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Pnas201214605 1.5

Pain relief produces negative reinforcement throughactivation of mesolimbic reward–valuation circuitryEdita Navratilovaa,1, Jennifer Y. Xiea,1, Alec Okuna, Chaoling Qua, Nathan Eydea, Shuang Cia, Michael H. Ossipova,Tamara Kingb, Howard L. Fieldsc, and Frank Porrecaa,2 aDepartment of Pharmacology, Arizona Health Sciences Center, University of Arizona, Tucson, AZ 85724; bDepartment of Physiology, University of NewEngland, Biddeford, ME 04005; and cErnest Gallo Clinic and Research Center, University of California at San Francisco, Emeryville, CA 94608 Edited by Leslie Lars Iversen, University of Oxford, Oxford, United Kingdom, and approved November 2, 2012 (received for review August 22, 2012) Relief of pain is rewarding. Using a model of experimental post- (avoidance of touching the floor with the injured area) and surgical pain we show that blockade of afferent input from the thermal hypersensitivity (decreased response latencies to a nox- injury with local anesthetic elicits conditioned place preference, ious thermal stimulus). As demonstrated previously (22), evoked activates ventral tegmental dopaminergic cells, and increases dopa- pain hypersensitivity was prominent at 24 h and still present, mine release in the nucleus accumbens. Importantly, place prefer- although diminished, at 96 h postincision. Peripheral nerve block ence is associated with increased activity in midbrain dopaminergic (PNB) with popliteal fossa (PF) lidocaine injection given 24 h neurons and blocked by dopamine antagonists injected into the postincision resulted in strong preference for the chamber paired nucleus accumbens. The data directly support the hypothesis that with PNB, demonstrating negative reinforcement. In contrast, in relief of pain produces negative reinforcement through activation of sham-operated animals or at 96 h postincision, when evoked the mesolimbic reward–valuation circuitry.
hypersensitivity is still present, pairing PNB with the context didnot produce CPP (Fig. 1 A and B). PNB at the site contralateral motivated behavior incision in vivo microdialysis to the injured hind paw did not result in CPP con- immunohistochemistry ventral tegmental area firming that lidocaine at the dose used for PF injection does not produce systemic effects on pain relief, as demonstrated pre- Reinforcement of behaviors that maximize benefit (positive re- viously (23). These data suggest that relief of postsurgical, on- inforcement) and reduce loss or injury (negative reinforcement) going (i.e., spontaneous) pain is rewarding.
is crucial for survival. Whereas positive reinforcement can be To determine whether the mesolimbic reward circuit is neces- produced by activation of mesolimbic dopaminergic pathways, sary for PNB-induced CPP, we investigated whether CPP would the neural circuits that underlie negative reinforcement are not be prevented by inactivation of dopaminergic neurons in the well understood. Ongoing pain can be "unmasked" in animals VTA. Microinjection of lidocaine (4%, wt/vol; 0.5 μL per side) to using conditioned place preference (CPP). Thus, in the presence of block neuronal activity, including inhibition of fibers of passage, in ongoing pain, pairing manipulations that are not rewarding in the the VTA 10 min before PNB prevented PNB-induced CPP (Fig.
absence of pain, such as peripheral nerve block (PNB) or in- 1C). Moreover, VTA baclofen, a GABAB receptor agonist (25 ng/ trathecal administration of ω-conotoxin or clonidine, with a pre- 0.2 μL per side), known to inhibit firing of dopaminergic neurons viously neutral context elicits CPP (1–3). CPP resulting from pain and reduce NAc dopamine release (24–26), also abolished PNB- relief is a measure of negative reinforcement.
induced CPP. In the absence of PNB, pairing VTA baclofen Human functional imaging studies have shown that offset of an treatment with a chamber had no effect on place preference ( acute noxious stimulus (4, 5) or placebo analgesia (6) activates ). In contrast, although endogenous opioids in the VTA un- brain regions that overlap extensively with those implicated in derlie the positive reinforcing effects of addictive drugs (27), appetitive rewards, in particular the ventral tegmental area (VTA), pretreatment of the VTA with a nonselective opioid receptor and its dopaminergic projections to the nucleus accumbens (NAc) antagonist, naloxone (3 μg/0.5 μL per side), did not prevent PNB- (5, 6). Manipulations that disrupt mesolimbic dopamine trans- induced CPP (Fig. 1C).
mission attenuate food or drug reward-induced CPP (7, 8). Elec- Activation of VTA dopaminergic neurons in injured rats fol- trophysiological recordings from dopaminergic neurons in the lowing PNB was investigated using immunohistochemistry and VTA demonstrate phasic neuronal activation by primary food or expression of c-FOS, a marker of neuronal activity (28). In cor- liquid rewards, by rewarding drugs, and reward-predicting cues (9).
onal brain sections the number of FOS-positive cells was counted Similarly, immunohistochemical studies show increased expression along the anteroposterial axes of the VTA identified by staining of the immediate early gene cFOS in the VTA in response to re- of catecholaminergic (dopaminergic) neurons with tyrosine hy- warding drugs, providing further support for an enhanced neuronal droxylase (TH) (Fig. 2 and ). PNB in injured rats increased activity (10–13). The NAc can be anatomically and functionally the number of FOS-expressing cells in posterior regions of the divided into core and shell regions that respectively receive pro- VTA at −5.8 to −6.3 mm from bregma. Incision injury itself, or jections from the lateral and medial VTA (14). In vivo micro- PNB in sham rats, did not change FOS expression (sham/saline, dialysis measurements or fast-scan voltammetry demonstrate that 37 ± 8; sham/lidocaine, 41 ± 10; incision/saline, 39 ± 6; incision/ appetitive rewards promote an efflux of dopamine in the NAc (15, lidocaine, 62 ± 8; n = 4–5 rats). Confocal images acquired within 16). It has been suggested that NAc neurons signal reward valueand participate in behavioral decision making (17–21).
We hypothesized that relief of ongoing pain would activate the Author contributions: T.K., H.L.F., and F.P. designed research; E.N., J.Y.X., A.O., C.Q., N.E., mesolimbic dopamine pathway and that such activation is nec- and S.C. performed research; E.N., J.Y.X., M.H.O., and T.K. analyzed data; and E.N., J.Y.X., essary for negative reinforcement. We tested this hypothesis di- H.L.F., and F.P. wrote the paper.
rectly in rats with incisional injury-induced pain (22) subsequently The authors declare no conflict of interest.
relieved by peripheral nerve block.
This article is a PNAS Direct Submission.
1E.N. and J.Y.X. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: Incision of the skin and underlying hind-paw tissue induced This article contains supporting information online at time-dependent, observable pain behaviors, including guarding PNAS Early Edition 1 of 5

PNB in rats with incisional pain produced VTA- and NAc-dependent CPP. PNB in injured rats 24 h, but not 96 h, following incision of the hind paw induced CPP as demonstrated by (A) increased time spent in chambers paired with popliteal fossa (PF) lidocaine and (B) difference scores. (C) Pretreatmentwith lidocaine or baclofen, but not naloxone, in the VTA 10 min before PNB abolished CPP. (D) Pretreatment with NAc lidocaine or flupenthixol blocked CPP.
Mean ± SEM, n = 9–13, *P < 0.05, Student's paired t test.
the posteromedial VTA at bregma −5.8 mm (rectangle in Fig.
difficult to evaluate in laboratory animals. In agreement with our 2A) demonstrate that increased FOS immunoreactivity occurred previous findings in other models of experimental pain (1, 3, 30, preferentially in TH-positive (dopaminergic) neurons (sham/sa- 31), our current behavioral data demonstrate that the removal of line, 20 ± 5; sham/lidocaine, 17 ± 7; incision/saline, 18 ± 2; in- the aversive state resulting from ongoing postsurgical nociceptive cision/lidocaine, 32 ± 8; n = 4–5 rats).
input by PNB elicits CPP. Importantly, no CPP was observed In vivo microdialysis in awake and freely moving rats was following PNB in sham-treated rats or 96 h after the incision performed to investigate potential efflux of dopamine in the NAc surgery, when injury-induced ongoing pain was likely to be greatly shell of incised rats following PNB. Basal NAc dopamine levels diminished. Thus, as we have previously shown, ongoing (i.e., did not differ between incised and sham-treated animals [4.68 ± spontaneous) pain can be unmasked by demonstrating that its 0.45 pg/30 μL (n = 15) and 4.71 ± 0.45 pg/30 μL (n = 13), re- removal is rewarding using CPP (1, 3, 30, 31).
spectively]. An increase from baseline was detected in injured rats Findings that relief of ongoing incisional, neuropathic, osteo- during the 120-min time period following PNB, with peak levels arthritic, and inflammatory pain (1, 3, 30) produces CPP confirms in the 30- to 60-min fraction elevated by 63 ± 24% (Fig. 3A). The that pain relief elicits reward. However, direct neurochemical area under the curve of percent change from baseline was sig- evidence for the activation of the brain reward circuitry during nificantly higher only in incised rats given PNB 24 h, but not 96 h, pain relief has not previously been established directly. Here, we postincision (Fig. 3B). Administration of cocaine (20 mg/kg, i.p.) show that (i) peripheral nerve block at 24 h, but not 96 h, pro- after the testing induced a robust increase (about 500%) in ex- duces CPP in animals with hind-paw incision consistent with tracellular dopamine in both incised and sham animals and served ongoing surgery-induced pain; (ii) activation of dopaminergic as a positive control.
projections from the VTA to NAc is necessary for PNB-induced In line with dopamine efflux in the NAc following pain relief, CPP; (iii) dopaminergic transmission in the NAc is required for we further investigated whether inhibiting the dopaminergic ac- PNB-induced CPP; and (iv) in contrast, PNB-induced CPP does tivity of the NAc would prevent PNB-induced CPP (Fig. 1D).
not require endogenous opioids in the VTA. These findings are Microinjection of lidocaine (4%, wt/vol; 0.5 μL per side) into the consistent with functional magnetic resonance imaging studies in NAc shell 10 min before PNB in injured rats abolished CPP.
humans showing increased activation of the NAc at the offset of More specifically, blockade of dopamine signaling by a non- an acute noxious stimulus (pain relief) (5).
selective dopamine receptor antagonist, flupenthixol (3 μg/0.5 μL Behavioral, electrophysiological (9), and in vivo microdialysis per side) (29), also prevented CPP. This directly demonstrates (15, 16) studies have consistently demonstrated activation of that dopaminergic transmission in the nucleus accumbens is re- mesolimbic dopaminergic neurons by natural rewards, rewarding quired for the negative reinforcement (manifested as CPP) that drugs, and reward-predicting cues (11, 32, 33). Distinct patterns results from pain relief. In the absence of PNB, pairing NAc of neuronal activity are consistent with the role of the VTA in flupenthixol treatment with a chamber had no effect on place positive reinforcement and learned appetitive behavior (34). Our preference (These results implicate mesolimbic dopa- data showing activation of VTA dopaminergic neurons and do- minergic signaling in pain-relief–induced CPP.
pamine efflux in the NAc indicate that a population of midbraindopamine neurons is activated following termination of an aver- sive state and may underlie negative reinforcement. Importantly, Pain is a subjective and multidimensional experience with sen- because PNB-induced CPP is prevented by blockade of meso- sory, affective, and cognitive components. The subjective un- limbic dopamine neurotransmission, activation of VTA neurons pleasantness of pain is essential to the human experience but appears to be necessary for reinforced learning.
Navratilova et al.

PNB increased the number of FOS-positive dopaminergic neurons in the VTA. (A) A coronal brain section at bregma −5.8 mm outlining the VTA identified by tyrosine hydroxylase labeling (red). (B) The average number of FOS+ cells (green) per section was calculated between bregma −5.2 and −6.8 mm.
(C) The number of FOS+ cells increased significantly at bregma −5.8 to −6.3 mm only in incised rats following PNB. (D) Confocal images within the medial VTA(rectangle in Fig. 2A). The number of dopaminergic FOS+/TH+ cells (E) increased in incised rats after PNB (F); the number of nondopaminergic FOS+/TH- cells(G) did not change (H). Mean ± SEM, n = 4–5 rats/group, *P < 0.05; two-way ANOVA with Bonferroni multiple comparisons test (B), one-way ANOVA withTukey's multiple comparisons test (C and F).
The midbrain VTA is an anatomically and functionally heteroge- these neurons, dopamine levels were increased in the medial shell of neous region composed of several nuclei including the parabrachial the NAc. Thus, pain relief reward appears to share many similarities pigmented area (PBP), paranigral nucleus (PN), rostral linear nu- with appetitive rewards. However, as indicated by our findings with cleus raphe (RLi), interfascicular nucleus (IF), and caudal linear VTA naloxone, activation of a reward circuit by pain relief does not nucleus raphe (CLi) (Fig. 2A). The activation of posteromedial VTA appear to involve an endogenous opioid system in the VTA, sug- dopaminergic neurons, which primarily target the medial accumbens gesting that there may be important differences as well (27, 35, 36).
shell, appears to promote positive affective states (14). Accordingly, Acute noxious stimuli have been reported to excite some and the present study found enhanced cFOS expression in the poster- inhibit other midbrain dopamine neurons (37), suggesting multi- omedial division of the VTA. In agreement with cellular targets of ple populations of neurons support different aspects of aversive PNB increased the extracellular dopamine (DA) levels in the NAc shell of rats with incisional pain. (A) Extracellular dopamine levels increased above basal levels following PNB only in incised animals. (B) Area under the curve (AUC) of percent change from baseline demonstrated significant dopamine releasewithin 120 min after PNB given 24 h, but not 96 h, following incision. Mean ± SEM, n = 3–8 rats/group, *P < 0.05; one-way ANOVA with Tukey's multiplecomparisons test.
Navratilova et al.
PNAS Early Edition 3 of 5

processing (38). These diverse patterns of neuronal activity are 200 μL of saline or lidocaine (4%, wt/vol; Qualitest Pharmaceuticals) into PF likely reflected in the observed complexity of dopaminergic under light isofluorane anesthesia (30).
signaling in experimental models of tonic pain (39, 40). In thepresent study, we found no change in the number of FOS-posi- Brain Microinjection. Bilateral intracranial microinjections of 4% (wt/vol) li-docaine hydrochloride (0.5 μL per side), baclofen (25 ng/0.2 μL per side; tive neurons in incised versus sham rats. Further, the basal do- Sigma), flupenthixol (3 μg/0.5 μL per side; Sigma), or naloxone hydrochloride pamine levels in the NAc of incised and sham rats did not differ.
(3 μg/0.5 μL per side; Tocris) in the VTA or NAc were done using injectors Thus, 24 h following incisional surgery, before PNB-induced pain extended 1 mm beyond the guide cannula. All animals were euthanized via relief, we saw no net change in mesolimbic dopamine transmission.
CO2 overdose at the end of the experiments and the placement of the guide Numerous factors may account for this observation, including cannulas/injection sites was confirmed with histology methods. Data from (i) the sensitivity of our analytical technique, (ii) the non- animals with misplaced cannulas were removed from the analyses.
chronic nature of the injury, or (iii) involvement of VTA neu-rons that project to other sites beyond the NAc.
Guarding Behavior. Assessment of guarding behavior was done as previously How afferent nociceptive pathway(s) modulate the reward cir- described (30). Rats were observed for 10 s each at 1-min intervals and scored cuit is not known. In the rat, major ascending nociceptive pathways 0–2 (0, injured hind-paw area was touching the mesh and the area wasblanched or distorted by the mesh; 1, the injured hind paw touched the mesh from lamina I of the lumbar spinal cord terminate in the lateral without blanching or distortion; 2, the injured hind paw was completely off parabrachial nucleus (PBN) or in the thalamic nuclei (41, 42).
the mesh). For each rat a cumulative score was obtained by adding 30 scores Midbrain dopaminergic neurons receive direct nociceptive inputs during the 30-min testing period. All testing was done by an experimenter from projections in the PBN (43). The anterior cingulate cortex blinded to the treatment conditions.
(ACC) receives prominent inputs from nociceptive neurons in thethalamus, and activation of the ACC through the spinothalamic Thermal Hypersensitivity. Nociceptive withdrawal thresholds to noxious ra- pathway contributes to the aversiveness of nociceptive stimuli.
diant heat were determined using the Hargreaves test. Rats were allowed to Accordingly, functional imaging studies have implicated the ACC acclimate within a Plexiglas enclosure on a clear glass plate for 30 min. A in processing the unpleasant affective aspects of pain in humans radiant heat source was directed onto the plantar surface of the left hindpaw. A motion detector halted both heat lamp and timer when the paw was (44). The ACC projects directly or via other limbic regions such as withdrawn. Baseline latencies were established at 20 s. A maximal cutoff of the amygdala to the mesolimbic reward system (45, 46). Recent 30 s was used to prevent tissue damage.
work has identified a circuit that includes the lateral habenulaneurons that indirectly inhibit midbrain dopamine neurons (47).
Conditioned Place Preference Procedures. A single trial conditioning protocol Inhibition of this input pathway would disinhibit VTA dopa- was used for CPP as previously described (2, 30). All rats underwent handling mine neurons.
by the experimenter before the preconditioning phase. On preconditioning In conclusion, we demonstrate that relief of ongoing post- day, rats were placed into the CPP boxes with access to all chambers; time surgical pain following PNB produces CPP and activates the mes- spent in each chamber was determined by an automated process and ana- olimbic dopaminergic circuit implicated in positive reinforcement.
lyzed across 15 min to verify no preconditioning chamber preference. Fol-lowing preconditioning, rats received incision or sham surgeries and were PNB elicits efflux of dopamine in the NAc shell only in the setting placed back into their home cages overnight. On conditioning day (24 h of injury-induced, ongoing pain. Additionally, inhibition of do- postincision), rats received a saline (200 μL) injection into the PF and were paminergic neurons in the VTA and dopamine signaling in their immediately (within 2 min) placed into the appropriate pairing chamber.
projection target in the NAc shell prevented PNB-induced CPP, Four hours later, rats received a lidocaine injection (4%, wt/vol; 200 μL) into providing direct evidence for a causal relationship between acti- the PF and were placed into the opposite chamber. Chamber pairings were vation of this mesostriatal circuit and the negative reinforcing counterbalanced. To determine the role of the VTA or NAc in PNB-induced effect of pain relief. These data indicate that activation of the CPP, rats with VTA or NAc cannulas received saline injection into the VTA or VTA to NAc dopamine signaling contributes to both positively NAc followed 10 min later by PF saline injection and immediate placement and negatively reinforced behavior.
into the appropriate pairing chambers. Four hours later, rats received treat-ment drug injection into the VTA or NAc followed in 10 min by PF lidocaine Materials and Methods injection and placement into the opposite chambers. The conditioning timewas 30 min in each chamber. On test day, 20 h following the afternoon Animals. Adult, male Sprague-Dawley rats (250–350 g; Harlan) were used. All pairing, rats were placed in the CPP box with access to all chambers and procedures were performed in accordance with the policies of the National behavior was recorded for 15 min for analysis for chamber preference. Dif- Institutes of Health guidelines for laboratory animals under protocols ap- ference scores were calculated as test time minus preconditioning time spent proved by the University of Arizona Institutional Animal Care and Use in the PF–lidocaine paired chamber.
Committee. Rats were housed three per cage on a 12-h light–dark cycle withfood and water provided for ad libitum consumption.
Immunohistochemistry. Two hours after treatment rats were anesthetizedby i.p. ketamine/xylazine and transcardially perfused with 4% (wt/vol) Intracranial VTA and NAc Cannulation. Stereotaxic surgeries were performed paraformaldehyde. Coronal brain sections (30 μm thick) were cut in a Microm in anesthetized rats (i.p. ketamine/xylazine 80/12 mg/kg; Western Medical HM 525 cryostat and mounted on Superfrost Plus microscope slides. Brain Supply/Sigma) according to the brain atlas. Two 26-gauge guide cannulas tissue was permeabilized with 0.2% Triton X-100, blocked with 5% (wt/vol) (Plastics One) were directed toward the following coordinates: VTA [ante- normal goat serum and incubated overnight with the mixture of primary roposterior (AP), bregma −5.8 mm; mediolateral (ML), midline ±0.6 mm; antibodies of rabbit polyclonal anti-cFOS (sc-52, 1:25,000; Santa Cruz) and dorsoventral (DV), skull −8.0 mm], NAc shell (AP, bregma +1.5 mm; ML, mouse monoclonal anti-tyrosine hydroxylase (MAB55280, 1:3,000; Milli- midline ±1.0 mm; DV, skull −6.5 mm). For microdialysis, a single guide can- pore). The sections were incubated with biotinylated anti-rabbit antibody nula (AG-8; EICOM Corp.) was implanted into the left NAc (AP, bregma +1.7 followed by the ABC complex (Vectastain Elite ABC kit; Vector Laboratories, mm; ML, midline −1.0 mm; DV, skull −6.0 mm). Stainless steel dummy can- Inc.) and tyramide signal amplification detection (TSA Plus Fluorescein Kit; nulas were inserted to keep the guide free of debris. After surgery, rats were Perkin-Elmer). TH was visualized with anti-mouse Alexa Fluor 555 (1:1,000; housed individually and allowed to recover for 5–7 d.
Molecular Probes, Invitrogen). Slides were mounted in Vectashield mountingmedium (Vector Laboratories, Inc.) and examined under an Olympus BX51 Incisional Injury Pain Model. Incision injury of the skin plus deep tissue, in- microscope equipped with a Hamamatsu C8484 digital camera. Confocal cluding fascia and underlying muscle, was done as described by Brennan et al.
images were obtained with a Zeiss LSM520 laser scanning confocal micro- (22). Rats were anesthetized with 2% (vol/vol) isoflurane, a 1-cm longitudi- scope using the 488- and 543-nm excitation wavelengths. Micrographs of nal incision was made through the skin of the left hind paw, and the 10–15 sections per rat 150 μm apart within the bregma −5.20 to −6.80 mm plantaris muscle was elevated and incised longitudinally. The cut skin was were analyzed for FOS expression. In the ImageJ software the VTA area stitched with two 5–0 nylon sutures and the wound site treated with neo- including the PBP, PN, RLi, IF, and CLi was outlined according to the TH mycin. Sham animals were anesthetized and the left hind paw was cleaned, staining. FOS-positive nuclear puncta within the outlined area were counted but no incision was made. Peripheral nerve block was achieved by injecting manually by an observer blinded to the treatment conditions. Coexpression Navratilova et al.

of FOS and TH was evaluated within the medial VTA at bregma −5.80 mm column and Coulochem III 5014B electrochemical detector (ESA). The guard (rectangle in Fig. 2A) in two to four sections per each rat using the Zeiss cell was set at 350 mV, electrode 1 at −150 mV, and electrode 2 at 250 mV.
LSM520 confocal microscope images. Cells with nuclear FOS staining and Standard curve was obtained from seven serial dilutions of dopamine a visible cytoplasmic TH staining (Fig. 2E) were counted as dopaminergic; (2.5–160 pg in 20 μL aCSF plus antioxidant mixture). The limit of detection FOS-positive cells with no clear cytoplasmic TH staining (Fig. 2G) were (LOD) and limit of quantification (LOQ) were calculated according to the counted as nondopaminergic.
formulas LOD = 3.3 (SDr/S) and LOQ = 10 (SDr/S), where the SD of the responseSDr (SD of y intercepts of regression lines) and the slope of the standard curve In Vivo Microdialysis and HPLC Quantification of Dopamine. Microdialysis was S was determined from the measurements of 10 independent standard curves done in awake, freely moving animals. The microdialysis probe (A-I-8-02; (The data from rats that failed to generate dopamine efflux following Eicom) was inserted into the NAc with 2 mm of semipermeable membrane cocaine treatment were excluded. Dopamine concentrations were expressed projecting beyond the guide cannula (and perfused at 1.25 μL/min as percent of the corresponding baseline level.
with artificial cerebrospinal fluid (aCSF: 147.0 mM NaCl, 2.8 mM KCl, 1.2mM MgCl2, and 1.2 mM CaCl2). After a 90-min washout period, two base-line and four treatment fractions (30 min/fraction) were collected into Statistical Analysis. Statistical analyses were calculated using GraphPad Prism prechilled (4 °C) Eppendorf tubes containing 1.0 μL of 40× antioxidant so- 5 software. Results were expressed as mean ± SEM. Two-way ANOVA with lution [6.0 mM L-cysteine, 2.0 mM oxalic acid, and 1.3% (vol/vol) glacial Bonferroni multiple comparisons test or one-way ANOVA with Tukey's acetic acid] (48). All rats were then injected with cocaine (20 mg/kg, i.p.) multiple comparison post hoc tests was used for between-groups compari- and dialysates were collected for additional 90 min. Fractions were ana- son. Student's paired t test was used to analyze the difference scores for the lyzed using an Agilent 1100 HPLC system with a 5020 guard cell, MD-150 CPP data. Significance was set at P < 0.05.
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