Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development
June 9th, 2021
Here we describe a method to visualize synaptogenesis of granule neurons in the mouse cerebellum over the time course of postnatal brain development when these cells refine their synaptic structures and form synapses to integrate themselves into the overall brain circuit.
The protocol studies cerebellar granule neurons across different stages of development in order to visualize key morphological growth. The advantages of the technique include cell specific targeting, fast expression of transfected constructs, and sparse labeling of cells which allows for the study of cell autonomous effects. Start with cutting an 11.2 millimeter segment from a loading pipette tip and place the cut part over the tip of the Hamilton syringe as a spacer to limit the injection depth to 1.5 millimeters.
Secure the spacer on the syringe tip with adhesive or parafilm. To study the morphology of single electroporated CGNs from sagittal brain sections of the experimental pup, take Z-stack images at 0.5 micrometers per stack on a confocal microscope. Image one cell per image window to allow for easy image analysis and 3D reconstruction.
Analyze neurite length and dendritic claw formation in a blinded manner using simple neurite tracer. Upload single channel Z-stack images of electroporated CGNs into Fiji and click on Plugins"Segmentation"and Simple Neurite Tracer"Select Create New 3D Viewer"from the dropdown menu. Scroll to the base of a dendrite connecting to cell soma and start a path by clicking on the junction.
Manually trace the path by clicking through the sections where the cell fill signal is brightest and pressing Y to keep the trace. Trace until the end of the dendrite and confirm the path by pressing F.Alternatively, trace until the base of the claw. Next, trace the claw from the base of the structure until the end of the longest neurite.
Trace secondary and tertiary branches by holding down Control on windows or ALT on a macOS and clicking the path. Confirm the path by pressing F.Observe that measurements for the traces are visible on a separate window. Add up all the sizes of the claw branches to obtain the total length for each claw.
In the representative analysis, projection images of electroporated CGNs from 3 to 14 days post-injection showed a progressive decrease in number of dendrites. CGNs underwent a phase of dendritic growth followed by refinement from 3 DPI to 7 DPI that resulted in the pruning of more than 50%of excess dendrites. This event coincides with the gradual lengthening of the remaining arbors in the formation of claw-like structures at the end of each dendrite indicating that these developmental processes are happening concurrently.
By 7 DPI, claws were found on roughly 75%of dendrites. Each labeled CGN was reconstructed in NMRS to quantify the total somato-dendritic surface area and volume. No significant difference in CGN size was observed across development.
Though at 7 DPI, CGNs exhibited a significant 20%decrease in volume compared to 3, 5, and 10 DPI. The most important thing in the procedure is to accurately locate the cerebellum before the injection. The method can be adapted to genetically manipulate genes in vivo to study their role in granule neuron development by transfection of either shRNAs, siRNAs, or Cre Recombinase.
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Overview
This article presents a protocol for studying the morphological development of cerebellar granule neurons (CGNs) in the developing mouse cerebellum using in vivo electroporation. The technique enables sparse labeling and genetic manipulation of CGNs, allowing detailed analysis of dendritic growth, claw formation, and synaptogenesis during key developmental stages. The method combines precise injection, confocal imaging, and quantitative morphometric analysis to track CGN maturation over time.
Key Study Components
Area of Science
- Neuroscience
- Developmental Biology
- Cell Biology
Background
- Neurons undergo significant structural and functional changes during brain development.
- The rodent cerebellum is a model system for studying single-cell morphogenesis, particularly of CGNs.
- CGNs form specialized dendritic claws that are critical for synaptic input.
- Traditional labeling methods often lack cell specificity or temporal resolution.
Purpose of Study
- To visualize and quantify the morphological development of individual CGNs in vivo.
- To track dendritic growth, pruning, and claw formation across developmental time points.
- To provide a platform for cell-autonomous genetic manipulation of CGNs.
Methods Used
- In vivo electroporation of granule neuron progenitors in the developing mouse cerebellum.
- Sparse labeling of CGNs for clear morphological analysis.
- Confocal microscopy with Z-stack imaging for 3D reconstruction.
- Manual tracing and quantification of neurite length and dendritic claw structures using Fiji and Simple Neurite Tracer.
Main Results
- Projection images from 3 to 14 days post-injection (DPI) revealed a progressive decrease in dendrite number per CGN.
- Between 3 and 7 DPI, CGNs underwent dendritic pruning, eliminating over 50% of excess dendrites.
- Claw-like structures formed at the ends of remaining dendrites, with claws present on ~75% of dendrites by 7 DPI.
- CGN size remained largely consistent, though a significant 20% decrease in volume was observed at 7 DPI compared to other time points.
Conclusions
- The protocol enables detailed, cell-specific analysis of CGN morphological development in vivo.
- Dendritic pruning and claw formation are concurrent processes during CGN maturation.
- The method is adaptable for genetic manipulation to study gene function in CGN development.
Frequently Asked Questions
Chapters in this video
0:05
Introduction
0:39
Preparing the Injection Syringe for Electroporation
2:34
Results: Analysis of Granule Neuron Morphology and Size During Cerebellar Development
3:45
Conclusion
1:00
Morphological Analyses of Cerebellar Granule Neurons (CGNs): 3D Reconstruction
