Two distinct mechanisms for experiencedependent

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1 SUPPLEMENTARY INFORMATION Articles In the format provided by the authors and unedited. Two distinct mechanisms for experiencedependent homeostasis Michelle C. D. Bridi 1,5, Roberto de Pasquale 1,5, Crystal L. Lantz 2,5, Yu Gu 3, Andrew Borrell 3, Se-Young Choi 1, Kaiwen He 1, Trinh Tran 1, Su Z. Hong 1, Andrew Dykman 1, Hey-Kyoung Lee 1,4, Elizabeth M. Quinlan 2,3 * and Alfredo Kirkwood 1,4 * 1 Mind/Brain Institute, Johns Hopkins University, Baltimore, MD, USA. 2 Department of Biology, University of Maryland, College Park, MD, USA. 3 Neuroscience and Cognitive Sciences Program, University of Maryland, College Park, MD, USA. 4 Department of Neuroscience, Johns Hopkins University, Baltimore, MD, USA. 5 These authors contributed equally: Michelle C. D. Bridi, Roberto de Pasquale and Crystal L. Lantz. * equinlan@umd.edu; kirkwood@jhu.edu Nature Neuroscience Nature America Inc., part of Springer Nature. All rights reserved.

2 Supplementary Figure 1 Single-unit recordings in awake, head-fixed animals (A,B) Representative waveforms from units recorded before and after DE (A) and BS (B) (see Figure 1A, D). Thin lines represent individual waveforms; black lines represent the average waveform. (C) Single unit firing rates in naïve mice at baseline and following DE with diazepam treatment. There was no significant difference between groups (P=0.676, 2-tailed Wilcoxon rank sum test), confirming that diazepam prevents the DE-mediated increase in spontaneous firing (see Figure 1A). Data are shown as individual points; dashed lines and error bars represent mean±sem. Sample size is indicated in parentheses as (cells, mice).

3 Supplementary Figure 2 Effects of visual deprivation and modulation of inhibition on single-unit firing properties (A) Spontaneous bursting increased after DE and after BS with flumazenil treatment. *P (U(60)=211.0), #P=0.047 (U(60)=235.0), P=0.006 (U(64)=328.0), P=0.013 (U(49)=193.0), 2-tailed Wilcoxon rank sum test. Sample size is shown as (units, mice). (B) Non-burst firing rates increased after DE. *P (U(60)=235.0), 2-tailed Wilcoxon rank sum test. No significant differences were observed between the recovery and DE+diazepam (U(60)=363.0, P=0.100), baseline and BS (U(64)=403.0, P=0.071), or recovery and BS+flumazenil (U(49)=238, P=0.103) conditions. Sample size as shown in panel (A). Data are shown as individual points; dashed lines and error bars represent mean±sem. (C) Raster plots of two example units from awake, head-fixed mice viewing a grey screen. Spikes were classified as non-burst (blue) or part of a burst (magenta) (see Methods). A portion of the raster plots is enlarged (below) to show more detail.

4 Supplementary Figure 3 Effects of acute drug treatment on spontaneous single-unit firing properties (A) Single unit recordings were obtained during presentation of a grey screen in awake, head-fixed mice. V1 firing rates were measured at baseline (open circles) and 20 minutes after drug administration i.p. (filled circles). Diazepam and THIP decreased, and flumazenil increased, spontaneous firing rate. *P 0.001, #P=0.002, 2-tailed Wilcoxon signed rank test. injection did not alter spontaneous firing rate (Z(15)=0.646, P=0.528). Sample size is indicated as (units, mice). (B) To demonstrate the long-lasting effects of a single drug injection i.p., single unit recordings were made from V1 under isoflurane anesthesia. Diazepam decreased and flumazenil increased (4 units, 4 mice per group) spontaneous neuronal firing rates for at least 2h after the injection. The dotted gray line indicates baseline firing rate for reference. *P vs. -30 minute time point, One-way repeated measures ANOVA with Holm-Sidak post-hoc test. Sample size is shown as (units, mice). (C,D) Single unit firing properties during acute drug administration. Diazepam and THIP decreased, and flumazenil increased, both spontaneous bursting (C; 2-tailed paired t test) and the inter-burst firing rate (D, 2- tailed Wilcoxon signed rank test). *P 0.001, #P= injection did not alter these parameters (bursting, 2-tailed paired t test t(15)=-0.313, P=0.759; inter-burst firing rate, 2-tailed Wilcoxon signed rank test Z(15)=0.267, P= Sample size as shown in (A). For all panels, individual unit firing parameters at each time point are connected by grey lines. Colored symbols and lines indicate the mean and error bars represent SEM.

5 Supplementary Figure 4 Diazepam administered i.c.v. prevents the DE-mediated increase in mepsc amplitude (A) Average mepsc traces after diazepam or vehicle infusion directly into the lateral ventricle via osmotic minipump during DE. (B) mepsc amplitude was larger in vehicle- than diazepam-infused animals. *P=0.013, 2-tailed t test. Dashed lines and error bars indicate mean±sem. For clarity, some data points are displaced horizontally. Sample size is shown as (cells, mice).

6 Supplementary Figure 5 Activation of G i -DREADD in PV cells elevates spontaneous activity in neighboring regular-spiking neurons (A) CNO (5 mg/kg i.p.) increased the firing rate of regular spiking neurons in hemispheres where Gi-DREADD was expressed in PV cells. *P 0.001, 2-tailed paired t test. Data are from 31 units recorded from 3 awake, head-fixed animals viewing a grey screen. Grey lines represent individual units before and after CNO administration; green line and error bars represent mean ±SEM. (B) For mepsc recordings (Fig. 3C, D), patch electrodes were targeted to pyramidal cells neighboring mcherry-labeled, Gi-DREADD expressing PV cells.

7 Supplementary Figure 6 Diazepam administered i.c.v. prevents the DE-mediated increase in GluN2B function (A) DE increased the percentage of NMDAR EPSC blocked by ifenprodil (*P=0.021, 2-tailed t test) and the NMDA receptor decay constant ( w ) ( P=0.002, 2-tailed t test) in animals with no drug administration. (B) In DE animals, diazepam prevented the GluN2B increase compared to vehicle (*P=0.039; P=0.030; 2-tailed t test). Solid lines: average baseline NMDAR current; dashed lines: after ifenprodil wash-in. Traces are normalized to baseline. Data are displayed as individual points; dashed lines indicate average (±SEM); sample size is shown as (neurons, mice).

8 Supplementary Table 1. Group 1. NR (11, 6) 2. DE (13, 4) Amplitude (pa) Frequency (Hz) Rise Time (ms) Decay Time (ms) 11.77± ± ± ±0.22 *1, 3, ± ± ± ±0.29 RMS, 4 Ri (M ) Ra (M ) 1.55± ± ±0.93 *1 1.84± ± ± NR Diazepam (12, 5) 12.02± ± ± ± ± ± ± DE Diazepam (14, 5) 12.49± ± ± ±0.34 *1 1.80± ± ±0.7 ANOVA P F or H R R 0.06 R R Effects of DE and diazepam treatment on the mepsc properties (amplitude, frequency and kinetic parameters) and recording conditions (root mean square noise (RMS), membrane input resistance (Ri) and access resistance (Ra)). Sample size (cells, mice) is indicated. P and F or H s for one-way ANOVAs (F) or one-way ANOVAs on ranks (H) (denoted by R ) are shown. Degrees of freedom= 3, 46. *P<0.05, Holm-Sidak post-hoc test. Values are average ± SEM.

9 Supplementary Table 2. Treatment Amplitude (pa) Frequency (Hz) Rise Time (ms) Decay Time (ms) RMS Ri (M ) Ra (M ) 1. NR (18, 5) * ± ±0.63 *4 0.84± ± ± ± ± BS (21, 6) 3. NR Flumazenil (22, 6) * ± ± ±0.03 * ± ± ±0.04 *1, 3, ± ± ± ± ± ± ± ± BS Flumazenil (22, 6) *1, 2, ± ±0.24 *1 0.70± ± ± ± ±0.5 ANOVA P R R R R F or H Effects of BS and flumazenil treatment on the mepsc properties (amplitude, frequency and kinetic parameters) and recording conditions (root mean square noise (RMS), membrane input resistance (Ri) and access resistance (Ra)). Sample size (cells, mice) is indicated. P and F or H s for one-way ANOVAs (F) or one-way ANOVAs on ranks (H) (denoted by R ) are shown. Degrees of freedom= 3, 79. *P<0.05, Holm-Sidak or Dunn s post-hoc test. Values are average ± SEM.

10 Supplementary Table 3. Treatment Value Ri (M ) Ra (M ) % NMDAR current blocked by ifenprodil NR (18, 5) DE (20, 5) NR (33, 5) 10.56±2.2% ± ± ±1.8% ± ±0.47 t(36)=2.360, P=0.024 U(36)=167.0, P=0.715 t(36)=0.147, P= ±2.6 ms ± ±0.37 w (ms) DE (35, 5) ±2.5 ms ± ±0.42 t(66)=4.277, P U(66)=573.5, P=0.966 t(66)=0.655, P=0.515 % NMDAR current blocked by ifenprodil NR Diazepam (20, 5) DE Diazepam (19, 5) 10.12±1.2% ± ± ±2.1% ± ±0.59 U(37)=180.0, P=0.790 t(37)=0.691, P=0.494 U(37)=183.0, P=0.855 w (ms) NR Diazepam (32, 5) DE Diazepam (37, 5) 84.96±3.0 ms 341.5± ± ±2.0 ms 314.9± ±0.37 U(67)=488.0, P=0.213 U(67)=465.0, P=0.128 U(67)=579.0, P=0.880 Summary of the effects of DE and diazepam on two measures of GluN2B levels (mean±sem), and recording conditions (input resistance, Ri; access resistance, Ra) in each group. Sample size (cells, mice) is indicated under each treatment. U statistics (2-tailed Wilcoxon rank sum test), t statistics (2-tailed t test), and P s are shown.

11 Supplementary Table NR (19, 6) 2. DE (21, 7) 3. NR Ro (29, 6) 4. DE Ro (35, 6) ANOVA P Amplitude (pa) Frequency (Hz) Rise Time (ms) Decay Time (ms) RMS Ri (M ) Ra(M ) 13.68± ± ± ± ± ± ±0.5 *1, 3, ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±7 15.6± R R R R R F or H Effects of DE and Ro treatment (delivered subcutaneously via osmotic minipump) on mepsc properties (amplitude, frequency, and kinetic parameters) and recording conditions (root mean square noise (RMS), membrane input resistance (Ri) and access resistance (Ra)), displayed as mean ±SEM. Sample size (cells, mice) is indicated. P s for one-way ANOVAs or one-way ANOVAs on ranks (denoted by R ) and the corresponding F or H s are shown. *P<0.05, Holm-Sidak post-hoc test; P<0.05, Dunn s post-hoc test. Degrees of freedom=3, 100.

12 Supplementary Table 5. Amplitude (pa) Frequency (Hz) Rise Time (ms) Decay Time (ms) RMS Ri (M ) Ra(M ) 1. NR (32, 5) 2. DE (31, 5) * 2, 3, ±0.04 * 1, ± ± ± ± ± ± ± ± ± ± ± ± ± NR THIP (30, 5) 4. DE THIP (27, 5) ANOVA P F or H * 1, ± ± ± ± ± ± ±0.3 * 1, 2, ± ± ± ± ± ± ± R R R R R Effects of DE and THIP treatment on mepsc properties (amplitude, frequency and kinetic parameters) and recording conditions (root mean square noise (RMS), membrane input resistance (Ri) and access resistance (Ra)); s are presented as mean ±SEM. Sample size (cells, mice) is indicated. P s for one-way ANOVAs or one-way ANOVAs on ranks (denoted by R ) and corresponding F or H s are shown. *P<0.05, Holm-Sidak post-hoc test; P<0.05, Dunn s post-hoc test.