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• W2547032004 abstract "•A system for optogenetic control of spinal circuits using off-the-shelf components•Bidirectional control of SOM+ neurons indicates primary role in mechanosensation•Temporally sparse SOM+ neuron stimulation reveals role in itch sensation Spinal dorsal horn circuits receive, process, and transmit somatosensory information. To understand how specific components of these circuits contribute to behavior, it is critical to be able to directly modulate their activity in unanesthetized in vivo conditions. Here, we develop experimental tools that enable optogenetic control of spinal circuitry in freely moving mice using commonly available materials. We use these tools to examine mechanosensory processing in the spinal cord and observe that optogenetic activation of somatostatin-positive interneurons facilitates both mechanosensory and itch-related behavior, while reversible chemogenetic inhibition of these neurons suppresses mechanosensation. These results extend recent findings regarding the processing of mechanosensory information in the spinal cord and indicate the potential for activity-induced release of the somatostatin neuropeptide to affect processing of itch. The spinal implant approach we describe here is likely to enable a wide range of studies to elucidate spinal circuits underlying pain, touch, itch, and movement. Spinal dorsal horn circuits receive, process, and transmit somatosensory information. To understand how specific components of these circuits contribute to behavior, it is critical to be able to directly modulate their activity in unanesthetized in vivo conditions. Here, we develop experimental tools that enable optogenetic control of spinal circuitry in freely moving mice using commonly available materials. We use these tools to examine mechanosensory processing in the spinal cord and observe that optogenetic activation of somatostatin-positive interneurons facilitates both mechanosensory and itch-related behavior, while reversible chemogenetic inhibition of these neurons suppresses mechanosensation. These results extend recent findings regarding the processing of mechanosensory information in the spinal cord and indicate the potential for activity-induced release of the somatostatin neuropeptide to affect processing of itch. The spinal implant approach we describe here is likely to enable a wide range of studies to elucidate spinal circuits underlying pain, touch, itch, and movement. A key virtue of the optogenetic approach to the control of neural circuitry has been the ability to directly link neural activation to behavior and, in so doing, test predictions of proposed circuit models. While this approach has been very powerful in the brain (Adamantidis et al., 2015Adamantidis A. Arber S. Bains J.S. Bamberg E. Bonci A. Buzsáki G. Cardin J.A. Costa R.M. Dan Y. Goda Y. et al.Optogenetics: 10 years after ChR2 in neurons—views from the community.Nat. Neurosci. 2015; 18: 1202-1212Crossref PubMed Scopus (87) Google Scholar, Boyden, 2015Boyden E.S. Optogenetics and the future of neuroscience.Nat. Neurosci. 2015; 18: 1200-1201Crossref PubMed Scopus (107) Google Scholar, Deisseroth, 2015Deisseroth K. Optogenetics: 10 years of microbial opsins in neuroscience.Nat. Neurosci. 2015; 18: 1213-1225Crossref PubMed Scopus (734) Google Scholar) and peripheral nervous system (Copits et al., 2016Copits B.A. Pullen M.Y. Gereau 4th, R.W. Spotlight on pain: optogenetic approaches for interrogating somatosensory circuits.Pain. 2016; (Published online October 1, 2016)https://doi.org/10.1097/j.pain.0000000000000620Crossref Scopus (25) Google Scholar, Iyer et al., 2014Iyer S.M. Montgomery K.L. Towne C. Lee S.Y. Ramakrishnan C. Deisseroth K. Delp S.L. Virally mediated optogenetic excitation and inhibition of pain in freely moving nontransgenic mice.Nat. Biotechnol. 2014; 32: 274-278Crossref PubMed Scopus (151) Google Scholar, Montgomery et al., 2016Montgomery K.L. Iyer S.M. Christensen A.J. Deisseroth K. Delp S.L. Beyond the brain: Optogenetic control in the spinal cord and peripheral nervous system.Sci. Transl. Med. 2016; 8: 337rv5Crossref PubMed Scopus (104) Google Scholar), the application of optogenetic control in the mammalian spinal cord has been largely restricted to ex vivo slice preparations (Carr and Zachariou, 2014Carr F.B. Zachariou V. Nociception and pain: lessons from optogenetics.Front. Behav. Neurosci. 2014; 8: 69Crossref PubMed Scopus (39) Google Scholar, Dougherty et al., 2013Dougherty K.J. Zagoraiou L. Satoh D. Rozani I. Doobar S. Arber S. Jessell T.M. Kiehn O. Locomotor rhythm generation linked to the output of spinal shox2 excitatory interneurons.Neuron. 2013; 80: 920-933Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, Foster et al., 2015Foster E. Wildner H. Tudeau L. Haueter S. Ralvenius W.T. Jegen M. Johannssen H. Hösli L. Haenraets K. Ghanem A. et al.Targeted ablation, silencing, and activation establish glycinergic dorsal horn neurons as key components of a spinal gate for pain and itch.Neuron. 2015; 85: 1289-1304Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, Hägglund et al., 2010Hägglund M. Borgius L. Dougherty K.J. Kiehn O. Activation of groups of excitatory neurons in the mammalian spinal cord or hindbrain evokes locomotion.Nat. Neurosci. 2010; 13: 246-252Crossref PubMed Scopus (196) Google Scholar, Hägglund et al., 2013Hägglund M. Dougherty K.J. Borgius L. Itohara S. Iwasato T. Kiehn O. Optogenetic dissection reveals multiple rhythmogenic modules underlying locomotion.Proc. Natl. Acad. Sci. USA. 2013; 110: 11589-11594Crossref PubMed Scopus (130) Google Scholar, Talpalar et al., 2011Talpalar A.E. Endo T. Löw P. Borgius L. Hägglund M. Dougherty K.J. Ryge J. Hnasko T.S. Kiehn O. Identification of minimal neuronal networks involved in flexor-extensor alternation in the mammalian spinal cord.Neuron. 2011; 71: 1071-1084Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, Wang and Zylka, 2009Wang H. Zylka M.J. Mrgprd-expressing polymodal nociceptive neurons innervate most known classes of substantia gelatinosa neurons.J. Neurosci. 2009; 29: 13202-13209Crossref PubMed Scopus (101) Google Scholar, Yang et al., 2015Yang K. Ma R. Wang Q. Jiang P. Li Y.-Q. Optoactivation of parvalbumin neurons in the spinal dorsal horn evokes GABA release that is regulated by presynaptic GABAB receptors.Neurosci. Lett. 2015; 594: 55-59Crossref PubMed Scopus (22) Google Scholar, Zhang et al., 2014Zhang Y. Yue J. Ai M. Ji Z. Liu Z. Cao X. Li L. Channelrhodopsin-2-expressed dorsal root ganglion neurons activates calcium channel currents and increases action potential in spinal cord.Spine. 2014; 39: E865-E869Crossref PubMed Scopus (10) Google Scholar), which do not allow for direct analysis of the behavioral consequences of neural control. One of the most influential circuit models in the spinal cord is the “gate control circuit,” proposed by Melzack and Wall in 1965 (Melzack and Wall, 1965Melzack R. Wall P.D. Pain mechanisms: a new theory.Science. 1965; 150: 971-979Crossref PubMed Scopus (7277) Google Scholar) to explain empirical observations related to acute and chronic pain perception, specifically, the emergence of allodynia in chronic pain, and the dampening of pain sensation by innocuous touch. In this model, light touch fibers preferentially synapse not only on an inhibitory interneuron (a “gate” cell) in the dorsal spinal cord but also on an excitatory projection neuron (known as a “T” cell). Pain fibers synapse only on the T cell, which also receives inhibitory drive from the gate cell. Thus in non-pathological conditions, light touch acts to dampen pain sensation, through activation of the gate cell. This is in contrast to chronic pain conditions, wherein the efficacy of gate cell inhibition is reduced and thus the activation of light touch fibers induces, instead of attenuates, pain. Fifty years after the proposal of this circuit, neurons whose electrophysiological response properties agree with major testable predictions of this model have been identified. Specifically in mechanosensation, somatostatin-positive (SOM+) interneurons, glutamatergic neurons in layer 2/3 of the superficial dorsal horn, have been proposed as the T cell in the Melzack and Wall model (Duan et al., 2014Duan B. Cheng L. Bourane S. Britz O. Padilla C. Garcia-Campmany L. Krashes M. Knowlton W. Velasquez T. Ren X. et al.Identification of spinal circuits transmitting and gating mechanical pain.Cell. 2014; 159: 1417-1432Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). While behavioral responses to ablation of these neurons (i.e., a reduction in mechanical sensitivity and mechanical allodynia) agree with Melzack and Wall’s predictions (Duan et al., 2014Duan B. Cheng L. Bourane S. Britz O. Padilla C. Garcia-Campmany L. Krashes M. Knowlton W. Velasquez T. Ren X. et al.Identification of spinal circuits transmitting and gating mechanical pain.Cell. 2014; 159: 1417-1432Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar), researchers have lacked the tools to test predictions associated with the activation of these neurons in vivo. Here, we developed a method to optogenetically activate dorsal horn neurons in awake, behaving rodents that was compatible with typical pain assays and relied only on off-the-shelf products. Using this system, we find that activation of SOM+ neurons results in strong nocifensive behavior in an array of pain assays, in broad agreement with gate control model predictions. We also report that this neural population plays a role in regulating pruritoception, most likely through activity-dependent release of the neuropeptide somatostatin. While these results indicate that activation of SOM+ neurons is generally consistent with predictions from the gate control model, they also suggest that this population of neurons may play a broader role in the regulation and processing of peripheral somatosensory signals, highlighting the complex and interwoven nature of spinal circuitry. We first developed a surgical implantation procedure to attach a standard fiber optic ferrule (commonly used in the brain) to the thoracic or lumbar spinal column (Figures 1A and S1; see Supplemental Experimental Procedures). Two primary constraints guided procedure development. First, the implant should be secured to only a single vertebral segment to allow for free movement of the spinal column (Figures 1B and 1C). Second, the implant should not penetrate the spinal parenchyma but instead must remain superficial to the cord, due to the relative motion between the cord and its vertebral housing (Figures 1D and 1E). After cannula implantation, mice remained housed in a group and displayed no visible signs of distress or pathology. To assess this, we quantified mouse behavior in affective, motor, and somatosensory tasks and observed no implantation related deficits (Figures 1H–1K and S2). To verify cannula placement, we performed in vivo anesthetized recordings from the spinal cord segment in which we implanted our cannula (lumbar segment 4) and confirmed that dorsal horn neurons in that region had a receptive field on the plantar surface of the ipsilateral hindpaw (Figure S2). As a proxy for implant-induced damage, we stained sections from implanted mice for markers for microglia (Iba1) or astrocytes (GFAP) (Figures 1F and 1G) (Canales et al., 2015Canales A. Jia X. Froriep U.P. Koppes R.A. Tringides C.M. Selvidge J. Lu C. Hou C. Wei L. Fink Y. Anikeeva P. Multifunctional fibers for simultaneous optical, electrical and chemical interrogation of neural circuits in vivo.Nat. Biotechnol. 2015; 33: 277-284Crossref PubMed Scopus (406) Google Scholar). We compared these sections to those from control mice that had received a sham surgery but did not have a cannula implanted, and we observed no difference between sections from the two sets of mice. We performed this characterization ∼14 days after cannula implantation, the time point at which we generally began our behavioral experiments. Of the over 150 cannulations we have performed since developing the procedure, cannulae have become dislodged prior to the sacrifice of the animal in only 6 cases, comparable to the failure rates observed in standard brain cannula implantation procedures. To verify that we could drive behavioral changes by optogenetically activating neurons in the spinal cord with light delivered through our implanted cannula, we expressed ChR2 in a non-specific population of spinal cord neurons through intraparenchymal injection of AAVDJ:CaMKIIa:ChR2-eYFP in the spinal cord. The transduced population included dorsal and ventral horn neurons, such that optical activation caused visible hindlimb contraction and nocifensive behavior (Figure S2; Movie S1). We histologically verified optical activation of neurons through staining for c-Fos (Figure S2). In addition to verifying that light delivered through implanted cannulae could drive behavioral responses, these results also demonstrated potential applications of this technique to studying ventral horn circuits underlying spinal motor control. We performed a Monte Carlo simulation of light propagation (Stujenske et al., 2015Stujenske J.M. Spellman T. Gordon J.A. Modeling the Spatiotemporal Dynamics of Light and Heat Propagation for In Vivo Optogenetics.Cell Rep. 2015; 12: 525-534Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar) to determine the expected depth of functional optogenetic excitation (Figure S2). We observed that sufficiently high levels of light intensity (>0.5 mW/mm2) could be achieved at depths up to 1 mm in the spinal cord with 10 mW of light output from the fiber. Having validated the utility of the spinal cord implant for in vivo neuromodulation, we then turned to the gate control circuit model. If SOM+ interneurons are homologous to T cells, the model (Figure 2A) predicts that their activation would directly engage ascending pain pathways. This would be reflected behaviorally both through an immediate reflexive nocifensive response and through behavioral manifestations of aversion representing the affective dimension of pain (associated negative emotional valence) (Duan et al., 2014Duan B. Cheng L. Bourane S. Britz O. Padilla C. Garcia-Campmany L. Krashes M. Knowlton W. Velasquez T. Ren X. et al.Identification of spinal circuits transmitting and gating mechanical pain.Cell. 2014; 159: 1417-1432Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, Melzack and Wall, 1965Melzack R. Wall P.D. Pain mechanisms: a new theory.Science. 1965; 150: 971-979Crossref PubMed Scopus (7277) Google Scholar). To test this prediction, we injected transgenic mice expressing Cre in somatostatin neurons (SOM-Cre mice) with AAV:Ef1a:DIO:ChR2-eYFP. The extent and distribution of ChR2-eYFP expression was largely as previously described (Duan et al., 2014Duan B. Cheng L. Bourane S. Britz O. Padilla C. Garcia-Campmany L. Krashes M. Knowlton W. Velasquez T. Ren X. et al.Identification of spinal circuits transmitting and gating mechanical pain.Cell. 2014; 159: 1417-1432Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar), with an enrichment of cell bodies dorsal to, but not overlapping with, protein kinase C gamma (PKCγ) neurons (which denote the lamina II/III border (Malmberg et al., 1997Malmberg A.B. Chen C. Tonegawa S. Basbaum A.I. Preserved acute pain and reduced neuropathic pain in mice lacking PKCgamma.Science. 1997; 278: 279-283Crossref PubMed Scopus (585) Google Scholar) and a dense network of axons and dendrites throughout the dorsal horn (Figure 2B). Importantly, we saw no retrograde expression of ChR2-eYFP in DRG neurons (Figure 2B). We verified with slice electrophysiology that blue light illumination of SOM-ChR2 neurons evoked light-induced current and subsequent action potentials at intensities as low as 0.002 mW/mm2 (Figure S3). Light-evoked current increased with increasing light power density, as did the probability of action potential generation. ChR2-expressing neurons faithfully followed light pulse trains with frequencies ranging from 1 to 10 Hz (10 ms pulse width). Consistent with known properties of ChR2, probability of action potential generation decreased at higher frequencies (Figure S2). Next, we optogenetically stimulated SOM-ChR2 interneurons in awake mice. Consistent with model predictions, mice showed an immediate nocifensive response to blue light stimulation (Figure 2C; Movie S2). Mice consistently licked the appropriate dermatomes and, with variation in lumbar implant site, engaged in licking that ranged in location from the ipsilateral thigh to the plantar surface of the ipsilateral hindpaw. We then assessed whether a negative emotional valence was associated with activation of SOM+ neurons by testing whether optogenetic activation was sufficient to generate conditioned place aversion (Figure 2G). We found SOM-ChR2 mice spent significantly less time in the chamber in which light was delivered after a training period, while control mice expressing YFP did not show any preference. We then determined the light level that would elicit a behavioral response from each mouse. This level ranged from 40 μW to 3 mW and was less than 0.5 mW in four out of six tested mice. Higher intensity thresholds are likely due to obstruction of the cannula-spinal cord interface (Figures 2D and S2M). We used each mouse’s individual “threshold” to determine light levels for future behavioral experiments. We tested whether blue light stimulation below this threshold would change the response of the mice to mechanical and thermal stimuli. If SOM+ neurons form the output of a purely mechanosensory gait circuit, then sub-threshold activation of these neurons would decrease mechanical, but not thermal, thresholds. Consistent with these predictions, we found that sub-threshold activation of somatostatin interneurons significantly reduced mechanical thresholds on the von Frey test but did not alter thermal thresholds on the Hargreaves test (Figures 2E and 2F). Previous studies examining SOM+ interneurons have used genetic ablation strategies to characterize the function of these neurons. However, recent reports indicate that the results of transient neural silencing may differ dramatically from the results of permanent ablation (Otchy et al., 2015Otchy T.M. Wolff S.B.E. Rhee J.Y. Pehlevan C. Kawai R. Kempf A. Gobes S.M.H. Ölveczky B.P. Acute off-target effects of neural circuit manipulations.Nature. 2015; 528: 358-363Crossref PubMed Scopus (220) Google Scholar). We therefore tested whether transient inhibition of SOM+ interneuron activity would confirm model predictions, recapitulating aspects of previously observed behavior (Duan et al., 2014Duan B. Cheng L. Bourane S. Britz O. Padilla C. Garcia-Campmany L. Krashes M. Knowlton W. Velasquez T. Ren X. et al.Identification of spinal circuits transmitting and gating mechanical pain.Cell. 2014; 159: 1417-1432Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). Two major strategies have been used to reversibly inhibit neural circuits: optogenetic inhibition (Adamantidis et al., 2015Adamantidis A. Arber S. Bains J.S. Bamberg E. Bonci A. Buzsáki G. Cardin J.A. Costa R.M. Dan Y. Goda Y. et al.Optogenetics: 10 years after ChR2 in neurons—views from the community.Nat. Neurosci. 2015; 18: 1202-1212Crossref PubMed Scopus (87) Google Scholar, Boyden, 2015Boyden E.S. Optogenetics and the future of neuroscience.Nat. Neurosci. 2015; 18: 1200-1201Crossref PubMed Scopus (107) Google Scholar, Deisseroth, 2015Deisseroth K. Optogenetics: 10 years of microbial opsins in neuroscience.Nat. Neurosci. 2015; 18: 1213-1225Crossref PubMed Scopus (734) Google Scholar) and the use of chemogenetic Gi-coupled designer receptors exclusively activated by designer drugs (DREADDs) (Armbruster et al., 2007Armbruster B.N. Li X. Pausch M.H. Herlitze S. Roth B.L. Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand.Proc. Natl. Acad. Sci. USA. 2007; 104: 5163-5168Crossref PubMed Scopus (1276) Google Scholar, English and Roth, 2015English J.G. Roth B.L. Chemogenetics: a transformational and translational platform.JAMA Neurol. 2015; 72: 1361-1366Crossref PubMed Scopus (26) Google Scholar, Iyer et al., 2016Iyer S.M. Vesuna S. Ramakrishnan C. Huynh K. Young S. Berndt A. Lee S.Y. Gorini C.J. Deisseroth K. Delp S.L. Optogenetic and chemogenetic strategies for sustained inhibition of pain.Sci. Rep. 2016; 6: 30570Crossref PubMed Scopus (51) Google Scholar, Urban and Roth, 2015Urban D.J. Roth B.L. DREADDs (designer receptors exclusively activated by designer drugs): chemogenetic tools with therapeutic utility.Annu. Rev. Pharmacol. Toxicol. 2015; 55: 399-417Crossref PubMed Scopus (404) Google Scholar). Here, we adopted a chemogenetic strategy for two reasons: (1) lamina II neurons exhibit large rostro-caudal patterns of activation in response to primary afferent input (Nishida et al., 2014Nishida K. Matsumura S. Taniguchi W. Uta D. Furue H. Ito S. Three-dimensional distribution of sensory stimulation-evoked neuronal activity of spinal dorsal horn neurons analyzed by in vivo calcium imaging.PLoS ONE. 2014; 9: e103321Crossref PubMed Scopus (21) Google Scholar), and we were concerned that the narrow field of illumination provided by an implanted fiber-optic ferrule would not be sufficient to drive behavior; and (2) optogenetic inhibition typically requires high-intensity constant light, which, given the high density of TRPV1 expression in nociceptor terminals in the dorsal spinal cord, poses a significant heating-related activation confound. We injected SOM-Cre mice intraspinally with AAV5:hSyn:DIO:hM4D(Gi)-mCherry and assessed nociception with mechanical and thermal assays. hM4D(Gi)-mCherry expression was consistent with previous results (Figures 2H and S4A). If SOM+ neurons are the T cells in the gate control model, then they should relay input from Aδ and C-HTMR (high-threshold mechanoreceptors) primary afferents to ascending pain pathways. Therefore, chemogenetic inhibition of these neurons would be predicted to transiently increase mechanical withdrawal thresholds. Gate control also predicts that in naive animals, input from light-touch afferents (Aβ-, Aδ- and C-LTMRs) should not be sufficient to drive activation of T cells, and therefore, chemogenetic inhibition of SOM+ cells should not have any effect on light touch. However, in mice exhibiting mechanical allodynia, input from light-touch afferents is relayed through T cells, and therefore, chemogenetic inhibition of SOM+ cells should meaningfully reduce mechanical hypersensitivity. Our behavioral results were consistent with these predictions. In naive SOM-hM4Di mice, we observed that following intraperitoneal injections of clozapine-N-oxide (CNO), but not saline, mice showed a significant increase in mechanical withdrawal thresholds, as well as a slight increase in heat withdrawal latency, but no reduction in sensitivity to a measure of light touch (cotton swab assay; Figures 2I–2K). To examine the effects of chemogenetic inhibition on mechanical allodynia, we injected the paws of the animals with complete Freund’s adjuvant (CFA), a pro-inflammatory agent. As expected, CFA injection caused dynamic mechanical allodynia (Figure 2K). Consistent with model predictions, intraperitoneal injection of CNO now significantly reduced allodynia, restoring light-touch sensitivity to baseline pre-CFA levels (Figure 2K). In addition, mice expressing hM4Di displayed no differences in measures of locomotion following CNO administration, as compared with following saline injection, suggesting these results are not due to motor-related confounds (Figure S4B). In recent work, it has been demonstrated that intrathecal administration of the somatostatin analog octreotide results in strong scratching behavior that can be eliminated by ablation of Bhlhb5 neurons, which are a subpopulation of neurons that express the 2A isoform of the somatostatin receptor (SST2AR) (Kardon et al., 2014Kardon A.P. Polgár E. Hachisuka J. Snyder L.M. Cameron D. Savage S. Cai X. Karnup S. Fan C.R. Hemenway G.M. et al.Dynorphin acts as a neuromodulator to inhibit itch in the dorsal horn of the spinal cord.Neuron. 2014; 82: 573-586Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). When we immunostained spinal cord sections from mice expressing ChR2 in SOM+ neurons for SST2AR, we noticed significant spatial overlap between regions of strong ChR2-eYFP expression and regions of strong SST2AR expression (Figure 3A). Thus, we were curious if, in addition to their role in gate control, SOM+ neurons may contribute to regulation of itch through activity-dependent release of somatostatin. We designed an experiment to test this hypothesis. We injected histamine intradermally into the thigh of SOM+ mice expressing either ChR2 or mCherry and concurrently performed intrathecal (IT) injections of either saline or the SST2R antagonist CYN-154806. We then optogenetically stimulated these mice with a temporally sparse 1-Hz, 100-ms pulse train, titrating light intensity on a mouse-by-mouse basis to minimize stimulation evoked paw-licking behavior, and we then video recorded histamine-evoked thigh-biting behavior. We observed that in SOM-ChR2 mice, but not in SOM-mCherry controls, optogenetic stimulation paired with IT saline resulted in high rates of histamine-evoked thigh biting, as compared to optogenetic stimulation paired with IT CYN-154806 (Figure 3B). In contrast, when temporally sparse optogenetic stimulation and IT saline/CYN-154806 were paired with measures of mechanical and thermal sensitivity, no significant differences were observed upon optogenetic stimulation in either IT saline or IT CYN-154806 conditions (Figures 3C and 3D). A critical prediction of gate theory is that activation of T cells results in recruitment of ascending pain pathways. Although previous work (Duan et al., 2014Duan B. Cheng L. Bourane S. Britz O. Padilla C. Garcia-Campmany L. Krashes M. Knowlton W. Velasquez T. Ren X. et al.Identification of spinal circuits transmitting and gating mechanical pain.Cell. 2014; 159: 1417-1432Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar) suggested that SOM+ interneurons relay mechanosensory information to neurons in laminae I and II, the genetic identity of these neurons, and whether they project to supraspinal centers, was unknown. We assayed downstream activity induced by optogenetic activation of SOM+ neurons through c-Fos immunohistochemistry. We found c-Fos+ neurons in both superficial laminae and deep dorsal horn laminae, including neurons that express NK1R, a subset of which are known to relay pain information to supraspinal centers (Todd, 2002Todd A.J. Anatomy of primary afferents and projection neurones in the rat spinal dorsal horn with particular emphasis on substance P and the neurokinin 1 receptor.Exp. Physiol. 2002; 87: 245-249Crossref PubMed Scopus (133) Google Scholar; Figures 4A–4H). In the past decade, researchers have begun to employ increasingly sophisticated in vivo genetic tools to piece together subsets of spinal cord sensory circuits responsible for processing external stimuli. These tools have enabled the investigation of behavioral responses to ablation of subsets of dorsal horn neurons either during development (Duan et al., 2014Duan B. Cheng L. Bourane S. Britz O. Padilla C. Garcia-Campmany L. Krashes M. Knowlton W. Velasquez T. Ren X. et al.Identification of spinal circuits transmitting and gating mechanical pain.Cell. 2014; 159: 1417-1432Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar) or in adulthood (Foster et al., 2015Foster E. Wildner H. Tudeau L. Haueter S. Ralvenius W.T. Jegen M. Johannssen H. Hösli L. Haenraets K. Ghanem A. et al.Targeted ablation, silencing, and activation establish glycinergic dorsal horn neurons as key components of a spinal gate for pain and itch.Neuron. 2015; 85: 1289-1304Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). However, these approaches are not without limitations. Genetic knockout strategies can introduce interpretational confounds due to the absence of knocked out genes during development, while cellular ablation is permanent and may differ in its effects from transient silencing (both due to the potentially toxic byproducts of cellular ablation and recently characterized differences between the effects of transient and chronic neural silencing; Otchy et al., 2015Otchy T.M. Wolff S.B.E. Rhee J.Y. Pehlevan C. Kawai R. Kempf A. Gobes S.M.H. Ölveczky B.P. Acute off-target effects of neural circuit manipulations.Nature. 2015; 528: 358-363Crossref PubMed Scopus (220) Google Scholar). Here, we have described how standard optogenetic tools can be co-opted for use in the spinal cord, enabling direct selective control of spinal circuits in freely moving mice. The implantation strategy we describe does not impede locomotion, alter baseline responses to measures of somatosensory sensitivity, or induce significant anxiety. The optogenetic tools we use in this work are currently available for ∼$20 per implant and require no additional fabrication or construction after purchase. They therefore may enable a substantial cost and time savings for the large fraction of experiments in which wirelessly powered implants (Ho et al., 2015Ho J.S. Tanabe Y. Iyer S.M. Christensen A.J. Grosenick L. Deisseroth K. Delp S.L. Poon A.S.Y. Self-tracking energy transfer for neural stimulation in untethered mice.Phys. Rev. Appl. 2015; 4: 024001Crossref Scopus (30) Google Scholar, Montgomery et al., 2015Montgomery K.L. Yeh A.J. Ho J.S. Tsao V. Mohan Iyer S. Grosenick L. Ferenczi E.A. Tanabe Y. Deisseroth K. Delp S.L. Poon A.S.Y. Wirelessly powered, fully internal optogenetics for brain," @default.
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• W2547032004 date "2016-11-01" @default.
• W2547032004 modified "2023-10-11" @default.
• W2547032004 title "In Vivo Interrogation of Spinal Mechanosensory Circuits" @default.
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