Hi Mohammad,

We usually take this distance into account, and adjust coordinates accordingly (we also use SD rats). However, I've found that anatomical landmarks on the skull and brain (e.g., blood vessels) are very important for hitting specific targets, so I take that into account when following coordinates (whether absolute or adjusted).

Good luck!

Hey Roni!

So for example if you wanna go 4mm anterior from the Lambda of a rat with 8mm of distance between Bregma-Lambda you go 3.5 (4*8/9) instead of 4? I did not get your point about the landmarks and I'd appreciate if you elaborate on that. 

Thanks for your response.

Yes, in general that is what I do. However, hitting the right location sometimes requires more than blindly following coordinates, and therefore I find it important to get to know some features of the skull (e.g., sutures) and brain (e.g., blood vessels on the surface of the cortex) around my area of interest. I then use the surgical microscope to visually validate that things look right when I follow coordinates. Sometimes you come across a "weird" rat, in which you would have to make a decision which landmarks to follow. For example, you may decide you don't want to adjust your coordinates according to the bregma-lambda distance, because the cortical blood vessels don't look right when you do that.

Hope this helps

One has to take into account, that one can only validate the target by post-mortem histology. I am aware, that nobody talks about this, but precision below 1/10mm is not easyly achieved with current surgical systems. If you are honest about the procedure, one has to add errors of the atlas, the precision (and stability) of the stereotax frame, the biological spread, brain shift (at least in bigger animals) and individual anatomy into account. That means, a linear correction of target coordinates (as you calculated above) is sufficient - given that you check afterwards. 

I add a paper trying to improve the hardware precision by a robotic system - and we work on an in vivo electrophysiological atlas for the rat brain.... It'l take a wile. 

Hi,

It's true that you will allways fall into an error of 100 um. And also that Paxinos works with this error for rats weighting 270-310 g. The authors argue the atlas can be successfully used also in rats weighting 250-350 g. Take also in account that  the brain does not change that much in adult rats and even if you're correcting by size you'll commit the same kind of error of 100 um.  In the atlas they argue same type of error is commited independently of sex and strain, including spargue-dawley. Hope it helps!!

سلام. با تقسیم فاصله برگما لامبدا موش جراحی بر 9 در اطلس اعداد شما اصلاح می شوند

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Roni! I are there any other landmarks other than the coronal suture, lambdoid suture, sagittal suture and the gusher artery beneath the sagittal suture that can be helpful?

It really depends on your area of interest. I usually work in the cerebellum, where I have gotten to know the folia and the blood vessels on the surface of the cerebellar cortex. Exactly which vessels you see depends on where specifically you're looking (the craniotomy I perform is usually not wider than 1 mm). Of course, this requires some trial and error - I start by going according to atlas coordinates, I take notes of what I'm seeing under the surgical microscope (sometimes I also try to learn the way in terms of electrophysiological activity, but that's another story...), and then I do the histology and see where I actually was. Next time I will know better where to go, and thus slowly learn the way and become more accurate.

I hope these explanations make sense - hitting a very specific spot can definitely be somewhat of an art...

Dr. Hofmann! Thank you very much for your answer. On a different note! I was wondering where are the discrepancies between the Watson-Paxinos atlas, for rats, and what we actually get are coming form. For example, the coordinates where you can record DA neurons of the VTA, is AP: 3.2-3.6 from Lambda and laterality is 0.6-1.0. But the VTA in the atlas is more medial than that! I am also very curious to know how the in vivo electrophysiological atlas, that you mentioned, works.

Note that there are even substantial differences between different editions of Paxinos and Watson....

Dear Marcel! I did not get your point and I'd really appreciate if you elaborate on that.

Good to know Roni! So does it mean the more recent its been published it's better or it depends on the structure that you are targeting? I'm using a 3rd edition printed in 1998.

I did not notice that one edition is substantially better than others, but the most recent I have is 2001...

The most recent one is 2007 edition

I am agreeing with the view of Susan that even for rats of the same weight/size there can be slight differences in neuroanatomy from batch to batch. Moreover, each individual has different perception towards measurements of co ordinates. Basically the reported co ordinates we can just use for reference and we have to standardize by ourselves how we are hitting our target area. Anyways we have to spare few rats for this important part.

Yes, the parameters are to be modified accordingly.

We usually measure the depth of the bregam and lamda to ensure leveled skull. We use rats that are > 250 g for brain surgeries when the skull length barely changes as body weight increases. We don't normally adjust the coordinates unless the rats are substantially smaller. And we always verify the placement histologically after each experiment. I agree with Susan that the "offsite" control group actually makes your data stronger.

The 2007 atlas, in my opinion, is not as accurate as the old versions at some locations, while other parts may be better. You really have to compare the dissected brain sections with different versions to make that judgement call.

I agree with previous answers: the best way to reach the target is to do some pilot studies (with microinfusion of comonly used dyes and post-mortem analyses) . However, if the distance between bregma and lambda is a problem with only one or two animals then just apply the traditional method for benchmarks determination (http://www.springer.com/medicine/surgery/book/978-2-8178-0471-2).

Thanks Barbara! I did not get the second part your argument. What I am trying to figure out are the coordinates you start with, the phase before the post-mortem verification. So when you want to start with a rat, and lets say the B-L distance is 1mm off, is it a good idea to modify the coordinates accordingly or not?

First: What is the weigh of your animals?

They are Sprague-Dawley rats  and they weigh 300g (+-20) but once in a while there is a rat in the range of 280g that have a bregma-lambda distance of around 8.2. 

Mo

Are you abolsutely sure you take the right point of reference for lambda ?

In my experience, a distance of 8.2 mm in smaller Sprague-Dawleys is not so rare (although in 90% of the cases I would expect the distance to be in the range of 8.5-9.3 mm for rats weighing 200-600 grams - with weight correlating with distance, obviously)

So Barbara! they way I measure the lambda is by getting the average of these two points:

1- Where the lambdoid suture intersects with the mid-line

2- Where an imaginary line that cuts the distance of the mid-line and the left/right margin of the skull into half, intersects with the lamboid suture

That average represents the AP of my lambda but for the laterality  of lamda I analyze the mid-line.   

Mo

Give me your professional email address so I can send you a figure.

Barbara

Thanks Barbara! It's so kind of you! It is [email protected]. I have attached a figure, to this comment, showing the two points ,that I mentioned in my previous comment, in red circles. 

Mo

I am gonna send you a note 2morrow morning with a coment on the way you calculate your coordinates;  I see your references are not rightly taken (see the book I gave you the references of , it is very helpful in that case). Talk to you on tuesday

Barb

Dear Mohammad,

I am sorry to be so late. I was just reinforcing Ulrich's point about the limitations of stereotaxic measures.

Regards

For adult rats, the distance between bregma and lamda is 9 mm (Paxinos). From my experience, this distance is between 8 to 8.5 mm for 3 or 4 weeks old SDs. So the scaling factor = measured bregma-lamda distance/9. Then multiply with this factor to the coordinates of an brain structure mentioned in the adult rat atlas. It works quite well for me. I think i gave the answer of your question in this example. Good luck

I work with Female Wistar rats (230-250g). I usually measure the distance between bregma and lamba which is always in between 8 to 8.6 mm and rarely it is above 8.6mm. I used to clibrate the coordinates according to bregma-lambda distance. It was working quite good in my case. I used to calibrate like this..bregma-lambda distance/9 * x-coordinate (X=AP; ML; DL). Hope this helps you.

am also interested in Bryony's question if anyone has an answer, is the linear correction needed for all X,Y,Z coordinates..thanks

Yes, it needs to be adjusted as per equation you gave. 

Hi Dr. Barbara Ferry and Dr. Ebrahimzadeh,

Would you mind sharing with me the reference and or figure that Dr. Ferry sent you to calculate the coordinates. This is my professional email [email protected].

Thank you,

Juan Vasquez

I was thinking of using a similar method in mice and found this thread. Does anyone know the bregma lamda distance used in the Paxinos mouse atlas ("X1")? I'll then divide my measured bregma lamda distance (X2) by X1. This factor (X2/X1) will be multiplied by all the coordinates in Paxinos.

Yes, injections are not perfect, but certainly we can improve!

I'm also wondering how people control for skull thickness (mainly the distance between the outermost surface of the skull and brain). Most coordinates are relative to the outermost surface of the brain at bregma, but it is more of challenge to determine the brain's surface than say skull. It would therefore be helpful to know roughly how big the space is. I'm guessing at least 1 millimeter.