www.sciencemag.org/content/350/6260/550/suppl/DC1

Supplementary Materials for

Gate control of mechanical itch by a subpopulation of spinal cord

interneurons

Steeve Bourane, Bo Duan, Stephanie C. Koch, Antoine Dalet, Olivier Britz, Lidia Garcia-

Campmany, Euiseok Kim, Longzhen Cheng, Anirvan Ghosh, Qiufu Ma,* Martyn

Goulding*

*Corresponding author. E-mail: goulding@salk.edu (M.G.); qiufu_ma@dfci.harvard.edu (Q.M.)

Published 30 October 2015, Science 350, 550 (2015)

DOI: 10.1126/science.aac8653

This PDF file includes:

Materials and Methods

Figs. S1 to S8

Full Reference List

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MATERIALS AND METHODS

Mouse Lines

The RH26 NPY::Cre transgenic mouse line was generated by the Gene Expression Nervous System Atlas

(GENSAT) project (27). The specificity of Cre recombination and reporter expression in NPY::Cre INs

was determined by crossing NPY::Cre mice with a R26LSL-tdTomato mouse line (28). Other mouse lines used

in this study were the GAD1::GFP (29), GlyT2::GFP (30), Thy1LSL-YFP (31), ROSA26ds-HTB (32), Tauds-DTR

(33), mouse lines. The JS66 Pitx2-EGFP transgenic mouse line was generated by GENSAT. The R26ds-

hM4D-tdTomato and Lbx1FlpO mouse lines are described below.

Lbx1FlpO mice were generated by homologous recombination (18). A Bluescript-derived backbone

containing PGK-DTA was used to capture a genomic fragment encompassing the mouse Lbx1 gene from a

129SV BAC. A Not1-fragment containing a FlpO-floxed PGK-Neo cassette was then inserted in-frame

into a unique Not1 site at amino acid 62 of the mouse Lbx1 protein. R26ds-hM4D-tdTomato mice were generated

by homologous recombination by inserting a CAG-double stop-hM4D-tdTomato cassette into the R26

locus (see Fig. S5)

Diphtheria Toxin Ablation

To ablate NPY::Cre expressing neurons, 6-10 week old mice were injected intraperitoneally with

diphtheria toxin (DTX, 50 mg/kg) on day 1 and then again on day 4. Behavioral analyses were performed

7 days post DTX injection, prior to the development of spontaneous scratch.   Littermates lacking the

Lbx1FlpO allele were used as controls. All animals received DTX injection.

Immunohistochemistry

Mice were euthanized with a cocktail of ketamine (10 mg/ml), xylazine(1 mg/ml): 10 µl/g body weight)

prior to perfusion with 4% paraformaldehyde in PBS. The brain and spinal cord, with DRGs attached,

were dissected and post-fixed for 2 hours at 4°C. Tissues were washed 3 times (10 min each) in cold PBS

and cryoprotected overnight in 30% sucrose-PBS. Tissues were embedded in OCT and cryostat sections

were then cut, collected and dried at room temperature. Sections were permeabilized with PBT (PBS,

0.1% Triton X-100), blocked for 1 h at room temperature with PBT containing 10% donkey serum and

then incubated overnight at 4°C with primary antibodies in PBT containing 1% donkey serum. Primary

antibody staining was detected and visualized with fluorophore–conjugated secondary antibodies. Images

were captured using a Zeiss LSM 700 microscope. Quantitative analysis was determined by analyzing 3-6

spinal cords for each genotype (5-10 sections per cord).

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The following primary antibodies were used: rabbit anti-Ds-Red (1:1000, Clontech), guinea pig anti-

Lmx1b (1:5000), rabbit anti-Pax2 (1:500, Zymed), rabbit anti-NPY (1:1000, Peninsula Lab), guinea pig

anti-PKCγ (1:1000, Frontier Institute Co.), mouse anti-NeuN (1:500, Chemicon), guinea pig anti-vGluT1

(1:1000, Millipore), sheep anti-CGRP (1:1000, Abcam), isolectin IB4 Cy5-conjugated (1:500, Invitrogen),

goat anti-GFP (1:1000, Abcam), rabbit anti-tyrosine hydroxylase (1:1000, Protos Biotech), rabbit anti-

cMaf (1:1000, Bethyl Laboratories), goat anti-TrkC (1:1000, R&D systems), chicken anti-TrkB (gift from

Louis Reichardt, UCSF), goat anti-c-Ret (1:50, R&D Systems), rabbit anti-nNOS (1:1000, Invitrogen),

goat anti-CTB (1:4000, List Laboratories), rabbit anti-GRPR (1:100, MBL).

In situ Hybridization

For in situ hybridization spinal cord were cryosectioned at 14 µm and stored at -20 °C until needed. Spinal

cord sections were hybridized overnight at 65°C. Sections were washed twice in 1 X SSC, 50%

formamide, and 0.1% Tween-20 at 65 °C for 30 min and blocked with a solution of MABT, 2% blocking

reagent and 20% inactivated sheep serum for 2 hours at room temperature. Sections were then incubated

overnight with anti-DIG-alkaline-phosphatase (AP)-conjugated antibody, washed twice in 1X MABT and

revealed with NBT/BCIP staining solution. For double staining analyses of tdTomato fluorescence

coupled with in situ hybridization, the tdTomato fluorescent signal was photographed prior to performing

in situ hybridization. After in situ hybridization each section was re-photographed and the in situ signals

were pseudo-colored and superposed onto the tdTomato signal with Adobe Photoshop software.

Quantitative analysis was determined by analyzing 3-6 spinal cords (5-10 sections each) per genotype.

Only cells with clearly visible nuclei were scored.

Surgery

For CFA-induced inflammation, mice were briefly anesthetized with isofluorane (3–5 min at 2%), and 20

µl of Complete Freund’s Adjuvant (CFA, Sigma-Aldrich) was injected into the plantar surface of the left

hindpaw. Mechanical threshold using von Frey and dynamic touch tests were measured 1 and 3 days after

CFA injection.

Behavioral Testing

All behavioral tests were performed blind to the genotype of the animals. Animal experiments were

conducted according to NIH guidelines using protocols approved by the Institutional Animal Care and

Use Committee at Salk Institute for Biological Studies and Dana-Farber Cancer Institute. Animals were

acclimatized to the behavioral testing apparatus for 3 to 5 days for 30 min prior to experimentation and

data collection. After habituation, baseline measures were recorded on two consecutive days for each

  4  

behavioral test prior to surgery or chemical injection. Behavioral tests were then performed at defined

intervals as outlined in the text. Five different groups of mice were used for the behavioral tests with the

order indicated. 1) touch-evoked itch, von Frey, brush and Hargreaves, 2) pruritogen-evoked itch, 3) cold

plate sensitivity, 4) hot plate sensitivity and 5) Randall-Selitto and acetone-cooling.

Accelerating Rotarod Test

To investigate motor performance, mice were trained on the accelerating rotarod. Training sessions

consisted of mice being placed on a rotarod moving at 5 rpm for 5 min so that they could stay on the

rotarod for the entire 5 min. If a mouse fell, it was placed back on the rotarod and the 5 min trial was

started again. Training took place on two consecutive days. Two days later, mice were subjected to a full

rotarod test, with the rotorod accelerating from 4 rpm to 40 rpm over 5 min. The time to fall was

automatically recorded. The rotarod latency was determined as the average of 3 trials per animal

performed at 20 min intervals.

Randall-Selitto Test

Noxious mechanical pain testing was undertaken with a Randall-Selitto device. Prior to testing, mice were

placed in a restraining plastic tube and allowed 5 min to acclimatize. Slowly increasing pressure was

applied to a point midway along the tail until the animal showed clear signs of discomfort or tried to

escape. This pressure was taken as the pain threshold. The overall pain threshold was determined as the

average of 6 trials per animal taken at 2 min intervals.

Dynamic Touch Test

To measure light touch sensitivity, mice were placed on an elevated wire grid and habituated for 15 min

on the day of the experiment. The plantar hindpaw was stimulated by light stroking with a paintbrush, in a

heel to toe direction. The test was repeated three times, with intervals of 10 sec. For each test, no evoked

movement was scored as 0, and walking movement or brief paw lifting (~1 sec or less) was scored as 1.

For each mouse, the cumulative score from three tests was used as a measure of the touch response.

To assess dynamic mechanical allodynia, the plantar region of the left hindpaw was stimulated by light

brush strokes. Each test comprised of three episodes of stimulation performed at 10 sec intervals. Each test

was repeated three times at intervals of at least 3 min to obtain the averaged score for each individual

mouse. A score of 0 indicates walking away or occasionally very brief paw lifting (this response was

scored as 1 for dynamic touch test, but as 0 for allodynia assay); a score of 1 indicates a sustained lifting

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(more than 2 sec) of the stimulated paw. A score 2 of indicates a strong lateral lifting above the level of

the body. A score of 3 indicates flinching or licking of the affected paw.

Acetone Evaporative Cooling Test

To measure sensitivity to cooling, the acetone evaporation assay was performed (34). Briefly, mice were

acclimated for 15 min in an elevated chamber with a mesh floor, a syringe with a piece of rubber tubing

attached to the end was filled with acetone, and the plunger was depressed so that a small drop of acetone

formed at the tip. The syringe was raised from below, depositing the acetone drop on the paw. Mice were

tested with an interstimulation period of 4 min per mouse, alternating paws between stimulations.

Responses were video recorded for later quantification by an observer blind to the experimental

conditions. Behaviors were scored according to the magnitude of the response using the following scale:

0, no response; 1, brief lift, sniff, flick, or startle; 2, jump, paw shake; 3, multiple lifts of paw, paw lick; 4,

prolonged paw lifting, licking, shaking, or jumping; 5, paw guarding.

Cold Sensitivity Test

To measure cold pain, mice were placed on a cold plate and the latency to forepaw flinching and hindpaw

licking was measured as previously described (34). All animals were tested sequentially with a minimum

of 5 min between tests. To avoid tissue damage, a 60 sec cutoff time was set.

Hargreaves Test

To measure radiant heat pain by the Hargreaves test, we placed mice in a plastic chamber and the plantar

paw surface was exposed to a beam of radiant heat according to the Hargreaves method. The latency to

paw withdrawal was averaged for 5 trials per animal, with a 10 min interval between each trial. A cutoff

time of 30 sec was set to prevent tissue damage.

Hot Plate Test

Mice were placed on a hot plate and the latency to hindpaw flinching and licking was measured. The hot

plate was set at 46ºC, 50ºC or 54ºC. All animals were tested sequentially with a minimum of 5 min

between each test. To avoid tissue injury, a cutoff time was set at 60 sec for assays at 46ºC and 50ºC, and

30 sec for 54ºC.

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von Frey Test

For the von Frey test, mice were placed on an elevated wire grid and the lateral plantar surface of the

hindpaw was stimulated with calibrated von Frey monofilaments (0.008-1.4 g). The paw withdrawal

threshold for the von Frey assay was determined by Dixon’s up-down method (35).

Pruritogen-induced Itch Test

Pruritogen-induced itch behavioral tests were performed as previously described (36). All animals were

acclimatized to the behavioral testing apparatus during three to five ‘habituation’ sessions. On the day of

the experiment, mice were placed in a chamber and habituated for 15 min. The behavior of the mice was

video-recorded for 30 min before (baseline behavior) and 30 min after injecting each animal. Compound

48/80 (100 µg), or chloroquine (200 µg) dissolved in 50 µl of sterile saline was injected intradermally into

the nape of the neck using a 0.5 ml insulin syringe with a 28G1/2 needle. For the injections into the cheek,

10 µl of vehicle (7% Tween-80 in sterile saline) containing varying amounts of capsaicin was used for

each mouse. Scratching and wiping behaviors were monitored by video recording. The number of bouts in

the 30 min following each injection were counted.

Mechanical Alloknesis Test

To measure mechanical alloknesis, the fur on the nape of mice was shaved 5 days after first injecting

DTX. Mice were habituated for 2 days, during which time there was no evidence of spontaneous

scratching or skin lesions in NPY::Cre IN-ablated mice. Mice then received five separate mechanical

stimuli at 10 sec intervals at separate randomly selected sites on the nape of the neck. Mechanical stimuli

were delivered with von Frey filaments ranging from 0.008 g to 1.0 g. The presence or absence of a

positive response (hindlimb scratch directed to the site of mechanical stimulation) was noted for each

stimulus prior to the next one being given. The alloknesis score was the total number of positive scratch

responses elicited by the 5 stimuli.

For the NPY::Cre IN silencing experiments, the fur on the nape of NPY::Cre; Lbx1FlpO, R26ds-hM4D-tdTomato

mice and their littermate controls (NPY::Cre, R26ds-hM4D-tdTomato) was shaved 2 days before each

experiment. Animals were placed in a plastic chamber and acclimatized for 30 min during three

‘habituation’ sessions. On the day of the experiment, mice were acclimatized for 30 min and then briefly

removed from the chamber for intraperitoneal injection of clozapine-N-oxide (CNO, 1 mg/kg) (37). All

animals received an injection of CNO. Mice were then returned to the chamber. 30 to 40 min after CNO

injection, which coincides with the time of maximal neuronal silencing, each mouse received five separate

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mechanical stimuli at 10 sec intervals delivered with von Frey filaments (0.04 g and 0.07 g) at separate,

randomly selected sites on the nape. Experiments with high-threshold von Frey filaments (0.6 g and 1.0 g)

were performed 2 days later as previously described. The presence or absence of a positive response

(hindlimb scratching directed to the site of mechanical stimulation) was noted for each stimulus before the

next stimulus was given. The alloknesis score was the total number of positive responses elicited by 5

stimuli.

Drug Administration

H1 receptor antagonist diphenhydramine (50 mg/kg) and H4 receptor antagonist JNJ7777120 (30 mg/kg)

were administered orally as a 20 min pretreatment in a volume of 300 ml sterile saline. GRPR antagonist

RC-3095 (0.3 nmol) was administered intrathecally 10 min prior to testing, in a volume of 10 µl sterile

saline.

Mice were given a single intrathecal injection of either bombesin-saporin (400 ng in 10 µl sterile saline) or

sterile saline (10 ml). The first DTX injection was performed at 7 days after the treatment, with a second

injection 3 days later. Mice were then used for behavioral testing experiments 7 days after the first DTX

injection.

Electrophysiology

Spinal cord slice preparation

Spinal cord slices were prepared from postnatal P21 to P28 NPY::Cre; R26LSL Tomato and NPY::Cre; Thy1-

YFP mice. Data was pooled as the results were indistinguishable between the reporter expressions.

Following anesthesia, induced by ketamine and xylazine intraperitoneal mixture injection, mice were

transcardially perfused with oxygenated ice-cold dissecting ACSF solution (dACSF - NaCl, 95mM; KCl,

2.5mM; NaHCO3, 26mM; NaH2PO4H2O, 1.25mM; MgCl2, 6mM; CaCl2, 1.5mM; Glucose, 20mM;

Sucrose, 50mM; Kynurenic Acid, 1mM; Ethyl Pyruvate, 5mM). The spinal cords were then quickly

isolated in oxygenated dACSF at 4°C. Meningeal membranes were removed and particular attention was

given to avoid dorsal root damage. T12-S2 segments were embedded in low-melting agarose at 33°C

(Lonza) and sectioned transversely at 500-600µm in 4°C dACSF using a vibratome (Leica VT1000S).

Slices were then allowed to recover an hour at 32°C in oxygenated recording ACSF (rACSF - NaCl,

125mM; KCl, 2.5mM; NaHCO3, 26mM; NaH2PO4H2O, 1.25mM; MgCl2, 1mM; CaCl2, 2mM; Glucose,

20mM; Ethyl Pyruvate, 5mM). Selected slices were finally placed in a recording chamber at room

temperature and perfused constantly by oxygenated rACSF.

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Whole-cell patch clamp recordings

Reporter positive neurons were identified using an upright microscope with a 40x water-immersion

objective, fluorescence and infrared differential interference contrast. Whole cell patch clamp recordings

were performed using 5-6MΩ pipettes filled with intracellular solution (K-gluconate, 130mM; NaCl,

5mM; CaCl2 2H2O, 1mM; MgCl2, 1mM; HEPES 10mM; Mg-ATP, 4mM; pH 7.3). Data were acquired

using pClamp 9.2 at 50 kHz and filtered at 10 kHz. Neuronal firing was elicited by injecting depolarizing

currents ranging from 0 to 200 pA in 20 pA increments for 1 sec every 10 sec. All tests were made at the

neurons resting potential. A calculated 14 mV liquid junction potential was corrected offline.

Dorsal root stimulation

Voltage clamp evoked currents were recorded in neurons maintained at -65 mV. The L4 or L5 dorsal roots

were stimulated using a homemade suction pipette and a stimulus isolator delivering a constant current.

The following stimulation intensities were used to discriminate Aβ (0 µA to 25 µA, 0.1 msec duration),

Aδ (25 µA to 100 µA, 0.1 msec duration) and C (up to 500 µA, 0.5 msec duration) fiber type input. The

response threshold was assessed and a 20 sweep high frequency stimulation performed (20 Hz for Aβ, 2

Hz for Aδ and 1 Hz for C fiber) to classify monosynaptic and polysynaptic responses. Neurons showing

no failure and latencies variance (“jitter”) below 0.6 ms were considered monosynaptic.

In Vivo Single Unit Extracellular Recordings

Mice of both sexes weighing between 20-35 g were used. In vivo single unit extracellular recordings were

performed out as outlined elsewhere (38). Briefly, mice were anesthetized using urethane (1.5 µg/g in

0.9% NaCl), which was supplemented as needed throughout the experiment. Dexamethasone (10 µg/g)

and atropine (1.5 µg/g in 0.9% NaCl) were injected s.c. before the start of the experiment to minimize

spinal cord swelling and bronchial secretions respectively. The animal was tracheotomized and mounted

in a custom-made stereotaxic frame. A laminectomy was performed to expose spinal lumbar cord at

L4/L5, and a vertebral clamp used to secure the vertebral column at the thoracic spine before the pia and

dura mater were removed. The dorsal horn was then covered in mineral oil to prevent drying of the tissue.

To isolate individual dorsal neurons in the spinal cord, a 7 µm tipped glass-coated carbon fiber

microelectrode (Carbostar-1, Kation Scientific) was lowered into the spinal cord. Stroking of the

hindlimb, and/or hindpaw was used as a search stimulus. Wide dynamic range neurons responding to both

brush and to pinch, and located at a depth of 170 µm or less (laminae I-III) from the surface of the spinal

cord were used for this analysis. Once isolated, an individual neuron’s receptive field was mapped and

characterized using a Windsor and Newton 0 brush (brush) and Dumont fine tooth forceps (pinch).

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Brushing of the receptive field was repeated 5 times in succession, while pinch was repeated three times,

with each 2 sec stimulus interrupted by a 2 sec gap. Brushing of the hairy skin comprised of light strokes

to displace the hair with touching the skin. The glabrous skin was gently stroked on the plantar surface of

the hindpaw. The mean number of spikes fired during each stimulus, as well as the mean number of

spikes fired between each stimulus (the afterdischarge) was used for analysis.

Recording and analysis was performed using a Digidata series 1322A digitizer and pClamp 10.4 software.

Statistical analyses and graphing were performed using GraphPad Prism. One-way Kruskall Wallis

ANOVAs followed by a Dunn’s multiple comparison posttest or a two-way ANOVAs followed by

Bonferroni posttest were performed to determine significant values. A 95% confidence interval was used

as a measure of statistical significance for all data. All animals were euthanized with an overdose of

anesthetic at the end of the experiment.

Retrograde Cholera Toxin-B Labeling of Cutaneous Sensory Neurons

P30 NPY::Cre; R26LSL-tdTomato mice were anesthetized by isoflurane and 0.5 µl of CTB-488 (2.5 µg/µl) was

injected into the hairy skin of the hindlimb with a fine glass capillary. 3 days after injecting CTB, the

spinal cord and dorsal root ganglia (DRG) were dissected out and used to visualize CTB-labeled sensory

neurons and their afferent terminals in the lumbar dorsal horn.

Rabies Virus Tracing

NPY::Cre; Lbx1FlpO; R26ds-HTB mice (P7) were injected at the lumbar spinal cord. Mice were anesthetized

by isoflurane and an incision was made in the skin above the upper lumbar region of the spinal cord and a

laminectomy was performed at the T13-L1 level. After removing the dura mater with a fine needle and

exposing the spinal cord, a fine glass capillary was inserted on the left side of the dorsal spinal cord. Focal

injections of EnvA-pseudotyped, G-deleted-mCherry rabies virus (250 nl; ~1x109 units per ml) were made

into the dorsal cord to target NPY::Cre neurons in laminae II-IV. The skin was then closed using tissue

adhesive and a Reflex skin closure system. Animals were perfused 6 days post-injection and processed for

immunostaining.

Statistical Analysis

All data are presented as the mean ± standard error of the mean (SEM) with n indicating the number of

mice analyzed. Statistical analyses were performed by two-tailed, unpaired Student’s t test or ANOVAs.

P< 0.05 was considered to be statistically significant.

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SUPPLEMENTARY FIGURES

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Fig. S1. Selectivity of NPY::Cre IN ablation.

Sections through the lumbar dorsal spinal cord of P60 control and NPY::Cre IN-ablated mice showing the

quantification of tdTomato/NeuN, Pax2, NPY, nNOS, dynorphin and parvalbumin cells in laminae I-IV.

(A-C) Analysis of NPY::Cre ablation showing a ~70% reduction in NPY-tdTomato+/NeuN+ INs in

NPY::Cre IN-ablated mice (control, 99.20 ± 3.9 cells; NPY::Cre IN-ablated, 28.82 ± 3.8 cells; n=3 cords;

*** P<0.001). This closely matches the number of NPY::Cre INs that display Lbx1FlpO mediated

recombination (74.3%). (D-F) Pax2+ cell numbers were reduced by ~53% (control, 111.6 ± 4.50;

NPY::Cre IN-ablated, 52.5 ± 4.12; n=3 cords; ***P<0.001). (G-I) NPY expression in lamina I-IV was

reduced by ~63%. NPY expression was determined by densitometry scanning of sections stained with an

antibody against the NPY peptide. (J-R) nNOS+ (control, 38.86 ± 4.33; NPY::Cre IN-ablated, 37.64 ±

3.07; n=3 cords; P>0.05), dynorphin+ (control, 29.59 ± 2.16; NPY::Cre IN-ablated, 25.29 ± 2.49; n=6

cords; P>0.05) and parvalbumin+ (control, 41.40 ± 1.29; NPY::Cre IN-ablated, 45.01 ± 1.03; n=3 cords;

P>0.05) cell numbers are unchanged. P values were calculated using the Student’s unpaired t-test. Scale

bars: 50µm

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Fig. S2. Excitatory dorsal horn IN cell types are spared following NPY::Cre IN ablation.

(A-L) Sections through the lumbar dorsal spinal cord of P60 control and NPY::Cre IN-ablated mice

showing Lmx1b (antibody), somatostatin (in situ), Tac2 (in situ) and PKCγ (antibody) expression.

Lmx1b+ cell numbers (control: 167.4 ± 6.35; NPY::Cre IN-ablated: 159.8 ± 3.87; n=3 cords; P>0.05).,

somatostatin+ cell numbers (control: 135.4 ± 5.78; NPY::Cre IN-ablated: 129.1 ± 6.13; n=3 cords;

P>0.05), Tac2+ cell numbers (control: 31.68 ± 1.68; NPY::Cre IN-ablated: 30.39 ± 0.03; n=3 cords;

P>0.05) and PKCγ+ cell numbers (control: 27.32 ± 2.22; NPY::Cre IN-ablated: 27.98 ± 1.00; n=4 cords;

P>0.05) were all unchanged. P values were calculated using the Student’s unpaired t-test. p values above

0.05 are not significant (ns). Scale bars: 50µm.

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Fig. S3. Ablation of dorsal horn NPY::Cre INs does not affect sensory afferent innervation or other

NPY::Cre expressing neuronal cell types.

(A) The central afferents terminals of nociceptive peptidergic (CGRP+) and non-peptidergic (IB4+)

sensory neurons show similar terminal arborization patterns in control and NPY::Cre IN-ablated mice. (B)

tdTomato+ cells marked by NPY::Cre-tdTomato that lie outside of the medulla and spinal cord are not

affected in the NPY::Cre IN-ablated mice due to the restricted expression of Lbx1. Note the reduced

expression of tdTomato in the spinal trigeminal nucleus (arrow) which is due to the expression of Lbx1 in

these INs during development (39). The residual band of tdTomato expression in the NPY::Cre IN-ablated

hindbrain is due to the expression of NPY::Cre in trigeminal sensory neurons. Me5, mesencephalic

trigeminal sensory nucleus. Scale bars: 50 µm (A), 100 µm (B).

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Fig. S4. Behavioral analysis of NPY::Cre IN-ablated mice.

(A) General motor coordination as assessed by the accelerating rotarod test is unchanged in NPY::Cre IN-

ablated mice compared to control littermates (control, n=12, ablated, n=12; P>0.05). Responses to acute

mechanical pain using the Randall-Selitto test and light mechanical pain using the von Frey test do not

differ between control and NPY::Cre IN-ablated mice (control n=17, ablated n=12; P>0.05). The response

to dynamic light touch stimuli with the brush test is similar in control and NPY::Cre IN-ablated mice

(control n=17, ablated n=12; P>0.05). Responses to radiant heat pain by using the Hargreaves test do not

  15  

differ between control and NPY::Cre IN-ablated mice (control n=17, ablated n=12; P>0.05).

Thermosensitivity tested using the hot plate test do not differ between control and NPY::Cre IN-ablated

mice (control n=13, ablated n=8; P>0.05). Responses (forepaw flinching and hindpaw licking) to noxious

cold by using the cold plate test do not differ between control and NPY::Cre IN-ablated mice (control

n=13, ablated n=8; P>0.05). Responses to cooling using the acetone evaporative cooling test do not differ

between control and NPY::Cre IN-ablated mice (Control n= 10, ablated n=9; P>0.05). (B) The response to

capsaicin as measured by the cheek assay does not differ between control and NPY::Cre IN-ablated mice

(control n= 4, ablated n=4; P>0.05).

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Fig. S5. R26ds-hM4D-tdTomato allele and behavioral analysis of NPY::Cre IN-silenced mice.

Map showing the targeting vector used to generate the knockin R26ds-hM4D-tdTomato allele in mice. (A)

Targeting vector. (B) Targeted R26 allele. (C) The response to light mechanical pain using the von Frey

test does not differ between control and NPY::Cre IN-silenced mice (control n=10, silenced n=8; P>0.05).

  17  

The response to dynamic light touch stimuli with the brush test was similar in control and NPY::Cre IN-

silenced mice (control n=9, silenced n=8; P>0.05). Response to radiant heat pain as assessed by the

Hargreaves test does not differ between control and NPY::Cre IN-silenced mice (control n=10, silenced

n=8; P>0.05). (D-E) After peripheral inflammation by CFA treatment, control and NPY::Cre IN-silenced

mice show similar withdrawal thresholds to static stimuli as assessed by the von Frey assay and dynamic

allodynia score as measured by the brush assay (control n=4, silenced n=4; P>0.05). Data: mean ±SEM, P

values were calculated using the Student’s t-test and are not significant (ns) above 0.05.

Fig. S6. Ablation of GRPR-expressing neurons with bombesin-saporin.

(A and B) Sections through the dorsal spinal cord of mice 2 weeks after intrathecal injection of saline (A)

or bombesin-saporin (B). Note the absence of GRPR expression in mice injected with bombesin-saporin

(BOM-saporin). Scale bar: 50 µm.

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Fig. S7. NPY::Cre INs are innervated by low-threshold mechanoreceptors.

(A-B) Whole-mount immunostaining of hairy skin from Pitx2-EGFP mice showing Pitx2-EGFP+ neurons

are Aβ-LTMs that form longitudinal and transverse lanceolate endings associated with hair follicles. (C)

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Section through dorsal root ganglion of Pitx2-EGFP mice stained with antibodies to GFP and Ret showing

that GFP is expressed in Ret+ LTMs. (D-F) Dorsal root ganglia sections from a P30 NPY::Cre-tdTomato

mouse 3 days after injecting CTB into the hairy skin. Sections were stained with the indicated markers.

CTB (green) efficiently labeled cMaf+, TrkB+ and TH+ LTM subtypes that are known to innervate hairy

skin. (G-Gb) Section of lumbar dorsal spinal cord of P30 NPY::Cre-tdTomato mice 3 days post injection

of CTB in hairy skin stained with indicated markers. CTB+/vGluT1+ sensory terminals of Aβ- and Aδ-

LTMs are found in laminae III/IV in close apposition to NPY::Cre-tdTomato+ cell bodies (a).

CTB+/vGluT1– C-LTM terminals form putative contacts with NPY::Cre-tdTomato+ neurons in lamina II

(b). Arrows mark putative A-LTM terminals. Arrowheads indicate putative C-LTM terminals. (H).

Section through the lumbar dorsal spinal cord of P20 NPY::Cre; Lbx1FlpO; R26ds-HTB stained with

antibodies to GFP (green) and NeuN (blue) showing expression of GFP in superficial dorsal horn. These

cells express nuclear GFP (derived from the R26ds-HTB reporter allele, (32)), the TVA receptor and the

rabies B19 glycoprotein. They are thus competent for infection and transsynaptic transport of EnvA-

pseudotyped rabies virus. (I) Strategy used to infect NPY::Cre INs in dorsal spinal cord. (J) Section of

dorsal spinal cord of P13 NPY::Cre; Lbx1FlpO; R26ds-HTB stained with an antibody to mCherry (red), GFP

(green) and PKCγ (blue). Note that the infected NPY::Cre INs (GFP+/mCherry+) are primarily located in

lamina III/IV (arrows). Scale bars, 20 µm (A,B), 50 µm (C,J), 10 µm (D-G), 100 µm (H).

  20  

Fig. S8. In vivo recording from dorsal horn neurons.

(A) In vivo recordings from lumbar dorsal spinal cord neurons in laminae I-III with glabrous or hairy skin

receptive fields. There is no significant increase in spontaneous activity between control and NPY::Cre

IN-ablated mice, as measured prior to stimulation of the receptive field (RF) for 5 minutes. Control

(glabrous RF), 0.52±0.18 spikes/sec, n= 6; NPY::Cre IN-ablated (glabrous RF), 0.61±0.4 spikes/sec, n=8;

control (hairy RF), 0.21±0.18 spikes/sec, n=6; NPY::Cre IN-ablated (hairy RF), 1.51±1.02 spikes/sec,

n=8; Kruskal-Wallis non-parametric one-way ANOVA: P= 0.31). (B) In vivo recordings from dorsal

  21  

spinal cord neurons in laminae I-III with hairy skin receptive fields showing the response to noxious

stimulation (pinch). These recordings demonstrated an equivalent mean number of spikes fired during

active pinch (control: 19.7 ± 3.7 spikes/sec, n=7; NPY::Cre IN-ablated: 16.8 ± 3.3 spikes/sec, n=7,

Kruskall-Wallis one way ANOVA, P=0.7) and afterdischarge firing (control: 6.1 ± 6.1 spikes/sec, n=7;

NPY::Cre IN-ablated: 6.3 ± 0.6 spikes/sec, n=7, two way ANOVA, P=0.2) in NPY::Cre IN-ablated and

control mice. (C) In vivo recording from dorsal spinal cord neurons with a glabrous skin receptive field

showing the response to noxious stimulation (pinch). These also show an equivalent mean number of

spikes fired during active pinch (control: 21.6 ± 6.2 spikes/sec, n=7; NPY::Cre IN-ablated: 14.8 ± 3.2

spikes/sec, n=5, Kruskall-Wallis one way ANOVA, P=0.63) and afterdischarge firing (control: 0.4 ± 0.2

spikes/sec, n=5; NPY::Cre IN-ablated: 0.2 ± 0.2 spikes/sec, n=7, two way ANOVA, P=0.51) in NPY::Cre

IN-ablated mice as compared to control mice.

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