Supplementary Figure 1. GABA depolarizes the majority of immature neurons in the upper cortical layers at P3 4 in vivo. (a b) Cell-attached current-clamp recordings illustrate responses to puff-applied GABA (100 mm, 5 s; application interval highlighted in grey) for all cells included in the analysis [n = 12 cells in the cortical plate (a), n = 3 cells in the marginal zone (b)]. Each trace represents a single trial. Electromagnetic artefacts due to valve 1
opening/closure were clipped for clarity. Cell numbers refer to those in Fig. 1f. Depolarizing responses are shown in red, hyperpolarizing responses in blue. Grey traces refer to trials with amplitudes lower than 3 times the standard deviation of the baseline noise. Scale bars, 10 mv, 5 s. 2
Supplementary Figure 2. Polarity of GABA A R-induced membrane potential changes in layer 2/3 neurons at P25 27 in vivo. (a,c) Cell-attached current-clamp recordings illustrate responses to puff-application of the specific GABA A R agonist muscimol (5 mm, 1 s; application interval highlighted in grey) in the presence (a) and absence (c) of the membranepermeable carbonic anhydrase (CA) inhibitor EZA (150 µm) at P25 27 in vivo. Scale bars, 10 mv, 2.5 s. Note that intense GABA A R activation in mature cortical neurons may be depolarizing due to GABA A R-dependent HCO 3 continuous replenishment of intracellular HCO 3 flux which is strictly dependent on the by cytosolic CA activity (commencing at ~P10) and suppressed by membrane-permeable CA inhibitors 1-3. (b,d) Quantification for cells recorded in the presence (b) and absence (d) of EZA. Each symbol represents a single trial. For cells #1 and #3 (in two out of four trials) shown in d, muscimol-application time was set 5 s. In all other cells/trials, muscimol was puff-applied for 1 s. In all experiments, the tip of the application pipette (tip diameter ~2 µm) was positioned in the subarachnoid space. Note that 3
muscimol was used in order to avoid activation of metabotropic GABA B receptors which, at this age, may induce hyperpolarizing postsynaptic responses 4,5. Alexa Fluor 594 was added to the application solution in order to confirm outflow (see Methods). In the presence of EZA, the mean ΔV was significantly lower than at P3 4 (P25 27: +1.2 ± 1.2 mv, n = 10; P3 4: +6.7 ± 1.5 mv, n = 15; P < 0.05, two-sample t test). (a d) Depolarizing responses are indicated by red traces/symbols, hyperpolarizing responses by blue traces/symbols. Grey traces/symbols refer to trials with amplitudes lower than 3 times the standard deviation of the baseline noise. 4
Supplementary Figure 3. GABA induces somatic Ca 2+ transients in cortical plate cells at P3 4 in vitro. (a f) Loading with the Ca 2+ indicator OGB1 was performed in vivo only. Experiments were carried out first in vivo (see also Fig. 2a-d) and second in acute brain slices in vitro. (a) Experimental arrangement. (b) Two-photon fluorescence image of OGB1-stained CP cells in vitro. Scale bar, 10 µm. (c) In vitro single-cell Ca 2+ responses to puff-applied glutamate (Glu; 1 mm) and GABA (1 mm). Scale bars, 1.5 ΔF/F 0, 2.5 s. (d) ΔF/F 0 amplitude distributions. Each symbol represents a single cell (n = 407 cells from 10 slices and 5 mice). (e) Cumulative single-cell ΔF/F 0 amplitude distributions for glutamate- and GABA-induced responses (in vivo: n = 204 cells, in vitro: n = 407 cells). (f) Mean ΔF/F 0 amplitudes per animal (n = 5 mice). (g) Two-photon fluorescence image of OGB1-stained CP cells in vitro. Scale 5
bar, 10 µm. (h) In vitro single-cell Ca 2+ responses to puff-applied GABA (1 mm) in the absence (Control) and presence of BayK 8644 (20 µm). Scale bars, 0.5 ΔF/F 0, 2 s. (i) Singlecell GABA-induced ΔF/F 0 amplitude distributions (n = 129 cells from 5 slices and 3 mice). (j) Mean GABA-induced ΔF/F 0 amplitudes per slice (P < 0.01, n = 5 slices, paired t test). (f,j) Mean ± SEM. 6
Supplementary Figure 4. Two-photon Ca 2+ imaging in CP neurons at P1 in vivo. (a) Twophoton fluorescence image of OGB1-stained CP cells at P1. Scale bar, 10 µm. (b) Single-cell Ca 2+ responses to puff-applied glutamate (Glu 100 mm, 400 ms) and GABA (100 mm, 400 ms). Scale bars, 1.0 ΔF/F 0, 10 s. (c) Distributions of single-cell responses to glutamate (Glu), GABA and muscimol (Musci). Each symbol represents a single-cell (n = 117 cells from three mice for all groups). The subscript long refers to puff durations that were 5 times the reference duration used for glutamate. Experiments were performed in the absence of BayK 8644. (d) Cumulative ΔF/F 0 amplitude distributions in response to glutamate, GABA and muscimol (n = 117 cells). 7
Supplementary Figure 5. Correlation between GABA- and muscimol-induced responses in the presence of BayK 8644 at P3 4 in vivo. In the presence of BayK 8644 (20 µm), ΔF/F 0 amplitudes induced by puff-application of GABA and muscimol displayed significant positive correlation (Spearman s ρ = 0.67, P < 0.001). Each symbol represents a single cell (n = 90 cells). Note that only GABA-responsive cells (ΔF/F 0 > 0.13) were included in the analysis. 8
Supplementary Figure 6. Local GABA application within the cortical plate fails to elicit somatic Ca 2+ transients at P3 4 in vivo. (a) Two-photon fluorescence image of OGB1- stained CP cells in vivo. The pipette used for puff application is indicated schematically. Cells close to the pipette tip were discarded from analysis due to puff-associated movement artefacts (dotted line). Scale bar, 10 µm. (b) Single-cell Ca 2+ responses to puff-applied glutamate (Glu 10 mm, 40 ms) and GABA (10 mm, 40 ms). Scale bars, 1.0 ΔF/F 0, 5 s. (c) Distributions of normalized rise slopes for glutamate-induced responses evoked by epidural (n = 406 cells) or local (n = 150 cells) application in vivo and local application in vitro (n = 203 cells). Note significantly faster rise kinetics of responses induced by local application of glutamate in vivo as compared to epidural application. Also note large overlap with rise kinetics obtained with local application in vitro (see Supplementary Fig. 3c). (d) Single-cell ΔF/F 0 amplitude distributions in response to local application of glutamate (n = 150 cells), GABA (n = 150 cells) or ACSF (n = 74 cells) in vivo and GABA in vitro (n = 406 cells, same as in Supplementary Fig. 3e). Note that distinct GABA-mediated (10 mm, 30 40 ms) CaTs were not observed in glutamate-responsive cells (GABA-induced ΔF/F 0 : 0.01 ± 0.00, glutamate- 9
induced ΔF/F 0 : 1.55 ± 0.04, n = 150 cells from six recording sites and three mice). For comparison, ΔF/F 0 distributions in response to GABA in vitro for cells with normalized rise slopes of glutamate-induced responses <1.3 s 1 (n = 212 cells) as well as to epidural application of glutamate in vivo (n = 204 cells, same as in Supplementary Fig. 3e) are also shown. 10
Supplementary Figure 7. Basic properties of Ca 2+ cluster activity in the neonatal 11
occipital neocortex at P3 4 in vivo. (a) Two-photon fluorescence image of GCaMP3- expressing CP cells in vivo. Scale bar, 10 µm. (b) Sample traces (ΔF/F 0 ) from six individual cells indicated in a. Scale bars, 1.0 ΔF/F 0, 25 s. (c) Cumulative distribution of the frequency of spontaneous somatic Ca 2+ transients. Mean (solid line) ± SEM (shaded area) from 5 mice. (d) Mean frequency of spontaneous somatic Ca 2+ transients per cell and animal (n = 5 mice). Each symbol represents a single animal. Mean ± SEM. (e) In vivo view (under normal light) of the recording chamber (ch) glued to the intact skull. The field of view during recording is indicated by a dotted rectangle (trans, transverse sinus; sag, transition into superior sagittal sinus). Scale bar, 500 µm. (f) GCaMP3 fluorescence (measured through the intact skull) overlaid with area plots of four spatially confined cluster events (colour-coded). Scale bar, 200 µm. (g) Sample traces (ΔF/F 0 ) from individual ROIs. Inset indicates positions of ROIs from which sample traces were obtained. Scale bars, 0.25 ΔF/F 0, 5 s. (h) Sample raster plots from a single animal (same as in e g) 60 min after withdrawal of isoflurane (Control, left), in the presence of N 2 O (middle) and N 2 O plus 1% isoflurane (right). Periods of mechanical instability that were discarded from analysis are highlighted by grey vertical lines. (i k) Quantification (mean ± SEM). Note that, in the presence of N 2 O, the parameters analyzed were similar whether or not a craniotomy was performed. Also note that isoflurane completely blocked spontaneous Ca 2+ cluster activity. Grey bars reflect pooled data from animals shown in Fig. 5 (n = 21 mice). (l) In vivo view of a craniotomy (~1.2 mm²) over the occipital cortex as used for experiments analyzed in Fig. 5. The field of view during recording is indicated by a dotted rectangle. Scale bar, 500 µm. (m) Ex vivo transmission image of the isolated brain overlaid with emission from a red fluorescent position marker (JPW 1114) injected in vivo after the imaging session. The projected field of view during imaging is indicated by a dotted rectangle. Scale bar, 1 mm. (n) Raster plot of times of peak of Ca 2+ transients for all ROIs in the 12
presence of gabazine (top) and gabazine plus TTX (3 µm, bottom). N 2 O was administered throughout the experiment. Data are from the same animal shown in (l m) and Fig. 5i. For clarity, periods of mechanical instability are not indicated here. (o) Quantification of TTX effects on Ca 2+ transient frequency per ROI. Each pair of symbols represents a single animal (n = 2 in bumetanide, n = 2 in gabazine). 13
Supplementary Figure 8. Spontaneous neocortical network activity in vivo depends on intact GABA A R-mediated transmission. (a d) Quantification of cluster frequency as a function of the number of active ROIs per cluster event in control versus bumetanide (n = 5, a), control versus diazepam (n = 6, b), control versus gabazine (n = 5, c) and control versus L-655,708 (n = 5, d). Bin size is 10 ROIs (except for the last bin with range 211 221). Bars and error bars indicate mean ± SEM. 14
Supplementary Figure 9. GABA A R inhibition enhances NMDA-induced somatic Ca 2+ transients in the upper cortical plate at P3 4 in vivo. (a) Two-photon fluorescence image of OGB1-stained CP cells in vivo. Scale bar, 10 µm. (b) Single-cell Ca 2+ responses to NMDA (10 mm, 150 ms) in the absence (Control) and presence of gabazine (40 µm) under isoflurane anaesthesia. NMDA was focally applied from an epidurally positioned pipette in order to preferentially activate neurons in the upper CP. Scale bars, 1.0 ΔF/F 0, 5 s. (c) Cumulative NMDA-induced ΔF/F 0 amplitude distributions (n = 199 cells from 4 mice). (d) Mean NMDAinduced ΔF/F 0 amplitudes per animal (P < 0.05, n = 4 mice, paired t test). Mean ± SEM. *P < 0.05. 15
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