Nov 12, 2024
6-Hydroxydopamine lesion and virus injection
- 1Alcala University
Protocol Citation: Cristina Alcacer 2024. 6-Hydroxydopamine lesion and virus injection. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v929bql3e/v1
Manuscript citation:
Abnormal hyperactivity of specific striatal ensembles encodes distinct dyskinetic behaviors revealed by high-resolution clustering
Cristina Alcacer, Andreas Klaus, Marcelo Mendonça, Sara F. Abalde, Maria Angela Cenci, Rui M. Costa
bioRxiv 2024.09.06.611664; doi: https://doi.org/10.1101/2024.09.06.611664
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: October 21, 2024
Last Modified: November 12, 2024
Protocol Integer ID: 110446
Keywords: ASAPCRN, 6-OHDA
Abstract
Injection of 6-OHDA(6-Hydroxydopamine) or vehicle and virus injection protocol used in Alcacer et al.
Guidelines
All procedures were done in aseptic conditions.
Materials
-Stereotaxic surgery set up required (this protocol uses Kopf instruments)
-Nanojet II Injector (Drummond Scientific, USA)
-Surgical Tools
-6-Hydroxydopamine hydrochloride (Sigma Aldrich, Portugal) was dissolved in 0.02% ice-cold L-ascorbic acid/saline (3.2 μg free-base 6-OHDA/μL)
-AAV5.CAG.Flex.GCaMP6f.WPRE.SV40 virus (made by University of Pennsylvania Vector Core, Addgene Cat# 100835-AAV5)
Before start
Mice were kept in deep anesthesia using 1-3% isoflurane and oxygen (at 1L/min flow rate) and mouse body temperature was maintained at 37 degrees Celsius using a temperature controller (ATC1000, World Precision Instruments). Surgeries were performed on a stereotactic frame (David Kopf Instruments, Model 962LS) with a mouse adaptor (David Kopf Instruments, Model 923-B Mouse gas anesthesia heads holder).
Stereotaxic Surgery
Stereotaxic Surgery
Anesthetize mouse with isoflurane.
Shave head and transfer to stereotaxic apparatus and secure the head and keep mouse anesthetized for length of procedure.
Disinfect surgical area of the mouse head with 70% ethanol and iodine.
Make a small incision in the skin to expose the skull.
Carefully remove any connective and muscle tissue from the exposed area.
Level the skull surface to be at less than 0.05mm by comparing the height of bregma and lambda, and also in medial-lateral directions.
6-OHDA or Vehicle Injection
6-OHDA or Vehicle Injection
Find the injection coordinates (coordinates used for medial forebrain bundle : AP= - 0.7, ML= - 1.2, DV= - 4.7) and mark area for drilling.
Drill Burr-hole in marked areas carefully.
Load glass pipette into the pipette holder (Nanojet II (Drummond Scientific)) and affix to the stereotaxic frame.
Set rate of injection to 4.6 nL per pulse every 5 seconds.
Leave pipette in place for 2 minutes before injection.
Inject 1 ul of 6-OHDA or vehicle solution then wait 10 minutes after injection before removing.
Virus Injection
Virus Injection
The capillary was replaced with a new one to inject the virus.
Inject 600 nL AAV5.CAG.Flex.GCaMP6f.WPRE.SV40 into right dorsal striatum, ipsilateral to the lesion (coordinates AP= + 0.5, ML= -2.3, DV= 2.3).
Post-operative Care
Post-operative Care
Close incision with tissue glue (Vetbond tissue adhesive, 3M, USA).
Inject carprofen (5mg/kg; 10uL/10g body weight) as an analgesia. Then inject 0.6 mL sterile glucose-ringer acetate (subcutaneously) to prevent dehydration.
Add that the mice in a warming cabinet at 27 °C to keep a constant body temperature avoiding hypothermia immediately after the surgery and during the first 2 weeks post-surgery.
During the first 2 to 3 weeks of surgery, mice receive daily subcutaneous injections of sterile glucose-ringer acetate solution (0.1 mL/10g vody weight) and dietary supplementations.