Image Courtesy: Bob Jacobs, Ph.D. , Colorado College

With the release of its new version on November 28, NeuroMorpho.org adds 9,987 new images to its archive, bringing its impressive collection of digitally reconstructed neurons to 80,012.

Scientists used MBF Bioscience’s software, Neurolucida and Neurolucida 360, to reconstruct the majority of these cells. In fact, 64 times more neurons were reconstructed with MBF Bioscience software than those imaged by our closest commercial competitor – that’s 42,121 reconstructions compared to 656. This metric demonstrates that Neurolucida and Neurolucida 360 are truly the gold standards for neuron reconstruction.

Featuring contributions from hundreds of laboratories from around the globe, NeuroMorpho.Org is the world’s leading database of publicly accessible 3D neuronal reconstructions and associated metadata. From the dragonfly to the humpback whale, researchers have access to accurate and verified data from an array of different organisms. Arranged by animal species, brain region, cell type, or contributing laboratory, each file contains specific details about the cell’s morphology such as age, developmental stage, soma volume, and number of branches – all of which are searchable.

Recently, NeuroMorpho.Org hit the 8 million download mark, with researchers in 166 different countries accessing this valuable resource, and more than one thousand published articles referencing its data

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The four protocols describe best practices for imaging and analyzing dendritic spines and entire neurons. Clearly laid out procedures specify necessary materials, image acquisition techniques, and analysis procedures with Neurolucida 360.

Imaging technique is crucial to obtaining unbiased, reproducible results. Clear, crisp images captured with an appropriate z-interval will make analysis with Neurolucida 360 easier and more accurate. Throughout the paper, the authors emphasize the importance of image scaling parameters and unbiased sampling for achieving repeatable results. They also discuss the benefits of correcting optical distortion, especially in the Z-plane, with deconvolution to acquire clear images – a process critical to getting the most accurate representation of dendrites and spines.

Dendritic spine analysis is traditionally performed through tedious, time-consuming manual techniques. According to the paper, this has spawned a growing interest in a more efficient solution for spine quantification and morphological analysis like the one Neurolucida 360 provides. A software platform for automatic neuron reconstruction and spine detection in a 3D environment, Neurolucida 360 offers a variety of benefits, including:

• Fast and accurate spine detection and neuron reconstruction
• Accurate spine classification and length quantification using a five-point segment that more accurately models the spine backbone.
• 3 user-guided and automatic algorithms to accurately model neurons visualized with multiple methodologies and imaging techniques.
• A large number of metrics, including volume, length, and surface area.

“We believe that the new quantitative software package, Neurolucida 360, provides the neuroscience research community with the ability to perform higher throughput automated 3D quantitative light microscopy spine analysis under standardized conditions to accelerate the characterization of dendritic spines with greater objectivity and reliability,” (Dickstein, et al. 2016)

The full paper can be found here.

An infographic quickly outlines Protocol 1: Imaging of fluorescently labeled dendritic segments. Use this as a quick reference tool in your lab (right-click on it to save as an image):

Dickstein, D.L., Dickstein, D.R., Janssen, W.G.M., Hof, P.R., Glaser, J.R., Rodriguez, A., O’Connor, N., Angstman, P., and Tappan, S.J. 2016. Automatic dendritic spine quantification from confocal data with Neurolucida 360. Curr. Protoc. Neurosci. 77:1.27.1-1.27.21. doi: 10.1002/cpns.16

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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

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This figure illustrates the separate and combined effects of acute stress and fear conditioning/extinction on dendritic morphology of pyramidal neurons in the infralimbic region of medial prefrontal cortex. Each neuron shown is a composite made up of apical (blue) and basilar (orange) arbor near the mean of the group. The apical and basilar arbors of each composite are from different neurons. Image courtesy of Cara Wellman, PhD.

A soldier jumps at the sound of fireworks. Though there is no threat to his or her life, the blasts mimic the ones heard on the battlefield, and that fear response is not easy to forget. The process of shedding a fear response like this one is called fear extinction. Scientists think patients suffering from stress-sensitive psychopathologies, like Post-Traumatic Stress Disorder, aren’t able to suppress certain fear responses because of deficits in their brain circuitry induced by stress.

A recent study by researchers at Indiana University and the University of Haifa, in Israel, describes significant differences between the brains of stressed rats and unstressed rats.

Using Neurolucida to analyze neurons in the infralimbic cortex (IL) – a region of the brain associated with fear extinction – the research team found that stressed rats had shorter dendrites and less dendritic branching in pyramidal neurons of the IL. They also found that while stress had no affect on spine density, rats that underwent fear conditioning and extinction had decreased spine density on apical terminal branches, providing evidence that dendritic morphology in this region is sensitive to stress, while spine density may be a reflection of learning.

“Having helped colleagues set up procedures for neuron reconstructions and spine counts in labs that aren’t equipped with Neurolucida, I can tell you with complete confidence that my lab wouldn’t be nearly as productive without our Neurolucida system,” said Dr. Cara Wellman. “It makes mapping out regions of interest, identifying neurons for reconstruction, and reconstructions, and data analysis a simple and streamlined process. My students and I especially appreciate the Lucivid, which allows us to trace neurons while looking through the oculars – so much easier and clearer in my opinion than on a video monitor.”

To achieve their results, the researchers subjected rats to fear conditioning, where they learned to associate a certain tone with a footshock. Some of the rats were then exposed to an elevated platform in a brightly lit room for 30 minutes (stressed) while others returned to their home cages (unstressed). Next came extinction sessions. In a test to see if they would be able to shed the fear response associated with the stimulus, rats were placed in a space where they heard a tone but did not experience a footshock. The scientists observed that stressed rats exhibited freezing during the extinction sessions at a much higher rate than unstressed rats, leading them to believe that rats exposed to acute stress were resistant to fear extinction.

Further quantification of apical and basilar dendritic branching in the pyramidal neurons of the IL, measured with three-dimensional Sholl analysis, confirmed differences between the stressed and unstressed rats’ brains that correlated with fear behavior.

“The main findings of the current study were that acute stress, concurrent with producing resistance to extinction, produced changes in morphology of pyramidal neurons in IL,” the authors say in their paper. “These findings provide evidence that alterations in IL pyramidal neuron morphology occur quickly and differentially in response to acute stress and fear conditioning/extinction.”

Moench KM, Maroun M, Kavushansky A, Wellman C. Alterations in neuronal morphology in infralimbic cortex predict resistance to fear extinction following acute stress. Neurobiology of Stress. 3: 23-33. doi:10.1016/j.ynstr.2015.12.002

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The U.S. Small Business Administration recently published a success story about MBF Bioscience. The article highlights our success using federal Small Business Innovation Research (SBIR) grants to develop innovative products that help advance science. MBF Bioscience has a distinguished R&D program that develops cutting-edge tools for scientific research.

The SBIR grants have helped MBF Bioscience develop tools such as Neurolucida, the most widely used system for neuron reconstruction and analysis, and Stereo Investigator, the gold standard for quantifying the number, area, and volume of neurons. MBF Bioscience has grown significantly since it was awarded its first SBIR grant in 1987. It is now a global company helping scientists around the world discover new information about the brain. The full success story written about MBF Bioscience can be found here.

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“Neurolucida 360 is a technological revolution” says Jack Glaser, President of MBF Bioscience. “It is the state-of-the-art tool for neuroscientists to analyze the shape and connectivity of neurons more quickly and accurately than has ever been possible, so that we can better understand the brain and the mechanisms behind diseases such as Alzheimer’s and Parkinson’s. With the unique ability to automatically detect and analyze dendrites, dendritic spines, axons, somas, and synapses, Neurolucida 360 is now the standardized platform for the global neuroscience research community to perform unprecedented investigations into the functioning of the brain.”

Using automated tools in Neurolucida 360, researchers generate high-resolution, digital 3D reconstructions of neurons imaged with numerous microscopy techniques, including light sheet, 2 photon, confocal and brightfield. Using the most advanced algorithms for neuron reconstruction, researchers instantly receive hundreds of analyses about the size, shape, and complexity of neurons. The reliable data from Neurolucida 360 is integral to learning how injury, disease, or chemicals change neuronal structure, discovering how neuronal structure affects function, finding out which brain regions neurons communicate with, and more.

The National Institute of Mental Health provides funding to support the development of Neurolucida 360. It is the latest development in the renowned legacy of neuron tracing tools that started with Neurolucida – the most widely cited tool for neuron reconstruction and analysis.

For more information on Neurolucida 360, please visit our website or watch this short video.

The post New Software Released for Automated Neuron Reconstruction and Analysis appeared first on MBF Bioscience.

Representative dendrites of dentate gyrus neurons of Siberian hamsters injected with melatonin (stained with Cresyl violet). Ikeno et al found hamsters injected with melatonin displayed decreased spine density on neurons in the dentate gyrus. Image courtesy of Tomoko Ikeno, Ph.D.

Night falls and a powerful hormone called melatonin kicks in. The gears of the circadian clock are turning as you get ready for bed and soon drift off to dreamland. But all is not quiet in the brain. In response to the circadian rhythm, neurons are transforming.

A new study published in the journal Hippocampus found that melatonin prompts dendrites to grow longer in one part of the brain, while in another part the hormone causes dendritic spine loss.

In their study, scientists at Ohio State University injected Siberian hamsters with a dose of melatonin in the afternoon, several hours before a natural increase in the hormone would normally occur. Four hours after the injection, they used Neurolucida to examine sections of their brains, reconstructing neurons in two areas of the hippocampus – the CA1 and dentate gyrus. They then used the software to calculate the number of branch points and length of dendrites in their reconstructions. What they saw was longer, more complex dendrites in the CA1 region of the hippocampus of hamsters that received melatonin versus those that received a placebo. Then they analyzed spine density, finding that hamsters that received melatonin had decreased spine density in the dentate gyrus than the control group.

“By using Neurolucida, we found that melatonin treatment induced rapid remodeling of hippocampal neurons and induced a nighttime state of the hippocampal neuronal morphology,” said Dr. Tomoko Ikeno, who worked with Dr. Randy Nelson on the study.

The “nighttime state” she refers to is characterized by the presence of certain hormones produced during the dark hours of night. In their analysis, the researchers saw elevated levels of Period1 and Bmal1 after melatonin injection. These hormones are expressed by genes associated with the circadian clock, and their presence offers evidence that “melatonin functions as a nighttime signal to coordinate the diurnal rhythm” and that this rhythm compels hippocampal neurons to change structurally, according to the paper.

Ikeno, T. and Nelson, R. J. (2014), Acute melatonin treatment alters dendritic morphology and circadian clock gene expression in the hippocampus of Siberian Hamsters. Hippocampus. doi: 10.1002/hipo.22358

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Golgi-stained human brain tissue from the dorsolateral prefrontal cortex.

Schizophrenia and bipolar disorder are very different mental illnesses, but researchers are discovering evidence that the two disorders have some common pathologies. According to a recent study, a shared characteristic appears to be dendritic spine loss.

The researchers used Neurolucida to study pyramidal cells in human brain tissue from individuals with schizophrenia (n=14), individuals with bipolar disorder (n=9) and unaffected control participants (n=19). The pyramidal cells were located in the dorsolateral prefrontal cortex – a region that plays a key role in working memory. Bipolar patients showed significantly reduced spine density (10.5 percent) compared to control. Schizophrenic patients also showed lower spine density (6.5 percent), but this number just missed significance when compared to control patients. Individuals with both illnesses showed a lower number of spines per dendrite, as well as reduced dendritic length compared to controls.

To obtain these results, researchers analyzed 15 Golgi-stained pyramidal cells in each tissue sample. They used Neurolucida to reconstruct the longest dendrite on the pyramidal cells and to mark spines. After the researchers finished reconstructing the cells, Neurolucida provided them with important data about the dendrites and spines.

“It would have been very difficult, if not impossible, to obtain the measurements for this study without Neurolucida,” said lead investigator Dr. Glenn Konopaske. “Other programs I am aware of allow measurements of dendritic spine density along only a portion of the dendrite. Neurolucida allowed an assessment of spine density along the whole dendritic branch.”

Dr. Konopaske’s research confirms previous studies, which have shown spine loss on pyramidal cells in the dorsolateral prefrontal cortices of individuals with schizophrenia, but according to the paper, the level of reduction is lower (6.5 percent) than previously reported (23 percent) – a finding, which the authors say suggests “variability in spine pathology in SZ.”

“The current study suggests that spine pathology is common to both SZ and BP. Moreover, the study of the mechanisms underlying the spine pathology might reveal additional similarities and differences between the two disorders, which could lead to the development of novel biomarkers and therapeutics,” the authors say.

Konopaske, G.T., Lange, N., Coyle, J.T., Benes, F.M. (2014). Prefrontal Cortical Dendritic Spine Pathology in Schizophrenia and Bipolar Disorder. JAMA Psychiatry. doi: 10.1001/jamapsychiatry.2014.1582.

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Drs. May-Britt and Edvard Moser Image from GEIR MOGEN / NTNU

Drs. May-Britt and Edvard Moser were awarded the 2014 Nobel Prize in Physiology or Medicine for discovering the cells that form a network for spatial navigation in the brain, and we’re proud to say they are MBF Bioscience customers and used Neurolucida in their research.

In 2006, the Norwegian husband and wife team published a paper in the journal Science entitled “Conjunctive Representation of Position, Direction, and Velocity in Entorhinal Cortex” – a pivotal step in a line of research initiated in 1971 by co-laureate Dr. John O’Keefe (The Hippocampus as a Spatial Map). In their study, the scientists used Neurolucida to create 3D reconstructions of a complex network of neurons that make it possible for rats, and other animals, including humans, to navigate the world around them.

Building upon Dr. O’Keefe’s discovery of place cells – neurons located in the hippocampus that fire when an animal is in a particular place, thereby forming an internal neural map of a particular environment – the Mosers went a step further. By using electrodes to analyze the activity of specific neurons deep inside the rat brain, they identified a multilayered network of neurons located in a region called the entorhinal cortex. They found that these cells are what triggered the “place cells” to fire. And they also discovered that these cells, which they called “grid” cells, fired when a rat crossed certain physical points in the space in which it was being tracked, much like “place cells.”

This honeycomb pattern is the result of connecting the centers of the grid cells’ firing fields. Image from Wikipedia

However, when the points were connected, the researchers realized that they formed perfect hexagonal shapes, and that this geometric “honeycomb” pattern was initiated within the brain by the complex network of grid cells firing in a spatial pattern corresponding to the rat’s location. In other words, they form an “inner GPS” that helps us know where we are, and how to find our way home.

“Together, the hippocampal place cells and the entorhinal grid cells form interconnected nerve cell networks that are critical for the computation of spatial maps and navigational tasks. The work by John O’Keefe, May-Britt Moser and Edvard Moser has dramatically changed our understanding of how fundamental cognitive functions are performed by neural circuits in the brain and shed new light onto how spatial memory might be created” – Ole Kiehn and Hans Forssberg, Karolinska Institute 

MBF Bioscience wholeheartedly congratulates the work of Drs. May-Britt and Edvard Moser!

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This image stack was used in the study to analyze spine density. Image courtesy of Tara Chowdhury, Ph.D. first author of the study.

To find out how anorexia nervosa changes the brain, scientists at New York University are studying a rat model of the disease called activity-based anorexia (ABA). Previously, they discovered that ABA rats develop unusually robust dendritic branching of neurons in part of the hippocampus. Their new study takes those findings a step further, illuminating more differences between the brains of healthy versus ABA rats, and offering evidence that ABA rats may be developing too early, closing a critical period of development too soon.

But before making any conclusions about ABA brains, the researchers made some interesting discoveries about normal brain development. Using Neurolucida to analyze CA1 pyramidal cells in the stratum radiatum layer of the ventral hippocampus, they found that after puberty, around postnatal day 51, dendrites go through a growth spurt, more than doubling the number of branches seen seven days earlier. This growth spurt is followed by a decrease, or a pruning, which the researchers say is part of the normal maturation process.

They also used Neurolucida to examine dendritic spines – parts of the neuron that receive excitatory input from other neurons. While they saw no change in spine density as adolescence progressed, the researchers did see a larger proportion of mature spines – a finding, which suggests that weaker, immature spines are eliminated in favor of the stronger, more mature ones as time passes, according to the paper.

“Using Neurolucida’s built-in tools to analyze dendritic complexity and the density of different spine types, we learned that the hippocampus is continuing to develop and mature during adolescence,” said first author Tara Chowdhury.

Surprisingly, when the scientists analyzed the brains of ABA rats, they saw the same developmental patterns they saw in healthy rats occurring, but they happened earlier. They noted that the increase in dendritic branching at P41 in ABA rats was comparable to the increase in healthy rats at P51. They also witnessed a correlation in pruning occurring several days later in both rat populations. And analysis of dendritic spines showed that 44-day-old ABA rats had about the same proportion of mature dendritic spines as healthy, 55-day-old rats. Taken together, this information lead the researchers to conclude that the ventral hippocampus of ABA rats is developing too soon.

“We suggest that adolescence is a period of robust development of the CA1 hippocampus, puberty opens a critical period for extensive, experience-dependent modifications in the dendritic structure of the CA1 hippocampus; and that ABA induction, consisting of food restriction and excessive exercise, accelerates the closure of the critical period,” the authors say.

Chowdhury, T. G., Rios, M. B., Chan, T. E., Cassataro, D.S., Barbarich-Marsteller, N. C., & Aoki, C. (2014).Activity-based anorexia during adolescence disrupts normal development of the CA1 pyramidal cells in the ventral hippocampus of female rats. Hippocampus. doi: 10.1002/hipo.22320.

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