Electrophysiological recordings were performed as described previously15 (link). All electrophysiological recordings were performed in ACSF at 26 °C. ACSF was exchanged at a rate of 1 ml/min. A bipolar tungsten-stimulating electrode (WPI) was placed in the CA1 stratum radiatum region. fEPSPs were recorded from the CA1 stratum radiatum following 0.05-Hz test pulses. The recording electrode was set in a glass pipette (Harvard Apparatus) filled with ACSF. Electrical signals were amplified using a MultiClamp 700A (Molecular Devices) and digitized at 10 kHz and filtered at 2 KHz using a Digidata 1440 system with pCLAMP10 software (Molecular Devices).
Glass pipette
Manufactured by Harvard Apparatus
Sourced in United States
A glass pipette is a laboratory equipment used for measuring and transferring precise volumes of liquids. It consists of a narrow, calibrated glass tube with one end open and the other end tapered to a point. The pipette is designed to draw in and dispense liquids accurately, making it a crucial tool for various scientific applications.
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5 protocols using glass pipette
1
CA1 Electrophysiological Recordings in ACSF
2
Anterograde Cre-dependent Optogenetic Inhibition
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Anterograde Cre-dependent rAAV2-CAG-FLEX-ArchT-GFP (UNC Vector Core, 2.0 × 1012 titer; n = 5 VGATCre mice and 3 PVCre mice) or rAAV8/PAAV-hSyn-DIO-EGFP (UNC Vector Core; 8 × 1012 titer; n = 1 PVCre mouse) were pressure injected (400 nl/mouse) using a glass pipette (5 μl; Harvard Apparatus) with tip diameter of 10–15 µm into the medial septum of mice (stereotaxic coordinates relative to Bregma: 0.86 mm anterior, 0.39 mm lateral and 3.75 mm ventral with 5° angle). Pressure was applied using a 1-μl syringe at a rate of ~ 100 nl/min. To minimise tissue damage and dorsal diffusion, the electrode was lowered into place 5 min before and was retracted 10 min after the injection. After a minimum of 28 days and allowing optimal virus expression, mice were perfusion fixed (2% PFA) and the brains were processed (see below).
3
Optogenetic Manipulation of MSDB Neurons
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After performing a small craniotomy at 0.86 mm anterior and 0.39 mm lateral of bregma, a glass pipette (tip diameter: 12–20 μm, 5 μl; Harvard Apparatus) was lowered at a 5° lateromedial angle to 3.75 mm ventral of the dura mater into the MSDB. Anterograde Cre-dependent AAV2-CAG-FLEX-ArchT-GFP (n = 4 mice; 400 nl/mouse; UNC Vector Core) or pAAV2-EF1a-DIO-EYFP (n = 3 mice; same mice used in Unal et al., 2015 (link)) was pressure injected using a 1 μl syringe at a rate of ∼100 nl/min. Mice were perfuse-fixed >28 d after injections to ensure optimal viral expression.
4
Extracellular Recordings of Hippocampal Slices
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Extracellular field recordings were performed in the CA3 pyramidal layer of hippocampal slices of P16 mice with glass pipettes (Harvard Apparatus, MA, USA) containing ACSF. Spike frequency was recorded for 10 min (control period), then isoguvacine (10 μM; Sigma-Aldrich, MO, USA) was applied in the bath solution for 90 s (isoguvacine period). Following isoguvacine application, spike frequency was recorded for an extra 15 min (wash-out period). Signals were recorded with a low-noise multichannel DAM-80A amplifier (WPI, UK; low-pass filter 1 Hz; high-pass filter 3 kHz; gain ×1000) and digitized online with a Digidata 1400A digitizer (Molecular Devices, CA, USA). Recordings were analyzed using Clampfit 10.4 software (Molecular Devices, CA, USA). The spike detection threshold was defined as three times the standard deviation of the noise recorded in the bath solution. Spike frequency was calculated for control, isoguvacine, and wash-out periods. Slices for which wash-out spike frequency did not come back to control levels (±20% of control) were excluded from this study.
5
Patch-Clamp Recordings of Hippocampal Neurons
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For ex vivo electrophysiological recordings, 0.01 mM bare AuNPs, PEG (mw: 800) or PEG-AuNPs were added into the perfusion solution and recordings were obtained from hippocampal CA1 neurons during 5–60 min of application. Spontaneous action potentials were measured by whole-cell patch configuration at -65mV. Glass pipettes (Harvard Apparatus) having 3–5 MΩ tip resistance were filled with a potassium gluconate based internal solution during whole-cell recordings. Pipette solution contained: 145 mM K-gluconate, 1 mM MgCl2, 10 mM HEPES, 1.1 mM EGTA, 2 mM Mg-ATP, 0.5 mM Na2-GTP, and 5 mM Na2-phosphocreatine (pH 7.3 with KOH; 290–295 mOsm). Recordings were corrected for liquid junction potential.
Membrane potentials and stimulated action potentials were generated by current injection protocol, which was set for 500 ms pulses of 50 pA steps starting from −150 pA to +150 pA by whole cell patch configuration. Average of 500 ms were used to plot current-voltage (I–V) relationship.
Membrane potentials and stimulated action potentials were generated by current injection protocol, which was set for 500 ms pulses of 50 pA steps starting from −150 pA to +150 pA by whole cell patch configuration. Average of 500 ms were used to plot current-voltage (I–V) relationship.
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