P 97 pipette puller

 Two-photon guided in-vivo whole-cell recordings were performed as described in Polack et al. (2013) (link). Long-tapered micropipettes made of borosilicate glass (1.5 mm outer diameter, 0.86 mm inner diameter, Sutter Instrument) were pulled on Sutter Instruments P-97 pipette puller to a resistance of 3–7 MΩ, and filled with an internal solution containing 115 mM potassium gluconate, 20 mM KCl, 10 mM HEPES, 10 mM phosphocreatine, 14 mM ATP-Mg, 0.3 mM GTP, and 0.01–0.05 mM Alexa-594. Whole-cell current-clamp recordings were performed using the bridge mode of an Axoclamp 2A amplifier (Molecular Devices), further amplified and low-pass filtered at 5 kHz using a Warner Instruments amplifier (LPF 202A). Series of current pulses of small intensity (typically −100 pA) were used to balance the bridge and compensate the pipette capacitance. The membrane potential was not corrected for liquid junction potentials (estimated to be about 10 mV).

Electrophysiological recordings were performed in whole-cell mode, and the synaptic currents were monitored with a Multiclamp 700A amplifier (Molecular Devices). Patch pipettes were pulled from borosilicate glass capillary tubes (World Precision Instruments) using a P-97 pipette puller (Sutter Instrument). The resistance of the pipettes filled with intracellular solution varied between 3 and 5 MΩ. The pipette solution contained 145 mM KCl, 5 mM NaCl, 10 mM HEPES, 5 mM EGTA, 0.3 mM Na₂GTP, and 4 mM MgATP (pH 7.2, adjusted with KOH). The bath solution contained 150 mM NaCl, 4 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES, and 10 mM glucose (pH 7.4, adjusted with NaOH). The miniature EPSCs were monitored in the presence of tetrodotoxin (TTX, 1 μM) and 100 μM picrotoxin. The miniature IPSCs were recorded in the presence of 1 μM TTX and 10 μM CNQX. The mEPSCs and mIPSCs were analyzed using Clampfit 10 software (Molecular Devices).

Electrophysiological recordings were performed as described elsewhere^{21 (link)} using a HEKA EPC10 amplifier in whole-cell patch-clamp mode. Patch pipettes were prepared from borosilicate glass capillary tubes (World Precision Instruments, Inc.) by using a P-97 pipette puller (Sutter). The whole-cell pipette was filled with the solution, which contained 120 mM CsCl, 10 mM HEPES, 10 mM EGTA, 0.3 mM Na-GTP, and 3 mM Mg²⁺-ATP (pH 7.2, adjusted with CsOH). The cell bath solution contained 140 mM NaCl, 5 mM KCl, 2 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES-NaOH, and 10 mM glucose (pH 7.4). The miniature IPSCs were monitored in the presence of tetrodotoxin (TTX, 1 μM) and 20 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). The evoked IPSCs were recorded in the presence of 20 μM CNQX. Single extracellular stimulus pulses were given by 1 ms current injection (90 μA) with an Isolated Pulse Stimulator (Model 2100, A-M Systems Inc.). The RRP was measured by application of hypertonic sucrose (0.5 M). Synaptic responses were all monitored at a holding potential of −70 mV. The data were digitized at 10 kHz with a 2-kHz low-pass filter.

Whole-cell patch clamp measurements were performed as described earlier (Badheka et al., 2015 (link)). Measurements were carried out on YFP positive cells, in an extracellular solution containing (in mM) 137 NaCl, 5 KCl, 1 MgCl₂, 10 HEPES and 10 glucose, pH 7.4. The intracellular solution contained (in mM) 135 Cs-Metanesulfonate, 1 MgCl₂, 10 HEPES, 5 EGTA, 4 NaATP (pH 7.25). Patch clamp pipettes were prepared from borosilicate glass capillaries (Sutter Instruments) using a P-97 pipette puller (Sutter Instrument) and had a resistance of 4–6 MΩ. In all experiments after formation of gigaohm-resistance seals, the whole-cell configuration was established and currents were recorded using a ramp protocol from −100 mV to +100 mV over 500 ms preceded by a −100 mV step for 200 ms; the holding potential was −60 mV, and this protocol was applied once every 2 s. The currents were measured with an Axopatch 200B amplifier, filtered at 5 kHz, and digitized through the Digidata 1440A interface. In all experiments, cells that had a passive leak current more than 100 pA were discarded. Data were collected and analyzed with the PClamp10.6 (Clampex) acquisition software (Molecular Devices, Sunnyvale, CA), and further analyzed and plotted with Origin 8.0 (Microcal Software Inc, Northampton, MA, USA).

The Lin28a pooled siRNA and non-targeting pooled siRNA (Dharmacon Germany) were resuspended in RNase free water according to the manufacturer’s instructions and stored in single-use aliquots at −80 °C. siRNAs were microinjected using an Eppendorf FemtoJet microinjector and Narishige micromanipulators. Microinjection pipettes were pulled with a Sutter P-97 pipette puller. siRNA solution (2 μL of 50 μmol/L) was loaded into the pipette and ~5 pL was injected into the cytoplasm of each zygote. A relatively consistent amount was carefully injected each time. After injection, zygotes were rinsed and cultured at 37 °C with 5% CO₂. The sequences of siRNA used are listed in Table S3.