Name: adeno-associated virus-mediated transgene expression in genetically defined neurons of the spinal cord
Uploaded: 2018-05-12T13:00:00
Description: Watch this Scientific Journal Video about Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord at JoVE.com

We thank Hanns Ulrich Zeilhofer for generously supporting this work. Hendrik Wildner was supported by the Olga Mayenfisch foundation. We thank Carmen Birchmeier for the Lmx1b antibody.

All animal experiments were approved by the Swiss cantonal veterinary office (Zurich) and are in accordance and compliance with all relevant regulatory and institutional guidelines.
NOTE: All materials along with respective manufacturers and/or vendors are listed in the Table of Materials.
1. Generation of Cre-dependent AAV Vectors
NOTE: A variety of Cre-dependent vectors with different promoters can be purchased (see <stro...

<ol>
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J., Dymecki, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptic+boundaries+in+roof+plate+and+choroid+plexus+identified+by+intersectional+gene+activation.">Cryptic boundaries in roof plate and choroid plexus identified by intersectional gene activation.</a> Nat Genet. 35 (1), 70-75 (2003).</li><li>Kim, J. C., 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=Linking+genetically+defined+neurons+to+behavior+through+a+broadly+applicable+silencing+allele.">Linking genetically defined neurons to behavior through a broadly applicable silencing allele.</a> Neuron. 63 (3), 305-315 (2009).</li><li>Kim, J. C., Dymecki, S. 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Intraspinal injection of AAVs may become a powerful technique in a research laboratory, enabling the analysis of spinal cells with a high temporal and spatial solution. This protocol enables the transduction of the three main spinal segments innervated by sensory neurons extending their peripheral axons to the hindlimb. Transducing three segments produces robust and reproducible behavioral data. It also enables testing of a larger sensory area than possible after a single intraspinal injection. For example, the same inje...

The dorsal spinal cord is essential for information exchange between the periphery of the body and the brain. Sensory stimuli such as heat, cold, touch, or noxious stimuli are detected by specialized peripheral neurons, which convey this information to neurons of the spinal cord dorsal horn. Here, a complex network of inhibitory and excitatory interneurons modulates and eventually relays sensory information via spinal projection neurons to supraspinal sites1,2. The computations carried out by spinal inter- and projection neurons gate sensory information, thus determining which information is suppressed or relayed at which intensity. Changes in the integration of sensory stimuli, such as an altered balance between inhibition and excitation, can cause sensory dysfunctions such as hypersensitivity or allodynia (painful sensations after normally non-painful stimulation). These changes are thought to be the underlying cause of various chronic pain states3,4. Thus, spinal circuits are of high importance in sensory processing and consequently in the perception of an organism&#39;s environment and self. With the recent advent and combination of molecular, genetic, and surgical techniques that allow the precise manipulation of genetically identified spinal neuron subpopulations, scientists are now beginning to understand the underlying spinal circuits responsible for the processing of distinct sensory modalities.
Intraspinal injection of rAAV into wild-type or transgenic mice has greatly contributed to the manipulation, analysis, and understanding of the function of specific subsets of spinal neurons5,6,7,8,9,10,11. This technique allows the delivery of marker proteins (such as GFP/ GFP fusion proteins), reporter proteins (such as GCaMP), or effector proteins (such as bacterial toxins, channelrhodopsin, or pharmacogenetic receptors) in a spatially restricted manner to spinal neurons. Local injection of Cre-dependent rAAVs into transgenic mice expressing Cre recombinase in a specific subset of spinal neurons allows the specific analysis of the respective neuronal population. We have employed this technique to label, ablate, inhibit or activate spinal glycinergic neurons demonstrating that they are an essential part of the spinal gate controlling pain and itch transmission7. In these experiments, intraspinal injection of Cre-dependent rAAV into GlyT2::Cre mice enabled the selective manipulation of glycinergic neurons in the lumbar spinal cord. Thereby, simultaneous manipulation of supraspinal circuits that contain glycinergic neurons critical for the survival of the animal can be avoided.
While an intraspinal injection of rAAVs limits infection to the site of injection, viral transduction can occur not only in local neurons but also in neurons that connect to the injection site via axonal projections. The latter is often used to trace CNS areas providing neuronal input to a particular nucleus in the brain. The infection of axonal projections can, however, also be a confounding factor when a defined population of neurons shall be studied at a particular site. To address these issues, we have recently conducted a comprehensive analysis of AAV serotypes and expression cassettes to identify serotypes and promoters that can be used to either minimize or maximize retrograde transduction. In the context of this specific research in spinal circuits, we analyzed the ability of different serotypes and promoters to retrogradely transduce neurons in the dorsal root ganglia (DRG), the rostral ventromedial medulla (RVM), and the somatosensory cortex12. The technique outlined in this protocol can therefore be used either to analyze spinal neurons at the injection site or to analyze projection neurons that provide input to the injected site of the spinal cord. In the protocol described here, three injections of rAAV into the left side of the lumbar spinal cord are performed to enable transduction of neurons in the three lumbar segments (L3-L5). The L3-L5 segments receive the majority of the sensory input from the hindlimb ipsilateral to the injection site. We demonstrate that functional manipulation of genetically labeled neurons in L3-L5 is sufficient to evoke robust behavioral changes, thus providing functional evidence for the circuit function of such a genetically labeled neuron subtype.

<table>
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>micropipette puller: DMZ-Universal-Electrode-Puller</td>
			<td>Zeitz</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>anesthesia unit: Oxymat3 oxygen concentrator</td>
			<td>Weinmann</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>anesthesia unit: VIP 3000 Veterinary Vaporizer</td>
			<td>Midmark</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat mat: Mio Star Thermocare 100</td>
			<td>Migros</td>
			<td>717614700000</td>
			<td></td>
		</tr>
		<tr>
			<td>Electric shaver</td>
			<td>Philips</td>
			<td>BT9290</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical microscope (OPMI pico)</td>
			<td>Zeiss</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Small animal stereotaxic apparatus</td>
			<td>Kopf</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurostar StereoDrive (optional)</td>
			<td>Neurostar</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Model 51690 Cunningham mouse spinal adaptor</td>
			<td>Harvard Apparatus</td>
			<td>72-4811</td>
			<td></td>
		</tr>
		<tr>
			<td>PHD Ultra syringe pump with nanomite</td>
			<td>Harvard Apparatus</td>
			<td>70-3601</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton 701 RN 10 &#956;l glass microliter syringe</td>
			<td>Hamilton</td>
			<td>7635-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton Removable needle (RN) compression fitting 1 mm</td>
			<td>Hamilton</td>
			<td>55750-01</td>
			<td></td>
		</tr>
		<tr>
			<td>fine dentistry drilling apparatus: Osada success 40</td>
			<td>Osada</td>
			<td>OS-40</td>
			<td></td>
		</tr>
		<tr>
			<td>spherical cutter, 0.5mm</td>
			<td>Busch</td>
			<td>12001005B</td>
			<td></td>
		</tr>
		<tr>
			<td>electronic von Frey anesthesiometer</td>
			<td>IITC</td>
			<td>23905</td>
			<td></td>
		</tr>
		<tr>
			<td>flexible von Frey hairs</td>
			<td>IITC</td>
			<td>#7</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM710 Pascal confocal microscope</td>
			<td>Zeiss</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>0.8 NA &#215; 20 Plan-apochromat objective</td>
			<td>Zeiss</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>1.3 NA &#215; 40 EC Plan-Neofluar oil-immersion objective</td>
			<td>Zeiss</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Surgical Tools</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Handle #4, 13cm</td>
			<td>Fine Science Tools</td>
			<td>10004-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Extra Fine Bonn Scissors</td>
			<td>Fine Science Tools</td>
			<td>14084-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Adson forceps, 1 x 2 teeth, 12 cm</td>
			<td>Fine Science Tools</td>
			<td>11027-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Friedman-Pearson rongeurs, curved, 0.7 mm cup</td>
			<td>Fine Science Tools</td>
			<td>16121-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #2 laminectomy forceps</td>
			<td>Fine Science Tools</td>
			<td>11223-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Olsen-Hegar needle holders, serrated, 8.5 mm clamp length</td>
			<td>Fine Science Tools</td>
			<td>12002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps #5</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Consumables and Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thin-wall glass capillary, 1mm outside diameter</td>
			<td>World Precision Instruments</td>
			<td>TW 100-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes (1, 5 and 20 ml)</td>
			<td>B. Braun</td>
			<td>(9166917V, 4606051V, 4606205V)</td>
			<td></td>
		</tr>
		<tr>
			<td>26G beveled needle</td>
			<td>B. Braun</td>
			<td>4665457</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile scalpel blades</td>
			<td>B. Braun</td>
			<td>BB523</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical sutures Safil Quick+ 4/0, absorbable</td>
			<td>B. Braun</td>
			<td>C1046220</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical sutures Premilene 5/0, non-absorbable</td>
			<td>B. Braun</td>
			<td>C0932191</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile PBS or saline (0.9%)</td>
			<td></td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, 70% (disinfectant)</td>
			<td></td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodine solution (e.g. Braunol)</td>
			<td>B. Braun</td>
			<td>18380</td>
			<td></td>
		</tr>
		<tr>
			<td>Anaesthetics&#160; (e.g. Attane isoflurane)</td>
			<td>Provet</td>
			<td>2222</td>
			<td></td>
		</tr>
		<tr>
			<td>Aldasorber</td>
			<td>Provet</td>
			<td>333526</td>
			<td></td>
		</tr>
		<tr>
			<td>analgesics (e.g. buprenorphine: temgesic)</td>
			<td>Indivior</td>
			<td>GTIN: 7680419310018</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic ointment (e.g. vita-pos)</td>
			<td>Pharma medica</td>
			<td>GTIN: 4031626710635</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton swabs (e.g. from)</td>
			<td>IVF Hartmann</td>
			<td>1628100</td>
			<td></td>
		</tr>
		<tr>
			<td>Facial tissues (e.g. from)</td>
			<td>Uehlinger AG</td>
			<td>2015.10018</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost plus microscope slides</td>
			<td>ThermoScientific</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J mice&#160; (wildtype)</td>
			<td>The Jackson Laboratory</td>
			<td>RRID:IMSR_JAX:000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Rorbtm1.1(cre)Hze/J mice (ROR&#946;Cre)</td>
			<td>The Jackson Laboratory</td>
			<td>RRID:IMSR_JAX:023526</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J mice (R26Tom)</td>
			<td>The Jackson Laboratory</td>
			<td>RRID: IMSR_JAX:007914</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Viral vectors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AAV1.CB7.CI.eGFP.WPRE.rBG (AAV1.CAG.eGFP)</td>
			<td>Penn Vector Core</td>
			<td>AV-1-PV1963</td>
			<td></td>
		</tr>
		<tr>
			<td>AAV1.CAG.flex.eGFP.WPRE.bGH (AAV1.CAG.flex.eGFP)</td>
			<td>Penn Vector Core</td>
			<td>AV-1-ALL854</td>
			<td></td>
		</tr>
		<tr>
			<td>AAV1.CAG.flex.tdTomato.WPRE.bGH (AAV1.CAG.flex.tdTomato)</td>
			<td>Penn Vector Core</td>
			<td>AV-1-ALL864</td>
			<td></td>
		</tr>
		<tr>
			<td>AAV1.EF1a.flex.DTA.hGH (AAV1.EF1a.flex.DTA)</td>
			<td>Penn Vector Core</td>
			<td>Custom production</td>
			<td></td>
		</tr>
		<tr>
			<td>AAV1.hSyn.DIO.hM3D(Gq)-mCherry.hGH (AAV.flex.hM3D(Gi))</td>
			<td>Penn Vector Core</td>
			<td>Custom production</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Plasmids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pAAV.hSyn.flex.hM3D(Gq)-mCherry</td>
			<td>Addgene</td>
			<td>44361</td>
			<td></td>
		</tr>
		<tr>
			<td>pAAV.EF1&#945;.flex.hChR2(H134R)-eYFP</td>
			<td>Addgene</td>
			<td>20298</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Bacteria</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MDS42</td>
			<td>ScarabGenomics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stbl3</td>
			<td>ThermoScientific</td>
			<td>C737303</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit</td>
			<td>Quiagen</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr>
			<td>NucleoBond PC 500</td>
			<td>Machery &#38; Nagel</td>
			<td>740574</td>
			<td></td>
		</tr>
		<tr>
			<td>clozapine-N-oxide (CNO)</td>
			<td>Enzo Life Sciences</td>
			<td>BBL-NS105-0025</td>
			<td></td>
		</tr>
		<tr>
			<td>chloroquine diphosphate salt</td>
			<td>Sigma</td>
			<td>C6628</td>
			<td></td>
		</tr>
		<tr>
			<td>histamine</td>
			<td>Sigma</td>
			<td>H7125</td>
			<td></td>
		</tr>
		<tr>
			<td>Dapi</td>
			<td>Invitrogen</td>
			<td>D3571</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Antibodies (dilution)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-GFP (1:1000)</td>
			<td>Molecular Probes</td>
			<td>RRID:AB_221570</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-NeuN (1:3000)</td>
			<td>Abcam</td>
			<td>RRID:AB_10711153</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Pax2 (1 : 200)</td>
			<td>R &#38; D Systems</td>
			<td>RRID:AB_10889828</td>
			<td></td>
		</tr>
		<tr>
			<td>Guinea pig anti-Lmx1b (1 : 10 000)</td>
			<td>Dr Carmen Birchmeier</td>
			<td>Muller et al. 2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-GFAP (1 : 1000)</td>
			<td>DakoCytomation</td>
			<td>RRID:AB_10013382</td>
			<td></td>
		</tr>
		<tr>
			<td>Secondary antibodies raised in donkey (1:800)</td>
			<td>Jackson ImmunoResearch Laboratories</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

adeno-associated virus-mediated transgene expression in genetically defined neurons of the spinal cord

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

In order to illustrate the expression levels that can be obtained by the intraspinal injection of rAAV encoding a marker protein, we first injected AAV1.CAG.eGFP into the lumbar spinal cord of wild-type mice. Three injections spaced approximately 1 mm apart produced a nearly continuous infection of lumbar spinal segments L3 to L5 (Figure 1A-C). Virus injection at a depth of 300 &#181;m from the spinal surface leads to predominant infection of...

Watch this Scientific Journal Video about Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord at JoVE.com

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Intraspinal injection of recombinase dependent recombinant adeno-associated virus (rAAV) can be used to manipulate any genetically labelled cell type in the spinal cord. Here we describe how to transduce neurons in the dorsal horn of the lumbar spinal cord. This technique enables functional interrogation of the manipulated neuron subtype.

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...

Research

JoVE Journal

Neuroscience

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

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Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

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Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...