A complete guide to imaging and analyzing spines and neurons with Neurolucida 360


Following a well-designed protocol is essential to achieving accurate and consistent results in scientific research. Now, scientists using Neurolucida 360 for dendritic spine and neuron analysis can follow a published set of guidelines to ensure optimal confocal data series for proper dendritic spine quantification and neuron reconstruction. The paper, written by MBF Bioscience scientists and researchers from the Icahn School of Medicine at Mount Sinai in New York, was published in Current Protocols in Neuroscience.

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

Researchers Explore Spatial Memory with Stereo Investigator

The researchers quantified c-Fos positive cells in the CA1 region of the hippocampus. Image provided by Dr. Matthew Holahan.

The researchers quantified c-Fos positive cells in the CA1 region of the hippocampus. Image provided by Dr. Matthew Holahan.

Spacial memories help us navigate places like the office, the local coffee shop, or the supermarket. The hippocampus plays a key role in processing and recalling spacial memory, but as time passes, there is evidence that the anterior cingulate cortex (ACC) becomes more involved in recalling these memories. A recent paper published in PLOS ONE further investigates the ACC and found that taxing the hippocampus with spacial memory tasks accelerates the recruitment of the ACC for spacial memory recall.

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Hippocampal Neurons Change After Melatonin Injection

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.

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

 

Anorexia Accelerates the Development of the Rat Hippocampus

spine_edited

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.

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New Neurons Erase Memories

Dentate gyrus

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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3D Reconstructions of Neurons Reveal More Branching in Sedentary Rats

Left: A neuron from the brain of a rat that exercised for two hours each day. Right: A neuron from the brain of a sedentary rat.

Left: A neuron from the brain of a rat that exercised for two hours each day.
Right: A neuron from the brain of a sedentary rat. Scientists saw greater branching in inactive versus active rats. (Image courtesy of Dr. Patrick Mueller)

Scientists discovered that inactivity makes brain cells grow, but not in a good way. In a study published in the Journal of Comparative Neurology, researchers found more neuronal branching in sedentary rats compared to active rats. The growth occurred in a region of the brain that controls blood pressure, leading the scientists to hypothesize that these changes may be part of the reason inactivity is linked to an increased risk of heart disease.

Using Neurolucida to reconstruct neurons in 3D, the scientists at Dr. Patrick Mueller’s lab at Wayne State University School of Medicine, in Detroit, saw structural differences between the brains of active and inactive rats.

Focusing on the rostral ventrolateral medulla (RVLM) – an area that controls several critical biological processes that rats as well as humans do unconsciously, like swallowing, breathing, and regulating blood pressure, the scientists saw longer dendrites, more dendritic branching, and more intersections with other neurons in sedentary rats.

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Scientists Discover Anorexia-Driven Changes to Dendrites With Neurolucida

A digital reconstruction of a CA1 pyramidal cell from the ventral hippocampus, traced using Neurolucida with Sholl spheres at 20 micron intervals. Cells in this region featured greater dendritic length and branching versus controls.

A digital reconstruction of a CA1 pyramidal cell from the ventral hippocampus of a rat with activity-based anorexia, traced using Neurolucida with Sholl spheres at 20 micron intervals. Cells in this region featured greater dendritic length and branching versus controls.

Gaunt facial features and a frighteningly thin figure are physical hallmarks of anorexia nervosa, an eating disorder that predominantly affects adolescent girls. But in addition to extreme weight loss, changes take place that aren’t as visually apparent. For the first time, scientists in New York have found evidence of brain plasticity in the activity-based anorexia (ABA) mouse model.

Led by Dr. Chiye Aoki of New York University, the research team used Neurolucida to analyze pyramidal neurons in the rat brain. Since anorexia is linked to elevated stress hormones and anxiety, the researchers focused on the hippocampus, a region that regulates anxiety and is known to change structurally in response to hormones and stress.

“Using Neurolucida, we were able to collect, store, and analyze large amounts of data with more precision and accuracy than would have been possible without the digital interface,” said Tara Chowdhury, a graduate student working in Dr. Aoki’s lab, and first author of the paper.

“Additionally, with its very approachable interface, the software allowed us to trace dendrites, get precise thickness measurements, and categorize spine types easily during tracing. The built-in Sholl analysis and spine analysis tools resulted in quick quantification of all the measurements that would have taken hours to achieve without Neurolucida.”

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Ohio State Neuroscientists Use Neurolucida to Analyze Brain Cells in Sexually Active Hamsters

A Golgi stained human neocortical pyramidal neuron. Morris et al studied cells like this to determine the affect of sexual experience on the adult brain. Using Neurolucida, they saw shorter, less extensive dendrites in hamsters which mated during adolescence versus controls.

A Golgi-stained human neocortical pyramidal neuron. Morris et al. studied cells like this to determine the effect of sexual experience on the adult brain. Using Neurolucida, they saw shorter, less extensive dendrites in hamsters which mated during adolescence versus controls.

Scientists hypothesize that during puberty, experiences influence brain development in ways that shape brain structure and even behavior in adulthood. One type of experience that often arises in the minds of pubescent teens and adolescents is sex. But a study published in the journal Hormones and Behavior suggests engaging in sexual activity too soon could be detrimental to the adult brain, and may lead to depression.

In their study of Siberian hamsters, scientists at the Wexner Medical Center at Ohio State University say adolescent sexual experiences alter brain structure.

“We used Neurolucida to reconstruct the morphology of prefrontal cortical neurons in the brains of Siberian hamsters that were exposed to sexual experience during early adolescence, later in young adulthood, or left socially isolated,” said Dr. Zachary M. Weil, an author of the study. “Interestingly, hamsters that engaged in sexual experience during early adolescence but not during other developmental periods exhibited reduced branching and dendritic length in the prefrontal cortex.”

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MBF Bioscience attends the International Conference of the Society of Neuroscientists of Africa (SONA)

Dr. Jose talks with a customer at the MBF Bioscience booth

Dr. Jose talks with a customer at the MBF Bioscience booth

The 11th International Conference of the Society of Neuroscientists of Africa (SONA) was held in Rabat, Morocco from June 7-12, 2013. Neuroscientists from Africa and around the world gathered to discuss the latest advances in Neuroscience. The program filled with lectures, symposia, posters, and oral sessions was sponsored by the International Brain Research Organization (IBRO) and the Society for Neuroscience (SfN).

Neuroscientists gather at the SONA conference held in Rabat, Morroco

Neuroscientists gather at the SONA conference held in Rabat, Morroco

Dr. Jose Maldonado, Head of Operations for MBF Bioscience Latin America and Africa, attended the conference. “This year’s SONA meeting in Morocco was a great opportunity to introduce MBF Bioscience Inc. to the neuroscience community in Africa. Quantification of anatomical regions, as well as cell populations, was a common theme among the posters. It was a dynamic meeting and I look forward to attending again in the future,” said Dr. Maldonado. Attendees who stopped by the MBF Bioscience booth were particularly interested in Stereo investigator, MBF’s system for stereology that gives accurate, unbiased estimates of the number, length, area, and volume of cells or biological structures in tissue specimens.

Thank you to everyone who attended the conference and visited us at our booth.

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