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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

Neurons undergo dynamic changes in their structure and function during brain development to form appropriate connections with other cells. The rodent cerebellum is an ideal system to track the development and morphogenesis of a single cell type, the cerebellar granule neuron (CGN), across time. Here, in vivo electroporation of granule neuron progenitors in the developing mouse cerebellum was employed to sparsely label cells for subsequent morphological analyses. The efficacy of this technique is demonstrated in its ability to showcase key developmental stages of CGN maturation, with a specific focus on the formation of dendritic claws, which are specialized structures where these cells receive the majority of their synaptic inputs. In addition to providing snapshots of CGN synaptic structures throughout cerebellar development, this technique can be adapted to genetically manipulate granule neurons in a cell-autonomous manner to study the role of any gene of interest and its effect on CGN morphology, claw development, and synaptogenesis.

Introduction

Brain development is a prolonged process that extends from embryogenesis into postnatal life. During this time, the brain integrates a combination of intrinsic and extrinsic stimuli that sculpt the wiring of synapses between dendrites and axons to ultimately guide behavior. The rodent cerebellum is an ideal model system to study how synapses develop because the development of a single neuron type, the cerebellar granule neuron (CGN), can be tracked as it transitions from a progenitor cell to a mature neuron. This is due, in part, to the fact that a majority of the cerebellar cortex develops postnatally, which allows for easy genetic manipulation and cell labeling afte....

Protocol

NOTE: All procedures were performed under protocols approved by Duke University Institutional Animal Care and Use Committee (IACUC).

1. DNA preparation for in vivo electroporation or IVE (1 day before surgery)

  1. Gather the following materials: purified DNA (0.5-25 µg per animal), 3 M sodium acetate, ethanol, Fast Green dye, ultrapure distilled water, phosphate buffer solution (PBS) (see the Table of Materials).
    ​NOTE: For DNA, a construct expre.......

Representative Results

figure-representative results-25
Figure 4: Analysis of granule neuron morphology during cerebellar development. (A) Maximum projection images of electroporated CGNs from 3-DPI to 14-DPI (postnatal age P10 to P21), nuclei (blue) and GFP (green); arrowheads indicate individual dendrite, and scale bar is 10 µm. (B) Average number of dendrites. (C) Average dendrite le.......

Discussion

Cerebellar granule neurons are the most abundant neurons in the mammalian brain, making up almost 60-70% of the total neuron population in the rodent brain1,14. The cerebellum has been extensively utilized to elucidate mechanisms of cellular proliferation, migration, dendrite formation, and synapse development6,9,10,11,

Acknowledgements

The work was supported by NIH grants R01NS098804 (A.E.W.), F31NS113394 (U.C.), and Duke University's Summer Neuroscience Program (D.G.).

....

Materials

NameCompanyCatalog NumberComments
BetadinePurdue Production67618-150-17
Cemented 10 µL needleHamilton1701SN (80008)33 gauge, 1.27 cm (0.5 in), 4 point style
Chicken anti-GFPMillipore SigmaAB16901Our lab uses this antibody at a 1:1000 concentration
Cotton-tip applicator
Donkey anti-chicken Cy2Jackson ImmunoResearch703-225-155Our lab uses this antibody at a 1:500 concentration
Ethanol (200 proof)KoptecV1016
Electroporator ECM 830BTX Harvard Apparatus45-0052
Fast Green FCFSigmaF7252-5G
FUGW plasmidAddgene14883
Glass slidesVWR48311-703Superfrost plus
GlycerolSigma-AldrichG5516
Heating padSoftheat
Hoescht 33342 fluorescent dyeInvitrogen62249
ImarisBitplane
IsofluranePatterson Veterinary07-893-1389
Micro cover glassVWR48382-138
Nail polishSally HansenColor 109
Normal goat serumGibco16210064
O.C.T. embedding compoundTissue-Tek4583
Olympus MVX10 Dissecting ScopeOlympusMVX10
P200 pipette reach tipFisherbrand02-707-138Used for needle spacer
ParafilmBemisPM-996
PBS pH 7.4 (10x)Gibco70011-044
Simple Neurite TracerFIJIhttps://imagej.net/Simple_Neurite_Tracer:_Basic_
Instructions
SucroseSigmaS0389
Surgical toolsRWD Life ScienceSmall scissors and tweezers
Triton X-100Roche11332481001non-ionic detergent
TweezertrodesBTX Harvard Apparatus45-04895 mm, platinum plated tweezer-type electrodes
Ultrapure distilled waterInvitrogen10977-015
Vectashield mounting mediaVectashieldH1000
Vetbond tissue adhesive3M1469SB
Zeiss 780 Upright ConfocalZeiss780

References

  1. Altman, J., Bayer, S. A. . Development of the cerebellar system : in relation to its evolution, structure, and functions. , (1997).
  2. Rahimi-Balaei, M., Bergen, H., Kong, J., Marzban, H. Neuronal migration during development of the cerebellum. Frontiers in Cellular Neur....

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