Zhang et al., Supplemental information, 1

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1 Zhang et al., Supplemental information, 1

2 Zhang et al., Supplemental information, 2 Figure S1: Electrophysiological properties of layer V pyramidal cells and PVINs in normal and SNI mice (relates to Figure 1) (A) Resting membrane potential, (B) action potential voltage threshold, (C) input resistance and (D) current threshold in prelimbic cortex layer V pyramidal neurons in slices from sham operated and SNI injured mice. Note the decrease in input resistance and the accompanying increase in current threshold after nerve injury. (E) Inter-event interval, (F) amplitude and (G) decay time of miniature synaptic events recorded in prelimbic pyramidal cells from SNI and SHAM mice in the presence of TTX (1 µm). The inset shows the experimental configuration for panels E-G (triangle = pyramidal cell, circle = GABA neuron). (H) Mean inter-event interval (Left) and its cumulative probability distribution (Right) of spontaneous excitatory postsynaptic current (sepsc, Vh = -70 mv) recorded (for 4 min) in PVINs of layer V of the prelimbic cortex in SHAM and SNI mice. Note the frequency (inverse of inter-event interval) in SNI mice was increased compared to SHAM control. (I) Mean amplitude (Left) and its cumulative probability distribution (Right) of sepscs in PVINs in SHAM and SNI mice. Note the amplitude was not altered in SNI mice. The inset in H shows the experimental configuration for both H and I (circle = GABA neuron).

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4 Zhang et al., Supplemental information, 4 Figure S2: Verification of the specificity of optogenetic targeting of ChR2 mice (relates to Figures 2, 3 and 4) Confocal image of a brain slice showing eyfp-expression in ChR2 mice (upper, eyfp) and parvalbumin immunostaining (middle, PV). The overlap of eyfp and parvalbumin (lower, Merge) indicates that the parvalbumin neurons express eyfp. Scale bars are 20 µm.

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6 Zhang et al., Supplemental information, 6 Figure S3: Verification of activation and inactivation of prefrontal cortical neurons by optogenetic techniques (relates to Figures 3 and 4) (A) Brain section showing the locus of optic fiber implantation in the PFC (over prelimbic cortex) (Left, arrow heads) and c-fos expression (Middle, red; Right Lower), visualized by immunostaining in a PFC slice after in vivo blue light (ChR2) activation of PVINs in ChR2 mice. The arrows highlight stained round nuclei, most of which overlapped with EYFP fluorescence in the plasma membrane (Right Lower) suggesting activated PVINs. There are no c-fos stained neurons present in areas that are not directly underneath fiber optic tip (Middle blue, Right Upper). (B) Brain section showing the locus of optic fiber implantation in another mouse (Left, arrow heads) and c-fos expression (Middle red; Right Lower), visualized by immunostaining in a PFC slice after in vivo yellow light (Arch3) inhibition of PVINs in Arch3 mice. The arrows highlight c-fos stained round nuclei that were never surrounded by GFP fluorescence (Right Lower), suggesting c-fos expression in activated pyramidal cells but not PVINs. Similarly to ChR2 mice, there are no c-fos stained neurons present in areas that are not directly underneath fiber optic tip (Middle blue, Right Upper). Note that eyfp and GFP fluorescence in both panels is confined to the plasma membrane, due to the fact that these fluorophores are attached to the ChR2 and Arch3 constructs rather than ubiquitously expressed in the cytoplasm.

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8 Zhang et al., Supplemental information, 8 Figure S4: Verification of the locus of optic activation and inactivation in the mpfc (relates to Figures 3 and 4) (A) Schematic diagram of a PFC section illustrating the placement of the light fiber for optical stimulation and inhibition of PVINs in behavioral experiments. Circles denote the location of optogenetically activated or inhibited neurons. PrL: prelimbic cortex; InL: infralimbic cortex. Adapted from Paxinos et al. (2001). The prelimbic cortex spans a wide range in the rostral-posterior dimension of the mpfc (Bregma ~ to mm). In the present study, we targeted the prelimbic cortex at Bregma to mm using a 200 µm core optic fiber. (B) Quantification of activated neurons in prefrontal area after in vivo stimulation of ChR2 with blue light. c-fos stained (red) nuclei were counted underneath the tip of optic fiber path in 20 µm brain slices from ChR2-expressing mice and pooled in 300 µm bins. The majority of activated neurons are scattered in an area underneath the optic fibers within a depth of 0.8 mm (prelimbic cortex, shaded area) (blue line, plane at optic fiber, n = 8 mice). Fewer c-fos stained cells are found at brain sections 180 μm (red line, n = 4) and 240 μm (green line, n = 4) anterior to fiber optic plane (center of 200 µm core optic fiber). (C) Quantification of c-fos stained, putative pyramidal cells in prefrontal cortex after in vivo activation of Arch3 with yellow light. Similarly to blue light, the activated neurons (due to inhibition of GABA neurons) are scattered in an area underneath the optic fibers within a depth of 0.8 mm (prelimbic cortex, shaded area). Very few c-fos stained cells are found at brain sections 180 μm (red line, n = 4) and 240 μm (green line, n = 4) anterior to fiber optic plane. *P < 0.05, **P < 0.01 (relative to data point at 1200 μm).

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10 Zhang et al., Supplemental information, 10 Table S1. Intrinsic neuronal properties of PVINs (relates to Figure 1) There is no difference in cell size (Cm), resting membrane potential (RMP), input resistance (Rinput), current (rheobase) and voltage threshold (Vthreshold) of AP in SHAM and SNI mice. Recorded in EYFP-expressing ChR2 mice. Ambient light was turned off during experiments.

11 Zhang et al., Supplemental information, 11 Supplemental experimental procedures: Animals All experiments were performed following approval of an animal protocol by the Institutional Animal Care and Use Committee and all efforts were made to minimize animal suffering according to the policies and recommendations of the International Association for the Study of Pain. Adult male C57BL/6 (wild-type) mice (> 8 weeks old) were purchased from Jackson Laboratories. Channelrhodopsin-2 (ChR2) or archaerhodopsin-3 (Arch3) expressing mice (> 8 weeks old) were generated by crossing parvalbumin (PV)-Cre knock-in mice strain B6;129P2-Pvalb tm1(cre)arbr /J (Stock: ) with strains B6;129S-Gt (ROSA)26 Sor tm32(cag-cop4*h134r/eyfp)hze /J (ai32, Stock: ) and B6;129S-Gt(ROSA)26Sor tm35.1(cag-aop3/gfp)hze /J (ai35d, Stock: ), respectively. Homozygous parents were purchased from Jackson Laboratories and bred in house. Only male mice were used for the experiments. Animals were kept at a maximum of five mice per cage ( cm) and with free access to food and water. The room temperature was controlled (23 ± 1 C) on a 12 h light/dark cycle. Different cohorts of mice were used for the various behavioral tests. The observer was blind to the experimental groups when measuring mechanical and thermal hyperalgesia. Electrophysiology Acute brain slices were prepared from the various mouse strains by cutting coronal sections (300 µm) with a vibratome (Leica VT1200, Buffalo Grove, IL) in ice-cold sucrose-based solution containing (in mm): 75 sucrose, 67 NaCl, 25 glucose, 26 NaHCO 3, 1.25 NaH 2 PO 3, 2.5 KCl, 0.5 CaCl 2, 7 MgCl 2-6H 2 O. Brain slices were then transferred into normal extracellular solution (bubbled with 95% O 2, 5% CO 2 ) containing (in mm): 124 NaCl, 3 KCl, 1.25 NaH 2 PO 4, 26 NaHCO 3, 1.6 CaCl 2, 1.8 MgCl 2, 10 glucose at 33 C for min, and then kept at room temperature until use. The intracellular solution used in current-clamp was (in mm): 130 K-gluconate, 10 HEPES, 0.2 EGTA, 10 KCl, 10 Na 2 -

12 Zhang et al., Supplemental information, 12 phosphocreatine, 4 Mg-ATP, 0.3 Na-GTP (ph adjusted to 7.3). For voltage-clamp we used: 130 Cs Methanesulphonate, 4 CsCl, 2 EGTA, 4 Mg-ATP, 0.3 Na-GTP, 10 HEPES, 5 QX-314. The recording temperature was 33 ± 1 ºC. Whole cell patch clamp recordings were performed using a MultiClamp 700B amplifier and a Digidata 1440A converter (Molecular Devices). In current clamp experiments, after stabilization, a small bias current was injected through patch pipette to maintain a resting membrane potential at -70 mv for all recorded cells. Short positive current steps (2 ms) with an increment of 10 pa were injected every 0.4 s through the recording electrode until action potential was constantly activated. Current threshold was defined as the minimum amount of current needed to induce the first action potentials. Voltage threshold is defined as the most negative voltage that is achieved by the current injection for an all-or-none action potential firing to occur. To determine the input resistance, hyperpolarizing currents (1 s duration, from 0 to 225 pa, with 25 pa increments) were injected into the cell. Input resistance was taken at the linear part of the I-V curve. Steady state firing frequency and current injection relationship (F-I curve) were determined from a series of 10 current injections (1 s duration, 50 pa steps) in pyramidal neurons. To examine the input-output relationship of inhibitory synaptic transmission, a concentric bipolar stimulating electrode (FHC Bowdoinham ME) was placed adjacent to the recording electrode ( µm) in layer V of the prelimbic cortex. Incremental voltage pulses (500 µs, from 1 to 10 V, 1 V increment) were applied to pyramidal cells every 10 s via a Digitimer DS2A voltage stimulator (AutoMate Scientific Inc, Berkeley CA). eipscs were recorded at holding voltage of 0 mv from brain slices obtained from both SHAM and SNI mice. For eipsc recordings, we used a low stimulus intensity at twice the threshold value to prevent unspecific current spreading, with the stimulating electrode placed in layer V of the infralimbic cortex (600 ~ 800 µm away from recording electrode). For basic neuronal properties and spontaneous EPSC (sepsc) recording in PVINs, we recorded EYFP-expressing neurons from brain slices of ChR2 mice in the absence of TTX in the perfusion medium. A cell with membrane potential

13 Zhang et al., Supplemental information, 13 less negative than 60 mv (e.g., 55 mv) was discarded. APV, DNQX, QX 314 chloride and bicuculline were purchased from Tocris Bioscience, TTX was purchased from Abcam. All other chemicals were purchased from Sigma. Optogenetics ChR2- and Arch3-expressing neurons were stimulated, respectively, with blue (473 nm) and yellow (589 nm) light from DPSS laser systems (Laserglow Technologies, Ontario) in both slice recording and behavioral experiments. In some brain slice recordings, we also used a LED blue light source (473 nm, Thorlabs Inc, New Jersey) to activate GABA neurons. Light was delivered to brain slices in a focal area (1 mm 2 ) around the recording electrode via a 200 µm core, 0.37 NA, multimode optic fiber. Fiberoptic cannulae (2.5 mm SS Ferrule, Doric Lens, Quebec) with a 200 µm core fiber (0.37 NA, Length 2.5 mm) were used for brain implantation in the prelimbic area of the PFC. In behavioral experiments, the fiberoptic cannula was connected to DPSS laser generator through two patch cables (200 µm core, 0.37 NA, multimode) linked by a rotary joint (Doric Lens, Quebec). Optical power was measured before each experiment using a power sensor (S130C, Thorlabs, New Jersey). The power at the fiber tip was mw (continuous blue or yellow light) for activation of ChR2 and Arch3 in brain slice recording and 3 to 3.5 mw for the activation of both ChR2 and Arch3 in behavioral experiments. To mimic γ frequency neuronal activity in behavioral experiments, GABA neurons were activated by blue light pulses (10 ms) at 40 Hz (Cardin et al., 2009), whereas, GABA neurons were inhibited using continuous solid yellow light. Spared nerve injury animal model Surgery was performed on wild type (C57BL/6) or ChR2- and Arch3-expressing mice. Briefly, under isoflurane anesthesia a 0.5 cm skin incision was made on the left thigh to expose the three branches of

14 Zhang et al., Supplemental information, 14 the sciatic nerve (i.e., common peroneal, tibial and sural nerves). Common peroneal and tibial nerves were tightly ligated with a 6-0 silk suture (Ethicon, USA) and transected together. A piece of 1 mm nerves were removed. Precaution was taken to avoid damage to the sural nerve. After surgery, the overlaying muscles and skin were separately closed with 6-0 and 4-0 silk sutures, respectively. After surgery animals were put in a new cage for recovery at least days before further brain cannula implantation or brain slice recordings. In SHAM operated animals, we performed exactly the same procedure but without ligation and transection of the nerves. Most of the mice showed neuropathic pain behavior (reduced thermal withdrawal latency and mechanical threshold) 7 days after SNI surgery. Animals that did not exhibit a neuropathic phenotype after 10 days were discarded without further experiments. Cannula implantation A fiberoptic cannula was implanted in the right prelimbic cortex 10 days after SNI/SHAM surgery. Briefly mouse was placed in a stereotaxic frame under isoflurane anesthesia. After the skull was exposed, a checker pattern was etched into the surface of the skull with a 21 gauge needle. The surface was then cleaned with hydrogen peroxide and water. A burr hole (< 1 mm) was drilled with a handheld drill above the prefrontal area. A fiberoptic cannula (2.5 mm fiber length) was slowly inserted into prelimbic area at coordinates: AP 1.8 mm, RL 0.5 mm and DV 2.2 mm (at the upper edge of the prelimbic cortex). A thin layer of C&B Metabond adhesive (Parkel Inc, NY, USA) was applied to the skull and dental cement was then applied to secure the fiberoptic cannula. After the implantation, the cannula was protected by a plastic cap. Animals were housed individually after the implantation. Behavioral experiments began at least 14 days after implantation surgery. Before behavioral experiments, animals were handled by the experimenter daily for at least 5-6 days (3-5 min/day) to habituate them to human handling. On the day of experiment, animals were transported to the testing

15 Zhang et al., Supplemental information, 15 facility at least 2hrs before experimentation. In the presence of 0.1 ml isoflurane, the implanted cannula was connected to a patch cable with a 2.5 mm zirconia mating sleeve. The patch cable was then connected to the laser generator via a rotary joint. Animals were placed in small enclosed testing arenas, while minimizing patch cable tension to ensure free mobility for at least 90 min before testing. Measurements of pain threshold in mice Measurements of mechanical and thermal withdrawal threshold/latencies were taken between 2-3 weeks after nerve injury and under optogenetic stimulation. Before experiments, mice were allowed to acclimate for a period of at least 90 minutes. For evaluation of thermal hyperalgesia, we analysed the latency to withdrawal of the hind paws from a focused beam of radiant heat (IR = 30) using a Plantar Test apparatus (UgoBasile, Varese, Italy) (Garcia-Caballero et al., 2014). For measurements of mechanical hyperalgesia, a digital plantar anesthesiometer was used (UgoBasile, Varese, Italy) as described by us previously (see also Garcia-Caballero et al, 2014). Animals were tested individually before and after the lights were turned on by a blinded-experimenter to the assigned groups, with a 1 hour interval between measurements. Measurements of thermal and mechanical hyperalgesia were performed with the same animals within a 48h period interval. The light was turned on 3 minutes before the measurements began and was kept on until all the measurements were finished. Conditioned place preference testing (CPP) Experiments started with a 2-day pre-conditioning phase where the animal with a patch cable attached to the implanted cannula was placed in a three chamber CPP box (two side chambers with dimension LWH 10 x 8 ¼ x 12 inch; middle chamber 5½ x 8¼ x 12 inch) with no restricted access to all chambers for 30 min each day. Animal behavior was recorded to verify no pre-conditioning preference. Animals that spent more than 70% (or less than 30% time)(he et al., 2012) in one chamber

16 Zhang et al., Supplemental information, 16 were excluded from the experiments. A pretest of 15 min was recorded on the second day of preconditioning for data analysis. After pre-conditioning, mice underwent a 2-day conditioning phase that consisted of a 15 min session in the morning and a 15 min session in the afternoon with the same room environment (temperature, humidity and light). During the sessions, animals were restricted to one side chamber by a transparent plexiglass partition placed vertically between this side chamber and the middle chamber. The animals were able to see the middle and the other side chamber through the partition without access to them. In the morning session, blue or yellow laser stimulation was paired with one of the two side chambers (horizontally and vertically-striped walls, respectively) for 15 min, while non-stimulation (no light) was paired with the opposing side chamber for 15 minutes 4 hrs later in the afternoon. Light stimulation and no light stimulation were randomly alternated in the morning and afternoon. On the test day (day 5), mice were treatment-free and placed in the middle of the CPP box with free access to all chambers for 15 min, and the time spent in each chamber was recorded. The CPP score was calculated as the time in the light conditioned side chamber on the test day minus the time spent in the same chamber during the pre-test. Place Escape/Avoidance Paradigm (PEA) Place escape/avoidance testing (LaBuda and Fuchs, 2000; LaGraize and Fuchs, 2007) was performed 2-3 weeks after brain cannula implantation and 3-4 weeks after SHAM or SNI surgery. Both ChR2 and Arch3 mice were divided randomly into 4 different groups (SHAM/light off, SHAM/light on, SNI/light off and SNI/light on). No training is necessary for this behavioral test. Animals were placed within a PEA box (LWH 25 ½ x 8 ¼ x 12 inch, without top and bottom) that was positioned on top of a mesh screen. One half of the wall of the box is painted white (light area), and the other half is painted black (dark area). During behavioral testing, animals were allowed unrestricted movement in the test chamber for the duration of a 30-min test period. Fiber optic patch cable was

17 Zhang et al., Supplemental information, 17 attached to the cannula under slight isoflurane anesthesia. The animals were allowed to habituate to the patch cable for 1~2 hrs before testing which began immediately after the animal was placed in the middle of the test box. Mechanical stimulation (von Frey filament 0.4g) was applied to the lateral plantar surface of the hindpaws at 15-s intervals. The mechanical stimulus was applied to the left paw (ipsilateral to injury) when the animal was within the preferred dark area of the test chamber and the right paw (contralateral to injury) when the animal was within the non-preferred light area of the test chamber. Some animals were tested with light off (yellow or blue laser, SHAM/light off and SNI/light off group); other animals were tested with light on (SHAM/light on and SNI/light on group). SHAM and SNI animals were mechanically stimulated in an identical manner. The location of the animal (within light side or dark side) was recorded and converted to a percentage of time spent in the light side of the chamber. Immunohistochemistry To verify fiberoptic and cell activation location in behavioral experiments, we did c-fos immunostaining (Daou et al., 2013). Animals were placed in the same environment and received the same laser stimulation as in CPP experiments. We then waited for 3hr to allow for c-fos protein synthesis. Animals were then transcardially perfused with normal saline and then 4% paraformaldehyde in PBS. The anterior part of the brain containing the prefrontal cortex was dissected and stored in the same fixative overnight, and transferred to 30% sucrose in PBS until cryosectioning. Brain sections (14-30 µm) were cut from frozen tissue samples using a cryostat (Leica CM3050 S) and collected on positively charged microscope slides for immunohistochemistry. After permeabilization and blocking with Triton X-100 and 10% normal goat serum, tissue sections were treated with a rabbit polyclonal c-fos antibody (1:500) (sc-52, Santa Cruz Biotechnology) and/or a mouse monoclonal parvalbumin antibody (1:2000) (MAB1572, EMD Millipore) for 24 hrs. On the following day tissue

18 Zhang et al., Supplemental information, 18 sections were treated with Alexa Fluor 594 goat anti-rabbit secondary antibody (A-11037, Invitrogen) or Alexa Fluor 633 goat anti-rabbit secondary antibody (A-21070, Life Technologies) for c-fos staining and Alexa Fluor 546 goat anti-mouse secondary antibody (A-11030, Life Technologies) for parvalbumin staining for 1.5 hrs in a dark room, and then were mounted on coverslips and analyzed using slide scanner (Olympus, System VS110, Model BX61VSF, Japan). Statistics Statistical analysis was performed using Student s t-tests or two-way ANOVA (Prism 3.0 software; GraphPad Software). Miniature or evoked ISPC (mipsc or eipsc) data were analyzed using the Mini Analysis Program (Synaptosoft). Clampfit (Molecular Devices) was used for analysis of all electrophysiological data. Statistical significance was accepted at the level of p < Data are presented as mean ± SEM. Additional references not cited in the main document: Daou, I., Tuttle, A.H., Longo, G., Wieskopf, J.S., Bonin, R.P., Ase, A.R., Wood, J.N., De Koninck, Y., Ribeiro-da-Silva, A., Mogil, J.S., et al. (2013). Remote optogenetic activation and sensitization of pain pathways in freely moving mice. The Journal of neuroscience : the official journal of the Society for Neuroscience 33, Paxinos, G., Franklin, K.B.J. (2001) The mouse brain in stereotaxic coordinates. 2. Academic; San Diego.