MBF Bioscience research team contributes novel dendritic spine analysis in study published in Science

Posted on January 18, 2019 by Celeste Sunderland

Combination of new microscopy and expansion tissue preparation methods facilitate better and faster analysis of subcellular neural elements.

Today, the journal Science published a paper authored by a research team led by Dr. Ed Boyden of MIT and Nobel Prize recipient Dr. Eric Betzig of Janelia Research Campus. Among the authors are MBF Bioscience Scientific Director Dr. Susan Tappan and Senior Software Engineer Alfredo Rodriguez. In the paper, the researchers introduce new analyses for neural circuits at nanoscale resolutions.

Combining microscopy methods that create high resolution 3D images from whole brains and tissue that have been made physically larger, the researchers imaged a mouse cortex and fruit fly brain in their study “Cortical column and whole-brain imaging of neural circuits with molecular contrast and nanoscale resolution (Gao et al, 2019).”

By creating enhanced processing and analysis tools in MBF Bioscience’s Stereo Investigator and Neurolucida 360 software, Dr. Tappan and Mr. Rodriguez analyzed these images to obtain comprehensive morphometrics of delicate dendritic spines at a greater accuracy than ever before.

GAO ET AL./SCIENCE 2019

“We combined expansion microscopy and lattice light sheet microscopy (ExLLSM) to image the nanoscale spatial relationships between proteins across the thickness of the mouse cortex or the entire Drosophila brain, including synaptic proteins at dendritic spines, myelination along axons, and presynaptic densities at dopaminergic neurons in every fly neuropil domain.” (Gao et al, 2019)

While several forms of microscopy exist that have the ability to image subcellular neural elements, scientists say that each of these methods is lacking in one way or another. According to the paper, the combination of expansion microscopy with lattice-light sheet microscopy gives the most effective results, while considerably decreasing the time spent carrying out the experiment.

“I believe this type of imaging represents a major milestone in terms of the accuracy that can be achieved in dendritic spine morphometry from light microscopy,” Mr. Rodriguez said.

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Diet Restriction Slows Neurodegeneration and Extends Lifespan of DNA-Repair-Deficient Mice

Posted on September 26, 2017 by Celeste Sunderland

DNA damage occurs in human cells at a constant rate. These cells are usually able to repair themselves, but sometimes deficiencies in certain genes cause the repair process to shut down. When damaged DNA isn’t fixed, mutations can occur that cause accelerated aging or cancerous tumors to form (Hoeijmakers, 2009). Scientists at Erasmus University Medical Center in Rotterdam have found a way to slow down the process – at least in mice.

In a study published in Nature, the researchers report that when mice deficient in the DNA-repair genes Ercc1 or Xpg are put on a restricted diet, they experience better overall health and increased lifespans compared to DNA-repair-deficient mice fed a normal diet. They also found significantly lower levels of neurodegeneration in the brains and spinal cords of diet restricted animals compared to controls.

“Here we report that a dietary restriction of 30 percent tripled the median and maximal remaining lifespans of these progeroid mice, strongly retarding numerous aspects of accelerated aging Mice undergoing dietary restriction retained 50 percent more neurons and maintained full motor function far beyond the lifespan of mice fed ad libitum,” (Vermeij, et al 2016).

Since the DNA-repair-deficient mice were already smaller and weaker than normal mice, the Rotterdam researchers wondered whether diet restriction would be beneficial or detrimental to their health. They found that gradually restricting the diets of DNA-repair-deficient mice starting at age seven weeks increased their median lifespans from 10 to 35 weeks in males and 13 to 39 weeks in females as compared to controls.

They also saw significant differences in the levels of neurodegeneration between these two populations. Using Stereo Investigator, they found 50 percent more neurons in the brains of diet-restricted mice compared to those fed a normal diet. They also saw lower levels of cells expressing p53 – a protein expressed in response to DNA damage – in diet-restricted mice.

According to the authors, dietary restriction may not fix defects in DNA repair mechanisms, but it may help to reduce the severity and speed at which the damage occurs.

“Our findings establish the Ercc1 mouse as a powerful model organism for health-sustaining interventions, reveal potential for reducing endogenous DNA damage, facilitate a better understanding of the molecular mechanism of dietary restriction and suggest a role for counterintuitive dietary-restriction-like therapy for human progeroid genome instability syndromes and possibly neurodegeneration in general,” (Vermeij, et al 2016).

Vermeij W.P., Dollé M.E.T., Reiling E., Jaarsma D., Payan-Gomez C, Bombardieri C.R., Wu H., Roks A.J.M., Botter S.M., van der Eerden B.C., Youssef S.A., Kuiper R.V., Nagarajah B., van Oostrom C.T., Brandt R.M.C., Barnhoorn S., Imholz S., Pennings J.L.A., de Bruin A., Gyenis Á., Pothof J, Vijg J, van Steeg H., and Hoeijmakers J.H.J. (2016) Restricted diet delays accelerated aging and genomic stress in DNA repair deficient mice. Nature 537, 427-431, doi:10.1038/nature19329

Hoeijmakers JH (2009) DNA Damage, aging, and cancer. N Engl J Med; 361:1475-1485, DOI: 10.1056/NEJMra0804615

Stock image of DNA used in accordance with the CC0 public domain license.

Uncovering the role of microglia in fetal alcohol spectrum disorders

Posted on January 11, 2017 by Celeste Sunderland

Representative images of Iba-1+ microglia in the postnatal day 10 rat hippocampus. Image courtesy of Anna Klintsova, PhD.

Children born with fetal alcohol spectrum disorders face a range of physical and cognitive impairments including long-term deficits in learning, behavior, and immune function. In a paper published in Neuroscience, Dr. Anna Klintsova and her lab at the University of Delaware report that activation of the brain’s immune response may contribute to some of the damage caused by fetal alcohol spectrum disorders.

In their study, the researchers used Stereo Investigator and Neurolucida to examine the hypothesis that exposure to alcohol while the brain is growing rapidly is associated with abnormal microglial activation and high levels of pro-inflammatory proteins which impair learning-related plasticity; leading to neuro-developmental and psychopathological disorders.

“My lab has been using both Stereo Investigator and Neurolucida for more than a decade in all quantitative neuroanatomical studies, including the featured one,” said Dr. Anna Klintsova. “We find this software to be user-friendly, reliable and essential for obtaining unbiased results.”

They used Stereo Investigator to quantify the number of microglia in the hippocampus of neonatal rats who were exposed to alcohol during the equivalent of the third trimester of a human pregnancy. The researchers expected to see an increased number of microglia in alcohol-exposed neonatal rats, however they found a decreased number of microglia. Despite the decrease in microglia number, there was a significant increase in pro-inflammatory proteins expressed by microglia and an increase in microglial activation.

To measure microglial activation, the researchers quantified the area of cell territory using Neurolucida. Activated microglia have a smaller cell territory than resting microglia, so the smaller cell territory found in alcohol exposed rats indicates a more active state.

This research supports the hypothesis that abnormal microglia activation plays a role in fetal alcohol spectrum disorders, however more research is needed to further understand the relationship.

Boschen, K., Ruggiero, M.J., Klintsova, A.Y., (2016) Neonatal binge alcohol exposure increases microglial activation in the developing rat hippocampus. Neuroscience 324: 355–366. DOI: 10.1016/j.neuroscience.2016.03.033

Stereological Study Reveals Neuron and Glia Proliferation in Hippocampus of Lithium-Treated Mice

Posted on September 15, 2016 by Celeste Sunderland

The optical fractionator probe was used to quantify the number of neurons and glia in the dentate gyrus

Doctors have used lithium to treat patients with bipolar disorder since the 1970s. Known for its efficacy in stabilizing patients’ moods by regulating manic episodes, lithium is also associated with a decreased risk of suicide. But while this naturally occurring element is the most widely prescribed medication for those suffering from bipolar disorder, scientists still have much to learn about how lithium physically affects the brain.

A recent study published in the journal Bipolar Disorders adds to the growing body of evidence that says lithium contributes to cell proliferation in parts of the brain. Conducted by scientists at the University of Mississippi and the VU University Medical Center in Amsterdam, the study revealed an increased number of neurons and glia, and increased astrocyte density in the dentate gyrus of lithium-treated mice versus controls treated with a placebo.

Using the optical fractionator probe in Stereo Investigator, the researchers quantified the number of Nissl stained neurons and glial cells, and calculated astrocyte density. The results showed twenty-five percent more neurons and twenty-one percent more glia in the denate gyrus of lithium-treated mice. They also performed a stereological examination of another brain region – the medial prefrontal cortex (mPFC), but did not witness significant differences between lithium-treated and control mice in this area.

“In this study, particular cortical regions, ie. the fascia dentata in the hippocampus and the mPFC in the cerebral cortex needed to be selected in histological sections of the mice brains,” explained Dr. Harry B.M. Uylings, “therefore the stereological counting procedure applied was the best one. Stereo Investigator greatly assisted in the counting of cells, and the software’s excel data-output was especially beneficial.”

According to the paper, the findings present a more detailed picture of lithium-induced alterations in the dentate gyrus cellular phenotype than previously available, and provide the first evidence for lithium-induced increases in glia and astrocytes.

The authors also explain that while cell number increased in the dentate gyrus of lithium-treated mice, the region’s overall volume as well as that of the greater hippocampus was unaffected by the element. The volume of the dentate gyrus and the hippocampus as a whole was measured with the Cavalieri method in Stereo Investigator. The researchers describe the dissociation between cell proliferation and volume as “an interesting observation that warrants further investigation.”

Rajkowska, G., Clarke, G., Mahajan, G., Licht, C.M., van de Werd, H.J., Yuan, P., Stockmeier, C.A., Maji, H.K., Uylings, H.B., Differential effect of lithium on cell number in the hippocampus and prefrontal cortex in adult mice: a stereological study. Bipolar Disord. 2016 Feb;18(1):41-51. doi: 10.1111/bdi.12364.

Iron Deficiency Worsens Fetal Alcohol Spectrum Disorders

Posted on April 5, 2016 by Celeste Sunderland

An immunostained image of myelin basic protein in the cerebella of a mouse brain with an iron-sufficient diet compared with the brain of a mouse exposed to alcohol and fed an iron-insufficient diet. It shows the reduced cerebellar size due to the ID-alcohol combination. Green is MBP immunostain, blue is DAPI for nuclei. Image courtesy of Susan Smith, PhD.

If a pregnant woman drinks alcohol, she risks giving birth to a baby with physical and cognitive deficits – characteristics of fetal alcohol spectrum disorders. In a new study, researchers say that when the mother is low in iron, the consequences are even worse.

The scientists examined two groups of pregnant rats – one group was fed an iron sufficient diet while the other was fed a diet with insufficient iron levels. The offspring from both groups were exposed to alcohol from 4 to 9 days after birth – a time when their brains are going through a growth spurt and are particularly sensitive to alcohol. They were compared to offspring who received an iron-sufficient diet but were not exposed to alcohol. This growth spurt correlates to a growth spurt in humans that occurs during the third trimester of pregnancy.

The researchers used delay and trace eye blink classical conditioning methods to assess the offspring’s learning and memory. Learning impairments were reported in both alcohol-exposed groups regardless of their iron status, but more extreme impairments were seen in iron deficient rats compared to iron sufficient rats. After the behavioral tests were completed, the researchers studied the cerebellum and hippocampus – brain regions involved in learning and memory – at a cellular level.

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Genetic Mutation Accelerates CTE Pathology

Posted on February 11, 2016 by Celeste Sunderland

Phosphorylated tau pS422 immunoreactive profiles in the cortex of P301Smice after repetitive mild TBI. Image courtesy of Dr. Leyan Xu.

Phosphorylated tau pS422 immunoreactive profiles (dark brown) in the cortex of P301S mice after repetitive mild TBI. Image courtesy of Dr. Leyan Xu, Department of Pathology, Johns Hopkins University.

Over the course of a football game or a boxing match, athletes may experience a series of mild concussions. Some of these athletes develop a condition known as chronic traumatic encephalopathy (CTE), a neurodegenerative disease characterized by the build-up of abnormal tau protein that eventually leads to dementia. But not every athlete develops CTE after repetitive mild traumatic brain injury, and scientists think genetic factors are involved.

In a recent study, researchers at the Johns Hopkins University School of Medicine found that the density of abnormal tau protein increased exponentially in mice that had a genetic mutation thought to cause neurodegenerative diseases. Their findings contrast with previous studies of mice without genetic mutation, where abnormal tau protein build-up did not occur. This evidence leads the scientists to infer that genetic factors play a role in the onset of CTE.

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Dying neurons in Alzheimer’s patients show signs of improvement after gene therapy

Posted on November 11, 2015 by Celeste Sunderland

Micrograph of cholinergic neurons in the nucleus basalis of Meynert. Image from Wikipedia.

Cholinergic neurons degenerate at devastating rates in Alzheimer’s disease, but Dr. Mark Tuszynski and his team at the University of California, San Diego may have found a way to slow the decline.

Their study, published in JAMA Neurology, reports that nerve growth factor gene therapy increased the size, axonal sprouting, and signaling of cholinergic neurons in 10 Alzheimer’s disease patients.

The patients were enrolled in a clinical trial between 2001 and 2012. Ex vivo and in vivo methods of gene therapy were used to deliver nerve growth factor – a protein that protects neurons and stimulates growth – to the patients. Eight received an implant of their own skin cells that were genetically modified to express nerve growth factor (ex vivo ) and two patients received injections that induced neurons already in the brain to express nerve growth factor (in vivo). In all 10 patients, gene therapy was delivered to the nucleus basalis of Meynert – part of the basal forebrain rich in cholinergic neurons that undergoes degeneration during Alzheimer’s disease.

The patients’ survival time ranged from one to 10 years. After they had died, researchers analyzed the effects of nerve growth factor on cholinergic neurons.

The axons of cholinergic neurons, labeled with p75, grew toward the source of the nerve growth factor in all 10 patients. To determine if there was a change in cell size, researchers used the nucleator probe in Stereo Investigator to analyze cholinergic neurons of 3 patients who received gene therapy via the ex vivo method in one hemisphere – the other hemisphere was used as a control. Results from Stereo Investigator showed that cell bodies were larger in the treated hemisphere vs. the untreated hemisphere.

Finally, to find out if nerve growth factor induced signaling within cells, the researchers analyzed the amount of CREB and c-fos – markers for cell activation – in 2 patients who received nerve growth factor in vivo. An elevated amount of CREB and c-fos was found when compared to control regions. Neurons exhibiting tau pathology also expressed nerve growth factor, indicating that degenerating cells could respond to nerve growth factor gene therapy.

A phase 2 clinical study is currently under way to report cognitive outcomes in patients with Alzheimer’s disease.

“Collectively, these anatomical findings support the rationale for clinical trials to test the hypothesis that sustained growth factor delivery over time can reduce cell degeneration and stimulate cell function in chronic neurodegenerative disorders, thereby slowing functional decline,” Tuszynski, et al.

Tuszynski, M.H., Yang, J.H., Pay, M.M., Masliah, E., Barba, D., U, H.S., Conner, J.M., Kobalka, P., Roy, S., and Nagahara A.H. (2015). Nerve Growth Factor Gene Therapy: Activation of Neuronal Responses in Alzheimer Disease. JAMA Neurology, published online August 24, 2015. DOI: 10.1001/jamaneurol.2015.1807.

Delayed loss of neurons occurs in mice with mild TBI and anxiety

Posted on September 4, 2014 by Celeste Sunderland

Almeida-Suhett et al saw delayed loss of GABAergic interneurons in the BLA within the first week after mild CCI. (Representative photomicrographs of GAD-67 immunohistochemically stained GABAergic interneurons in the BLA of sham (left), 1-day CCI (middle), and 7-day CCI (right) animals. Total magnification is 630x; scale bar, 50 µm.)

Soldiers, athletes, and other individuals who suffer a traumatic brain injury often develop anxiety disorders, but scientists aren’t sure why. Some speculate that fear about future health or the stress of the trauma itself contributes to elevated anxiety, while others suspect changes happening inside the brain as a result of the injury are to blame.

Researchers at Maria Braga’s lab at the Uniformed Services University of the Health Sciences in Bethesda, Maryland, recently found direct evidence that physical changes happen in the brain after TBI that coincide with increased anxiety levels.

She and her team studied a rat model of mild TBI, focusing on the basolateral amygdala (BLA) – a brain region often damaged by TBI, which has also been associated with increased fear and anxiety in instances of hyperactivity.

To find out what happens in the BLA that might be causing anxiety after a mild TBI, the researchers analyzed changes in synaptic activity in this region. Using Stereo Investigator with the optical fractionator probe to perform a stereological quantification of Nissl-stained and GAD-67 immunostained brain cells, they found that many of the inhibitory neurons – the cells that quiet activity – were lost seven days after injury. Whole cell recordings from principal neurons confirmed that the inhibitory cells’ synaptic transmissions were impaired during this period, resulting in increased excitability and “open field tests” showed elevated anxiety levels in post-injury rats at the exact same time point. Continue reading “Delayed loss of neurons occurs in mice with mild TBI and anxiety” »

Higher levels of pTau found in Alzheimer’s disease patients with psychosis

Posted on July 23, 2014 by Celeste Sunderland

An image of neurofibrillary tangles and neuropil threads

People with Alzheimer’s disease suffer from severe memory loss and often have problems focusing, reasoning, and communicating. About half of all Alzheimer’s patients also experience delusions and hallucinations, this is called Alzheimer’s disease with psychosis, and scientists at the University of Pittsburgh are learning more about this severe version of the disease.

In a recent study, researchers at Dr. Robert Sweet’s lab zeroed in on a protein called tau, which forms tangles in the brains of Alzheimer’s patients, and along with amyloid plaques is one of the major hallmarks of the disease. But despite being involved in these pathological conditions, tau and amyloid may instigate other processes as well – namely, synaptic toxicity, which the authors say is “the strongest correlate of cognitive decline in Alzheimer’s disease.”

Recent research suggests that amyloids (misfolded proteins) drive the deterioration of synapses, but phospho-tau (tau, which has undergone phosphorylation), enables the process. So in their study the Pittsburgh research team analyzed the presence of tau in the prefrontal cortex, a region of the brain involved in higher processes, of 45 Alzheimer’s disease patients with and without psychosis.

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New Neurons Erase Memories

Posted on July 3, 2014 by Celeste Sunderland

Neurogenesis occurs in the dentate gyrus, pictured here, from birth through adulthood.

A baby laughs at an elephant at the zoo. A toddler runs across a beach. Small children make memories all the time, but how many will they recall as the years pass? Maybe none at all. The phenomenon is called “infantile amnesia,” and scientists may have pinpointed a reason for why it occurs – neurogenesis.

Researchers at the Hospital for Sick Children in Toronto say that when new brain cells integrate into existing circuitry, they remodel the structure of networks already in place, wiping out the information previously stored there. This process is prevalent in infancy and early childhood because this is the time when new brain cells are being generated faster and more frequently than at any other time in a human being’s life. Humans and other mammals spawn new neurons throughout their lifespans, although the rate of neurogenesis decreases significantly with age.

In their paper, published in Science, the researchers explain how recent studies have focused on how new brain cells can lead to new memories, but the Toronto team speculated that neurogenesis could also wipe away memories. To test their hypothesis, they conducted a series of studies on populations of newborn and adult mice. Neuron development in mice occurs in much the same way as in humans, with rapid cell genesis in infancy that tapers off with age.

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