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.

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Research

JoVE Journal

Neuroscience

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

Embryonic neurons are born in the ventricular zone of the brain, but subsequently migrate to new destinations to reach appropriate targets. Deciphering the molecular signals that cooperatively guide neuronal migration in the embryonic brain is therefore important to understand how the complex neural networks form which later support postnatal life. Facial branchiomotor (FBM) neurons in the mouse embryo hindbrain migrate from rhombomere (r) 4 caudally to form the paired facial nuclei in the r6-derived region of the hindbrain. Here we provide a detailed protocol for wholemount ex vivo culture of mouse embryo hindbrains suitable to investigate the signaling pathways that regulate FBM migration. In this method, hindbrains of E11.5 mouse embryos are dissected and cultured in an open book preparation on cell culture inserts for 24 hr. During this time, FBM neurons migrate caudally towards r6 and can be exposed to function-blocking antibodies and small molecules in the culture media or heparin beads loaded with recombinant proteins to examine roles for signaling pathways implicated in guiding neuronal migration.

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

Embryonic neurons are born in the ventricular zone of the neural tube, but migrate to reach appropriate targets. Facial branchiomotor (FBM) neurons are a useful model to study neuronal migration. This protocol describes the wholemount ex vivo culture of mouse embryo hindbrains to investigate mechanisms that regulate FBM migration.

Embryonic neurons are born in the ventricular zone of the brain, but subsequently migrate to new destinations to reach appropriate targets. Deciphering ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.