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.

<ol>
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E., 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=The+Cellular+and+Synaptic+Architecture+of+the+Mechanosensory+Dorsal+Horn.">The Cellular and Synaptic Architecture of the Mechanosensory Dorsal Horn.</a> Cell. 168 (1-2), 295-310 (2017).</li><li>Wildner, 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=Genome-wide+expression+analysis+of+Ptf1a-+and+Ascl1-deficient+mice+reveals+new+markers+for+distinct+dorsal+horn+interneuron+populations+contributing+to+nociceptive+reflex+plasticity.">Genome-wide expression analysis of Ptf1a- and Ascl1-deficient mice reveals new markers for distinct dorsal horn interneuron populations contributing to nociceptive reflex plasticity.</a> J Neurosci. 33 (17), 7299-7307 (2013).</li><li>Inquimbert, P., Moll, M., Kohno, T., Scholz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotaxic+injection+of+a+viral+vector+for+conditional+gene+manipulation+in+the+mouse+spinal+cord.">Stereotaxic injection of a viral vector for conditional gene manipulation in the mouse spinal cord.</a> J Vis Exp. (73), e50313 (2013).</li><li>Kohro, 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=A+new+minimally-invasive+method+for+microinjection+into+the+mouse+spinal+dorsal+horn.">A new minimally-invasive method for microinjection into the mouse spinal dorsal horn.</a> Sci Rep. 5, 14306 (2015).</li><li>Bourane, S., 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=Gate+control+of+mechanical+itch+by+a+subpopulation+of+spinal+cord+interneurons.">Gate control of mechanical itch by a subpopulation of spinal cord interneurons.</a> Science. 350 (6260), 550-554 (2015).</li><li>Bourane, S., 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=Identification+of+a+spinal+circuit+for+light+touch+and+fine+motor+control.">Identification of a spinal circuit for light touch and fine motor control.</a> Cell. 160 (3), 503-515 (2015).</li><li>Duan, B., 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=Identification+of+spinal+circuits+transmitting+and+gating+mechanical+pain.">Identification of spinal circuits transmitting and gating mechanical pain.</a> Cell. 159 (6), 1417-1432 (2014).</li><li>Gutierrez-Mecinas, 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=Preprotachykinin+A+is+expressed+by+a+distinct+population+of+excitatory+neurons+in+the+mouse+superficial+spinal+dorsal+horn+including+cells+that+respond+to+noxious+and+pruritic+stimuli.">Preprotachykinin A is expressed by a distinct population of excitatory neurons in the mouse superficial spinal dorsal horn including cells that respond to noxious and pruritic stimuli.</a> Pain. 158 (3), 440-456 (2017).</li><li>Awatramani, R., Soriano, P., Rodriguez, C., Mai, J. 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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The authors have nothing to disclose.

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 injection regime allows for testing the paw (von Frey, Hargreaves, etc.) and the thigh (intradermal injections, mechano-receptor stimulation), thus expanding the sensory modalities that can be addressed.
Critical Steps:
There are several steps critical for obtaining robust morphological and behavioral data and for avoiding artefacts. The design of the viral vector and the choice of the promoter and serotype can have a strong impact on transduction efficiency and extent of the transduced area, and therefore on behavioral results (for details on viral spread and transduction efficiencies, see 12). High quality viral preparations are required to minimize side effects of the virus injection, which can occur if contaminants or cell debris are present in the virus solution. The surgery that is required to inject the spinal cord has to be undertaken with great care in order to avoid morphological and behavioral artefacts introduced by the surgery. Particular attention is required in handling the spinal cord, especially during the laminectomy and during the perforation of the dura with the needle. Damage to the spinal cord could impair somatic sensations and motor coordination of the mouse, thus compromising any planned behavioral experiments. In addition, it is important to assemble the syringe carefully. Improper assembly or clogging of the micropipette can hamper the experiment. Spinal tissue at the injection site has to be examined at the end of the experiment in order to verify successful injection and transduction of the injected area and to exclude excessive tissue damage as a result of the surgery. This protocol has used viral titers up to a concentration of 1x1013 GC/mL without apparent toxicity, yet higher titers of virus might become toxic at some time point. In addition, toxicity will most likely also depend on the protein encoded by the injected AAV.
Modifications:
Several parameters in the protocol can be adapted to the neuronal population and research question to be studied. Injection of 300 nL virus solution at a depth of 300 &#181;m leads to transduction of neurons throughout, and predominantly in the dorsal horn. If the aim is to target neurons further ventral, the depth can be adjusted. Injection of larger volumes of virus (we have used up to 500 nL per injection) will lead to a larger spread in dorsoventral and rostrocaudal directions. This may be beneficial to achieve targeting of additional cells in the rostrocaudal extent, but can increase the spillover to the contralateral side, which may hamper the use of the contralateral side as an internal control. In addition, the dorsoventral extent of transduced cells will increase, which may be the desired effect or should be avoided. Injection of 300 nL at a depth of 300 &#181;m avoided side effects on motor control in GlyT2::Cre mice, while injection of 500 nL or injection at a greater depth occasionally produced motor phenotypes, such as spastic extension of the hindlimb (data not shown). Injection of smaller volumes can be used to restrict targeting to even more confined areas5. The viral titer is another parameter that can be modified and should in fact be determined for each virus. For bacterial toxins, such as the highly efficient DTA, low expression levels may be sufficient to induce the desired effect, whereas for fluorescent reporters and DREADDs, higher titers may be necessary for detectable or effective expression.
Significance of the Method with Respect to Existing/Alternative Methods:
To date, mainly two techniques have been used to functionally interrogate the neuronal circuits required for transmission of sensory signals. Many researchers have used intraspinal injections of rAAV coding for reporter and effector proteins into recombinase-expressing mice in order to label and manipulate spinal neuronal subpopulations. Intraspinal injection as a technique to manipulate spinal cells has previously been described15,16. The protocol outlined here was specifically designed to transduce genetically labeled neurons in three consecutive segments of the lumbar spinal cord while minimizing surgical manipulation. This is achieved by placing two of the three injections in the intervertebral space rostral and caudal of the T13 vertebrae and second, by drilling a hole into T13 for the third injection instead of removing the vertebrae. The targeted L3-L5 segments are the main termination area of sensory neurons innervating the hindlimb. We demonstrate that this type of transduction is sufficient to produce robust behavioral changes after the manipulation of glycinergic neurons and suggest that it is suitable for the analysis of a variety of different genetically labeled spinal neurons.
The second technique that has been used in order to manipulate the function of spinal neuron subsets is based on crossing transgenic animals. Here, mice expressing a recombinase in a specific subset of spinal neurons have to be crossed to reporter mice in order to achieve expression of the desired marker or effector protein in the respective neuronal subset17,18,19. To illustrate some of the differences that can occur when comparing results obtained by using reporter mice or intraspinal virus injections we either crossed ROR&#946;Cre knock-in animals to a Cre-dependent reporter mouse or injected ROR&#946;Cre mice with a Cre-dependent reporter rAAV. We compared reporter gene expression obtained from the rAAV to reporter gene expression obtained in ROR&#946;Cre; R26Tom mice. Reporter gene expression obtained from the intraspinal injection of a Cre-dependent rAAV was restricted to neurons of the spinal cord, while R26Tom reporter mouse-evoked tdTomato expression is also found in astrocytes. This suggests that ROR&#946; is transiently expressed in astrocytes. Similarly, Gutierrez-Mecinas et al. found a more restricted reporter expression (spatially and cell-type specific) after viral injection compared to mouse-evoked reporter expression in Tac1Cre mice20. By using intraspinal injections of a rAAV reporter into the spinal cord of adult mice, reporter and/or effector gene expression can efficiently be restricted to the population of cells that express the gene in the adult and thus avoid recombination/expression in those populations with earlier transient gene activity.
A second main advantage of intraspinal rAAV injections is the ability to restrict the expression of the rAAV-encoded genes to the injection site. In transgenic mice, Cre driver-mediated expression is often not only found in the neuronal subpopulation of interest, but also in other populations within the nervous system. Dymecki and colleagues, as well as the groups of Ma and Goulding, have developed elegant ways of using intersectional reporter mouse-based approaches that avoid recombination in parts of the nervous system that shall remain unaltered17,18,19,21,22,23. Using knock-in mice to obtain expression of marker or effector proteins can also be assumed to be less variable, as there is only one genomic copy of the respective expression cassette per cell and the expression cassette is present in all cells in the region of interest. In contrast, the presence of a respective expression cassette and also the copy number of the expression cassette after viral transduction is dependent on the transduction efficiency, which may vary from cell to cell, from injection to injection, and depends on the virus lot. Finally, the main disadvantage of virus-mediated gene expression over traditional genetic methods is the necessity of surgery. Surgery-mediated injury/inflammation may affect neurons and circuits directly. Inclusion of control mice that have undergone the same surgery is therefore mandatory.
Future Applications:
Intraspinal injection can be used to analyze and manipulate any genetically identified spinal subpopulation through overexpression of marker and effector proteins. It is therefore likely in the future that many will adopt this technique to study neuronal populations in their specific focus. Moreover, besides using intraspinal injection of rAAVs to achieve overexpression of exogenous effector proteins, this technique can also be used to silence or overexpress endogenous proteins and therefore to study the function of any given gene with high spatial and temporal resolution.

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 cells in the spinal cord dorsal horn. However, infected cells could also be found in the ventral horn (Figure 1D). Next, the goal was to illustrate the difference in rAAV-mediated expression when injecting a Cre-dependent rAAV into a Cre transgenic mouse. We therefore injected the Cre-dependent AAV1.CAG.flex.eGFP vector into the spinal cord of GlyT2::Cre transgenic mice. As before, eGFP expression was observed in the dorsal and ventral horn. However, as expected, the expression of eGFP became more restricted, reflecting the distribution of GlyT2+ neurons, i.e. the relatively sparse expression in the superficial dorsal horn and dense expression in the deep dorsal horn (Figure 1E).
Next, to demonstrate the feasibility of addressing circuit function of spinal neuronal subpopulations, two different Cre-dependent AAVs coding for different effector proteins (DTA and hM3Dq) were injected into GlyT2::Cre transgenic mice. Analogous to the viral expression observed after three injections of AAV1.CAG.eGFP into wild-type mice, there was robust ablation of inhibitory neurons (Pax2+) in lumbar segments L3-L5 after the injection of AAV1.EF1a.flex.DTA into Glyt2::Cre mice (Figure 2A,C). Loss of glycinergic inhibitory neurons in these segments evoked a marked mechanical hypersensitivity (Figure 2D) and spontaneous aversive behavior directed towards the ipsilateral hindlimb (Figure 2E). The aversive behavior led to self-inflicted lesions, which could be observed on the paws, calf, and thigh (for data, see Foster et al.7) Opposite effects were observed when activating glycinergic neurons through injection of AAV1.hSyn.flex.hM3Dq and subsequent intraperitoneal injection of CNO (Figure 2B). Mice became desensitized to noxious mechanical stimulation (Figure 2F) and other noxious stimuli (see Foster et al.7) In addition, when treated with the pruritogens histamine or chloroquine, hM3Dq-mediated activation of glycinergic neurons efficiently suppressed prurifensive responses (Figure 2G). These results demonstrate that three injections of rAAV into the lumbar spinal cord are able to transduce an area of the lumbar spinal cord sufficient to observe robust behavioral changes evoked by stimulation of the corresponding hindlimb.
In a final set of experiments, we wanted to demonstrate the potential differences in using Cre reporter mice compared to Cre reporter viruses. Therefore, a Cre driver gene was chosen that has previously been described as displaying a restricted expression pattern in the mouse spinal cord. The gene ROR&#946; has been suggested to be expressed predominantly in inhibitory interneurons of the deep dorsal horn13,14. This study used ROR&#946;Cre knock-in mice and crossed them to Rosa26lox-STOP-lox-tdTomato (R26Tom) Cre reporter mice, which lead to the expression of tdTomato in all cells displaying Cre expression at any time point before analysis (Figure 3A). Characterization of tdTomato+ cells in ROR&#946;Cre; R26Tom mice revealed expression in neurons and astrocytes of the dorsal horn (Figure 3B,C). In fact, quantification of the tdTomato+ cells suggested that the majority of cells undergoing Cre-mediated recombination (58%) were non-neuronal. We then injected a Cre-dependent tdTomato reporter rAAV (AAV1.CAG.flex.tdTomato) into the spinal cord of P40 ROR&#946;Cre mice (Figure 3D). In contrast to R26Tom-mediated reporter expression, we found all tdTomato+ cells labeled by the reporter virus to be neurons (Figure 3E,F). Finally, the identity of the tdTomato+ neurons was analyzed in both sets of mice (ROR&#946;Cre; R26Tom and ROR&#946;Cre injected with AAV1.CAG.flex.tdTomato). In both cases, the majority of neurons were inhibitory (&#62;85%) and the minority of neurons&#160;excitatory (&#60;20%) (Figure 3 G-I), which is in agreement with previous assessments of the identity of ROR&#946;+ neurons13,14.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57382/57382fig1.jpg" /> 
Figure 1: Intraspinal Injection of AAV1-eGFP/AAV1-flex-eGFP 
(A) Schematic illustration of an intraspinal injection of an AAV1.CAG.eGFP (AAV1-eGFP) into the lumbar spinal cord, which is innervated from the hindlimb. (B) Anatomical location of the lumbar spinal cord segments L3-L4 can be seen in a top down view of the back of a mouse. The skin was opened to expose the vertebral column. Spinal processes of vertebrae T13-L6 are colored as anatomical references and an arrow indicates the iliac crest. (C) Representative image of a whole mount lumbar spinal cord. Green fluorescence indicates virus-transduced areas of the spinal cord. (D) Representative image of a cross section through an AAV1-eGFP-transduced lumbar spinal cord from a wild-type mouse. (E) Representative image of a cross section of an AAV1.CAG.flex.eGFP-transduced spinal cord of a GlyT2::Cre mouse. Dashed lines represent outline of the gray matter and superficial dorsal horn of the spinal cord. Scale bars C = 1 mm, D = 100 &#181;m. <a href="/files/ftp_upload/57382/57382fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57382/57382fig2.jpg" /> 
Figure 2: Functional Manipulation of Spinal Cre-expressing Glycinergic Neurons 
(A+B) Schematic illustration of an intraspinal injection of an AAV1.EF1a.flex.DTA (A) or an AAV1.EF1a.flex.hM3Dq (B) into the lumbar spinal cord. Cre-dependent expression of diphtheria toxin fragment A (DTA) will lead to ablation of the glycinergic neurons (GlyT2+) (A), while Cre-dependent expression of the pharmacogenetic designer receptor hM3Dq will render GlyT2 neurons activatable by clozapine-N-oxide (CNO) (B). (C) Three injections of AAV1.EF1a.flex.DTA into the L3-L5 segments of GlyT2::Cre mice led to a marked loss of inhibitory neurons in the respective segments of the ipsilateral but not of the contralateral side. (D) Loss of GlyT2 neurons evoked a long-lasting hypersensitivity to mechanical von Frey stimulation in the ipsilateral hind paw of GlyT2::Cre mice injected with AAV1.EF1a.flex.DTA, but no change was observed if Cre-negative mice were injected. (E) Loss of GlyT2 neurons evoked spontaneous aversive behavior reminiscent of chronic itch. (F) hM3Dq-mediated activation of GlyT2 neurons alleviated noxious mechanical pain evoked by pinprick stimulation. (G) hM3Dq-mediated activation of GlyT2 neurons reduced pruritogen (chloroquine or histamine)-evoked aversive behavior. Data are represented as mean &#177; SEM. *** p &#60; 0.001; ** p &#60; 0.01. Scale bar C = 1 mm. g = grams. Images are re-used and modified from Foster et al.7 <a href="/files/ftp_upload/57382/57382fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57382/57382fig3.jpg" /> 
Figure 3: Genetic and Virus-mediated Labeling of ROR&#946;-expressing Cells. (A) Diagram showing the strategy for Cre-dependent expression of fluorescent tdTomato reporter in ROR&#946;Cre; Rosa26lox-STOP-lox-tdTomato (R26Tom) mice. (B) Immunostaining on spinal cord sections of ROR&#946;Cre; R26Tom mice revealed that NeuN+ ROR&#946;-Tom neurons can be found in laminae I-IV.&#160;(C) Cre-dependent expression of tdTomato was also observed in GFAP+ cells of ROR&#946;Cre; R26Tom mice, suggesting that ROR&#946; is expressed in astrocytes during development. Arrowheads indicate double-labeled astrocytes in lamina III.&#160;(D) Diagram showing intraspinal injection of AAV-flex-Tom (rAAV1.CAG.flex.tdTomato) virus into ROR&#946;Cre mice to drive local Cre-dependent expression of tdTomato. (E) Immunostaining on spinal cord sections of ROR&#946;Cre mice injected with AAV-flex-Tom virus. ROR&#946;-Tom neurons were localized to the superficial laminae of the spinal dorsal horn and expression of tdTomato was absent from astrocytes. (F) Percentage of ROR&#946;-Tom cells expressing the neuronal marker NeuN in spinal sections of ROR&#946;Cre; R26Tom mice and ROR&#946;Cre mice injected with AAV-flex-Tom virus. (G-H) Immunostaining on spinal cord sections of (G) ROR&#946;Cre; R26Tom mice and (H) ROR&#946;Cre mice injected with AAV-flex-Tom virus showing colocalization between ROR&#946;-Tom neurons and the inhibitory marker Pax2 or the excitatory marker Lmx1b. (I) Percentage of ROR&#946;-Tom neurons expressing Lmx1b and Pax2 in ROR&#946;Cre; R26Tom mice and ROR&#946;Cre mice injected with AAV-Tom virus. Data are represented as mean &#177; SEM. Data are from 2-3 mice and 1-3 sections per mouse. Scale bars represent 100 &#181;m (B,E) and 20 &#181;m (C,E in high magnification images, G and H). <a href="/files/ftp_upload/57382/57382fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Variable</td>
			<td>Set value</td>
			<td>Unit</td>
		</tr>
		<tr>
			<td>heat&#160; (H)</td>
			<td>450</td>
			<td>- (value proportional to power of the radiated heat)</td>
		</tr>
		<tr>
			<td>force preliminary pull (F(TH))</td>
			<td>20</td>
			<td>- (value proportional to voltage applied to force coil)</td>
		</tr>
		<tr>
			<td>distance threshold (s(TH))</td>
			<td>25</td>
			<td>0.12 mm</td>
		</tr>
		<tr>
			<td>delay heatstop (t(H))</td>
			<td>30</td>
			<td>0.5 ms</td>
		</tr>
		<tr>
			<td>distance heatstop (s(H))</td>
			<td>0</td>
			<td>0.12 mm</td>
		</tr>
		<tr>
			<td>delay pull 1 (t(F1))</td>
			<td>200</td>
			<td>0.5 ms</td>
		</tr>
		<tr>
			<td>force pull 1 (F1)</td>
			<td>300</td>
			<td>- (value proportional to voltage applied to force coil)</td>
		</tr>
		<tr>
			<td>distance pull 2 (s(F2))</td>
			<td>30</td>
			<td>0.12 mm</td>
		</tr>
		<tr>
			<td>force pull 2 (F2)</td>
			<td>600</td>
			<td>- (value proportional to voltage applied to force coil)</td>
		</tr>
		<tr>
			<td>adjust (AD)</td>
			<td>0</td>
			<td>-</td>
		</tr>
	</tbody>
</table>
Table 1: Puller Settings

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 ...

adeno-associated-virus-mediated-transgene-expression-genetically

Nanyang Technological University

Research

JoVE Journal

Neuroscience

17.9K Views.  University of Zurich. 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.

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

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Viral Tracing of Genetically Defined Neural Circuitry

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus (RABV), and more recently vesicular stomatitis virus (VSV), for studying circuitry, is becoming increasingly popular. These higher throughput methods use viruses that transmit between neurons in either the anterograde or retrograde direction.
Recently, a modified RABV for monosynaptic retrograde tracing was developed. (Figure 1A). In this method, the glycoprotein (G) gene is deleted from the viral genome, and resupplied only in targeted neurons. Infection specificity is achieved by substituting a chimeric G, composed of the extracellular domain of the ASLV-A glycoprotein and the cytoplasmic domain of the RABV-G (A/RG), for the normal RABV-G1. This chimeric G specifically infects cells expressing the TVA receptor1. The gene encoding TVA can been delivered by various methods2-8. Following RABV-G infection of a TVA-expressing neuron, the RABV can transmit to other, synaptically connected neurons in a retrograde direction by nature of its own G which was co-delivered with the TVA receptor. This technique labels a relatively large number of inputs (5-10%)2 onto a defined cell type, providing a sampling of all of the inputs onto a defined starter cell type.
We recently modified this technique to use VSV as a transsynaptic tracer9. VSV has several advantages, including the rapidity of gene expression. Here we detail a new viral tracing system using VSV useful for probing microcircuitry with increased resolution. While the original published strategies by Wickersham et al.4 and Beier et al.9 permit labeling of any neurons that project onto initially-infected TVA-expressing-cells, here VSV was engineered to transmit only to TVA-expressing cells (Figure 1B). The virus is first pseudotyped with RABV-G to permit infection of neurons downstream of TVA-expressing neurons. After infecting this first population of cells, the virus released can only infect TVA-expressing cells. Because the transsynaptic viral spread is limited to TVA-expressing cells, presence of absence of connectivity from defined cell types can be explored with high resolution. An experimental flow chart of these experiments is shown in Figure 2. Here we show a model circuit, that of direction-selectivity in the mouse retina. We examine the connectivity of starburst amacrine cells (SACs) to retinal ganglion cells (RGCs).

A method of tracing synaptically connected neurons is described. We use TVA specificity of an upstream cell to probe whether a cell population of interest receives synaptic input from genetically defined cell types.

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

Diffusion Imaging in the Rat Cervical Spinal Cord

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a diagnostic and prognostic tool in cases of disease or injury. Diffusion weighted imaging (DWI), is sensitive to the thermal motion of water molecules and allows for inferences of tissue microstructure. This report describes a protocol to acquire and analyze DWI of the rat cervical spinal cord on a small-bore animal system. It demonstrates an imaging setup for the live anesthetized animal and recommends a DWI acquisition protocol for high-quality imaging, which includes stabilization of the cord and control of respiratory motion. Measurements with diffusion weighting along different directions and magnitudes (b-values) are used. Finally, several mathematical models of the resulting signal are used to derive maps of the diffusion processes within the spinal cord tissue that provide insight into the normal cord and can be used to monitor injury or disease processes noninvasively.

The goal of this protocol is to obtain high-quality diffusion weighted magnetic resonance imaging (DWI) of the rat spinal cord for noninvasive characterization of tissue microstructure. This protocol describes optimizations of the MRI sequence, radiofrequency coil, and analysis methods to enable DWI images free from artifacts.

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a ...

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 organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

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

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

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

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

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 adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

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

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

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

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

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

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Chapters in this video

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Viral Tracing of Genetically Defined Neural Circuitry

Spinal Cord Transection in the Larval Zebrafish

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

Diffusion Imaging in the Rat Cervical Spinal Cord

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

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

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

Laminectomy and Spinal Cord Window Implantation in the Mouse