1. Clean and rinse the NET device in isopropanol, and blow dry.

2. Use a pair of tweezers to attach the PEEK tube near the base of the NET bonding pad [Fig. 3(a)]. The PEEK tube is $\sim 6\text{-}\mathrm{mm}$ long, extending from the bottom of the bonding pad. Add a small amount of adhesive epoxy on the outside of the tube to fully fix its position and secure it to the NET carrier chip.

3. Retrieve the sharpened tungsten-wire shuttle using a pair of forceps or micro-pincers. Grip and slowly guide the wire shuttle through the PEEK tube. Gently push the wire forward and toward the tip of the NET electrode. Keep driving the shuttle wire through the tube and across the glass surface until reaching the end of the device. The shuttle should stop $\sim 300$ to $400\mu \mathrm{m}$ before reaching the tip of the unreleased NET [Figs. 3(a) and 3(b)].

4. Apply PEG (8 k molecular weight) as the dissolvable adhesive between the tungsten microwire and the PEEK tube. This step will fix the tungsten shuttle to its current position, preventing any unintended movements during future handling and insertion.

5. Remove the sacrificial layer Ni on the NET device to release the flexible section of NET by applying Ni etchant for $\sim 10$ to 30 min [Fig. 3(c)]. Once all the Ni is etched away and the device is released, the device will float in the Ni etchant. Wash away any excess Ni etchant using deionized (DI) water. Keep the released NET section in DI water before attaching it to the shuttle wire.

1. Immerse the released device in DI water. Use a fine needle and carefully place the flexible NET section at least 0.5 mm away from the cleaving mark on the carrier chip [Fig. 4(a)].

2. Break off the bottom piece of glass by applying concentrated pressure on the cleaving mark using a pair of wafer cleaving/glass breaking pliers [Fig. 4(b)].

3. Manually align the ultraflexible section of NET onto the tungsten wire shuttle [Figs. 4(c) and 4(d)]. Keep the device fixed in place using adhesive tape. Leverage the surface tension of water to wrap the ultraflexible NET onto the tungsten wire. Use a 31-gauge needle to control a single drop of water and the associated water-air surface tension to align the electrode thread on the tungsten shuttle [Fig. 4(e) and supplementary video]. The alignment between the flexible section of NET and the shuttle wire needs to satisfy the following criteria: (a) The individual recording sites on NETs face away from the tungsten wire so they will be in intimate contact with the brain tissue during and after implantation. (b) The tip of the NET thread should not extend beyond the tip of the tungsten wire to decrease the chance of premature detachment during implantation.

4. Once the flexible section of NET and the shuttle wire is appropriately aligned, apply 10% PEG aqueous solution using a 31-gauge needle to attach NET with the wire. We choose PEG because PEG is biocompatible and biodegradable. The dissolution time by the cerebrospinal fluid is a few minutes, which is conveniently short for prompt implantations, and sufficiently long to support implantations into mouse cortex and hippocampus. Start at the tip of the shuttle device and work along the thread to the end of the flexible section. The thickness of the PEG adhesion layer should not exceed two microns³² and therefore not easily visible under the microscope [Fig. 4(d)].

Note: If the flexible device is placed too short or too long from the sharpened end of the shuttle, adjust the wire’s position by first dissolving the PEG attaching the shuttle to the PEEK tube. Reposition the shuttle using a pair of tweezers to the proper position and reapply PEG to re-fix its location.

5. Sterilize the assembled device by ethylene oxide low-temperature sterilization. The total weight of the implant is $<1\mathrm{g}$.

1. Anesthetize the animal using isoflurane (3% for induction and 1% to 2% for maintenance). Place the animal on a homeothermic blanket at 37°C to maintain body temperature stable. Monitor the depth of anesthesia by periodic toe-pinch tests (every 15 min) and the breath rate (55 to $65\text{breaths}/\min$).

2. Administer Ethiqa-XR ($3.25\mathrm{mg}/\mathrm{kg}$) for analgesia and dexamethasone ($2\mathrm{mg}/\mathrm{kg}$, subcutaneous) for anti-inflammation after induction. Apply ophthalmic ointment on the eyes to prevent drying of the cornea. Administer warmed lactated ringers subcutaneously $1\mathrm{ml}/100\mathrm{g}$ body weight per anesthesia hour to minimize postoperative dehydration.

3. Shave fur on the surgical site with a clipper.

4. Fix the mouse on the stereotaxic apparatus. Position the intra aural positioning studs to symmetrically fix the position of the animal’s head in the apparatus. Use only blunted style ear studs for survival procedures. To insert the incisor adapter, use small forceps to pull down the animal’s lower jaw and slowly move the incisor adapter into the mouth until the incisors fit in the opening; gently pull back slightly and fix the adapter in place. Gently position the nose clamp by resting on top of the nose using very low pressure. Adjust the incisor screw to make the head level so that bregma and lambda are equal in height. Level the head horizontally in the caudal-to-rostral direction.

5. Sterilize the surgical site via alternating ($3\times$) scrubbing of iodine (betadine) and 70% isopropyl alcohol. Administer 0.5% Lidocaine ($7\mathrm{mg}/\mathrm{kg}$) underneath the scalp.

6. Incise into the skin of the scalp and resect to expose the skull between bregma and lambda skull sutures [Fig. 5(a)]. Remove the periosteum and score the skull with the tip of a scalpel blade to roughen the skull.

7. Measure the desired coordinates with a pair of calipers and use a scalpel blade to mark the position. Thin the bone along the marked craniotomy perimeter using a surgical drill. Drill a circular craniotomy with a diameter of 3 mm over the target brain areas [Fig. 5(b)].

Note: Regularly rinse the skull with sterile saline during drilling to prevent heating. The thickness of the bone along the drilled perimeter can be checked by gently pressing on the bone. Drill until the central piece of the bone is detached from the rest of the skull.

8. Using a pair of forceps, carefully lift the central piece of the skull. If dura is attached to some part of the skull, apply sterile saline, slowly pry the edge, and follow around.

9. Immediately after removing the central piece of the skull, place a piece of saline-soaked gel foam in the craniotomy to keep the exposed brain moist and promote coagulation.

10. Carefully remove any remaining thin layer of bone on the edge of the craniotomy. This prevents rapid bone regrowth under the window that impedes optical clarity for chronic studies. Remove small residues of bone dust and blood clots.

11. Drill a burr hole at the contralateral hemisphere of the brain and insert a metal wire (made of platinum, silver, or medical-grade stainless steel) as the grounding reference.

1. Fix the assembled NET device onto a stereotaxic arm using a 3D-printed case and a pair of screws [Fig. 6(a)].

2. Remove any gel foam from the craniotomy. Aim the device to the insertion site. To avoid surface vasculature, move in z-direction toward the brain and fine adjust the implantation location [Fig. 6(b-I)].

Note: Avoid prolonged periods of the device in close proximity to the brain before insertion. Water vapor and condensation from the brain surface may weaken the adhesion between the NET and the insertion shuttle, leading to premature detachment prior to or during insertion.

3. Insert the device quickly. For a thin layer (1 to $2\mu \mathrm{m}$) of PEG as we demonstrate here, the insertion time should be within 5 s to avoid pre-mature separation between the shuttle wire and PEG. To achieve slower insertion speed, thicker layer of PEG can be used at the expense of larger implantation footprint. After insertion, dissolve PEG outside the brain by gently dripping body-temperature saline on the device where PEG is adhered to the shuttle needle. Wait until PEG outside the brain is completely dissolved ($<10\min$). The device will float in saline and separate from the shuttle needle, indicating it is ready for retraction [Fig. 6(b-II)]. By this time, PEG inside the brain should have also been dissolved by cerebrospinal fluid.

4. Grip the PCB end of the shuttle wire using a pair of tweezers and retract the shuttle wire manually. During retraction, carefully inspect the interface between the implanted NET and the shuttle needle through the surgical microscope. The implanted section of NET should not move with the shuttle wire.

5. To achieve precise implantation depth after shuttle needle is retracted, slowly retract the stereotaxic arm, straighten the flexible section outside the brain, and pull out the implanted section to reach the designated depth. Depth markers are fabricated on NET as guidance [Fig. 6(b-III)].

6. Rotate the stereotaxic arm to reduce the mounting angle of the carrier chip on the skull. Depending on the rotation axis, this may involve iterative steps of rotation and moving the micromanipulator on the stereotaxic arm. Avoid straightening the flexible section of NET outside the brain to minimize uncontrolled stress on it [Fig. 6(b-III)]. The carrier chip should be more than 2 mm away from the edge of the craniotomy and at an angle of 45 deg or shallower to accommodate the optical objectives for two-photon imaging [Fig. 6(c)]. The flexible section of NET outside the brain should have some slack (at least $500\mu \mathrm{m}$) to accommodate possible tissue motion as initial implantation damage subsides with time. Fix the stereotaxic arm when the carrier chip reaches the final location and angle.

1. After implantation of NET, add sterile saline to the brain. Place a glass window on the exposed area. If there is space between the window and the remaining skull, fill it with Kwik-sil.

2. Dry the skull. Apply a layer of low viscosity super glue on the remaining skull. Apply a layer of high viscosity super glue around the edge of the window, ensuring contact with the skull.

3. Apply a layer of Metabond dental cement over the high-viscosity super glue and the Kwik-sil, and apply additional cement to fix the NET carrier chip to the skull [Fig. 7(a)].

4. Mount a lightweight titanium headbar on the skull for head-restrained experiment by applying another layer of Metabond cement [Fig. 7(b)].

5. Cap the glass window using a piece of silicone to protect the window from mechanical scratches and tissue from photobleaching by ambient light.