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Neuroscience

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

Published: June 9th, 2021

DOI:

10.3791/62568

1Department of Neurobiology, Duke University Medical Center

ERRATUM NOTICE

Important: There has been an erratum issued for this article. Read more …

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.

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.

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....

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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.......

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Figure 4
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.......

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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,

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

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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

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Erratum

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

An erratum was issued for: Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development. A figure was updated.

Figure 2 was updated from:

Figure 2
Figure 2: In vivo cerebellar electroporation of granule neuron progenitors in P7 wildtype mouse pups. (A) Pups are anesthetized with 4% isoflurane delivered at a rate of 0.8L/min to ensure anesthesia throughout the injection of the DNA solution. Isoflurane is delivered at a rate of 0.8 L/min. (B) After sterilizing the mouse 3 times with betadine and 70% ethanol, an incision is made that spans the distance of the ears, revealing the hindbrain. (C) A magnified image of a white demarcation on the cranium, a landmark for the injection site. DNA construct should be injected within 1 mm above the mark; dotted lines outline the demarcation, and black arrow denotes the injection site. The ridges of the cerebellar vermis may be visible and can be useful for finding the injection site. (D) Tweezer-type electrode orientation for efficient electroporation. Plus (+) end must be oriented downwards to pull negatively charged DNA into the cerebellar parenchyma prior to administration of electrical pulses. (E) Test injection of 1 µL of a 0.02% Fast Green dye shows injection is localized to the middle of the cerebellar vermis between lobules 5-7. Please click here to view a larger version of this figure.

to:

Figure 2
Figure 2: In vivo cerebellar electroporation of granule neuron progenitors in P7 wildtype mouse pups. (A) Pups are anesthetized with 4% isoflurane delivered at a rate of 0.8L/min to ensure anesthesia throughout the injection of the DNA solution. Isoflurane is delivered at a rate of 0.8 L/min. (B) After sterilizing the mouse 3 times with betadine and 70% ethanol, an incision is made that spans the distance of the ears, revealing the hindbrain. (C) A magnified image of a white demarcation on the cranium, a landmark for the injection site. DNA construct should be injected within 1 mm above the mark; dotted lines outline the demarcation, and black arrow denotes the injection site. The ridges of the cerebellar vermis may be visible and can be useful for finding the injection site. (D) Tweezer-type electrode orientation for efficient electroporation. Plus (+) end must be oriented downwards to pull negatively charged DNA into the cerebellar parenchyma prior to administration of electrical pulses. (E) Test injection of 1 µL of a 0.02% Fast Green dye shows injection is localized to the middle of the cerebellar vermis between lobules 5-7. Please click here to view a larger version of this figure.

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