National Natural Science Foundation of China (grant NO. 82101249 to XY Sun), Postdoctoral Research Foundation of China (grant NO. 2022M722125 to XY Sun). Shanghai Sailing Program (grant NO. 21YF1425100 to SH Chen). Special Project for Clinical Research of Shanghai Municipal Health Commission (grant NO. 202340088 to J Zhou). National Natural Science Foundation of China (grant NO. 82101262 to X Zhang, grant NO. 82101287 to SH Chen).

All animal experiments were conducted in compliance with the Animal Research Reporting In Vivo Experiments (ARRIVE) guidelines and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The present study was approved by the Animal Care and Use Committee of Renji Hospital, Shanghai Jiaotong University School of Medicine. Eight-week-old C57BL/6J male mice were used for the present study. The animals were commercially obtained (see Table of Materials) and housed in standard cages (22 &#176;C &#177; 2 &#176;C, 12 h/12 h light/dark cycle, food, and water ad libitum).
1. Selection of target brain regions
NOTE: This is a sterile procedure. Ensure all surgical instruments are sterilized. Spray the gloves with alcohol prior to contact with surgical instruments. Avoid touching the tip of the instruments or the surgical area with gloves. Ensure the mouse is adequately anesthetized before starting the procedure by checking for the loss of righting reflexes and no response to cutaneous stimulation. Before the drilling and injection, closely monitor the respiratory rate (60-220 breaths per min, average 100-150 breaths per min) and the breathing amplitude (should be consistent and not excessively shallow or deep). Any significant changes in the respiratory rate or amplitude should be noted.
<ol>
	<li>Anesthetize the mouse with an intraperitoneal injection of 1.25% tribromoethanol (250 mg/kg) using a 29 G needle. Shave the hair on the skull once the righting reflex disappears. Secure the mouse in the stereotaxic frame (see Table of Materials) by fixing the upper incisors with a nose clamp and stabilizing the head using two ear bars.</li>
	<li>Apply ophthalmic ointment to prevent eye dryness. Disinfect the scalp by swabbing it three times with Anerdian (disinfectant solution; see Table of Materials). Inject 0.2 mL of 1% lidocaine subcutaneously using a 29 G insulin needle to provide local analgesia.</li>
	<li>Make a 0.5 cm incision using eye scissors to expose the cranium. Utilize a cotton swab dipped with 3% H2O2 to scrub the exposed skull to remove the periosteum. Allow the cranium to dry.</li>
	<li>Affix the drill to the stereotaxic instrument. Adjust the stereotaxic arms&#39; knobs to position the drill tip precisely at the bregma with the help of a magnifier and record the z-axis value using the LCD digital display.
	<ol>
		<li>Adjust the knobs and position the drill tip at the lambda and record the value of the z-axis. Adjust the nose clamp and ear bars to make two z-values closer, and repeat the steps above until the difference between the two z-values is less than 0.1 mm. 
		NOTE: The x, y, and z axes on the LCD digital display correspond to mediolateral (ML), anteroposterior (AP), and dorsoventral (DV), respectively. The bregma and lambda can be considered to be on the same horizontal plane once the difference between the two z-values is less than 0.1 mm.</li>
	</ol>
	</li>
	<li>Ensure the left-right levelness of the brain. Adjust the ear bars attached to the stereotaxic frame with the same scale. Take the laterodorsal tegmental nucleus, ventral part (LDTgV), for example; its stereotaxic coordinates are -5.2 for x, +0.8 for y, -4.0 for z, according to Paxinos and Franklin&#39;s mouse brain atlas16.
	<ol>
		<li>Adjust the ear bar to make the drill at (-5.2, +0.8) to measure the z value in the dorsal-ventral direction and move the drill to (-5.2, -0.8) to measure the z value. 
		NOTE: The left-right is considered to be at the equal level when the difference between the two z-values is less than 0.2 mm. The ear bars need to be inserted into the right place because the external auditory meatus is symmetric. If the difference between the two z-values is more than 0.5 mm, the ear bar may be inserted into the wrong place.</li>
	</ol>
	</li>
	<li>Position the drill to the bregma again and set three coordinates on the LCD digital display to zero. Position the drill above the region of interest using reference coordinates. Start the drill.
	<ol>
		<li>Gradually lower the drill using the vertical arm until it penetrates the cranium. Clear away debris and blood using cotton swabs. 
		NOTE: Be careful not to damage brain tissue.</li>
	</ol>
	</li>
</ol>
2. Preparing dye solution and brain microinjection
<ol>
	<li>Take 25 &#181;L of SDS-PAGE loading buffer containing bromophenol blue (see Table of Materials) into a 200 &#181;L microcentrifuge tube. Add 50 &#181;L of ddH2O to the tube to prepare the dye solution.</li>
	<li>Cover the cranial window with a saline-moistened cotton pad. Ensure microsyringe patency by aspirating and expelling saline repeatedly. Load 0.3 &#181;L of dye into the micro syringe. Attach the syringe and the motorized stereotaxic microinjector to the stereotaxic arm.</li>
	<li>Position the syringe tip at the bregma and set the coordinates to zero on the LCD digital display. Adjust the needle into the region of interest according to reference coordinates. Set the injection speed to 0.1 &#181;L/min and start the motorized microinjector, delivering 0.3 &#181;L of blue dye into the target region. Leave the syringe in position for at least 10 min before slowly withdrawing the injector. 
	NOTE: Different color dyes can be used in the same mice to distinguish closely located regions. Test other dyes for suitable concentrations based on their physical properties. It&#39;s recommended to use the bromophenol blue formula and thoroughly rinse the syringe before and after use to prevent blockages. Adjust injection volumes based on dye diffusion.</li>
</ol>
3. Brain tissue harvest and cryosectioning
<ol>
	<li>Release the mouse from the stereotaxic frame. Anesthetize the mouse with an intraperitoneal injection of 50 mg/kg pentobarbital sodium using a 29 G needle. Secure the mouse on a foam board with tape.</li>
	<li>Open both sides of the thorax to induce rapid suffocation. Make a 1-2 mm incision in the right atrial appendage. Insert an infusion needle into the left ventricle. Infuse 20 mL of pre-cooled saline and 20 mL of iced 4% paraformaldehyde solution.</li>
	<li>Decapitate the mouse with tissue dissecting scissors (following institutionally approved protocols). Remove the skin by making an incision along the midline from the neck to the nose and then expose the skull. Clear off residual muscle on the skull with forceps or scissors.</li>
	<li>Position the tip of one blade of the fine scissors between the foramen magnum and the brain, with the sharp edge facing the bone, at the rostral end of the mid-sagittal suture. Proceed to slide and cut the cranium along the mid-sagittal suture. Employ forceps to remove the skull, revealing the exposed brain. 
	NOTE: Exercise caution to avoid causing any damage to the brain during the operation. Before removing the bone chips, carefully peel off any meninges that may be attached to the skull. This precaution is crucial to prevent inadvertent slicing of the brain tissue during the process.</li>
	<li>Turn on the power of the rotary microtome cryostat (see Table of Materials) and set the chamber temperature to -20 &#176;C. Pour a little OCT compound to cover the surface of the specimen disc evenly. Place it into the cryo-chamber and allow the OCT to solidify.</li>
	<li>Attach the disc to the specimen head. Bring the blade closer to the block and trim the OCT block to make a flat plane that is parallel to the disc.</li>
	<li>Cut the brain in a coronal direction away from the injection site in a brain slice mold by a shaving blade. Place the brain tissue containing the injection site with the cut side down on the OCT flat plane.
	<ol>
		<li>Pour OCT onto the brain and put the disc into the cryo-chamber, allowing the specimen block to solidify. Pour OCT repeatedly onto the brain until the brain is fully embedded with OCT. 
		NOTE: Add the OCT compound layer by layer. Once the previous layer of OCT is frozen, pour the subsequent layer of OCT compound onto the brain.</li>
	</ol>
	</li>
	<li>Trim the specimen block until the target region is approaching. Make several sections from the injection site level. Collect the coronal brain slices into a 6-well plate containing room-temperature PBS using a small writing brush kept at -20 &#176;C. 
	NOTE: Set the cutting thickness to a larger one to trim the block, and then set the cutting thickness to 40 &#181;m as the blade approaches the injection site. If neural circuit tracers are used, the mice are not sacrificed immediately. Place the mouse in a sterile, postoperative recovery cage over a heating blanket for recovery from anesthesia. Return the mice to the original cage or a new cage once awake.</li>
</ol>
4. Imaging
<ol>
	<li>Wash the OCT with room temperature PBS. Use a brush to pick up brain sections and place them on a slide. Allow sections to air dry at room temperature.</li>
	<li>Observe the injected region visually or use a microscope when the target nuclei are minuscule. Compare the location of the injected dye with the corresponding region in the brain atlas. 
	NOTE: Adjust the values of the x, y, and z axes according to the direction and distance of the injection point deviation. It may be necessary to repeat all steps in this protocol multiple times to determine the coordinates accurately.</li>
</ol>

Optimizing Stereotaxic Microinjection Accuracy in Mice

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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anterior+hypothalamic+parvalbumin+neurons+are+glutamatergic+and+promote+escape+behavior.">Anterior hypothalamic parvalbumin neurons are glutamatergic and promote escape behavior.</a> Curr Biol. 33 (15), 3215-3228 (2023).</li><li>Perens, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodal+3D+mouse+brain+atlas+framework+with+the+skull-derived+coordinate+system.">Multimodal 3D mouse brain atlas framework with the skull-derived coordinate system.</a> Neuroinformatics. 21 (2), 269-286 (2023).</li><li>Adhikari, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basomedial+amygdala+mediates+top-down+control+of+anxiety+and+fear.">Basomedial amygdala mediates top-down control of anxiety and fear.</a> Nature. 527 (7577), 179-185 (2015).</li><li>Tao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projections+from+infralimbic+cortex+to+paraventricular+thalamus+mediate+fear+extinction+retrieval.">Projections from infralimbic cortex to paraventricular thalamus mediate fear extinction retrieval.</a> Neurosci Bull. 37 (2), 229-241 (2021).</li><li>Haggerty, D. 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The authors declare no competing interests.

This article has described a stable strategy to verify the accuracy of stereotaxic brain injections5,6more quickly and simply before viral tracing, but the irreplaceable aspect of reporter gene expression in the brain region is crucial for brain region labeling. The blue dye we used allowed for the immediate visualization of the injection site.
Several critical steps in this protocol contribute to improving the accuracy of the injectio...

Nearly all modern neuromodulation tools, including in vivo calcium recording, optogenetic, and chemogenetic tools, require the use of stereotaxic coordinates to target the brain area of interest1,2,3, forming the foundation of neural manipulation. Stereotaxic coordinates for mouse brain regions are defined in relation to bregma and lambda, the bony landmarks on the cranium, forming the so-called skull-derived stereotaxic coordinate system. Either bregma or lambda can serve as the zero point of the three-dimensional coordinates. The three axes are anteroposterior (AP), mediolateral (ML), and dorsoventral (DV), representing the y, x, and z axes on the digital display of stereotaxic instruments. For well-known brain regions, the stereotaxic coordinate parameters of a specific area can be obtained from mouse brain atlases4 (e.g., Paxinos and Franklin&#39;s mouse brain in stereotaxic coordinates) and/or the published literature5,6. However, further validation is necessary due to variations in stereotaxic equipment and the age/populations of mice used by different researchers.
Structure is the basis of function. Neural circuits form the foundation for many brain functions, such as cognitive activities, emotion, memory, sensory, and motor functions1. Labeling the structure and manipulating the activity of neural circuits are vital for understanding the function of a specific neural circuit. Over the past decades, neural tracers have evolved through many generations; early research adopted wheat germ agglutinin (WGA) and phaseolus vulgaris agglutinin (PHA) as anterograde tracers, and fluorogold (FG), cholera toxin subunit B (CTB), carbocyanine as retrograde tracers. However, unlike viral tracers, these traditional neural tracers cannot integrate exogenous genes into the host, nor do they have cell type selectivity. Nowadays, the viral strategy has become an important proposition during basic neuroscience research. For different research purposes, various viral tools can be selected7,8. There are non-transsynaptic viruses, trans-multisynaptic viruses (retrograde and anterograde), and trans-monosynaptic viruses (retrograde and anterograde). Each category contains several types with respective characteristics.
The process of viral administration and expression is highly time- and resource-intensive, often taking weeks or even longer. Among various viral vectors, adeno-associated virus has been identified as a promising means for gene delivery, providing a wide window ranging from 3 to 8 weeks post-injection for the experimental procedure7,8. As AAV evolves, analysis can be performed 2-3 weeks after administration9,10. Other neural circuit tracers, such as pseudorabies virus (PRV) and rabies virus (RV), also require a tracing period of at least 2-7 days11,12,13,14,15. Thus, a preliminary verification of the injection site before observing fluorescence signals is both time- and cost-effective.
To facilitate a simple and rapid verification of stereotaxic injections, in this study, a dye is administered before viral vectors, and cryosectioning enables researchers to observe the injection site and track it within 30 min post-injection.

<table><tbody><tr><td>1.0 &amp;micro;L, Neuros Syringe, Model 7001 KH, 32 G, Point Style 3</td><td>Hamilton</td><td>65458-01</td><td></td></tr><tr><td>200 &amp;mu;L pipette tips</td><td>biosharp</td><td>BS-200-T</td><td></td></tr><tr><td>20 mL syringe</td><td>Kindly group</td><td></td><td></td></tr><tr><td>3%H2O2 solution</td><td>Lircon Company</td><td></td><td></td></tr><tr><td>6-well plate</td><td>Shengyou Biotech</td><td>20006</td><td></td></tr><tr><td>Anerdian</td><td>Likang High-tech</td><td>31001002</td><td></td></tr><tr><td>Anti roll plate</td><td>Leica</td><td>14047742497</td><td></td></tr><tr><td>BD insulin syringe</td><td>Becton,Dickinson and Company</td><td>328421</td><td></td></tr><tr><td>Bend toothed dissecting forceps</td><td>Jinzhong</td><td>JD1050</td><td></td></tr><tr><td>Cellsens dimension software</td><td>Olympus</td><td></td><td></td></tr><tr><td>Cotton swab</td><td>Fisher Scientific</td><td>23-400-122</td><td></td></tr><tr><td>Dapi-Fluoromount-G</td><td>Southernbiotech</td><td>0100-20</td><td></td></tr><tr><td>Drill</td><td>Longxiang</td><td></td><td></td></tr><tr><td>Fine brushes</td><td>HWAHONG</td><td></td><td></td></tr><tr><td>Fine scissors</td><td>Jinzhong</td><td>y00030</td><td></td></tr><tr><td>Fluorescent microscopy</td><td>Olympus</td><td>BX63</td><td></td></tr><tr><td>freezing microtome</td><td>Leica</td><td>CM1950</td><td></td></tr><tr><td>Hemostatic forceps straight with tooth</td><td>Jinzhong</td><td>J31010</td><td></td></tr><tr><td>Infusion needle 0.7 mm</td><td>Kindly group</td><td></td><td></td></tr><tr><td>Lidocaine hydrochloride injection</td><td>Harvest Pharmaceutical Company</td><td>71230803</td><td></td></tr><tr><td>Magnifying glass</td><td>M&amp;amp;G Chenguang Stationery</td><td></td><td></td></tr><tr><td>Male C57/BL mice</td><td>The Shanghai Institute of Planned Parenthood Research&amp;ndash;BK Laboratory</td><td></td><td></td></tr><tr><td>Mice coronal brain slice mold</td><td>RWD Life Science</td><td>68713</td><td></td></tr><tr><td>Microcentrifuge tube</td><td>biosharp</td><td>BS-02-P</td><td></td></tr><tr><td>Microtome blades</td><td>Leica</td><td>819</td><td></td></tr><tr><td>Ophthalmic ointment</td><td>Cisen Pharmaceutical Company</td><td>G23HDM9M4S5</td><td></td></tr><tr><td>paraformaldehyde</td><td>Biosharp</td><td>BL539A</td><td></td></tr><tr><td>Peristaltic pumps</td><td>Harvard Apparatus</td><td>70-4507</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Servicebio</td><td>G4202</td><td></td></tr><tr><td>Piette 2-200 &amp;mu;L</td><td>thermofisher</td><td>4642080</td><td></td></tr><tr><td>SDS-PAGE sample loading containing bromophenol blue</td><td>Beyotime</td><td>P0015A</td><td></td></tr><tr><td>Shaving blades</td><td>BFYING</td><td>91560618</td><td></td></tr><tr><td>Slides</td><td>Citotest Scientific</td><td>188105</td><td></td></tr><tr><td>Stereotaxic apparatus</td><td>RWD Life Science</td><td>68807</td><td></td></tr><tr><td>Straight toothed dissecting forceps</td><td>Jinzhong</td><td>JD1060</td><td></td></tr><tr><td>Syringe Holder</td><td>RWD Life Science</td><td>68206</td><td></td></tr><tr><td>Tissue scissors</td><td>Jinzhong</td><td>J21040</td><td></td></tr><tr><td>Tissue-Tek O.C.T compound</td><td>Sakura</td><td>4583</td><td></td></tr><tr><td>Tribromoethanol</td><td>Aibei Biotechnology</td><td>M2910</td><td></td></tr></tbody></table>

preliminary validation of stereotaxic injection coordinates via cryosectioning

Stereotaxic injection of a specific brain region constitutes a fundamental experimental technique in basic neuroscience. Researchers commonly base their choice of stereotaxic injection parameters on mouse brain atlases or published materials that employed various populations/ages of mice and different stereotaxic equipment, necessitating further validation of the stereotaxic coordinate parameters. The efficacy of calcium imaging, chemogenetic, and optogenetic manipulations relies on the precise expression of reporter genes within the region of interest, often requiring several weeks of effort. Thus, it is a time-consuming task if the coordinates of the target brain region are not verified in advance. Using an appropriate dye instead of a virus and implementing cryosectioning, researchers can observe the injection site immediately following dye administration. This facilitates timely adjustments to coordinate parameters in cases where discrepancies exist between the actual injection site and the theoretical position. Such adjustments significantly enhance the accuracy of viral expression within the target region in subsequent experiments.

This study successfully identified the injection site within 30 min using the demonstrated method. Initially, an SDS-PAGE sample loading solution containing bromophenol blue was injected into the LDTgV in the C57/BL mice. Figure 1A shows a schematic representation of the dye solution injection. The distribution of the blue dye in the LDTgV is illustrated in Figure 1B.
Bromophenol blue was also injected into the mPFC prelimbic cortex, ...

Author Spotlight: Accelerating Cognitive Impairment Research in Mice Through Stereotaxic Injection and Cost-Effective Dyes

The present protocol describes a practical strategy to expedite the verification step of stereotaxic injection coordinates before conducting viral tracing using dyes and frozen sections.

Preliminary Validation of Stereotaxic Injection Coordinates via Cryosectioning

Stereotaxic injection of a specific brain region constitutes a fundamental experimental technique in basic neuroscience. Researchers commonly base their ...

preliminary-validation-stereotaxic-injection-coordinates-via

Research

JoVE Journal

Neuroscience

1.5K Views.  Shanghai Jiaotong University, School of Medicine. Ons onderzoek richt zich voornamelijk op het onderzoeken van het neurale circuitmechanisme dat ten grondslag ligt aan cognitieve stoornissen bij muizen. En we gebruiken vaak stereotaxische technieken voor de hersenen in deze studies. Ons doel is om het verificatieproces voor stereotaxische injectieplaatsen met kleurstoffen te optimaliseren en te versnellen, zodat het een eenvoudige en snelle procedure wordt.Verschillende strategieën zijn cruciaal geworden in neurowetenschappelijk onde...

Video: Auteur in de schijnwerpers: onderzoek naar cognitieve stoornissen bij muizen versnellen door middel van stereotaxische injectie en kosteneffectieve kleurstoffen

Rodent Stereotaxic Surgery and Animal Welfare Outcome Improvements for Behavioral Neuroscience

Stereotaxic surgery for the implantation of cannulae into specific brain regions has for many decades been a very successful experimental technique to investigate the effects of locally manipulated neurotransmitter and signaling pathways in awake, behaving animals. Moreover, the stereotaxic implantation of electrodes for electrophysiological stimulation and recording studies has been instrumental to our current understanding of neuroplasticity and brain networks in behaving animals. Ever-increasing knowledge about optimizing surgical techniques in rodents1-4, public awareness concerning animal welfare issues and stringent legislation (e.g., the 2010 European Union Directive on the use of laboratory animals5) prompted us to refine these surgical procedures, particularly with respect to implementing new procedures for oxygen supplementation and the continuous monitoring of blood oxygenation and heart rate levels during the surgery as well as introducing a standardized protocol for post-surgical care. Our observations indicate that these modifications resulted in an increased survival rate and an improvement in the general condition of the animals after surgery (e.g. less weight loss and a more active animal). This video presentation will show the general procedures involved in this type of stereotaxic surgery with special attention to our several modifications. We will illustrate these surgical procedures in rats, but it is also possible to perform this type of surgery in mice or other small laboratory animals by using special adaptors for the stereotaxic apparatus6.

Stereotaxic surgery on rodents allows for targeted administration of drugs or electrical stimulation and recordings in awake, behaving animals. In this video presentation we will demonstrate recent procedural refinements to this long-standing procedure that successfully improved survival rate and reduced post-surgical weight loss.

Stereotaxic surgery for the implantation of cannulae into specific brain regions has for many decades been a very successful experimental technique to ...

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

Operative Technique and Nuances for the Stereoelectroencephalographic (SEEG) Methodology Utilizing a Robotic Stereotactic Guidance System

The SEEG methodology has gained favor in North America over the last decade as a means of localizing the epileptogenic zone (EZ) prior to epilepsy surgery. Recently, the application of a robotic stereotactic guidance system for implantation of SEEG electrodes has become more popular in many epilepsy centers. The technique for the use of the robot requires extreme precision in the pre-surgical planning phase and then the technique is streamlined during the operative portion of the methodology, as the robot and surgeon work in concert to implant the electrodes. Herein is detailed precise operative methodology of using the robot to guide implantation of SEEG electrodes. A major limitation of the procedure, namely its heavy reliance on the ability to register the patient to a preoperative volumetric magnetic resonance image (MRI), is also discussed. Overall, this procedure has been shown to have a low morbidity rate and an extremely low mortality rate. The use of a robotic stereotactic guidance system for the implantation of SEEG electrodes is an efficient, fast, safe, and accurate alternative to conventional manual implantation strategies.

Operative Technique and Nuances for the Stereoelectroencephalographic SEEG Methodology Utilizing a Robotic Stereotactic Guidance System

The SEEG methodology is simplified and made faster with a stereotactic robot. Careful attention must be paid to the registration of the preoperative volumetric MRI to the patient prior to use of the robot in the OR. The robot streamlines the procedure, leading to decreased operative times and accurate implantations.

The SEEG methodology has gained favor in North America over the last decade as a means of localizing the epileptogenic zone (EZ) prior to epilepsy ...

Behavior

Investigating the Function of Deep Cortical and Subcortical Structures Using Stereotactic Electroencephalography: Lessons from the Anterior Cingulate Cortex

Stereotactic Electroencephalography (SEEG) is a technique used to localize seizure foci in patients with medically intractable epilepsy. This procedure involves the chronic placement of multiple depth electrodes into regions of the brain typically inaccessible via subdural grid electrode placement. SEEG thus provides a unique opportunity to investigate brain function. In this paper we demonstrate how SEEG can be used to investigate the role of the dorsal anterior cingulate cortex (dACC) in cognitive control. We include a description of the SEEG procedure, demonstrating the surgical placement of the electrodes. We describe the components and process required to record local field potential (LFP) data from consenting subjects while they are engaged in a behavioral task. In the example provided, subjects play a cognitive interference task, and we demonstrate how signals are recorded and analyzed from electrodes in the dorsal anterior cingulate cortex, an area intimately involved in decision-making. We conclude with further suggestions of ways in which this method can be used for investigating human cognitive processes.

Stereotactic Electroencephalography (SEEG) is an operative technique used in epilepsy surgery to help localize seizure foci. It also affords a unique opportunity to investigate brain function. Here we describe how SEEG can be used to investigate cognitive processes in human subjects.

Stereotactic Electroencephalography (SEEG) is a technique used to localize seizure foci in patients with medically intractable epilepsy. This procedure ...

A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and contributes significantly to the development of vascular dementia. Few models of focal white matter stroke exist and this lack of appropriate models has hampered understanding of the neurobiologic mechanisms involved in injury response and repair after this type of stroke. The main limitation of other subcortical stroke models is that they do not focally restrict the infarct to the white matter or have primarily been validated in non-murine species. This limits the ability to apply the wide variety of murine research tools to study the neurobiology of white matter stroke. Here we present a methodology for the reliable production of a focal stroke in murine white matter using a local injection of an irreversible eNOS inhibitor. We also present several variations on the general protocol including two unique stereotactic variations, retrograde neuronal tracing, as well as fresh tissue labeling and dissection that greatly expand the potential applications of this technique. These variations allow for multiple approaches to analyze the neurobiologic effects of this common and understudied form of stroke.

Here we present methodology for the production of a focal stroke in murine white matter by local injection of an irreversible endothelial nitric oxide synthase (eNOS) inhibitor (L-Nio). Presented are two stereotactic variations, retrograde neuronal tracing, and fresh tissue labeling and dissection that expand the potential applications of this technique.

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and ...

Medicine

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

Simultaneous Cryosectioning of Multiple Rodent Brains

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In neuroscience, as well as a plethora of other scientific fields, these techniques are used. Immunohistochemistry can be done on slide mounted tissue or free-floating sections. Preparing slide-mounted samples is a time intensive process. The following protocol for a technique, called the Megabrain, reduced the time taken to cryosection and mount brain tissue by up to 90% by combining multiple brains into a single frozen block. Furthermore, this technique reduced variability seen between staining rounds, in a large histochemical study. The current technique has been optimized for using rodent brain tissue in downstream immunohistochemical analyses; however, it can be applied to different scientific fields that use cryosectioning.

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In ...

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional investigation of long-range connections requires targeted recordings of single neurons combined with the specific stimulation of identified distant inputs. This is often difficult to achieve with conventional and electrical stimulation techniques, because axons from converging upstream brain areas may intermingle in the target region. The stereotaxic targeting of a specific brain region for virus-mediated expression of light-sensitive ion channels allows selective stimulation of axons originating from that region with light. Intracerebral stereotaxic injections can be used in well-delimited structures, such as the anterior thalamic nuclei, in addition to other subcortical or cortical areas throughout the brain.
Described here is a set of techniques for precise stereotaxic injection of viral vectors expressing channelrhodopsin in the mouse brain, followed by photostimulation of axon terminals in the brain slice preparation. These protocols are simple and widely applicable. In combination with whole-cell patch clamp recording from a postsynaptically connected neuron, photostimulation of axons allows the detection of functional synaptic connections, pharmacological characterization, and evaluation of their strength. In addition, biocytin filling of the recorded neuron can be used for post-hoc morphological identification of the postsynaptic neuron.

This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo brain slices.

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional ...

Adaptable Angled Stereotactic Approach for Versatile Neuroscience Techniques

Stereotactic surgery is an essential tool in the modern neuroscience lab. However, the ability to precisely and accurately target difficult-to-reach brain regions still presents a challenge, particularly when targeting brain structures along the midline. These challenges include avoiding of the superior sagittal sinus and third ventricle and the ability to consistently target selective and discrete brain nuclei. In addition, more advanced neuroscience techniques (e.g., optogenetics, fiber photometry, and two-photon imaging) rely on targeted implantation of significant hardware to the brain, and spatial limitations are a common hindrance. Presented here is a modifiable protocol for stereotactic targeting of rodent brain structures using an angled coronal approach. It can be adapted to 1) mouse or rat models, 2) various neuroscience techniques, and 3) multiple brain regions. As a representative example, it includes the calculation of stereotactic coordinates for targeting of the mouse hypothalamic ventromedial nucleus (VMN) for an optogenetic inhibition experiment. This procedure begins with the bilateral microinjection of an adeno-associated virus (AAV) encoding a light-sensitive chloride channel (SwiChR++) to a Cre-dependent mouse model, followed by the angled bilateral implantation of fiberoptic cannulae. Using this approach, findings show that activation of a subset of VMN neurons is required for intact glucose counterregulatory responses to insulin-induced hypoglycemia.

Described here is a stereotactic procedure that can target challenging and difficult-to-reach brain regions (due to spatial limitations) using an angled coronal approach. This protocol is adaptable to both mouse and rat models and can be applied to diverse neuroscientific applications, including cannula implantation and microinjections of viral constructs.

Stereotactic surgery is an essential tool in the modern neuroscience lab. However, the ability to precisely and accurately target difficult-to-reach brain ...

Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice

Stereotaxic surgery to target brain sites in mice is commonly guided by skull landmarks. Access is then obtained via burr holes drilled through the skull. This standard approach can be challenging for targets in the caudal brainstem and upper cervical cord due to specific anatomical challenges as these sites are remote from skull landmarks, leading to imprecision. Here we outline an alternative stereotaxic approach via the cisterna magna that has been used to target discrete regions of interest in the caudal brainstem and upper cervical cord. The cisterna magna extends from the occipital bone to the atlas (i.e., the second vertebral bone), is filled with cerebrospinal fluid, and is covered by dura mater. This approach provides a reproducible route of access to select central nervous system (CNS) structures that are otherwise hard to reach due to anatomical barriers. Furthermore, it allows for direct visualization of brainstem landmarks in close proximity to the target sites, increasing accuracy when delivering small injection volumes to restricted regions of interest in the caudal brainstem and upper cervical cord. Finally, this approach provides an opportunity to avoid the cerebellum, which can be important for motor and sensorimotor studies.

Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice

Stereotaxic surgery to target brain sites in mice commonly involves access through the skull bones and is guided by skull landmarks. Here we outline an alternative stereotaxic approach to target the caudal brainstem and upper cervical spinal cord via the cisterna magna that relies on direct visualization of brainstem landmarks.

Stereotaxic surgery to target brain sites in mice is commonly guided by skull landmarks. Access is then obtained via burr holes drilled through the skull. ...

Preliminary Validation of Stereotaxic Injection Coordinates via Cryosectioning

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