Science topic

Neuroanatomy - Science topic

Study of the anatomy of the nervous system as a specialty or discipline.
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Hello, I would like to ask from everyone's perspective what is the biological relevance and impact if the neurons that are being affected by an exogenous stimulus is (1) peptidergic or non-peptidergic neuron, (2) and their respective class of nerve fibers?
Currently, I am still consolidating and distinguishing these concepts because I think these are important research questions in molecular and cellular neuroscience projects.
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If these are people, then a clinical response to the administration of naloxone is likely. If the experiment... is a microelectrode neuronal response also using blockade of opiate receptors.
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After decades of teaching Neuroanatomy for Behavioral Scientists and using many other sources, I have concluded that the functions of the central nervous system (CNS) can be reduced to thee types of processing: sensory, effector or motor, and memory. Agree or disagree? if disagree, please explain.
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Sorry. Here are files I failed to attach first time.
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Dear all,
I am organizing online activities with M2 students to explore neuroanatomy using Allen Brain Atlas. I have already some ideas and prepared some activities around the expression of a protein implicated in Parkinson's disease but I would like to know if you have already used this Atlas for teaching and how.
Many thanks,
Patrick Pla
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This is what we have studied as regards PD:
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I would like to scan calcium-indicator-dye-loaded neurons after fixation, just to know, which cells have been loaded. Fura and Carbodiimide fixation have been reported to retain fluorescence but I prefer paraformaldehyde fixation for several reasons. Is there a single calcium dye, which is not quenched after paraformaldehyde fixation?
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May I ask if you continue your experiment and find a good agent? I am also thinking about fixing my cells on a coverglass right after treating with Fura-2 AM and I am wondering if fixation processes by 4% PFA may effect the membrane permeability and cause Ca2+ fluorescence to be lost.
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I am trying to discern whether, in the brain (structure & function) of people with bipolar disorder/MDD/schizophrenia who experienced childhood trauma, there are:
1. already differences in children's brain structure & function that trauma further modifies and the person develops a mental illness OR (genetics first then trauma)
2. trauma changes the structure and function of the brain that the child's genetics further modifies and the person develops a mental illness (trauma first then genetics)
Has anyone researched this & if so, can you please share your findings or references you know of?
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Thank you for your insightful comment. I have read Cassiers et al (2018) as well as 3 other insightful articles: 1) Nemeroff CB.(2016). Paradise Lost: The Neurobiological and Clinical Consequences of Child Abuse and Neglect. In Neuron; 2) Aas M et al. (2019) Childhood maltreatment and polygenic risk in bipolar disorders. In Bipolar Disorders; and 3) Stevelink et al. (2019) Childhood abuse and white matter integrity in bipolar disorder patients and healthy controls. In European Neuropsychopharmacology.
  1. In Cassiers et al (2018), their review of the literature supports the hypothesis that the brain changes are a protective adaptation in response to abuse, they did not discuss the genetic components that may be predisposing to or responsive to abuse so it doesn't directly answer the question.
  2. In Nemeroff (2016), he suggests (based on my assessment) some brain changes are due to the moderating effects of genetics (so genetics first) (eg, carriers of 1 or 2 copies of the "short" allele of the serotonin transporter promoter polymorphism have greater rates of depression vs those with the "long" allele homozygotes with equal childhood trauma) and other changes are in response to the trauma and predispose the person to the mental illness (eg, reduced hippocampal volume in depressed women with a history of childhood maltreatment but not in equally depressed women without maltreatment).
  3. Aas M et al (2019) reported that polygenic risk score (PRS) and CTQ were inversely correlated so those with lower PRS reported more severe abuse and vice versa, so the based on this (my interpretation) is that in a brain/body with more "imbalances" due to genetics (brain structure/function and physiology that is predisposed to BD), abuse does not need to be severe to make the changes sufficient to cause BD wherein people with low PRS have brain structures and physiology is more "balanced", greater severity of abuse is needed to sufficiently change the brain structures and physiology to cause BD. They did not also look at brain or physiology so we can only infer based on genetics.
  4. In Stevelink et al (2019), their study findings suggest "that childhood abuse results in poor white matter integrity in a subset of people who are then possibly more vulnerable to development of psychiatric disorders, including bipolar disorder" and "that childhood abuse, in particular, is associated with FA, possibly due to its effects on HPA-axis activity." But as pointed out in the limitations, their cross-sectional study design does not allow for establishing causation "Based on our results, we cannot differentiate whether decreased integrity of white matter in patients is caused by childhood abuse, or whether this decreased integrity was already present prior to experiencing childhood abuse."
I am interested in causation and the interplay of genetics and childhood trauma as I am trying to write a book on childhood trauma and how the different types of trauma at different times explains the behaviors of survivors, the mental health challenges caused by childhood trauma and toxic stress, and how people can "overcome" (I hate this word but can't think of another) trauma. My story of childhood trauma, developing several mental illnesses including bipolar disorder, and how I was able to get a PhD and have a successful career while growing and healing is being used as the backdrop to tell the scientific story to a lay audience.
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I am searching for a detailed description of the blood vessel in the rat brain, and it would be very helpful if someone could indicate a good reference (atlas, book, papers).
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Dear Fabiola,
I was in the need of the same thing. I had found this atlas for free. See if suits you.
Best regards,
Vicente
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Hi!
I am looking at cortical neurons in coronal sections from mice expressing Thy-YFP and a top secret genetic manipulation :)
I noticed these vacuoles in the apical dendrites of most of the neurons, and was wondering if anyone with an experienced eye can tell me if these look like some sort of artifact? The brains were perfused, fixed, sunk in sucrose, frozen, and sectioned on a microtome. Ice crystals? Freeze thaw damage?
I don't have a wildtype to compare it to right this second due to technical difficulties, but am impatient and thought I'd ask all of you first, ESPECIALLY because of what I found out when I googled apical dendrite vacuoles. Thanks!
Sara
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using DAPI to do counter staining
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Background:
Professional-school student with extensive ACE (child adverse events) history along with severe depression and anxiety diagnosed over previous year, presented with recent severe ADHD (I-Type) diagnosis at age 26.
Documentation confirmed maximum dose step therapy for various Amphetamine-based stimulants was completed but still not found to be fully affective.
Unexpectedly, they are currently prescribed daily 50mg Mydayis (Mixed salts of single-entity amphetamine product) along with 80mg Prozac, and consumming 300-400mg of caffeine.
Due to initial medication-only use producing very minimal stabilizing effects, but found to increase at re-introduction of SSRI and further increase with Caffeine reintroduction.
No adverse effects (cardiac, neuromuscular, neurocognitive) have been reported/measured in 4 months of aforementioned therapeutic combination.
NOTE: Adverse reaction to methylphenidate-based medications were identified early on.
Assessment of (remaining) presenting symptoms seems to overlap with tentatively defined SCT Criteria.
NOTE: Student has never been prescribed Strattera (only presently confirmed SCT-symptom relief medication)
Specific question:
Recent research has shown SCT + ADHD to correlate with much greater impairment in adults, do you think a combination of severe ADHD + SCT may result in required use of excess pharmacotherapy dosages that surpass established safe therapeutic/combination parameters?
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Remember both PTSD and major depression can produce significant cognitive impairment including issues with attention equal to ADHD.
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In my research I have analysed structural volumes in MRI scans by manual tracing measures and correlated against automated measures, with particular interest in subcortical grey matter structures. As I am still a student it is interesting to gauge the overall need for the skill in the international community, either as a diagnostic tool or as an inter-rater reliability measure.
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Excellent opinions ...I think all in respect to the general goal behind the question...
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I am interested in scaling my coordinates for stereotaxic injections by measuring the distance from bregma to lamda in each of my mice and then scaling this to the "typical" value used in mouse brain atlases. This scaling factor should reduce some error that results from differences in mouse brain sizes. I would be most interested in knowing the bregma-lamda distance in the "average" or "typical" mouse used to map brain coordinates in the Paxinos Mouse Brain Atlas. Does anyone know this value? I see other people have used this method successfully in rats, where the Bregma-Lamda distance is 9mm.
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Is that information not in the mouse brain atlas? I know the scaling works reasonably in rats. However, if you are trying to hit a very small deep structure I would take a different approach. (1) Make a best estimate of the coordinates based on the atlas. (2) Take animals of the same age within a very narrow weight range. (3) Do a few injections with ink or just make a track with a needle to mark where you end up. (4) Perfuse and analyze the brains. If you are consistently too superficial, lateral or anterior, adjust your coordinates and repeat the cycle untill you get the correct ones.
Finally, mouse brains are so small in comparison to the inherent errors in our hands, eyes, stereotactic equipment etc. that it is to be expected to miss a small structure more often than you would in rats. Just doing more injections and keeping the ones that worked is the solution. This only becomes a problem if the injection is just a small step in a long line of experiments, e.g. extended behavioral training, electrophysiological recordings etc., as the investment in animals that you will not be able to use once you post-mortem check if you injection was successful.
good luck
Nils
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I am performing stereotaxic injection in mice. In the Paxinos Mouse Brain Atlas it appears that the depth is relative to the brain surface at bregma (z=0). The hole I drill in the skull is tiny, so I cannot see the surface of the brain. I want to account for the distance between the skull surface and then add this value to my depth. This would include skull thickness, dura, and subarachnoid space, etc. Does anyone know what a typical value is? How much does it vary between mice and at different locations within mice? I'm injecting roughly 5mm rostral to bregma and 1mm lateral from the midline.
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If you use the mouse brain atlas ( Paxinos, George, and Keith B.J. Franklin. The mouse brain in stereotaxic coordinates: hard cover edition. Access Online via Elsevier, 2001 ) to get your coordinates the 0 value is the skull level at bregma, not brain surface as you state in your question. So you actually do not need to know what the distance is between skull level and brain surface is.
However, judging from my experience and the brain atlas I would say it is approximately 700 um. Take a look at this image from the brain atlas
look at the coronal section at 0.02mm from bregma. As you can see the 0 point in the dorsal ventral axis is above the brain surface.
good luck
Nils
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Now I'm planning about a clinical research about brainstem infarct. And I want an useful atlas of the brainstem.
I know of the two following atlas':
1. Stereotaxic atlas of the human brainstem and cerebellar nuclei : a variability study
F. Afshar , E. S. Watkins , J. C. Yap -- Raven Press, c1978.
2. Atlas of the human brainstem
George Paxinos, Xu-Feng Huang -- Academic Press, c1995.
I think textbook No.2 is better than No.1, but there is a small description about basis pontis.
I'd like to have a more detailed atlas. Does anybody have any information about a new brainstem atlas?
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I found this MNI atlas that some may find helpful. I haven't tried it out yet, though. https://www.martinos.org/resources/aan-atlas
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I'm stuck on some basic mouse anatomy for a project I'm doing looking at descending motor pathways that are involved in forelimb function. I'm trying to figure out if the medullary reticular formation in the mouse brain contains the lateral reticular nucleus. I'm also trying to tease apart which areas contain the gigantocellular, parvoceullar, and magnocellular areas. 
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Terminations of reticulospinal fibers originating from the gigantocellular reticular formation in the mouse spinal cord.
I think, this is a good reference for your question.
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as we know the specific frequency of sound stimulate the specific part of cochlea of ear that was named cilia(hair cells). subsequently ,the cilia convert the amplitude of the sound to corresponding frequency. nevertheless, is the brain sense the rhythm of the sound?
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May not be relevant but there have been many studies of 'sonic driving' during trance experience and other situations. Personally, I was trained in facilitating shamanic trance postures using rattle by Felicitas Goodman, Spirits Ride the Wind, etc. In my search at least in English Neher 1961 was a pioneer on this topic. Goodman also mentions in her books EEG studies of practitioners of shamanic trance. Somewhere I read a study recently that sonic driving is an independent variable for trance; not necessary.
Best regards,
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I need to find neutral cue words for my research on autobiographical memory and future thinking. I would really appreciate any information to have access to the word lists mentioned in the question. Thanks.
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Hi Meymune.
I suggest you contact the authors directly. E-mail the corresponding author in each of the articles. If not available in the article, a google search will give you their e-mail addresses. Good luck.
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Neural circuitry underlying nocifensive response depends on the type of reflex. But, when we talk about withdrawal response, what are the neural elements involved?
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It is obvious that there has to be reflex arc between nociceptive input and the spinal motor neurons, in other words, as you mentioned between fibers of lamina I and the motor neurons. Maybe we, to whom you have posed the question, do not know the interneurons involved. However, since you have mentioned "neuroanatomy" as your area of specialty or interest, you can enlighten us with the answer to your question which has gone unanswered for 4 years. thanks.
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Can anyone suggest me some articles about the neural basis of Theory of Mind?
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Books and articles by Antonio Damasio.
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I do stereotaxic injections into mPFC of mice brains. I've been trying to figure out a way to overlay a mouse brain atlas outline onto images of brain slices in order to determine the accuracy and precision of injections and if our expression pattern is within our target area. 
I think there's a way to use ImageJ to do it, but I haven't had any luck as of yet.
I was wondering what programs and methods others have used and if they could provide either a brief walk through or provide a link to a paper or source that describes the process. 
The image I've attached is a great example of what my end result should look like. I just don't know how to get there.
Thanks in advance.
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While, it is possible to create this type of image in ImageJ through threshold masking and overlays or possibly even stacks, I personally find that using ImageJ for line drawing overlays is too complicated, in terms of the number of steps, though I wouldn't be surprised if plug-ins or macros exist to more easily facilitate the process.
The way I would overlay anatomical line drawings onto neural tissue micrographs is by using layers and color transparency in the GNU Image Manipulation Program (GIMP). This is a powerful, free, and open source image editing program you can download at www.gimp.org.
I wrote out simplest form of the procedure:
1. Open your black and white line drawing and scale the dimensions to approximately match those of your histological micrograph. Use the menu command 'Image --> Scale Image.'
2. In the menu select 'Colors --> Color to Alpha' and choose white as the color. Your line drawing should now be a black outline with a checkerboard background pattern.
3. Copy your line drawing and paste it as a new layer onto the histological section image.
4. Using the 'Scale' tool, click the image and then click and drag the middle circle in the grid to move the line drawing overlay into position at the edge of the brain or corner of some identifiable landmark or region, for example caudate putamen in the bottom-most section of the image you uploaded.
5. Pulling on the corners of the scaling grid, adjust the size of the line drawing to match the anatomy of your histological section image. Keep track of the percentage you are scaling your overlay since you can probably reuse the relative adjustment from one coronal plane to others. For example, if you scale the atlas overlay 85% in the bottom-most image, start with an 85% scaled line-drawing overlay for the other two images when you need to demarcate the PFC.
6. You can export a flattened version of the overlay in a variety of formats.
Hope that helps. Tell us if it works! 
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I study adult neurogenesis in the dentate gyrus of the hippocampus and performed a double staining (4Month mouse brain, 40µm, free floating) for BrdU, and NeuN using flourescence ( Alexa Fluor 488, 555) conjugated secondaries.
I refer to one paper for quantification but problem is the total number is significantly different.(about 1/10)
In the paper, 10-12 sections (every 5th section of slides from each animal)
including the DG area were selected. The entire dentate gyri were scanned using a confocal microscope. Z series stacks of confocal images were obtained. The number of double stained cells were counted using the Image Pro Plus software.
I also used 10 sections and got Z stack images and counting a double positive cells using ImageJ.  
Is there any things I miss?
I found another BrdU quantification method relative to the stereology. However, It is very complicated to me.   
How to I easily count the cells that is representative of the entire DG. I've never done this type of quantification before.
Please let me know, if you have useful suggestions/links/protocols to learn quantification.
Thanks a lot! 
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Several factors may contribute to the different number of BrdU+/NeuN+ cells that you obtained, not least of which is the strain of mouse. Even substrains on the same background may differ. Second is the environment and living conditions. Then of course there are experimental conditions. First, how long did you wait before euthanizing the mice after injecting with BrdU? Typically you won't get very many double labeled cells anyway, and this number is time dependent. You may see fewer cells than the published paper because they collected longer after BrdU administration allowing for more new neurons to mature. Second, as mentioned before BrdU requires antigen retrieval so you may not be getting full penetration of the antibody. HCl is one option, heat-mediated retrieval with a citrate buffer is another. How many BrdU+ cells are you getting in the granule layer? Make sure your protocol is optimized before doing double labeling.  NeuN can ge tricky, too, especially if using a mouse on mouse antibody. Finally, quantification varies by lab/publication. How did the other group report their numbers? Absolute number counted, number per field, per section, per mm2? You may not be as far off as you think. But as mentioned unbiased design based stereology, which incorporates random sampling and volumetric analysis to generate a population estimate is the gold standard. If you are confident that you have double labeled cells and that your coubt reflects the number and distribution you obtained, then don't worry too much about the number not matching the other publication. There are so many factors that modulate neurogenesis and there is a wide range of reported values out there. 
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I have some rat brain tissue already horizontally sectioned that contain the spinal trigeminal nucleus, facial nucleus, and paratrigeminal nucleus. I am hoping to use anatomical landmarks to quantify various cell types in these regions, but all of the papers I am finding that examine these regions use coronal sections. 
Ideally, I would like to be able to differentiate subnuclei, but I would be fine with just having landmarks to differentiate the region borders. 
I have tried using various brain atlases to determine landmarks, but my sections are much more thin (40 microns) then those typically included in atlases.
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I think you can try Immunohistochemistry 
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There are papers describing the population of cones and RGCs and their variation with eccentricity in the primate retina. I however cannot find a similar paper on bipolar cells. I would like to know the relationship (in terms of numbers or ratios) between cones and bipolar cells as well as bipolar cells and RGCs as a function of eccentricity. Thank you
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Martin, Paul R., and Ulrike Grünert. "Spatial density and immunoreactivity of bipolar cells in the macaque monkey retina." Journal of Comparative Neurology 323.2 (1992): 269-287: used the differential immunoreactivity to antisera against glutamate, glycine, GABA, parvalbumin, calbindin (CaBP D-28K), and the L7 protein from mouse cerebellum to distinguish bipolar cells can be from amacrine and horizontal cells in macaque retina.
Grünert, U., Martin, P. R., & Wässle, H. (1994). Immunocytochemical analysis of bipolar cells in the macaque monkey retina. Journal of Comparative Neurology, 348(4), 607-627; used a variety of anitbodies including the β isozyme of protein kinase C (PKCAβ), a monoclonal antibody raised against rabbit olfactory bulb glutamate transporter protein (GLT-1), calbindin (CaBP D-28K), and α isozyme of PKC to label different bipolar subtypes.   Their comparison of the spatial density of cone bipolar cell populations with that of photoreceptors suggested that each bipolar cell class provides a complete coverage of the cone array.
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Two papers are using the method.
One attached and the other cited here: "Single rodent mesohabenular axons release glutamate and GABA" Root et al  2014
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Supplemental figure 3 and supplemental text from "Cho JH, Deisseroth K, Bolshakov VY. Synaptic encoding of fear extinction in mPFC-amygdala circuits. Neuron. 2013 Dec 18;80(6):1491-507." have a very nice explanation of the logic behind using TTX and 4-AP isolating monosynaptic inputs. They also show a positive control in which disynaptic inputs are not rescued by 4-AP. Here is a link to their supplemental materials: 
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I am pursuing a project that will require me to quantify excitatory synapses in rat spinal cord slices (fixed in PFA). I have a number of references and protocols which describe methods for quantification, but they all use tissue that is 5-15 uM thick. 
I would like to use 20 uM thick slices (for a number of other standardized outcome measures), but am concerned that this could be too thick and create too much background/reduce resolution. If you have experience quantifying synapses in fixed tissue, do you believe that this would be an issue? 
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Hi Khalid,
Look at this paper: 10.1007/s00429-016-1278-x ( i have helped for the quantif and it was ok, as long as you do the deconvolution step!). have also a look to this one: 10.3389/fncir.2013.00153
Hope that helps!
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I'm using 35um horizontal brain cuts, spinning disc microscopy and Fiji to analyze stacks of 35 slices. I'm trying to meassure differences between two types of neuronal projections, in one condition they are very neat and parallel to each other, but in the other, they are very intrincated and more projections can be seen in the tissue. I have compared them using z projections, but I'm not sure what pluggins can I use. Not Sholl, because neuronal projections are not radial, and nuclei are not in the pictures. Any sugestions?
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Thanks a lot for your time and help, Dan! I'm going to check that. I thought about something like that, but as an histogram of pixels used for the projections. I'm not sure what kind of projections are (axon/dendrites, or more cells or more projections of the same number of cells, etc). It was not my first question, but I noticed this difference between two conditions....and now I want to know what is happening
Good luck with your stuff :)
Ana
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Hi everyone,
I'm currently using the Allan Brain Atlas's Mouse Brain Connectivity tool (http://connectivity.brain-map.org/) to look at the connectivity between certain sensory and association brain regions. However, most of the literature on neural tracer studies uses different nomenclature for brain regions, and more specifically almost the entire literature uses different nomenclature than the Allan Brain's Mouse Brain Reference Atlas. 
For example in the literature, the primary auditory cortex is often referred to as A1, the Auditory Core areas (A1 + AAF), Brodmann area 41, or Te1. Yet, the Mouse Brain Reference Atlas uses the name 'Auditory Areas', which they subdivide in the 'Dorsal auditory area', 'Primary auditory area', 'Posterior auditory area', 'Ventral auditory area'. In this case, I figure, 'Primary auditory area' is the region that I'm looking for, as it probably corresponds to A1 (and maybe AAF too?).
If this is the case then I can of course estimate for each brain region described in the literature its relative position to the primary sensory areas and then sort of guess in what Reference Atlas region it most likely is.. However, this seems rather imprecise, and also it will take a very long time.
So my question here is:
1) How can I find out what the regions of the Mouse Reference Atlas comprise of?
2) Is there potentially a mouse brain anatomy database, which allows you to quickly look up all/most of the different names used for certain brain regions?
3) Is there a database to identify homologues for brain regions across species?
Also, other tips to tackle this problem would be most welcome. 
Best wishes and many thanks!
Jesse
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Dear all,
Thank you for your replies. Together with all these different sources and the individual papers, I am making much progress!
Thanks,
Jesse
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We already have a paraffin section of the rat brain and are sagittal.
Is there any staining or labeling that can measure the hypothalamic area?
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I am now approaching to the neurophatological analysis. I have to detect differences in microglial morphology in brain slices stained in IHC. The aim is to distinguish microglial cells with different shapes and dimension with major objectivity. Is there a free software that can do that?  
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I used Neurolucida neuron tracing Software (MBF Bioscience)
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I am planning to quantify the expression level of a receptor in certain brain region. The technical challenges for me are:
1. IHC or in situ: is it valid to use the normalized fluorescence intensity of the receptor to DAPI for quantification?
2. Of course, the more accurate methods are Western blot or qRT-PCR. However, my interested brain region is difficult to isolate for RNA extraction due to anatomical location and small area size. Is any good way to extract RNA from certain neuron types from a brain region?
Any advise or comment is welcome. Thank you!
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Immunohistochemical study with measuring the optical density for the photographs is an easy method for the small hardly dissected areas in the brain. In my work; on doing qPCR for the hippocamus, nearly the same results as immunostain expression were obtained.
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Hello,
I am interested in the segmentation of the corpus callosum (CC) into its 5 regions: genu, body, isthmus and splenium. I was wondering, based on the Mouse Brain Atlas (Paxinos & Franklin), what coordinates would you use to separate these regions for coronal sections. I found one paper by Steelman et al. (2012) that gives the coordinates for each region (i.e. 1.34 to 1.44 from bregma for the genu etc.). However, most other papers I found either looked at the human brain, or just briefly mentioned they looked at the genu or splenium, without giving any other details.
Thank you.
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I agree with Shafagat Mahmudova good references. thanks
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Hi,
Can Somebody help me on how I know how I can micro-direct the somatosensory representative area in Adult Rat Brain ?
Thanks.
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It depends on what method you are going to use to analyze the area.  If you are doing immuno, the perfusion would be sufficient, and provide you the best method to localize the area.  If you are doing something like western where you will be homogenizing the tissue, you'll want to decap, and punch the tissue directly.  To do this you will need to use steel matrices like the one linked.  Then you make precise cuts, identifying major structures within your R.O.I. I would also suggest flash freezing the remainder of the tissue as well just in case your cut turns out to not be as accurate as you wanted. 
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Hi everyone,
I would like to find a relation between several cortical parameters I extracted from sMRI data (as gyrification, sulci depth, thickness of grey matter) and behavior. Does anyone know if there is an atlas or something similar (papers, references,...) that I can use?
Thank you very much!
Edoardo
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Dear Edoardo,if I understand well your question, you are asking if anybody ever described data such as "length of ascending branch of left sylvian fissure correlates with verbal fluency" or "degree of convexity of the precentral knob predicts dexterity in finger movements". Well, as far as I know there are no descriptions of relations between normal anatomical variants of macroscopic sulcal anatomy and behavior. If I had to bet I would probably think that there is no clear relation, but it's just my opinion. Sorry for negative answer. I hope I'm wrong and that some neuroanatomists provide more acurate responses!
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There is a huge clinical literature on this mental disorder, but the problem stubbornly and tragically persists. I wonder if it is strongly grounded in amygdala pathology. 
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Paul- before proceeding, I have to point out that the term "intractable" is  an illusional way of referring to an ineffective therapy - major source of the treatment difficulty is sloppy approach to research of brain biology- decades of advances in brain imaging confirm glitches in  brain systems that are implicated in OCD - most psychologists, social workers, counselors and others who practice psychotherapy have been content with shifting brain biology issues to medicine knows physicians - even undertake sometimes to encourage patients to comply with medication regimens- need to address the fact that  even the FDA  relies only on evidence that medications reduce the symptoms per DSM listings - no requirement to demonstrate corrections of brain impairments- I know of no evidence from any neuropharmaceutical that corrects defects in brain biology- We are all  aware, however, of the numerous examples of neurological damage like EPS and TD as well as serious endocrine disruptions- the only study I know that identifies brain pathology in mental illness, is the landmark project of Dr. Jeffrey Schwartz and his team in the UCLA  department of Psychiatry. They designed a psychotherapy protocol based on discoveries of neuroplasticity in the discipline of neuroscience-neuroplasticity refers to the process in which every learning experience includes biological brain changes. Part of therapy involved cognitive-behavioral methods for changing pathologies of thinking and behavior, which most psychotherapists will recognize.  (f)MRIs were applied to assess brain benefits .  Structures that were not doing what they are designed to do, were in overactive areas. Mindfulness skills ,along with other benefits, normalize activity levels. The resulting effects were that malfunctioning structures started working as designed.  Successful psychotherapy is always a learning experience - hundreds of studies of neuroplasticity confirm this phenomenon. A giant leap in the UCLA  project was designing a therapy where the brain changes came in the form of correcting defective functioning.  Highly recommend thew book describing the project- includes details of research references, which provides good update for modern advances in neuroscience
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We are studying the neurobiological basis of ethnic variations in responses to similar life events.
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Dear Adejoke,
We humans are all genetically very, very similar. The variations found within an ethnic group are large compared with the differences between the means of various ethnic groups. Most humans have genetic components from many, many ethnic groups - we are a mixture. (See National Geographic's Genome Project.)
The differences in values and behaviors are principally due to differences in culture.
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They are frequently referred to but not commonly imaged.
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Hi,
Please find the conceptual image attached. It is taken from the book Neuroglia edited by Kettenmann and Ranson.  It states there that the P2X4R channels function differently in the presence or absence of the Ca2+ (i.e., as a caution channels or as a macropores). Shinozaki et al (citation below) provided an image of the ATP–induced structural change in the P2X4Rs.
Shinozaki Y, Sumitomo K, Tsuda M, Koizumi S, Inoue K, Torimitsu K. Direct observation of ATP-induced conformational changes in single P2X(4) receptors. PLoS Biol. 2009;7(5):e1000103. doi: 10.1371/journal.pbio.1000103
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I observed such an anomaly on a dried skull, and wonder if related to any neuro-anatomical subsequent/associated anomaly. Thanks+
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Allargamento impronta vasi meningei (maggiore spessore e profondità) rispetto al normale. Maggiore profondità e grandezza delle foveole o granulazioni del Pacchioni .oltre l'interno del cranio guarda anche se alterazioni arrivano tavolato esterno; tutti questi caratteri possono indicare presenza di tumore meningi (meningioma). Bibl: potresti vedere M. Rubini 2008 elementi di paleopatologia; Ortener and Putschar identification of pathological  conditions in human skeletal remains; per altra bibl. guarda Minozzi and Canci Archeologia dei resti umani. 
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I am doing cerebellar organotypic slice culture from mice p8 and would like to know if there is a way of making sure the slices are happy and alive? 
I would also like to know if it is normal to see migration of cells (probably microglia??) outside the slice boundaries? 
Thanks,
Orli
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Several ways to assess their health, depending on what your end point measure is. The simplest would be to simply gauge that they maintain some semblance of their native morphology - I have no experience with cerebellar slices, but certainly hippocampal ones should look like they look in schematic figures for at least a couple of weeks in vitro.
If you or a neighbouring lab has access to electrophysiology set-ups, you can place them under DIC optics, and should be able to visualize the neurons clearly; you can go further by recording some functional electrical activity.
If you're interested in morphology, you should be able to find a way to label a subset of the cells - a transgenic mouse with fluorescent cells would be the simplest, but can also use lipohilic dyes like DiI. Can also simply label nuclei with Propidium Iodide, where labelling should be minimal in a good healthy slice (perhaps compare to a slice that you induce toxicity in).
In answer to your last question, yes it is normal for there to be cells outside the original boundary of the tissue you placed on the membrane - these cultured slices "thin out" with time, and there is glial migration.
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We want to observe by fluorescence microscopy or light microscopy the parallell fibers in rat cerebellum. Is it better to use sagital or coronal sections? Any suggestion for a marker?
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Dear Natacha,
If you want to see a long stretch of PF (intervaricosity distance study ?) a coronal or transversal section seems more appropriate than parasagital. As for marker, you probably do not want to label ALL the PFs, it would become too messy to see anything. I would suggest to place a very small DiI crystal on the GL or inject the GL of your fixed slice with a bolus of Fast-red (slightly less lipophilic than DiI). If the crystal/bolus is small enough and you if you leave your slices rest at 10C long enough (w/ azide?) you should get a subset of brightly stained PF. I hope this helps. Good luck    
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I usually look at microglia isolated from mice brain using the standard percol gradient method without using Collagenase/DNAse steps i.e. just meshing the brain with 10 ml syringe plunger and then layering 30% brain cell suspension on 70% percol. This is the way I look for infiltrating lymphocytes (CD45 Hi)  and microglia (CD45 Int CD11b+ cells) by Flow cytometry. The method works well for analyzing infiltrating lymphocytes but for microglia, I get the fluctuating data for the mice even within the same group. Is there any other clean and better way to isolate and characterize microglia from mice brain? Waiting for your suggestions.
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Dear Tabasum
There are specific products for microglia isolation which can save you lots of time and make your life easier. 
Here's a tutorial video which might be helpful for you. The entire procedure is shown step by step for three adult mouse brains, and it is the same for human brain tissue.  For young mice until P7 you can skip the myelin removal step. For a small number of mice the enrichment may also be done manually with a magnetic separator.
Kind regards
Sandra
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I usually cut cryo sections of mouse sciatic nerve at servile postnatal stages, but detaching happens only with P1 slices. I guess it is because those sections are extremely small and have low adhesive force, but I really need to keep them on the slide since I have to run FISH on them.
Thank you for the help.
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Thank you for all your suggestions, I think I should give them all a try.
I usually fix my tissues in 4% paraformaldehyde  and then 20% sucrose and keep my slides at -20°C after cutting. Before staining I let the slices dry 1h at RT.
Thanks once more for your kind suggestions and for the time spent on this issue!
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Hi guys, I am doing experiment of stereotactic intracranial surgery. I wander how to adjust the orientation of rat head in stereotactic to make sure I can injection drug precisely into the relative small area in rat brain as indicated in Paxinos rat atlas?
Thank you very much!
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Hi Wenchong,
It depends on what animal you are using , body weight and what target in the brain you are aiming for, But, in general the standard way of head positioning in a Kopf stereotaxic instrument, for adult male rat is done so to keep the heights of Lambda & Bregma equal. and the line passing between the incisor bar in the horizontal plane through the interaural line , and the line between the Lambda and interaural bar in the lateral plane are making a 90 degree angle , (i.e.) the result is a flat skull position with straight line falling from Lambda trough the ear . accordingly in order to achieve this you need to lower the incisor bar  3. 0 mm below horizontal zero (-3 mm).  and according to The Rat Brain in Stereotaxic Coordinates, 4th Edition by George Paxinos (1998) | ISBN: 0125476175, in Male wistar 290 gm rat, the incisor bar is lowered to 3.3 ± 0.4 mm below the horizontal zero.
if you are working with different strain of rats and different body weight , and aiming for deeper areas in the brain like VMH or Nucleus Tractus Solitarius, you need to calibrate and adjust your measurements accordingly.
"please see schematics below"
hope this is helping,
kind regards,
Ghanim.
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Is anyone aware of a paper where cuprizone is injected into the brain or spinal cord? I was just wondering if it was possible to induce local demyelination this way.
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Ya, you can try this recently published paper 'Anatomical Distribution of Cuprizone-Induced Lesions in C57BL6 Mice'. Journal of Molecular Neuroscience 57(2) · June 2015. DOI 10.1007/s12031-015-0595-5.
I strongly believed that it may be of help to you.
Good luck! 
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Dear colleagues,
I was wondering if anyone know the specific or unique molecular markers for the following ANS fibers (I would like to do some immunohistology to distinguish between the non-ANS nerve fibers and the ANS nerve fibers, and between the ANS nerve fibers):
1) sympathetic fibers
2) parasympathetic fibers
3) enteric fibers
Thank you very much in advance.
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Hi Ping,
In what tissue are you trying to distinguish ANS fibers from the background? Brain? Peripheral NS? In which organ? It might help others assist you if you are more specific.
Best,
jg
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What is the intracellular distribution of Matrix Metalloproteinase (MMP)-9 in fast motor neurons (if possible, which organelle)? Do they found in the axon terminal?
Additionally, do microglia express MMP-9 (it seems to me that I found some "localized" expression)?
I have a picture for your references (where green is the MMP-9 immunopositive reactions; red is IBA-1).
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It appears that primary murine microglia produce MMP-9 precursors (proMMP-9) when exposed to vitronectin and fibronectin.
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Hi, I am looking for data sources and/or references that describe brain mass increases in mammals with age. For context, I recently read a paper that shows captive mink brain mass peaks at approximately 3 months of age; i.e., the brain mass of the adult is less than that of a 3-month old. I want to know how common this brain mass growth pattern is, so I am searching for other references and sources for comparisons. Thank you all in advance.
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An old classic book which includes information on humans is: The Human Brain in Figures and Tables, By, Samuil M. Blinkov and   Il'ya Glezer, Translated from the Russian by Basil Haigh.  Basic Books, Inc Publishers Plenum Press,1968. What is also good about this book, is that is goes over the different methods of quantitative analysis.This might be helpful when you review the above very interesting papers.
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I have tried making 10 um cryocut brain sections stained by H&E. I can measure the total cortical thickness, but it is really difficult to distinguish between the different cortical layers. The H&E staining procedure works great in the lab, so I wondered whether it the thickness of the slices that are the problem, or if I should choose another histochemical technique?
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Dear Peter, in what species are you working?
In mouse, for example, identifying cortical layer boundaries is quite subjective and may depend significantly on what markers / stains / illumination methods are used. The Allen Institute's database (or similar resources) can be quite helpful for a gene/antibody-based approach. For a paper demonstrating multiple complementary techniques of laminar identification, please see our recent work (attached).
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I´m working with 20 microns sections and whenever I mount them and leave them to dry, they shred. If I don´t let them dry, they´re fine, but they don´t stick to the slide, falling off in the staining process. I´m losing a lot of samples and I´m trying my best to find the perfect timing between mounting and staining, but nothing seems to work. Does anyone already worked with this and had the same problem?
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Have you tried to do a free floating staining and mount the stained slides?
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I would like to perform RNA FISH and/or IHC on sections from mouse spinal cord and dorsal root ganglia...i was thinking about sectioning spinal cord within the vertebral column and a part of surrounding tissue where the dorsal root ganglia are present, but i have read the bone must be decalcified and that this procedure can affect histologic quality :( Does somebody have experience with this kind of tissue preparation? Thanks
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 Wow, thank you very much Sarah. It's a very useful information for me, as I am new in this field...and frankly the first time I assisted at the dissection and DRG extraction I thought it was impossible to localize and identify specific DRGs, but with this protocol there is a hope :) 
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I am confused with the score for body symmetry and Whisker response. By the way, I use intraluminal MACo as a model and do it on  left MCA.
About the body symmetry
score 2- Moderate asymmetry: body lies on ipsi-lateral side, legs at the ischemic side are extended laterally, the tail is bent. My question is, do both ipsi-lateral side and ischemic side both mean left side or ischemic side mean right side? Since, left hemisphere controls the right part of the body.
And Whisker response
score 1- no response on the ipsi-lateral side , normal on the contra-lateral. Again, does ipsi-lateral means left side and contra-lateral means right side ? 
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Although listed as New Q, I had answered it couple of months back. Some works are referred again.
1. Cognitive Deficits After Focal Cerebral Ischemia in Mice
by K Hattori - ‎2000 
Cognitive Deficits After Focal Cerebral Ischemia in Mice .... As a means of assessing adequacy of occlusion, a neurological score was assigned to each ..... Clark W et al. Monofilament intraluminal middle cerebral ...
2. Conditionally immortalised neural stem cells promote ...
by S Patkar - ‎2012 -
Acute neurological deficit, evaluated using Clark's deficit score, was significantly greater in both the general and focal scores post-MCA
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Does anyone have an idea why sometimes gray/white matter increase over time due to skill learning, and sometimes they decrease?
I've heard people say that maybe it decreases because the structures are becoming more efficient, but do we know what distinguishes or predicts increase versus decrease due to learning?
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@ G. Mello: Gray matter is NOT mainly cell bodies! Pericaria make up less than 3 % in volume in grisea, glial cells about 1 %, the rest is dendrites and axons.
To my knowledge absolute increases in grey matter are only seen in oedema, reversible reduction in volume only in dehydration. Cortical fields can show relative increase by extensive training ("remapping"). This is because existing neurons are recruited for a specific task, not because of new neurons.
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In fact, I am have a good struggling with coinjection of fluorogold &BDA in the same brain region. My protocol for this combination is quite similar to which L. Swanson reported in PNAS, 2010 (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2930585/), but with smaller injection current (+1-2 µA) and longer injection time (15-20 min). I can, however, get barely BDA labelled neurons or dendrites, but always very nice retrograde FG labelled cell bodies.
I almost always oberseve FG gets precipitated, especially up to first 5mm of glass pipette from its tip. I consider this might be a reason for a lack of BDA labeling in my case.
Any suggestions or comments? Thanks in advance!
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In my experience, the best choice for dissolving fluoro-gold is to use 0.1 M cacodylate buffer. This results in a crystalline solution without any trace of precipitates. By going this way you can inject FG either by iontophoresis or by pressure. This buffer also works for BDA. Just search PubMed for my earlier papers.
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I hope you all will be doing well. I have a question regarding cortical sample collection after MCAo surgery. I have operated 48hrs Permanent MCAo in rats. But after collecting the samples some of tissues did not clearly show apoptotic behavior when I applied different Ab. After surfing on PUBMED I have found the following from  
“Overall, middle cerebral artery occlusion led to a remarkably consistent pattern of necrosis. The rostral limit was at the level of the intermediate olfactory tract; all animals displayed an infarction dorsal to the rhinal fissure which could attain the frontal and primary olfactory cortices. More caudally (+ 10. 05 mm) the pattern was the same; the nucleus accumbens was very rarely infarcted. At the level of the caudatoputamen (which was invariably damaged), the highest incidence of the infarction was seen at the ventral surface of the brain, corresponding to the primary olfactory cortex. The frontoparietal somatosensory cortex was affected to a greater or lesser degree. The infarction reached its maximum extent, in terms of necrotic surface area, at the level of the globus pallidus (+ 6. 28 mm). Again, the caudatoputamen was systematically infarcted though the degree of cortical involvement was variable. More caudally, the infarction size tended to decrease at the coronal section that corresponded to the level of the anterior hypothalamus. The tail of the caudatoputamen and the external capsule were frequently involved, whereas the involvement of the frontoparietal somatosensory cortex was more erratic. Thereafter, especially in the normotensive strains, the infarcted surface area decreased rapidly and was limited to the dorsal convexity of the cortex and the external capsule”. Journal of Cerebral Blood Flow and Metabolism 8:449-461 © 1988
From this I am confident that my sample collections region is mostly the upper layer of somatosensory cortex and according to this article it is the 2nd layer of primary olfactory cortex (piriform cortex). So I want to ask that which particular areas in cortex do I collect the sample/sampling and will be more grateful  if somebody give me hints  by mentioning some paper or some site.
Thankx alot
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Dear Fawad.
In my experience, the infarct area depends on the age, weight, sex, and strain of the rat.  Even when all of these are controlled for, there is still some variation in the damaged area because there is some animal-to-animal variability in the branching patterns of the MCA.  It is important to validate the damage created by your surgical technique in some way, rather than to just rely on the descriptions of the damaged areas in the literature.
What my colleagues and I did was to verify the damaged areas using TTC staining:
J Pharmacol Methods. 1986 Nov;16(3):201-14.
Morphometric evaluation of brain infarcts in rats and gerbils.
Lundy EF, Solik BS, Frank RS, Lacy PS, Combs DJ, Zelenock GB, D'Alecy LG.
The Levine rat preparation, the gerbil stroke model, and appropriate control
animals were used to determine if the 2,3,5-triphenyltetrazolium chloride (TTC)
would selectively identify noninfarcted versus infarcted cerebral tissue. The TTC
is frequently used to quantify infarcted myocardial tissue and has been shown to
have great specificity, reproducibility, and efficacy. The TTC produces a red
product upon reaction with the respiratory enzymes (dehydrogenases) present in
non-infarcted tissues. Irreversibly damaged tissues, lacking dehydrogenases, do
not form red reaction products. Six gerbil brains and seven rat brains were
incubated with the TTC, and the unreacted areas were macroscopically identified.
The brains were fixed and sectioned for routine hematoxylin and eosin staining to
determine the specificity of the TTC. The TTC was found to react selectively only
with non-infarcted cerebral tissue. The gross brain sections were evaluated by
macroscopic morphometric analysis, and the unreacted area was always ipsilateral to ligation and correlated with histologic identification of infarct. The brains from neurologically intact animals demonstrated neither macroscopic nor
histological evidence of infarction. This technique allows macroscopic
quantification of infarct size by planimetry. The average area of infarct for the
neurologically impaired rats was 34.7% and it was 31.4% for the impaired gerbils.
The percentage of surface area of each infarcted slice was found to correlate
with the severity of the neurologic deficit. We conclude that TTC staining is
effective for macroscopically delineating cerebral infarcts in rats and gerbils,
thus permitting quantification of infarct size.
PMID: 2431224 [PubMed - indexed for MEDLINE]
The technique is very easy, since it is performed on live brain tissue rather than fixed.  We invested in a stainless steel brain matrix in order to reproducibly slice the brains into thin sections, and used alternate sections for TTC staining and biochemical analysis.  In order to do this, you must order the brain matrix that matches the weight of your rats.
After the TTC stained sections were photographed, we fixed them in cold paraformaldehyde overnight, cryoprotected them in sucrose, and cut 20-40 um free-floating frozen sections for other types of histology (NOT apoptotic markers).  Once we had this data to confirm the damaged regions, we could then analyze new surgical groups without using the TTC.  We would either perfuse the animals for traditional immunohistochemistry, or snap-freeze fresh brains for frozen sections, depending on the markers we were interested in.
Good Luck!
Jill
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I'll preface this post by indicating that I work primarily in bioinformatics, but I need to demonstrate some wet-lab skills in order to graduate with a degree in neuroscience.  
I'm proposing a study that seeks to count various immunohistochemically-labeled cell counts and to quantitatively assess dendritic morphology/spine features from serial sections of the **same individuals**.  I'm insistent on using the same individuals, because the opposite hemisphere is being fresh-frozen and used for RNAseq studies, so we want to correlate these changes with morphological/neuroanatomical features.   The tissue in question will be saline perfused, immersion fixed in 4% PFA.   I had planned to section the tissue at roughly 40-50 microns; hopefully just large enough to permit the study of a few intact dendritic arbors per slice (and also thin enough to permit antibody penetration for IHC).  
Ideally, some variation of Golgi staining seems would have been great for dendritic arbors (e.g. Sholl analysis)  and spine quantification.   However, I've read that these approaches are generally poorly suited for pre-sectioned slices at my planned thickeness (e.g. they tend over-stain and become very rigid).  
So I'm wondering if:   
1) Anyone is aware of a modified Golgi protocol for thin sections (e.g. 40 microns; they can be free floating or mounted as needed to accomodate).  
2) Anyone can suggest an alternative approach to Golgi-type staining that would facilitate the quantitative morphological study of the dendritic arbor and dendritic spine density/features.  
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Hi Daniel,
I did extensive work on reconstructing the dendritic morphology of layer V pyramidal neurons in mice for my masters work. I performed sparse single cell iontophoresis in 200um sections to label neurons with fluorescent dye. This enabled faster and more accurate reconstruction. I subsequently performed IHC to determine the genotype of the cells that were filled with dye.
I agree that depending on the cell type you want to reconstruct, 40um will likely not be sufficient.
I now work in biotech but I wrote up a blog about the free programs I used to trace and extract complex morphological parameters. I highly suggest you checkout those programs. They were a lifesaver for a lab with no budget for fancy software. Here's the link:
The protocol for the single cell electroporation can be found in my paper just published. http://journal.frontiersin.org/article/10.3389/fncel.2015.00145/abstract
Let me know if you have any questions!
Best of luck!
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I would like to apply a stain that will really make the diagonal band in the pigeon brain "pop-out". I can identify it in cresyl violet-stained sections, but sometimes it is quite difficult to make out boundaries. I'm thinking luxol fast blue (stain for myelin) may be a good. Suggestions, please?
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I have work on guinea fowl, rock pigeon and domestic Fowl. Cresyl fast violet is good. i will suggest luxol fast blue to you. the stain will mark out the diagonal band in the pigeon according to my understanding.
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  1. These regions are nucleus accumbens and ventromedial prefrontal cortex. Is there a method to locate them?
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We've found that the most useful method for fluorescently tagging neurons is to use IHC with a primary against neuronal nuclear protein (NeuN). The NeuN can be processed for fluorescence or light-field and makes a fantastic counterstain, allowing the sections to be matched easily with atlas plates. If you're interested in delineating the shell and core subregions of the accumbens (and you probably should be), you might want to try processing the tissue for calcium-binding protein, which is differentially distributed in these compartments.
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Can someone inform me very briefly (or indicating the reference) about symmetries, sides or lateralization of default mode network (DMN) in human brain?
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Dear Slobodan,
there were some papers published recently (one very new, one a bit older) that might help you: 
1. Agcaoglu O, Miller R, Mayer AR, Hugdahl K, Calhoun VD.: Lateralization of resting state networks and relationship to age and gender. NeuroImage. 2015 Jan;104:310-25. doi: 10.1016/j.neuroimage.2014.09.001. Epub 2014 Sep 18.
2. Saenger VM, Barrios FA, Martínez-Gudiño ML, Alcauter S.: Hemispheric asymmetries of functional connectivity and grey matter volume in the default mode network. Neuropsychologia. 2012 Jun;50(7):1308-15. doi: 10.1016/j.neuropsychologia.2012.02.014. Epub 2012 Mar 1.
Hope this helps in some way!
Best,
Angelique
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I am recently planning to do some stereotaxic injections to some mice brain nuclei. Due to the various choices (in particular the G number) of Hamilton needles, I was wondering which one would be the best for my surgery.
If available, may I also have the "cat no" please? (I am thinking of purchasing one)
Thank you very much in advance once again!
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What is the aim?
Lesion studies, large (like 32G or 33G) might be fine,
looking at fine scale cell morphology in the target region everything below 34G is too large. Blunt needles are prone to clogging, Bvld more difficult to calculate the injection depth.
For many applications a glass capillary is equally well suited or can even be better, since you can work with an even much smaller diameter.
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Hi,
I am injecting a virus (AAV9.Syn.GCaMP6s.WPRE.SV40) to express GCaMP6s in the mouse hippocampus. I am using young animals (around P20), and as most of the atlases I know base their stereotaxic coordinates in P56 animals, I cannot use them, so I'm injecting with no coordinates. 
So, the first question would be: is there any atlas for young animals that I can use to inject more precisely? I haven't been able to find any so far.
Even though I am using no coordinates, I'm not having problems to hit the hippocampus. However, I almost exclusively see expression in the DG. Is this something common? Discussing this with colleagues, we came to the conclusion that even though the virus infects non-diving cells, it might be more effective infecting dividing cells, and in that case that would explain why the granule cells are more effectively expressing the protein. Does this make sense to you?
My last question (sorry this is getting so long) is regarding two-photon microscopy. I am trying different wavelengths for imaging the GCaMP (I know 920 nm is the optimal, but this is part of my experiment) and I think the shapes of the cells look different when I image them at different wavelengths. I don't really understand why this could happen. Does any one know anything about this?
Needless to say, I will be really grateful for any opinion or information regarding any of the points. Thank you for taking the time for reading the post. I hope to be able to help you in the future. 
Kind Regards,
Julieta
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Dear Julieta,
Regarding expression of the GCaMP6, over-expression may (i guess) alter calcium homeostasis and I could imagine that that would be harmful for cells. If the expression is not so very strong, however, it should be fine I think. Secondly, the brightness of the GCaMP6 depends on how strongly it is expressed and on whether it is bound by calcium. What I would suggest to do is to inject a virus (of preferentially the same serotype and titer) that expresses GFP (or any other FP) and make sure that your virus injection does make it as far lateral as CA1/CA3.
How long do you wait to check your virus expression and do you check in vivo or in brain slices?
There are also some papers describing two-photon imaging of hippocampal CA1 cells (From David Tank's lab) with GCaMP, and the virus they used was aav1 or aav2.1, maybe try that serotype, maybe that will solve the expression problem. See e.g. these papers: Rickgauer JP, Deisseroth K, Tank DW. (2014) Simultaneous cellular-resolution optical perturbation and imaging of place cell firing fields. Nat Neurosci. 17: 1816-24. Dombeck DA, Harvey CD, Tian L, Looger LL, Tank DW. (2010) Functional imaging of hippocampal place cells at cellular resolution during virtual navigation. Nat Neurosci. 13: 1433-40. PubMed
Regarding the shapes of the cells, i'm not sure through how much mirrors and glass you are imaging, but dispersion is wavelength dependent and maybe this effects your image. Since you indicate to be not sure if the cells really look different: Try imaging fluorescent beads (making a zstack of a single bead at high zoom) and you can calculate your point spread function. This will probably be more telling than looking at an actual living sample.
A colleague of mine showed me the following imageJ plugin & paper on how to to this, which I find useful: 
Hope this helps and cheerios,
Pieter
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Are there any articles published about cerebral dominance in rodents (rats) and histological changes based on the cerebral dominance?
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I am planning to do a stereotaxic injection to the medial forebrain bundle (MFB) of mice. I have tried AP:-1.2; ML:-1.1; DV:-5.0mm, but this does not seem to work.
I was wondering whether there is any preferences from anyone please?
Thank you very much in advance!
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I would highly recommend to check the coordinates with dye injections and keep mouse strains, weight and age constant. Always measure your Bregma-Lambda distance and check for flat head. We found some small variations in the same strain but different breeders as well as between laboratories. The coordinates that Moises reports we have used repeatedly and they work well in our hands, with male C57/Bl6 mice. Pay attention to the post-operative care as many mice will lose a lot of weight starting 2-3 days after surgery and some people have reported high mortality rates.
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I have a question regarding the use of control as follows:
Although it is quite usual to setup 2 groups of animals (one for experiment, and the other for control), there is some paper/studies comparing between the 2 brain hemispheres (i.e. using one side of the brain as the experimental group while the contra-lateral side used as a control).
I was wondering what would be the advantages of using the other hemisphere as the control? Wouldn't the differences between 2 hemispheres (e.g. the functional differences between 2 brain hemispheres in human) make this control invalid?
If there is a choice between using individual or contra-lateral hemisphere as the control (while the study is not mainly comparing the differences between the cerebral hemispheres), then which way would be the preferred choice?
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I agree that the key issue is the hypothesis being tested.  If you wish to test whether an intervention on one hemisphere causes a measureable change in that hemisphere, then comparing an appropriate untreated or sham-treated animal certainly gets around the problem that your intervention may well (in fact, likely) affect both sides.  If, however, you are studying a naturally-occuring event which is rare (for example, Rasmussen's encephalitis or hemimegencephaly in humans), a comparison of one side versus the other might be the most appropriate (if you were looking at, say, metabloism by PET or white matter volume by MRI).  In such a case, the comparison of one side against the other allows within-subject statistical power, and automatically controls for age, gender, and many genetic factors.
For the situation your question suggests, comparison with another individual brain seems most appropriate.
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As M1 and M2 lie very close to each other, if I am going to do an implant to gather signals from only M1 neurons, is there any established way for me to locate accurately the M1 or M2?
The anatomical location of M1 and M2 can be referenced by the following:
Thank you very much in advance!
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From which parts of M1 do you wish to record? Vibrissal (whisker) M1? Or somatic (e.g. forelimb, hindlimb) M1?
Vibrissal M1 is medial to somatic M1. Secondary motor cortex (M2) appears to be anterior to somatic M1, in frontal cortex. This is according to a variety of recent studies (although how to define M2 functionally is not entirely clear). This is different from some atlas, e.g. Paxinos, which shows M2 medial to M1: the region labeled "M2" in many an atlas appears functionally to be vibrissal M1 (vM1). See for example papers by Svoboda and Petersen groups for location of vM1.
If you wish to record from vibrissal M1, rough coordinates are 1.0 anterior, 0.9 lateral to bregma. For somatic M1, you'd need to be more lateral, say 1.5 lateral to bregma.
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I'm looking for anatomical changes in parietal and occipital cortices in patients with MDD, but there isn't a lot of literature about it.
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Dear Daniela,
This maybe should be helpful.
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It is said that dendritic spines can naturally occur among primary mouse neurons and their density or morphology can be thoroughly assayed. So can anybody provide any information?
At the moment we want to evaluate effect of some chemicals at synaptic level, but we wonder how valid such effect on cultured spines is, given that synapses need extra modulation in vivo.
Thanks a lot if you could help!
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If you have access to a good microscope (confocal, 2-photon) you can try to measure the number of spines over some period of time. From this you can extract data such as spine density (spines per micrometer), fractions of dynamic spines (new spines, lost spines, transient spines) and their evolution over time etc.
You could also measure intensities which are found to correlate with actual spine volume, as validated with electron microscopy (see link).
One step further is to construct models based on these measured spine parameters in order to make predictions and extract fundamental characteristics of your system.
As you eluded to it is known that primary cultures do make an excess of connections as compared to the in vivo situation, so ultimately any results will have to be validated in vivo. It stills makes sense to start with cultures if your lab is adjusted to those methods, as setting up in vivo studies can take a considerable amount of time (years).
I hope this helps!
Vassilis
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We are looking for somebody who is able to isolate the sympathetic trunk (chain) from mouse or rat and is willing to give us a privat lesson. We'll bring the cake you the know how.
cheers
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Please search.
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Additionally, I find varicosities in some neurons. I am puzzled as to what they amount to?
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Dendrites with varicosities along their length are sometimes referred to as moniliform.  Electon microscopic examination shows that these varicosities are filled with mitochondria.
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I am interested in sagittally representing rat brain structure, I was woundering if there were brain atlases for that. Please let me know if there are any?
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The most known brain rats atlas contain also sagittal sections. As said by Fernando, you have to be very familiar with anatomy (neuroanatomy) and spatial disposition of nuclei and aresa (notably those of interest) to worh with them. In general, we use this plans of sections to dectect fastly if we have a labelling in sommes structures before working on coronal sections. Sagittal sections are also useful for tracing methods.
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I'm searching for brainstem atlas, especially Nifti format one. I've already checked MNI-ICBM and SUIT. Anyone knows more detail ones?
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Hello Yoshiyuki,
This is a hard one to get, I agree.  The best I could find was this site from Harvard University.  Hope this helps.
M Diaz-Rios
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I am performing patch-clamp recordings of cells from red nucleus in slice preparations. I tried both horizontal and coronal slices but I still encounter troubles with optical visualization. I can barely see cells since the huge amount of fibers passing through the red nucleus. The thickness of slices is 200-250 um. Do you have any suggestions to get a better visualization of cells?
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My dear, not really (difficult, nowadays, to catch the post-doc dealing with similar issue in late 80s!). Try to speak, in case, with physiologists of the Scarnati's team (Capozzo, Florio), involved also recently into brainstem recordings. Another leader, I believe, is K Takakusaki (albeit mostly in vivo...)
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How does brain sense teh temperature through scalp? Any ruote map? 
From skin, we have fibres running from skin through spinal cord and to brain reaching somatosensory cortex. Is it same when first event occurs at scalp and not skin?
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Hi Kalpita,
They are the same. To give you a short answer: External temperature (like a hot water bath) is sensed by thermoreceptors under the skin. The only thermoreceptors between the skull and skin are those located right under the skin. Simply put, there are two types of thermoreceptors: those that increase firing rate with warming, and those that increase firing rate with cooling (see my previous reply).
A more in-depth answer:
We can differentiate thermoreceptors based on their location.
1. Peripheral thermoreceptors are located under the skin and cause our perception of external temperature. Most thermoreceptors by far are peripheral. There are 2 temperature types: warm and cold, see above. My textbook mentions 6 different channels, two of which increase firing rate with cooling, and four that increase firing rate with warming.
2. Central thermoreceptors are located more internally in the body, namely in the medial pre-optic area of the hypothalamus. These change their firing rate in response to small changes in core temperature (3 temperature types: see source).
So, in your example of a hot water head bath, due to the sudden change in temperature near the skin surface the peripheral thermoreceptors under the skin would dramatically change their firing rate (increase or decrease depending on type of receptor). After a few seconds pass and temperature stays the same, the initial receptor response is attenuated as the receptors adapt to the new temperature.
The central thermoreceptors only change their firing rate with a change in core body temperature, and I am unsure whether this is the case with a hot water head bath. I am not a hot water bath expert, but maybe core temperature is only affected after an extended period of time or with a very hot bath! I guess you could test this by measuring core temperature during the hot water bath.
Hope this helps! Good luck.
If you need to know more, like ion channel properties or their exact location under the skin, I suggest you look at some textbooks or articles.
General source with a few pages on this subject: Neuroscience: Exploring the Brain (3rd ed.) by Bear, Connors, and Paradiso (2007).
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We are writing up on discussions for a paper. I was quite unsuccessful to find the actual sizes of these neurons (range). Can anyone with significant expertise suggest what the sizes are of these cell types on average. Even a ratio would be okay eg, cortical are x-times smaller than dopamine.... etc.,
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reliable information on cortical neuros in the classical paper of Haug: Am J Anat. 1987 Oct;180(2):126-42.
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Hi. I am planning on doing unbiased counting of TH positive neurons in the substantia nigra region in our animal model. I would like to know if there are any good software out there to do unbiased counting. 
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There are some plugins for ImageJ that allow automatic cell counting and, best of all, they are free so might be worth trying out in your system. The ones I know of are:
I'm not sure how well either package works. I used automated detection once in the Fiji distribution of ImageJ (
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I just posted this in the CLARITY resource center forum, but posting it here too. We found this hydrogel perfused/embedded right hemisphere of a rat cortex to be nearly completely clear after sitting in clearing solution in 37 degree incubator for ~5 weeks completely unattended. Next to zero tissue damage and it looks by far better than any brain that we have run through our ETC chamber. Has anyone else seen this?
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I have done passive clearing on rodent brain slices, 2-3mm thick. Works like a charm. Electeophoresis induced too much damage, I found. I tried electrophoresis on a half hemisphere, and it just looked like a yellow loogie in the end, despite temperature and voltage being low (~35C and 20V respectively). I also did passive clearing on a larger chunk (about 2cm^3), and a half hemisphere, but at a 4% bis-acrylamide concentration it took almost 4 months. However, it was not at 37C most of the time, and I somewhat neglected it. No damage, looks great, just slow. The slices took about 3 weeks at 37C if it was below 3% (closer to 4-5 weeks for 3-4%), and it worked beautifully (Without changing the clearing solution at all). I do antibody staining for about 4 days on the slices.
I tried a 0.5% concentration on entire half hemispheres passively, but its not really clearing, just slowly disintegrating..
So far the passive approach on thick slices is a clear winner for me. 
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CHALLENGE to my Neuroscience, Neurophysiology, Neuroanatomy, Neuroengineering friends: I am looking for some papers that quantify the spinal subarachnoid space geometry in primates using MRI or other methods? Any suggestions? I can't find a single paper with information on this on google scholar or Pubmed after quite some time searching.
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Can you be a little more specific about what information you are seeking? We have conducted several MRI studies for stereotaxic targeting, and in general the cisterns resemble those of humans. Are you trying to determine the CSF volume?
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