Post on 13-Feb-2020
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
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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
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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).
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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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