Supplementary Information

The supplemental sections are numbered to indicate corresponding sections in the main text.

S4.4 Electrophysiological Identification of Layer IVCurrent source density (CSD) was used to identify layer IV in the visual cortex (Fig. 7a). Immediately

following an ‘ON’ stimulus, a strong electrical current sink (neural source) can be observed using CSD as large populations of layer IV input neurons’ cell bodies depolarize, which is then followed by an electric source. In addition, LFP polarity inversion can typically be observed around the border of layer IV to help identify its depth(Schroeder et al., 1998). Delayed sources in layer II/III and layer V also help contrast the location of layer IV. After the stimulation turns ‘OFF,’ weaker sinks can be observed in layer II/III and layer V (Fig. 7b). Note, both ‘ON’ and ‘OFF’ CSDs have a ~50 ms delay before the first current sink. The depth of layer IV on the day of the surgery is defined as 0 µm in Figure 3c-f.

Examining the changes in the depth of layer IV over time shows that while there are some subtle fluctuations early on, the depth is mostly stable (Fig. 7c). Since the probes were mechanically fixed (cemented) to the skull instead of floating, any change in the depth of Layer IV should be attributed to movement of the brain tissue instead of movement of the electrode. After 100+ days there is a mean decrease in the depth position of layer IV as well as an increase in variation. Individual analysis of layer IV depth (Fig. 7d) showed that in three mice, layer IV became more superficial during the first 2 days following the implant. In two other mice, the depth of layer IV remained stable throughout the experiment. One mouse showed a small decrease in depth, and then recovered over the first 2-3 weeks. Layer IV of another mouse sank deeper during the first week, but recovered over the next two weeks. In the last animal, layer IV sank 400 microns during the first five days, then partially recovered by the end of the week, and then stabilized until 120 days when it began to sink again. Examination of the magnitude of depth change over time shows that most of the depth fluctuation occurs during the first week, stabilizing by 14 days (Fig. 7e). Therefore, the average depth of layer 4 between day 14 and day 120 was used for depth related analysis.

Lastly, examining the individual SU yield of the animal with the dramatic layer IV depth change shows that the drop and recovery of SU yield trends very closely with the drop and recovery of layer IV depth (Fig. 7f). After sacrificing this animal, it was noted that the brain had lost substantial volume compared to the other animals, though no signs of infection were detected.

S4.5 Depth Dependent Chronic Electrode Performance AnalysisProbe implant depths were aligned across animals at their average layer IV depth between day 14 and

day 100 (Fig. 3). Layer IV was determined with CSD following the visual stimulus. Of immediate note is that cortical layers play a critical role in chronic SU yield (Table S1, Fig. 8a). For example, the mid-cortical layers have the greatest yield acutely, but the shallow layers have the best cortical yield in chronic time points. In contrast, visually evoked MU (including SU) yield was much greater across all depths. However, depth dependent features can still be observed (Table S2-3, Fig. 8b). While SUs were not detected in the most superficial layer, visually evoked MU activity was detected ~30±26% throughout the experimental period.

Signal quality and signal quality changed over time was also dependent on recording layer. The SU SNAR follows the same trend as the SU yield (Table S4, Fig. 8a). To better display the average SNAR of the detected units across depth (Fig. 9a), channels that did not detect SUs were considered to have an SNAR equal to the noise floor (2 standard deviations). To quantify functional MU activity, a new evoked SNFRR metric was developed. Here, the change in firing rate between ‘ON’ and ‘OFF’ was measured as “signal” while the average standard deviation of all the ‘ON’ and ‘OFF’ firing rate was calculated as “noise” (Table S5-6, Fig. 9b). To better display the average evoked SNFRR, firing rates were only averaged if significant activity was detected. A value of zero signifies that no significant activity was detected on a certain day at that certain depth.

In addition, signal strength and signal strength change over time showed dependence on recording depth. The average SU amplitude (Table S7, Fig. 10a) followed a similar trend to the SU yield. MU amplitudes on channels detecting significant evoked activity showed slower amplitude decreases over time compared to SU amplitude (Table S8-9, Fig. 10b). Together these data show that rate of charge across electrophysiolgical performance metrics are dependent on the layer they record from.

Similarly, longitudinal impedance changes occur over time in a depth dependent manner (Table S10, Fig. 11a). Impedance generally increased over the first week, but at different times in different layers; the

impedance appeared to increase from the tip of the electrode as well as from the surface, leaving Layer II/III to experience the increase in impedance last. It also becomes apparent that the impedance drastically changes in the cortex and subcortical white matter region over the first 77 days, both increasing and decreasing. On the other hand, impedance in the CA1 shows a dramatic increase in impedance over the first week, but decrease again over the next 21 days. While subtle changes occur within a layer after day 77, these changes are much more muted than during the first 77 days. Noise also showed initial increase to be greatest in layer I and the most superficial recording site (Fig. 11b).

Supplementary Tables

Table S1: Summary of Chronic SU Yield1-7 Days 14-42 Days 49-77 Days 84-182 Days

0-100 µm 2.4±1.7 % 2.9±2.9 % 2.0±2.0 % 0.0±0.0 %200-300 µm 31.3±4.3 % 36.3±6.6 % 18.8±4.3 % 12.9±0.7 %400-500 µm 61.6±3.9 % 43.8±4.7 % 32.5±3.3 % 16.4±1.8 %600-700 µm 67.9±2.8 % 38.8±3.5 % 15.0±2.5 % 5.5±1.2 %800-900 µm 54.5±2.2 % 20.0±3.3 % 15.0±3.6 % 3.1±1.2 %

1,000-1,100 µm 27.7±5.1 % 21.3±4.2 % 10.0±2.5 % 5.5±1.1 %1,200-1,300 µm 25.9±4.6 % 20.0±2.0 % 15.0±3.1 % 7.4±1.5 %1,400-1,500 µm 78.6±2.9 % 46.9±5.3 % 12.6±3.9 % 1.0±0.7 %1,600-1,700 µm 57.1±11.3 % 36.7±13.6 % 23.3±11.2 % 4.2±2.0 %

Table S2: Summary of Chronic MU Yield1-7 Days 14-42 Days 49-77 Days 84-182 Days

0-100 µm 64.3±4.2 % 30.7±5.6 % 19.3±5.7 % 19.4±3.8 %200-300 µm 81.0±3.5 % 63.3±6.5 % 48.3±6.3 % 38.3±3.3 %400-500 µm 88.1±3.2 % 68.3±3.9 % 58.3±4.5 % 41.1±3.7 %600-700 µm 88.1±2.5 % 63.3±4.2 % 55.0±5.6 % 35.7±3.2 %800-900 µm 83.3±3.9 % 58.3±5.7 % 45.0±4.3 % 30.7±3.9 %

1,000-1,100 µm 78.6±4.2 % 58.3±6.7 % 48.3±5.2 % 38.8±3.8 %1,200-1,300 µm 76.2±6.1 % 71.7±8.3 % 63.3±4.8 % 46.6±4.4 %1,400-1,500 µm 68.9±4.6 % 63.5±10.9 % 52.5±8.7 % 27.7±4.3 %1,600-1,700 µm 35.7±9.6 % 53.3±12.4 % 76.7±8.7 % 55.2±8.0 %

Table S3: Summary of Bonferroni Corrected Chronic MU Yield1-7 Days 14-42 Days 49-77 Days 84-182 Days

0-100 µm 17.1±5.6 % 12.0±4.4 % 2.0±2.0 % 1.1±1.1 %200-300 µm 44.8±4.8% 38.3±3.6 % 25.0±5.1 % 21.7±0.9 %400-500 µm 58.3±3.7% 48.3±3.8 % 40.0±5.7 % 23.6±2.3 %600-700 µm 57.3±3.7 % 46.7±3.3 % 26.7±5.1 % 13.1±2.2 %800-900 µm 42.7±3.0 % 28.3±4.3 % 8.3±3.7 % 3.9±1.5 %

1,000-1,100 µm 26.0±4.6 % 18.3±3.9 % 11.7±2.5 % 9.2±1.8 %1,200-1,300 µm 21.9±3.9 % 25.0±6.2 % 31.7±7.2 % 14.4±3.4 %1,400-1,500 µm 31.3±4.0 % 25.0±7.1 % 8.5±3.5 % 1.8±1.3 %1,600-1,700 µm 6.3±3.4 % 10.0±10.0 % 30.0±15.3 % 14.4±6.3 %

Table S4: Summary of Chronic SU SNAR1-7 Days 14-42 Days 49-77 Days 84-182 Days

0-100 µm 2.03±0.031 2.02±0.02 2.01±0.01 -200-300 µm 2.49±0.10 2.49±0.11 2.47±0.11 2.53±0.04400-500 µm 3.28±0.09 2.92±0.15 2.56±0.05 2.44±0.06600-700 µm 3.45±0.18 2.53±0.11 2.13±0.03 2.11±0.03800-900 µm 2.85±0.06 2.26±0.03 2.16±0.06 2.05±0.02

1,000-1,100 µm 2.72±0.14 2.32±0.09 2.19±0.05 2.09±0.021,200-1,300 µm 2.40±0.09 2.32±0.03 2.25±0.06 2.12±0.021,400-1,500 µm 3.73±0.13 2.54±0.05 2.13±0.04 2.01±0.011,600-1,700 µm 2.81±0.18 2.35±0.12 2.21±0.08 2.04±0.02

* For the purpose of illustrating Figure 5a, this table is calculated with the signal equaling the noise floor for channels that do not detect SUs

Table S5: Summary of Chronic SNFRR1-7 Days 14-42 Days 49-77 Days 84-182 Days

0-100 µm 0.99±0.06 1.06±0.12 0.61±0.11 0.38±0.03200-300 µm 1.73±0.08 2.09±0.12 2.21±0.12 3.12±0.13400-500 µm 2.36±0.09 2.51±0.09 2.36±0.16 2.18±0.12600-700 µm 2.19±0.08 2.98±0.20 1.61±0.13 1.45±0.11800-900 µm 1.67±0.06 1.85±0.13 0.90±0.04 0.65±0.04

1,000-1,100 µm 1.26±0.04 1.09±0.04 0.99±0.04 1.03±0.041,200-1,300 µm 1.14±0.03 1.25±0.07 1.41±0.08 1.17±0.051,400-1,500 µm 1.66±0.12 1.47±0.24 0.75±0.07 0.54±0.041,600-1,700 µm 0.47±0.07 0.81±0.11 0.98±0.05 0.83±0.06

* For the purposes of illustrating Figure 5b, this table only averages the channels detecting significant changes in firing rate.

Table S6: Summary of Bonferroni Corrected Chronic SNFRR1-7 Days 14-42 Days 49-77 Days 84-182 Days

0-100 µm 0.30±0.11 0.42±0.15 0.18±0.18 0.02±0.02200-300 µm 1.21±0.08 1.38±0.13 1.70±0.13 2.14±0.13400-500 µm 1.54±0.10 1.65±0.16 1.44±0.18 1.31±0.16600-700 µm 1.47±0.12 1.80±0.19 1.14±0.28 0.80±0.16800-900 µm 1.25±0.11 1.44±0.19 0.35±0.15 0.15±0.06

1,000-1,100 µm 1.00±0.15 0.67±0.15 0.52±0.13 0.46±0.091,200-1,300 µm 0.80±0.13 0.91±0.20 0.88±0.10 0.44±0.091,400-1,500 µm 1.15±0.17 0.83±0.27 0.36±0.18 0.07±0.051,600-1,700 µm 0.17±0.09 0.11±0.11 0.20±0.10 0.15±0.06

* For the purposes of illustrating Figure 5b, this table only averages the channels detecting significant changes in firing rate.

Table S7: Summary of Chronic SU Amplitude1-7 Days 14-42 Days 49-77 Days 84-182 Days

0-100 µm 42.6±8.2 µV * * -200-300 µm 48.7±5.2 µV 53.4±6.4 µV 69.6±10.8 µV 82.8±3.9 µV400-500 µm 68.3±5.8 µV 86.3±8.9 µV 69.2±5.2 µV 58.3±5.4 µV600-700 µm 69.5±7.3 µV 62.1±3.8 µV 38.6±3.4 µV 22.3±6.5 µV800-900 µm 57.9±3.2 µV 56.9±7.1 µV 42.0±7.2 µV 14.6±6.3 µV

1,000-1,100 µm 62.0±5.7 µV 62.7±4.7 µV 56.6±8.0 µV 56.6±3.5 µV1,200-1,300 µm 60.0±7.2 µV 73.3±3.6 µV 47.3±6.5 µV 29.6±3.0 µV

1,400-1,500 µm 89.2±5.1 µV 61.7±5.1 µV 27.8±5.2 µV 2.9±3.3 µV **1,600-1,700 µm 45.5±6.1 µV 26.6±5.1 µV 14.0±3.7 µV 3.1±1.4 µV **

* Only detected one SU 49.5 µV, 36.9 µV, and 43.2 µV on days 14, 45, and 70 respectively** Averages of days 84-140. No SU were detected after day 140

Table S8: Summary of Chronic MU Amplitude1-7 Days 14-42 Days 49-77 Days 84-182 Days

0-100 µm 43.2±2.0 µV 57.6±6.9 µV 39.8±11.3 µV 28.6±4.7 µV200-300 µm 50.9±3.3 µV 87.9±5.0 µV 82.8±3.5 µV 95.8±4.3 µV400-500 µm 63.5±7.3 µV 98.9±7.8 µV 95.4±4.9 µV 85.2±5.7 µV600-700 µm 84.3±7.3 µV 87.5±2.4 µV 68.7±2.5 µV 56.8±2.8 µV800-900 µm 64.0±2.9 µV 67.5±4.2 µV 69.8±3.9 µV 40.7±4.9 µV

1,000-1,100 µm 67.0±2.9 µV 70.7±3.5 µV 63.0±5.4 µV 51.1±4.6 µV1,200-1,300 µm 64.7±1.9 µV 69.2±2.6 µV 56.7±a.9 µV 48.7±3.1 µV1,400-1,500 µm 88.2±5.1 µV 61.6±7.5 µV 57.5±10.0 µV 32.7±4.7 µV1,600-1,700 µm 52.1±14.2 µV 40.0±9.4 µV 47.7±4.9 µV 25.7±3.2 µV

Table S9: Summary of Bonferroni Corrected Chronic MU Amplitude1-7 Days 14-42 Days 49-77 Days 84-182 Days

0-100 µm 15.7±5.4 µV 34.0±11.7 µV 7.4±7.4 µV 1.8±1.8 µV200-300 µm 57.1±6.0 µV 93.6±5.3 µV 100.1±4.3 µV 111.6±4.5 µV400-500 µm 73.5±6.6 µV 114.7±9.0 µV 102.9±9.2 µV 90.5±9.1 µV600-700 µm 95.4±10.9 µV 92.2±3.6 µV 66.1±8.3 µV 38.0±5.6 µV800-900 µm 64.2±3.9 µV 67.5±3.7 µV 24.8±10.5 µV 10.4±4.3 µV

1,000-1,100 µm 73.0±9.6 µV 59.8±12.8 µV 27.4±6.4 µV 18.7±4.1 µV1,200-1,300 µm 47.7±6.0 µV 61.8±12.4 µV 47.4±7.8 µV 20.6±4.4 µV1,400-1,500 µm 63.6±4.8 µV 48.7±10.9 µV 25.4±13.0 µV 2.1±1.4 µV1,600-1,700 µm 15.9±8.7 µV 3.5±3.5 µV 10.2±5.2 µV 5.1±2.1 µV

Table S10: Summary of Chronic Impedance1-7 Days 14-42 Days 49-77 Days 84-182 Days

0-100 µm 906±72 kΩ 1,198±44 kΩ 1,228±62 kΩ 1,168±54 kΩ200-300 µm 794±55 kΩ 1,261±45 kΩ 1,540±69 kΩ 1,541±83 kΩ400-500 µm 764±42 kΩ 1,164±45 kΩ 1,370±103 kΩ 1,566±92 kΩ600-700 µm 803±39 kΩ 1,329±73 kΩ 1,653±134 kΩ 1,576±84 kΩ800-900 µm 828±39 kΩ 1,351±103 kΩ 1,435±131 kΩ 1,411±67 kΩ

1,000-1,100 µm 896±42 kΩ 1,683±101 kΩ 1,386±150 kΩ 1,108±67 kΩ1,200-1,300 µm 913±46 kΩ 952±135 kΩ 1,074±61 kΩ 910±51 kΩ1,400-1,500 µm 989±52 kΩ 952±51 kΩ 1,074±52 kΩ 760±49 kΩ1,600-1,700 µm 1,071±80 kΩ 809±78 kΩ 540±34 kΩ 449±34 kΩ

Table S11: Summary of Chronic Electrophysiology Noise Floor1-7 Days 14-42 Days 49-77 Days 84+ Days

0-100 µm 14.7±0.7 µV 18.1±0.3 µV 18.1±0.5 µV 16.7±0.4 µV200-300 µm 13.2±0.7 µV 19.2±0.7 µV 19.6±0.3 µV 19.8±0.5 µV400-500 µm 14.4±0.7 µV 19.9±0.6 µV 20.0±0.5 µV 21.1±0.7 µV600-700 µm 15.1±0.7 µV 19.9±0.3 µV 19.7±0.5 µV 20.2±0.5 µV800-900 µm 15.3±0.5 µV 18.6±0.4 µV 18.5±0.7 µV 19.8±0.6 µV

1,000-1,100 µm 15.3±0.5 µV 19.5±0.3 µV 18.5±0.7 µV 18.0±0.7 µV1,200-1,300 µm 16.4±0.5 µV 19.0±0.5 µV 18.1±0.4 µV 17.9±0.6 µV

1,400-1,500 µm 21.5±0.8 µV 19.1±0.9 µV 18.5±1.2 µV 15.8±0.5 µV1,600-1,700 µm 21.4±1.0 µV 17.0±1.8 µV 12.6±1.1 µV 12.0±0.6 µV

Table S12: Summary of Chronic Unit Yield (SU+MU)1-7 Days 14-42 Days 49-77 Days 84-182 Days

0-100 µm 66.7±4.2 % 34.7±6.1 % 22.7±5.5 % 20.7±4.0 %200-300 µm 84.4±3.9 % 68.3±4.6 % 48.3±6.3 % 38.3±3.5 %400-500 µm 88.5±3.3 % 71.7±3.6 % 60.0±3.7 % 48.3±3.4 %600-700 µm 93.8±2.1 % 68.3±3.9 % 60.0±5.7 % 39.4±2.9 %800-900 µm 91.7±3.0 % 66.7±5.0 % 53.3±6.0 % 34.7±4.0 %

1,000-1,100 µm 86.5±3.8 % 70.0±4.8 % 55.0±5.6 % 45.3±3.1 %1,200-1,300 µm 86.5±4.6 % 78.3±7.0 % 70.0±4.8 % 50.0±3.4 %1,400-1,500 µm 97.2±1.9 % 88.5±3.9 % 52.5±8.7 % 29.6±4.3 %1,600-1,700 µm 64.6±11.6 % 76.7±13.2 % 80.0±7.4 % 60.0±7.9 %

Table S13: Summary of Bonferroni Corrected Chronic Unit Yield (SU+MU)1-7 Days 14-42 Days 49-77 Days 84-182 Days

0-100 µm 19.6±6.0 % 16.0±6.5 % 5.3±3.7 % 1.1±1.1 %200-300 µm 53.1±4.1 % 48.3±3.9 % 30.0±4.8 % 23.1±1.4 %400-500 µm 64.6±3.7 % 53.3±3.3 % 48.3±4.6 % 34.2±2.8 %600-700 µm 70.8±4.4 % 56.7±4.4 % 35.0±3.9 % 16.7±2.3 %800-900 µm 70.8±3.6 % 46.7±4.8 % 25.0±3.7 % 8.9±2.0 %

1,000-1,100 µm 44.8±5.4 % 40.0±5.1 % 46.7±5.4 % 23.9±3.5 %1,200-1,300 µm 45.8±5.6 % 40.0±5.1 % 46.7±5.4 % 23.9±3.5 %1,400-1,500 µm 88.1±4.0 % 75.5±4.2 % 24.5±6.6 % 2.6±1.5 %1,600-1,700 µm 56.3±11.7 % 46.7±14.2 % 53.3±14.2 % 18.9±6.3 %

Supplemental Figures

Figure S1. Calculation of Voltage. Blue is the raw LFP voltage signals following the ON or OFF evoked trigger. The red region is the calculated value over the first 1000 ms following the trigger. The equation used in the calculation and the variable name is listed for each metric.

Figure S2: Peak stability of LFP. Shown is LFPON (blue), LFPOFF (red), RSpseudoON (gray) and RSpseudoOFF (black dashed) a) on day 0 and b) day 189 for Mouse D; c) day 0 and d) day 182 for Mouse E; e) day 0 and f) day 182 for Mouse F. Note: minimal drift in peak between first and last days of recording for LFPON and LFPOFF.

Figure S3: Power in Depth vs. Frequency. a) Bone screw reference (left) and same data with CAR (right) recorded on day 6. Mouse A with high power activity in shallow layers. Power is increased with CAR and band of low power is visible at -600µm. b) Bone screw reference (left) and same data with CAR (right) recorded on day 126. Mouse E with high power activity in deep layers. Power activity in Layers I and II/II is increased and band of low power seen at -1000µm in this animal.

Figure S4. Calculation of Power. Blue is the normalized LFP power spectrum of the ON-RS, OFF-RS, or |ON-OFF| response. The red region is the calculated value. The equation used in the calculation and the variable name is listed for each metric.

Figure S5. Division Power Spectra Normalization. a-b) Results from power spectrum normalization by dividing with resting state activity using the contralateral bone screw reference (a: BSR), and common average reference (b: CAR). c-e) Yield calculation comparison between BSR (black) and CAR (blue), and subtraction

normalization (dark), division normalization (middle), and without normalization (light). c) Evoked Power Area (Mean). d) Evoked Median Power. e) Evoked Peak Power.

Figure S6: Recording Yield as a function of depth and time. a) SU. b) Bonferroni corrected activity dependent unsorted units (p<0.05, α<1.645x10-5). Note: there are greater yields around layer IV and in CA1. c) LFP yield using peak ON response using BSR (without CAR).

Figure S7: Signal Quality: SNR as a function of depth and time. a) SU SNAR (voltage). b) Bonferroni corrected evoked MU SNFRR of Activity dependent unsorted units (p<0.05, α<1.645x10-5). Note: greater SNR is detected around layer IV and in CA1. c) LFP SNLPR of power ON response using BSR (without CAR).

Figure S8: Signal Strength as a function of depth and time. a) Mean SU voltage amplitude. b) Bonferroni corrected evoked MU mean voltage amplitude + 2*STD of activity dependent unsorted units (p<0.05, α<1.645x10-5). Note: greater amplitude is detected around layer IV and in CA1. c) LFP peak power (strength) of ON using BSR (without CAR).

Figure S9: a) 1 kHz impedance. b) Noise floor voltage. Note: early increases in impedance in the region bordering hippocampus and cortex as well as increases in impedance of the deeper layers. Also note: high impedance in region bordering hippocampus and cortex of later time points. c) Stability of LFP ON response using BSR (without CAR) reported as frequency shift (Hz).

Figure S10: Impedance and noise floor over the two weeks. a) 1 kHz impedance. b) Noise floor voltage. Note: early increases in impedance in the region bordering hippocampus and cortex as well as increases in impedance of the deeper layers. Also note: high impedance in region bordering hippocampus and cortex of later time points.

Reference

Schroeder CE, Mehta AD, Givre SJ. A spatiotemporal profile of visual system activation revealed by current source density analysis in the awake macaque. Cerebral cortex, 1998; 8: 575-92.