Name: utilizing in vivo postnatal electroporation to study cerebellar granule neuron morphology and synapse development
Uploaded: 2021-06-09T14:30:00
Description: Watch this Scientific Journal Video about Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development at JoVE.com

The work was supported by NIH grants R01NS098804 (A.E.W.), F31NS113394 (U.C.), and Duke University&#39;s Summer Neuroscience Program (D.G.).

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)
<ol>
	<li>Gather the following materials: purified DNA (0.5-25 &#181;g per animal), 3 M sodium acetate, ethanol, Fast Green dye, ultrapure distilled water, phosphate buffer solution (PBS) (see the&#160;Table of Materials). 
	&#8203;NOTE: For DNA, a construct expre...

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+precursor+cells+from+the+rhombic+lip+are+specified+to+a+cerebellar+granule+neuron+identity.">Embryonic precursor cells from the rhombic lip are specified to a cerebellar granule neuron identity.</a> Neuron. 17 (3), 389-399 (1996).</li><li>Hatten, M. E., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+neural+patterning+and+specification+in+the+developing+cerebellum.">Mechanisms of neural patterning and specification in the developing cerebellum.</a> Annual Review of Neuroscience. 18, 385-408 (1995).</li><li>Ben-Arie, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Math1+is+essential+for+genesis+of+cerebellar+granule+neurons.">Math1 is essential for genesis of cerebellar granule neurons.</a> Nature. 390 (6656), 169-172 (1997).</li><li>Borghesani, P. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BDNF+stimulates+migration+of+cerebellar+granule+cells.">BDNF stimulates migration of cerebellar granule cells.</a> Development. 129 (6), 1435-1442 (2002).</li><li>Espinosa, J. S., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timing+neurogenesis+and+differentiation:+insights+from+quantitative+clonal+analyses+of+cerebellar+granule+cells.">Timing neurogenesis and differentiation: insights from quantitative clonal analyses of cerebellar granule cells.</a> Journal of Neuroscience. 28 (10), 2301-2312 (2008).</li><li>Markwalter, K. H., Yang, Y., Holy, T. E., Bonni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensorimotor+coding+of+vermal+granule+neurons+in+the+developing+mammalian+cerebellum.">Sensorimotor coding of vermal granule neurons in the developing mammalian cerebellum.</a> Journal of Neuroscience. 39 (34), 6626-6643 (2019).</li><li>Shalizi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PIASx+is+a+MEF2+SUMO+E3+ligase+that+promotes+postsynaptic+dendritic+morphogenesis.">PIASx is a MEF2 SUMO E3 ligase that promotes postsynaptic dendritic morphogenesis.</a> Journal of Neuroscience. 27 (37), 10037-10046 (2007).</li><li>Shalizi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Calcium-regulated+MEF2+sumoylation+switch+controls+poststynaptic+differentiation.">A Calcium-regulated MEF2 sumoylation switch controls poststynaptic differentiation.</a> Science. 311 (5763), 1012-1017 (2006).</li><li>Konishi, Y., Stegmuller, J., Matsuda, T., Bonni, S., Bonni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdh1-APC+controls+axonal+growth+and+patterning+in+the+mammalian+brain.">Cdh1-APC controls axonal growth and patterning in the mammalian brain.</a> Science. 303 (5660), 1026-1030 (2004).</li><li>Holubowska, A., Mukherjee, C., Vadhvani, M., Stegmuller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulation+of+cerebellar+granule+neurons+in+vitro+and+in+vivo+to+study+neuronal+morphology+and+migration.">Genetic manipulation of cerebellar granule neurons in vitro and in vivo to study neuronal morphology and migration.</a> Journal of Visualized Experiments: JoVE. (85), e51070 (2014).</li><li>Yang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+remodeling+inactivates+activity+genes+and+regulates+neural+coding.">Chromatin remodeling inactivates activity genes and regulates neural coding.</a> Science. 353 (6296), 300-305 (2016).</li><li>Herculano-Houzel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinated+scaling+of+cortical+and+cerebellar+numbers+of+neurons.">Coordinated scaling of cortical and cerebellar numbers of neurons.</a> Frontiers in Neuroanatomy. 4, 12 (2010).</li><li>Wilson, P. M., Fryer, R. H., Fang, Y., Hatten, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astn2,+a+novel+member+of+the+astrotactin+gene+family,+regulates+the+trafficking+of+ASTN1+during+glial-guided+neuronal+migration.">Astn2, a novel member of the astrotactin gene family, regulates the trafficking of ASTN1 during glial-guided neuronal migration.</a> Journal of Neuroscience. 30 (25), 8529-8540 (2010).</li><li>Kokubo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BDNF-mediated+cerebellar+granule+cell+development+is+impaired+in+mice+null+for+CaMKK2+or+CaMKIV.">BDNF-mediated cerebellar granule cell development is impaired in mice null for CaMKK2 or CaMKIV.</a> Journal of Neuroscience. 29 (28), 8901-8913 (2009).</li><li>Schwartz, P. M., Borghesani, P. R., Levy, R. L., Pomeroy, S. L., Segal, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+cerebellar+development+and+foliation+in+BDNF-/-+mice+reveals+a+role+for+neurotrophins+in+CNS+patterning.">Abnormal cerebellar development and foliation in BDNF-/- mice reveals a role for neurotrophins in CNS patterning.</a> Neuron. 19 (2), 269-281 (1997).</li><li>Segal, R. A., Pomeroy, S. L., Stiles, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+growth+and+fasciculation+linked+to+differential+expression+of+BDNF+and+NT3+receptors+in+developing+cerebellar+granule+cells.">Axonal growth and fasciculation linked to differential expression of BDNF and NT3 receptors in developing cerebellar granule cells.</a> Journal of Neuroscience. 15 (7), 4970-4981 (1995).</li><li>Zhou, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarized+signaling+endosomes+coordinate+BDNF-induced+chemotaxis+of+cerebellar+precursors.">Polarized signaling endosomes coordinate BDNF-induced chemotaxis of cerebellar precursors.</a> Neuron. 55 (1), 53-68 (2007).</li><li>Dhar, M., Hantman, A. W., Nishiyama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+pattern+and+structural+factors+of+dendritic+survival+in+cerebellar+granule+cells+in+vivo.">Developmental pattern and structural factors of dendritic survival in cerebellar granule cells in vivo.</a> Scientific Reports. 8 (1), 17561 (2018).</li><li>Ito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+plasticity+in+the+cerebellar+cortex+and+its+role+in+motor+learning.">Synaptic plasticity in the cerebellar cortex and its role in motor learning.</a> Canadian Journal of Neurological Sciences. 20, 70-74 (1993).</li><li>Jorntell, H., Hansel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+memories+upside+down:+bidirectional+plasticity+at+cerebellar+parallel+fiber-Purkinje+cell+synapses.">Synaptic memories upside down: bidirectional plasticity at cerebellar parallel fiber-Purkinje cell synapses.</a> Neuron. 52 (2), 227-238 (2006).</li><li>Nakanishi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulation+study+of+information+processing+in+the+cerebellum.">Genetic manipulation study of information processing in the cerebellum.</a> Neuroscience. 162 (3), 723-731 (2009).</li><li>Chang, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atoh1+controls+primary+cilia+formation+to+allow+for+SHH-triggered+granule+neuron+progenitor+proliferation.">Atoh1 controls primary cilia formation to allow for SHH-triggered granule neuron progenitor proliferation.</a> Developmental Cell. 48 (2), 184-199 (2019).</li></ol>

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,<sup c...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62568">Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development</a>. A figure was updated.
Figure 2 was updated from:
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62568/62568fig02.jpg" /> 
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&#160;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 &#181;L of a 0.02% Fast Green dye shows injection is localized to the middle of the cerebellar vermis between lobules 5-7. <a href="https://www.jove.com/files/ftp_upload/62568/62568fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62568/62568fig2v2.jpg" /> 
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&#160;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 &#181;L of a 0.02% Fast Green dye shows injection is localized to the middle of the cerebellar vermis between lobules 5-7. <a href="https://www.jove.com/files/ftp_upload/62568/62568fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62568">Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development</a>. A figure was updated.

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

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 after birth1.
In mammals, CGN differentiation begins at the end of embryonic development when a subset of proliferative cells in the hindbrain migrates over the rhombic lip to form a secondary germinal zone on the surface of the cerebellum2,3,4. Although they are fully committed to a granule neuron progenitor (GNP) identity, these cells continue to proliferate within the outer portion of the external granule layer (EGL) until postnatal day 14 (P14). Proliferation of this layer results in a massive expansion of the cerebellum as these cells give rise exclusively to CGNs5. Once newborn CGNs exit the cell cycle in the EGL, they migrate inwards towards the internal granule layer (IGL), leaving behind an axon that will bifurcate and travel in the molecular layer of the cerebellum, forming parallel fibers that synapse onto Purkinje cells6. The position of these fibers within the molecular layer is dependent on the timing of cell-cycle exit.
CGNs that differentiate first leave their parallel fibers towards the bottom of the molecular layer, whereas the axons of CGNs that differentiate later are clustered at the top7,8. Once the CGN cell bodies reach the IGL, they begin to elaborate dendrites and form synapses with nearby inhibitory and excitatory neurons. The mature dendritic tree of a CGN exhibits a stereotyped architecture with four main processes. Over the course of CGN maturation, the structures at the end of these dendrites form a claw that becomes enriched with postsynaptic proteins9,10. These specialized structures, called dendritic claws, contain the majority of the synapses onto granule neurons and are important for receiving both excitatory inputs from mossy fiber innervations originating from the pons, as well as inhibitory inputs from local Golgi cells. Once fully configured, the synaptic connections of CGNs allow these cells to relay inputs from pre-cerebellar nuclei to Purkinje cells, which project out of the cerebellar cortex to the deep cerebellar nuclei.
In vivo postnatal electroporation of GNPs is advantageous over other labeling-based methods, such as viral infection and generation of transgenic mouse lines, because the expression of desired constructs can be achieved on a fast timeline, and the method targets a small population of cells, useful in studying cell-autonomous effects. This method has been used in prior studies to study morphological development of CGNs; however, these studies have focused on either a single time point or a short window of time9,10,11,12,13. This labeling method was paired with image analysis to document the changes in CGN morphology that occur across the entire time course of CGN differentiation over the first three weeks of postnatal life. These data reveal the dynamics of CGN dendrite development that underlie construction of cerebellar circuits.

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			<td>Betadine</td>
			<td>Purdue Production</td>
			<td>67618-150-17</td>
			<td></td>
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		<tr>
			<td>Cemented 10 &#181;L needle</td>
			<td>Hamilton</td>
			<td>1701SN (80008)</td>
			<td>33 gauge, 1.27 cm (0.5 in), 4 point style</td>
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			<td>Chicken anti-GFP</td>
			<td>Millipore Sigma</td>
			<td>AB16901</td>
			<td>Our lab uses this antibody at a 1:1000 concentration</td>
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			<td>Cotton-tip applicator</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>Donkey anti-chicken Cy2</td>
			<td>Jackson ImmunoResearch</td>
			<td>703-225-155</td>
			<td>Our lab uses this antibody at a 1:500 concentration</td>
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			<td>Ethanol (200 proof)</td>
			<td>Koptec</td>
			<td>V1016</td>
			<td></td>
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			<td>Electroporator ECM 830</td>
			<td>BTX Harvard Apparatus</td>
			<td>45-0052</td>
			<td></td>
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		<tr>
			<td>Fast Green FCF</td>
			<td>Sigma</td>
			<td>F7252-5G</td>
			<td></td>
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		<tr>
			<td>FUGW plasmid</td>
			<td>Addgene</td>
			<td>14883</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>VWR</td>
			<td>48311-703</td>
			<td>Superfrost plus</td>
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			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
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			<td>Heating pad</td>
			<td>Softheat</td>
			<td></td>
			<td></td>
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		<tr>
			<td>Hoescht 33342 fluorescent dye</td>
			<td>Invitrogen</td>
			<td>62249</td>
			<td></td>
		</tr>
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			<td>Imaris</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
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			<td>Isoflurane</td>
			<td>Patterson Veterinary</td>
			<td>07-893-1389</td>
			<td></td>
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			<td>Micro cover glass</td>
			<td>VWR</td>
			<td>48382-138</td>
			<td></td>
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			<td>Nail polish</td>
			<td>Sally Hansen</td>
			<td>Color 109</td>
			<td></td>
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			<td>Normal goat serum</td>
			<td>Gibco</td>
			<td>16210064</td>
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			<td>O.C.T. embedding compound</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td></td>
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			<td>Olympus MVX10 Dissecting Scope</td>
			<td>Olympus</td>
			<td>MVX10</td>
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			<td>P200 pipette reach tip</td>
			<td>Fisherbrand</td>
			<td>02-707-138</td>
			<td>Used for needle spacer</td>
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			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM-996</td>
			<td></td>
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			<td>PBS pH 7.4 (10x)</td>
			<td>Gibco</td>
			<td>70011-044</td>
			<td></td>
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			<td>Simple Neurite Tracer</td>
			<td>FIJI</td>
			<td></td>
			<td>https://imagej.net/Simple_Neurite_Tracer:_Basic_ 
			Instructions</td>
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			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td></td>
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			<td>Surgical tools</td>
			<td>RWD Life Science</td>
			<td></td>
			<td>Small scissors and tweezers</td>
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			<td>Triton X-100</td>
			<td>Roche</td>
			<td>11332481001</td>
			<td>non-ionic detergent</td>
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			<td>Tweezertrodes</td>
			<td>BTX Harvard Apparatus</td>
			<td>45-0489</td>
			<td>5 mm, platinum plated tweezer-type electrodes</td>
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			<td>Ultrapure distilled water</td>
			<td>Invitrogen</td>
			<td>10977-015</td>
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			<td>Vectashield mounting media</td>
			<td>Vectashield</td>
			<td>H1000</td>
			<td></td>
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			<td>Vetbond tissue adhesive</td>
			<td>3M</td>
			<td>1469SB</td>
			<td></td>
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			<td>Zeiss 780 Upright Confocal</td>
			<td>Zeiss</td>
			<td>780</td>
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utilizing in vivo postnatal electroporation to study cerebellar granule neuron morphology and synapse development

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&#160;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.

<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62568/62568fig04.jpg" /> 
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 &#181;m. (B) Average number of dendrites. (C) Average dendrite le...

Watch this Scientific Journal Video about Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development at JoVE.com

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

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.

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

Neurons undergo dynamic changes in their structure and function during brain development to form appropriate connections with other cells. The rodent ...

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.

Research

JoVE Journal

Neuroscience

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