Supplementary Information
The supplemental sections are numbered to indicate corresponding sections in the main text.
S4.4 Electrophysiological Identification of Layer IV
Current 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 Analysis
Probe 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 Yield
1-7 Days
14-42 Days
0-100 µm
2.4±1.7 %
2.9±2.9 %
200-300 µm
31.3±4.3 %
36.3±6.6 %
400-500 µm
61.6±3.9 %
43.8±4.7 %
600-700 µm
67.9±2.8 %
38.8±3.5 %
800-900 µm
54.5±2.2 %
20.0±3.3 %
1,000-1,100 µm
27.7±5.1 %
21.3±4.2 %
1,200-1,300 µm
25.9±4.6 %
20.0±2.0 %
1,400-1,500 µm
78.6±2.9 %
46.9±5.3 %
1,600-1,700 µm
57.1±11.3 % 36.7±13.6 %
49-77 Days
2.0±2.0 %
18.8±4.3 %
32.5±3.3 %
15.0±2.5 %
15.0±3.6 %
10.0±2.5 %
15.0±3.1 %
12.6±3.9 %
23.3±11.2 %
84-182 Days
0.0±0.0 %
12.9±0.7 %
16.4±1.8 %
5.5±1.2 %
3.1±1.2 %
5.5±1.1 %
7.4±1.5 %
1.0±0.7 %
4.2±2.0 %
Table S2: Summary of Chronic MU Yield
1-7 Days
14-42 Days
0-100 µm
64.3±4.2 %
30.7±5.6 %
200-300 µm
81.0±3.5 %
63.3±6.5 %
400-500 µm
88.1±3.2 %
68.3±3.9 %
600-700 µm
88.1±2.5 %
63.3±4.2 %
800-900 µm
83.3±3.9 %
58.3±5.7 %
1,000-1,100 µm
78.6±4.2 %
58.3±6.7 %
1,200-1,300 µm
76.2±6.1 %
71.7±8.3 %
1,400-1,500 µm
68.9±4.6 %
63.5±10.9 %
1,600-1,700 µm
35.7±9.6 %
53.3±12.4 %
49-77 Days
19.3±5.7 %
48.3±6.3 %
58.3±4.5 %
55.0±5.6 %
45.0±4.3 %
48.3±5.2 %
63.3±4.8 %
52.5±8.7 %
76.7±8.7 %
84-182 Days
19.4±3.8 %
38.3±3.3 %
41.1±3.7 %
35.7±3.2 %
30.7±3.9 %
38.8±3.8 %
46.6±4.4 %
27.7±4.3 %
55.2±8.0 %
Table S3: Summary of Bonferroni Corrected Chronic MU Yield
1-7 Days
14-42 Days
49-77 Days
0-100 µm
17.1±5.6 %
12.0±4.4 %
2.0±2.0 %
200-300 µm
44.8±4.8%
38.3±3.6 %
25.0±5.1 %
400-500 µm
58.3±3.7%
48.3±3.8 %
40.0±5.7 %
600-700 µm
57.3±3.7 %
46.7±3.3 %
26.7±5.1 %
800-900 µm
42.7±3.0 %
28.3±4.3 %
8.3±3.7 %
1,000-1,100 µm
26.0±4.6 %
18.3±3.9 %
11.7±2.5 %
1,200-1,300 µm
21.9±3.9 %
25.0±6.2 %
31.7±7.2 %
1,400-1,500 µm
31.3±4.0 %
25.0±7.1 %
8.5±3.5 %
1,600-1,700 µm
6.3±3.4 %
10.0±10.0 % 30.0±15.3 %
84-182 Days
1.1±1.1 %
21.7±0.9 %
23.6±2.3 %
13.1±2.2 %
3.9±1.5 %
9.2±1.8 %
14.4±3.4 %
1.8±1.3 %
14.4±6.3 %
Table S4: Summary of Chronic SU SNAR
1-7 Days
14-42 Days
84-182 Days
49-77 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±0.04
400-500 µm
3.28±0.09
2.92±0.15
2.56±0.05
2.44±0.06
600-700 µm
3.45±0.18
2.53±0.11
2.13±0.03
2.11±0.03
800-900 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±0.02
1,200-1,300 µm
2.40±0.09
2.32±0.03
2.25±0.06
2.12±0.02
1,400-1,500 µm
3.73±0.13
2.54±0.05
2.13±0.04
2.01±0.01
1,600-1,700 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 SNFRR
1-7 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±0.03
200-300 µm
1.73±0.08
2.09±0.12
2.21±0.12
3.12±0.13
400-500 µm
2.36±0.09
2.51±0.09
2.36±0.16
2.18±0.12
600-700 µm
2.19±0.08
2.98±0.20
1.61±0.13
1.45±0.11
800-900 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±0.04
1,200-1,300 µm
1.14±0.03
1.25±0.07
1.41±0.08
1.17±0.05
1,400-1,500 µm
1.66±0.12
1.47±0.24
0.75±0.07
0.54±0.04
1,600-1,700 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 SNFRR
1-7 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±0.02
200-300 µm
1.21±0.08
1.38±0.13
1.70±0.13
2.14±0.13
400-500 µm
1.54±0.10
1.65±0.16
1.44±0.18
1.31±0.16
600-700 µm
1.47±0.12
1.80±0.19
1.14±0.28
0.80±0.16
800-900 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±0.09
1,200-1,300 µm
0.80±0.13
0.91±0.20
0.88±0.10
0.44±0.09
1,400-1,500 µm
1.15±0.17
0.83±0.27
0.36±0.18
0.07±0.05
1,600-1,700 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 Amplitude
1-7 Days
14-42 Days
0-100 µm
42.6±8.2 µV
*
200-300 µm
48.7±5.2 µV
53.4±6.4 µV
400-500 µm
68.3±5.8 µV
86.3±8.9 µV
600-700 µm
69.5±7.3 µV
62.1±3.8 µV
800-900 µm
57.9±3.2 µV
56.9±7.1 µV
1,000-1,100 µm
62.0±5.7 µV
62.7±4.7 µV
1,200-1,300 µm
60.0±7.2 µV
73.3±3.6 µV
49-77 Days
*
69.6±10.8 µV
69.2±5.2 µV
38.6±3.4 µV
42.0±7.2 µV
56.6±8.0 µV
47.3±6.5 µV
84-182 Days
82.8±3.9 µV
58.3±5.4 µV
22.3±6.5 µV
14.6±6.3 µV
56.6±3.5 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 Amplitude
1-7 Days
14-42 Days
0-100 µm
43.2±2.0 µV
57.6±6.9 µV
200-300 µm
50.9±3.3 µV
87.9±5.0 µV
400-500 µm
63.5±7.3 µV
98.9±7.8 µV
600-700 µm
84.3±7.3 µV
87.5±2.4 µV
800-900 µm
64.0±2.9 µV
67.5±4.2 µV
1,000-1,100 µm
67.0±2.9 µV
70.7±3.5 µV
1,200-1,300 µm
64.7±1.9 µV
69.2±2.6 µV
1,400-1,500 µm
88.2±5.1 µV
61.6±7.5 µV
1,600-1,700 µm
52.1±14.2 µV
40.0±9.4 µV
49-77 Days
39.8±11.3 µV
82.8±3.5 µV
95.4±4.9 µV
68.7±2.5 µV
69.8±3.9 µV
63.0±5.4 µV
56.7±a.9 µV
57.5±10.0 µV
47.7±4.9 µV
Table S9: Summary of Bonferroni Corrected Chronic MU Amplitude
1-7 Days
14-42 Days
49-77 Days
0-100 µm
15.7±5.4 µV
34.0±11.7 µV
7.4±7.4 µV
200-300 µm
57.1±6.0 µV
93.6±5.3 µV
100.1±4.3 µV
400-500 µm
73.5±6.6 µV
114.7±9.0 µV
102.9±9.2 µV
600-700 µm
95.4±10.9 µV
92.2±3.6 µV
66.1±8.3 µV
800-900 µm
64.2±3.9 µV
67.5±3.7 µV
24.8±10.5 µV
1,000-1,100 µm
73.0±9.6 µV
59.8±12.8 µV
27.4±6.4 µV
1,200-1,300 µm
47.7±6.0 µV
61.8±12.4 µV
47.4±7.8 µV
1,400-1,500 µm
63.6±4.8 µV
48.7±10.9 µV
25.4±13.0 µV
1,600-1,700 µm
15.9±8.7 µV
3.5±3.5 µV
10.2±5.2 µV
Table S10: Summary of Chronic Impedance
1-7 Days
14-42 Days
0-100 µm
906±72 kΩ
1,198±44 kΩ
200-300 µm
794±55 kΩ
1,261±45 kΩ
400-500 µm
764±42 kΩ
1,164±45 kΩ
600-700 µm
803±39 kΩ
1,329±73 kΩ
800-900 µm
828±39 kΩ
1,351±103 kΩ
1,000-1,100 µm
896±42 kΩ
1,683±101 kΩ
1,200-1,300 µm
913±46 kΩ
952±135 kΩ
1,400-1,500 µm
989±52 kΩ
952±51 kΩ
1,600-1,700 µm
1,071±80 kΩ
809±78 kΩ
49-77 Days
1,228±62 kΩ
1,540±69 kΩ
1,370±103 kΩ
1,653±134 kΩ
1,435±131 kΩ
1,386±150 kΩ
1,074±61 kΩ
1,074±52 kΩ
540±34 kΩ
Table S11: Summary of Chronic Electrophysiology Noise Floor
1-7 Days
14-42 Days
49-77 Days
0-100 µm
14.7±0.7 µV
18.1±0.3 µV
18.1±0.5 µV
200-300 µm
13.2±0.7 µV
19.2±0.7 µV
19.6±0.3 µV
400-500 µm
14.4±0.7 µV
19.9±0.6 µV
20.0±0.5 µV
600-700 µm
15.1±0.7 µV
19.9±0.3 µV
19.7±0.5 µV
800-900 µm
15.3±0.5 µV
18.6±0.4 µV
18.5±0.7 µV
1,000-1,100 µm
15.3±0.5 µV
19.5±0.3 µV
18.5±0.7 µV
1,200-1,300 µm
16.4±0.5 µV
19.0±0.5 µV
18.1±0.4 µV
84-182 Days
28.6±4.7 µV
95.8±4.3 µV
85.2±5.7 µV
56.8±2.8 µV
40.7±4.9 µV
51.1±4.6 µV
48.7±3.1 µV
32.7±4.7 µV
25.7±3.2 µV
84-182 Days
1.8±1.8 µV
111.6±4.5 µV
90.5±9.1 µV
38.0±5.6 µV
10.4±4.3 µV
18.7±4.1 µV
20.6±4.4 µV
2.1±1.4 µV
5.1±2.1 µV
84-182 Days
1,168±54 kΩ
1,541±83 kΩ
1,566±92 kΩ
1,576±84 kΩ
1,411±67 kΩ
1,108±67 kΩ
910±51 kΩ
760±49 kΩ
449±34 kΩ
84+ Days
16.7±0.4 µV
19.8±0.5 µV
21.1±0.7 µV
20.2±0.5 µV
19.8±0.6 µV
18.0±0.7 µV
17.9±0.6 µV
1,400-1,500 µm
1,600-1,700 µm
21.5±0.8 µV
21.4±1.0 µV
19.1±0.9 µV
17.0±1.8 µV
Table S12: Summary of Chronic Unit Yield (SU+MU)
1-7 Days
14-42 Days
0-100 µm
66.7±4.2 %
34.7±6.1 %
200-300 µm
84.4±3.9 %
68.3±4.6 %
400-500 µm
88.5±3.3 %
71.7±3.6 %
600-700 µm
93.8±2.1 %
68.3±3.9 %
800-900 µm
91.7±3.0 %
66.7±5.0 %
1,000-1,100 µm
86.5±3.8 %
70.0±4.8 %
1,200-1,300 µm
86.5±4.6 %
78.3±7.0 %
1,400-1,500 µm
97.2±1.9 %
88.5±3.9 %
1,600-1,700 µm
64.6±11.6 % 76.7±13.2 %
18.5±1.2 µV
12.6±1.1 µV
49-77 Days
22.7±5.5 %
48.3±6.3 %
60.0±3.7 %
60.0±5.7 %
53.3±6.0 %
55.0±5.6 %
70.0±4.8 %
52.5±8.7 %
80.0±7.4 %
15.8±0.5 µV
12.0±0.6 µV
84-182 Days
20.7±4.0 %
38.3±3.5 %
48.3±3.4 %
39.4±2.9 %
34.7±4.0 %
45.3±3.1 %
50.0±3.4 %
29.6±4.3 %
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 |ONOFF| 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.