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 cited MBF systems in 27 papers during the week of 03/13/2017

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Ambrosi, G., Kustrimovic, N., Siani, F., Rasini, E., Cerri, S., Ghezzi, C., . . . Blandini, F. (2017). Complex Changes in the Innate and Adaptive Immunity Accompany Progressive Degeneration of the Nigrostriatal Pathway Induced by Intrastriatal Injection of 6-Hydroxydopamine in the Rat. Neurotoxicity research, 1-11. doi: 10.1007/s12640-017-9712-2.

Chen, B. K., Madigan, N. N., Hakim, J. S., Dadsetan, M., McMahon, S. S., Yaszemski, M. J., & Windebank, A. J. (2017). GDNF Schwann cells in hydrogel scaffolds promote regional axon regeneration, remyelination and functional improvement after spinal cord transection in rats. Journal of Tissue Engineering and Regenerative Medicine, n/a-n/a. doi: 10.1002/term.2431.

Cheng, L., Lau, W. K. W., Fung, T. K. H., Lau, B. W. M., Chau, B. K. H., Liang, Y., . . . Lee, T. M. C. (2017). PM2.5 Exposure Suppresses Dendritic Maturation in Subgranular Zone in Aged Rats.  Neurotoxicity research, 1-8. doi: 10.1007/s12640-017-9710-4.

Herrera-Soto, A., Díaz-Veliz, G., Mora, S., Muñoz, P., Henny, P., Steinbusch, H. W. M., & Segura-Aguilar, J. (2017). On the Role of DT-Diaphorase Inhibition in Aminochrome-Induced Neurotoxicity In Vivo.  Neurotoxicity research, 1-7. doi: 10.1007/s12640-017-9719-8.

Kaufling, J., Girard, D., Maitre, M., Leste-Lasserre, T., & Georges, F. (2017). Species-specific diversity in the anatomical and physiological organization of the BNST-VTA pathway. European Journal of Neuroscience, n/a-n/a. doi: 10.1111/ejn.13554.

Keiner, S., Niv, F., Neumann, S., Steinbach, T., Schmeer, C., Hornung, K., . . . Redecker, C. (2017). Effect of skilled reaching training and enriched environment on generation of oligodendrocytes in the adult sensorimotor cortex and corpus callosum. BMC Neuroscience, 18(1), 31. doi: 10.1186/s12868-017-0347-2.

Mikrogeorgiou, A., Sato, Y., Kondo, T., Hattori, T., Sugiyama, Y., Ito, M., . . . Kazama, T. (2017). Dedifferentiated Fat Cells as a Novel Source for Cell Therapy to Target Neonatal Hypoxic-Ischemic Encephalopathy. Developmental Neuroscience.

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Researchers cited MBF systems in 28 papers during the week of 03/06/2017

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Akkhawattanangkul, Y., Maiti, P., Xue, Y., Aryal, D., Wetsel, W. C., Hamilton, D., . . . McDonald, M. P. (2017). Targeted deletion of GD3 synthase protects against MPTP-induced neurodegeneration. Genes, Brain and Behavior, n/a-n/a. doi: 10.1111/gbb.12377.

Chung, Y. C., Baek, J. Y., Kim, S. R., Ko, H. W., Bok, E., Shin, W.-H., . . . Jin, B. K. (2017). Capsaicin prevents degeneration of dopamine neurons by inhibiting glial activation and oxidative stress in the MPTP model of Parkinson/’s disease. Experimental and Molecular Medicine, 49, e298. doi: 10.1038/emm.2016.159.

Hajheidari, S., Sameni, H. R., Bandegi, A. R., & Miladi-gorji, H. (2017). Effects of prolonged abstinence from METH on the hippocampal BDNF levels, neuronal numbers and apoptosis in methamphetamine-sensitized rats. Neuroscience Letters, 645, 80-85. doi: http://dx.doi.org/10.1016/j.neulet.2017.02.051.

Krishnasamy, S., Weng, Y.-C., Thammisetty, S. S., Phaneuf, D., Lalancette-Hebert, M., & Kriz, J. (2017). Molecular imaging of nestin in neuroinflammatory conditions reveals marked signal induction in activated microglia. Journal of neuroinflammation, 14(1), 45. doi: 10.1186/s12974-017-0816-7.

Langley, M., Ghosh, A., Charli, A., Sarkar, S., Ay, M., Luo, J., . . . Kanthasamy, A. (2017). Mito-apocynin Prevents Mitochondrial Dysfunction, Microglial Activation, Oxidative Damage and Progressive Neurodegeneration in MitoPark Transgenic Mice. Antioxidants & redox signaling. doi: 10.1089/ars.2016.6905.

Leal-Campanario, R., Alarcon-Martinez, L., Rieiro, H., Martinez-Conde, S., Alarcon-Martinez, T., Zhao, X., . . . Macknik, S. L. (2017). Abnormal Capillary Vasodynamics Contribute to Ictal Neurodegeneration in Epilepsy.  Scientific Reports, 7, 43276. doi: 10.1038/srep43276

Mao, Z., Liu, C., Ji, S., Yang, Q., Ye, H., Han, H., & Xue, Z. (2017). The NLRP3 Inflammasome is Involved in the Pathogenesis of Parkinson’s Disease in Rats. Neurochemical Research, 1-12. doi: 10.1007/s11064-017-2185-0.

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Researchers cited MBF systems in 12 papers during the week of 02/27/2017

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Cao, M., Wu, Y., Ashrafi, G., McCartney, A. J., Wheeler, H., Bushong, E. A., . . . De Camilli, P. (2017). Parkinson Sac Domain Mutation in Synaptojanin 1 Impairs Clathrin Uncoating at Synapses and Triggers Dystrophic Changes in Dopaminergic Axons. Neuron, 93(4), 882-896.e885. doi: http://dx.doi.org/10.1016/j.neuron.2017.01.019.

Makinson, C. D., Tanaka, B. S., Sorokin, J. M., Wong, J. C., Christian, C. A., Goldin, A. L., . . . Huguenard, J. R. (2017). Regulation of Thalamic and Cortical Network Synchrony by Scn8a. Neuron. doi: http://dx.doi.org/10.1016/j.neuron.2017.01.031.

Mor, D. E. (2016). The toxic interaction of dopamine and alpha-synuclein: Implications for Parkinson’s disease. University of Pennsylvania. Retrieved from http://search.proquest.com/openview/28281453309e818989ee516dd6262df1/1?p….

Patzlaff, N. E., Nemec, K. M., Malone, S. G., Li, Y., & Zhao, X. (2017). Fragile X related protein 1 (FXR1P) regulates proliferation of adult neural stem cells. Human Molecular Genetics.

Shepherd, D. (2016). Examining the effects of anti-Nogo-A immunotherapy on post-stroke neurogenesis in the adult rat. Loyola University Chicago. Retrieved from http://search.proquest.com/openview/f196ea3a12bdd002c1a443d44292c2a4/1?p….

Shobin, E., Bowley, M. P., Estrada, L. I., Heyworth, N. C., Orczykowski, M. E., Eldridge, S. A., . . . Rosene, D. L. (2017). Microglia activation and phagocytosis: relationship with aging and cognitive impairment in the rhesus monkey. GeroScience, 1-22. doi: 10.1007/s11357-017-9965-y.

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Exercise changes astrocytes and eases symptoms of neurodegenerative disorders

Astrocytes (GFAP) in the dentate gyrus of a mouse hippocampus. Image courtesy of Dr. Ahmad Salehi, Stanford University. 

It is well known that physical exercise eases the symptoms of neurodegenerative disorders like Alzheimer’s disease and helps to prevent their onset. Researchers at Stanford University are working on figuring out how it happens.

In their study, published in the journal Brain Structure and Function, scientists in Dr. Ahmad Salehi’s lab examined the effects of physical exercise on astrocytes in a region of the mouse brain that is critical for cognition – the dentate gyrus of the hippocampus. Previous studies have shown that an increase in the expression of brain-derived neurotrophic factor (Bdnf) occurs in this region after exercise (Philips, Salehi et al 2014). Bdnf is a protein that supports the survival of existing neurons and encourages new growth, playing an important role in cognitive function.

While the current study reconfirms that exercise generates increased levels of Bdnf (more than a fourfold increase in exercised mice versus non-exercised mice), it also describes several new findings including increased synaptic load in the dentate gyrus, alterations in the morphology of astrocytes, and changes in the orientation of astrocytic projections toward dentate granule cells.

The authors speculate that the changes they observed may be attributed to increased expression of a receptor called TrkB, which astrocytes express in response to increases in Bdnf levels. According to the paper, TrkB binds to Bdnf, activating the mechanisms behind neuronal development.

“Our study suggests that astrocytes actively respond and could indeed mediate the positive effects of physical exercise on the central nervous system and potentially counter degenerative processes during aging and neurodegenerative disorders,” (Fahimi, et al 2016).

The researchers used Neurolucida to determine the location, the extent, and orientation of astrocytic projections, finding a significant increase in the length of astrocytic projections in exercised mice.

“Neurolucida is one of the very few systems that combines complex morphometrical quantification with beautiful display of the results,” said Dr. Salehi, Clinical Professor, Department of Psychiatry and Behavioral Sciences at Stanford Medical School.

Since astrocytes help prevent excitotoxicity in the brain by removing excess glutamate from extracellular space, the researchers speculate that the increased length of astrocytic projections they observed in exercised mice could make this process more efficient.

Differences in the orientation of astrocytic projections were also reported, with the majority of projections of exercised mice directed toward the dentate granule cell layer – a region featuring increased levels of Bdnf release and synthesis after exercise.

The number of astrocytes in the molecular layer of the dentate gyrus in exercised and non-exercised mice was quantified with Stereo Investigator, however, there was no significant difference in astrocyte populations between the two groups.

“In summary, our study suggests that astrocytes constitute an important element in mediating the positive effects of physical exercise in the dentate gyrus of the hippocampus. Furthermore, it appears that physical exercise-induced release of Bdnf by the DG leads to a significant alteration in structure and function of astrocytes in protection against glutamate toxicity during aging and a number of neurodegenerative disorders,” (Fahimi et al 2016)

Fahimi, A., Baktir, M.A., Moghadam, S., Mojabi, F.S., Sumanth, K., McNerney, M.W., Ponnusamy, R., Salehi, A. Brain Struct Funct (2016). doi:10.1007/s00429-016-1308-8

Phillips, C., Baktir, M.A., Srivatsam, M., Salehi, A. Front. Cell. Neurosci., (2014) https://doi.org/10.3389/fncel.2014.00170

NeuroArt Image Contest Celebrates the Beauty of the Brain

MBF Bioscience is sponsoring a new image contest that encourages scientists and artists to share their views of the brain

Williston, VT— The NeuroArt image contest brings together scientists, artists, and neuroscience enthusiasts from around the world to share their view of the brain. Any image of the brain is accepted, including but not limited to microscope images, pencil drawings, and paintings.

Eligible contestants can visit neuroart.com/image-contest to submit images. Entries will be submitted through the NeuroArt website where people can vote on images. You can also vote for your favorite entry even if you don’t submit an image.

“The NeuroArt image contest is a way to recognize and foster an appreciation for the artistic aspect of neuroscience,” said Jack Glaser, President of MBF Bioscience. “The diverse entries make for an interesting collection of images celebrating the beauty of the brain.”

There is a new contest each month with two winners per month. Judging consists of two rounds of evaluation. Round one is peer-reviewed: the five images with the highest number of votes proceed to round two. In round two, a judging panel consisting of neuroscientists and artists choose the two winners. First place wins $250 towards the purchase of MBF Bioscience products and second place wins $100 towards the purchase of MBF Bioscience products. The judging panel will choose the annual grand prize winners from the monthly winners. The three Grand prize winners receive $3,000, $2,000 and $1,000 towards the purchase of MBF Bioscience products, respectively.

For more information, visit neuroart.com or watch this short video.

Researchers cited MBF systems in 14 papers between 1/20/2017 and 1/27/2017

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Doerr, J., Schwarz, M. K., Wiedermann, D., Leinhaas, A., Jakobs, A., Schloen, F., . . . Brüstle, O. (2017). Whole-brain 3D mapping of human neural transplant innervation. Nature Communications, 8, 14162. doi: 10.1038/ncomms14162

Fields, J. A., Metcalf, J., Overk, C., Adame, A., Spencer, B., Wrasidlo, W., . . . Masliah, E. (2017). The anticancer drug sunitinib promotes autophagyand protects from neurotoxicity in an HIV-1 Tat model of neurodegeneration. Journal of Neurovirology, 1-14. doi: 10.1007/s13365-016-0502-z.

Haidar, M., Guèvremont, G., Zhang, C., Bathgate, R. A. D., Timofeeva, E., Smith, C. M., & Gundlach, A. L. (2017). Relaxin-3 Inputs Target Hippocampal Interneurons and Deletion of Hilar Relaxin-3 Receptors in ‘Floxed-RXFP3′ Mice Impairs Spatial Memory. Hippocampus, n/a-n/a. doi: 10.1002/hipo.22709.

Kelly, S. C., He, B., Perez, S. E., Ginsberg, S. D., Mufson, E. J., & Counts, S. E. (2017). Locus coeruleus cellular and molecular pathology during the progression of Alzheimer’s disease. Acta Neuropathologica Communications, 5(1), 8. doi: 10.1186/s40478-017-0411-2.

Shen, X.-L., Song, N., Du, X.-X., Li, Y., Xie, J.-X., & Jiang, H. (2017). Nesfatin-1 protects dopaminergic neurons against MPP+/MPTP-induced neurotoxicity through the C-Raf–ERK1/2-dependent anti-apoptotic pathway. Scientific Reports, 7, 40961. doi: 10.1038/srep40961

Turner, R. C., Naser, Z. J., Lucke-Wold, B. P., Logsdon, A. F., Vangilder, R. L., Matsumoto, R. R., . . . Rosen, C. L. (2017). Single low-dose lipopolysaccharide preconditioning: neuroprotective against axonal injury and modulates glial cells. [Lipopolysaccharide preconditioning, oncostatin M receptor, diffuse axonal injury, gliosis, neuroprotection]. Neuroimmunology and Neuroinflammation, 4(1), 6-15.

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Researchers cited MBF systems in 25 papers between 1/13/2017 and 1/20/2017

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Allegra, M., Spalletti, C., Vignoli, B., Azzimondi, S., Busti, I., Billuart, P., . . . Caleo, M. (2017). Pharmacological rescue of adult hippocampal neurogenesis in a mouse model of X-linked intellectual disability. Neurobiology of Disease, 100, 75-86.

Anan, J., Hijioka, M., Kurauchi, Y., Hisatsune, A., Seki, T., & Katsuki, H. (2017). Cortical hemorrhage-associated neurological deficits and tissue damage in mice are ameliorated by therapeutic treatment with nicotine. Journal of Neuroscience Research, n/a-n/a. doi: 10.1002/jnr.24016.

Ardestani, P. M., Evans, A. K., Yi, B., Nguyen, T., Coutellier, L., & Shamloo, M. (2017). Modulation of neuroinflammation and pathology in the 5XFAD mouse model of Alzheimer’s disease using a biased and selective beta-1 adrenergic receptor partial agonist. Neuropharmacology.

Estrada, L. I., Robinson, A. A., Amaral, A. C., Giannaris, E. L., Heyworth, N. C., Mortazavi, F., . . . Rosene, D. L. (2017). Evaluation of Long-Term Cryostorage of Brain Tissue Sections for Quantitative Histochemistry. Journal of Histochemistry and Cytochemistry, 0022155416686934. doi: 10.1369/0022155416686934.

Klocke, C., Allen, J. L., Sobolewski, M., Mayer-Pröschel, M., Blum, J. L., Lauterstein, D., . . . Cory-Slechta, D. A. (2017). Neuropathological Consequences of Gestational Exposure to Concentrated Ambient Fine and Ultrafine Particles in the Mouse. Toxicological Sciences. doi: 10.1093/toxsci/kfx010.

Loris, Z. B., Pieper, A. A., & Dalton Dietrich, W. (2017). The neuroprotective compound P7C3-A20 promotes neurogenesis and improves cognitive function after ischemic stroke. Experimental Neurology, 290, 63-73. doi: http://dx.doi.org/10.1016/j.expneurol.2017.01.006.

Luo, L., Chen, J., Su, D., Chen, M., Luo, B., Pi, R., . . . Wang, R. (2017). L-F001, a Multifunction ROCK Inhibitor Prevents 6-OHDA Induced Cell Death Through Activating Akt/GSK-3beta and Nrf2/HO-1 Signaling Pathway in PC12 Cells and Attenuates MPTP-Induced Dopamine Neuron Toxicity in Mice. Neurochemical Research, 1-10. doi: 10.1007/s11064-016-2117-4.

Moore, B. A., Tyrrell, L. P., Pita, D., Bininda-Emonds, O. R. P., & Fernández-Juricic, E. (2017). Does retinal configuration make the head and eyes of foveate birds move? . Scientific Reports, 7, 38406. doi: 10.1038/srep38406

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Uncovering the role of microglia in fetal alcohol spectrum disorders


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


Researchers cited MBF systems in 21 papers between 12/16/2016 and 1/6/2017

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Campolo, M., Casili, G., Biundo, F., Crupi, R., Cordaro, M., Cuzzocrea, S., & esposito, e. (2016). The neuroprotective effect of dimethyl fumarate in a MPTP-mouse model of Parkinson’s disease: involvement of reactive oxygen species/nuclear factor-κB/nuclear transcription factor related to NF-E2. Antioxidants & redox signaling. doi: 10.1089/ars.2016.6800.

Charvet, C. J., Stimpson, C. D., Kim, Y. D., Raghanti, M. A., Lewandowski, A. H., Hof, P. R., . . . Sherwood, C. C. (2016). Gradients in cytoarchitectural landscapes of the isocortex: diprotodont marsupials in comparison to eutherian mammals. Journal of Comparative Neurology, n/a-n/a. doi: 10.1002/cne.24160.

Embury, C. M., Dyavarshetty, B., Lu, Y., Wiederin, J. L., Ciborowski, P., Gendelman, H. E., & Kiyota, T. (2016). Cathepsin B Improves ß-Amyloidosis and Learning and Memory in Models of Alzheimer’s Disease. Journal of Neuroimmune Pharmacology, 1-13. doi: 10.1007/s11481-016-9721-6.

Güleç, A., Bakkalbaşı, B. Ç., Cumbul, A., Uslu, Ü., Alev, B., & Yarat, A. (2017). Effects of local platelet-rich plasma injection on the rate of orthodontic tooth movement in a rat model: A histomorphometric study. American Journal of Orthodontics and Dentofacial Orthopedics, 151(1), 92-104. doi: http://dx.doi.org/10.1016/j.ajodo.2016.05.016.

Hoeijmakers, L., Ruigrok, S. R., Amelianchik, A., Ivan, D., Dam, A.-M. v., Lucassen, P. J., & Korosi, A. (2017). Early-life stress lastingly alters the neuroinflammatory response to amyloid pathology in an Alzheimer’s disease mouse model. Brain, Behavior, and Immunity. doi: http://dx.doi.org/10.1016/j.bbi.2016.12.023.

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