After all polymer probes were affixed, the saline filling the 3d-printed base piece was then removed and silicone gel (Dow-Corning 3-4680) was used to fill the 3d-printed base piece, providing a means to seal the durectomies and craniectomies, and also provide added support for the polymer arrays. Additional custom 3d-printed pieces were used to construct a protective case around the polymer devices and active electronic components of the implant. Silicone elastomer (Quik-sil, WPI) was then added to the remainder of the exposed polymer, with special attention to the soft polymer – rigid printed circuit board interface, and 3d-printed casing was affixed to the skull using dental acrylic.

Once at final depth, the polymer array was affixed to the 3d-printed base piece using light-curable dental acrylic (Vivid Flow, Pearson Dental; 2 to 4 min). Next, the 3d-printed base piece was filled with saline and additional saline was dripped onto the silicon stiffener until the PEG dissolved (1 to 2 min). The silicon stiffener was retracted using the second micromanipulator at a rate of ∼5 μm / second for the first 1 mm, and then at a rate of ∼25 μm / second until the silicon stiffener was above brain surface (3 to 4 min). Gentle bends were allowed to form below the anchoring points on the polymer arrays, acting as strain relief. The time from mounting the insertion device to the stereotax to completion of stiffener retraction typically took 10 to 25 min, and largely depended on the surface vasculature and nearby arrays. Insertion was repeated for all targeted locations.

Polymer probes attached to silicon stiffeners by polyethylene glycol (PEG) were then inserted to the brain (). Probe insertion involved several steps prior to the surgery, covered below in the Methods section “ Preparation of stiffeners and arrays for insertion .” During the surgery, each of the custom 3d-printed insertion pieces (one connected to the stiffener and polymer array, one to the stiffener only) was attached to two one-axis micromanipulators (MO-10, Narshige). In turn, the insertion device (two 3d-printed pieces and two micromanipulators) was connected to the stereotax. The array was moved to the target and adjusted to avoid surface vasculature and other arrays (1 to 15 min). Next, it was quickly (< 5 s) lowered ∼0.5 mm into the brain using the stereotax. A quick initial insertion was required to prevent premature softening of the PEG and detachment of stiffener and array in the cerebrospinal fluid above the brain. Next, a micromanipulator was used to advance the stiffener and the polymer array simultaneously, until reaching the final targeted depth. The stiffener and array were lowered at a rate of ∼25 μm / second until within 1 mm of the final depth, at which time the rate was reduced to ∼5 μm / second (3 to 6 min).

The skull was cleaned, targets were marked, and all drilling was completed. Commercially-pure titanium (CpTi) 0-80 set screws (United Titanium, OH), selected for their well-known ability to osseo-integrate (), were then placed around the perimeter of the implant. Bone dust was cleared from the skull, and craniectomies and durectomies were completed. The skull was briefly allowed to dry and a custom 3d-printed base piece (RGD837 Stratasys, MN) was then fixed to the skull using 4-META/MMA-TBB () (C&B Metabond). This base piece serves a multitude of functions, including acting a reservoir for saline or silicone gel, an anchoring point for the polymer arrays, and a standardized interface from which the rest of the implant can be affixed and constructed during the implantation.

Male Long-Evans rats (RRID: RGD_2308852), were implanted with polymer probe(s) at 6-12 months of age. Polymer arrays were targeted to a variety of targets (all coordinates given in millimeters relative to bregma: medial prefrontal cortex (mPFC, including prelimbic and anterior cingulate cortices; ± 1.2 ML, +1.5 to +4.5 AP, −2.0 to −4.0 DV, arrays implanted 6-8° transverse from sagittal plane, perpendicular to coronal plane), ventral striatum (VS, primarily nucleus accumbens shell; ± 0.7 to +1.9 ML, +0.8 to +1.9 AP, −7.2 DV, arrays implanted parallel to midline and perpendicular to coronal), orbitofrontal cortex (OFC, primarily lateral orbitofrontal cortex; ± 3.5 to 3.7 ML, +2.6 to +3.4 AP, −4.0 DV, arrays implanted at 54° sagittal from coronal plane), dorsal hippocampus (dHPC, ± 2.3 to 2.8 ML, −3.5 to −4.0 AP, −4.0 to −6.0 DV, arrays implanted 45° coronal from sagittal plane). Also see Figure S1 C for view of insertion angles. For some subjects, stimulating electrodes and tetrode microdrives were also implanted at the same time, targeted to the ventral hippocampal commissure (vHC, ± 1.0 ML, −1.2 or −2.0 AP) and dHPC.

Reagents and data acquisition

Polymer arrays Tooker et al., 2012a Tooker A.

Tolosa V.

Shah K.G.

Sheth H.

Felix S.

Delima T.

Pannu S. Optimization of multi-layer metal neural probe design. Tooker et al., 2012b Tooker A.

Tolosa V.

Shah K.G.

Sheth H.

Felix S.

Delima T.

Pannu S. Polymer neural interface with dual-sided electrodes for neural stimulation and recording. The polymer arrays were fabricated at the Lawrence Livermore National Laboratory nanofabrication facility as described previously (). Briefly, devices have three trace metal layers and four polyimide layers with a total device thickness of 14 μm. All arrays had a tip angle of 45°. Devices with an LFP configuration had 20 μm contacts in a single-line with a center-to-center distance of 100 μm, tapered shank width of 61 μm to 80 μm, 21 or 22 contacts per shank, and an edge-of-shank to edge-of-shank distance of 420 μm (center-of-shank to center-of-shank distance or pitch of 500 μm). Devices with a 4-shank, 64-channel single-unit configuration are diagrammed in Figure 1 and had an edge-of-shank to edge-of-shank distance of 250 μm (center-of-shank to center-of-shank distance or pitch of 330 μm). This design was used in the 1024-channel rat implant, and one module was used in a 352-channel implant (one 4-shank 64-channel module alongside six 2-shank 32-channel arrays, and 24 tetrodes). Devices with a 2-shank, 32-channel single unit configuration had an identical shank layout to the 4-shank configuration with the notable reduction in edge-of-contact to edge-of-shank distance from 12 μm (4-shank design) to 6 μm (2-shank design). This device design was used for the majority of the data shown, used in the 128-channel implant (data shown in Figure 3 ), and all 288-channel implants (six, two-shank, 32-channel polymer arrays and 24 tetrodes). The device with a 2-shank, 36-channel single-unit configuration (featured in Figure S2 ) had a similar dual-line, staggered design to the other single-unit configurations with a few notable exceptions. The shank width was 100 μm, edge-of-contact to edge-of-shank distance was 12 μm, and 3 of the 18 contacts were placed closer to the tip of the shank.

PEDOT-PSS application and site impedance The solution used for PEDOT-PSS application consisted of 0.14% by weight 3,4-ethylenedioxythiophene (EDOT) and 0.08% by weight poly(sodium 4-styrenesulfonate) (PSS) in deionized water. Constant current was applied to microelectrodes for PEDOT-PSS deposition at a current density of 3 mA/cm2 for 50 s. The typical impedances of the 20 μm circular contacts after PEDOT-PSS deposition was less than 100 kOhm at 1 kHz measured in-vitro in the following manner. Electrochemical impedance (EIS) measurements were made using a potentiostat (Princeton Applied Research, AMETEK) using vendor-supplied software. All measurements were made in a three-electrode cell using a Pt counter electrode, an Ag/AgCl reference electrode, and phosphate-buffered saline (pH 7.5) as the electrolyte.

Silicon stiffeners The silicon stiffeners had different dimensions than the polymer arrays that they were coupled to. The stiffeners were 30 μm thick and 60 μm wide and centered relative to the 80 μm wide polymer array shanks. This was done to help prevent overflow of the polyethylene glycol from the bond interface to the top of the polymer array. The array and stiffener were both aligned at their tips, although the stiffener had a sharper tip angle of 25° compared to the polymer array’s tip angle of 45°.

Preparation of stiffeners and arrays for insertion Polymer arrays had a 2 cm long x 1 mm thick strip or tube of polyimide attached perpendicular to the length of the array, 10 – 20 mm above the shank tips using UV curable epoxy (Loctite 3974, Henkel). This strip of polyimide was later used for tethering the probe to the implant. The silicon stiffener and polymer array were bonded together using PEG. This involved PEG application to the silicon stiffener’s reservoir and heating of the stiffener using a hot plate. The PEG wicked down the channel etched into each shank of the stiffener. Next, the stiffener and array were aligned such that the tips were overlapping and the array’s shanks were centered relative to the stiffener. The stiffener-probe device was then allowed to cool and the PEG bonded them together. Next, a drop of PEG was placed on top of the polymer probe and silicon stiffener, 2 to 4 mm above the top of the shanks. The stiffener was fixed to a custom 3d-printed insertion piece (RGD837 Stratasys, MN) using an ethyl cyanoacrylate-based adhesive (Loctite 1363589, Henkel) and the Omnetics connector or PCB bonded to the polymer array was attached to a second custom 3d-printed insertion piece (RGD837 Stratasys, MN). The two 3d-printed pieces were connected using a screw. Once affixed to the 3d-printed insertion pieces, the stiffener and array were sterilized using ethylene oxide (Anprolene AN74i, Andersen products).

16-module, 1024-channel implant The 16-modules were distributed equally across both hemispheres. Of the 16 modules implanted, 2 were targeted to dHPC. These two arrays were designed for sampling local field potentials and had an electrode pitch of 100 μm (center-to-center distance of 100 μm) with 20 μm circular contacts. Data from these arrays were not used for spike sorting. Of the remaining 14 modules, 4 were targeted to OFC, 4 were targeted to VS, and 6 were targeted to mPFC. Of the 6 devices targeted to mPFC, 4 were implanted too superficially. There were device failures on 2/4 targeted to VS, with one module having an intermittent connection and one module having highly correlated signal, possibly due to a short at the level of the polymer or the PCB.

160 day periodic recordings Polymer probes were targeted to mPFC or OFC. In one implant, two two-shank 36-channel arrays were implanted into mPFC and recorded from for 263 days, the termination of the experiment due to animal approaching end of life expectancy. This animal was recorded from using the NSpike data acquisition system (L.M.F. and J. MacArthur, Harvard Instrumentation Design Laboratory) in a 13’’ x 13’’ rest box, and was returned to its home cage. The second implant consisted of four 2-shank 32-channel arrays, all targeted to OFC (128-channel implant). The third animal was implanted with six 2-shank 32-channel polymer arrays targeted to mPFC, alongside two stimulating electrodes targeted to vHC, and 24 tetrodes targeted to dHPC bilaterally, for a total of 288-channels of recording. For the longevity analyses, the second and third animals were also recorded from in a 13’’ x 13’’ rest box, but on some unanalyzed days, recordings were also carried out while the animal ran in a spatial environment.

10-day continuous recording in mPFC Three animals were implanted with six, two-shank, 32-channel polymer arrays targeted to mPFC, alongside two stimulating electrodes targeted to vHC, and 24 tetrodes targeted to dHPC bilaterally. One of the three animals also had one four-shank, 64-channel polymer array targeted to right OFC. This same animal had a device failure resulting in two functional 32-channel polymer arrays in mPFC and one 64-channel polymer array in OFC. Another animal had a commutator failure on day 4 of recording, causing intermittent data loss, and firing rates from this animal’s day of recording were not used for firing rate analyses. Recordings were carried out while animals were housed in their home cages and in alternating epochs of exposure to a familiar rest box and one of two spatial environments in different rooms. Data was not collected when the animal was being moved between rooms with gaps in recording of 15 to 20 min in length, with one instance of a 45-min gap when the commutator failure was discovered. Animals ran 600 – 1000 m per day in these spatial environments and provided a challenging experimental setting in which to assess recording stability. On the first day of continuous recording, animals stayed in one room, room A, where they had been performing the same spatial task for several weeks, and performed three behavioral sessions, each lasting 30 - 40 min. On the second day of recording, animals performed two 30 - 40-min behavioral sessions in room B, their first time being exposed to that room, and then one in room A. On days three through eleven, animals performed two or three sessions of behavior in room B followed by one in room A. Recording was stopped half an hour after the animal finished the session of behavior in room A on day eleven (animals A and B), or day twelve (animal C). In animal C, a twelfth day of recording was carried out with all behavioral sessions occurring in room A. Animals had red/green tracking LED arrays attached to the implant, allowing their position to be extracted from video recorded by a camera mounted to the ceiling.