UV–Vis spectra

ATG was dissolved to a concentration of 50 μM in buffer containing (in mM) 138 NaCl, 1.5 KCl, 2.5 CaCl 2 , 1.2 MgCl 2 , 10 Glucose and 5 HEPES, adjusted to pH 7.4. UV–VIS spectra were taken in a 100 μl cuvette with the switching light (monochromator) introduced through a glass fibre from the top of the cuvette, perpendicular to the light path of the spectrometer (Varian, Cary 50). The kinetics of the trans- to cis-conversion were recorded at the maximal absorption wavelength of trans-ATG (330 nm). To achieve fast switching rates, we used high power LEDs at 365 and 460 nm (Prizmatix) for trans-cis and cis-trans isomerization, respectively.

Cortical slice preparation and external solutions

Cortical coronal slices were prepared from C57Bl6JRj mice (postnatal day 10–15, both male and female animals were used without known experimenter bias). Following decapitation, the brain was rapidly removed and transferred to an ice-cold saline solution composed of (in mM) 2.5 KCl, 1.25 NaH 2 PO 4 , 25 NaHCO 3 , 0.5 CaCl 2 , 7 MgCl 2 , 25 glucose, 75 sucrose saturated with carbogen (95% O 2 /5% CO 2 ). Slices (300 μm thick) were made using a Campden vibratome 7,000 smz-2 (NPI Electronic). Slices were incubated at 34 °C for 30 min in ACSF composed of (in mM) 125 NaCl, 2.5 KCl, 1.25 NaH 2 PO 4 , 26 NaHCO 3 , 2 CaCl 2 , 1 MgCl 2 , 20 glucose saturated with carbogen (95% O 2 and 5% CO 2 ). After incubation, slices were stored at room temperature from 30 min to five hours before being recorded. Experiments were carried out at room temperature. Unless stated otherwise, ATG was added from a 200 mM dimethyl sulfoxide stock to the ACSF to yield a final concentration of 200 μM. The ACSF was heated to 40 °C to improve the solubility of the ATG stock. The solution was not filtered because ATG adheres to filter materials.

For the identification of target receptors and calcium imaging experiments, the iGluR antagonists NBQX (25 μM) and D-AP-5 (40 μM), and the channel blockers TTX (1 μM) and felodipine (40 μM) were bath-applied, whereas MK-801 (50 μM) (all from Abcam) was loaded into the patch pipette. NMDA (1 mM, Sigma-Aldrich) and cis-STG (200 μM) were puff-applied through a glass pipette using a pressure ejection system (PDES, NPI Electronic). For voltage-clamp recordings, TTX was added to the ACSF.

Patch clamp recordings of cortical layer 2/3 neurons

Pyramidal neurons were patched using fire-polished glass electrodes with a resistance of 6–9 MΩ. Current-clamp recordings were carried out using the following intracellular solution (in mM): 140 K-gluconate, 10 HEPES, 12 KCl, 4 NaCl, 4 Mg-ATP, 0.4 Na 2 -GTP. For whole-cell voltage-clamp recordings, we used (in mM) 110 Cs-gluconate, 15 NaCl, 10 HEPES, 5 TEA, 0.16 EGTA, 4 Mg-ATP, 0.4 Na 2 -GTP. Recordings were made with an EPC 10 USB amplifier, controlled by the Patchmaster software (HEKA). Data was filtered at 2.9–10 kHz and digitized at 50 kHz. Holding potential was corrected for a 14 mV liquid junction potential. Cells were rejected if leak currents were >200 pA or series resistance >25 MΩ. Data was analysed using the Patcher’s Power Tools (MPI Göttingen) and routines written in IgorPro (Wavemetrics).

For antidromic stimulation, glass electrodes (5 MΩ) filled with ACSF were placed within 20 μm of the axon hillock and the stimulus pulse was applied through an isolated stimulation unit (A-M Systems). The stimulation intensity was set to be subthreshold. The temporal pattern of the antidromic and ATG light stimuli were controlled through the Patchmaster software (HEKA).

Hippocampal slice preparation and external solutions

Hippocampal coronal slices were prepared from C57Bl6JRj mice (Janvier Labs) (postnatal day 15-55, both male and female animals were used without known experimenter bias) following decapitation and rapid removal of the brain37. A Leica VT1200S vibratome was used to make 250 μm thick slices while the brain was immersed in an ice-cold saline solution composed of (in mM) 2.5 KCl, 1.25 NaH 2 PO 4 , 25 NaHCO 3 , 0.5 CaCl 2 , 8 MgCl 2 , 25 glucose, 230 sucrose and 0.5 ascorbic acid saturated with 95% O 2 /5% CO 2 . After 30 min incubation at 33 °C in solution composed of (in mM) 125 NaCl, 2.5 KCl, 1.25 NaH 2 PO 4 , 25 NaHCO 3 , 2 CaCl 2 , 1 MgCl 2 , 25 glucose and 0.5 ascorbic acid saturated with 95% O 2 /5% CO 2 , slices were stored at room temperature from 30 min to 5 h in the same solution before being recorded. NMDAR currents were recorded in the presence of (in μM) 10 SR95531 (Abcam Biochemicals) to block GABARs, 0.3 strychnine (Sigma-Aldrich) to block glycine receptors, 5 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) (Abcam Biochemicals) to block AMPARs, and 1 TTX (Abcam Biochemicals) to minimize spontaneous activity. 50 μM D-serine (Sigma-Aldrich) was also included in the bath solution to saturate the co-agonist binding site of NMDAR. GABAR currents were isolated through the addition of (in μM) 0.3 strychnine, 5 NBQX, 10 D-(-)-2-Amino-5-phosphonopentanoic acid (Abcam Biochemicals) and 20 7-chlorokynurenic acid (Abcam Biochemicals). ATG or 4-methoxy-7-nitroindolinyl-caged L-glutamate (MNI-glutamate; Tocris Bioscience) was perfused locally using a 3–6 μm tip-diameter patch pipette. The ATG and MNI-glutamate perfusion solutions contained (in mM) 110 NaCl, 2.5 KCl, 2 NaHCO 3 , 1.25 NaH 2 PO 4 , 30 HEPES, 10 Glucose, 2 CaCl 2 , 1 MgCl 2 , 0.05 Alexa Fluor 488 (Life Technologies), 0.01 SR95531, 0.0003 strychnine, 0.005 NBQX, 0.001 TTX, 0.05 D-serine, and where noted, 0.05–0.1 D, L-threo-β-Benzyloxyaspartic acid (Tocris Bioscience, Bristol, UK). Alexa Fluor 488 was used to visualize the perfusion and ensure its regularity over the course of the experiment. The pH of the final perfusion solution was adjusted to 7.3 after dilution. As ATG is not readily soluble in water, a stock solution (10 mM) was prepared in 0.1N NaOH.

Patch clamp recordings in hippocampal CA1 pyramidal neurons

Whole-cell voltage-clamp was performed from visually identified hippocampal CA1 pyramidal cells at 32 °C using an Axopatch 700B, Molecular Devices. Fire-polished patch electrodes had a tip resistance of 4-6 MΩ, and contained a Cs-methanesulfonate-based internal solution composed of (in mM) 105 CsCH 3 O 3 S, 10 EGTA, 3 CaCl 2 , 4 MgCl 2 , 10 Hepes, 4 NaCl, 4 NaATP, 0.4 NaGTP and 5 phosphocreatine for recording NMDAR currents. To record GABA A R currents, we used a similar solution except that we did not use NaCl and we added (in mM) 70 CsCl and 35 CsCH 3 O 3 S. 40-100 μM Alexa Fluor 594 was also added to the solution to visualize dendritic morphology during whole-cell experiments for all experiments except GABA A R recordings. Extracellular stimulation of EPSCs and IPSCs was performed at 0.1 Hz using a constant voltage stimulator (20–60 V, 50 μs, Digitimer) and a 4 MΩ resistance pipette. We recorded IPSCs for 10 min in ACSF, then 20 min in ACSF plus 400 μM ATG. Currents were filtered at 4–10 kHz then digitized at 100 kHz (NI PCI-6052E, National Instruments) using the software Neuromatic (www.neuromatic.thinkrandom.com/). Offline, traces were filtered between 4–10 kHz. Holding potentials were corrected for a −7 mV liquid junction potential (JPCalcW), and then set at −30 mV, except for GABA A R currents which were recorded at −70 mV.

Photoswitching of ATG and calcium imaging

A Poly-V monochromator (FEI Systems) controlled through the Patchmaster software was used to toggle between trans- and cis-ATG at varying wavelengths (370 and 420 nm unless otherwise indicated). During calcium imaging experiments, the monochromator was controlled by the Live Analysis software (FEI). The calcium indicator Quest-Fluo-8-AM (50 μM MoBiTec) was added to the ACSF, and acute hippocampal slices were incubated for at least 20 min at 37 °C to facilitate uptake of the indicator. Calcium changes were recorded at 480 nm, digitized at 10 Hz, background corrected and the ΔF/F 0 ratio was calculated using IgorPro routines and ImageJ (NIH).

Laser-mediated photoswitching and uncaging

Visually guided patch experiments were performed using differential interference contrast or Dodt contrast. Dendrites were visualized using either a confocal (592 nm excitation of Alexa Fluor 594) or a 2P imaging system (810 nm, Ultima, Prairie Technologies). We coupled a 405 nm diode laser (Model PhoxX 405-120, Omicron Laserage) and a 375 nm diode laser (Model PhoxX 375, Omicron Laserage) into the photoactivation galvanometers of the scanhead (for rapid spot positioning) using single-mode optical fibres (Part QPMJ-A3S,A3S-400-3/125-3-2-1, Oz Optics). For one-photon photoswitching, we used 150 μW (bath application) or 375 μW (local application) of 375 nm and 10.5 mW of 405nm, measured after the objective. For two-photon photoactivation, a pulsed Ti:Sapphire laser (Chameleon Ultra II, Coherent) tuned to either 725nm or 740 nm was directed into a photoactivation path. Photoactivation was performed on proximal dendrites within 100 μm of the soma to minimize possible effects of dendritic filtering. Illumination spots were typically placed <0.5 μm from the tip of the spine head, except when longer duration pulses were used (>500 ms), in which case the spot was placed 1 μm away.

In vitro transcription and preparation of cRNA

For expression in Xenopus laevis oocytes, clones (derived from Rattus norvegicus) of GluN1-1a (genebank accession number: U08261), GluN2A (AF001423), GluN2B (U11419.1), GluN2C (U08259.1) and GluN2D (U08260.1), each in the X. laevis oocyte expression vector pSGEM, were transcribed to cRNA in vitro using the T7 mMESSAGE mMACHINE Kit (Ambion) according to the protocol provided. Transcribed cRNA was isolated with a spin-column kit (Clean & Concentrator 25, Zymo) and cRNA integrity was checked via denaturing agarose gel electrophoresis. RNA concentration was determined photometrically with a NanoPhotometer (Implen) and the concentrations of all samples were adjusted to 200 ng μl−1 with nuclease-free water.

Expression and two-electrode recordings in Xenopus oocytes

Frog oocytes were surgically removed from the ovaries of X. laevis (Nasco) anesthetized with ethyl 3-aminobenzoate methanesulfonate (2.3 g l−1; Sigma). The lumps of oocytes were incubated with 300 U ml−1 (10 mg ml−1) collagenase type I (Worthington Biochemicals) for 3 h at 21 °C in Ca2+-free Barth's solution (in mM) 88 NaCl, 1.1 KCl, 2.4 NaHCO 3 , 0.8 MgSO 4 , 15 HEPES, pH adjusted to 7.6 with NaOH) with slow agitation to remove the follicular cell layer, and then washed extensively with Barth's solution (in mM) 88 NaCl, 1.1 KCl, 2.4 NaHCO 3 , 0.8 MgSO 4 , 0.4 CaCl 2 , 0.3 Ca(NO) 3 15 HEPES, pH adjusted to 7.6 with NaOH). Oocytes were maintained in Barth's solution supplemented with 100 μg ml−1 gentamycin, 40 μg ml−1 streptomycin and 63 μg ml−1 penicillin. Intact oocytes of stages V or VI were selected and cRNA was injected with a Nanoliter 2010 injector (WPI) within 8 h after surgery. For expression of GluN1/GluN2 heteromers, 20 nl (4 ng) of cRNA for each subunit were injected. Electrophysiological recordings were carried out 5 days after injection. Two-electrode voltage clamping was performed using a TurboTec-10CX amplifier (npi electronic) controlled by Pulse software (HEKA). For photoswitching experiments, the recording chamber was illuminated using LEDs (365 and 460 nm, Prizmatix) coupled to a light guide, which was placed directly above the oocyte. LEDs were controlled via the TTL outputs of the ADC/DAC (ITC-16, Instrutech). Borosilicate glass capillaries (Harvard Instruments) were pulled to resistances of 0.1–1 MΩ with a vertical puller (PIP5, HEKA) and filled with 3 M KCl.

Oocytes were clamped at −70 mV. All recordings were performed in continuously superfused with Barium Ringer (BaR, in mM) 115 NaCl, 2.5 KCl, 1.8 BaCl 2 , 10 HEPES-NaOH, pH 7.2). When a stable holding current was attained, the recording protocol was started. To further prevent opening of calcium-induced chloride channels, niflumic acid (NFA, 250 μM) was added to the BaR. All agonist solutions contained 10 μM glycine. In addition, the BaR used for recording photo-currents was supplemented with ATG (200 μM). For recording of cis-ATG mediated currents, the microscope light was switched-off and the photoswitching recording sequence was started. A 5 s pulse of blue light (460 nm) was followed by 5 s of UV light (365 nm) and, again, 5 s of blue light. These protocols were carried out first in the absence of ATG to control for possible artifacts, and then repeated in the presence of 200 μM ATG. For the recording of NMDA-induced currents, 1 mM NMDA in BaR supplemented with 10 μM glycine was perfused. After steady-state currents were achieved, the NMDA was washed out until a stable baseline was reached. The application of 1 mM NMDA was repeated until two consecutive applications resulted in similar steady-state amplitudes. For analysis, light-induced ATG-independent currents were subtracted from cis-ATG-mediated currents. The steady-state amplitude of the corrected cis-ATG-mediated currents were then normalized to steady-state amplitude of NMDA-induced currents.

Data analysis

Data analysis was performed using the Neuromatic analysis package and custom routines within the IgorPro environment (Wavemetrics). Cells were rejected from analysis if the leak current was >−100 pA at −30 mV holding potential or the series resistance was over 15 MΩ, except for the GluN2A KO animals in which larger leak currents were accepted. Cis-ATG-mediated current peak amplitudes from one-photon photoswitching were measured over a 1 ms window around the peak. Error bars are presented as the mean±s.e.m. unless otherwise indicated. Current decays were estimated from a fit with a double exponential equation y=A 1 exp{−(x−x 0 )}/τ 1 }+A 2 exp{−(x−x 0 }/τ 2 }. The weighted decay time constant (τ weighted ) was calculated as . Isochronal amplitudes represent averages of the currents in a 200 μs window centred at the time point at which the on-spine response had reached 75% of its peak value.

To better estimate the NMDAR current decay following 405 nm illumination without contamination from partial cis-activation of ATG, we performed a subtraction protocol. 405 nm-induced currents were fit to an empirical function that describes the rising phase and dual exponential decay38:

The fits of each individual cell were scaled by eye to maximally overlay the slow decay component of 405 nm only and 375/405 nm-induced currents. We justified such a scaling because the population traces overlapped (Fig. 4a,b). The scaled fits for each cell were then subtracted from the associated average current trace induced by 375 nm illumination followed by 405 nm illumination. Cells were only analysed if three or more recordings were performed under each illumination condition (375 nm only, 375 nm followed by 405 nm, 405 nm only).

GABAR spontaneous events (Supplementary Fig. 6) were detected using an event detection algorithm within the Neuromatic analysis software based on initial 10 pA threshold detection. Events were refined using two additional search features: (1) event onset detection using a 1.5 ms backward sliding window to ensure the point of threshold detection was 7 × s.d. of background noise; and (2) event peak detection using a 0.2 ms forward sliding window to ensure the event peaked within 2 ms of the time point of event threshold detection with a current greater than that of the point of event threshold by at least 2 × s.d. of background noise. Parameters were determined empirically, but were held constant for all analysis.

Peak amplitudes of IPSCs (Supplementary Fig. 6) were estimated from a 200 μs window centred at the time point of the peak value of the average of all events for a particular condition (number of events were typically between 50 and 300 per cell). Each synaptic current peak amplitude was corrected for series resistance error, as well as for a slight (∼5%) decay in peak amplitude of IPSCs over the duration of the recording, which was not due to ATG activity (estimated from sham experiments, n=5 cells). The coefficient of variation of the GABAR IPSCs was calculated as where σ represents the s.d. over a 200 μs window. Background variance was measured as the standard deviation over a 200 μs window immediately before the stimulation pulse.

Statistical analyses requiring multiple comparisons were first examined with a nonparametric one-way analysis of variance (Kruskal–Wallis) followed by nonparametric tests between specific values. We considered comparisons to be significantly different if P-values were <0.05.