DOI: 10.5061/dryad.bnzs7h4m5
title: Data computational model parameters of nociceptive medullary dorsal horn projection neuron from: Opposite regulation of medullary pain-related projection neuron excitability in acute and chronic pain

description: Pain hypersensitivity is associated with increased activity of peripheral
 and central neurons along the pain neuroaxis. We show that at the peak of
 acute inflammatory pain, superficial medullary dorsal horn projection
 neurons (PNs) that relay nociceptive information to the parabrachial
 nucleus reduce their intrinsic excitability and, consequently, action
 potential firing. When pain resolves, the excitability of these neurons
 returns to baseline. Using electrophysiological and computational
 approaches, we found that an increase in potassium A-current (IA)
 underlies the decrease in the excitability of medullary dorsal horn PNs in
 acute pain conditions. In chronic pain conditions, no changes of IA were
 observed, and medullary dorsal horn PNs exhibit increased intrinsic
 excitability and firing. Our results reveal an opposite modulation of the
 excitability of medullary dorsal horn projection neurons in acute and
 chronic pain conditions, suggesting a regulatory mechanism that, in acute
 pain conditions, fine tunes the output from the dorsal horn network and,
 if lacking, could facilitate pain chronification.
description: <strong>Methods</strong>
 <strong>Animals </strong> Adult
 (9-13 weeks) C57BL/6J mice were used in all experiments unless stated
 otherwise. To specifically express Channelrhodopsin 2 (ChR2) in transient
 receptor potential vanilloid receptor 1 (TRPV1) expressing neurons, we
 used homogeneous TRPV1<sup>cre</sup> mice
 (<em>48</em>) that were purchased from the Jackson Laboratory
 (#017769) and bred them in-house. Animals were group-housed under a
 controlled temperature (21 ± 2°C) and humidity (55 ± 10%) on a 12h-12h
 light/dark cycle, with <em>ad libitum</em> access to food and
 water. All experimental procedures were approved by the Institutional
 Animal Care and Use Committees at the Hebrew University of Jerusalem
 (study approval number MD-21-16152).
 <strong> </strong>
 <strong>Pain models</strong>
 <em>Acute pain model: UV-photokeratitis
 model</em> Corneal photokeratitis was induced as
 previously described (<em>28</em>, <em>29</em>).
 Briefly, mice were anesthetized with a combination of 70 mg/kg ketamine
 and 0.8 mg/kg medetomidine, intraperitoneally (i.p.). Mice were placed on
 a heating pad, and one eye was exposed to UV-C radiation (254 nm, 450
 mJ/cm<sup>2</sup>), delivered through a UV light source
 (VL-4.C 230V 50/60Hz; Viber Lourmoat, Marne-la-Vallee, France), from a
 distance of 17 cm for 22 minutes. Following UV radiation, mice were
 injected with 1.3 mg/kg atipamezole for anesthesia reversal. Sham mice
 underwent the same procedure but without the UV radiation.
 <em>Chronic pain model: Chronic constriction injury of the
 distal infraorbital nerve (CCI-dION)</em> The
 CCI-dION model was performed as previously described
 (<em>49</em>, <em>50</em>). In short, mice were
 anesthetized with a combination of 70 mg/kg ketamine and 0.8 mg/kg
 medetomidine (i.p.) and subcutaneous injection of 1-2 mg/kg meloxicam for
 analgesia. Then, mice were placed on a heating pad, and the skin below the
 infraorbital foramen (3 mm) was shaved and cleansed using 10% betadine. A
 0.5 cm incision was made, and the nerve was gently exposed by blunt
 preparation. A chromic gut ligature (5-0) was loosely tied around the
 distal part of the ION. The skin incision was sutured, and mice were
 injected with 1.3 mg/kg atipamezole for anesthesia reversal. Sham mice
 underwent the same procedure of nerve exposure but without
 ligation. <strong>Behavioral
 tests</strong> All mice were handled for 5
 consecutive days before the behavioral experiments. For 3 days before the
 experiments, as well as during experimental days, mice were habituated to
 head restriction by the experimenter and to the behavioral room (≥30 min)
 and recording chambers used (≥10 min). The experimenter
 was blinded to mice groups in all the experiments and analyses.
 <em> </em><em>Assessment of ongoing pain:
 Eye-closing ratio </em> The eye-closing ratio is
 a measurement of the orbital tightening used to quantify ongoing pain
 <em>(51–53)</em>. The eye-closing ratio was calculated as the
 ratio of the height and width of the tested eye. The height was defined as
 the distance between the edge of the upper and lower eyelids, and the
 width as the distance between the internal and external canthus. For
 eye-closing ratio measurements over the course of corneal
 UV-photokeratitis, mice were head-restricted and photographed. For
 eye-closing ratio measurements in the CCI-dION model, measurements were
 done 4 weeks following the CCI-dION / sham procedure. On a separate set of
 experiments, measurements were taken for baseline, 1, 2, and 7 days
 following the CCI-dION / sham procedure.  
 <em>Assessment of nociceptive
 hyperalgesia</em> To assess the changes in
 sensitivity to a noxious stimulus, behavioral responses produced by
 corneal instillation of capsaicin were recorded under control and acute
 pain conditions. After habituation, mice were treated with a 10 µl drop of
 vehicle (0.1% ethanol in standard extracellular solution, SES) onto the
 tested eye and video recorded for 5 minutes in the recording chamber (a
 custom-made 10×20×25 cm transparent plexiglass). SES was composed of (in
 mM): 145 NaCl, 5 KCl, 2 CaCl<sub>2</sub>, 1
 MgCl<sub>2</sub>, 10 glucose, and 10 HEPES. One hour later, 10
 µl of 1 µM capsaicin (in SES) was applied to the tested eye, and the mouse
 was video recorded for 5 minutes in the recording chamber. Eye wipes were
 manually counted post hoc. Mice (N=2) that showed apathetic behavior,
 manifested by reduced motor activity, were noted by the blinded
 experimenter and were excluded from the analysis. Mice (N=2) that showed
 no eye wipe responses to both vehicle and capsaicin in baseline (day 0)
 were excluded from the analysis. In the trials in which mice were not in
 front of the camera, preventing correct detection of eye wipes for at
 least 30 seconds out of the first minute following capsaicin application,
 were excluded from analysis. <em>Assessment of
 mechanical allodynia </em> To assess changes in
 sensitivity to innocuous mechanical stimulus, withdrawal responses to von
 Frey filaments applied to the middle area of the whisker pad were measured
 in mice in sham and CCI-dION conditions. Mechanical sensory thresholds
 were assessed using von Frey filaments of 0.07g to 6g by the simplified
 up-down (SUDO) method (<em>54</em>) while immobilizing the
 mice through neck pinch. A positive response was recorded if the mouse
 withdrew its head in 3 out of 5 trials for each filament.
 <em>Assessment of nocifensive behavior using optogenetics
 in freely behaving mice</em> 4-6 weeks after TG
 injections, for 3 consecutive days prior to the experiment, mice were
 taken to the behavioral room, where we inserted into their home cage a
 custom-made miniature netted apparatus (size of 11×6×6 cm) for mice
 acclimatization with the apparatus and the room. After 1 hour, each
 individual mouse was put inside the apparatus for an additional 1 hour.
 2-3 days prior to the experiment, the mice's cranium was shaved for
 the experiment. First, mice were anesthetized with a combination of 70
 mg/kg ketamine and 0.8 mg/kg medetomidine (i.p.). Mice's cranium was
 carefully shaved, and the depilation was completed with hair removal
 cream. Finally, mice were injected with 1.3 mg/kg atipamezole for
 anesthesia reversal. On an experimental day, mice were taken to the
 behavioral room and acclimatized to the testing apparatus, as explained
 above. A 470-nm ultra high power collimated LED (Prizmatix, Israel)
 coupled to a multimode optical fiber (1 mm core diameter, 1-m length,
 prizmatix, Israel) was used for light stimulation (0.5-5
 mW/mm<sup>2</sup>) onto the cranium of the tested mouse. The
 optical fiber tip was carefully positioned above the cranium with extra
 care so as not to touch the skin. We waited at least 30 seconds between
 light stimulations. Each combination of light intensity and duration was
 repeated 3-5 times. Experiments were video recorded and analyzed post hoc.
 For analysis, the percentage of withdrawal response was calculated for
 each combination of light intensity and duration. 1-3 days after the
 experiment, all tested mice were validated for successful viral infection
 by imaging the expression of EYFP in the fibers innervating the cornea
 using <em>in-vivo </em>corneal imaging
 (<em>55</em>, <em>56</em>). We used the expression
 of the EGFP in the corneal afferents as evidence for the successful
 expression of the virus in TG neurons and nerve endings. Mice that did not
 show expression of the fluorophore in the corneal terminals were excluded
 from the analysis. During behavioral experiments and analysis, the
 experimenter was blinded to ChR2 / control injections.
 <strong> </strong>
 <strong>Stereotactic injections</strong>
 5-6 weeks old mice were anesthetized with a combination of 60
 mg/kg ketamine, 0.5 mg/kg medetomidine, and 0.4-0.6% isoflurane (i.p.),
 pretreated with a subcutaneous injection of 1-2 mg/kg meloxicam and placed
 on a heating pad in a stereotactic frame (KOPF instruments). The cranium
 was shaved, and the skin was cleansed using 70% ethanol and 10% betadine.
 A 1-1.5 cm midline incision was made, followed by drilling a small hole in
 the skull using a fine drill burr. Unilateral injections to the PBN were
 delivered at the following coordinates (from bregma): ML: 1.3 mm, AP: −5.2
 mm, DV: −3.75 mm using a microliter syringe (35g nanofil needle, World
 Precision Instruments, Sarasota, FL) loaded with retro-AAV-CaMKII-tdTomato
 (titer 4.5×10<sup>12</sup> genome copies per ml, ELSC vector
 Core Facility, Israel). 250 nl of the virus was injected at 100nl/min via
 UltraMicroPump (World Precision Instruments, Sarasota, FL). Injections to
 the TG were delivered at the following coordinates (from bregma): ML: 1.35
 mm, AP: 0.4 mm, DV: −6.38. For TG injections, a pulled glass pipette was
 loaded with AAV1-EF1a-double floxed-hChR2(H134)-EYFP-WPRE-HGHpA or
 AAV1-EF1a-DIO-EYFP (titer 6×10<sup>11</sup> -
 7×10<sup>11</sup> genome copies per ml, gc
 ml<sup>-1</sup>) (Addgene, #20298, #27056 and ELSC vector Core
 Facility, Israel), and ~2µL/ganglion were injected using pressure. For
 both TG and PBN injections, pipette/needle were left at the injection site
 for an additional 5-10 minutes before being slowly retracted. Finally, the
 incision was sutured using chromic gut ligature (5-0) or glued by tissue
 adhesive (VetBond), and 1 mg/kg atipamezole was injected for anesthesia
 reversal.<strong> </strong>For both electrophysiological and
 immunohistological experiments, we injected the retro-AAV into either the
 right or left PBN and examined the corresponding ipsilateral medullary
 dorsal horn (<em>24</em>,
 <em>47</em>).<strong> </strong>Dual injections to
 the PBN and TG (<strong>fig. S1D</strong>) were performed
 unilaterally, such that both injections were performed on the same side
 (right or left). For optogenetics behavioral experiments, the virus
 containing ChR2/EYFP was injected bilaterally.
 <strong> </strong><strong>Electrophysiological
 recordings</strong> <em>Slice
 preparation</em> 3-7 weeks after stereotactic
 injections, mice were deeply anesthetized with isoflurane, decapitated and
 the brain stem was quickly removed into a warm (34°C) artificial
 cerebrospinal fluid (aCSF) solution containing (in mM): 126 NaCl, 3 KCl,
 1.3 MgSO<sub>4</sub>, 1.2
 KH<sub>2</sub>PO<sub>4</sub>, 26
 NaHCO<sub>3</sub>, 10 glucose, 2.4
 CaCl<sub>2</sub>, saturated with 95%
 O<sub>2</sub>/5% CO<sub>2</sub>. 300 µm coronal
 slices of caudal brain stem containing the medullary dorsal horn were
 prepared using a vibratome (VT 1200S, Leica). Slices were incubated in
 warm (34°C) aCSF solution for 1 hour, after which they were maintained at
 room temperature (23 ± 2°C) until recording.
 <em>Whole-cell recordings</em>
 Slices were transferred into a recording chamber and continuously
 perfused with oxygenated aCSF (the same as cutting solution). PNs of the
 medullary dorsal horn were visualized using differential interference
 contrast infrared microscopy (BX61WIF, Olympus, Tokyo, Japan) and
 identified by location and tdTomato expression using a CoolLed
 fluorescence excitation system. Borosilicate fire-polished patch pipettes
 (4-6 MΩ, pulled on a P-1000 puller, Sutter Instrument, USA) were used for
 all experiments. Signals were amplified using Multiclamp 700B (Molecular
 Devices) and digitized at 20-50 kHz (and low-pass filtered at 3 kHz) with
 Digidata 1440A (Molecular Devices) interfaced with pClamp 10.3 (Molecular
 Devices). Electrode series resistance (R<sub>s</sub>) was
 routinely checked, and recordings were discarded if: (1) R<sub>s
 </sub>was more than 30 MΩ; (2) R<sub>s</sub> changed
 &gt;15% during recordings, and (3) if resting membrane potential (RMP)
 was higher than −50 mV. <em>Current clamp
 recordings</em> The intrinsic properties of
 medullary dorsal horn PNs were recorded using internal solution containing
 either (in mM): 130 K-gluconate, 4 Na<sub>2</sub>ATP, 0.5
 NaGTP, 20 HEPES, 0.5 EGTA, 5 KCl (internal solution 1, 283 mOsm, pH was
 adjusted to 7.3 with KOH), or (in mM): 130 K-gluconate, 4 MgATP, 0.3
 NaGTP, 10 HEPES, 0.5 EGTA, 5 KCl, 10
 Na<sub>2</sub>-phosphocreatine (internal solution 2, 291 mOsm,
 pH was adjusted to 7.3 with KOH). The recordings with either solution 1 or
 2 produced similar results, and we combined the data from both
 experimental sets. <em>The resting membrane
 potential </em>(<em>RMP)</em> was measured within 1-2
 minutes after seal breaking. Once the membrane potential was stable, 3-10
 seconds of the membrane potential was averaged and determined as the
 RMP. To calculate <em>input resistance
 (R<sub>in</sub>)</em>, the neuron was held approximately
 at −60 mV, and a hyperpolarizing step of −10 pA for 400 ms was given.
 R<sub>in</sub> was calculated by dividing the voltage response
 at the steady state by the current injection amplitude. At least 2
 repetitions were used and averaged to calculate
 R<sub>in</sub>. For the
 <em>f-I</em> (frequency-intensity) curve, the membrane of
 medullary dorsal horn PNs was held approximately at −70 mV, and a 500 ms
 current step was given at 20 pA increments every 10 seconds. Firing type
 was classified by the following criteria (<em>57</em>): (1)
 tonically firing cells showed AP discharge throughout the current
 injection; (2) delayed firing cells had a delayed onset to the first AP
 and a hyperpolarizing notch after the initial depolarization at the start
 of the current injection; (3) phasically firing cells ceased AP firing in
 the second half of the current injection or showed only an initial burst
 of APs; (4) H-current cells showed a sag potential that peaked at ~55 ms
 following termination of the current injection; (5) Cells that could not
 be classified by one of the criteria above, were named unidentified. Only
 the properties of medullary dorsal horn PNs exhibiting delayed firing
 patterns were analyzed. <em>Action potential (AP)
 frequency</em> was calculated by dividing the number of APs by the
 duration of the current injection (500 ms).
 <em>Instantaneous firing frequency</em> was
 calculated as the reciprocal for the inter-spike intervals (ISIs) and
 averaged for each current step. <em>Regularity
 index </em>was calculated by dividing the average instantaneous
 firing frequency by the regular firing frequency described
 above. <em>Coefficient of variation (CV) of
 firing </em>was calculated by dividing the standard deviation of the
 ISIs by the average of the ISIs. <em>Adaptation
 ratio </em>was calculated as the ratio of the first and last
 instantaneous firing frequency.
 <em>f-I</em> <em>curve slope</em> was
 calculated by extracting the slope of the linear fit for the firing
 frequencies starting in the pulse preceding the first action potential
 firing. <em>Latency to the first AP</em>
 was calculated as the time interval between the beginning of the current
 injection and the time point of the first AP threshold.
 To calculate <em>AP threshold, half-width, and
 after-hyperpolarization (AHP), </em>the<em> </em>first
 spike of the first suprathreshold voltage response in the
 <em>f-I</em> protocol was analyzed. <em>AP
 threshold</em> was calculated using a phase plot analysis as
 previously described (<em>14</em>). The point in the phase
 plot where the increase in dV/dt was larger than 8 mV/ms was determined as
 the AP threshold. <em>AP half-width</em>
 was determined as the width at half amplitude between the AP threshold and
 peak. <em>AP afterhyperpolarization
 (AHP)</em> was determined as the difference between the AP threshold
 and the minimal membrane potential 5-10 ms after the AP peak.
 <em>AP amplitude</em> and
 <em>dV/dt<sub>max</sub></em> were calculated for
 APs evoked by a short, 10 ms, current steps increasing by 10 pA increments
 from a holding potential of approximately −70 mV. The AP of the first
 suprathreshold voltage response was analyzed. The AP Amplitude was
 determined as the amplitude between the AP threshold and peak.
 <em>dV/dt<sub>max</sub></em> was determined as the
 maximal point of phase plot analysis.
 <em>Capacitance </em>was calculated by first
 extracting the time constant from the protocol used to measure input
 resistance. To determine the time constant, we fitted a 1-exponential fit
 on the voltage response. Capacitance was calculated by dividing the time
 constant by input resistance. Recordings of AP firing
 before and after application of 5 mM 4-aminopyridine (4-AP) were performed
 in the presence of 10 µM NBQX to block excitatory postsynaptic potentials
 (EPSPs). The recordings were performed at least 5 minutes following the
 perfusion of 4-AP. <em>Voltage-clamp
 recordings</em> For voltage-clamp recordings,
 data were sampled at 50 kHz and low pass filtered at 2 kHz. Recordings
 commenced at least 3 minutes after breaking the seal. Capacitive transient
 and leak currents were not compensated, and recordings were discarded if
 R<sub>s </sub>was &gt;20 MΩ or changed &gt;15% during
 recordings. For I<sub>A</sub> recordings,
 0.5 µM tetrodotoxin (TTX), 100 µM CdCl<sub>2</sub>, 10 µM
 NBQX, and 100 µM picrotoxin were added to bath solution to block
 voltage-gated Na<sup>+</sup> currents,
 Ca<sup>2+</sup> currents, excitatory and inhibitory
 postsynaptic currents (EPSCs and IPSCs), respectively. The internal
 solution contained (in mM): 130 K-gluconate, 4 MgATP, 0.3 NaGTP, 10 HEPES,
 0.5 EGTA, 5 KCl, 10 Na<sub>2</sub>-phosphocreatine (291 mOsm,
 pH was adjusted to 7.3 with KOH). <em>To isolate
 I<sub>A</sub></em>, membrane voltage was held at –80 mV
 in voltage-clamp mode. A two-step voltage protocol was used to determine
 the voltage-dependence of activation with a series of 500 ms depolarizing
 voltage steps in +10 mV increments to a maximum of +40 mV at 8-second
 intervals (<strong>fig. S11A</strong>,<em>
 left</em>). Next, a corresponding paired voltage step was given,
 consisting of the same voltage command but with a 150 ms prepulse to −10
 mV (<strong>fig. S11A</strong>,<em> right</em>).
 I<sub>A </sub>was extracted by the digital subtraction of each
 pair of pulses. I<sub>A</sub> amplitudes were calculated from
 the isolated I<sub>A</sub> traces as the current difference
 between the minimal current after the transient capacitive current and the
 peak of the current. <em>Voltage-dependence of
 I<sub>A</sub></em> <em>activation</em>:
 I<sub>A</sub> amplitudes were converted to conductances using
 the equation g = I/(V<sub>m</sub> –
 V<sub>rev</sub>) where V<sub>m</sub> is the
 membrane voltage at the voltage step, and V<sub>rev</sub> is
 the calculated K<sup>+</sup> reversal potential (−88.85 mV).
 Normalized conductances were fitted with a Boltzmann equation:
 g/g<sub>max</sub> = 1/(1+exp((V<sub>1/2</sub> – V)
 /<em>k</em>)), where g<sub>max</sub> is the
 maximal conductance, V<sub>1/2</sub> is the half-maximal
 voltage and <em>k</em> is the slope factor.
 <em>Voltage-dependence of
 I<sub>A</sub></em> <em>inactivation</em> was
 determined using incremental conditioning command of 150 ms prepulses from
 −100 mV to +20 mV, followed by a voltage step to +40 mV for 500 ms at
 8-second intervals (<strong>fig. S11B</strong>). All traces
 were subtracted with the trace obtained at the final voltage step to zero
 residual currents. Current amplitudes were calculated as the difference
 between the peak amplitude and the mean steady-state current in the last
 20 ms of the voltage command. Normalized currents were fitted similarly
 with the Boltzmann equation: I/I<sub>max</sub> =
 1/(1+exp((V<sub>1/2</sub> – V) /<em>k</em>)),
 where I<sub>max</sub> is the maximal current,
 V<sub>1/2</sub> is the half-maximal voltage and
 <em>k</em> is the slope factor.
 <em>I<sub>A </sub>window current </em>was
 calculated as the area under the activation and inactivation curves for
 each cell.  I<sub>A </sub>kinetics were
 quantified for the isolated I<sub>A</sub> traces at the peak
 conductance (+10 mV). Decay current was fitted with 1 or 2 exponents,
 depending on the fit with the minimal residual sum of squares between the
 data and the fit. For most cells, 2 exponents resulted in the best fit
 (Control: 7/9; Inflammation: 6/10; Sham: 8/14; CCI-ION: 5/7).
 <em>I<sub>A</sub> charge</em> was
 calculated as the area under the curve (integral) of the smoothed current
 response to the +10 mV voltage step. For recording
 excitatory and inhibitory synaptic inputs following optogenetic
 stimulation of TRPV1-expressing afferents, we used an internal solution
 that contained (in mM): 115 Cs<sup>+</sup>-methansulfonate, 4
 MgATP, 0.3 NaGTP, 10 HEPES, 1 EGTA, 1.5 MgCl<sub>2</sub>, 10
 Na<sub>2</sub>-phosphocreatine, 2 QX 314-Cl, 10 BAPTA
 tetracesium (297 mOsm, pH was adjusted to 7.3 with CsOH). To isolate
 evoked excitatory and inhibitory synaptic responses, the membrane voltage
 was held at −70 mV and 0 mV, respectively. A 470-nm ultra high power
 collimated LED (Prizmatix, Israel) was used for light stimulation under
 the control of Master-9 (A.M.P.I, Israel). For the stimulation, we used
 the parameters that evoked nocifemsive responces <em>in
 vivo</em> (10 ms, 5 mW/mm<sup>2</sup>). Light was
 delivered through the optical path of the microscope and the ×40
 objective. For each holding potential, five optogenetic stimulation were
 given with &gt;30 seconds intervals between stimulation. We classified
 the response type to optogenetic stimulation by the following criteria:
 (1) an excitatory response was determined for neurons that showed 5/5
 responses of inward currents (at a holding potential of −70 mV) with peak
 amplitudes that were &gt;15 pA and &lt;20 ms latencies (2) an
 inhibitory response was determined for neurons that showed 5/5 responses
 of outward currents (at a holding potential of 0 mV) with peak amplitudes
 that were &gt;5 pA and because of the polysynaptic nature of the
 events latencies of &lt;100 ms (3) excitatory and inhibitory response
 was determined for cells that passed both criteria 1 and 2.
 All electrophysiological data were analyzed using custom scripts
 in Matlab (MathWorks, Natick, MA, USA).
 <strong>Validation of functional expression of ChR2 in
 TRPV1-expressing TG neurons</strong> ChR
 expression (conjugated with EYFP, see above the “<em>Stereotactic
 injections</em>” section) and function in TRPV1-expressing TG
 neurons were evaluated by recording from acutely dissociated
 EYFP-expressing TG neurons and behavioral assays (detailed above in the
 “<em>Behavioral tests</em>” section).
 <em>Electrophysiological recordings from EYFP-expressing
 acutely dissociated TG neurons</em>
 <em>1.     </em><em>Trigeminal ganglion cell
 culture preparation</em> Primary trigeminal
 ganglion (TG) neurons were isolated from TRPV1Cre-ChR2 mice 4-6 weeks
 following stereotactic injections. TG neurons (of both right and left TGs)
 were removed and placed into DMEM with 1% penicillin-streptomycin, then
 digested in 5 mg/ml collagenase, 1 mg/ml Dispase II (Roche). Cells were
 triturated with Pasteur pipettes in the presence of DNase I (250 U) and
 centrifuged through 15% BSA. The cell pellet was re-suspended in 1 ml
 Neurobasal media, containing B27 supplement (Invitrogen), 1%
 penicillin-streptomycin, 10 mM AraC, 2.5S NGF (100 ng/ml, Promega), and
 GDNF (2 ng/ml). Cells were plated onto poly-D-lysine (100 g/ml) and
 laminin (1 mg/ml) coated 35 mm tissue culture dishes. Cells were kept in
 the incubator at 37°C with 5% carbon dioxide.
 <em>2.     </em><em> Whole-cell
 recordings</em> Recordings were performed from
 dissociated EYFP-expressing TG neurons, up to 24 hours after plating.
 Cells were visualized using an inverted eclipse Ti Nikon. Whole-cell
 membrane voltages were recorded using a Multiclamp 700B amplifier
 (Molecular Devices), at room temperature (24 ± 2°C). Data were sampled at
 20 kHz and were low-pass filtered at 500 Hz (−3 dB, 8 pole Bessel filter).
 Patch pipettes (2-5 MΩ) were pulled from borosilicate glass capillaries
 (1.5 mm/1.1 mm OD/ID, Sutter Instrument, USA) on a P-1000 puller (Sutter
 Instrument, USA) and fire-polished (LWScientific). Data were digitized
 with Digidata 1440A A/D interface (Molecular Devices), interfaced with
 pClamp 10.3 (Molecular Devices). Internal solution for
 whole cell patch recordings contained (in mM): 130 K-gluconate, 8 NaCl, 6
 KCl, 10 HEPES, 2 Mg-ATP (290 mOsm, pH 7.25 adjusted with KOH).
 The extracellular solution contained (in mM): 145 NaCl, 5 KCl, 1
 MgCl2, 2 CaCl2 10 HEPES, 10 D-Glucose, pH was adjusted to 7.4 with
 NaOH. Electrode series resistance (Rs) was routinely
 checked, and recordings were discarded if: (1) Rs was more than 20 MΩ; (2)
 Rs changed &gt;15% during recordings, and (3) if resting membrane
 potential (RMP) was higher than −50 mV. A 470-nm
 ultra-high power collimated LED (Prizmatix, Israel) was used for light
 stimulation under the control of pClamp 10.3 (Molecular
 Devices). Action potential firing was examined in
 current clamp mode, using short (5 ms) optogenetic stimulation (5
 mW/mm<sup>2</sup>), which was delivered through the optical
 path of the microscope and the ×40 objective. Next, we switched to voltage
 clamp mode in −70 mV holding potential and recorded light-evoked inward
 current in response to long (1 s) optogenetic stimulation. Finally,
 capsaicin (1 µM) was applied to determine that the neurons were
 TRPV1-sensitive nociceptors. 4 out of 5 EYFP-expressing neurons that
 responded to optogenetic stimulation by inward current and action
 potential firing also showed capsaicin-evoked inward current
 (<strong>fig. S1B</strong>).  
 <strong>Immunofluorescence
 staining</strong> For spontaneous c-Fos
 expression, mice perfused 2 days after sham or UV radiation. For evoked
 c-Fos expression, 2 days after sham or UV radiation, 10 µl of 1 µM
 capsaicin (in SES) was applied to the eye, and mice were put in a separate
 cage for 5 minutes before putting back in the home cage. Mice were
 perfused 90 minutes after capsaicin instillation. In both spontaneous and
 evoked c-Fos expression experiments, mice were not habituated or handled
 before the experiments. Mice were deeply anesthetized
 with isoflurane and perfused transcardially with 20 ml of cold
 phosphate-buffered saline (PBS) followed by 20 ml of cold 4%
 paraformaldehyde (PFA). Trigeminal ganglia (TGs) and brains were extracted
 and post-fixed in 4% PFA 2 hours and overnight, respectively, at 4°C.
 Next, tissues were rinsed in PBS (3×10 min), followed by cryoprotection in
 30% (w/v) sucrose in PBS for at least 2 nights at 4°C. Next, brains and
 TGs were frozen in OCT for sectioning. For TG staining, 15 µm
 free-floating TG sections were washed (3x10 min) in PBS. Following the
 initial washing steps, the sections were blocked with CAS-Block universal
 blocking agent (#008120, ThermoFisher Scientific) for 10 min at room
 temperature. The sections were then incubated in goat anti-c-Fos primary
 antibody (1:500, #sc-52-G, Santa Cruz Biotechnology) diluted in the same
 blocking buffer overnight at 4°C. On the next day, the slices were washed
 (3x10 min) in PBS and incubated with Alexa Fluor 488-conjugated donkey
 anti-goat secondary antibodies (1:1000, #705545147, Jackson
 ImmunoResearch) diluted in freshly prepared PBS in 0.3% Triton X-100
 (#X100, Sigma Aldrich) for 2 hours at room temperature. Final wash steps
 were performed (3x10 min) in PBS. All the above steps were carried out on
 a shaker with gentle shaking at 70 rpm. Finally, the sections were mounted
 onto the microscope slides and air-dried before covering them with
 VECTASHIELD antifade mounting media with DAPI (#EW9395224, Vector
 Laboratories). The slices were then coverslipped for imaging. Imaging was
 done with a confocal microscope (Nikon Ti Eclipse) using a 20X objective
 at a resolution of 2048 pixels. c-Fos expressing cells were identified
 using custom scripts in Cellprofiler (<em>58</em>) and
 validated by eye. For medullary dorsal horn staining, 30 µm free-floating
 sections (3-5 slices for each mouse) were washed in PBS and blocked in a
 solution containing 10% normal donkey serum and 0.3% Triton X-100 in PBS
 for 1 hour at room temperature. Rabbit anti-c-Fos primary antibody (1:500,
 #226 003, Synaptic Systems) was diluted in a solution containing 5% normal
 donkey serum and 0.3% Triton X-100 in PBS and incubated with sections
 overnight at 4°C. The following day, sections were rinsed in PBS (3x10
 min) and incubated with donkey anti-rabbit Alexa Fluor 647 (1:1000,
 ab150063, abcam) secondary antibody diluted in a solution containing 5%
 normal donkey serum and 0.3% Triton X-100 in PBS for 2 hours at room
 temperature. Next, slices were incubated in DAPI (2 µg/ml, 10236276001,
 Roche Merck) in PBS (10 min), followed by two additional washes in PBS
 (2x10 min). Finally, slices were mounted (Dako fluorescence mounting
 medium) and coverslipped. Slices were imaged using Olympus IX83P2ZF with a
 10x objective lens. For analysis, the medullary dorsal horn region was
 cropped for each slice using ImageJ. Using Cellprofiler custom scripts,
 c-Fos and tdTomato expressing cells were identified and validated by eye.
 For each mouse, the percentage of co-labeled cells was calculated as the
 number of c-Fos+ and tdTomato expressing cells from all slices out of the
 total number of tdTomato expressing cells or out of the total number of
 c-Fos+ cells. TG and medullary dorsal horn c-Fos stainings were done by an
 experimenter blinded to mice groups.
 <strong> </strong>
 <strong>Computational modeling</strong>
 Projection neurons were simulated using NEURON simulation
 environment. The mathematical parameters model are detailed in the
 Matherials and Methods section of the article and in the code in the
 database All model parameters and the complete code
 used for simulation are available on Git Hub.
  
 <strong>Statistics</strong>
 Experiments were performed with side-by-side control and in a
 random order. No power analysis was used to determine experimental sample
 sizes in advance, but sample sizes were similar to those reported
 previously. The number of mice, slices, and cells are indicated in the
 Supplementary Tables accompanying each figure. Parametric and
 non-parametric tests were used where appropriate. All statistical tests,
 statistics (<em>F</em> or <em>t</em>), and
 <em>P</em> values are reported in figure legends or in the
 Supplementary Tables accompanying each figure. For the statistical
 analysis of eye wipes over time (<strong>Fig. 2C</strong>,
 <em>middle</em>) we fitted a linear mixed-effects model
 (MATLAB, function <em>fitlme</em>) using <em>group, day,
 </em>and the interaction of them for fixed effects and
 <em>mouse ID </em>for a random effect. For the statistical
 analysis of <em>f</em>-I curves, latency to
 1<sup>st</sup> AP, and intrinsic properties between 3 groups
 (<strong>Fig. 4</strong>), we fitted a linear mixed-effects
 model using <em>group</em> or <em>condition</em>
 (for <strong>fig. S10B</strong>) for a fixed effect. In
 repeated measurements structured experiments (<em>f</em>-I
 curves and latency to 1<sup>st</sup> AP), we usually used
 <em>mouse ID</em>, <em>neuron ID, </em>and
 <em>current injection</em> for random effects unless stated
 otherwise in the statistics table. For intrinsic properties analysis in
 <strong>Fig. 4, B </strong>(<em>inset</em>),
 <strong>D</strong> (<em>right</em>)<strong>
 </strong>and <strong>E</strong>, we used <em>mouse
 ID </em>for a random effect. The random effects were varied
 specifically for each case to improve the fit of the model to the data.
 This was examined using <em>compare</em> function in
 MATLAB<em>.</em> All models are depicted in the statistics
 table. All statistical analyses were conducted with Matlab (MathWorks,
 Natick, MA, USA).    
description: # Source data for main and supplementary figures and computational model
 parameters of nociceptive medullary dorsal horn projection neuron from:
 Opposite regulation of medullary pain-related projection neuron
 excitability in acute and chronic pain ## Description of the data and file
 structure **For the Source DATA Dataset** **Title of Dataset:**\
 Electrophysiological and Behavioral Source Data for the Article: Opposite
 Regulation of Medullary Pain-Related Projection Neuron Excitability in
 Acute and Chronic Pain **Dryad DOI:**\
 [https://doi.org/10.5061/dryad.sqv9s4ncp](https://doi.org/10.5061/dryad.sqv9s4ncp) --- **Summary** This dataset contains the raw data underlying the figures in the manuscript, derived from experiments examining the biophysical regulation of medullary dorsal horn projection neurons in murine models of acute and chronic pain. The measurements include intrinsic electrophysiological properties, potassium A-type current characteristics, and behavioral indicators of pain hypersensitivity. ### Files and variables #### File: source\_data.xlsx **Description:**  **Structure:**\ Each sheet in the Excel file corresponds to a specific figure panel (e.g., 2B, 3E, S7A). Data include numerical values for action potential firing frequency, membrane properties, IA current parameters, and pain behaviors. Rows correspond to individual neurons or animals; columns represent specific measured features. **Naming Conventions:** * Sheet names correspond to figure panels (e.g., 2C, 5F, S4F) * Columns typically include experimental group (e.g., Sham, UV, CCI), measurement values, and sometimes metadata such as cell ID or mouse ID. **Missing Data Explanation:** Blank rows and missing labels in the header rows are intentional formatting elements used to delineate the title of each data table and to distinguish it from the associated dataset entries Missing values are designated as: * **NaN (Not a Number)** * **n/a:** (**not applicable**)  *Detailed* *Explanation of all missing cells:* Fig. 2C left – n/a represents points excluded by criteria depicted in methods, or due to camera recording failure. Fig. 2C right – n/a is added due to unequal number of mice between groups. Fig. 3B left – NaN (Not a Number) is filled in F-I data points that were not examined for some cells, due to the fear of losing the cell with overexcitation. Fig. 3B right – n/a is added due to unequal number of cells between groups. Fig. 3C – NaN (Not a Number) is filled in latency to first AP data points that either didn’t have latency (because trial wasn’t accompanied with firing) or it was not examined due to the fear of losing the cell with overexcitation. Fig. 3D – n/a is added due to unequal number of cells between groups. Fig. 3E – n/a is added due to unequal number of cells between groups. Fig. 4B left - NaN (Not a Number) is filled in F-I data points that were not examined for some cells, due to the fear of losing the cell with overexcitation. Fig. 4B right – n/a is added due to unequal number of cells between groups. Fig. 4C – NaN (Not a Number) is filled in latency to first AP data points that either didn’t have latency (because trial wasn’t accompanied with firing) or it was not examined due to the fear of losing the cell with overexcitation. Fig. 4D – n/a is added due to unequal number of cells between groups. Fig. 4E – n/a is added due to unequal number of cells between groups. Fig. 5E left - NaN (Not a Number) is filled in F-I data points that were not examined for some cells, due to the fear of losing the cell with overexcitation. Fig. 5E right – n/a is added due to unequal number of cells between groups. Fig. 5F – NaN (Not a Number) is filled in latency to first AP data points that either didn’t have latency (because trial wasn’t accompanied with firing) or it was not examined due to the fear of losing the cell with overexcitation. Fig. 5G – n/a is added due to unequal number of cells between groups. Fig. 5H – n/a is added due to unequal number of cells between groups. Fig. 6E – n/a is added due to unequal number of cells between groups. Fig. 6G – n/a is added due to unequal number of cells between groups. Fig. 6H – n/a is added due to unequal number of cells between groups. Fig. 7B – n/a is added due to unequal number of cells between groups. Fig. 7C – n/a is added due to unequal number of cells between groups. Fig. 8D – NaN (Not a Number) is filled in latency to first AP data points that didn’t have latency (because trial wasn’t accompanied with firing). Fig. 8E – NaN (Not a Number) is filled in latency to first AP data points that didn’t have latency (because trial wasn’t accompanied with firing). Fig. S1C – NaN represents points that were not collected. Fig. S3 – n/a is added due to unequal number of mice between groups. Fig. S4F – n/a is added due to unequal number of mice between groups. Fig. S5A - NaN (Not a Number) is filled in F-I data points that were not examined for some cells, due to the fear of losing the cell with overexcitation or because frequency could not be calculated since trial results with single AP without inter-spike-interval. Fig. S5B - NaN (Not a Number) is filled for regularity index points that couldn’t be calculated because either the numerator were NaN, or point was not examined due to the fear of losing the cell with overexcitation. Fig. S5C - NaN (Not a Number) is filled for adaptation ratio points that couldn’t be calculated because there were not at least 2 ISIs, or point was not examined due to the fear of losing the cell with overexcitation. Fig. S5D - NaN (Not a Number) is filled for coefficient of variation points that couldn’t be calculated because there was not at least 1 ISI, or point was not examined due to the fear of losing the cell with overexcitation. Fig. S5E – n/a is added due to unequal number of cells between groups. Fig. S6 – n/a is added due to unequal number of cells between groups. Fig. S7A - NaN (Not a Number) is filled in F-I data points that were not examined for some cells, due to the fear of losing the cell with overexcitation or because frequency could not be calculated since trial results with single AP without inter-spike-interval. Fig. S7B - NaN (Not a Number) is filled for regularity index points that couldn’t be calculated because either the numerator were NaN, or point was not examined due to the fear of losing the cell with overexcitation. Fig. S7C - NaN (Not a Number) is filled for adaptation ratio points that couldn’t be calculated because there were not at least 2 ISIs, or point was not examined due to the fear of losing the cell with overexcitation. Fig. S7D - NaN (Not a Number) is filled for coefficient of variation points that couldn’t be calculated because there was not at least 1 ISI, or point was not examined due to the fear of losing the cell with overexcitation. Fig. S7E – n/a is added due to unequal number of cells between groups. Fig. S8C left – NaN (Not a Number) is filled in F-I data points that were not examined for some cells, due to the fear of losing the cell with overexcitation. Fig. S8C right – n/a is added due to unequal number of cells between groups. Fig. S8D – NaN (Not a Number) is filled in latency to first AP data points that either didn’t have latency (because trial wasn’t accompanied with firing) or it was not examined due to the fear of losing the cell with overexcitation. Fig. S8E – n/a is added due to unequal number of cells between groups. Fig. S10B – NaN (Not a Number) is filled in latency to first AP data points that either didn’t have latency (because trial wasn’t accompanied with firing) or it was not examined due to the fear of losing the cell with overexcitation. Fig. S11C – n/a is added due to unequal number of cells between groups. Fig. S12 – n/a is added due to unequal number of cells between groups. Fig. S13 – n/a is added due to unequal number of cells between groups. Fig. S15A– n/a represents points excluded by criteria depicted in methods, or due to camera recording failure. Fig. S15B left – NaN (Not a Number) is filled in F-I data points that were not examined for some cells, due to the fear of losing the cell with overexcitation. Fig. S15B right – n/a is added due to unequal number of cells between groups. Fig. S15C – n/a is added due to unequal number of cells between groups. **Abbreviations** | **Abbreviation** | **Full Term** | | :--------------- | :---------------------------------------------------------------------------- | | AP | Action Potential | | AHP | After-Hyperpolarization | | CCI-dION | Chronic Constriction Injury of the Distal Infraorbital Nerve | | IA | A-type Potassium Current | | f-I | Frequency–Current Relationship | | Rin | Input Resistance | | RMP | Resting Membrane Potential | | EPSC | Excitatory Postsynaptic Current | | IPSC | Inhibitory Postsynaptic Current | | ISI | Inter-Spike Interval | | TG | Trigeminal Ganglion | | PBN | Parabrachial Nucleus | | PN | Projection Neuron | | DAPI | 4′,6-Diamidino-2-Phenylindole (nuclear stain) | | 4-AP | 4-Aminopyridine (IA channel blocker) | | ChR2 | Channelrhodopsin-2 | | EYFP | Enhanced Yellow Fluorescent Protein | | UV | Ultraviolet (refers to UV photokeratitis model) | | SUDO | Simplified Up-Down Method | | SES | Standard Extracellular Solution | | NaN | Not a Number (missing measurement due to technical or biological constraints) | | n/a | Not Applicable |   #### For a Computational Model dataset #### File: Computational\_Model\_PN.zip **Description:** This dataset contains the full source code and simulation scripts for the point-neuron computational model described in the Figure 8 of the manuscript. The model simulates the intrinsic excitability of medullary dorsal horn projection neurons, with a focus on IA potassium current modulation in acute pain states. Simulations reproduce the firing frequency, latency to first action potential, and other electrophysiological features under normal and inflammatory conditions. **Files Included:** * main.py: Defines the PointNeuron class, including morphology and ion channel mechanisms * parameter_sweep.py: Runs simulations and parameter sweeps including: * Validation of membrane properties * Input resistance and resting potential * Frequency and latency curves * Vhalf parameter sweep for IA modulation * DOCS: file containing additional explanation of model parameters * destination_path.eps: Exported simulation figure (used for publication) ## Code/software **Simulation Details:** * Simulation environment: [NEURON](https://www.neuron.yale.edu/neuron) (version ≥7.8) * Stimulus protocol: Current injection (100–500 ms, 0.06–0.14 nA) * Outputs: Membrane potential traces, frequency-intensity (FI) curves, latency plots * Vhalf modulation: IA inactivation curves simulated between -49.32 mV (baseline) and -45.2 mV (inflammation)   **How to Run:** 1. Install NEURON and Python (≥3.7) 2. Compile the .mod files if present using nrnivmodl 3. Run parameter_sweep.py to: * Validate firing under various current amplitudes * Simulate control vs. inflammatory shifts in IA * Plot and visualize FI curves, latency, and frequency ---   1\. Clone the current repository to your local machine. 2\. Open NEURON and load the project files. 3\. Modify the parameters and settings as needed for your experiments. 4\. Run the simulations and analyze the results. --- **Abbreviations** | **Abbreviation** | **Full Term** | | :--------------- | :---------------------------------------------------- | | AP | Action Potential | | FI | Frequency–Current Relationship | | IA | A-type Potassium Current | | RMP | Resting Membrane Potential | | Rin | Input Resistance | | EPS | Encapsulated PostScript (figure format) | | NEURON | Neural simulation environment | | SEClamp | Single-electrode clamp | | IClamp | Intracellular current clamp | | Vhalf | Voltage at half-maximal inactivation of IA | | vhalfm\_borgka | NEURON parameter controlling IA inactivation midpoint | | NaN | Not a Number |   ## Access information Other publicly accessible locations of the data: * The computational model parameters and the code are also available on Git Hub [https://github.com/hujiroyyanai/Guardians-of-passage](https://github.com/hujiroyyanai/Guardians-of-passage) Data was derived from the following sources: * This dataset originates from experimental and computational work performed in the lab of Prof. Alexander Binshtok at the Hebrew University of Jerusalem. The **experimental data** include electrophysiological recordings and behavioral assays conducted in murine models of acute (UV photokeratitis) and chronic (chronic constriction injury of the distal infraorbital nerve) pain. These data were used to generate all quantitative results reported in the manuscript *"Opposite regulation of medullary pain-related projection neuron excitability in acute and chronic pain"*. * The **computational model** was developed in parallel to simulate the firing properties of medullary dorsal horn projection neurons, incorporating experimental findings on IA current modulation. Model parameters and validation procedures were directly informed by the experimental measurements, ensuring that simulation outputs reflected observed biological phenomena.