Neuroanatomy - Science topic

David J Daegling oddly it seems to be missing nearly all the sesamoid bones!

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.

Sorry. Here are files I failed to attach first time.

This is what we have studied as regards PD:

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.

Hans-Ulrich Iselin

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.

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.

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

Interesting discussion that I just found!

When I was performing stereotaxic surgeries on my rats either for cannulation or for finding the exact coordination to insert my recording and stimulation electrodes, I came across the intricate difference of lambda and so called true lambda.

As I had been told that no significant difference would it make to choose either, I was dubious enough to do my own trial and error. I removed the brains of those that I performed surgery with experimental lambda, dissected 50 um coronal sections through the region of cannulae and electrode insertion, and compared them with those that I did surgery with true lambda under a microscope. Even though the difference was insignificant for large areas like hippocampal formation, it was remarkable for small nuclei like the VTA or NAc (core).

Unsurprisingly, the anatomical difference that is something around 0.9 um-1 mm on the skull surface, is considered significant in terms of brain size and scale ratio of rat brain.

I think that considering lambda or true lambda is the matter of the ratio or scale of the target region size to the whole brain. But to be meticulous the middle point of the interaural line or the imaginary midline of the lamdoid suture is a safe and sure point to estimate the distance to bregma and lay your calculations for the rest of the procedure.

using DAPI to do counter staining

Remember both PTSD and major depression can produce significant cognitive impairment including issues with attention equal to ADHD.

Excellent opinions ...I think all in respect to the general goal behind the question...

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

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

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,

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.

Encyclopedia of Infant and Early Childhood Development

2008, Pages 343–356

Fractionating theory of mind: A meta-analysis of functional brain imaging studies. Matthias Schurz. Neuroscience & Biobehavioral Reviews. Volume 42, May 2014, Pages 9–34

Best regards

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! 

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. 

I think you can try Immunohistochemistry 

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.

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: 

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!

Have you tried searching pubmed? There are over 100 articles related to this:

https://www.ncbi.nlm.nih.gov/pubmed/?term=medial+entorhinal+cortex+grid 

https://www.ncbi.nlm.nih.gov/pubmed/?term=medial+entorhinal+cortex+grid+navigation 

https://www.ncbi.nlm.nih.gov/pubmed/?term=medial+entorhinal+cortex+grid+position+orientation 

Jerome

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

Dear all,

Thank you for your replies. Together with all these different sources and the individual papers, I am making much progress!

Thanks,

Jesse

Hello Yiran Xu

please check the pdf and links pertaining to your topic

I used Neurolucida neuron tracing Software (MBF Bioscience)

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.

I agree with Shafagat Mahmudova good references. thanks

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!

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

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.

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

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. 

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.

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    

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

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!

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.

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! 

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

It appears that primary murine microglia produce MMP-9 precursors (proMMP-9) when exposed to vitronectin and fibronectin.

(http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3345906/)

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.

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).

 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 :) 

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

@ 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.

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.

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!

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.

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.

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

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.

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

These might be helpful

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.

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.

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.

Dear Daniela,

This maybe should be helpful.

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

Please search.

Dendrites with varicosities along their length are sometimes referred to as moniliform.  Electon microscopic examination shows that these varicosities are filled with mitochondria.

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.

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

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...)

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).

Source for central thermoreceptors: http://link.springer.com/article/10.1007%2Fs11055-007-0167-4#page-2

reliable information on cortical neuros in the classical paper of Haug: Am J Anat. 1987 Oct;180(2):126-42.

Hi! You may use ImageJ software, which is free and essentially performing as well as expensive commercial solutions. Following links on the website you will find multiple tool packages for different applications including automated cell counting. Certainly, documentation and support is better in commercial software. The same often applies to user-friendlyness. You will have to decide, whether this is worth several thousand dollars. I would suggest to download some free trial versions and compare them to the free imageJ as a basis for your decision.

Best, Just

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. 

I think you will find these articles interesting

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?

For many years I used S35 radioactive or digoxigenin labeled probes but in the past 6 months have been using the RNAscope technology that Justin Smith mentioned above.  This is not an inexpensive method but it is undoubtedly the very best in terms of specificity and simultaneous localization of multiple RNA targets in the same cell. I do not think that I will ever use S35 or digoxigenin again.

Thanks, I am going to try this, sounds very reasonable.