Stereotactic Surgery - Science topic

I need to make two consecutive stereotactic injections in the same area of the midbrain of a mouse. How can I do this correctly without traumatizing the mice? Are there tools to maintain drilled holes in the mouse skull for repeated injections?

I will attempt to keep this as concise as possible, as this has been a toilsome endeavor.

1st issue:

Our coordinates never exactly match up with what the brain atlas says. The virus I inject almost always appears to be more dorsal than intended. It is to the extent that I have aimed towards the bottom of the basolateral amygdala and I have barely reached the top of the BLA. I purposefully picked -1.3mm for the anterior/posterior coordinates and +- 3.0 mm for the medial/lateral coordinates as this part of the brain atlas showed smaller fiber tracts to make it through and it was easier to make it around the ventricle. This also gets me some wiggle room as the BLA is fairly large here and neighboring anterior/posterior coordinates.

2nd issue:

We also seem to have sporadic unilateral injections even though we are doing bilateral injection.

I draw up 950nl into a Hamilton syringe. I eject 50 nl to check virus has been drawn up by the virus. After injecting over 5 minutes, I let it diffuse for 10 minutes. Then I pull the needle out and eject 50 nl to ensure that the needle wasn't clogged. I do my 2nd injection. Repeat injection and diffusion times. Eject 50 nl and check to ensure it wasn't clogged. So I dont understand, how it is even possible to have a unilateral injection.....unless it is getting caught in a ventricle?

If anyone has any thoughts about what it could be or even thoughts about how I could better troubleshoot, please let me know.

We tried different methods. The problem consists of the absorption of our colour marker by the brain tissue. We want to reach to the basal forebrain, 5.5 mm deep from above by cranial stereotactic surgery with canulas of 0.43 mm diameter. For those trials we kill the mice and do the surgery post mortem.

Coating the glass fibre canulas with ink or rhodamine solution didn't work so far, it seems to have been wiped off while entering the brain tissue. We cannot see the hole/tunnel made by the glass fiber canula, probably because the brain tissue wobbles back into place after ejection of the canula.

I recently started learning stereotaxic injections in mice, we will be targeting the Medulla but I started with practicing ICV to get used to the process using Evan's Blue Dye 1:100, and I had a very few success and too many failures. I don't have trouble with fixating the head as when I started, and I think I don't have trouble reading the coordinates measurements in the Vernier scale.

The coordinates we use for right ICV injection are, from the Bregma:

Most of the time I get results as below, what might be the reason?

Thanks a lot in advance.

Hi, I am Joe and currently, I am working in a neuroscience lab using the optogenetic technique. And I have some technical problem with that, which the AAV always injected off target, for example when I inject the AAV in the BLA. However, there is always a lack to the CeA and the injected passage which bothers me a lot.

Therefore I would like to ask if there are any tips to prevent the situation that I mentioned? Thank you so much for your help.

Surgical lesioning, like thalamotomy, is usually targeted using AC-PC coordinates. Does anyone know of thalamotomy coordinates in MNI space?

Hi All,

I have been running into a technical issue when I try to cannulate the prelimbic cortex of mice. I find that I am about .1-.2 off from being even (see the attached pictures). I am using bilateral cannula with .8 mm between the tubing. I found coordinates (AP: 1.78, ML: ± 0.4, DV: -1.5) that get me to the right place A/P-wise but my problem is M/L. I am careful with how I place the mouse in the stereotaxic frame and I check bregma and lambda prior to drilling my screw and cannula holes to make sure the brain is level. I suspect I am setting my reference (i.e. bregma) incorrectly but I don't see how. Has anyone experienced this and if so, how did it you fix it?

Hi, I am currently learning to do electrophysiological recording on mice visual cortex, and sometimes the cortex bleeds and swells after the craniotomy and removal of the meninges. I want to know if bleeding is the major cause of the swelling cortex, and how to prevent the cortex from swelling.

Hello, I'm recording neurons in vivo in the ventral tegmental area of rat brains using stereotaxic equipment. What is an effective way of determining whether my equipment is accurately horizontal and/or perpendicular with relation to the animal other than just "eye-balling" it? The VTA is 7-8 mm deep and I'm attempting to target an area that is roughly 0.4mm wide and 0.8 mm long so even small deviations from being being perfectly perpendicular will have an effect.

A related question: how can I ensure that the head of the animal is properly secured in a position that is perfectly horizontal?

Any help is greatly appreciated. Thank you.

Hi everyone, 

I am performing stereotactic surgery for mPFC in CD1 mice. The coordinates that I am trying to use is AP: +1.9, ML: +/- 0.3, DV: -2.5. Apart from this antero-posterior coordinates, I have also tried +1.6, +1.7 and +1.8. But in all these cases, I see enormous blood while inserting the cannula or even the injector. I see so many papers injecting drugs, lentivirus etc in mPFC using same coordinates, but none say a word about the blood. If anyone has experience on this, please share your experience here. Thanks! 

Instrument would be used for experimentation in mice.

I am trying to find a reference describing the accurate method to locate the lateral ventricles for a female Sprague dawley rats using stereotaxic apparatus.

The setup is isoflurane with passive scavenging through an activated charcoal filter. These mice are Parvalbumin Cre (+) and (-). Mice are placed on a heating pad throughout surgery. There also is a lamp about a foot and a half above the surgery table that can get pretty hot. I'm not sure if this is the problem though. The mice have no problem going down. Their breathing is normal up until about an hour to an hour and a half. Their breathing becomes labored, they start pooping, and their muscles become rigid. Although their breathing gets lighter, they do not respond to a tail or toe pinch reflex. After about 5-10 minutes of this labored breathing, they die. The isoflurane level is between 0.5 and 2 during surgery.

Do you think it is a problem with the isoflurane chamber, hyperthermia, buildup up isoflurane waste, or something else? Any help would be greatly appreciated. I have attached a video of the labored breathing with the mouse not responding to a touch pinch.

I am injecting 200nL of a vasoconstrictor into mouse brains, trying to hit a very precise area.  Can I inject a small amount of india ink with my vasoconstrictor as a way to see post-tissue processing if the injection is exactly where I need it?  I am doing survival up to 3 weeks, so would india ink be stable and not interfere with flourescent IHC over that time frame? 

Has anyone performed stereotaxic surgeries into the BLA from an angle rather than straight down? I would like to angle the injection so that i do not damage the striatum, but don't know a good angle to approach the BLA. 

I need to make sure whether I did correctly in the surgery. I need to implant tetrodes in the mPFC of 4 month old rats. I have a confusion about dura removal. After removing a small piece of dura, I could see the thick white layer shown in the attached file. And when I removed the white layer, I could see clearly the vessels. Here comes the problem. Sometimes, even after removing the white layer, my tetrodes still couldn't get through. I have to remove another transparent thin layer containing vessels. But sometimes, my tetrodes could get through without removing the transparent vessel layer. So I am thinking whether below the white thick layer, there are acturally two transparent layers. For tetrodes implantation, I need to remove the first transparent layer and leave the second transparent vessel layer?  But in my operation, sometimes I remove both transparent layers and cause bleeding.  I searched the structure of meninges, and I got to know dura consisted of two parts. So I am curious the white thick layer is only one part of dura or what? And what about the arachnoid and pia mater? I couldn't see them under microscope and I don't need to remove them for my tetrodes, right?

I am seeking a protocol or advice on expression in vivo. I am currently using a GEG Tech lentiviral vector containting GFP and a NMDAR subunit gene. I have a physical titer of 4.5E6 RNA genome/uL and an efficient titer of 1.9 E9 Transduction Units/mL. I have not been able to detect any GFP expression, and would like to rule out delivery as the problem. I have been experimenting with volumes ranging from .5, 1, 1.5, 2ul per injection site. Injections are done manually using a micropipette with a manual delivery system. I typically allow 5-10 minutes for viral absorbtion before removing the needle. In histology of the injection site, I see granules that fluoresce in all spectra, but these do not seem to be cells. Any advice or wisdom about lentiviral injection is appreciated.

For example, if I inject 1 uL of drug solution directly to the primary visual cortex, what is the dispersal volume for which I can expect a drug effect?

1 mm^3 ?  

5 mm^3 ?

References appreciated.

I am injecting AAV cre into the dorsal hippocampus in 4-5 week old mice. The coordinates I have are -1.5mm [AP], 1mm [lat], 1.5 mm [ventral]. However these coordinates are for adult mice, and I want to inject in 4-5 week old mice. I'd like a reference point to where I should first try injecting for the dorsal hippocampus. 

It would help me if there was a paper that did stereotaxic injections in young, and adult mice and what the coordinates were for those two mice. I had a difficult time finding a paper that had this.  

Need to inject ASOs into 4 weeks mice. Any suggestions?

I am working on an endogenous brain tumor model and will induce tumor formation by stereotaxic injection.

Part of the treatment regimen will also be delivered via stereotaxic injection into the tumor tissues - is it possible to perform this procedure twice in the same area within 2-6 weeks? 

I am planning to perform a pharmacological study to assess hippocampal neurogenesis via IGF-1 receptor inhibitor. But most studies were performed  in vitro or in vivo (systemic). So whether this inhibitor of IGF-1R (such as AG-1024 or picropodophyllin) can be  intracranially injected by stereotaxic localization and what is the optimal dose? Thank you!

Thanks in advance for your replies.

I will use it for functional and stereotactic surgery planning.

I use a CRW frame with which I use a very simple software for planning, and I am in need of a more sophisticated and low priced software.

I'm trying to find a device to be attached to my stereotactic to perform bilateral injections at once.

Our lab is about to purchase several new microscopes for implantations and microinjections to mouse brain, and we've never really been thrilled with the scopes we've had at our disposal. I'd appreciate if anyone has recommendations of scopes they enjoy using.

We are developing such system and we are intersted in similar developments.