Jul 16, 2024
Dendritic spine analysis
- 1Northwestern University, Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Protocol Citation: Chuyu Chen 2024. Dendritic spine analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw1879lmk/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: July 16, 2024
Last Modified: July 30, 2024
Protocol Integer ID: 103538
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's [ASAP-020600] through the Michael J. Fox Foundation for Parkinson's Research (MJFF)
Grant ID: ASAP-020600
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Abstract
Haloperidol is reported to induce a series of homeostatic synaptic and intrinsic adaptations primarily in the indirect pathway circuits. These adaptations include dendritic spine loss in iSPNs, which occurs after 5 days and persists for up to 14 days of daily treatment. We examined the haloperidol-induced dendritic spine density patterns in iSPNs after 7 days of treatment
Stereotaxic Surgeries
Stereotaxic Surgeries
P4-5-day-old LRRK2GS/WT-Adora2aCre pups were cryoanesthetized and received ketoprofen for analgesia
Pups were placed on a cooling pad on a stereotaxic frame
200 nl of the AAV-DIO-EGFP virus (7×10¹² vg/mL) were delivered into the dorsal striatum at a rate of 100 nl min-1 using Micro-4 controller (WPI).
In order to ensure the dorsal striatum was targeted, the needle was placed 1 mm anterior to the midpoint between the ear and eye, 1.5 mm from the midline, and 1.8 mm ventral to the brain surface
After the injection, the pups were warmed on a heating pad before returning to their home cages.
Confocal microscopy
Confocal microscopy
Confocal images of fixed 80-um-thick brain sections of P30 pups injected with the AAV-DIO-EGFP were obtained with the Nikon A1R microscope
Fluorescence projection images of dendrites and the corresponding dendritic spines were acquired with a 60x oil immersion objective (NA = 1.4) at 0.1 um intervals with 1,024 x 1,024 pixel resolution
Dendritic spine analysis
Dendritic spine analysis
Segments from secondary and tertiary dendrites without overlap with other neurons or discontinuities were chosen for analysis
Dendritic spine density was assessed using Imaris 10.1 software
Dendritic segments were traced using autopath mode of the filament tracer at default settings
Settings used for spine detection
Thinnest diameter of seed points at 0.45 μm; maximum length at 2.5 μm; no branched spines allowed; and seed point threshold at auto