Ethical statement: All experimental procedures suggested below were approved a prior by an independent animal ethical committee (DEC-Consult, Soest, The Netherlands) as required by Dutch law and conform the relevant institutional regulations of the Erasmus MC and Dutch legislation on animal experimentation.
Preparation of the probe (the evening before the surgery)
Mount the mini-amp 64 headstage to the protective mini-amp-64 plastic case. First, put a thin layer of Kwik-Cast™ on the Intan chip of the headstage; superglue may be used as an alternative, in order to adhere the headstage to the case. Gently flip and press the headstage this inside the plastic case (see figure 1).
Prepare the stereotaxic nano-Drive holder and firmly insert the nano-Drive screw-head in to the holder tool; rotate the thumbwheel on the holder tool to provide clamping force on the nano-Drive. Apply a small amount of BluTack to the holder tool body as illustrated in figure 2 (this will temporarily secure the headstage during surgery).
Note: The nano-Drive is supplied with the movable shuttle in the ‘home’ position – depending on your implant trajectory and probe geometry, you can adjust the shuttle position to your desired depth by using the stereotaxic holder tool to rotate the drive screw; anti-clockwise = down in increments of 250 microns per turn. Additionally, the nano-Drive should be sterilized before the placement by soaking in 100% Ethanol for a few minute , or by standard autoclave cycle.
Apply Vaseline to the internal screw mechanism of the nano-Drive using a Low-Temperature Cautery (LTC) to melt the Vaseline and lubricate the shaft. Taking care not to get Vaseline onto the moveable shuttle.
Note: Make sure the shaft is fully covered, specifically at the bottom. The nano-Drive screw can be used to move the shuttle when applying the Vaseline, to provide better access to the internal part of the nano-Drive.
Prepare the probe for coupling to the headstage (see figure 3):
(1) Gently remove the tape above the probe from the packaging (peel the tape to the side).
(2) Gently remove the tape from the interface chip component of the probe, pay attention that the tape does not stick to the interface chip!
Connecting the probe to the headstage. Using gloved fingers, take hold of the Molex connector. Lift the plastic packaging box, and slowly invert, allowing the probe to naturally fall away from the packaging into mid-air. The packaging box may now be put aside. Particular attention is required to ensure the correct orientation of the probe connector with respect to the headstage connector so that GND and REF inputs are appropriately connected; as detailed in the headstage user guide. Bring the Mini-Amp 64 up to the probe connector and ensure the SPI connector is to the right of the flex cable, ensuring correct channel mapping (as also stated above). Use gentle thumb / finger pressure to secure the connector in place. A small click may be audible, or felt.
Note: When using a sacrificial connector interposer board, this should be mated with the headstage prior to connecting the probe. Gently press on the Molex connector until a soft click is felt; visually inspect the connection to ensure no observable gaps and that the connectors are well mated.
Place a drop of superglue gel (Powerflex Super Glue-3) on the nano-Drive shuttle. Holding the probe / headstage assembly by the headstage, carefully support the interface chip part of the probe using a fine spatula (or similar) and slowly drag the interface across the shuttle surface until the surface tension of the glue takes hold of the probe (in a upwards motion). Adjust the probe position on the drive shuttle as desired, by carefully pulling on the headstage; once complete gently press the headstage in to the BluTack to hold it during surgery. Finally, bend the grounding wires upwards facing away from the probe end (see figure 4).
Cover all edges and exposed contacts of the headstage with a thin layer of Kwik-Cast™, including the accelerometer. Make sure that the “topside” with the SPI-connector remains uncovered (see figure 6). The aim of this step to prevent cement making direct contact with the electronics so as to facilitate their removal and recovery once the experiment is concluded.
Cover all moving parts of the nano-Drive/probe assembly in Vaseline using the LTC. Make sure the following parts are covered: all parts of the drive shuttle, the probe interface chip along with the proximal section of the probe flex cable and surround sides of the headstage (see figure 7).
Note: Use the LTC as less a possible when applying the Vaseline to the flex cable; Vaseline melts at 40 Celsius so temperatures higher than this are not required and will prevent potential heat-related damage to the probe flex cable.
Now the probe preparation is done. Make sure to place the prepared probe and holder in a safe spot where nobody will touch or interfere with it!
Immediately prior to implantation, you may clean/sterilize the probe shanks with either 1% Trypsin solution, or isopropyl Alcohol, followed by a rinse with dH2O. Further information can be obtained from Cambridge NeuroTech.
Implantation of the probe, for cerebellar targeting (surgery day)
Anesthetize the mouse according to normal surgical procedure for Moderate discomfort. This includes injection of 0.05 mL of Rimadyl (5 mg/kg) and 0.05 mL Buprenorphine (50 μg/kg).
Do not forget to apply Duratears to the eyes, to prevent the eyes from drying out.
Remove the hair from the head of the mouse and sterilize the surgical skin field with Povidone / Iodine solution.
Take the angled forceps and open up the skin, from between the eyes ~15 mm in a caudal direction (almost between the ears).
Apply a few drops of Lidocaine onto the periosteum (bone membrane). Thoroughly remove the membranes on the skull. (This can be done by either twisting the membrane around a paper point and then cutting with the spring scissors or if possible, by grappling the membrane with the angled forceps and then cutting with the spring scissors).
Note: Membranes are easier to remove when kept wet, use the micro curette to remove any additional pieces.
Remove the muscles necessary to reach the target (in this case Crus I/II). Do this by carefully cutting with the spring scissors. Apply additional lidocaine before removing the last layer of muscle (3/3) and if necessary, remove the last part of muscle from the skull by gently scraping this away with a scalpel (10/11 blade) or micro curette.
Use the small drill to drill a craniotomy above the target area (see figure 7a for Crus I/II), the later steps will be easier if the craniotomy drilled a bit bigger than the width of the probe itself.
Use the small drill to drill the initial hole for the bone screw, compare the sizes of the craniotomy and screw under the microscope and drill the additional space with the bigger drill tip.
Note: The reference electrode skull screw should be located ideally in a brain area that does not directly communicate with the target region being recorded. If using the headstage with independent GND and REF inputs, you will need to insert two skull screws accordingly.
Stabilize the screw using a small amount of dental cement, ensuring there is enough space to wind the grounding wire around the screw (see figure 7b).
Sharpened probes are able to penetrate mouse dura, but for standard probes, removal of the dura may be required. A durotomy can be performed by using a modified 25G needle to carefully cut and grab the dura, starting from a corner of the craniotomy. To modify the 25G needle, take a fresh needle point and carefully apply pressure to the tip to form a small curved hook; the hook can be used to snag, drag and slice the dura accordingly (as described above).
Briefly rinse the probe shanks using 70% ethanol solution, as a final cleaning step immediately prior to implantation. Attach the prepared stereotaxic holder to the stereotaxic frame and carefully lower the probe above the target entry area. (see figure 7) Slowly drive the probe in to the tissue, noting the surface depth for reference (see figure 8). Ideally, you should implant the probe a short distance above your desired target, then use the nano-Drive to slowly lower to target over the next few days of post-surgery recovery.
Note: If you observe bending in one or more of the probe shanks ( see figure 8). Check and clear obstructions (debris, fibrous tissue, blood vessel) or refine penetration positioning as needed.
Use a 20G needle to mix a small amount of Dura-Gel, mix until a “mutti/putti” structure (comparable with glue). Apply the Dura-Gel directly to the craniotomy This should take ~10 -15min.
Using the LTC, apply Vaseline all around the probe and the drive shuttle with the aim being to build a ‘column’ of Vaseline around the pathway of the probe and shuttle as they will descend. Apply molten Vaseline to the interface chip and allow gravity to take that Vaseline downwards, as desired. Making sure no dental cement can obstruct the shuttle travel (see figure 9).
Apply a small amount of dental cement sufficient to partially secure the anchor point(s) on the rear of the nano-Drive to the skull ( (see figure 9 b-d).
Carefully wind the grounding wire around the skull screw and use dental cement to secure it (see figure 9 b-d).
Carefully release headstage from the BluTack and now position the headstage on the skull, taking care not to crimp or pinch the probe flex cable. Place the headstage in a position where you will have sufficient ‘loosenes’ in the flex cable (see next) and cement in place (see figure 10 a-f).
Note: Make sure everything is covered SMOOTH in dental cement, to ensure the mouse cannot reach below or around the implant.
Remove the stereotaxic holder and cut the additional reference wire (if not operating the headstage with independent GND and REF inputs. Carefully apply more dental cement to all the electronic components (see figure 11).
Note: In the end, everything should be covered with dental cement also the left side of the head stage.
When the implant is fully covered in cement, standard post-surgery recovery procedures can be followed (Recovery under a heat lamp for example).