The authors obtained post-mortem human brain samples from the Brodmann area 28 (BA28) entorhinal cortex (EC) of subjects exhibiting a range of Alzheimer’s disease (AD) pathology and categorized into 3 groups based on cognition and AD pathology: normal cognition, noAD pathology; normal cognition with moderate to severe AD pathology, and definite AD cases. Synaptosome fractions were characterized biochemically, and proteomic profiles were determined using liquid chromatography coupled to mass spectrometry. Weighted gene co-expression network analysis was used to generate a protein co-expression network and identify protein modules (co-expressed proteins) that were present in the different cognition/AD pathology categories.

In parallel, tissue samples from the same brain area were fixed and processed for dendrite imaging using Golgi-Cox staining. Dendritic segments of pyramidal neurons from layers 2 and 3 of BA28 from each category of cognition/AD pathology were imaged with a 60X/1.40 NA oil-immersion objective using a brightfield microscope. The resulting 3D image stacks were opened in Neurolucida 360 and dendrite and dendritic spine morphologies were reconstructed using semi-automatic and automatic functions. Spines were automatically classified as stubby, mushroom, or filopodia. Volumetric measurements of the spine, as well as the density of each spine type per dendrite length were extracted with Neurolucida Explorer.

Figure: Overview of workflow. Synaptosomes were isolated from postmortem human BA28 entorhinal cortex (EC) and subjected to liquid chromatography tandem mass spectrometry-based proteomics. Weighted Gene Co-Expression Network Analysis (WGCNA) was used to generate a network of protein co-expression modules. BA28 EC samples were also Golgi stained and z-stacks of dendritic segments were imaged and digitally reconstructed to obtain measurements of dendritic spine density and morphology. Module eigenprotein values were correlated with dendritic spine metrics. The hub protein of a module significantly correlated with a dendritic spine metric would be selected for functional validation by CRISPR activation in rat primary hippocampal neurons.

The authors then correlated dendritic spine measurements with module eigenprotein expression from the proteomic analysis to integrate the two data categories. Among the results, one particular protein module stood out; it was consistently present in both AD and non-AD tissue, and was positively correlated with thin dendritic spine length, especially thin spines. Twinfilin2, the hub protein within this module, has a well-established role in modulating the cytoskeleton, specifically the protein actin. When the authors looked at neurons from rats grown in culture with different amounts of TWF2, they found those with more TWF2 grew longer thin-spines. This was the only type of spine affected by TWF2, demonstrating what the authors call a remarkable specificity regarding the ability of their cross-platform analysis to identify the functions of proteins.

Looking ahead, the researchers have identified many proteins organized in modules with hub-proteins, some that are expressed equally in AD and non-AD cases, and some that are not. They are in a good position to determine which of these hub proteins merit further study using functional analyses.

The comprehensive workflow employed by the researchers opens up new possibilities for unraveling the mysteries of neuronal function and holds immense potential for advancing our knowledge of diverse neurological conditions. As we delve deeper into the complex world of neuroscience, the connection between technology and scientific inquiry continues to illuminate the path towards groundbreaking discoveries.

Reference:

Walker, C. K., Greathouse, K. M., Tuscher, J. J., Dammer, E. B., Weber, A. J., Liu, E., Curtis, K. A., Boros, B. D., Freeman, C. D., Seo, J. V., Ramdas, R., Hurst, C., Duong, D. M., Gearing, M., Murchison, C. F., Day, J. J., Seyfried, N. T., & Herskowitz, J. H. (2023). Cross-platform synaptic network analysis of human entorhinal cortex identifies TWF2 as a modulator of dendritic spine length. The Journal of Neuroscience. https://doi.org/10.1523/jneurosci.2102-22.2023

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Researchers used Neurolucida to reconstruct a newly discovered type of neuron found only in the human brain, according to a study published in the journal Nature Neuroscience. Known as “rosehip” neurons because of the way they resemble a rose after its petals have fallen off, these cells feature compact, bushy axonal arborizations.

Found in the first layer of the cerebral cortex, a highly complex brain region that is thought to play an important role in consciousness, “rosehip neurons” have not been seen in mice or other laboratory animals, and scientists suggest that they may exist only in humans. Classified as inhibitory neurons, these brain cells form synapses with pyramidal neurons in layer 3 of the cerebral cortex, according to the study.

Led by Dr. Ed Lein, of the Allen Institute for Brain Science, and Dr. Gábor Tamás, a neuroscientist at the University of Szeged in Szeged, Hungary, the research team used Neurolucida to reconstruct rosehip neurons in 3D. Their reconstructions revealed that these cells display morphological characteristics that differ significantly from other types of cells found in this region of the brain.

Scientists used Neurolucida and Neurolucida Explorer to reconstruct and analyze a rosehip neuron. Image Credit: Tamas Lab, University of Szeged

Using Neurolucida Explorer to quantitatively analyze their cell reconstructions, the researchers observed similar numbers of primary dendrites in both rosehip neurons and basket cells, but fewer compared to neurogliaform cells. Meanwhile, they calculated similar total dendritic length and frequency of dendritic nodes in rosehip neurons and neurogliaform cells, but recorded differences in basket cells.

Also, their analysis revealed that the axonal branching of rosehip neurons was more robust than any other type of cell observed in this brain region, with the volume of axonal terminations, or boutons, measuring four times larger than NGFC boutons.

Furthermore, the researchers say that the rosehip neuron has a molecular marker signature of (GAD1+CCK+, CNR1–SST–CALB2–PVALB–), a signature not seen in the mouse cortex.

According to the paper, the researchers still have much to learn about the function of rosehip neurons in the human brain. Because they observed rosehip neurons connecting to their partner neurons – pyramidal neurons, in very specific places, they hypothesize that rosehip neurons might be controlling the flow of information in a distinctive way.

One next step will be to see if postmortem brains from patients with neuropsychiatric disorders display rosehip neurons with alterations, to begin investigating whether or not these newly discovered cells play a role in mental illness.

Boldog E, Bakken TE, Hodge RD, Novotny M, Aevermann BD, Baka J, Bordé S, Jennie L. Close, Diez-Fuertes F, Ding SL, Faragó N, Kocsis AK, Kovács B, Maltzer Z, McCorrison JM, Miller JA, Molnár G, Oláh G, Ozsvár A, Rózsa M, Shehata SI, Smith KA, Sunkin SM, Tran DN, Venepally P, Wall A, Puskás LG, Barzó P, Steemers FJ, Schork NJ, Scheuermann RH, Lasken RS, Lein ES, Tamás G (2018) Transcriptomic and morphophysiological evidence for a specialized human cortical GABAergic cell type. Nature Neuroscience doi.org/10.1038/s41593-018-0205-2

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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.”

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.

To test their hypothesis that adolescence is a sensitive period for brain development and that social stimuli influence changes that show up in adulthood, the researchers studied 40-day-old and 80-day-old hamsters, a period equivalent to approximately 13 and 16.5 human years, respectively.

Using Neurolucida, they traced six pyramidal neurons randomly selected from layer three of the prefrontal cortex of each animal, and then used Neurolucida Explorer to analyze each cell’s dendritic length and branching. They found that in adulthood, the hamsters which had mated during adolescence (40 days) had neurons with shorter dendrites and a lower number of dendritic intersections compared to hamsters that had not mated at all, and hamsters that mated after puberty (at 80 days). These hamsters also showed signs of depression in behavioral tests.

“These data indicate that sexual experience during key developmental epochs produces long lasting adjustments in neuronal morphology and behavioral phenotype,” said Dr. Weil.

Morris, J. S., Weil, Z. M., & Nelson, R. J. (2013). Sexual Experience and Testosterone During Adolescence Alters Adult Neuronal Morphology and Behavior. Hormones and Behavior(0). doi:http://dx.doi.org/10.1016/j.yhbeh.2013.08.001

Image of Golgi stained human neocortical pyramidal neuron licensed under the Creative Commons Attribution-Share Alike 2.5 Generic license, via wikimedia.

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Recent research focuses on a key element of this circuit – the nucleus accumbens (NAc), part of the brain region known as the ventral striatum, which is thought to regulate motivation and reward processing. In a new study of stress-induced depression in rats, researchers at the University of Minho in Braga, Portugal saw morphological changes in the dendrites of medium spiny neurons in the NAc, alongside disturbances in gene expression in this region. They also saw these changes reversed after administering antidepressants.

By using Neurolucida Explorer to analyze 3D reconstructions of medium spiny neurons generated with Neurolucida, the researchers observed longer than normal dendrites and greater spine density in the depressed rats. According to the paper, these findings contrast with studies of the hippocampus and prefrontal cortex, where chronic stress leads to shorter dendrites.

Nucleus Accumbens

The authors say the morphological changes are related to changes in gene expression, including increased brain-derived neurotrophic factor (BDNF) expression and other genes related to neuroplasticity.

“These observations add to the evidence that neuroplastic changes in the NAc contribute to the pathophysiology of depression and its pharmacologically-induced recovery, and point to the role of the NAc in the regulation of (an)hedonia [diminished interest or pleasure],” the authors say in their paper published last month in Translational Psychiatry.

To see how stress-induced depression affected cell volume in the NAc, the research team conducted a stereological analysis of the region. But after quantifying neurons with Stereo Investigator, they did not see significant differences in the number of neurons in depressed rats versus controls.

“Given that this region is remarkably interconnected with the hippocampus and the prefrontal cortex, it remains to be demonstrated whether the changes herein described are causal or a mere consequence of the neurodegenerative effects triggered by stress in these ‘cortical’ regions,” the authors say.

Bessa, J., Morais, M., Marques, F., Pinto, L., Palha, J., Almeida, O., & Sousa, N. (2013). Stress-induced anhedonia is associated with hypertrophy of medium spiny neurons of the nucleus accumbens. Translational Psychiatry, 3(6), e266. doi:10.1038/tp.2013.39

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A monkey spots a mango and part of its brain lights up. The action takes place in the inferior temporal cortex, part of the brain that’s essential to object recognition. Using retrograde tracing and anatomical imaging, scientists at the National Institute of Neuroscience, and the RIKEN Brain Science Institute in Japan identified two interwoven, yet distinct, systems within the region’s complex circuitry.

“Our anatomical findings provide evidence for a recurrent network of at least two parallel systems,” the authors say in their paper published last December in Scientific Reports.

One system may send information about an object’s visual characteristics rapidly from one part of the inferotemporal cortex to the other, while the second system might work on a more local level, possibly helping to “compute multipart shape configurations,” the authors hypothesize.

To visualize the patching and circuitry of the cortical cells, the researchers developed a two-step method with retrograde tracing and fluorescent in vivo surface connection imaging. They first injected red fluorescent tracer into the anterior inferotemporal cortex (TE) of rhesus macaques. This revealed patches of feedforward neurons that projected from the posterior inferotemporal cortex (TEO), and provided a guide for a second tracer injection in green. Once stained, the circuits offered a clearer visual so that the researchers could examine the spatial relationship between the two systems more efficiently.

What they saw was a pair of “parallel, spatially intermingled circuits,” with one circuit projecting from the TEO to the TE, and a second circuit projecting “widely in the intrinsic network,” but not venturing to the other side of the inferior temporal cortex. “These parallel systems might be specialized for, respectively, fast vs. highly processed signals,” they explain.

The researchers quantified and plotted neurons with Neurolucida, and calculated distances between patches with Neurolucida Explorer, according to the paper.

Ichinohe, N., Borra, E., Rockland, K., (2012). Distinct Feedforward and Intrinsic Neurons in Posterior Inferotemporal Cortex Revealed by in Vivo Connection Imaging, Sci. Rep., 2(934). doi:10.1038/srep00934

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Scientific research shows that women are twice as likely as men to develop stress disorders. Why are women more sensitive than men to stress? A recent research study presents new evidence that estrogen could play a role.

The symptoms of disorders like major depressive disorder and post traumatic stress disorder lead neuroscientists to speculate that a dysfunction occurs in the way the medial prefrontal cortex connects to the amygdala–regions of the brain associated with the regulation of memory and behavior. Following research published in 2009 determining resilience against changes in dendritic morphology in this region in male rats, scientists at the Mount Sinai School of Medicine turned their focus to female rats. They discovered unexpected changes in dendritic length and spine density to the neurons in this region when both estrogen and stress are present.

After removing the ovaries from all subjects and implanting half of the rats with estrogen, the researchers exposed them to ten days of either immobilization stress (two hours in a rodent immobilization bag) or home cage rest. They then sectioned the rats’ brains and examined the neurons in question.

“We used Neurolucida and Neurolucida Explorer to measure dendritic length and branch point number in a set of pyramidal neurons that had been filled with the fluorescent dye Lucifer Yellow,” said lead author Dr. Rebecca Shansky. “The software was very user-friendly, and we were easily able to customize the settings to get just the analyses we wanted,” Dr. Shansky added.

What they found was increased dendritic arborization and spine density in the females treated with estrogen, “indicating that estrogen and stress can interact at the level of this circuit to produce a unique response to stress in females,” according to the paper “Estrogen Promotes Stress Sensitivity in a Prefrontal Cortex–Amygdala Pathway,” published earlier this year in Cerebral Cortex.

Read the free abstract, or download the full paper at Cerebral Cortex.

Rebecca M. Shansky, Carine Hamo, Patrick R. Hof, Wendy Lou, Bruce S. McEwen, and John H. Morrison, “Estrogen Promotes Stress Sensitivity in a Prefrontal Cortex–Amygdala Pathway” (Cereb Cortex 2010; 20: 2560-2567)

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When your mouth is dry, your joints are stiff, or your heart is inflamed because your immune system is attacking your own body, chances are you’re suffering from a little stress. A recent study shows that there may be physiological reasons why patients with autoimmune diseases experience increased levels of anxiety.

Scientists at the City University of New York Medical School, Columbia University, and the University of Messina suggest it may be brain inflammation that leads to elevated stress in patients with autoimmune diseases like systemic lupus erythematosus, rheumatoid arthritis, and Sjögren’s syndrome.

After modeling these diseases in a population of mice by introducing cytokine B-cell activating factor (BAFF), the research group examined their emotional behavior. They also checked for brain inflammation, stress-induced c-Fos protein, and the proliferation of progenitor cells in the hippocampus, using Neurolucida Explorer.

They found that the older mice produced anxiety-like characteristics associated with brain inflammation. These anxious mice responded to mild stress-inducing stimuli by displaying abnormal activity within the limbic system — the region of the brain that controls basic emotions.

During the course of the study, Neurolucida Explorer was used to calculate dendritic length. “I was very pleased with Neurolucida Explorer,” said Dr. Fortunato Battaglia. “I find the software very friendly and the quantitative data were crucial for our work. I am looking forward to using it again in future experiments.”

Read the free abstract or download the complete paper “Reduced Adult Neurogenesis and Altered Emotional Behaviors in Autoimmune-Prone B-Cell Activating Factor Transgenic Mice” at Biological Psychiatry.

Rosalia Crupi, Marco Cambiaghi, Linda Spatz, Rene Hen, Mitchell Thorn, Eitan Friedman, Giuseppe Vita, Fortunato Battaglia, “Reduced Adult Neurogenesis and Altered Emotional Behaviors in Autoimmune-Prone B-Cell Activating Factor Transgenic Mice” (Biological Psychiatry (2010) 67 6, 558-566)

{Illustration of a human brain and skull licensed under the Creative Commons Attribution 2.5 Generic license}

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Led by Dr. Jonathan Vinet of the Université Laval in Quebec, scientists have identified three different types of oligodendrocytes in the mouse hippocampus: “ramified,” “stellar,” and “smooth.”

Each type displayed varying morphological characteristics, mainly in shape, volume, and branching behavior, which led the researchers to believe that the three types represent different stages of maturation.

As described in the paper, “Subclasses of oligodendrocytes populate the mouse hippocampus,” published in the European Journal of Neuroscience, the “smooth,” or most simple type possibly morphs into the “stellar,” which eventually develops into the most complex of the three, the “ramified” oligodendrocyte.

The identification of these morphologically distinct oligodendrocyte populations in the hippocampus may help researchers determine which specific types of oligodendrocytes are affected in diseases such as schizophrenia and multiple sclerosis.

Using a Neurolucida system with an Olympus AX-50 microscope, the scientists formed 3D reconstructions of the hippocampal oligodendrocytes integral to their study. They then analyzed their tracings with Neurolucida Explorer.

“Without Neurolucida we couldn’t have carried out this study,” said Dr. Attila Sik, “it was an essential component. Nice piece of equipment, for sure.”

Read the free abstract, or access the full article (by subscription), at the European Journal of Neuroscience.

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