Regulating nociceptive transmission by VGluT2-expressing spinal dorsal horn neurons
Vesicular glutamate transporter-2 (VGluT2) mediates the uptake of glutamate into synaptic vesicles in neurons. Spinal cord dorsal horn interneurons are highly heterogeneous and molecularly diverse. The functional significance of VGluT2-expressing dorsal horn neurons in physiological and pathological pain conditions has not been explicitly demonstrated. Designer receptors exclusively activated by designer drugs (DREADDs) are a powerful chemogenetic tool to reversibly control neuronal excitability and behavior. Here we used transgenic mice with Cre recombinase expression driven by the VGluT2 promoter, combined with the chemogenetic approach, to determine the contribution of VGluT2-expressing dorsal horn neurons to nociceptive regulation. Adeno-associated viral vectors expressing double-floxed Cre-dependent G\xce\xb1q-coupled human M3 muscarinic receptor DREADD (hM3D)-mCherry or G\xce\xb1i-coupled \xce\xba-opioid receptor DREADD (KORD)-IRES-mCitrine were microinjected into the superficial spinal dorsal horn of VGluT2-Cre mice. Immunofluorescence labeling showed that VGluT2 was predominantly expressed in lamina II excitatory interneurons. Activation of excitatory hM3D in VGluT2-expressing neurons with clozapine N-oxide caused a profound increase in neuronal firing and synaptic glutamate release. Conversely, activation of inhibitory KORD in VGluT2-expressing neurons with salvinorin B markedly inhibited neuronal activity and synaptic glutamate release. In addition, chemogenetic stimulation of VGluT2-expressing neurons increased mechanical and thermal sensitivities in naive mice, whereas chemogenetic silencing of VGluT2-expressing neurons reversed pain hypersensitivity induced by tissue inflammation and peripheral nerve injury. These findings indicate that VGluT2-expressing excitatory neurons play a crucial role in mediating nociceptive transmission in the spinal dorsal horn. Targeting glutamatergic dorsal horn neurons with inhibitory DREADDs may be a new strategy for treating inflammatory and neuropathic pain.
Introduction<!>Animal models and drug administration<!>Behavioral tests<!>Viral vector microinjection<!>Immunohistochemistry<!>Spinal cord slice preparation and electrophysiological recordings<!>Data analysis<!>VGluT2-Expressing Neurons Are Predominantly Present in the Spinal Superficial Dorsal Horn<!>Distribution of hM3D-mCherry Dorsal Horn Neurons in VGluT2-Cre Mice<!>Activation of hM3D Potentiates the Firing Activity and Synaptic Glutamate Release of VGluT2\xe2\x80\x93Expressing Dorsal Horn Neurons<!>Chemogenetic Stimulation of VGluT2-Expressing Neurons in the Spinal Dorsal Horn Elicits Pain Hypersensitivity<!>Distribution of KORD-IRES-mCitrine Dorsal Horn Neurons in VGluT2-Cre Mice<!>Activation of KORD Inhibits the Firing Activity and Synaptic Glutamate Release of VGluT2-Expressing Dorsal Horn Neurons<!>Chemogenetic Silencing of VGluT2-Expressing Dorsal Horn Neurons Alleviates Neuropathic and Inflammatory Pain<!>Discussion
<p>Sensory stimuli such as pain, temperature, itch, and touch are transmitted and processed in the spinal cord dorsal horn. Glutamate is the major excitatory neurotransmitter in the spinal dorsal horn (Santos et al. 2007, Pan & Pan 2004, Yoshimura & Jessell 1990), and blocking glutamate receptors at the spinal cord level can effectively reduce pain induced by tissue and nerve injury (Chen et al. 2013, Chaplan et al. 1997). Glutamate transporters in the plasma membrane and in the membrane of synaptic vesicles maintain the homeostasis of the glutamatergic system. Glutamate is packaged into synaptic vesicles by the three vesicular glutamate transporter (VGluT) proteins, VGluT1, VGluT2, and VGluT3 (Brumovsky et al. 2011, Oliveira et al. 2003, Moechars et al. 2006). Each VGluT is localized within the nervous system, with only limited overlap. VGluT2 is expressed in both dorsal root ganglion (DRG) and spinal dorsal horn neurons (Brumovsky et al. 2007, Oliveira et al. 2003). In DRG neurons, VGluT2 is required for both acute and persistent pain (Rogoz et al. 2012, Scherrer et al. 2010). Disruption of one Vglut2 allele in mice does not affect the acute pain responses but reduces hypersensitivity after nerve injury (Moechars et al. 2006). The second-order neurons in the superficial dorsal horn primarily receive and modulate nociceptive information from primary afferent nerves (Chen et al. 2014a, Todd 2010, Pan & Pan 2004, Cervero & Iggo 1980). How discrete excitatory and inhibitory neuronal populations in the spinal dorsal horn participate in nociceptive integration is not fully known. Although excitatory interneurons in the spinal dorsal horn are generally considered to be important for nociception, the role of VGluT2-expressing spinal dorsal horn neurons in nociceptive transmission has not been demonstrated specifically.</p><p>Neurons in the spinal dorsal horn are functionally heterogeneous and molecularly diverse. On the basis of their physiological functions, these neurons are broadly divided into two classes: excitatory (glutamatergic) and inhibitory (GABAergic and/or glycinergic) neurons. At present, VGluT2 is the most commonly used molecular marker for excitatory glutamatergic neurons, although subpopulations of excitatory interneurons in the spinal dorsal horn may express cholescystokinin, somatostatin, neurotensin, calbindin-2/calretinin, TRPV1, and/or preprototachykinin 2 (Duan et al. 2014, Zhou et al. 2009, Antal et al. 1991, Huang et al. 2008, Smith et al. 2015, Xu et al. 2008). VGluT2 is abundantly expressed in the spinal dorsal horn, suggesting that VGluT2 expression levels depend on local sources (Alvarez et al. 2004, Brumovsky et al. 2007, Oliveira et al. 2003). Although lamina II interneurons in spinal cord slices are extensively used for recordings in studies of nociceptive transmission and synaptic plasticity in chronic pain (Li et al. 2002, Pan & Pan 2004, Santos et al. 2007, Yoshimura & Jessell 1990), whether lamina II interneurons play unique roles in physiological and pathological pain remains unclear.</p><p>The use of designer receptors exclusively activated by designer drugs (DREADDs) is a powerful chemogenetic strategy that slows researchers to remotely and noninvasively control neuronal signaling (Urban & Roth 2015, Whissell et al. 2016). DREADDs are modified G protein-coupled receptors typically introduced into cells using viral vectors. Because DREADDs can selectively modulate cellular activity upon application of selective ligands, they can be used to stimulate or inhibit specific populations of neurons (Alexander et al. 2009, Vardy et al. 2015). In the present study, we used transgenic mice with Cre recombinase expression driven by the VGluT2 promoter, combined with viral vectors to express DREADDs, to specifically determine the contribution of VGluT2-expressing dorsal horn neurons to nociceptive regulation. Our study provides substantial new evidence about the functional role of VGluT2-expressing spinal dorsal horn neurons in processing nociceptive information.</p><!><p>All of the surgical and experimental protocols were approved by the Animal Care and Use Committee of The University of Texas MD Anderson Cancer Center and conformed to the National Institutes of Health guidelines for the ethical use of animals (approval #1186-RN01). This study was not pre-registered. A total of 128 mice was used for the entire study, and 14 mice were excluded from data analysis due to incorrect injection sites. VGluT2-IRES-Cre knock-in mice (stock #028863, genetic background: C57BL/6J; RRID: IMSR_JAX:028863) and tdTomatoflox/flox mice (stock #007909; genetic background: C57BL/6J; RRID: IMSR_JAX:007909) were obtained from The Jackson Laboratory (Bar Harbor, ME). The VGluT2-Cre:tdTomatoflox/flox mice were generated by crossing male VGluT2-Cre mice with female tdTomatoflox/flox mice. Adult mice (9–11 weeks of age, males and females, sex and age matched) were used for behavioral and electrophysiological experiments. The animals were housed with 3 mice per cage and had free access to food and water. All tests and assays were done between 9 am and 6 pm.</p><p>Inflammatory pain was induced by injection of complete Freund's adjuvant (CFA) into the hindpaw. In brief, VGluT2-Cre mice were anesthetized using 2% isoflurane, and 20 μl of CFA (Sigma-Aldrich, St. Louis, MO) was injected subcutaneously into the plantar surface of the left hindpaw. Spared nerve injury (SNI) was used as an experimental model of neuropathic pain, as described previously (Laedermann et al. 2014). Briefly, mice were anesthetized with 2% isoflurane, and a small incision was made on the left lateral thigh to expose the sciatic nerve. The common peroneal and tibial nerves were ligated with a 5–0 silk suture under a surgical microscope, and the sural nerve was left intact. To minimize animal's suffering, 1% lidocaine was injected around the incision site during surgery.</p><p>Clozapine N-oxide (CNO) was purchased from Tocris Bioscience (#4936, Minneapolis, MN) or AK Scientific (#AMTA056, Union City, CA), and Salvinorin B (SALB) was obtained from Cayman Chemical (#92545–30-7, Ann Arbor, MI, USA). Both CNO and SALB were initially dissolved in saline and DMSO, respectively, and the effective doses of CNO (1 mg/kg) and SALB (10 mg/kg) have been showed in previous studies (Alexander et al. 2009, Vardy et al. 2015, Marchant et al. 2016).</p><!><p>The tactile threshold was quantified as previously described (Zhang et al. 2015). After mice were placed in plastic boxes on a mesh floor and allowed to acclimate for 30 min, a series of calibrated von Frey filaments (0.02 – 1.4 g) was applied to the plantar surface of the hindpaw with sufficient force to bend the filament for 6 s. Brisk withdrawal or paw flinching was considered to be a positive response. If there was no response, the filament of the next greater force was applied. When a response was observed, the filament of the next lower force was applied. Each trial was repeated 2 or 3 times at approximately 2-min intervals, and the mean value of each the trials considered the force needed to produce a withdrawal response. We used the "up-down" method (Chaplan et al. 1997) to determine the tactile stimulus force that produced a 50% likelihood of a withdrawal response.</p><p>For the thermal sensitivity assessments, mice were placed on the glass surface of a thermal testing apparatus (IITC Life Sciences, Woodland Hills, CA) and allowed to acclimate for 30 min. The temperature of the glass surface was maintained at a constant 30 °C. A mobile radiant heat source was focused onto the hindpaw of each mouse from under the glass. The paw withdrawal latency was recorded with a timer when the mouse abruptly moved the hindpaw away from the heat source. Each hindpaw was tested twice to obtain an average latency time. A maximum heat exposure time of 20 s was used to prevent tissue damage (Li et al. 2016).</p><!><p>We used 2 viral constructs, AAV8-hSyn-DIO-hM3Dq-mCherry and AAV8-hSyn-DIO-HA-KORD-IRES-mCitrine (#44361 and #65417, titer ≥ 4×1012 vg/mL; University of North Carolina Vector Core, Chapel Hill, NC and Addgene, Cambridge, MA), to induce Cre-dependent expression of hM3D-mCherry and KORD-IRES-mCitrine in spinal dorsal horn neurons of VGluT2-Cre mice. The viral vector microinjection was performed as in previous studies (Inquimbert et al. 2013, Kohro et al. 2015). Briefly, the mice were anesthetized with 2% isoflurane inhalation, and the lumbar spinal cord was exposed via hemilaminectomy. The viral solution was injected using a glass micropipette connected to a microinjector (Drummond Scientific Company, Broomall, PA) mounted on a stereotactic apparatus. Five injections of 100 nl each were administered into the left spinal cord dorsal horn at the L4 to L6 levels at a rate of 30 nl/min. After vector injection, the mice were allowed to fully recover in a warm cage and then returned to the housing facility. The spinal cords of mice used for final behavioral tests were inspected using a fluorescence microscope for the correct viral vector injection in the spinal dorsal horn.</p><!><p>Mice were deeply anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and then transcardially perfused with 4% paraformaldehyde in 0.1 M PBS. The spinal cord was dissected, postfixed for 2 h with the same fixative followed sequentially by 10%, 20%, and 30% sucrose solutions in PBS, and frozen in Tissue-Tek optimal cutting temperature compound. Twenty μm transverse sections were cut, collected onto Superfrost Plus glass slides, and air-dried for 1 h at room temperature. The sections were then washed with PBS, incubated for 20 min at room temperature in a blocking solution containing 1% BSA in PBS, incubated with the primary antibodies overnight at 4°C, and incubated with the corresponding Alexa 488- or Alexa 568-conjugated secondary antibodies (1:200, Invitrogen, Carlsbad, CA) for 1 h at room temperature. The antibodies used in this study were rabbit anti-GFP (1:300; #NB600–308, Novus Biologicals, Littleton, CO; RRID: AB_10003058), rabbit anti-Pax2 (1:200; #71–6000, Thermo Fisher Scientific.; RRID: AB_2533990), rabbit anti-mCherry (1:100; #ab167453, Abcam, Cambridge, MA; RRID: 2571870), and mouse anti-NeuN (1:200; #ab104224, Abcam; RRID: AB_10711040). Images were acquired with a confocal laser-scanning microscope (Zeiss, Jena, Germany). Quantification of colocalization was performed on 3 randomly selected sections from each mouse using the NIH ImageJ Cell Counter Plugin.</p><!><p>The lumbar spinal cords were quickly removed from isoflurane-anesthetized mice via laminectomy. We sliced the spinal cords into 400-μm-thick slices using a vibratome and continually superfused the slices with artificial cerebrospinal fluid containing (in mM) 117 NaCl, 3.6 KCl, 1.2 MgCl2, 2.5 CaCl2, 1.2 NaHPO4, 11 glucose, and 25 NaHCO3 (mixed with 95% O2 and 5% CO2). The slices were preincubated in the artificial cerebrospinal fluid solution at 34 °C for at least 1 h before recording.</p><p>Electrophysiological recordings were performed as described previously (Chen et al. 2014a, Li et al. 2002). A spinal cord slice was placed in a glass-bottomed chamber (Warner Instruments, Hamden, CT) and fixed with parallel nylon threads supported by a U-shaped stainless steel weight. The fluorescence-labeled neurons in lamina II were identified using a combination of epifluorescence illumination and infrared and differential interference contrast optics on an upright microscope (Olympus, Tokyo, Japan). The electrode for the whole-cell recordings was pulled from borosilicate glass capillaries with a micropipette puller (P-97; Sutter Instrument Co, Novato, CA). Perforated recordings were used to measure the firing activity of mCherry-labeled and mCitrine-labeled lamina II neurons (Li et al. 2016, Zhou et al. 2012). The perforated approach was used to minimize interference with the intracellular content (Chen et al. 2017). The internal solution contained (in mM) 130.0 potassium acetate, 15.0 KCl, 5.0 NaCl, 1.0 MgCl2, and 10.0 HEPES (pH adjusted to 7.2 with KOH to 296 mOsm). Gramicidin was freshly dissolved in DMSO and then diluted to a final concentration of 50 μg/mL using the pipette solution. Recording of the firing activity of lamina II neurons began when the firing activity had reached a steady state for 5 to 10 min.</p><p>We used conventional whole-cell voltage-clamp techniques to record spontaneous excitatory synaptic currents (sEPSCs) (Pan & Pan 2004, Zhou et al. 2009). The sEPSCs of mCherry-labeled and mCitrine-labeled lamina II neurons were recorded at a holding potential of –60 mV. The impedance of the glass electrode was 4 to 7 MΩ when the pipette was filled with the internal solution containing (in mM) 135 potassium gluconate, 5 KCl, 2.0 MgCl2, 0.5 CaCl2, 5.0 HEPES, 5.0 EGTA, 5.0 ATP-Mg, 0.5 Na-GTP, and 10 QX314 (280‒300 mOsm, pH: 7.25). The signals were processed using an amplifier (Molecular Devices, Foster City, CA), filtered at 1 to 2 kHz, digitized at 20 kHz using DigiData 1320A (Molecular Devices), and saved to the hard drive of a computer. At least 3 animals were used for each recording protocol, and only 1 neuron was recorded from each spinal cord slice.</p><p>All drugs were freshly prepared before the recording and delivered at their final concentrations using syringe pumps. The effective concentrations of CNO (30 μM) and SALB (100 nM) have been demonstrated previously (Inutsuka et al. 2014, Vardy et al. 2015, Marchant et al. 2016).</p><!><p>Data were expressed as means ± SEM. No statistical methods were used to predetermine sample sizes for the studies, but our sample sizes were similar to those generally employed in the field. The animals were assigned (1:1 allocation) to control and treatment groups, although a randomization was not used. The investigator performing the behavioral assessment was blinded to the experimental treatments. For proper exclusion of data points, the criteria were established before data collection. Mice with neurological deficits (e.g., motor weakness) after spinal cord microinjection were excluded from data analysis. In electrophysiological recording experiments, only one neuron was recorded in each spinal cord slice, and at least three mice were used in each group. We monitored cell capacitance, input resistance, and baseline holding current; the recording was discontinued if these parameters changed >15%. The firing activity and membrane potentials of each neuron were analyzed over a period of 3 to 5 min before, during, and 10 to 20 min after drug application. The firing rate and the amplitude and frequency of sEPSCs were analyzed offline using a peak detection program (MiniAnalysis; Synaptosoft, Leonia, NJ). Events were detected by setting a threshold above the noise level. Kolmogorov-Smirnov tests were used to compare the cumulative probability of the amplitude and inter-event interval of sEPSCs. Two-tailed Student's t tests were used to compare 2 groups, and one-way analysis of variance (ANOVA) followed by Dunnett's or Tukey's post hoc test was used to determine the differences between more than two groups. All statistical analyses were performed using Prism software (version 7, GraphPad Software Inc., La Jolla, CA). P values of less than 0.05 was considered to be statistically significant.</p><!><p>To determine the distribution of VGluT2-expressing neurons in the spinal dorsal horn, we crossed VGluT2-Cre mice (Vong et al. 2011) with tdTomatoflox/flox mice (Madisen et al. 2010) to obtain VGluT2:tdTomato mice (Fig. 1, A and B). In lumbar spinal cord sections of 3 VGluT2:tdTomato mice, immunofluorescence labeling was performed using an antibody against NeuN, a pan-neuronal marker, or an antibody against Pax2, a robust marker of inhibitory neurons in the mouse spinal dorsal horn (Punnakkal et al. 2014). Confocal microscopy images showed that tdTomato-expressing neurons were most abundant in the superficial dorsal horn, particularly in lamina II (Fig. 1C). The percentage of tdTomato-expressing neurons in the spinal cord was: lamina I, 6.21 ± 3.22%; lamina II, 57.10 ± 4.93%; lamina III, 18.30 ± 1.92%; lamina IV, 7.19 ± 1.33%; lamina V, 6.82 ± 2.06%; ventral horn, 4.42 ± 1.02%. Notably, tdTomato-expressing neurons were not co-labeled with Pax2 in the spinal dorsal horn (Fig. 1D).</p><!><p>To express the Gαq-coupled human M3 muscarinic receptor DREADD (hM3D) specifically in VGluT2 neurons in the spinal dorsal horn, we microinjected an adeno-associated viral (AAV) construct, hSyn-DIO-hM3Dq-mCherry (Fig. 2A), into the left superficial dorsal horn of VGluT2-Cre mice. This viral vector carries a FLEX system to enable Cre-dependent expression, ensuring cell-specific expression of hM3D in VGluT2-expressing neurons (Alexander et al. 2009, Urban & Roth 2015). Two weeks after the vector injection, we performed immunofluorescent labeling using anti-mCherry and anti-NeuN antibodies to examine the distribution of mCherry-labeled neurons in lumbar spinal cord sections from 3 mice. The vector injection was localized, and the mCherry expression was largely restricted to the superficial dorsal horn on the ipsilateral side (Supplementary Fig. S1A). mCherry-labeled neurons were largely confined to the spinal lamina II on the injected side (Fig. 2, B and C). In the spinal lamina II, 58.77 ± 6.03% of the NeuN-positive cells coexpressed mCherry. In contrast, no mCherry-labeled neurons were found in the spinal dorsal horn on the contralateral side.</p><!><p>Next, we performed electrophysiological recording of mCherry-labeled neurons in spinal cord slices from VGluT2-Cre mice that had undergone injection of AAV-hM3D-mCherry into the superficial dorsal horn. We used perforated whole-cell recording to test the effect of clozapine-N-oxide (CNO), a chemical ligand for muscarinic receptor DREADDs (Urban & Roth 2015, Alexander et al. 2009), on the excitability of mCherry-labeled lamina II neurons. As expected from the M3-mediated excitatory effect (Zhang et al. 2006, Zhu et al. 2016), bath application of 30 μM CNO for 3 min rapidly depolarized the membrane potential (Δ8.34 ± 2.01 mV) of all mCherry-labeled lamina II neurons and caused a large increase in the firing rate (from 0.54 ± 0.35 Hz to 3.13 ± 0.87 Hz, n = 15 neurons from 5 mice, P = 0.0027, F(1.46,20.47) = 9.44; Fig. 3, A and B). The excitatory effects of CNO were gradually reversed 20 min after washout of CNO. CNO had no effect on the firing activity of lamina II neurons in hM3D-free control mice (from 0.57 ± 0.38 Hz to 0.55 ± 0.33 Hz, n = 12 neurons from 4 mice; P > 0.05).</p><p>Furthermore, we sought to determine whether hM3D activation can stimulate synaptic glutamate release in VGluT2-expressing neurons in spinal cord slices. We used whole-cell voltage-clamp techniques to record spontaneous excitatory postsynaptic currents (sEPSCs), a measure of synaptic glutamate release (Pan & Pan 2004, Zhou et al. 2009). Bath application of 30 μM CNO significantly increased the frequency (P = 0.042, F(1.43,19.99) = 4.16), but not the amplitude, of sEPSCs in all mCherry-labeled neurons (n = 14 neurons from 4 mice; P < 0.05, Fig. 3, C-E). These data indicate that VGluT2-expressing neurons in the spinal dorsal horn are glutamate-releasing excitatory neurons.</p><!><p>Having established that activation of hM3D with CNO stimulates VGluT2-expressing glutamatergic dorsal horn neurons, we next determined how hM3D activation with CNO affects nociception in vivo. Because intrathecally injected viral vectors can transduce both DRG and spinal cord neurons (Li et al. 2016), we microinjected the AAV vector expressing Cre-dependent hM3D into the left spinal dorsal horn of VGluT2-Cre mice to specifically target VGluT2-expressing dorsal horn neurons. We measured the mechanical and thermal sensitivity of both hindpaws in response to von Frey filaments and a radiant heat stimulus 2 to 3 weeks after vector microinjection. Systemic administration of CNO (1 mg/kg, i.p.) (Alexander et al. 2009, Vardy et al. 2015) significantly reduced the mechanical (P = 0.0002, F(4,30) = 7.91) and thermal (P < 0.0001, F(4,30) = 10.14) withdrawal thresholds of the ipsilateral hindpaw (Fig. 4, A and B). CNO injection also elicited spontaneous flinching and licking of the hindpaw on the injected side (Supplementary Video S1). These pronociceptive effects were observed within 30 min after CNO injection and lasted for about 90 min.</p><p>Although it has been reported that systemically administered CNO may produce off-target effects through its metabolites (Gomez et al. 2017, MacLaren et al. 2016), we found that systemic treatment with CNO (1 mg/kg, i.p.) had no effect on the mechanical or thermal withdrawal thresholds of the contralateral hindpaw in VGluT2-Cre mice (Fig. 4, A and B). In additional VGluT2-Cre mice that received no viral vector injection, we tested whether CNO alone alters pain hypersensitivity induced by spared nerve injury (SNI) or by injection of complete Freund's adjuvant (CFA) into the hindpaw. At 7 to 10 days after SNI and 3 to 5 days after CFA injection, systemic injection of CNO had no significant effect on the mechanical (Fig. 4, C and E) or thermal withdrawal thresholds (Fig. 4, D and F) in these mice. Collectively, these results indicate that VGluT2-expressing glutamatergic neurons are important for nociceptive transmission in the spinal dorsal horn.</p><!><p>Our results indicating that VGluT2-expressing dorsal horn neurons play a critical role in physiological pain prompted us to determine whether inhibition of this population of dorsal horn neurons would alleviate pathological pain, such as nerve injury-induced neuropathic pain and CFA-induced inflammatory pain. The inhibitory DREADD, KORD, is based on the κ-opioid receptor and can be specifically activated by the endogenously inactive compound salvinorin B (SALB) (Vardy et al. 2015). Thus, SALB can selectively inhibit neurons that express KORD by stimulating Gαi-mediated signaling. We microinjected VGluT2-Cre mice with AAV-hSyn-DIO-HA-KORD-IRES-mCitrine (Fig. 5, A and B) in the left dorsal horn at the L3-L5 levels. mCitrine is a variant of yellow fluorescent protein, but its fluorescence signal is diminished in paraformaldehyde-fixed tissue sections. We therefore used an anti-GFP antibody to label and visualize mCitrine-expressing neurons in fixed spinal cord sections from 3 mice. Double immunofluorescent labeling with anti-GFP and anti-NeuN antibodies showed that mCitrine-expressing neurons were largely restricted to the superficial dorsal horn, particularly lamina II, of the injected side of the spinal cord (Fig. 5, C and D; Supplementary Fig. S1B). In the spinal lamina II, 60.37 ± 8.04% of NeuN-immunoreactive neurons expressed mCitrine. No mCitrine-expressing neurons were found on the contralateral side of the spinal dorsal horn.</p><!><p>We next performed perforated whole-cell recordings in acutely prepared spinal cord slices to test the ability of KORD, an inhibitory DREADD, to generate SALB-induced neuronal inhibition. One week after the AAV-KORD-IRES-mCitrine microinjection, spared nerve injury (SNI) was performed in VGluT2-Cre mice. The spinal cord slice recording was conducted two weeks after SNI (i.e., three weeks after viral vector injection). In mCitrine-labeled lamina II neurons from SNI mice, the baseline firing frequency was much higher than in lamina II neurons from control mice (2.10 ± 0.54 Hz vs. 0.54 ± 0.35 Hz; P < 0.05). Bath application of 100 nM SALB (Vardy et al. 2015) led to hyperpolarization of the membrane potential (Δ7.18 ± 2.65 mV) and a robust reduction in the firing rate of all mCitrine-labeled lamina II neurons (from 2.10 ± 0.54 Hz to 0.93 ± 0.29 Hz, n = 17 neurons from 5 mice; P = 0.0332, F(1.80,28.84) = 3.99; Fig. 6, A and B). SALB had no effect on the firing frequency of lamina II neurons from KORD-free control mice (from 2.21 ± 0.43 Hz to 2.28 ± 0.46 Hz, n = 15 neurons from 4 mice; P > 0.05).</p><p>To determine whether KORD activation can inhibit glutamatergic transmission, we recorded sEPSCs in mCitrine-labeled neurons in spinal cord slices from SNI mice. The baseline sEPSC frequency of lamina II neurons was much higher in SNI mice than in control mice (8.34 ± 1.14 Hz vs. 2.97 ± 0.52 Hz; P < 0.05; Fig. 6, C-E vs. Fig. 3, C-E). In VGluT2-Cre mice that had been injected with AAV-KORD-IRES-mCitrine into the superficial dorsal horn, bath application of 100 nM SALB markedly reduced the frequency (P = 0.0011, F(1.71,22.17) = 10.31), but not the amplitude, of sEPSCs of mCitrine-labeled lamina II neurons (n = 14 neurons from 4 mice, P < 0.05; Fig. 6, C-E). These data indicate that activation of KORD on VGluT2-expressing neurons reduces neuronal activity and synaptic glutamate release in the spinal dorsal horn.</p><!><p>Given that glutamate critically mediates nociceptive transmission in the spinal dorsal horn and that SALB induces KORD-mediated inhibition of VGluT2-expressing neurons, we next determined whether SALB-induced KORD activation attenuates chronic pain caused by nerve injury and tissue inflammation. One week after the AAV-KORD-IRES-mCitrine injection, SNI was performed in VGluT2-Cre mice. Final behavioral tests were conducted 10–14 days after SNI. Microinjection of the viral vectors alone had no effect on the baseline mechanical and thermal withdrawal thresholds before SNI. As reported previously (Shields et al. 2003), SNI profoundly reduced the mechanical withdrawal threshold but had no effect on the thermal sensitivity of the ipsilateral hindpaw in mice 10 days after the surgery (Fig. 7, A and B). Systemic administration of SALB (10 mg/kg, i.p.) significantly decreased SNI-induced tactile allodynia (P < 0.0001, F(4,25) = 23.48) but had no effect on the thermal withdrawal latency (Fig. 7, A and B). The effect of SALB was observed within 30 min and lasted for about 120 min after SALB injection. However, SALB treatment had no effect on the mechanical and thermal sensitivity of the hindpaw on the contralateral side (which received no AAV-KORD-IRES-mCitrine injection or SNI surgery) of VGluT2-Cre mice (Fig. 7, A and B).</p><p>To determine whether KORD activation affects pain hypersensitivity caused by tissue inflammation, 11 days after viral vector injection, we subcutaneously injected CFA into the left hindpaw of VGluT2-Cre mice. CFA-induced tissue inflammation caused a large reduction in the mechanical and thermal withdrawal thresholds 3 days after CFA injection. Systemic administration of SALB (10 mg/kg, i.p.) readily reversed CFA-induced mechanical (P < 0.0001, F(4,25) = 26.66, Fig. 7C) and thermal (P < 0.0001, F(4,25) = 9.33, Fig. 7D) sensitivities. In contrast, SALB had no effect on the mechanical and thermal sensitivity of the hindpaw on the contralateral side (which received no AAV-KORD-IRES-mCitrine or CFA injection) of VGluT2-Cre mice.</p><p>Furthermore, in additional VGluT2-Cre mice that received no AAV-KORD-IRES-mCitrine injection, systemic treatment with SALB (10 mg/kg, i.p.) had no significant effect on SNI- or CFA-induced reduction in mechanical and thermal withdrawal thresholds of the left hindpaw (Fig. 7, E-H). Taken together, these findings indicate that VGluT2-expressing glutamatergic dorsal horn neurons are critically involved in maintaining pain hypersensitivity induced by nerve injury and inflammation.</p><!><p>In this study, we used an excitatory and an inhibitory DREADD to determine the role of VGluT2-expressing dorsal horn neurons in the bidirectional modulation of nociception. In the spinal dorsal horn, about one-third of the total neuronal population are inhibitory GABAergic and glycinergic interneurons (Todd & Sullivan 1990, Todd et al. 2003, Foster et al. 2015). The majority of interneurons in the superficial dorsal horn appear to be glutamatergic based on their expression of VGluTs (Yasaka et al. 2010). Paired neuronal recordings in spinal cord slices indicate that more than 85% of lamina II neurons are glutamate-releasing excitatory interneurons (Santos et al. 2007). In the spinal dorsal horn, VGluT2 is mostly expressed in lamina II, whereas VGluT1 is present predominantly in deeper laminae (Todd et al. 2003). VGluT3 is also expressed in a small population of deep dorsal horn neurons and mediates mechanical pain (Peirs et al. 2015). To determine the distribution of VGluT2-expressing neurons in the spinal cord, we generated transgenic mice in which the reporter protein tdTomato was under the control of the VGluT2 promoter. Consistent with the notion that VGluT2 is selectively expressed in glutamatergic excitatory neurons, VGluT2-tdTomato neurons were not colocalized with inhibitory neurons labeled with Pax2 in the spinal dorsal horn.</p><p>Selective manipulation of cellular activity has been made possible with the development of DREADDs, the use of which is less invasive than the optogenetic approach (Urban & Roth 2015, Whissell et al. 2016). We used 2 AAV-mediated DREADDs, AAV-hM3D-mCherry and AAV-KORD-IRES-mCitrine, to specifically manipulate the activity of VGluT2-expressing dorsal horn neurons. The chemogenetic approach has advantages over the intersectional genetic ablation approach, which causes permanent loss of subpopulations of neurons (Duan et al. 2014), and thereby could induce rewiring of the local spinal circuitry. In contrast, the chemogenetic technique allows reversible manipulation of neuronal activity without neuronal damage. The ability to bidirectionally alter (i.e., activate and inhibit) neuronal activity offers an important advantage: it allows us to establish causal relationships between activity in specific neuronal populations and pain behaviors. In our study, localized viral vector injection restricted the DREADD expression to superficial dorsal horn neurons on the ipsilateral side. We demonstrated that activation of virally expressed hM3D robustly excited VGluT2 neurons and stimulated synaptic glutamate release in the spinal dorsal horn. Furthermore, peripheral nerve injury enhances glutamatergic input from primary afferent nerves to spinal lamina II neurons (Chen et al. 2014b, Li et al. 2016, Zhou et al. 2012). We found that nerve injury profoundly increased the firing activity and glutamatergic input of VGluT2-expressing lamina II neurons and that these effects were readily diminished by activation of the inhibitory KORD. Because SNI causes mechanical allodynia but does not alter thermal sensitivity in mice (Shields et al. 2003), our findings suggest that the SNI-induced increase in glutamatergic input to VGluT2 neurons predominantly mediates tactile allodynia. Thus, inhibiting VGluT2-expressing interneurons can largely blunt nerve injury-induced nociceptive input from primary afferent nerves. The dual excitatory input from primary sensory neurons and VGluT2-expressing interneurons likely serves as an intraspinal positive feedback circuitry (via glutamate release and tonic activation of both pre- and postsynaptic NMDA receptors) (Li et al. 2016, Zhou et al. 2012, Chen et al. 2014b) for central sensitization and for the maintenance of nerve injury-induced chronic pain.</p><p>Our study provides new insight regarding the role of VGluT2-expressing dorsal horn neurons in physiological and pathological pain conditions. We showed that stimulation of VGluT2-expressing dorsal horn neurons with hM3D-CNO in naive uninjured mice evoked mechanical and thermal hypersensitivity, suggesting that these neurons function as excitatory neurons that relay nociceptive information from primary sensory nerves to spinal projection neurons under physiological pain conditions. Furthermore, we demonstrated that inhibition of VGluT2-expressing dorsal horn neurons with KORD-SALB rapidly reversed nerve injury- and tissue inflammation-induced pain hypersensitivity. Interestingly, we observed that neither nerve injury nor KORD activation affected thermal sensitivity in the SNI model used in our study. It is possible that heat nociception may be mediated principally by thermal nociceptors and projection neurons in the spinal dorsal horn. Consistent with this view, it has been shown that loss of excitatory interneurons in the superficial dorsal horn diminishes mechanical pain but has no effect on the response to noxious heat (Wang et al. 2013). Nevertheless, inhibition of VGluT2 neurons with KORD-SALB reversed the CFA-induced reduction in both the mechanical and thermal withdrawal thresholds. Thus, VGluT2-expressing dorsal horn neurons are predominantly involved in nerve injury-induced tactile allodynia and tissue inflammation-induced mechanical and thermal hypersensitivities.</p><p>It should be acknowledged that because not all the VGluT2 neurons in the spinal dorsal horn can be transduced by the microinjected viral vectors, the importance of VGluT2-expressing dorsal horn neurons in transmitting nociception may be underestimated. It was reported recently that systemic injection of CNO could affect central nervous system-expressed DREADDs via converted clozapine and N-desmethylclozapine (Gomez et al. 2017, MacLaren et al. 2016). We did not observe any effects of CNO on the excitability of dorsal horn neurons or the withdrawal thresholds in DREADD-free control mice, indicating that CNO alone does not alter neuronal activity or nociception. Although the vast majority of excitatory neurons in the spinal dorsal horn express VGluT2, the diverse roles of subpopulations of excitatory dorsal horn neurons in physiological and pathological pain conditions remain to be established. Our validation of the hM3D and KORD DREADDs using electrophysiological recordings of transduced neurons shows that it is possible to use both CNO and SALB (i.e., multiplexing) to reversibly activate and inhibit the same population of neurons in the same animals to study nociceptive behaviors in future studies.</p><p>In summary, our study indicates that VGluT2-expressing dorsal horn neurons are glutamatergic excitatory neurons and mediate nociceptive transmission under physiological and pathological pain conditions. This new information advances our understanding of the spinal cord circuitry involved in nociceptive transmission. Current analgesic drugs for chronic pain have limited efficacy and often produce adverse effects. Our study suggests that VGluT2 neurons in the spinal dorsal horn could be a potential cellular target for clinical applications of virally mediated gene therapy for chronic pain. Targeting glutamatergic dorsal horn neurons via intrathecal delivery of inhibitory DREADDs may provide an alternative for the treatment of drug-resistant chronic pain.</p>