Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

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 expressing green fluorescent protein (GFP) under a human ubiquitin promoter was obtained from Addgene (FUGW, https://www.addgene.org/14883/). Any construct....

Results

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 brain^1,14. The cerebellum has been extensively utilized to elucidate mechanisms of cellular proliferation, migration, dendrite formation, and synapse development^6,9,10,11,

Materials

Name	Company	Catalog Number	Comments
Betadine	Purdue Production	67618-150-17
Cemented 10 µL needle	Hamilton	1701SN (80008)	33 gauge, 1.27 cm (0.5 in), 4 point style
Chicken anti-GFP	Millipore Sigma	AB16901	Our lab uses this antibody at a 1:1000 concentration
Cotton-tip applicator
Donkey anti-chicken Cy2	Jackson ImmunoResearch	703-225-155	Our lab uses this antibody at a 1:500 concentration
Ethanol (200 proof)	Koptec	V1016
Electroporator ECM 830	BTX Harvard Apparatus	45-0052
Fast Green FCF	Sigma	F7252-5G
FUGW plasmid	Addgene	14883
Glass slides	VWR	48311-703	Superfrost plus
Glycerol	Sigma-Aldrich	G5516
Heating pad	Softheat
Hoescht 33342 fluorescent dye	Invitrogen	62249
Imaris	Bitplane
Isoflurane	Patterson Veterinary	07-893-1389
Micro cover glass	VWR	48382-138
Nail polish	Sally Hansen	Color 109
Normal goat serum	Gibco	16210064
O.C.T. embedding compound	Tissue-Tek	4583
Olympus MVX10 Dissecting Scope	Olympus	MVX10
P200 pipette reach tip	Fisherbrand	02-707-138	Used for needle spacer
Parafilm	Bemis	PM-996
PBS pH 7.4 (10x)	Gibco	70011-044
Simple Neurite Tracer	FIJI		https://imagej.net/Simple_Neurite_Tracer:_Basic_ Instructions
Sucrose	Sigma	S0389
Surgical tools	RWD Life Science		Small scissors and tweezers
Triton X-100	Roche	11332481001	non-ionic detergent
Tweezertrodes	BTX Harvard Apparatus	45-0489	5 mm, platinum plated tweezer-type electrodes
Ultrapure distilled water	Invitrogen	10977-015
Vectashield mounting media	Vectashield	H1000
Vetbond tissue adhesive	3M	1469SB
Zeiss 780 Upright Confocal	Zeiss	780