GABAB receptors modulate NMDA receptor calcium signals in dendritic spines.

Preparation

Recordings were made from layer 2/3 pyramidal neurons in the medial prefrontal cortex (PFC) of acute slices from P21 - P28 C57/BL6 mice. Briefly, mice were anesthetized with a lethal dose of ketamine / xylazine and perfused intracardially with ice-cold external solution containing (in mM): 65 sucrose, 75 NaCl, 25 NaHCO 3 , 1.25 NaH 2 PO 4 , 25 glucose, 2.5 KCl, 1 CaCl 2 , 5 MgCl 2 , 0.4 Na ascorbate, 3 Na pyruvate, bubbled with 95% O 2 / 5% CO 2 . Coronal slices (300 μm thick) were cut in ice-cold external solution and transferred to ACSF containing (in mM): 119 NaCl, 25 NaHCO 3 , 1.25 NaH 2 PO 4 , 25 glucose, 2.5 KCl, 2 CaCl 2 , 1 MgCl 2 , 0.4 Na-ascorbate, 3 Na-pyruvate, bubbled with 95% O 2 / 5% CO 2 . After 30 minutes in ACSF at 35 °C, slices were stored for approximately 30 minutes at 24 °C, after which experiments were conducted at 33 - 34 °C. For experiments in 0 mM extracellular Mg, slices were incubated in ACSF containing NBQX, 2 mM Ca and 0 mM Mg for one hour before recording. In all experiments, 10 μM D-serine and 10 μM gabazine were present in the ACSF, to prevent NMDA-R desensitization and block GABA A -Rs, respectively. In some experiments, one or more of the following drugs were added to the ACSF (in μM): 10 NBQX, 10 (R)-CPP, 1 TTX, 1 ω-conotoxin-MVIIC, 0.3 SNX-482, 20 nimodipine, 10 mibefradil, 10 H89, 50 forskolin, 30 CPA, 1 or 5 (R)-baclofen. The cocktail of voltage-sensitive Ca channel (VSCC) blockers consisted of ω-conotoxin-MVIIC, SNX-482, nimodipine and mibefradil. This cocktail blocks Ca signals evoked by back-propagating action potentials ( Figure S1 ), which are readily observed in the dendrites of these neurons ( Larkum et al., 2007 ). In extracellular stimulation experiments ( Figure 1 ), (R)-CPP or NBQX was added to the bath to isolate AMPA-R or NMDA-R EPSCs, respectively.All chemicals were from Sigma or Tocris, with the exception of SNX-482 and ω-conotoxin-MVIIC (Peptides International, Inc.) and PKC inhibitor peptide (19-36) (PKC-I) (Calbiochem).

Physiology recordings

Whole-cell recordings were obtained from layer 2/3 pyramidal neurons identified with IR-DIC at 200 - 300 μm from the pial surface. Borosilicate recording pipettes (2 - 5 MΩ) were filled with 1 of 2 internal solutions. Current-clamp recordings used (in mM): 135 K-gluconate, 7 KCl, 10 HEPES, 10 Na-phosphocreatine, 4 Mg 2 -ATP, 0.4 NaGTP, 290-295 mOsm, pH 7.35 with KOH. Voltage-clamp recordings used (in mM): 135 Cs-gluconate, 10 HEPES, 10 Na-phosphocreatine, 4 Mg 2 -ATP, 0.4 NaGTP, 290-295 mOsm, pH 7.35 with CsOH. Solutions also contained Fluo-5F (1000 μM for voltage-clamp, 200 μM for current-clamp) to monitor Ca levels and Alexa Fluor-594 (20 μM) to image neuronal morphology. Neurons were filled via the patch electrode for at least 15 - 20 min before imaging. Dye concentrations were chosen to ensure that Ca signals were in the linear range of the indicators ( Yasuda et al., 2004 ). In some experiments, one of the following drugs were added to the internal solution (in mM): 3 Guanosine-5′-[β-thio]diphosphate trilithium salt (GDP-βS), 0.1 PKA inhibitor fragment (6-22) (PKI), or 0.1 PKC inhibitor peptide (19-36) (PKC-I). Recordings were made using a Multiclamp 700B amplifier, filtered at 5 kHz for current-clamp recordings and 2 kHz for voltage-clamp recordings, and sampled at 10 kHz. Action potentials were triggered with brief (2 ms) current injections (1200 - 2000 pA) through the recording pipette. Excitatory input fibers were stimulated with a theta-glass bipolar electrode (tip diameter 1 - 4 μm) filled with the extracellular ACSF, using brief (0.2 ms) and small (5 - 20 μA) current injections. The electrode was placed approximately 1 - 5 μm from the spine of interest, and the spatial resolution of this approach was found to be high.

Two-photon microscopy Intracellular

Ca imaging and glutamate uncaging was accomplished with a custom microscope that combines two-photon laser scanning microscopy (2PLSM) and two-photon laser uncaging (2PLU), as previously described ( Carter and Sabatini, 2004 ; Carter et al., 2007 ). For all experiments, proximal spines in the basal dendrites within a radial distance of 75 μm from the soma were chosen to reduce voltage-clamp errors. For 2PLSM, 810 nm light was used to excite Fluo-5F (green) and Alexa Fluor-594 (red), in order to monitor Ca signals and spine morphology, respectively. Reference frame scans were taken between each acquisition in order to correct for small spatial drift of the preparation over time. To measure Ca signals, green and red fluorescence were collected during 500 Hz line scans across a dendrite-spine or spine-spine pair. Ca signals were quantified as changes in green fluorescence to red fluorescence (ΔG/R), normalized to the maximal green fluorescence to red fluorescence (G sat /R), giving ΔG/G sat ( Bloodgood and Sabatini, 2007 ). The value of G sat /R was measured after each recording using a thin-walled pipette, containing a saturating concentration of Ca ( Yasuda et al., 2004 ), which was positioned directly above the recorded cell and used at the same recording temperature (33 - 34 °C). For two-photon optical quantal analysis, cells were voltage-clamped at +10 mV to relieve magnesium block and to inactivate VSCCs ( Oertner et al., 2002 ). Experiments were performed in the presence of (D)-serine, gabazine, and NBQX. Line-scans with electrical stimulation were interleaved with line-scans with no stimulation. Line-scans were acquired every 15 s for a total of 40 stimulation trials (20 baseline, 20 drug). Baclofen was washed-in for 5 minutes before the drug trials. To classify successful trials, the threshold for each spine was set to two standard deviations of the signal in the line-scans with no stimulation. To confirm that small amounts of drift did not result in losing the axon of interest, two stimuli (50 ms inter-stimulus interval) were occasionally given to confirm that the spine was activated. During normal (2 mM) Ca experiments, spines with Ca signals above the linear range of the indicator were excluded from analysis. During low (1 mM) Ca experiments, 4 out of 12 spines showed no synaptic responses after wash-in of 1 μM baclofen and were excluded from the analysis. In Figures 2 & 3 , individual ΔG/G sat trials are filtered for display purposes. For 2PLU, MNI-glutamate was bath applied at 2.5 mM in 6 - 12 ml of ACSF. All experiments were performed in the presence of (D)-serine, gabazine, and TTX. (R)-CPP or NBQX was added to isolate AMPA-R or NMDA-R uEPSCs, respectively. Experiments at −70 mV, 0 mM Mg and −20 mV, 1 mM Mg were performed in the presence of the VSCC blockers. Glutamate uncaging was achieved using a 1 ms pulse of 725 nm light. The uncaging location was chosen at 0.5 μm from the spine head. At this distance, photobleaching and photodamage are minimal but uEPSCs and Ca signals can be readily obtained ( Carter et al., 2007 ). The laser intensity was chosen to mimic the amplitude of spontaneous AMPA-R EPSCs or extracellularly evoked NMDA-R Ca signals. Similar to experiments using two-photon optical quantal analysis, line-scans were acquired every 15 s for a total of 40 stimulation trials (20 baseline, 20 drug). Baclofen was washed-in for 5 minutes before the drug trials. Baseline fluorescence was monitored and recordings were discarded if an increase was detected, which would indicate photodamage.

Data acquisition and analysis

Image and physiology data were acquired using National Instruments boards and custom software written in MATLAB (Mathworks). Off-line analysis was performed using custom routines written in Igor Pro (Wavemetrics). The amplitudes of EPSCs and uEPSCs are averages over a 1 ms time window around the peak. The amplitudes of NMDA-R Ca signals are averages over a 150 ms time period, starting 50 ms after the stimulus. In Figures 2 , 5 & 8 , images of spines and dendrites were treated with a 1.5-pixel-radius Gaussian filter for display purposes. Summary data are reported as median ± standard error. The standard error was calculated as the standard deviation of the medians calculated from 10,000 bootstrapped samples from the data. Electrophysiological and imaging data are shown as the arithmetic mean ± standard error from multiple trials of the experiment. There were occasionally small differences between the mean and median within a group, which explains some discrepancies between average traces in figures and medians reported in the text. Most summary data is in box plot form, showing the median, interquartile range, 10 - 90% range (whiskers), and including the data from individual experiments (open circles). Significance was defined as P < 0.05 (*) and determined using the non-parametric Wilcoxon-Mann-Whitney two-sample rank test or the Wilcoxon signed rank test for paired data (when appropriate), both of which make no assumptions about the data distribution.

Supplementary Material 01