This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (computer numeric controlled; CNC) stereotaxic instrument for around $1,000 (excluding a drill).
This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (CNC) stereotaxic instrument for around $1,000 (excluding a drill), using industry standard stepper motors and CNC controlling software. Each axis has variable speed control and may be operated simultaneously or independently. The robot's flexibility and open coding system (g-code) make it capable of performing custom tasks that are not supported by commercial systems. Its applications include, but are not limited to, drilling holes, sharp edge craniotomies, skull thinning, and lowering electrodes or cannula. In order to expedite the writing of g-coding for simple surgeries, we have developed custom scripts that allow individuals to design a surgery with no knowledge of programming. However, for users to get the most out of the motorized stereotax, it would be beneficial to be knowledgeable in mathematical programming and G-Coding (simple programming for CNC machining).
The recommended drill speed is greater than 40,000 rpm. The stepper motor resolution is 1.8°/Step, geared to 0.346°/Step. A standard stereotax has a resolution of 2.88 μm/step. The maximum recommended cutting speed is 500 μm/sec. The maximum recommended jogging speed is 3,500 μm/sec. The maximum recommended drill bit size is HP 2.
Stereotactic rodent surgery is used in a wide variety of neuroscience applications, including lesioning1, iontophoresis2, microwire implantation3, stimulation4, and thin skull imaging5. However, there are major hurdles facing those who wish to apply these techniques, including the steep learning curve for performing accurate stereotactic surgery and the high probability of human error. Human errors include measurement and calculation failures, as well as the low accuracy and replicability of human movements. In an effort to reduce these confounding errors, stereotactic surgeons would benefit from a system that ensures that all surgical procedures are performed identically across subjects. The reduction of errors is also one method by which investigators can minimize the use of animal subjects, a primary goal of the National Institutes of Health for animal experimentation6. In an ideal world, all stereotactic surgeries would be perfectly replicable within experiments, and between labs. To address this issue, companies have developed new ultra-precise stereotaxics, and digital displays for reading measurements. To remove human movement errors, motorized micro manipulators and stereotaxics were produced commercially, but their high cost can be prohibitive to a laboratory with a limited budget. Also, their software is fully proprietary, and cannot be modified by the researcher to accommodate a new type of surgery.
An affordable solution to the human error problem is to build a robotic stereotax from a lab's existing model, using industry standard CNC equipment. Because of a burgeoning CNC hobbyist community, the materials are significantly less expensive than scientific equipment. This allows one to build an accurate CNC stereotaxic instrument, which is also highly flexible and inexpensive. With a basic knowledge of CNC machining and G-Code, individuals can program any stereotactic surgery that they imagine, without the limitations of proprietary software. And, in order to expedite the production of g-code for simple surgeries, this protocol includes software that allows the user to design surgeries (sharp edge craniotomy, thin skull windowing, hole drilling & implant lowering) within point and click menus. These programs output a completed g-code that may be run directly from CNC software.
In all, a motorized stereotaxic upgrade is ideal for those who have an interest in increasing the accuracy and replicability of surgeries, while retaining the flexibility and low cost of an open source platform.
The end result of the surgery designed in the methods will be a rat skull with a sharp edge craniotomy, and 3 skull holes (Figure 17). Note that the skull used to demonstrate the surgery was much wider than the prototypical rat skull. The sharp edge craniotomy may be used to insert a microwire array into the brain, for high density neural recordings. The CNC stereotax may also be used to lower the array with great precision. Software is included in this protocol that allows the surgeon to define the parameters of a microwire, or cannula lowering. Sharp edge craniotomies could also be used to uncover large portions of motor cortex, for sensorimotor mapping studies.
The skull holes may be used to insert skull screws (Figure 18). These can be used as anchors for dental cement when affixing a head stage to the animal. The holes may also be used to insert single electrodes into the brain. These electrodes may be used for anesthetized or chronic recordings. However, when using the stereotax for anesthetized recording, ensure the power to the motors is off before collecting data. The motors electrical properties may induce noise in the recording.
The CNC stereotax may also produce thin skull windows with a great deal of accuracy (Figure 19). These windows may be used for in vivo optical imaging in anesthetized animals. The uniformity of the thin skull allows for even light penetration, which is necessary for comparative or quantitative analysis of optical imaging data.
Figure 1. Wiring diagram for stepper motor plug.
Figure 2. Assembly diagram for attaching the couplers to the stepper motors.
Figure 3. Assembly diagram for attaching the collars to the stereotaxic arm.
Figure 4. Assembly diagram for attaching the motors/couplers to the collars/stereotaxic arm.
Figure 5. Switch diagram for setting the stepper motor driver to half steps.
Figure 6. Wiring diagram for stepper motor driver.
Figure 7. Configuration diagram for output signals in CNC software.
Figure 8. Configuration diagram for input signals in CNC software.
Figure 9. Configuration diagram for motor outputs in CNC software.
Figure 10. Configuration diagram for motor tuning in CNC software.
Figure 11. Prompt for choosing surgery type in the "surgery designing" software (sharpedgecraniotomies.m).
Figure 12. Prompt for choosing custom or preset target coordinates in the "surgery designing" software.
Figure 13. Prompt for entering window coordinates (4 corners) in the "surgery designing" software.
Figure 14. Prompt for choosing the number of skull holes in the "surgery designing" software.
Figure 15. Prompts for choosing the method of placing skull holes, and for entering the hole coordinates in the "surgery designing" software.
Figure 16. Prompt for defining the drilling parameters in the "surgery designing" software.
Figure 17. A skull showing the end result of running the previously designed surgery.
Figure 18. A skull showing skull screws inserted into a hole produced during the example surgery. The drill bits size will determine the hole's diameter and consequentially the screw size.
Figure 19. Shows the skull from the example surgery, containinga thin skull window, indicated by the arrows. Note that in the right panel, (skull lit from within) light seems to penetrate the thin skull window uniformly. Also note that the window does not need to be square, or contain 90° corners.
The use of automated surgery equipment helps to eliminate some of the most common problems in neuroscience research. First, the tool paths are 100% reproducible. Every cut is guaranteed to be in the same location relative to Bregma. Second, it should reduce experimenter error. Although many researchers are highly skilled surgeons, it takes an exceptional amount of practice to become even a competent surgeon. This device will allow new students to quickly and easily perform highly accurate surgeries. Third, motorized surgery devices should reduce the number of animals needed to perform an experiment6. Surgeons will need less training, and mistakes will be made less frequently. Finally, the motorized stereotaxic is capable of making more precise and accurate movements than the human hand, allowing for more resolution in coordinate choice.
In today's neuroscience climate, there has been a push to increase the accuracy of surgical methods and techniques. It is no longer enough to target a brain region as a whole when it is clear that smaller subregions exist, and that they could be functionally distinct. One example comes from research focusing on microinjections into hippocampus. Not only do individuals desire to target subregions, like CA1 and CA3, but they wish to study dorsal and ventral subfields within these regions7. However, targeting these regions with a manual surgical technique is exceedingly difficult. The benefit of the motorized stereotaxic approach is that, once the correct coordinates of a target region are identified, every future surgery may be directed to the identical location. However, it's important to note that morphological differences in the skull and brains of animals will still contribute some error to surgeries.
Another field that would benefit from automating surgeries is chronic microelectrode implantations. Some labs are attempting to lower electrodes into subregions of nuclei, such as the subregions of globus pallidus or ventral pallidum8. Lowering electrodes with a motorized stereotaxic instrument will not only increase accuracy, but should increase recordable neuron yield. This is due to the fact that microwires cause damage as they are lowered. The robot is capable of lowering the electrodes slower than human hands and at a constant rate, minimizing damage to axons or neurons that very well may be afferent to the target region.
The robot's level of accuracy should also be beneficial to those who are imaging through skulls, to obtain quantitative measures of optical density9. The amount of light that may enter and exit through the skull is dependent on the skull thickness. Our motorized stereotax is capable of ensuring that the entire surface area of the thin skull window has identical depth. This helps light penetrate the window equally across its entire surface.
It is important to note the limitations of the included surgery generating software. First, all corners will be rounded off to the radius of the drill bit. For the hole drilling code, the diameter of each hole is dependent on the drills diameter. For the thin skull window and craniotomy codes, the center of the drill bit will follow the cutting path. This means that size of the window will increase on all sides by the radius of the drill bit. This may be remedied by subtracting the radius of the drill bit from the corner dimensions. Also for the thin skull window and craniotomy codes, the depth of drilling is static on each pass. This means that the entire window will be drilled to the same depth, regardless of the curvature of the skull. This is especially important to consider at extremely lateral coordinates, where the skull curves ventrally. However, there are no such limitations to the hardware, and researchers need not use the included software. With proper understanding of g-code, users may create surgeries from scratch that perfectly match the contour of the specific skull being used. Also, any hole larger than the current bit diameter may be made using simple circle interpolation. Movement in three dimensions is only constrained by the travel of the stereotax, and the user's proficiency at g-coding.
In all, automating surgeries provides a number of benefits for a modest cost, and as such, is becoming an increasingly popular technique10, 11. But it is important to recognize that the accuracy of the robot depends on quality machining, proper setup, and proper understanding of how CNC machines operate. As long as researchers are willing to take the time to understand the functioning of this motorized stereotaxic instrument, they may perform surgeries more accurately, with better replicability, and with less training. This makes integrating a motorized stereotaxic instrument into experiments a smart choice for any lab that performs a large number of surgeries.
The authors have nothing to disclose.
This study was supported by the National Institute on Drug Abuse Grants DA 006886, and DA 032270.
1x Standard U Frame Stereotax | Kopf | Kopf | This protocol should work with most existing stereotaxic devices. |
3x 12 V, 1.6 A, 233 oz-inch Geared Bipolar Stepper Motor | Phidgets | Robot Shop | Any high torque geared stepper motor should do. |
1x 3 Axis CNC Stepper Motor Driver Board Controller | Toshiba | Ebay | Any 3 Axis CNC driver should do. Linked Item includes Mach3 CNC software. |
2x Arm Couplers: medial-lateral (ML) & dorsal-ventral (DV) | custom machined | Part Drawings | These must be machined by your local machine shop. (costs will vary) |
1x anterior-posterior (AP) Coupler | custom machined | Part Drawings | These must be machined by your local machine shop. (costs will vary) |
3x Motor to Stereotax Collar | custom machined | Part Drawings | These must be machined by your local machine shop. (costs will vary) |
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12x NF10-32 Cup Point Set Screws | McMaster Carr | ½” Length | You will need 6 of each. |
¼” Length | |||
12x M3 Socket Head Screws (20 mm) | McMaster Carr | 20mm Length | You will need 4 for each motor |
1x Micro-Motor Drill | Buffalo Dental | X50 | Any Micromotor drill will work. At least 38,000 rpm recommended |
1x 12 V DC Power Supply | 12 Volt Adapters | 12v DC – 7 Amp | Any 12 V DC PSU should work (ensure amperage rating is higher than the sum of the motors’ amperage). |
1x Extra Large Probe Holder | Stoelting | Stoelting | |
1x Grade B Rat Skull | Skulls Unlimited | Skulls Unlimited | |
Mach 3 Mill | ArtSoft USA | Trial Download | Any Standard CNC controlling software should work. |
Surgery Designer | Kevin Coffey & David Barker | MATLAB File Exchange | These codes are available to modify. We accept no responsibility for your use or modification of code. |