Oct 09, 2024
Protocols for stereotaxic injections into mouse brain and ex-vivo electrophysiology
- Jyoti Gupta1,
- Michael J. Higley1
- 1Yale University
Protocol Citation: Jyoti Gupta, Michael J. Higley 2024. Protocols for stereotaxic injections into mouse brain and ex-vivo electrophysiology. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjn7qpgk5/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: September 23, 2024
Last Modified: October 10, 2024
Protocol Integer ID: 109187
Keywords: ASAPCRN, protocols for stereotaxic injection, stereotaxic injection, synuclin pff, mouse brain, cell patch clamp recording, method for injection, injection, mice, mouse
Funders Acknowledgements:
ASAP
Grant ID: ASAP-020616
Disclaimer
ETHICS DISCLAIMER
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board
Abstract
This protocol describes the method for injection of α-Synuclin PFF and monomer into the mouse brain. The second part of the protocol describes preparation of acute slices from these mice and whole-cell patch clamp recordings.
Materials
Oxygenated (95% O2/5% CO2) ice-cold choline artificial cerebrospinal fluid (Choline ACSF) containing (in mM):
| A | B | |
| choline | 110 | |
| NaHCO3 | 25 | |
| NaH2PO4 | 1.25 | |
| KCl | 2.5 | |
| MgCl2 | 7 | |
| CaCl2 | 0.5 | |
| glucose | 20 | |
| sodium ascorbate | 11.6 | |
| sodium pyruvate | 3.1 |
ACSF (32 °C ) containing (in mM):
| A | B | |
| NaCl | 127 | |
| NaHCO3 | 25 | |
| NaH2PO4 | 1.25 | |
| KCl | 2.5 | |
| MgCl2 | 1 | |
| CaCl2 | 2 | |
| glucose bubbled with 95% O2/5% CO2 | 20 |
Backfill glass electrodes (3-3.5 MΩ) with an internal solution containing (in mM):
| A | B | |
| cesium gluconate | 126 | |
| HEPES | 10 | |
| sodium phosphocreatine | 10 | |
| magnesium chloride | 4 | |
| Na2ATP | 4 | |
| Na2GTP | 0.4 | |
| EGTA (pH 7.3 with cesium hydroxide) | 1 |
Troubleshooting
Protocol for stereotaxic injections
Anesthetize the mouse with isoflurane by placing in the anesthesia chamber.
Place the anesthetized mouse in the stereotaxic frame and continually maintain isoflurane anesthesia using a vaporizer (Harvard Apparatus).
Locate the injection sites relative to bregma and make holes into the skull using a drill.
Lower the injection pipette into the brain at the predetermined coordinates. Perform the injection at a rate of 20 nL/s using a Nanoject III microinjector (Drummond Scientific).
Following the injection, leave the pipette in place for 10 minutes to prevent backflow while withdrawing.
Seal the incisions with sutures and allow the mice to recover on a heating pad until conscious.
Continue to monitor the mice and administer the post-operative analgesia.
Protocol for acute slice preparation and electrophysiology
30m
Anesthetize the mouse with isoflurane.
Transcardially perfuse the anesthetized mouse with oxygenated (95% O2/5% CO2) ice-cold choline artificial cerebrospinal fluid (Choline ACSF) containing (in mM):
| A | B | |
| choline | 110 | |
| NaHCO3 | 25 | |
| NaH2PO4 | 1.25 | |
| KCl | 2.5 | |
| MgCl2 | 7 | |
| CaCl2 | 0.5 | |
| glucose | 20 | |
| sodium ascorbate | 11.6 | |
| sodium pyruvate | 3.1 |
Decapitated the mouse and quickly remove the brain.
Mount the brain in the vibratome and cut 300 μm thick slices in the coronal plane.
Transfer the slices to warm ACSF (32 °C ) containing (in mM):
| A | B | |
| NaCl | 127 | |
| NaHCO3 | 25 | |
| NaH2PO4 | 1.25 | |
| KCl | 2.5 | |
| MgCl2 | 1 | |
| CaCl2 | 2 | |
| glucose bubbled with 95% O2/5% CO2 | 20 |
After incubating at 32 °C for 00:30:00 , transfer the slices to Room temperature (RT).
30m
Transfer the slices to the slice chamber that is perfused with oxygenated ACSF.
Identify layer 2/3 pyramidal cells in the primary somatosensory cortex.
Backfill glass electrodes (3-3.5 MΩ) with an internal solution containing (in mM):
| A | B | |
| cesium gluconate | 126 | |
| HEPES | 10 | |
| sodium phosphocreatine | 10 | |
| magnesium chloride | 4 | |
| Na2ATP | 4 | |
| Na2GTP | 0.4 | |
| EGTA (pH 7.3 with cesium hydroxide) | 1 |
Add 1 micromolar (µM) tetrodotoxin to the bath to record miniature postsynaptic currents.
Voltage-clamp the cells at -70mV to record miniature excitatory postsynaptic currents (mEPSCs) and at 0mV to record miniature inhibitory postsynaptic currents (mIPSCs).
Note
All protocols were carried out in accordance with Yale IACUC guidelines.