Uncovering the role of microglia in fetal alcohol spectrum disorders

microglia_alcohol

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

 

Scientists Observe Differences Between Brains of Stressed and Unstressed Rats After Fear Conditioning

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.

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

Researchers cited MBF systems in 24 papers between 11/2/2015 and 11/13/2015

Stereo Investigator:journal images sm

Bains, M., & Roberts, J. L. (2015). Estrogen protects against dopamine neuron toxicity in primary mesencephalic cultures through an indirect P13K/Akt mediated astrocyte pathway. Neuroscience Letters. doi:http://dx.doi.org/10.1016/j.neulet.2015.10.054.

Chen, M.-h., Liu, Y.-h., Xu, H., Xu, D.-w., Wang, C.-n., Wang, Y., . . . Wang, Y.-h. (2015). Lentiviral Vector-Mediated p27kip1 Expression Facilitates Recovery After Spinal Cord Injury. Molecular Neurobiology, 1-14. doi: 10.1007/s12035-015-9498-2.

Correa, M., Pardo, M., Bayarri, P., López-Cruz, L., San Miguel, N., Valverde, O., . . . Salamone, J. (2015). Choosing voluntary exercise over sucrose consumption depends upon dopamine transmission: effects of haloperidol in wild type and adenosine A2AKO mice. Psychopharmacology, 1-12. doi: 10.1007/s00213-015-4127-3.

Fragale, J. E. C., Khariv, V., Gregor, D. M., Smith, I. M., Jiao, X., Elkabes, S., . . . Beck, K. D. (2016). Dysfunction in amygdala–prefrontal plasticity and extinction-resistant avoidance: A model for anxiety disorder vulnerability. Experimental Neurology, 275, Part 3, 59-68. doi:http://dx.doi.org/10.1016/j.expneurol.2015.11.002.

Goodus, M. T., Kerr, N. A., Talwar, R., Buziashvili, D., Fragale, J. E. C., Pang, K., & Levison, S. W. (2015). LIF Haplodeficiency Desynchronizes Glial Reactivity and Exacerbates Damage and Functional Deficits After a Concussive Brain Injury. Journal of Neurotrauma. doi: 10.1089/neu.2015.4234.

Huang, Q., Du, X., He, X., Yu, Q., Hu, K., Breitwieser, W., . . . Li, M. (2015). JNK-mediated activation of ATF2 contributes to dopaminergic neurodegeneration in the MPTP mouse model of Parkinson’s disease. Experimental Neurology. doi: http://dx.doi.org/10.1016/j.expneurol.2015.10.010.

Jayasinghe, V. R., Flores-Barrera, E., West, A. R., & Tseng, K. Y. (2015). Frequency-Dependent Corticostriatal Disinhibition Resulting from Chronic Dopamine Depletion: Role of Local Striatal cGMP and GABA-AR Signaling. Cerebral Cortex. doi: 10.1093/cercor/bhv241.

Kohl, Z., Abdallah, N. B., Vogelgsang, J., Tischer, L., Deusser, J., Amato, D., . . . Winkler, J. (2015). Severely impaired hippocampal neurogenesis associates with an early serotonergic deficit in a BAC α-synuclein transgenic rat model of Parkinson’s disease. Neurobiology of Disease. doi:http://dx.doi.org/10.1016/j.nbd.2015.10.021.

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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

 

Dendritic Spine Loss Reported in Schizophrenia and Bipolar Disorder

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

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.

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Two 2014 Nobel Prize Laureates Used Neurolucida in their Groundbreaking Research

May-Britt and Edvard Moser

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.

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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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Scientists Use Neurolucida to Create 3D Reconstructions of Placental Villous Trees

(a,b) Comparison of the microscopic aspects of a thin (4–6 μm) histological section of a human placenta after staining with hematoxylin/eosin (a) with the microscopic aspects of a whole-mount isolated villous tree after staining with hematoxylin (b). The scale bars in a and b are 250 μm. (a) Various cross- and longitudinal sections of villi can be recognized. The stromal architecture inside the sectioned villi is visible. The cross-sections of branches belong to an unknown number of villous trees. (b) A single villous tree is visible, and branches are not sectioned. The hierarchical positions of nodes (branching points) and the branching topology can be recognized.

(a,b) Comparison of the microscopic aspects of a thin (4–6 μm) histological section of a human placenta after staining with hematoxylin/eosin.

When neuroscientists started studying neurons in 3D, it revolutionized brain science. Now, for the first time, scientists are using this same technology to study the human placenta, and they’ve made some fascinating new discoveries about its structure.

Using Neurolucida to create 3D reconstructions of villous trees – three-dimensional structures in the placenta that facilitate gas and nutrient exchange between the fetus and mother – researchers in Munich, Germany uncovered a wealth of information about their architecture.

For the first time, they analyzed the complexity of villous tree branches and branching, determined the number and location of nodes (branching points), and measured branch angles, discovering a surprising correlation between the branching angle of terminal branchesand the fetoplacental weight ratio (BW/PW) – a calculation commonly used to measure fetal health in prenatal medicine.

“The results show that 3D analysis with Neurolucida reaches beyond the horizons of 2D histology, the current gold standard in placenta morphology/pathology,” said Dr. Hans-Georg Frank, an author of the study. Continue reading “Scientists Use Neurolucida to Create 3D Reconstructions of Placental Villous Trees” »

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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