Astrocytes detect and upregulate transmission at inhibitory synapses of somatostatin interneurons onto pyramidal cells.

Mice

All experiments were approved by and performed in accordance with guidelines for maintenance and care of animals of the Canadian Council of Animal Care and Université de Montréal. To express the light-gated ion channel channelrhodopsin-2 in SOM-INs and PV-INs, heterozygous SOM or PV-IRES-Cre-ChR2(H134R)/EYFP mice (SOM or PV-ChR2/EYFP) were obtained by crossing SOM-IRES-Cre mice (kindly provided by Z.J. Huang—Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; JAX no. 013044) 36 or Pvalb tm1(cre)Arbr (PV-Cre; Jackson Labs; JAX no. 008069) with ChR2(H134R)/EYFP Ai32 mice (Jackson Labs; JAX no. 012569). Experiments were performed on 1–2 months old mice of either sex.

Slice preparation

Transverse hippocampal slices were obtained from 4 to 8-week-old SOM-ChR2/EYFP or PV-ChR2/EYFP mice 36 . Animals were anesthetized with isoflurane and the brain was rapidly excised and placed in ice-cold choline-based cutting solution saturated with 95% O 2 and 5% CO 2 containing the following (in m m ): 120 choline chloride, 3 KCl, 1.25 NaH 2 PO 4 , 26 NaHCO 3 , 8 MgCl 2 , 20 glucose, pH 7.4 and 295 mOsmol. A block of brain tissue containing the hippocampus was prepared and transverse hippocampal slices (300 μm thick) were cut on a vibratome (Leica VT1000S, Nussloch, Germany). Slices were transferred to oxygenated artificial CSF (ACSF) at 33 ± 0.5 °C containing the following (in m m ): 130 NaCl, 2.5 KCl, 1.25 NaH 2 PO 4 , 26 NaHCO 3 , 10 glucose, 1.3 MgCl 2 , 2 CaCl 2 , pH 7.3–7.4, and 305–310 mOsmol, and allowed to recover for at least 1 h before being placed in oxygenated ACSF at room temperature (RT). For experiments, the slices were transferred to a recording chamber where they were perfused (2.5 ml/min) with ACSF at 32–34 °C for the course of the experiment. The NMDA receptor antagonist AP-5 (20 μM) and the AMPA and Kainate receptor antagonist NBQX (10 μM) were present in the superfusate of all experiments. Slices were used for a maximum of 6 h after cutting. Cell identification CA1 pyramidal cells, astrocytes and SOM-INs and PV-INs were identified using an infrared camera (70 series; Dage-MTI, Michigan City, IN) mounted on an Zeiss LSM 510 confocal laser scanning microscope installed on a Zeiss Axioskop FS Upright Microscope (Carl Zeiss, Kirkland, Québec, Canada) and equipped with a 40× water immersion long-working distance objective (0.8 n.a.). CA1 pyramidal cells were visually identified based on their soma location and triangular shape. Astrocytes in the stratum radiatum (s.r.) were identified by their specific labeling with sulforhodamine 101 red fluorescent dye (SR101, 0.25 μM) 37 , 38 . Preliminary experiments showed that SR101 did not affect PC membrane properties and spontaneous IPSC frequency and amplitude (Supplementary Fig. 2c ), unlike previously suggested for EPSCs 39 . SOM-INs and PV-INs expressing ChR2 were identified by specific EYFP fluorescence and soma location in CA1 stratum oriens or near stratum pyramidale respectively 25 , 28 – 32 .

Electrophysiology

Whole-cell voltage-clamp recordings of CA1 pyramidal cells were obtained using borosilicate glass pipettes (3–5 MΩ) filled with intracellular solution containing the following (in m m ): 130 CsCl, 10 NaCl, 10 HEPES, 1 EGTA, 0.1 CaCl 2 , 10 creatine-PO 4 di(tris), 4 ATP-Mg, 0.4 GTP-Na and 5 lidocaine N -ethyl bromide (QX-314; voltage-gated Na + channel blocker) (pH 7.2 adjusted with CsOH; 285–290 mOsmol). Data was acquired using a Multiclamp 700B amplifier (Molecular Devices) and digitized using a Digidata 1320A digitizer and pClamp 10.3 (Molecular Devices). Recordings were low-pass filtered at 2 kHz and digitized at 20 kHz. Series resistance (Rs) was 10–25 MΩ and regularly monitored during experiments. Data were included only if the holding current and Rs were stable (