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In This Article

  • Summary
  • Abstract
  • Protocol
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing >95% loss of striatal dopamine and 90.63±-4.02 % ipsiversive rotational bias towards the side of the lesion.

Abstract

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise3,5.

In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)8, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer11. More recent studies have generated intra-nigral lesions with both a low mortality rate >80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse15. Whilst this model has proven useful in the assessment of potential neuroprotective agents16, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion17, 18.

Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes >95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.

Protocol

1. Housing and preparation of mice

  1. Maintain a colony of bacterial artificial chromosome (BAC) driven transgenic mice8 (Mutant Mouse Regional Resource Center (MMRRC) FVB in a 12:12 h light-dark cycle with free access to food and water. These mice are pure FVB mice, and it is not necessary to cross these mice with any other strain, either for breeding purposes, or to ensure success of the 6-OHDA lesion procedure.
  2. In order to generate a parkinsonian model, adult mice aged postnatal day 31-42.......

Discussion

This protocol describes a method for the generation of a stable unilateral 6-OHDA-lesioned mouse model of Parkinson's disease, which is extremely reproducible, with a high lesion success rate, and a low mortality rate. The success of 6-OHDA lesion surgery can be easily estimated by the measurement of spontaneous rotational behaviour with >70% ipsiversive rotations indicative of >95% dopamine depletion in the lesioned striatum27. Quantification of striatal dopamine levels is the most accurate measurement .......

Acknowledgements

This work was supported by the Department of Foreign Affairs and International Trade (Government of Canada), University of Toronto Connaught Fund, the Canadian Foundation for Innovation, NSERC, the Krembil Foundation and the Cure Parkinson’s Trust.

....

Materials

NameCompanyCatalog NumberComments
Name of the reagentCompanyCatalogue numberComments (optional)
desipramine HCl Sigma-Aldrich, Oakville, ON, CanadaD12525mg/kg
pargyline HCl Sigma-Aldrich, Oakville, ON, CanadaP80135mg/kg
6-OHDA HBr Sigma-Aldrich, Oakville, ON, CanadaH1163mg / mouse
stereotaxic Frame Kopf Instruments, Tujunga, CA, USAModel 900 
mouse ear cups Kopf Instruments, Tujunga, CA, USAModel 921 Zygoma Ear Cups 
mouse incisor bar Kopf Instruments, Tujunga, CA, USAModel 923B 
mouse anaesthesia mask Kopf Instruments, Tujunga, CA, USAModel 923B 
priming kit (containing 250ml syringe)Hamilton Company, Reno, NV, USAPRMKIT 81120 
RN compression fitting kit (1 mm) Hamilton Company, Reno, NV, USA55750-01    
PEEK tubing from RN compression fitting kit< (1/16th inch) Hamilton Company, Reno, NV, USA55751-01 
dual small hub RN Coupler Hamilton Company, Reno, NV, USA55752-01    
luer to small hub RN adaptorHamilton Company, Reno, NV, USA55753-01    
1ml 25S syringe model 7001KHHamilton Company, Reno, NV, USA80100  
*33G removable needle (RN) pack of 6. . Custom 1 inch with 45<° bevelHamilton Company, Reno, NV, USA7803-05  
Scissors Fine Science Tools, Vancouver, BC, Canada.14084-08 
Scalpel Fine Science Tools, Vancouver, BC, Canada10003-12 
Scalpel blades Fine Science Tools, Vancouver, BC, Canada10035-20 
Forcep Fine Science Tools, Vancouver, BC, Canada11608-15 
Hemostats Fine Science Tools, Vancouver, BC, Canada.13004-14 
Isoflurane Abbot022413152-3%
Suters (Vicryl 4.0) SynetureSS-683 
Steriliser Fine Science Tools, Vancouver, BC, Canada18000-45 
Infusion PumpHarvard ApparatusPhD 22/2000 
Needles (27G) Becton Dickinson305109 
Needles (25G) Becton Dickinson305127 
Syringes (1ml) BD syringe309692 
Anaesthesia trolley LEI medicalM2000 
BaytrilCDMV, St. hyacinthe, QC 102207 
LidocaineCDMV, St. hyacinthe, QC3914 
Betadine solutionCDMV, St. hyacinthe, QC19955 

References

  1. Costall, B., Naylor, R. J., Pycock, C. Non-specific supersensitivity of striatal dopamine receptors after 6-hydroxydopamine lesion of the nigrostriatal pathway. Eur. J. Pharmacol. 35, 276-283 (1976).
  2. Maneuf, Y. P., Mitchell, I. J., Crossman, A. R., Brotchie, J. M.

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