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.

UCLA Scientists use Stereo Investigator to Quantify Juvenile Neurogenesis in Mice

In the period of juvenile life, between birth and adulthood, a mouse brain adds a significant number of new neurons; nearly doubling their number in some regions. Researchers at the University of California Los Angeles published their findings last week in Frontiers in Behavioral Neuroscience.  Their findings showed that these new neurons may aid in the development of several cognitive skills.

Using a transgenic mouse model that lacked the ability to make new neurons after birth, the way a normal mouse does, the researchers were able to quantify the number of neurons contributed to the brain by postnatal, juvenile, and adult neurogenesis.

At age intervals between 14 days and 24 months, the researchers used the optical fractionator probe in Stereo Investigator to estimate cell numbers in the regions of the brain where new neurons are known to be continuously generated after birth. Their results show that during juvenile life parts of the olfactory bulb increase in cell number by 40%, while parts of the hippocampus, a brain structure known to be important in short term memory, grew by 25%. Additionally, in parts of the brain where no postnatal neurogenesis is known to occur cell numbers decreased significantly during this same period of life in all the mice tested.

MBF Staff Scientist Dr. Jose Maldonado, who is a co-author of the study, spoke to us about his methods: “Using Stereo Investigator I was able to quantify cells with high enough precision that we were able to clearly see changes in cell numbers (both up and down) in different parts of the mouse brain across the life of the animal. These cell number estimates describe the dynamic nature of cell numbers in the postnatal brain— in some areas neurons are added and in some they are lost. This shows that the brain of mice and perhaps other mammals is not really ‘done’ being built until the organism is in adulthood.”

The researchers administered behavioral tests dealing with sound, smell, fear, and new environments to see how the mouse’s ability to learn and adapt to its environment may have changed due to the inability to add postnatally generated neurons.

According to the study’s co-author Dr. Jesse Cushman, several cognitive deficits were observed in mice where juvenile neurogenesis was prevented, and males and females were affected differently. Not surprisingly they found the importance of smell in learning reduced in the transgenic mice, and transgenic male mice were unable to remember new environments. Additionally, mice lacking juvenile neurogenesis who were trained to be afraid of a particular sound were excessively afraid of new sounds—a behavior observed in people with anxiety disorders.  Dr. Cushman explained that we see this behavior, “particularly in post-traumatic stress disorder, where for example, any loud sound may trigger an excessive fear response once a soldier returns home to civilian life,” he said.

Read the full paper in Frontiers in Behavioral Neuroscience.

 

Reference

Cushman JD, Maldonado J, Kwon EE, Garcia AD, Fan G, Imura T, Sofroniew MV and Fanselow MS (2012) Juvenile neurogenesis makes essential contributions to adult brain structure and plays a sex-dependent role in fear memories. Front. Behav. Neurosci. 6:3. doi: 10.3389/fnbeh.2012.00003

 

DHA Supplementation Prior to Brain Injury May Reduce Severity

Helmet, neck roll, shoulder pads, thigh pads, knee pads, mouth guard…  A football player’s list of protective gear goes on and on. New research suggests adding one more item to the list: DHA.

Formally known as docosahexaenoic acid, DHA is one of the human brain’s primary fatty acids. Essential for proper brain function, the omega-3 fatty acid is known to benefit patients with heart disease, cancer, and traumatic brain injuries. Researchers at the West Virginia University School of Medicine say DHA may also help lessen the blow to the brain when taken prior to a head injury.

In their study, the scientists examined the brains of a population of rats, which had received dietary supplementation of DHA for 30 days prior to a traumatic brain injury. They used the Optical Fractionator with Stereo Investigator to quantify the amyloid precursor protein-positive axons, a marker of injury in the brain. A stereological count of injured axons revealed a significantly decreased amount of APP-positive axons in the rats who had received DHA supplements.

In addition to stereological analysis, the researchers assessed the brain damage with immunohistochemistry and water maze testing. Each trial revealed evidence that supplemental DHA was beneficial in reducing the injury response.

“Our findings suggest that meaningful public health benefits are likely from increasing currently low dietary DHA omega-3 intakes in our population overall and, in particular, our at-risk populations,” say the authors.

Read the free abstract or access the full article in Neurosurgery.

Mills, J. D MD; Hadley, K. PhD; Bailes, J. E MD; “Dietary Supplementation With the Omega-3 Fatty Acid Docosahexaenoic Acid in Traumatic Brain Injury” Neurosurgery. 68(2):474-481, February 2011: doi: 10.1227/NEU.0b013e3181ff692b

{Image: Public Domain via Wikipedia}

Washington University Researchers use Stereo Investigator to Study Axonal Transport in Parkinson’s Model

Journeying along axons, microscopic powerhouses known as mitochondria provide cells with the energy they need to function. When something goes wrong with the axonal transport and mitochondria isn’t delivered, the system fails, and the cell body dies.

Scientists in the Department of Anatomy and Neurobiology at the Washington University School of Medicine in St. Louis study the cellular bases of neurological disorders. Their recent research focuses on Parkinson’s disease and identifies impaired axonal transport as a possible key player in this neurological disorder.

The research group, of Dr. Karen L. O’Malley, Dr. Jeong Sook Kim-Han, and Dr. Jo Ann Antenor-Dorsey studied axonal transport in dopamine (DA) neurons in the mouse brain—the cells most affiliated with Parkinson’s disease. They localized cell axons by using a microchamber system that separated axons from cell bodies and dendrites. They also introduced 1-methyl-4-phenylpyridinium ion (MPP+), a toxin known to disrupt axonal function and mimic Parkinson’s disease.

Stereo Investigator helped them quantify TH-positive cell bodies and neurites. As early as 12 hours after MPP+ treatment, they saw breaks in axons. After 24 hours, they began to see a significant reduction in cell bodies. “This study underscores the necessity of developing therapeutics aimed at axons as well as cell bodies so as to preserve circuitry and function,” they say in their paper “The Parkinsonian Mimetic, MPP+, Specifically Impairs Mitochondrial Transport in Dopamine Axons, published in the Journal of Neuroscience.

The use of Stereo Investigator saved the researchers time and hard drive space, according to Dr. Kim-Han. “We used to count cells after acquiring images from a fluorescent microscope and saving it to a computer, which took long time and a lot of space on the computer,” she said. “Also, the data was more convincing since the area for counting was selected automatically with the number of fields assigned.”

Read the full paper “The Parkinsonian Mimetic, MPP+, Specifically Impairs Mitochondrial Transport in Dopamine Axons“, in the Journal of Neuroscience.

[The Journal of Neuroscience, 11 May 2011, 31(19): 7212-7221; doi: 10.1523/ JNEUROSCI.0711-11.2011]

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{IMG: Synapse Diagram – This file is licensed under the Creative Commons Attribution-Share Alike 1.0 Generic license}

University of Kansas Researchers use Stereo Investigator to Map Fetal Brain Hypoxia Sites

Long before a newborn baby takes its first breath, oxygen plays an integral role in its development. Oxygen-rich blood fed through the placenta facilitates the growth of a healthy fetus, powering cells to form organs and biological systems so that a healthy human emerges after nine months in utero.

However, when a fetus doesn’t receive enough oxygen, birth defects such as cerebral palsy can occur. Scientists at the University of Kansas Medical Center are researching perinatal brain injury. In one recent study, published in the American Journal of Obstetrics and Gynecology, the researchers determined that chronic hypoxia, a condition where the body is deprived of oxygen, causes selective brain injury as opposed to global, in a developing fetus. They also determined that the injury is associated with altered nitric oxide synthases, the enzymes that produce nitric oxide—key cell-signaling molecules in mammals, which contribute to the development of the nervous system.

Led by Dr. Yafeng Dong, the researchers examined the fetal brains of guinea pigs. Two third of the animals developed in an environment with low levels of oxygen. To test the role of inducible nitric oxide synthase (iNOS), half of this group received L-N6-(1-Iminoethyl)-lysine (L-NIL)—a selective pharmacologic inhibitor of iNOS—in their drinking water. The remaining third, the control group, developed in normal room air.

Brain sections were examined with 3,3’-diaminobenzidine–based immunostaining, multilabeled fluorescence immunostaining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, Nissl staining, and laser capture microdissection (LCM). Stereo Investigator helped quantify neuronal density in the Nissl stained sections.

Researchers used the resulting data to create a unique map showing the sites of injury in the cerebral cortex, hippocampus, and thalamic and hypothalamic nuclei. “In the present study, we map for the first time the geography of fetal brain injury that results from chronic fetal hypoxia and reveal specific injury to neuronal and glial cells at locations that are analogous to those reported in children with CP. This map provides a reference point for the future study of chronic hypoxia-induced fetal brain injury and the impact of therapeutic interventions to ameliorate or prevent the injury,” the authors said in their paper.

Read the free abstract, or download the full article “Chronic fetal hypoxia produces selective brain injury associated with altered nitric oxide synthases” at www.ajog.org.

Yafeng Dong, Zhiyong Yu, Yan Sun, Hui Zhou, Josh Stites, Katherine Newell, Carl P. Weiner, Chronic fetal hypoxia produces selective brain injury associated with altered nitric oxide synthases. Am J Obstet Gynecol 2011;204:254.e16-28.

{Image of the hippocampus of a fetal guinea pig brain from the control group courtesy of The University of Kansas Medical Center}

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Stereo Investigator Assists Stanford Stroke Center Scientists in Stem Cell Research

A stroke can leave its victim mentally and physically devastated. Recovery is demanding, and takes drive and determination. If the patient doesn’t receive medical attention within the small, critical window of time after the stroke occurs, chances of a quick recovery are slim. Developments in stem cell research find doctors optimistic about new possibilities for stroke rehabilitation.

Scientists at the Stanford Stroke Center, one of the leading centers for stroke research in the world, are working on figuring out how transplanted stem cells affect the host brain. They’re specifically working toward determining which trophic factors—substances secreted by cells—are necessary for stem cell transplantation to be effective. One recent study focused on vascular endothelial growth factor (VEGF), a factor associated with neurological recovery in stroke patients.

As described in their paper “Transplanted Stem Cell-secreted VEGF Effects Post-stroke Recovery, Inflammation, and Vascular Repair,” Drs. Nobutaka Horie, Gary Steinberg, and their team of researchers found VEGF to be essential for hCNS-SCns-induced recovery. The research team studied the brains of rats, which had undergone stroke surgery and were injected with human central nervous system stem cells (hCNS-SCns). After thorough analysis of various parameters, including stereological quantification of the brain inflammatory response and stem cell survival using Stereo Investigator, the Stanford team determined the stem cells helped suppress inflammation, helped form new blood vessels, and improved blood-brain barrier integrity.”

“Subacute cell transplantation therapy offers a multimodal strategy for brain repair that could significantly expand the therapeutic window for stroke,” say the authors in “Transplanted Stem Cell-secreted VEGF Effects Post-stroke Recovery, Inflammation, and Vascular Repair.”

Access the free abstract or download the full article at Stem Cells.

Horie, N., Pereira, M. P., Niizuma, K., Sun, G., Keren-Gill, H., Encarnacion, A., Shamloo, M., Hamilton, S. A., Jiang, K., Huhn, S., Palmer, T. D., Bliss, T. M. and Steinberg, G. K. (2011), Transplanted Stem Cell-Secreted Vascular Endothelial Growth Factor Effects Poststroke Recovery, Inflammation, and Vascular Repair. STEM CELLS, 29: 274–285. doi: 10.1002/stem.584

{Image of transplanted stem cells (pink) and blood vessels (green) courtesy of Stanford University}

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Toronto Scientists Get First Direct Measurement of Myometrial SMCs in Pregnant Rat Uterus With Stereo Investigator

During pregnancy, the uterus grows to accommodate the increasing size of the fetus within. Makes sense. But what is it exactly that compels the uterus to get bigger? If you said pregnancy hormones, you’re right. And if you said the growing fetus, stretching the uterine walls, you’re right too.

Researchers attribute the growth of the uterus during pregnancy to both hormones and mechanical stretch (the fetus pressing on the uterine walls). In early pregnancy, the smooth muscle cells (SMCs) in the middle layer of the uterine wall (myometrium) increase in number, causing the uterus to grow larger. And as the pregnancy progresses, these cells actually get bigger. Scientists say the pressure of the fetus pushing on the uterus causes this increase in growth, but until now, there hasn’t been any accurate numerical data to uphold these claims.

Previous studies used the protien:DNA ratio as a marker for cellular hypertrophy, but it is speculated that this technique may not reflect the true degree of muscle cell growth during gestation.

Scientists at Mount Sinai Hospital’s Samuel Lunenfeld Research Institute in Toronto examined uterine tissue from non-pregnant rats, normally pregnant rats, and rats only pregnant in one of the two horns found within the uterus.

Led by Dr. Oksana Shynlova, the research team used Stereo Investigator and the point sample intercept probe to conduct a thorough stereological analysis of smooth muscle cell volume in uterine tissue. They gathered direct data on the diameters of individual cells marked with immunohistochemical staining, and collected an unbiased average cell volume for each stage of gestation.

Their study confirmed low cell growth in early pregnancy, with an increase in cell size as gestation continued, and a threefold increase in muscle cell volume toward the end of the term. Since the cellular hypertrophy was only observed in the horn containing a fetus, the researchers suggest that mechanical stretch may be the signal for these cells to grow larger, and not hormones alone.

“Although we and other investigators have shown earlier hypertrophic changes of uterine myocytes, this study is the first to directly measure the volume of myometrial SMCs throughout pregnancy,” the authors explained in their paper “Mechanical stretch regulates hypertrophic phenotype of the myometrium during pregnancy.”

Oksana Shynlova, Ruth Kwong, and Stephen Lye, “Mechanical stretch regulates hypertrophic phenotype of the myometrium during pregnancy2009 (Reproduction 2010;139:247.)

{Image: Using Masking tool the area of interest (myometrium or decidua in each slide)(B) containing 4-6 uterine biopsies, A) is chosen. Then the software program randomly selects 2% of the total area representing 25-45 images per each slide. (C) In each image the stereology software applies 4 counting frames. (Courtesy of the Samuel Lunenfeld Research Institute)}

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Stereo Investigator Helps Uncover Autonomic Diabetic Neuropathy Link

Stomach pain, heartburn, and head rushes are frequent complaints of patients suffering from long-term diabetes. Doctors usually blame these symptoms on autonomic neuropathy, a dysfunction of the nerves that regulate blood pressure, heart rate, and digestion. But there’s really not all that much evidence out there to prove this diagnosis. Researchers at the University of Minnesota and Wake Forest University set out to discover the relationship between autonomic neuropathy and diabetes by studying mucosal nerves in the stomach.

Mucosa tissue biopsies were obtained from 15 healthy control subjects and 13 subjects with type 1 diabetes, according to the paper published last month in Neurology “Gastric mucosal nerve density – A biomarker for diabetic autonomic neuropathy?” All diabetic subjects had secondary complications, which included gastroparesis. The scientists used Stereo Investigator to conduct design-based stereology to quantify the mucosal nerves in the stomachs of each subject.

“This task proved challenging, as the mucosa is heavily innervated with a network of nerve fibers, and individual axons are difficult to isolate,” said lead author Dr. Mona Selim. “By using stereology and Stereo Investigator we were able to overcome this problem and get an estimate for nerve fiber length and volume density.”

Compared to the biopsies of the control subjects, which displayed healthy results, the diabetic subjects all showed abnormal mucosal nerve fibers, including discontinuous or disorganized networks, swollen nerves, and nerve loss. Also, after overnight fasting, the stomachs of nearly all the diabetic subjects contained residual food.

Read the free abstract, or download the full paper at neurology.org

M.M. Selim, MD, G. Wendelschafer-Crabb, MS, J.B. Redmon, MD, A. Khoruts, MD, J.S. Hodges, PhD, K. Koch, MD, D. Walk, MD and W.R. Kennedy, MD, “Gastric mucosal nerve density – A biomarker for diabetic autonomic neuropathy?” (Neurology 2010;75:973-981)

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