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Brain Matters from iBiology

Posted in Neuroscience, tagged blood brain barrier, brain, consciousness and books, culture, dreaming, dreams, Electrical Signaling, memory loss, mind, neurons, Neuroscience, retinoic acid, serotonin, sleep, Stress, synapses on March 19, 2016| Leave a Comment »

Brain Matters from iBiology

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

ADAM COHEN: VISUALIZING ACTIVITY IN THE BRAIN

http://www.ibiology.org/ibiomagazine/adam-cohen-visualizing-activity-brain.html

The pattern of electrical signals propagated through neuronal networks determines brain function. Adam Cohen examines the possibility of visualizing these signals inside an intact brain using fluorescent transmembrane proteins that are sensitive to voltage. Cohen discusses the barriers to this approach, something he predicts scientists from many disciplines will eventually overcome.

https://youtu.be/Zw8lWmGuXLU      Download: High ResLow Res     Recorded: 2014

Adam Cohen is Professor in the Departments of Chemistry and Physics at Harvard University and Investigator of the Howard Hughes Medical Institute. He develops biological tools and analytical approaches to investigate the behaviors of molecules and cells in vitro and in vivo. His lab merges protein engineering, optics, and physics, among other disciplines, on a variety of projects. For example, they have developed a fluorescent transmembrane protein that detects membrane voltage, which is useful in visualizing electrical activity in cells, such as cultured neurons.

Related —

• Jeffrey Lichtman iBioSeminar: Connectomics
• Karel Svoboda iBioSeminar: Optical Studies of Individual Synapses
• Mu-Ming Poo iBioSeminar: Learning and Memory: From Synapse to Perception
• William Catterall iBioSeminar: Electrical Signaling: Life in the Fast Lane

 

The Evolution of Neural Circuits and Behaviors​ Melina Hale (University of Chicago) Evolution can be defined as a change in heritable characteristics. In her fist talk, Hale does a excellent job of explaining how these changes occur. She uses examples, such as the variable color of the pepper moth, to explain selection of characteristics and she describes how geographic isolation can lead to the evolution of new species. In her second lecture, Hale describes work from her lab on the startle response, a highly conserved behavior found in fish and other vertebrates. Comparisons of the neurons which control the startle response, across many species of fish, have allowed Hale and her colleagues to determine how this neuronal circuit, and this behavior, have evolved over hundreds of millions of years. Part 1 is an outstanding video for high school or undergraduate educators looking for material to teach evolution. Watch Melina Hale’s iBioSeminar: Part 1: Introduction to Evolution Part 2: Neural Circuits and How They Evolve: A Startling Example!

 

Discovery of a ‘Neuronal Big Bang’

University of Geneva   http://www.biosciencetechnology.com/news/2016/03/discovery-neuronal-big-bang

 

This is an expression of all the genes of a neuron during the first hours after its birth. Each circle represents a development stage (6h, 12h, 24h), and the colored points within each circle represent the level of gene expression. (Credit: Jabaudon Lab/ UNIGE)

 

Our brain is home to different types of neurons, each with their own genetic signature that defines their function. These neurons are derived from progenitor cells, which are specialized stem cells that have the ability to divide to give rise to neurons. Neuroscientists from the Faculty of Medicine at the University of Geneva (UNIGE) shed light on the mechanisms that allow progenitors to generate neurons. By developing a novel technology called FlashTag that enables them to isolate and visualize neurons at the very moment they are born, they have deciphered the basic genetic code allowing the construction of a neuron. This discovery, which is published in Science, allows not only to understand how our brain develops, but also how to use this code to reconstruct neurons from stem cells. Researchers will now be able to better understand the mechanisms underlying neurological diseases such as autism and schizophrenia.

Directed by Denis Jabaudon, a neuroscientist and neuroscientist at the Department of Basic Neurosciences at UNIGE Faculty of Medicine and neurologist at the University Hospitals Geneva (HUG), the researchers developed a technology termed FlashTag, which visualizes neurons as they are being born. Using this approach, at the very moment where a progenitor divides, it is tagged with a fluorescent marker that persists in its progeny. Scientists can then visualize and isolate newborn neurons in order to dynamically observe which genes are expressed in the first few hours of their existence. Over time, they can then study their evolution and changes in gene expression. “Previously, we only had a few photos to reconstruct the history of neurons, which left a lot of room for speculation. Thanks to FlashTag, there is now a full genetic movie unfolding before our eyes. Every instant becomes visible from the very beginning, which allows us to understand the developmental scenario at play, identify the main characters, their interactions and their incentives”, notes Jabaudon. Working in the cerebral cortex of the mouse, the scientists have thus identified the key genesto neuronal development, and demonstrated that their expression dynamics is essential for the brain to develop normally.

A very precise primordial choreography

This discovery, by giving access to the primordial code of the formation of neurons, helps us to understand how neurons function in the adult brain. And it appears that several of these original genes are also involved in neurodevelopmental and neurodegenerative diseases, which can occur many years later. This suggests that a predisposition may be present from the very first moments in the existence of neurons, and that environmental factors can then impact on how diseases may develop later on. By understanding the genetic choreography of neurons, the researchers can therefore observe how these genes behave from the start, and identify potential anomalies predicting diseases.

After successfully reading this genetic code, the scientists we able to rewrite it in newborn neurons. By altering the expression of certain genes, they were able to accelerate neuronal growth, thus altering the developmental script. With FlashTag, it is now possible to isolate newborn neurons and recreate cerebral circuits in vitro, which enables scientists to test their function as well as to develop new treatments.

A website open to all

The UNIGE team posted a website where it is possible to enter the name of a gene and observe how it is expressed, and how it interacts with other genes. “Each research team can only focus on a handful of genes at a time, while our genome is made up of close to 20,000 genes. We therefore made our tool available for other researchers to use it, in a fully open way,” highlights Jabaudon.

Chronic Stress Causes Brain Inflammation, Memory Loss

A new study suggests that long-term stress can hurt short-term memory, in part due to inflammation brought on by an immune response.

Bevin Fletcher, Associate Editor    http://www.biosciencetechnology.com/news/2016/03/chronic-stress-causes-brain-inflammation-memory-loss

A new study suggests that long-term stress can hurt short-term memory, in part due to inflammation brought on by an immune response.

Researchers from Ohio State University performed experiments where mice were exposed to repeated social defeat by exposure to an aggressive, larger, alpha mouse.  The mice that were under chronic stress, had difficulty remembering where the escape hole was in a maze they had previously mastered before the stressful period.

The findings were published in The Journal of Neuroscience.

“The stressed mice didn’t recall it. The mice that weren’t stressed really remembered it,” lead researcher Johnathan Godbout, associate professor of neuroscience at Ohio State, said in statement.

The researchers noted that this kind of stress isn’t the once-in-a-while, acute stress someone might feel before a big meeting or presentation, but prolonged, continued stress.

The mice also displayed depressive-like behavior through social avoidance that continued after four weeks of observation.

Brain changes were also observed in the stressed mice, including inflammation associated with the presence of immune cells, known as macrophages, in the brain.  The researchers also recorded shortfalls in the development of new neurons at 10 days and 28 days after the chronic stress ended.

John Sheridan, associate director of Ohio State’s Institute for Behavioral Medicine said in a statement that there might be ways to interrupt the inflammation that occurs in the brain.

When the mice were given a chemical that inhibited inflammation, both memory loss and the inflammatory macrophages disappeared, leading researchers to conclude that post-stress memory deficits is directly tied to inflammation and the immune system. The depressive symptoms and the brain-cell problem did not go away.

“Stress releases immune cells from the bone marrow and those cells can traffic to brain areas associated with neuronal activation in response to stress,” Sheridan said. “They’re being called to the brain, to the center of memory.”

The team aims to understand the underpinnings of stress and responses that could one day lead to treatments for people that suffer from anxiety, depression, or post-traumatic stress disorder.

New information from this study could lead to immune-based treatments, Godbout said.

 

 A retinoic acid-enhanced, multicellular human blood-brain barrier model derived from stem cell sources Ethan S. Lippmann, Abraham Al-Ahmad, Samira M. Azarin, Sean P. Palecek &Eric V. Shusta

Scientific Reports 4, Article number: 4160 (2014)   http://dx.doi.org:/10.1038/srep04160

Blood-brain barrier (BBB) models are often used to investigate BBB function and screen brain-penetrating therapeutics, but it has been difficult to construct a human model that possesses an optimal BBB phenotype and is readily scalable. To address this challenge, we developed a human in vitro BBB model comprising brain microvascular endothelial cells (BMECs), pericytes, astrocytes and neurons derived from renewable cell sources. First, retinoic acid (RA) was used to substantially enhance BBB phenotypes in human pluripotent stem cell (hPSC)-derived BMECs, particularly through adherens junction, tight junction, and multidrug resistance protein regulation. RA-treated hPSC-derived BMECs were subsequently co-cultured with primary human brain pericytes and human astrocytes and neurons derived from human neural progenitor cells (NPCs) to yield a fully human BBB model that possessed significant tightness as measured by transendothelial electrical resistance (~5,000 Ωxcm²). Overall, this scalable human BBB model may enable a wide range of neuroscience studies.

The blood-brain barrier (BBB) is composed of brain microvascular endothelial cells (BMECs) which line brain capillaries and control molecular and cellular trafficking between the bloodstream and neural tissue. These properties are tightly regulated by the surrounding neurovascular microenvironment throughout BBB development and into adulthood. While this barrier is essential for preserving healthy brain activity, its dysfunction has been implicated in a number of neurological diseases¹. Moreover, an intact BBB serves as a major bottleneck for brain drug delivery². Studies regarding BBB development and regulation can be difficult and time-consuming to conduct in vivo and testing brain penetration of therapeutics in vivo is a low throughput endeavor. As such, in vitro BBB models have been widely implemented to study interactions between BMECs and other cells of the neurovascular unit and to conduct screens for prospective BBB-permeant drugs.

In vitro BBB models are typically constructed using primary BMECs isolated from animal brain tissue, including bovine, porcine, rat, and mouse (reviewed extensively in ref. 3). These BMECs are then co-cultured with combinations of other cells of the neurovascular unit, such as neurons, pericytes, and astrocytes, to upregulate BBB properties^4,5,6,7. Models derived from animal tissue have proven extremely useful in studying various aspects of the BBB, such as developmental and regulatory mechanisms^8,9,10,11,12 and assaying drug permeability, but it is generally well-accepted that owing to species differences, a robust human BBB model is vital to achieve a detailed understanding of human developmental pathways and to conduct relevant drug discovery and design studies¹³. Human BMEC sources for BBB models have previously consisted of either primary biopsied brain tissue^14,15 or immortalized cell lines¹⁶. Primary human BMECs typically possess moderate barrier properties but are of limited scale^14,15, and immortalized BMECs are clonal and readily scalable but often suffer from suboptimal barrier properties^16,17. From a co-culture perspective, human neurons, astrocytes, and pericytes can also be difficult to obtain from primary tissue sources in sufficient quantities for modeling purposes. These collective issues have hindered the development of in vitro human BBB models that are both high fidelity and scalable³.

We have recently demonstrated that stem cells may be attractive candidates to replace primary cells in human BBB models. Human pluripotent stem cells (hPSCs), including both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), can be differentiated into cells possessing both endothelial and BBB properties (coined hPSC-derived BMECs) via co-differentiation of neural and endothelial progenitors, followed by selection and subsequent culture of the endothelial cells¹⁸. The iPSC-derived BMECs co-cultured with rat astrocytes possessed reasonable barrier tightness as measured by TEER (860 ± 260 Ωxcm²)¹⁸, but the TEER remained below some primary bovine¹⁹ and porcine^20,21 models (800–2,000 Ωxcm²) and substantially lower than in vivo TEER (measured up to 5,900 Ωxcm²)²². In searching for candidates to improve the BBB phenotype, we identified all-trans retinoic acid (RA). BMECs in vivo have been shown to express retinol-binding protein and its membrane receptor STRA6²³,STRA6 expression has been detected in brain endothelium but not peripheral endothelium in adult mice²⁴, and STRA6 expression was increased during the differentiation of hPSC-derived BMECs in our previous work¹⁸. Moreover, RA has been shown to upregulate certain BBB properties in immortalized rodent^25,26 and human²⁷ BMEC lines. In this manuscript, we demonstrate maturation of hPSC-derived BMEC phenotypes following retinoic acid (RA) addition during the differentiation process, including enhanced adherens junction protein expression, barrier function, and multidrug resistance protein (MRP) efflux activity. We also demonstrated in previous work that primary human neural progenitor cells (NPCs) could be differentiated to a defined mixture of neurons and astrocytes capable of inducing BBB properties in rat BMECs in co-culture⁷. In this manuscript, it is shown that under optimized culture conditions, RA-treated hPSC-derived BMECs sequentially co-cultured with primary human brain pericytes and NPC-derived astrocytes and neurons can achieve physiologic TEER values, forming a scalable, fully human BBB model.

.…….

The purpose of this work was to construct a renewable, robust human BBB multicellular co-culture model employing hPSCs, NPCs, and pericytes. Using previous studies as guides^25,26,31, RA was identified as a significant modulator of BMEC properties during hPSC differentiation that greatly enhanced physical barrier characteristics as demonstrated by elevated TEER in BMECs cultured alone or with neurovascular cell co-culture. In recent work, RA treatment on the hCMEC/D3 human brain endothelial cell line served to increase occludin and VE-cadherin expression, and the authors suggested that RA secreted by radial glia may be involved in BBB development²⁷. In our study, when RA was added during the endothelial progenitor expansion phase of hPSC-derived BMEC differentiation, similar results were observed including an earlier onset of VE-cadherin expression and increased occludin expression. Moreover, the BMEC yield was increased 2-fold and the tightness of the hPSC-derived BMEC monolayers as measured by elevated TEER was significantly enhanced for three different hPSC lines. Somewhat unexpectedly, RA treatment resulted in decreased claudin-5 expression. However, the Western blotting analysis was conducted using whole-cell lysates and does not take into account the substantially improved intercellular claudin-5 junctional continuity upon RA treatment (Fig. 2C). We and others have previously observed a strong correlation between such junctional continuity and resultant barrier phenotype^6,29,32. In addition, previous work has demonstrated claudin-5 expression is relatively constant across peripheral and BBB endothelium while occludin expression is increased at the BBB relative to other vascular beds³¹. Thus, a combination of claudin-5 localization and elevated occludin expression may be the key phenotypic indicators of increased barrier function^31,33. RA treatment of hPSC-derived BMECs also selectively increased MRP efflux activity, which agrees with reports demonstrating that signaling via nuclear receptors can regulate efflux transporter expression and activity at the blood-brain barrier in vitro and in vivo^34,35,36,37. RA influences many aspects of brain development, such as anterior/posterior axis patterning in the hindbrain and anterior spinal cord^38,39,40 and regulation of neurogenesis^41,42,43. During BMEC differentiation, RA could trigger several modes of action. RA may act directly on the developing endothelial cells to upregulate BBB properties, it could induce changes in the neural cells to indirectly promote BBB differentiation, or it could act by a combination of these mechanisms. Future work will be necessary to deconvolute the RA signaling mechanisms affecting the hPSC-derived BMEC differentiation scheme.

 

In Your Dreams

Understanding the sleeping brain may be the key to unlocking the secrets of the human mind.

By David Gelernter | March 1, 2016

http://www.the-scientist.com/?articles.view/articleNo/45357/title/In-Your-Dreams

Many scientists who study the mind live in fantasyland. They ought to move back to reality: neuroscientists, psychologists, computer scientists pursuing artificial intelligence, and the philosophers of mind who are, in many cases, the sharpest thinkers in the room.

The mind makes us rational. That mind is the one we choose to study. When we study sleep or dreaming, we isolate them first—as the specialized topics they are. But, as I argue in my new book The Tides of Mind, we will never reach a deep understanding of mind unless we start with an integrated view, stretching from rational, methodical thought to nightmares.

Integrating dreaming with the rest of mind is something like being asked to assemble a car from a large pile of metal, plastic, rubber, glass, and an ocelot. Dreaming is hallucination, centering on a radically different self from our waking selves, within unreal settings and stories. Dreams can please or scare us far more vividly than our ordinary thoughts. And they are so slippery, so hard to grasp, that we start losing them the moment we wake up.

But dreaming fits easily into the big picture of mind; and we will make no basic progress on understanding the mind until we see how. Dreaming is the endpoint of the spectrum of consciousness, the smooth progression from one type of consciousness to the next, that we each experience daily.

The simplest approach to the spectrum centers on mental focus. The quality of our attention goes from concentrated to diffuse over the course of a normal day; from a state in which we can concentrate—we can think and remember in a relatively disciplined way—to one in which, with our minds wandering and memory growing increasingly vibrant and distracting, we approach sleep. Then our thinking becomes hallucinatory (as we pass through “sleep-onset thought”); and finally, we are asleep and dreaming. Usually, we oscillate down and up more than once during the day. We move partway down, come partway back, then finally slide slowly to the bottom, when we sleep and dream.

We can also describe the spectrum as a steady shift from a mind dominated by action to one dominated by passive mental experience; from mental doing to mental being. In the upper spectrum, we tend to ignore emotion as we pursue some mental object by means of reasoning or analysis. But the daydreams and fantasies that occupy us as we move down-spectrum are often emotional. And in dreaming we encounter the most saturated emotions, good and bad, that the mind can generate.

The spectrum clarifies important aspects of the mind. “Intentionality,” the quality of aboutness (“I believe that bird is a sparrow” is about “that bird”), is sometimes called “the mark of the mental”—the distinguishing attribute of mental states. But intentionality belongs strictly to the upper spectrum, and disappears gradually as we descend. At the bottom, our minds are dominated by experience, pure being. Happiness or pain or “the experience of seeing purple” are states that have causes but are about nothing.

Software simulations of the upper spectrum, of thinking-about, have grown steadily stronger over the years. That trend will continue. Being, however, is not computable. Software can no more reproduce “being happy” than it can reproduce “being rusty.” Such states depend on physical properties of particular objects. A digital computer resembles only the upper-spectrum mind. Software will never come close to reproducing the mind as a whole.
Leaving sleep outside our investigation is a good way not to see any of this. Arbitrarily hacking off one end of any natural spectrum is an invitation to conceptual chaos. There has been plenty of that in the science of mind. We must start by understanding sleep and dreaming, and go from there.

David Gelernter is a professor of computer science at Yale University. Read an excerpt from his latest book, The Tides of Mind: Uncovering the Spectrum of Consciousness at the-scientist.com.

Out in the Cold

Serotonin’s long-debated role in sleep promotion is temperature-dependent.

By Karen Zusi | March 1, 2016     http://www.the-scientist.com/?articles.view/articleNo/45346/title/Out-in-the-Cold

N.M. Murray et al., “Insomnia caused by serotonin depletion is due to hypothermia,” Sleep, 38:1985-93, 2015.

Sleepless nights
Early research into serotonin’s functions suggested that the neurotransmitter promotes sleep: lab animals deprived of the chemical often developed insomnia. More recent evidence indicated that serotonin plays a part in wakefulness instead, a theory that has gained significant traction. But explanations of the initial experimental data were scarce—so Nick Murray, then a research fellow at the University of Iowa Carver College of Medicine, started digging.

Faulty furnace?
“Over the past 5 or 10 years, we’ve found that serotonin is a key neurotransmitter for generating body heat,” says Murray. To investigate whether this role was related to serotonin’s impact on sleep, he and his colleagues injected para-chlorophenylalanine into mice to inhibit serotonin synthesis.

On ice
When kept at room temperature (20 °C), the mice with depleted serotonin slept less and developed a lower body temperature compared with their control counterparts. However, when housed at 33 °C—a thermoneutral temperature for mice—the sleep and body temperature of the treated mice stayed normal. “Serotonin isn’t a sleep-promoting neurotransmitter,” concludes Murray, now a resident at California Pacific Medical Center. He suggests that mice lacking serotonin had a tough time sleeping under early experimental conditions simply because the animals were cold, and that at higher temperatures other neurotransmitter systems in the brain would function to allow them a normal sleep-wake cycle.

Case closed
The study “solves a long-standing mystery” in the field, says Clifford Saper of Harvard University. “Not very many labs measure sleep and body temperature at the same time,” he adds. “It just basically escaped everybody’s notice for all these years.”

 

 

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