P 1000 pipette puller

A pipette internal solution comprised of (mM): 140 KCH₃OSO₃, 5 KCl, 0.5 EGTA, 1 MgCl₂, 10 HEPES, 5 MgATP, 0.25 NaGTP, ~292 mOsm, E_Cl = −78.8 mV was used. Pipettes 4–6 MΩ in resistance were pulled from borosilicate glass capillaries (World Precision Instruments, 1B150F-4) on a Sutter Instruments P-1000 pipette puller. Voltage-clamp traces 5 min in duration were recorded at a holding potential of −70 mV in the presence of 1 μM tetrodotoxin (Tocris). All recordings were conducted with access resistance of <20 MΩ, leak current of <100 pA, and an applied series resistance compensation of 80%. Cells that did not maintain these parameters for the duration of the recording were eliminated. Analysis of mEPSCs was performed using pCLAMP10 (Axon Instruments, Molecular Devices) using a variable-amplitude template method, generated from a stable recording of at least 50 mEPSC events. Each trace was first low-pass filtered at 1 kHz, and negative-going mEPSCs were detected using a template match threshold of 4, without fitting.

In order to record mIPSCs while maintaining a hyperpolarized membrane voltage, a high-chloride pipette internal solution (comprised of (mM): 100 CsCH₃O₃S, 50 CsCl, 10 HEPES, 0.2 BAPTA, 3 KCl, 1 MgCl₂, 0.25 GTP-Tris, 2.5 creatine phosphate disodium, 2 MgATP, ~296 mOsm, E_Cl = −23.7 mV) was used. Pipettes 4–6 MΩ in resistance were pulled from borosilicate glass capillaries (World Precision Instruments, 1B150F-4) on a Sutter Instruments P-1000 pipette puller. Voltage-clamp traces 3 min in duration were recorded at a holding potential of −70 mV in the presence of 1 μM tetrodotoxin (Tocris) and 2 mM kynurenic acid (Sigma Aldrich). All recordings were conducted with access resistance of <20 MΩ, leak current of <100 pA, and an applied series resistance compensation of 80%. Cells that did not maintain these parameters for the duration of the recording were eliminated. Analysis of mIPSCs was performed using pCLAMP10 (Axon Instruments, Molecular Devices) using a variable-amplitude template method, generated from a stable recording of at least 50 mIPSC events. Each trace was first low-pass filtered at 1 kHz, and negative-going mIPSCs were detected using a template match threshold of 4, without fitting.

A Nikon FN-1 microscope (Nikon, Tokyo, Japan) with a differential interference contrast optics and a video camera (Grasshopper 3 GS3-U3-23S6M-C; FLIR Integrated Imaging Solutions Inc., Wilsonville, OR, USA) was used to visualize the pyramidal neurons of CA1 hippocampus. Borosilicate glass pipettes (3–5 MΩ) were manufactured using a P-1000 pipette puller (Sutter Instrument, Novato, CA, USA). The pipette solution had the following composition: Cs-methanesulfonate—127 mM, HEPES—10 mM, NaCl—10 mM, EGTA—5 mM, QX314—6 mM, ATP-Mg—4 mM, and GTP—0.3 mM with pH adjusted to 7.25 by adding CsOH. The EPC 10 USB Double (HEKA Elektronik GmbH, Reutlingen, Germany) patch-clamp amplifier controlled with the HEKA Patchmaster software was used for the whole-cell patch-clamp recordings. The access resistance was typically 10–20 MΩ during the experiment.
The type of and the positioning of the stimulating electrode were the same as for the field potential recording. NMDAR-mediated currents were isolated by adding gabazine (10 µM, Alamone Laboratories, Jerusalem, Israel) and DNQX (10 µM, Tocris Bioscience, Bristol, UK) to the recording solution. The NMDAR-mediated eEPSCs were induced by the TBS protocol with the stimulus current strength set to 300–500 µA and recorded at −30 mV. The data were lowpass-filtered at 5 kHz and digitized at 40 kHz.

In
these experiments, LESA microextraction was followed by sample
collection in a well plate. The sample was then loaded into a gold-coated
borosilicate nanoelectrospray emitter, i.e., the LESA sampling and
ionization processes were decoupled. These experiments are referred
to as “nanoESI” throughout this Article. Details of
the microextraction are given in the Supporting Information.
Borosilicate glass capillaries were prepared
in house using a P-1000 pipette puller (Sutter Instrument) before
coating with gold using a sputter coater (Agar Scientific Ltd.).
Sample-loaded tips were inserted into a nanospray ion source equipped
with the static spray option (Thermo) attached to either of the mass
spectrometers described below. The electrospray voltage for the tips
was typically in the range of 1.0–1.2 kV and performed with
no additional backing pressure. The use of borosilicate emitters improved
nanoelectrospray stability, duration, and signal intensity when compared
with that of chip-based nanoESI. This observation can be attributed
to the narrower spray orifice (1–2 μm) and the tapered
geometry of the borosilicate emitter versus the square-cut geometry
of the chip-based nanoESI emitters.^{33 (link)}