Wisconsin Scientists Use Stereo Investigator to Quantify Neurons Formed From Stem Cells

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Researchers at the Waisman Center (University of Wisconsin-Madison) just took a big step in their quest to develop regenerative medicines for treating Parkinson’s, Alzheimer’s, and other neurodegenerative diseases. They used human embryonic stem cells to restore memory and learning in disabled mice.

The study, published last month in Nature Biotechnology, “is the first to show that human stem cells can successfully implant themselves in the brain and then heal neurological deficits,” senior author Su-Chun Zhang told the University of Wisconsin-Madison news department.

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Neurolucida & Stereo Investigator Help Uncover Cerebellar Granule Cells’ Role in Muscle Memory

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Learning a new dance routine or how to ride a bike is possible because of Cerebellar Granule Cells (GCs) according to Galliano and colleagues in The Netherlands. To find out more about the role of these abundant brain cells, and why we have so many of them, the scientists silenced most of the GCs in a group of mutant mice. They found the rodents could balance and run as well as they ever did, but when it came to learning new activities involving motor function, the mice had a harder time.

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Stereo Investigator Contributes to Study Showing Low Zinc Levels Associated With More Cell Deaths in Spinal Cord Injury

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Spinal cord injuries can result in a range of physical disabilities from slight loss of motor function to major paralysis, but little is known about the mechanisms underlying the damage. Scientists affiliated with the Miami Project to Cure Paralysis at the University of Miami are gaining knowledge about how the nervous system responds to spinal cord injuries. Their latest study, published last month in the Journal of Neuroscience Research, suggests that post trauma cell death is associated with low zinc levels.

“The expression of functional NF-kB signaling resulted in a reduction in extracellular zinc levels, thereby inducing glutamate-induced cell death,” the authors say in their paper “Reduced Extracellular Zinc Levels Facilitate Glutamate-Mediated Oligodendrocyte Death After Trauma.

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Stereo Investigator Helps Scientists Assess Damage in Rat Model of Ischemic Stroke

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A stroke patient is rushed to the hospital. Deprived of oxygen-rich blood, brain cells have already died, and more damage will probably occur in the hours and days to come. But researchers at the University of South Florida and the University of Padova in Italy say a two-part package administered through the body, rather than directly into the brain, may be the key to staving off some of the cell death that takes place after a stroke.

In their study, published in the Journal of Enzyme Inhibition and Medicinal Chemistry, the scientists saw a smaller region of damage in a rat model of focal cerebral ischemia, when the rats were treated with a combination of an anesthetic and a Caspase-3 inhibitor – a drug that suppresses a protein involved in brain cell death.

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Florida Researchers Study Traumatic Brain Injury With Stereo Investigator

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Figure 3 from “Hippocampal CA3 cell loss and downregulation of cell proliferation.”

If a head gets hit hard enough, the trauma occurs instantly. Neurons die, the brain swells as microglia cells rush to the damaged area, and the protective armor known as the blood brain barrier might even rupture. But it doesn’t end there. Long term effects include cognitive impairment, loss of sensory processing, and susceptibility to neurodegenerative diseases like Alzheimer’s.

Researchers at the University of South Florida say patients suffering from chronic Traumatic Brain Injury (TBI) experience a “cascade of events” marked by long-term neuroinflammation, cell loss, and impaired cell proliferation that may manifest over time.

“While TBI is generally considered an acute injury, a chronic secondary cell death perturbation (i.e., neuroinflammation) and a diminished endogenous repair mechanism (i.e., cell proliferation) accompany the disease pathology over long-term,” the authors say in their paper published this month in PLOS ONE.

The scientists used unbiased stereology to analyze activated microglia cells, cell proliferation, and differentiation into immature neurons in several regions of the brains of rats which had experienced TBI eight weeks prior.

They used Stereo Investigator with the Cavalieri estimator probe and the optical fractionator probe to estimate the quantity and volume of stained cells in the cortex, striatum, thalamus, fornix, cerebral peduncle, and corpus callosum, as well as the subgranular zone and the subventricular zone in both hemispheres of the brain.

Eight weeks after the TBI occurred, the researchers found an increased level of active microglia cells at the direct site of the TBI as well as surrounding regions. They also report a decrease in hippocampal neurons, and low levels of cell proliferation in the neurogenic niches.

“Our overarching theme advances the concept that a massive neuroinflammation after TBI represents a second wave of cell death that impairs the proliferative capacity of cells, and impedes the regenerative capacity of neurogenesis in chronic TBI,” the authors say in their paper.

They go on to suggest a “multi-pronged treatment targeting inflammatory and cell proliferative pathways” may help alleviate the pathological effects of chronic TBI.

Read the full paper “Long-Term Up-regulation of Inflammation and Suppression of Cell Proliferation in the Brain of Adult Rats Exposed to Traumatic Brain Injury Using the Controlled Cortical Impact Model” on PLOS ONE.

{Acosta S.A., Tajiri N., Shinozuka K., Ishikawa H., Grimmig B., et al. (2013). Long-Term Up-regulation of Inflammation and Suppression of Cell Proliferation in the Brain of Adult Rats Exposed to Traumatic Brain Injury Using the Controlled Cortical Impact Model. PLoS ONE 8(1): e53376. doi:10.1371/journal.pone.0053376}

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Scientists at Duke Say Mice Have Features Associated With Vocal Learning

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Dr. Erich Jarvis spends a lot of time with songbirds. At his Duke University lab, Jarvis, a Stereo Investigator user, studies the neurobiology of vocal communication. Since his feathered friends learn song much like humans learn speech, they’re a favorite model. But Dr. Jarvis says mice sing too, and new research says they can learn new tunes.

“We investigated the mouse song system and discovered that it includes a motor cortex region active during singing, that projects directly to brainstem vocal motor neurons and is necessary for keeping song more stereotyped and on pitch,” the authors said in their study published in PLoS ONE.

The discovery is controversial as it challenges longstanding beliefs that mice can not learn vocal behavior, but if Dr. Jarvis’ findings are true, it will be a breakthrough for scientists studying autism and anxiety disorders, according to a Duke University press release.

“The researchers who use mouse models of the vocal communication effects of these diseases will finally know the brain system that controls the mice’s vocalizations,” Dr. Jarvis said in the press release.

While studying speech evolution in humans, Dr. Jarvis and his colleagues tested male mice for vocal learning – an ability only believed to be shared by humans, songbirds, parrots and hummingbirds.

The research team used gene expression markers to see which neurons fired in the motor cortex when the mice sang. The researchers then damaged these neurons, and found the mice could no longer stay on pitch. Using an injectable tracer, they mapped a signal pathway from the motor cortex, through the brainstem, to the larynx muscles.

To test whether mice could learn vocalizations, the researchers placed pairs of male mice in a cage with one female mouse. After seven to eight weeks, the male mice had changed their tunes so they were singing the same pitch.

“Our results show that mice have the five features scientists associate with vocal learning. In mice, they don’t exist at the advanced levels found in humans and song-learning birds, but they also are not completely absent as commonly assumed,” he said.

Read about the study on Duke TODAY and CBS News, and read the full paper at PLoS ONE.

“Of mice, birds, and men: the mouse ultrasonic song system has some features similar to humans and song-learning birds,” Arriaga, G. et. al. (2012) PLOS ONE. 7(10): e46610. doi:10.1371/journal.pone.0046610

[Image via the Erich Jarvis Lab website]

Stereo Investigator Helps Harvard Scientists Study Social Isolation’s Effects on the Brain

Some children raised in orphanages grow up to develop social disorders, and there’s not all that much modern medicine can do about it. But scientists at Harvard Medical School are working on gaining a better understanding of how early isolation affects a developing brain. Their research gives new insight into the mechanisms at play, and indicates that timing and healthy myelination are crucial.

“Social isolation from P21 to P35 alters [medial Prefrontal Cortex] oligodendrocyte morphology, myelination, and mPFC-mediated behaviors,” the authors say in their paper, published in Science. “These effects persist even when isolated mice are re-exposed to social interactions, which suggests a link between the quality of mPFC myelination established during the juvenile period and adult behaviors.”

Led by Dr. Manabu Makinodan, the research team studied three groups of male mice. At 21-days-old, the mice were caged according to different scenarios: isolated environment (alone),  regular environment (with three other mice), or enriched environment (with seven other mice and a selection of toys). Four weeks later, testing showed deficits in social behavior and memory in the isolated mice.

To determine what went wrong in the brains of the isolated mice, the researchers examined the oligodendrocyte neurons in the prefrontal cortex, a brain region integral to social behavior. They determined that the density of oligodendrocytes was the same in all three groups, by using Stereo Investigator with the optical disector to perform a stereological count. Although density was consistent, the morphology of oligodendrocytes in the brains of the isolated mice was remarkably different. These mice displayed a simpler morphology that included “shorter processes, less branching, and fewer internodes.” Their myelin sheaths were thinner, resulting in decreased signaling between neurons and altered information processing.

Further trials showed that mice isolated later in life, after 35 days of age, showed the same morphology as normally reared mice, indicating that the critical period for development is before 35 days. They also noticed that mice isolated from 21 days, and which were later returned to normal environments, still showed abnormal morphology, implying that the detrimental effects of isolation could not be reversed.

“Our findings indicate that the effects of childhood isolation and neglect on adult mental health might be caused, at least in part, by alterations in oligodendrocytes and myelin development. Furthermore, we provide a cellular and/or molecular context and genetic models in which to begin to understand the effects of juvenile social experience on brain development in general and myelin maturation in particular. Our results also may be relevant to neuropsychiatric disorders such as schizophrenia and mood disorders” (Makinodan, et al, 2012).

Access the paper “A Critical Period for Social Experience–Dependent Oligodendrocyte Maturation and Myelination” at ScienceMag.org.

Manabu Makinodan, Kenneth M. Rosen, Susumu Ito, and Gabriel Corfas. “A Critical Period for Social Experience–Dependent Oligodendrocyte Maturation and Myelination.” Science, 2012; 337 (6100): 1357-1360 DOI: 10.1126/science.1220845

Image of Oligodendrocyte courtesy of Harvard Medical School.

Yale Researchers Make Breakthrough in Possible Depression Treatment

Commonly used as a human anaesthetic and animal tranquilizer, the experimental drug ketamine became famous in the last two decades as a hallucinatory club drug known as “Special K.” Now, researchers at Yale University say the drug is beneficial in treating depression by increasing synaptic connections in parts of the brain that regulate mood and cognition.

Dr. Ronald Duman, who uses Stereo Investigator and Neurolucida at his lab at the Yale School of Medicine was a co-author of the study. He and Dr. George Aghajanian studied rats exposed to stressful situations that produce symptoms similar to those found in human depression.

It appears that depression lowers the number of neuronal synaptic connections in the prefrontal cortex and hippocampus. Current antidepressants reverse these effects, but may take a long time to work, and aren’t successful in all cases. According to Drs. Duman and Aghajanian, ketamine “produces rapid (within hours) antidepressant responses in patients who are resistant to typical antidepressants,” by promoting new synaptic connections and reversing synaptic loss from stress.

“Ketamine works on an entirely different type of neurotransmitter system than current antidepressants, which can take months to improve symptoms of depression and do not work at all for one out of every three patients. Understanding how ketamine works in the brain could lead to the development of an entirely new class of antidepressants, offering relief for tens of millions of people suffering from chronic depression,” according to the Yale School of Medicine press release.

Learn more about the study on NPR.org, and read the free abstract or full paper (by subscription) at ScienceMag.org.

R. S. Duman, G. K. Aghajanian. Synaptic Dysfunction in Depression: Potential Therapeutic Targets. Science, 2012; 338 (6103): 68 DOI: 10.1126/science.1222939

 

UVM Scientists Use Neurolucida and Stereo Investigator to Study Neurons in the Avian Iris

During a chicken embryo’s twenty-one days of incubation, its eyes develop in astonishing ways. Muscles form, neurons branch, innervation occurs. Researchers at Dr. Rae Nishi’s lab at the University of Vermont, including two MBF Bioscience staff scientists Julie Simpson, Ph.D. and Julie Keefe, M.S. are studying the development of a chicken embryo’s nervous system. Their specific focus is on the behavior of neurons in the ciliary ganglion – a mass of nerve cells in the eye’s ciliary muscle.

Published last month in Developmental Neurology, their paper “Differential effects of RET and TRKB on axonal branching and survival of parasympathetic neurons” describes the multiple functions of several trophic factors in the development of ciliary ganglion neurons.

According to the paper, the researchers’ principal finding is that the neurotrophic factor receptors RET and TRKB work to ensure the survival of ciliary neurons and foster their axonal outgrowth as they innervate the striated muscle of the avian iris.

To come to this conclusion, the scientists first used Neurolucida to identify specific neurotrophic factors that are important in outgrowth and branching ciliary neurons. Next, they evaluated neuronal survival in the ciliary ganglion, and axonal branching in the iris after blocking neuromuscular transmission and signaling through RET and TRKB. They used Stereo Investigator with the Optical Fractionator probe to perform a design-based stereological count of the ciliary neurons.

“When the normal number of ciliary neurons is decreased by exogenous manipulations such as dTC and dnRET, axonal outgrowth increases to fill synaptic space. However, when neuromuscular transmission is blocked, the lack of activity causes the muscle to attract more axons through retrograde signaling mediated by RET, leading to a higher than normal axonal density,” the researchers said in their paper.

The study, which may be beneficial in neurodegenerative disease research,“suggests that interfering with neuromuscular transmission enhances retrograde signaling between muscle and nerve, which, in turn, promotes axonal branching, endplate formation, and neuronal survival.” (Simpson, Keefe, Nishi, 2012)

“It is always a pleasure to see hard work come to fruition in the form of a publication,” said Dr. Simpson. “I’d like to thank to Dr. Rae Nishi who was a wonderful advisor and mentor during my graduate career at the University of Vermont.”

Simpson, J., Keefe, J. and Nishi, R. (2012), Differential effects of ret and TRKB on axonal branching and survival of parasympathetic neurons. Devel Neurobio. doi: 10.1002/dneu.22036

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{Public domain illustration depicting ciliary muscle via Wikipedia.}

John Hopkins University Scientists Quantify Neurons with Stereo Investigator

 

Rats lose brain cells as they get older. But that doesn’t mean they can’t find their way through a water maze as quickly as their younger cohorts can.

Using unbiased stereology to quantify neurons in the prefrontal cortex of young and old rats, scientists at John Hopkins University in Baltimore found the total neuron number in the dorsal prefrontal cortex (dPFC) decreases with age. But despite the lost neurons, not all of the aged rats showed spatial learning impairment.

Led by Dr. Alexis Stranahan, the researcher team used Stereo Investigator with the Optical Fractionator to quantify total neuron number and the number of interneurons positively stained with antibodies to glutamic acid decarboxylase 67 (GAD67) in both the dorsal and ventral prefrontal cortex. They also used Stereo Investigator to outline cytoarchitectural boundaries in these regions of the rat brain.

To measure the efficiency of the rats’ spatial memory, the researchers used the Morris Water Maze. Trained to find a target platform while swimming in a pool of water, the rats were rated on their speed, distance traveled, and the time they spent in each area of the pool.

Their stereological analysis only revealed neuron count changes in the dPFC. No changes were observed in the vPFC; “and age-related neuronal loss was not associated with spatial memory performance,” the authors state in their paper, which was published online last February in the Journal of Comparative Neurology and will appear in the April 15 issue.

“We believe that when these data are taken together with the current observation that both aged-impaired and aged-unimpaired rats exhibit decreased neuron number in the dorsal prefrontal region, to the extent that such neuron loss is detrimental in this behavioral model, some compensatory mechanisms might be recruited to maintain the performance of unimpaired rats,” according to the study.

Read the full paper here.

 

Reference:

Stranahan, A. M., N. T. Jiam, A. M. Spiegel and M. Gallagher (2012).
“Aging reduces total neuron number in the dorsal component of the rodent prefrontal cortex.”
The Journal of Comparative Neurology 520(6): 1318-1326.