthy brain organization and brain disorders.
Relational neuroscience is at the intersection of multiple neuroscience fields that all focus on integrating information, but are diverse in their integrative approach:
Biological psychiatry. The interdisciplinary field of biological psychiatry studies mental disorders from a biological point of view. It aims to uncover the biological roots of psychiatric disorders for improved diagnosis, treatment, and prevention (Trimble and George 2010).
Cognitive science. Cognitive neuroscience bridges neuroscience and psychology in its investigation of the neural correlates of cognition (Gazzaniga et al. 2009).
Computational neuroscience. The field of computational neuroscience uses computer simulations to validate models of brain function (Sejnowski, Koch, and Churchland 1988). Computational neuroscience has the goal to make predictions about underlying brain’s mechanisms when the biological systems are too complex to solve analytically.
Integrative neuroscience. The field of integrative neuroscience proposes multidisciplinary research for the integration of brain theories to define general organizational principles of brain organization (Evian Gordon 2000).
Network neuroscience. Applying mathematical tools from graph theory, network neuroscience investigates the relations between brain components from a network perspective using tools from network science (Danielle S. Bassett and Sporns 2017).
Systems neuroscience. Regulatory processes in the brain are modelled in systems neuroscience as complex systems consisting of components, their structural relationships and their function (Kitano 2000).
Researchers have long investigated specific "functional units" within the brain to gain a deeper understanding of how distinct brain components facilitate diverse cognitive processes. On the microscopic scale, the "neuron doctrine" laid the foundation for this approach by stating that neurons constitute the functional units of the brain. At the macroscopic scale, the typological view of the mind directed the study of the brain by posting that individual regions were responsible for distinct cognitive functions and operated independently (Westlin et al. 2023). In addition to this specialist approach, there has always also been a tendency to consider the interrelationships among these units (Catani et al. 2013). On the microscopic scale, Hebb proposed in the 1940s that neural assemblies were the true functional units of the brain (Helfrich and Knight 2019). At the macroscopic scale, early neuro-anatomists Griesinger, Meynert and Wernicke already examined the role of white matter connectivity between brain regions in cognitive functioning and brain disorders in the nineteenth century (Collin, Turk, and van den Heuvel 2016).
Technological advances in the past twenty years have enabled the mapping and further examination of the extensive network of connections, leading to a surge in the field of "connectomics" that studies the network organization of the brain (Catani et al. 2013; Sporns, Tononi, and Kötter 2005). At the microscopic level, structural connections can now be obtained through high-resolution electron microscopy (Xu et al. 2020), viral tracing experiments (Oh et al. 2014) or light sheet microscopy (Ueda et al. 2020). At the macroscopic level, advances in MRI acquisition enable the reconstruction of structural connections from high-resolution diffusion-weighted imaging (Van Essen et al. 2012) and functional connections between brain regions can be obtained by calculating the dependencies in functional activity across regions measured using, for example, electroencephalography (EEG) or functional MRI (fMRI) (Van Essen et al. 2012).
Specific connections can be investigated for their relationship with behavior and disorders, but even more insightful is considering all connections in relation to each other and examining the topology of the whole brain network. Brain networks are naturally studied using graph theoretical tools from mathematics, which focus on the organizational features of the network, rather than individual regions or connections (D. S. Bassett and Sporns 2017). These methods have revealed that neuronal networks have a characteristic wiring architecture that can be described as a trade-off between minimizing wiring-cost and long-range connections important for efficient integration of information in the network (Bullmore and Sporns 2012). Wiring-efficient features such as densely connected clique of nodes, called modules, have been suggested to underpin specialized behavioral functions (Smith et al. 2009). Wiring-expensive features that promote integration of information in the brain have been hypothesized to form the backbone of the brain for information integration enabling general higher-order cognitive functions (Martijn P. van den Heuvel and Sporns 2013).
Connectomics has significantly contributed to our understanding of brain disorders (E. Gordon 2003; M. P. van den Heuvel, Scholtens, and Kahn 2019). At the microscale, connectome alterations are expected, as synaptic functions have been associated with psychiatric and neurological disorders in both genetic and in-vivo studies (Spronsen and Hoogenraad 2010). However, microscale connectome disease studies have been limited for a long time by the invasive methods required to map connectivity at the neuronal level. By using novel techniques to create induced pluripotent stem cells (iPSCs), it is now possible to explore the effects of disease risk genes on the microscale network structure of human neurons. For schizophrenia, this approach revealed that the previously reported morphological changes coincide with dysfunctional neuronal circuits (Sarkar et al. 2018). Efficient mapping methods for neuronal networks in animals, combined with gene editing techniques, further enabled the investigation of specific mutation effects on the function of neuronal circuits. For example, in a zebrafish model of Dravet syndrome, researchers provided in-vivo evidence that disease-specific sodium channel deficiency leads to neuron hyperexcitability and epileptiform seizure events (Brenet et al. 2019). At the macroscale, functional and structural connectome alterations have been observed across a wide variety of brain disorders (Griffa et al. 2013). Brain disorders are often associated with alterations within specific network modules and general network alterations that decrease the network efficiency of information processing (M. P. van den Heuvel and Sporns 2019). Investigating the brain as a network helped also to explain previously observed local gray matter disease alterations (Crossley et al. 2014).
Brain organization can be understood on a wide range of spatial scales, from the molecular scale of proteins, to the microscopic scale of individual neurons, to the macroscopic level of brain systems (Kandel et al. 2000). Linking information from multiple spatial scales makes it possible to reconstruct biological pathways from molecular mechanisms to behavioral outcomes (Betzel and Bassett 2017; D’Angelo and Wheeler-Kingshott 2017; Martijn P. van den Heuvel and Yeo 2017). At the smallest scales, research can provide direct measurements and manipulation to gain a causal understanding of a small section of the brain or molecular process associated with brain functions. Macroscale research is complementary as it provides a bird’s-eye perspective on how small (molecular) processes interact and form a complex system. Combining information from multiple scales is therefore often crucial to uncovering how the brain works. For example, in neurogenomics, microscale studies can demonstrate that biological processes (e.g. genetic loci or proteins) are causal to certain behavior and macroscale research can inform how these processes are related to behavior by identifying the biological pathways (e.g. cell types or brain regions) through which they act (Uffelmann et al. 2021).
Cross-scale research is however challenging due to technical and methodological difficulties in acquiring data simultaneously at multiple scales (Amunts et al. 2019). Innovative studies have taken on these challenges by acquiring datasets with high-resolution whole brain imaging in the Drosophila fly (Xu et al. 2020) and rodents (Ueda et al. 2020), which allows both microscopic and macroscopic analyses. In humans, studies have combined microscale functional cell recordings from brain tissue acquired during clinical surgery with macroscale functional activation time series from in-vivo whole brain fMRI to link microscale process to macroscale brain function and cognitive behavior (Goriounova et al. 2018). An alternative strategy is to link multiple scales in a stepping-stone approach, in which results are combined from multiple databases that describe small steps between intermediate scales, forming together a feasible path from the microscopic to the macroscopic level. Examples of this stepping-stone approach include studies that combine genetic, transcriptomic and connectome data to identify biological pathways from genetic variation, to spatial gene expression patterns to neuroimaging data (Wei et al. 2019) and studies that use transcriptomic data to find genetic underpinnings of structural and functional brain connectivity phenotypes (Fornito, Arnatkevičiūtė, and Fulcher, n.d.).
A neuroscience approach that integrates relations across scales offers opportunities for both neurology and psychiatry (E. Gordon 2003; M. P. van den Heuvel, Scholtens, and Kahn 2019). While the etiology of neurological diseases is typically characterized by specific cellular pathology, the addition of macroscale information linking regional neurodegeneration to cognitive functions can provide a more detailed understanding of disease progression (Yu, Sporns, and Saykin 2021). Furthermore, incorporating macroscale information from the white matter scaffold can also help model the spread of prions in neurodegenerative disorders and predict disease development (Jucker and Walker 2013; Raj 2021; Schmidt et al. 2016). In both neurological and psychiatric disorders, incorporating macroscale information can also help to understand the often small and dispersed disorder effects reported at the genomic level. The genetic architecture of brain disorders has been described using a watershed analogy, which posits that numerous microscale alterations, such as genetic predispositions, ultimately converge into abnormalities in ‘down-stream’ macroscale brain systems, which in turn give rise to the symptoms of these disorders (Keller and Miller 2006). To better understand these complex genetic interactions, researchers have developed tools for genomic annotation that incorporate higher level information (Watanabe et al. 2017; Lamparter et al. 2016; Ma et al. 2018). By clustering SNPs into genes, biological pathways, and tissue-enhanced groupings, these methods can help identify convergent genetic alterations that may be associated with particular endophenotypes or phenotypes of a given disorder (Ormel, Hartman, and Snieder 2019).
A broad set of imaging and measurement tools are available to measure functional activity and structures in the brain. The tools differ in the specific types of brain aspects, i.e. modalities, that they capture such as high frequency neuronal activation using EEG, slower brain activation patterns using fMRI or structural features using T1-weighted MRI. Integrating information from these different modalities in relational studies can provide a more comprehensive description of a biological pathway (Biessmann et al. 2011; Calhoun and Sui 2016).
Cross-modal studies can offer more insights than single-modality studies, as some brain processes can only be captured by the interaction between modalities (Sui et al. 2014). For example, the structure-function coupling of macroscale connections between brain regions is a well-known intermodal concept useful in understanding cognitive functions (Kringelbach et al. 2020). Similarly, the cross-modal relationship between the different modalities of regional activity and metabolic rate of glucose is suggested to capture a potentially unique aspect of the regional energy utilization and efficiency of brain regions that cannot be explained by the different modalities alone (Shokri-Kojori et al. 2019).
Disease studies have further demonstrated that brain alterations in disorders are often best described in a cross-modal framework. For example, in Alzheimer’s disease, combining information on grey matter morphology, functional connectivity, and energy consumption from different imaging methods improved the understanding of the disease by identifying multiple disease sub-populations with homogeneous pathophysiological signatures (Badhwar et al. 2019). Similarly, in Parkinson’s disease, multimodal phenotypic axes have been identified that provided a more nuanced interpretation of the pathology (Markello et al. 2021). In major depressive disorder, cross-modal meta-analyses have unveiled a set of brain regions that show a significant involvement across modalities, but not in single-modality analyses (Gray et al. 2020). The improved description of brain processes when multimodal relations are taken into consideration is also reflected in studies that show that diagnosis predictions based on multimodal data outperform unimodal predictions for Alzheimer’s disease (Rathore et al. 2017), amyotrophic lateral sclerosis (Burgh et al. 2016), and, in some cases, ADHD (Tulay et al. 2019).
In neuroscience, studies often focus on specific domains such as particular disorders in molecular psychiatry, specific species in translational neuroscience or specific cell-types in cellular neuroscience. Many of these domains are closely related and the studied biological processes overlap in practice across domains. Acknowledging and incorporating these relations in analyses would potentially improve the description of the examined components.
New approaches that conceptualize relational definitions of brain dysfunction are increasingly becoming a general consideration especially in psychopathology. These approaches allow for modelling of the overlap between disorders and heterogeneity within disorders of the involved biological processes (Yuan et al. 2019), genetics (Doherty and Owen 2014) and symptomatology (Craddock and Owen 2010). A first approach is to map the relations between symptoms to find symptom clusters that have genetically-similar architectures (Nagel et al. 2018) or brain endophenotype (Rashid and Calhoun 2020), or that are comorbid (Alexander-Bloch et al. 2020; Grisanzio et al. 2018). An alternative relational approach is to model brain disorders as the extremes on continuous neuropsychological scales (Craddock and Owen 2010). Within this approach, disorders can be described by a range of dimensions (Cuthbert and Insel 2013), scored according to internalizing and externalizing dimensions (Krueger and Eaton 2015), or described as a single general psychiatric disease factor (the p-factor) (Caspi et al. 2014). These factor models inhibit a relational structure between disorders that explains shared commonalities across multiple dimensions. In this line of thought, it has further been suggested to model disorders in an explicit network organization, describing mental disorders as inherently having relations in terms of feedback loops of symptoms (D. Borsboom 2017; Oude Maatman 2020).
Importantly, the relational view of mental disorders also paves the way for generating relational hypotheses regarding their origins and the shared biological mechanisms they may have (M. P. van den Heuvel and Sporns 2019). Furthermore, recognizing transdiagnostic components has already proven useful in patient care. Clinical trials have shown that addressing insomnia complaints in depressed patients not only improves sleep but also significantly uplifts their mood (Gebara et al. 2018). Similarly, treating insomnia complaints in anxiety patients with cognitive behavioral therapy has led to a reduction in their anxiety symptoms (Mason et al. 2022).
Similarities in brain mechanisms across animals and humans are a key assumption in modelling brain diseases in animals as done in translational research. However, studies have found that the mappings of mechanisms is complex, limiting the translatability of animal findings to human applications (Seok et al. 2013). Examining the relations between brain mechanisms across species can help to improve translational models by mapping both cross-species commonalities and differences (Normand et al. 2018; Seok et al. 2013).
Variation across species can also be utilized as a natural experimental contrast to identify mechanisms associated with species-specific features. Human-specific behavior such as schizophrenia disorder can for example be examined by contrasting human and primate white matter connectivity to highlight human-specific white matter connections that might be vulnerable to schizophrenia (Martijn P. van den Heuvel et al. 2019a). At the microscale, cross-species gene-expression data pointed out mechanisms related to human-specific general neurological and psychiatric disease susceptibility (Pembroke, Hartl, and Geschwind 2021). Investigating cross-species shared and differential relations in large-scale cross-species MRI acquisitions elucidated evolutionary conserved principles and the deviation from this trend in the human brain (Ardesch et al. 2021; Assaf et al. 2020; Milham et al. 2018).
The highlighted cross-disorder and cross-species fields are examples from a long list of cross-domain fields. Other such examples that show the potential of relational research include genetic studies that incorporate interactions between nature and nurture (Werme et al. 2021), relational approaches in cell biology to describe cell states and types as continuous rather than discrete entities (Escartin et al. 2021; Lahnemann et al. 2020; Shepherd et al. 2019) and the growing appreciation in neuroimaging studies that there is a dynamic system-level brain organization dependent on brain state rather than a single definitive subdivision of brain areas (Salehi et al. 2020).
In reviewing the limitations and opportunities of the relational studies presented in this text we observe that the progress made in relational studies was often facilitated by advances in data acquisition methods, enabling cross-scale and cross-modal studies, as well as the availability of large data sets that enable multi-domain data integration and the mapping of disease networks. Furthermore, relational studies often use relatively new analysis methods, such as network science and machine learning to analyze relational data. In the coming years, we believe that the availability of data and continued development of these methods will be crucial for the further advancement of relational neuroscience. In the following paragraphs, we will elaborate on possible directions that could improve data availability and development of relational analysis methods and further advance relational neuroscience.
Relational neuroscience studies require data that is measured simultaneously at multiple scales, modalities or domains to make direct inferences. Such measurements are scientifically very valuable, but also expensive and technically challenging. Publicly available data offers an alternative that lowers the methodological bar for relational neuroscience and enables not only the simple incorporation of multiple data sources but also the opportunity to relate more data sources than ever before. Open data constitutes both large initiatives that include many subjects as well as grass-root initiatives that concatenate unique locally sourced datasets into openly shared datasets (Thompson et al. 2020; Martijn P. van den Heuvel et al. 2019b). This upcycling of research data has the advantage of reusing data in unexpected ways in new studies (Saulnier et al. 2019). A large opportunity to further increase availability of deep, but also privacy-sensitive, data is in streamlining the sharing process: standardizing sharing-protocols of datasets across institutes would help the future availability of open data that can be integrated in relational studies (Saulnier et al. 2019). Large data sharing platforms provide here an opportunity as they can streamline data availability, data findability and data standardization that are all critical for efficient linking of datasets (Wilkinson et al. 2016).
We observe an urgent need for standardization of concepts across domains. Datasets need shared key properties to be cross-referenced, but differences in nomenclature, lack of consensus over specifics of brain regions, tracts and cell types, etcetera, limit integration efforts. Standardized acquisition and processing methods will strengthen the connection between datasets by providing more power in cross-referencing. The benefit of such standardization can already been seen with standardized GWAS methods (Murphy and Skene 2021) and the Human Connectome Project neuroimaging acquisition protocols (Glasser et al. 2016). Overcoming terminology differences across fields (such as the microscopic and macroscopic levels) would require the dialog between researchers from different fields to come up with language for expressing cross-scale components of brain organization.
The benefits of relational analyses come with the trade-off that the analyses are more complex and require more sophisticated statistical methods. The challenge is to develop methods that can represent interrelated and overlapping components in a pragmatic and tractable manner such that informative features can be extracted. Across relational studies different methods are applied, based on the different types of relations examined. Network representations are a natural description of, for example, structural brain connectivity, the comorbidity across diseases and multivariate psychological data (Denny Borsboom et al. 2021). Statistical methods have also been developed for datasets in which variables are in high covariance with each other but display no clear network structure. In multimodal datasets, cross-modal relations of interest can be extracted using joint independent component analysis (Miller et al. 2016), partial least squares (Wang et al. 2020), or canonical correlation analysis (Zhuang, Yang, and Cordes 2020). Single-cell sequencing also provides large amounts of data that needs to be categorized into cell classes for which tSNE plots are used (Kobak and Berens 2019). The used methodology is often based on historical customs; an opportunity lies in breaking methods free from their research silos such that methodological knowledge becomes shared across fields. In connectomics, such push is already seen in the search for hybrid representations that can describe data both as network data as well as continuous data with no clear network structure (Bijsterbosch et al. 2021).
Second, with the integration and combination of multiple resources, the size of the examined data increases, but the amount of possible relationships that can be explored grows even faster (Smith and Nichols 2018). To keep studying relations pragmatic, new statistics and or machine learning approaches will be necessary to deal with this large amount of data and number of hypotheses being tested. New methods can focus, for example, on data-driven pruning of the considered relations (Wang et al. 2020) or incorporate biological constraints on the relations considered in the models (Hammond et al. 2021).
Third, further complicating relational analyses is the inherent incorporation of data from multiple disciplines, requiring specialist knowledge in each domain. Collaboration between professionals from the different disciplines is key for efficient and reliable science. Software tools can also reduce the required specialized knowledge by automating cross-referencing datasets. In genomics, online tools made macro-scale function annotation of nucleotides and genes accessible to a wide range of scientists (Ashburner et al. 2000; Watanabe et al. 2017). In human brain imaging, useful tools enable a wide range of neuroscientists to link macroscale brain patterns to differential gene expression or behavioral functions (Wei et al. 2021; Yarkoni et al. 2011). Towards the ultimate goal of linking all datasets into one analysis suite, multi-domain tools have been developed cross-reference data from multiple “omics” (Glasser et al. 2016; Velde et al. 2019).
To cite this text, please refer to the full thesis:
de Lange, S. C. (2024). Relational analyses of the connectome. [PhD-Thesis - Research and graduation internal, Vrije Universiteit Amsterdam]. https://doi.org/10.5463/thesis.689
This text is an excerpt from the introduction and discussion chapters of the thesis “Relational analyses of the connectome”. I would like to thank my supervisors Eus van Someren and Martijn van den Heuvel for their input and help revising this thesis.
Text and figures are licensed under Creative Commons Attribution CC BY-NC-ND 4.0. The figures that have been reused from other sources don't fall under this license and can be recognized by a note in their caption: "Figure from ...".