Nov 04, 2024
Current clamp recordings
- Chuyu Chen1,
- Loukia Parisiadou1,
- Yevgenia Kozorovitskiy2
- 1Northwestern University, Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815;
- 2Department of Neurobiology, Northwestern University, Evanston, IL, USA
Protocol Citation: Chuyu Chen, Loukia Parisiadou, Yevgenia Kozorovitskiy 2024. Current clamp recordings. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldr3n8g5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 04, 2024
Last Modified: November 04, 2024
Protocol Integer ID: 111535
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's [ASAP-020600] through the Michael J. Fox Foundation for Parkinson's Research (MJFF)
Grant ID: ASAP-020600
Abstract
To better understand LRRK2 effects on intrinsic iSPNs adaptations, Drd2-eGFP BAC reporter mice were injected with haloperidol, haloperidol + MLi-2, or vehicle controls for 14 days. Then, we performed whole-cell current-clamp recordings on identified iSPNs in ex vivo striatal slices.
Acute Slice Preparation
Acute Slice Preparation
Drd2-eGFP mice were treated with either haloperidol or haloperidol plus LRRK2 inhibitor(MLi-2) for 14 days.
On day 15 or 16, mice were deeply anesthetized on isoflurane (3%) prior to perfusion with ice-cold aCSF containing in (mM): 127 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 20 glucose, 2 CaCl2, and 1 MgCl2.
Brains were dissected and sectioned on a Leica vibratome (VT1000S) to 300 um thickness.
Slices were transferred to a holding chamber and incubated at 34°C for 20 minutes prior to recordings.
Current clamp recordings
Current clamp recordings
Slices were placed in the recording chamber at RT and continually perfused with oxygenated aCSF with 1mM scopolamine to block cholinergic interneuron firing.
The dorsal striatum was located under a 10x air objective, then switched to a 60x water immersion objective was used to target neurons for recording (Olympus, Tokyo, Japan). Slices were visualized using DIC and a QIClick microscope camera (QImaging, Surrey, Canada). GFP-positive cells were selected using a CoolLED pe4000 system (CoolLED Ltd., Andover,UK).
Patch electrodes were pulled from borosilicate capillary glass to a resistance of 2.5–6 MΩ. Electrodes were filled with an intracellular solution comprised of (in mM) 135 K-gluconate, 4 KCl, 10 HEPES, 10 Na-phosphocreatine, 4 MgATP, 0.4 Na2GTP, 1 EGTA, 20 µM Alexa 594 (pH 7.28, 298-305 mOsm/L).
After break-in, cells were held for ~5-10 minutes prior to current clamp recordings.
Recordings were acquired using a Multiclamp 700B amplifier (Axon Instruments, Union City, CA), digitized at a rate of 20 kHz, and filtered at a rate of 3-4 kHz.
Five second-long sweeps were acquired using a version of the MATLAB-based (MathWorks, Natick, MA) acquisition suite, ScanImage.
A series of 500 ms-long current steps from -50 to 300 pA were pseudorandomized. Current steps occurred one second after the start of the sweep,with a 20 ms-long,-20 pA current step added at the end of the sweep to test input resistance throughout the recording.
Cells with input resistance that changed >10% over the recording were discarded. Sweeps occurred with an inter-sweep interval of 10-20 seconds. A minimum of five sweeps for each current step magnitude was obtained.
Current clamp recording analysis
Current clamp recording analysis
Average firing rate at each current step was determined using custom built MatLab code.
All values are reported as Hz. Firing rate values were then exported to GraphPad Prism (GraphPad, LaJolla, CA) for data visualization and statistics.