This work was supported by grants to JLRR from: Autism Speaks, Nina Ireland, Weston Havens Foundation, NIMH R01 MH081880, and NIMH R37 MH049428. PRW was supported by a fellowship from the National Science Council of Taiwan. SFS was supported by F32 (MH103003).

Ethics statement: The following procedures have been approved by our institution and animal protocol. Make sure to get approval for all procedures involving survival surgeries before beginning experiments and verify all protocols are up to date.
1. Lentivirus Preparation (Optional Step)
<ol>
	<li>Split three 10 cm plates of HEK293T cells for each lentivirus to be made, in the presence of 10 ml DMEM/10% FBS, and grow to ~60-70% confluence. Grow cells in an incubator at 37 &#176;C wit...

<ol>
	<li>Huang, Z. J., Di Cristo, G., Ango, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+GABA+innervation+in+the+cerebral+and+cerebellar+cortices.">Development of GABA innervation in the cerebral and cerebellar cortices.</a> Nat. Rev. Neurosci. 8 (9), 673-686 (2007).</li><li>Rubenstein, J. L. R., Merzenich, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model+of+autism:+increased+ratio+of+excitation/inhibition+in+key+neural+systems.">Model of autism: increased ratio of excitation/inhibition in key neural systems.</a> Genes, brain, and behavior. 2 (5), 255-267 (2003).</li><li>Anderson, S. A., Eisenstat, D. D., Shi, L., Rubenstein, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interneuron+migration+from+basal+forebrain+to+neocortex:+dependence+on+Dlx+genes.">Interneuron migration from basal forebrain to neocortex: dependence on Dlx genes.</a> Science. 278 (5337), 474-476 (1997).</li><li>Wonders, C. P., Anderson, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+and+specification+of+cortical+interneurons.">The origin and specification of cortical interneurons.</a> Nat. Rev. Neurosci. 7 (9), 687-696 (2006).</li><li>Gelman, D., 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+Wide+Diversity+of+Cortical+GABAergic+Interneurons+Derives+from+the+Embryonic+Preoptic+Area.">A Wide Diversity of Cortical GABAergic Interneurons Derives from the Embryonic Preoptic Area.</a> J. Neurosci. 31 (46), 16570-16580 (2011).</li><li>Miyoshi, G., 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=Genetic+fate+mapping+reveals+that+the+caudal+ganglionic+eminence+produces+a+large+and+diverse+population+of+superficial+cortical+interneurons.">Genetic fate mapping reveals that the caudal ganglionic eminence produces a large and diverse population of superficial cortical interneurons.</a> J. Neurosci. 30 (5), 1582-1594 (2010).</li><li>Batista-Brito, R., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+integration+of+cortical+interneurons+into+a+functional+network.">The developmental integration of cortical interneurons into a functional network.</a> Curr. Top. Dev. Bio. 87, 81-118 (2009).</li><li>Inan, M., Welagen, J., Anderson, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+Temporal+Bias+in+the+Mitotic+Origins+of+Somatostatin.">Spatial and Temporal Bias in the Mitotic Origins of Somatostatin.</a> and Parvalbumin-Expressing Interneuron Subgroups and the Chandelier Subtype in the Medial Ganglionic. 22 (4), 820-827 (2012).</li><li>Taniguchi, 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=A+Resource+of+Cre+Driver+Lines+for+Genetic+Targeting+of+GABAergic+Neurons+in+Cerebral+Cortex.">A Resource of Cre Driver Lines for Genetic Targeting of GABAergic Neurons in Cerebral Cortex.</a> Neuron. 71 (6), 995-1013 (2011).</li><li>Madisen, L., 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+robust+and+high-throughput+Cre+reporting+and+characterization+system+for+the+whole+mouse+brain.">A robust and high-throughput Cre reporting and characterization system for the whole mouse brain.</a> Nature Neuroscience. 13 (1), 133-140 (2010).</li><li>Hippenmeyer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+developmental+switch+in+the+response+of+DRG+neurons+to+ETS+transcription+factor+signaling.">A developmental switch in the response of DRG neurons to ETS transcription factor signaling.</a> PLoS Bio. 3 (5), e159 (2005).</li><li>Southwell, D. G., 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=Interneurons+from+embryonic+development+to+cell-based+therapy.">Interneurons from embryonic development to cell-based therapy.</a> Science. 344 (6180), 1240622 (2014).</li><li>Hunt, R. F., Girskis, K. M., Rubenstein, J. L., Alvarez-Buylla, A., Baraban, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+progenitors+grafted+into+the+adult+epileptic+brain+control+seizures+and+abnormal.">GABA progenitors grafted into the adult epileptic brain control seizures and abnormal.</a> Nature Neuroscience. 16 (6), 692-697 (2013).</li><li>Braz, J. 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=Forebrain+GABAergic+Neuron+Precursors+Integrate+into+Adult+Spinal+Cord+and+Reduce+Injury-Induced+Neuropathic+Pain.">Forebrain GABAergic Neuron Precursors Integrate into Adult Spinal Cord and Reduce Injury-Induced Neuropathic Pain.</a> Neuron. 74, 663-675 (2012).</li><li>Vogt, D., 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=Lhx6+Directly+Regulates+Arx+and+CXCR7+to+Determine+Cortical+Interneuron+Fate+and+Laminar+Position.">Lhx6 Directly Regulates Arx and CXCR7 to Determine Cortical Interneuron Fate and Laminar Position.</a> Neuron. 82 (2), 350-364 (2014).</li><li>Alvarez-Dolado, 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=Cortical+inhibition+modified+by+embryonic+neural+precursors+grafted+into+the+postnatal+brain.">Cortical inhibition modified by embryonic neural precursors grafted into the postnatal brain.</a> J. Neurosci. 26 (4), 7380-7389 (2006).</li><li>Du, T., Xu, Q., Ocbina, P. J., Anderson, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NKX2.1+specifies+cortical+interneuron+fate+by+activating+Lhx6.">NKX2.1 specifies cortical interneuron fate by activating Lhx6.</a> Development. 135 (8), 1559-1567 (2008).</li><li>Arguello, 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=Dapper+Antagonist+of+Catenin-1+Cooperates+with+Dishevelled-1+during+Postsynaptic+Development+in+Mouse+Forebrain+GABAergic+Interneurons.">Dapper Antagonist of Catenin-1 Cooperates with Dishevelled-1 during Postsynaptic Development in Mouse Forebrain GABAergic Interneurons.</a> PLoS ONE. 8 (6), e67679 (2013).</li><li>Lee, 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=Pyramidal+neurons+in+prefrontal+cortex+receive+subtype-specific+forms+of+excitation+and+inhibition.">Pyramidal neurons in prefrontal cortex receive subtype-specific forms of excitation and inhibition.</a> Neuron. 81 (1), 61-68 (2014).</li><li>Chen, Y. J. J., 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=Use+of+&#8220;MGE+Enhancers&#8221;+for+Labeling+and+Selection+of+Embryonic+Stem+Cell-Derived+Medial+Ganglionic+Eminence+(MGE)+Progenitors+and+Neurons.">Use of &#8220;MGE Enhancers&#8221; for Labeling and Selection of Embryonic Stem Cell-Derived Medial Ganglionic Eminence (MGE) Progenitors and Neurons.</a> PLoS ONE. 8 (5), e61956 (2013).</li><li>Pattabiraman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+of+enhancers+active+in+protodomains+of+the+developing+cerebral+cortex.">Transcriptional regulation of enhancers active in protodomains of the developing cerebral cortex.</a> Neuron. 82 (5), 989-1003 (2014).</li></ol>

The authors declare that they have no competing financial interests.

The use of GABAergic cortical interneuron precursors from the embryonic ganglionic eminences (GEs) for cell based therapies is showing promise for many conditions 12&#8211;14. Precise molecular techniques are needed to track, and express genes of interest in specific interneuron subgroups. Here we provide a detailed protocol for labeling embryonic MGE cells with lentiviruses before transplantation and show how this technique can be used to express genes of interest in specific cortical in...

GABAergic cortical interneurons comprise ~20-30% of neurons in the mammalian neocortex, while the rest correspond to excitatory, glutamatergic principal neurons. Interneurons are highly diverse in electrophysiological properties, axon and dendrite morphology and synaptic targeting 1, and imbalances in excitatory/inhibitory tone are hypothesized to underlie some phenotypes of neurological/neuropsychiatric disorders including autism, schizophrenia and epilepsy 2. The overall goal of the protocol described herein is to provide a means to efficiently genetically modify GABAergic cortical interneuron progenitors before transplantation for in vivo analyses.
Cortical GABAergic interneurons are born in the medial and caudal ganglionic eminences (MGE and CGE, respectively) 3,4 as well as the preoptic area 5. Cortical interneuron progenitors undergo long-distance tangential migration followed by radial migration to reach their final targets. Upon arrival at their destinations, these cortical interneurons must correctly integrate into the existing neuronal network, and each unique interneuron subgroup will contribute to cortical circuitry in specific ways. Four main subgroups can be distinguished by molecular markers: MGE-derived somatostatin (SST)+ and parvalbumin (PV)+ subgroups, and CGE-derived vasoactive intestinal peptide (VIP)+ and Reelin+;SST- subgroups 6. Different cortical interneuron subgroups are born over different times during embryonic development in the MGE and CGE 7, 8. These and other cortical GABAergic interneuron markers have been used to generate specific Cre-driver lines for many of these subgroups 9-11.
The transplantation of MGE progenitors has emerged as a potential cell-based therapy to treat disorders that may be caused by imbalances in excitation/inhibition 12&#8211;24. These therapeutic benefits may be due in part to the unique ability of MGE progenitors (to disperse, differentiate and integrate into a host brain), or potentially because many peri-somatic inhibitory PV+ cells are derived from the MGE. MGE cells can also be quickly and efficiently transduced with lentiviruses before transplantation 15, allowing cells that are genetically modified in vitro to be studied in vivo. The rationale for developing this approach was to overcome roadblocks in studying GABAergic cortical interneuron development and maturation. In particular, MGE transplantation allowed researchers to study the development of mutant cells in vivo, when the mutant mouse would have otherwise died at an early time point. Moreover, by introducing genes of interest before transplantation, the effects of specific genes on a mutant phenotype could be assessed in an efficient manner.
Here, we provide a detailed protocol to transduce MGE cells with lentiviruses prior to transplantation. In addition, we show how this technique can be adapted to express a gene of interest in specific interneuron subgroups from a heterogeneous group of cortical interneuron precursors, using a combination of Cre-dependent expression lentiviruses and available Cre-driver mouse lines. Moreover, this protocol introduces techniques and a platform for researchers to genetically modify GABAergic cortical interneuron precursors for in vivo studies in a unique way. One advantage of this technique over other current approaches is that the transplanted MGE cells will disperse away from the injection site. Also, unlike focal viral injections, after MGE cells disperse their morphology is easier to assess. This approach can be used to study the effect of introducing genes of interest into wild type or mutant cells, introducing a cell type specific reporter to assess morphology, or potentially to study the effect of disease alleles in vivo.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents and equipment for MGE cell transplantation</td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>REF 309602</td>
			<td colspan="2">BD, Becton Dickinson and company</td>
		</tr>
		<tr>
			<td>301/2 gauge needle</td>
			<td>305106</td>
			<td colspan="2">BD, Becton Dickinson and company</td>
		</tr>
		<tr>
			<td>Precision bore to deliver 5 &#181;l (comes with plunger)</td>
			<td>5-000-1005</td>
			<td colspan="2">Drummond scientific company</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>PM-999</td>
			<td colspan="2">Polysciences, Inc.</td>
		</tr>
		<tr>
			<td>Stereo microscope with boomstand **</td>
			<td>MZ6</td>
			<td>Leica</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital just for mice stereotaxic instrument **</td>
			<td>51725D</td>
			<td colspan="2">Stoelting company</td>
		</tr>
		<tr>
			<td>Single-axis oil hydraulic fine micromanipulator **</td>
			<td>MO-10</td>
			<td>Narishige</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond coated rotary beveler</td>
			<td>n.a.</td>
			<td colspan="2">made in house</td>
		</tr>
		<tr>
			<td>Needle pipette puller</td>
			<td>730</td>
			<td colspan="2">Kopf instruments</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>n.a.</td>
			<td colspan="2">Any drug store or pharmacy</td>
		</tr>
		<tr>
			<td colspan="2">Any pipette and tips that can reliaby measure 1 &#181;l of volume</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">** These are the particular models we use, but many other setups should work</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Optional Reagents (for making Lentiviruses)</td>
		</tr>
		<tr>
			<td>25 mm, 0.45 &#181;m filter</td>
			<td>09-719B</td>
			<td colspan="2">Fisher Scientific</td>
		</tr>
		<tr>
			<td>Ultra-clear centrifuge tubes (25 x 89 mm)</td>
			<td>344058</td>
			<td colspan="2">Beckman Coulter&#174;</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 (1.5 ml size)</td>
			<td>11668019</td>
			<td colspan="2">Invitrogen</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Other commercially available supplies</td>
		</tr>
		<tr>
			<td>pMDLg/pRRE plasmid encoding gag/pol</td>
			<td>12251</td>
			<td>Addgene</td>
			<td></td>
		</tr>
		<tr>
			<td>pRSV-Rev plasmid encoding Rev</td>
			<td>12253</td>
			<td>Addgene</td>
			<td></td>
		</tr>
		<tr>
			<td>pMD2.G plasmid encoding VSVG</td>
			<td>12259</td>
			<td>Addgene</td>
			<td></td>
		</tr>
		<tr>
			<td>pAAV-Flex-GFP</td>
			<td>28304</td>
			<td>Addgene</td>
			<td></td>
		</tr>
	</tbody>
</table>

viral-mediated labeling and transplantation of medial ganglionic eminence (mge) cells for in vivo studies

GABAergic cortical interneurons, derived from the embryonic medial and caudal ganglionic eminences (MGE and CGE), are functionally and morphologically diverse. Inroads have been made in understanding the roles of distinct cortical interneuron subgroups, however, there are still many mechanisms to be worked out that may contribute to the development and maturation of different types of GABAergic cells. Moreover, altered GABAergic signaling may contribute to phenotypes of autism, schizophrenia and epilepsy. Specific Cre-driver lines have begun to parcel out the functions of unique interneuron subgroups. Despite the advances in mouse models, it is often difficult to efficiently study GABAergic cortical interneuron progenitors with molecular approaches in vivo. One important technique used to study the cell autonomous programming of these cells is transplantation of MGE cells into host cortices. These transplanted cells migrate extensively, differentiate, and functionally integrate. In addition, MGE cells can be efficiently transduced with lentivirus immediately prior to transplantation, allowing for a multitude of molecular approaches. Here we detail a protocol to efficiently transduce MGE cells before transplantation for in vivo analysis, using available Cre-driver lines and Cre-dependent expression vectors. This approach is advantageous because it combines precise genetic manipulation with the ability of these cells to disperse after transplantation, permitting greater cell-type specific resolution in vivo.

Since MGE cells have the unique ability to migrate and integrate when transplanted into a host neocortex 16, they provide an excellent model system for genetic manipulation before in vivo studies. Herein, we show how one can isolate MGE tissue from E13.5 embryos (Figures 1 and 2), which can then be transduced with lentiviruses either in vitro or in a rapid manner before transplantation for in vivo studies. Labeling MGE via lentiviruses has been performed before using...

Watch this Scientific Journal Video about Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence MGE Cells for In Vivo Studies at JoVE.com

Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence MGE Cells for In Vivo Studies

GABAergic cortical interneuron progenitors disperse, develop and synaptically integrate into a host cortex after transplantation. These cells can be easily transduced before transplantation for in vivo studies of genetically modified GABAergic precursors. Here, we show viral labeling techniques to target specific interneuron subgroups using existing Cre lines and Cre-dependent reporters.

Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence (MGE) Cells for In Vivo Studies

GABAergic cortical interneurons, derived from the embryonic medial and caudal ganglionic eminences (MGE and CGE), are functionally and morphologically ...

viral-mediated-labeling-transplantation-medial-ganglionic-eminence

Research

JoVE Journal

Developmental Biology

11.4K Görüntüleme.  University of California San Francisco.  GABAerjik kortikal interneuron atalarıdır nakli sonrası bir konak korteks entegre sinaptik geliştirmek ve dağıtmak. Bu hücreler kolayca genetik olarak modifiye edilmiş GABAerjik ön in vivo çalışmalar için transplantasyonundan önce transduse edilebilir. Burada, biz mevcut Cre hatları ve Kre-bağımlı gazetecilere kullanarak belirli interneuron alt grupları hedef viral etiketleme teknikleri gösteriyor. 