Animal surgery

All experiments were approved by the Berlin animal ethics committee (LAGeSo) and carried out in accordance with European animal welfare law. Animal surgery, two-photon targeted whole-cell patch clamp recordings and histology were described in detail in a previous study16 and are summarized here. P21 to 30 C57BL6J, NEX-cre65 x Ai966, fosGFP67, GAD67-GFP68, PV-cre69, or SST-cre70 mice were initially anesthetized for surgery with 1.5–2% isoflurane in O 2 ; subsequent electrophysiological recordings were made under 1.5 g/kg urethane anesthesia. A lightweight metal head support was implanted onto the skull and next, a small (1 mm2) craniotomy was drilled over the C2 whisker barrel column define by intrinsic optical imaging response or stereotactic coordinates (−1.2 mm/3.5 mm lateral). Finally, the dura was carefully removed prior to recording. For awake experiments, mice were habituated to head restraint for 2–3 days and were given >2 h recovery time from isoflurane anesthesia in their home cage before recording.

Two-photon targeted whole-cell patch clamp recordings

A Femto2D in vivo two-photon laser-scanning microscope (Femtonics), illuminated with a Chameleon Ultra II (Coherent) pulsed laser, was used to image cortical layer 2/3 neurons via a ×40 Olympus water immersion objective (LUMPLFLN 40XW, NA 0.8, W.D. 3.3 mm). We used 2 mm borosilicate glass, resistance 5–7 MΩ, (Hilgenberg) to make whole-cell patch clamp recordings. Three to four pipettes were filled with intracellular solution containing, in mM: 135 potassium gluconate, 4 KCl, 10 HEPES, 10 phosphocreatine, 4 MgATP, 0.3 Na3GTP (adjusted to pH 7.3 with KOH), 30 μm Alexa-594 (Invitrogen) and 2 mg/ml biocytin. Current-clamp whole-cell recordings were made using an Axon Multiclamp 700b amplifier (Molecular Devices) and an Ag/AgCl ground electrode in the recording chamber. The membrane potential was not compensated for the liquid junction potential.

To insert the pipettes through the pia and avoid blood vessels, a positive pressure of 200–300 mbar was applied to the recording pipettes. As soon as the pipette went through the pia the pressure was reduced to 60 mbar and the pipettes moved to 100–150 μm from the pial surface and then reduced again to 30 mbar until reaching the target neurons. Cell somata were then approached to within 20 μm. During two-photon imaging, because of the dye Alexa 594 contained in the intracellular solution, neurons often look like dark cell bodies or “shadows” at excitation wavelength 820 nm. For targeting specific interneurons or in some experiments PYR neurons, we used the offspring of the mouse line Ai9, expressing the loxP-flanked STOP cassette before the sequence of the fluorophore td-Tomato (td-T), and PV-cre, SST-cre or NEX-cre mouse line. Recording pipettes were then pushed up against the cell soma and contact was monitored with resistance changes on an oscilloscope (Tektronix TDS2024C) and the live two-photon images. Next, a gigaseal was formed between the patch electrode and the cell membrane with negative pressure. To achieve whole-cell configuration we then ruptured each cell membrane with negative pressure.

Recordings were digitized at 20 kHz, filtered at 10 kHz and recorded via an ITC18 (Heka) analog to digital converter using IgorPro (Wavemetrics). The recording depth and distance between cell soma was noted at the start of recording. Next, −100 to +400 pA, 500 ms long current pulses were injected into each neuron to measure the evoked firing patterns and obtain trains of APs. The current threshold needed to trigger a single AP in each cell was then found manually using the Multiclamp stimulus command. Next, short square current pulses of low amplitude (10–20 ms, 100–400 pA) were injected into each cell at 1 or 0.5 Hz in sweeps of 60 s to trigger single APs. In some experiments, current pulses of 20–50 ms were used to evoke multiple APs. In vivo Z-stack images (2 μm/slice) were made after recording. All recorded neurons were confirmed online after the recording according to the morphology of the filled cells (shape of the cell body, dendritic apical truck and presence of spines) and their firing pattern. In vivo images shown in figures show pseudo-colored green cells (intracellular dye alexa 594) and yellow cells that represent the spectral overlap of the pseudo-colored intracellular dye with the genetically encoded fluorescent indicator td-T.

Histology

After recording, mice were deeply anesthetized by an additional i.p. injection of urethane and transcardially perfused with 4% paraformaldehdye (PFA). The brain was removed, fixed in 4% PFA overnight and stored in PB at 4 °C before histological processing for all brains. Next, 100 μm thick tangential slices were made using a Leica VT1000 S vibrating microtome. We stained for cytochrome oxidase to reveal the barrel cortex map and for biocytin, with a standard ABC kit (Vectastain), with DAB enhancement, to reveal the recorded neurons. Slices were mounted in Moviol, stored at 4 °C and reconstructed using NeuroLucida software (MicroBrightField).

Datasets and analysis software

The rates of connectivity shown between PYR neurons shown in Fig. 1d partly used the connectivity from a previous analysis during DOWNstate16. All other analysis on PYR to PYR neuron connections was during UPstates and was not previously reported. Data analysis was performed in Matlab (Mathworks, MA, USA), Igor Pro (Wavemetrics, OR, USA), and StatEL (ad Science, Paris, France) using custom written routines.

AP detection

To detect spontaneous or current evoked APs, we first split the data into epochs with and without current injection. APs thresholds were detected using the peak time of the third derivative of the V m . AP peak times were then detected by finding the maximum of the V m following an AP threshold crossing and this was used as the time point of the AP. Trials in which multiple APs were evoked by current injection and trials with spontaneous APs that occurred just before or after a current injection epoch were excluded.

Brain state classification

To classify brain state, each evoked AP was grouped into those triggered during epochs of low synaptic cortical activity with a hyperpolarized V m (DOWNstate), or those triggered during epochs of synaptic activity with depolarized V m (UPstate). The V m value of the DOWNstate/UPstate was estimated for each AP in a 10 s window around the AP time by averaging the bottom/top 10% of the V m . To classify each AP into either DOWNstate or UPstate, we estimated the amplitude of UPstate (UPstateAmp = V m difference between DOWNstate and UPstate) and the V m fluctuation in small window around the AP (−50 to 0 ms and 50 to 100 ms). APs were then characterized into UPstate when the mean V m around the AP was more depolarized than 20–30% of UPstateAmp and the V m fluctuations >6 mV. We excluded APs that were triggered at transitions between DOWNstate and UPstate. Spontaneous PYR neuron APs were not classified into different states as they only occurred during UPstate. For awake data, we analyzed postsynaptic responses evoked during depolarized synaptic activity.

Input resistance and Tau

Current pulses (100 ms, −100 pA) were used to test for input resistance in periods of DOWNstate. Access resistance was subtracted offline using an exponential fit of the V m from a 2 ms period after the start of current injection. The difference in V m between the baseline and the time point at which the fit crossed the onset time of current injection was taken as the access resistance. The input resistance was calculated from the difference in mV between the current injection response corrected for access resistance and the prestimulus V m . Tau was calculated from the exponential fit of the relaxation phase of the V m from 2 ms after the end of the hyperpolarizing pulse.

AP half-width

To measure the AP half-width (Supplementary Fig. 1), we averaged all spontaneous APs and measured the width at half height between AP threshold and AP peak.

Spontaneous AP triggered averaging

To study the V m depolarization and dynamics prior to spontaneous APs (Supplementary Fig. 5), we aligned APs at threshold (AP threshold = 0 ms). The V m depolarization was measured 50 ms prior to AP threshold and the V m dynamics prior to spiking were measured as the change in V m from −2 to 0 ms prior to AP threshold.

Evoked postsynaptic responses

To study the impact of an u EPSP on a postsynaptic neuron, we included pairs with a minimum number of single evoked AP in the presynaptic PYR cell during epochs of active cortical activity, UPstate (PYR n ≥ 20, STT n ≥ 50, PV ≥ 50). We then calculated the following measures of the sub- and supra- threshold postsynaptic responses.

Synaptic gain

To estimate the efficiency of a u EPSP driving a postsynaptic AP response (Fig. 3b), we counted the number of postsynaptic APs between 1 and 5 ms following the presynaptic AP and subtracted this number from the average baseline AP count (−40 to 0 ms). We termed this the synaptic gain after28. Due to the small temporal integration window, a maximum of one evoked AP was observed per trial. Because PYR neurons fire extremely few spontaneous spikes under urethane anesthesia, we were unable to measure the synaptic gain to spontaneous spikes in all pairs.

u EPSP V m peak

To estimate the membrane potential of the PV neuron postsynaptic response during epochs of network activity (Fig. 4e, f), we measured the u EPSP V m peak defined by the maximum V m value in the 1–5 ms following the presynaptic AP in the mean PV neuron response. PV neuron APs were truncated in this analysis.

AP latency and jitter

To estimate the temporal precision of the evoked AP in PV neurons (Fig. 5b, c), we measured the latency and jitter of the first APs occurring in the 5 ms following the presynaptic AP. AP latency is given as the average latency between the first APs following the presynaptic AP. AP jitter is given as the standard deviation of the first AP latencies across trials. We included PV neurons with n > 10 evoked APs.

PSTH peak time and half-width

To estimate the peak time and half-width of the evoked response (Fig. 5d, e), we calculated the PSTH of the AP times with 0.5 ms resolution. This high-resolution PSTH was then fitted with a Gaussian function in the interval between 0 and 6 ms following the presynaptic AP. In the population analysis, we included only responses with good fits (Goodness of fit > 0.6) and clear evoked AP rates (evoked AP rate > 15 Hz). From those cases, we extracted the PSTH peak time (mean of the Gaussian fit) and the PSTH halfwidth (2.3548 * sigma of the Gaussian fit).

Impact of single PYR neuron APs on neighboring PYR neurons

To study the effect of a single PYR neuron AP onto the local PYR neurons (including connected and unconnected PYR neurons), we analyzed 591 pairs of simultaneously recorded PYR neurons. We analyzed single evoked PYR neuron APs in UPstate. Pairs with >25 trials were included in the population average PSTH (Fig. 7: 113/591 pairs, in total 9513 trials). For the population average of the subthreshold response, we removed trials with spontaneous APs in the putative postsynaptic PYR neuron in a window −200 to 200 ms around the evoked AP (113/591 pairs, in total 7515 trials). To estimate the amplitude of the V m response, we measure the V m difference between the baseline (5–6 ms after the presynaptic AP time) and response (24–25 ms after the presynaptic AP time). The AP rate of the baseline was estimated in the window −30:0 ms and the AP rate of the response in the window 0:30 ms following the presynaptic AP.

Connectivity analysis

Connectivity analysis is described in detail in ref. 16. To identify the presence of an excitatory monosynaptic connection for the rate analysis (Fig. 1d), we measured the response to an evoked PYR neuron AP in DOWNstate. To identify the presence of an inhibitory monosynaptic connection from PV and SST to PYRs, we used the single evoked AP postsynaptic response in periods of UPstate. Because of the strongly facilitating PYR to SST connection, we used single APs as well as trains of 5 APs delivered in DOWNstate to identify the presence of a connection from PYR to SST neurons. Kinetics analysis for u EPSPs and u IPSPs was performed on single AP evoked PSPs during UPstate.

Kinetics of u EPSP and u IPSPs

The kinetics of single mean u EPSPs and u IPSPs were measured and visually confirmed from the averaged response to a single AP during network activity. Latency was measured as the crossing point of an extrapolation of two linear fits: the first from −5 to −2 ms prior to the presynaptic AP; the second between time points corresponding to 20–80% of the u EPSP/ u IPSP amplitude. Rise time was calculated as the difference in time between 20 and 80% of the peak of the u EPSP/ u IPSP on the rising phase. Amplitude was calculated as the difference in V m between the average V m ± 0.5 ms around the peak response and the average V m ± 0.5 ms around latency. Half-width was calculated as the difference in time between 50% of the rising phase and 50% of the decay phase of the response. Decay time was calculated as the difference between the time points on the decaying phase of the u EPSP/ u IPSP corresponding to 20 and 80% of the peak amplitude as measured on the rising phase.

Statistical analysis

A non-parametric, two-tailed, Wilcoxon Mann–Whitney two-sample rank test was used for unpaired data and a non-parametric, two-tailed, Wilcoxon signed-rank test was used for paired data. The statistical significance of a postsynaptic response was assessed using an unpaired Wilcoxon Mann–Whitney two-sample rank. To test for correlations between features of u EPSPs, we used Pearson’s correlation coefficient r with t statistics. To check for interactions between synaptic gain, u EPSP amplitude and APThr – u EPSP V m peak we used stepwise multiple linear regression. Values are given as mean ± s.e.m. unless otherwise stated. No statistical methods were used to predetermine the sample size. No randomization or blinding was performed in this study.

Data availability

All relevant data are available from the authors.