Some Galeomorph Sharks Express a Mammalian Microglia-Specific Protein in Radial Ependymoglia of the Telencephalon.

Ionized calcium-binding adapter molecule 1 (Iba1), also known as allograft inflammatory factor 1 (AIF-1), is a highly conserved cytoplasmic scaffold protein. Studies strongly suggest that Iba1 is associated with immune-like reactions in all Metazoa. In the mammalian brain, it is abundantly expressed in microglial cells and is used as a reliable marker for this cell type. The present study used multiple-label microscopy and Western blotting to examine Iba1 expression in the telencephalon of 2 galeomorph shark species, the swellshark (Cephaloscyllium ventriosum) and the horn shark (Heterodontus francisci), a member of an ancient extant order. In the swellshark, high Iba1 expression was found in radial ependymoglial cells, many of which also expressed glial fibrillary acidic protein. Iba1 expression was absent from most cells in the horn shark (with the possible exception of perivascular cells). The difference in Iba1 expression between the species was supported by protein analysis. These results suggest that radial ependymoglia of the elasmobranchs may be functionally related to mammalian microglia and that Iba1 expression has undergone evolutionary changes in this cartilaginous group.

A receptor-based analysis of local ecosystems in the human brain.

BACKGROUND: As a complex system, the brain is a self-organizing entity that depends on local interactions among cells. Its regions (anatomically defined nuclei and areas) can be conceptualized as cellular ecosystems, but the similarity of their functional profiles is poorly understood. The study used the Allen Human Brain Atlas to classify 169 brain regions into hierarchically-organized environments based on their expression of 100 G protein-coupled neurotransmitter receptors, with no a priori reference to the regions' positions in the brain's anatomy or function. The analysis was based on hierarchical clustering, and multiscale bootstrap resampling was used to estimate the reliability of detected clusters. RESULTS: The study presents the first unbiased, hierarchical tree of functional environments in the human brain. The similarity of brain regions was strongly influenced by their anatomical proximity, even when they belonged to different functional systems. Generally, spatial vicinity trumped long-range projections or network connectivity. The main cluster of brain regions excluded the dentate gyrus of the hippocampus. The nuclei of the amygdala formed a cluster irrespective of their striatal or pallial origin. In its receptor profile, the hypothalamus was more closely associated with the midbrain than with the thalamus. The cerebellar cortical areas formed a tight and exclusive cluster. Most of the neocortical areas (with the exception of some occipital areas) clustered in a large, statistically well supported group that included no other brain regions. CONCLUSIONS: This study adds a new dimension to the established classifications of brain divisions. In a single framework, they are reconsidered at multiple scales-from individual nuclei and areas to their groups to the entire brain. The analysis provides support for predictive models of brain self-organization and adaptation.

Functional associations among G protein-coupled neurotransmitter receptors in the human brain.

BACKGROUND: The activity of neurons is controlled by groups of neurotransmitter receptors rather than by individual receptors. Experimental studies have investigated some receptor interactions, but currently little information is available about transcriptional associations among receptors at the whole-brain level. RESULTS: A total of 4950 correlations between 100 G protein-coupled neurotransmitter receptors were examined across 169 brain regions in the human brain using expression data published in the Allen Human Brain Atlas. A large number of highly significant correlations were found, many of which have not been investigated in hypothesis-driven studies. The highest positive and negative correlations of each receptor are reported, which can facilitate the construction of receptor sets likely to be affected by altered transcription of one receptor (such sets always exist, but their members are difficult to predict). A graph analysis isolated two large receptor communities, within each of which receptor mRNA levels were strongly cross-correlated. CONCLUSIONS: The presented systematic analysis shows that the mRNA levels of many G protein-coupled receptors are interdependent. This finding is not unexpected, since the brain is a highly integrated complex system. However, the analysis also revealed two novel properties of global brain structure. First, receptor correlations are described by a simple statistical distribution, which suggests that receptor interactions may be guided by qualitatively similar processes. Second, receptors appear to form two large functional communities, which might be differentially affected in brain disorders.

Predicting the distribution of serotonergic axons: a supercomputing simulation of reflected fractional Brownian motion in a 3D-mouse brain model.

The self-organization of the brain matrix of serotonergic axons (fibers) remains an unsolved problem in neuroscience. The regional densities of this matrix have major implications for neuroplasticity, tissue regeneration, and the understanding of mental disorders, but the trajectories of its fibers are strongly stochastic and require novel conceptual and analytical approaches. In a major extension to our previous studies, we used a supercomputing simulation to model around one thousand serotonergic fibers as paths of superdiffusive fractional Brownian motion (FBM), a continuous-time stochastic process. The fibers produced long walks in a complex, three-dimensional shape based on the mouse brain and reflected at the outer (pial) and inner (ventricular) boundaries. The resultant regional densities were compared to the actual fiber densities in the corresponding neuroanatomically-defined regions. The relative densities showed strong qualitative similarities in the forebrain and midbrain, demonstrating the predictive potential of stochastic modeling in this system. The current simulation does not respect tissue heterogeneities but can be further improved with novel models of multifractional FBM. The study demonstrates that serotonergic fiber densities can be strongly influenced by the geometry of the brain, with implications for brain development, plasticity, and evolution.

An experimental platform for stochastic analyses of single serotonergic fibers in the mouse brain.

The self-organization of the serotonergic matrix, a massive axon meshwork in all vertebrate brains, is driven by the structural and dynamical properties of its constitutive elements. Each of these elements, a single serotonergic axon (fiber), has a unique trajectory and can be supported by a soma that executes one of the many available transcriptional programs. This "individuality" of serotonergic neurons necessitates the development of specialized methods for single-fiber analyses, both at the experimental and theoretical levels. We developed an integrated platform that facilitates experimental isolation of single serotonergic fibers in brain tissue, including regions with high fiber densities, and demonstrated the potential of their quantitative analyses based on stochastic modeling. Single fibers were visualized using two transgenic mouse models, one of which is the first implementation of the Brainbow toolbox in this system. The trajectories of serotonergic fibers were automatically traced in the three spatial dimensions with a novel algorithm, and their properties were captured with a single parameter associated with the directional von Mises-Fisher probability distribution. The system represents an end-to-end workflow that can be imported into various studies, including those investigating serotonergic dysfunction in brain disorders. It also supports new research directions inspired by single-fiber analyses in the serotonergic matrix, including supercomputing simulations and modeling in physics.

Brain serotonergic fibers suggest anomalous diffusion-based dropout in artificial neural networks.

Random dropout has become a standard regularization technique in artificial neural networks (ANNs), but it is currently unknown whether an analogous mechanism exists in biological neural networks (BioNNs). If it does, its structure is likely to be optimized by hundreds of millions of years of evolution, which may suggest novel dropout strategies in large-scale ANNs. We propose that the brain serotonergic fibers (axons) meet some of the expected criteria because of their ubiquitous presence, stochastic structure, and ability to grow throughout the individual's lifespan. Since the trajectories of serotonergic fibers can be modeled as paths of anomalous diffusion processes, in this proof-of-concept study we investigated a dropout algorithm based on the superdiffusive fractional Brownian motion (FBM). The results demonstrate that serotonergic fibers can potentially implement a dropout-like mechanism in brain tissue, supporting neuroplasticity. They also suggest that mathematical theories of the structure and dynamics of serotonergic fibers can contribute to the design of dropout algorithms in ANNs.

High-resolution spatiotemporal analysis of single serotonergic axons in an in vitro system.

Vertebrate brains have a dual structure, composed of (i) axons that can be well-captured with graph-theoretical methods and (ii) axons that form a dense matrix in which neurons with precise connections operate. A core part of this matrix is formed by axons (fibers) that store and release 5-hydroxytryptamine (5-HT, serotonin), an ancient neurotransmitter that supports neuroplasticity and has profound implications for mental health. The self-organization of the serotonergic matrix is not well understood, despite recent advances in experimental and theoretical approaches. In particular, individual serotonergic axons produce highly stochastic trajectories, fundamental to the construction of regional fiber densities, but further advances in predictive computer simulations require more accurate experimental information. This study examined single serotonergic axons in culture systems (co-cultures and monolayers), by using a set of complementary high-resolution methods: confocal microscopy, holotomography (refractive index-based live imaging), and super-resolution (STED) microscopy. It shows that serotonergic axon walks in neural tissue may strongly reflect the stochastic geometry of this tissue and it also provides new insights into the morphology and branching properties of serotonergic axons. The proposed experimental platform can support next-generation analyses of the serotonergic matrix, including seamless integration with supercomputing approaches.

Quantitative mRNA analysis of serotonin 5-HT4 and adrenergic ß2 receptors in the mouse embryonic telencephalon.

The adrenergic ß2 receptor (ß2-AR) gene is embedded (nested) within the serotonin 5-HT4 receptor (5-HT4-R) gene and these two receptors can interact at the transcriptional and post-transcriptional levels. The mouse 5-HT4-R gene contains a number of exons and codes at least four mRNA splice variants (5-HT(4(a))-R, 5-HT(4(b))-R, 5-HT(4(e))-R, 5-HT(4(f))-R), whereas the ß2-AR gene is intronless. Since 5-HT4-Rs and ß2-ARs can form homodimers and heterodimers and they increase intracellular cAMP levels, these receptors may be important for integrating serotonergic and noradrenergic signals at the single-neuron level. Both 5-HT4-R and ß2-AR have been implicated in autism spectrum disorders, depression, and Alzheimer's disease. In the fetal brain, these receptors may mediate the effects of stress on neurodevelopmental processes. We used quantitative reverse-transcription PCR (qRT-PCR) to investigate the developmental expression of 5-HT4-R and ß2-AR in the mouse telencephalon at embryonic days (E) 13-18. At E13-E14, the mRNA levels of all 5-HT4-R splice variants were very low, but by E17-E18 they increased 7-fold (5- HT(4(a))-R), 5-fold (5-HT(4(b))-R), 9-fold (5-HT(4(e))-R), and 11-fold (5-HT(4(f))-R). The expression of 5-HT(4(a))-R and 5-HT(4(b))-R was rapidly upregulated between E14 and E15, at the time when the thalamocortical projections arrive in the telencephalon. This pattern was not observed in the expression of 5-HT(4(e))-R and 5-HT(4(f))-R, the mRNA levels of which showed a steady, gradual increase from E13 to E18. The ß2-AR mRNA levels were relatively high throughout the studied period of development and increased only by 70% from E13-E14 to E17-E18. These findings suggest that 5-HT4-R splice variants and ß2-ARs are differentially regulated in the embryonic telencephalon and that their relative amounts may carry developmentally important information.

Serotonergic Axons as Fractional Brownian Motion Paths: Insights Into the Self-Organization of Regional Densities.

All vertebrate brains contain a dense matrix of thin fibers that release serotonin (5-hydroxytryptamine), a neurotransmitter that modulates a wide range of neural, glial, and vascular processes. Perturbations in the density of this matrix have been associated with a number of mental disorders, including autism and depression, but its self-organization and plasticity remain poorly understood. We introduce a model based on reflected Fractional Brownian Motion (FBM), a rigorously defined stochastic process, and show that it recapitulates some key features of regional serotonergic fiber densities. Specifically, we use supercomputing simulations to model fibers as FBM-paths in two-dimensional brain-like domains and demonstrate that the resultant steady state distributions approximate the fiber distributions in physical brain sections immunostained for the serotonin transporter (a marker for serotonergic axons in the adult brain). We suggest that this framework can support predictive descriptions and manipulations of the serotonergic matrix and that it can be further extended to incorporate the detailed physical properties of the fibers and their environment.

Reflected fractional Brownian motion in one and higher dimensions.

Fractional Brownian motion (FBM), a non-Markovian self-similar Gaussian stochastic process with long-ranged correlations, represents a widely applied, paradigmatic mathematical model of anomalous diffusion. We report the results of large-scale computer simulations of FBM in one, two, and three dimensions in the presence of reflecting boundaries that confine the motion to finite regions in space. Generalizing earlier results for finite and semi-infinite one-dimensional intervals, we observe that the interplay between the long-time correlations of FBM and the reflecting boundaries leads to striking deviations of the stationary probability density from the uniform density found for normal diffusion. Particles accumulate at the boundaries for superdiffusive FBM while their density is depleted at the boundaries for subdiffusion. Specifically, the probability density P develops a power-law singularity, P∼r^{κ}, as a function of the distance r from the wall. We determine the exponent κ as a function of the dimensionality, the confining geometry, and the anomalous diffusion exponent α of the FBM. We also discuss implications of our results, including an application to modeling serotonergic fiber density patterns in vertebrate brains.

Relationships among body mass, brain size, gut length, and blood tryptophan and serotonin in young wild-type mice.

BACKGROUND: The blood hyperserotonemia of autism is one of the most consistent biological findings in autism research, but its causes remain unclear. A major difficulty in understanding this phenomenon is the lack of information on fundamental interactions among the developing brain, gut, and blood in the mammalian body. We therefore investigated relationships among the body mass, the brain mass, the volume of the hippocampal complex, the gut length, and the whole-blood levels of tryptophan and 5-hydroxytryptamine (5-HT, serotonin) in young, sexually immature wild-type mice. RESULTS: Three-dimensional reconstructions of the hippocampal complex were obtained from serial, Nissl-stained sections and the gut was allowed to attain its maximal relaxed length prior to measurements. The tryptophan and 5-HT concentrations in the blood were assessed with high-performance liquid chromatography (HPLC) and the sex of mice was confirmed by genotyping. Statistical analysis yielded information about correlative relationships among all studied variables. It revealed a strong negative correlation between blood 5-HT concentration and body mass and a strong negative correlation between the brain mass/body mass ratio and gut length. Also, a negative correlation was found between the volume of the hippocampal complex and blood tryptophan concentration. CONCLUSION: The study provides information on the covariance structure of several central and peripheral variables related to the body serotonin systems. In particular, the results indicate that body mass should be included as a covariate in studies on platelet 5-HT levels and they also suggest a link between brain growth and gut length.

A stochastic approach to serotonergic fibers in mental disorders.

Virtually all brain circuits are physically embedded in a three-dimensional matrix of fibers that release 5-hydroxytryptamine (5-HT, serotonin). The density of this matrix varies across brain regions and cortical laminae, and it is altered in some mental disorders, including Major Depressive Disorder and Autism Spectrum Disorder. We investigate how the regional structure of the serotonergic matrix depends on the stochastic behavior of individual serotonergic fibers and introduce a new framework for the quantitative analysis of this behavior. In particular, we show that a step-wise random walk, based on the von Mises-Fisher probability distribution, can provide a realistic and mathematically concise description of these fibers. We also consider other stochastic models, including the fractional Brownian motion. The proposed approach seeks to advance the current understanding of the ascending reticular activating system (ARAS) and may also support future theory-guided therapeutic approaches.

Serotonergic Axons as 3D-Walks.

Experimental and theoretical research suggests that serotonergic axons (fibers) can be modeled as random walks or stochastic processes. This rigorous approach can support descriptive methods and dynamic control of the ascending reticular activating system, at the level of individual fiber trajectories.

Origin of the blood hyperserotonemia of autism.

BACKGROUND: Research in the last fifty years has shown that many autistic individuals have elevated serotonin (5-hydroxytryptamine, 5-HT) levels in blood platelets. This phenomenon, known as the platelet hyperserotonemia of autism, is considered to be one of the most well-replicated findings in biological psychiatry. Its replicability suggests that many of the genes involved in autism affect a small number of biological networks. These networks may also play a role in the early development of the autistic brain. RESULTS: We developed an equation that allows calculation of platelet 5-HT concentration as a function of measurable biological parameters. It also provides information about the sensitivity of platelet 5-HT levels to each of the parameters and their interactions. CONCLUSION: The model yields platelet 5-HT concentrations that are consistent with values reported in experimental studies. If the parameters are considered independent, the model predicts that platelet 5-HT levels should be sensitive to changes in the platelet 5-HT uptake rate constant, the proportion of free 5-HT cleared in the liver and lungs, the gut 5-HT production rate and its regulation, and the volume of the gut wall. Linear and non-linear interactions among these and other parameters are specified in the equation, which may facilitate the design and interpretation of experimental studies.

Serotonin in Space: Understanding Single Fibers.

All brain regions contain fibers that store and release 5-hydroxytryptamine (5-HT, serotonin). Since these fibers often do not have well-defined trajectories, most studies have focused on their overall densities, a measure that can be associated with local 5-HT tone in heathy and diseased brains. However, the observed fiber densities are the consequence of the behavior of single fibers. Evidence is presented as to why understanding single-fiber trajectories is important for basic and clinical neuroscience. In particular, serotonergic fibers can be viewed as natural, readily detectable stochastic probes that sample the invisible microarchitecture of brain tissue.

Serotonin 5-HT4 receptors modulate the development of glutamatergic input to the dorsal raphe nucleus.

The dorsal raphe nucleus (DRN) is a major serotonin (5-hydroxytryptamine, 5-HT)-producing region in the central nervous system. It receives glutamatergic inputs from several brain regions, which are reciprocally modulated by serotonergic signals. We investigated whether serotonin 5-HT4 receptors (5-HT4Rs) play a role in the development of glutamatergic control of the DRN, with an emphasis on cortical inputs. Double-label immunohistochemistry and confocal microscopy were used to quantify vesicular glutamate transporter 1 (vGluT1)-immunoreactive terminals in the DRN of mice with a null-mutation in the 5-HT4R gene. We found no significant change in the overall density of vGluT1-positive terminals in homozygous and heterozygous mice, but heterozygous mice had a significantly higher density of vGluT1-positive terminals contacting serotonergic neurons. These results suggest that altered 5-HT4R expression may affect the development of cortical glutamatergic control of the DRN.

Early serotonergic projections to Cajal-Retzius cells: relevance for cortical development.

Although the serotonergic system plays an important role in various neurological disorders, the role of early serotonergic projections to the developing cerebral cortex is not well understood. Because serotonergic fibers enter the marginal zone (MZ) before birth, it has been suggested that they may influence cortical development through synaptic contacts with Cajal-Retzius (CR) cells. We used immunohistochemistry combined with confocal and electron microscopy to show that the earliest serotonergic projections to the MZ form synaptic contacts with the somata and proximal dendrites of CR cells as early as embryonic day 17. To elucidate the functional significance of these early serotonergic contacts with CR cells, we perturbed their normal development by injecting pregnant mice with 5-methoxytryptamine. Lower reelin levels were detected in the brains of newborn pups from the exposed animals. Because reelin plays an important role in the cortical laminar and columnar organization during development, we killed some pups from the same litters on postnatal day 7 and analyzed their presubicular cortex. We found that the supragranular layers of the presubicular cortex (which normally display a visible columnar deployment of neurons) were altered in the treated animals. Our results suggest a mechanism of how serotonergic abnormalities during cortical development may disturb the normal cortical organization; and, therefore, may be relevant for understanding neurological disorders in which abnormalities of the serotonergic system are accompanied by cortical pathology (such as autism).

Statistical distribution of blood serotonin as a predictor of early autistic brain abnormalities.

BACKGROUND: A wide range of abnormalities has been reported in autistic brains, but these abnormalities may be the result of an earlier underlying developmental alteration that may no longer be evident by the time autism is diagnosed. The most consistent biological finding in autistic individuals has been their statistically elevated levels of 5-hydroxytryptamine (5-HT, serotonin) in blood platelets (platelet hyperserotonemia). The early developmental alteration of the autistic brain and the autistic platelet hyperserotonemia may be caused by the same biological factor expressed in the brain and outside the brain, respectively. Unlike the brain, blood platelets are short-lived and continue to be produced throughout the life span, suggesting that this factor may continue to operate outside the brain years after the brain is formed. The statistical distributions of the platelet 5-HT levels in normal and autistic groups have characteristic features and may contain information about the nature of this yet unidentified factor. RESULTS: The identity of this factor was studied by using a novel, quantitative approach that was applied to published distributions of the platelet 5-HT levels in normal and autistic groups. It was shown that the published data are consistent with the hypothesis that a factor that interferes with brain development in autism may also regulate the release of 5-HT from gut enterochromaffin cells. Numerical analysis revealed that this factor may be non-functional in autistic individuals. CONCLUSION: At least some biological factors, the abnormal function of which leads to the development of the autistic brain, may regulate the release of 5-HT from the gut years after birth. If the present model is correct, it will allow future efforts to be focused on a limited number of gene candidates, some of which have not been suspected to be involved in autism (such as the 5-HT4 receptor gene) based on currently available clinical and experimental studies.

Serotonergic paradoxes of autism replicated in a simple mathematical model.

The biological causes of autism are unknown. Since the early 1960s, the most consistent pathophysiological finding in autistic individuals has been their statistically elevated blood 5-hydroxytryptamine (5-HT, serotonin) levels. However, many autistic individuals have normal blood 5-HT levels, so this finding has been difficult to interpret. The serotonin transporter (SERT) controls 5-HT uptake by blood platelets and has been implicated in autism, but recent studies have found no correlation between SERT polymorphisms and autism. Finally, autism is considered a brain disorder, but studies have so far failed to find consistent serotonergic abnormalities in autistic brains. A simple mathematical model may account for these paradoxes, if one assumes that autism is associated with the failure of a molecular mechanism that both regulates 5-HT release from gut enterochromaffin cells and mediates 5-HT signaling in the brain. Some 5-HT receptors may play such a dual role. While the failure of such a mechanism may lead to consistent abnormalities of synaptic transmission with no alteration of brain 5-HT levels, its effects on blood 5-HT levels may appear paradoxical.

Subdivisions of the dorsal raphe nucleus projecting to the lateral geniculate nucleus and primary visual cortex in the Mongolian gerbil.

The mammalian dorsal raphe nucleus (DRN) is composed of sub-divisions with different anatomical and functional properties. Using cholera toxin subunit B as a retrograde tracer, DRN subdivisions projecting to the lateral geniculate nucleus and to the primary visual cortex were examined in the Mongolian gerbil. DRN neurons projecting to the lateral geniculate nucleus were observed in the lateral DRN (rostrally) and in the ventromedial DRN (caudally), while DRN cells projecting to the primary visual cortex were observed at all rostral-caudal levels in the ventromedial DRN. These results demonstrate a significant overlap between the DRN projections to the lateral geniculate and superior colliculus, and show that only the caudal ventromedial DRN projects to all three major visual targets: the lateral geniculate nucleus, primary visual cortex, and superior colliculus. Since the DRN is involved in depression and other neuropsychiatric disorders, as well as is affected by many psychotropic substances, these data may help to develop new treatments and therapies targeting specific DRN subdivisions.